Tumor vaccine and application thereof

Abstract

A tumor vaccine comprising and / or expressing an antigen molecule, a co-stimulatory signal molecule, a cytokine, and an NLRC5 molecule. The tumor vaccine can further comprise and / or express CIITA molecules. The invention also provides application of the tumor vaccine in prevention and / or treatment of tumors.

Description

Tumor vaccines and their uses Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a tumor vaccine and its use in preventing and / or treating tumors. Background Art

[0002] Currently, tumor vaccines as a new type of tumor treatment have attracted more and more attention.

[0003] Tumor vaccines mainly activate the patient's own immune system, using tumor cells or tumor antigens to induce the body to produce specific cellular and humoral immune responses against tumor-specific antigens or tumor-associated antigens, thereby enhancing the body's anti-cancer ability, and alleviating, preventing or controlling tumor growth, spread and recurrence.

[0004] Currently, only one cancer vaccine is available for prostate cancer, which has been shown to extend patient survival by three months. Currently, various tumor cell-based vaccines are undergoing preclinical validation and clinical trials, including peptide-based vaccines, tumor cell-based vaccines, recombinant protein vaccines, and mRNA-based vaccines. However, the development of these vaccines has encountered numerous obstacles. The low success rate of cancer vaccines is primarily due to two factors: 1) Lack of suitable tumor antigens and insufficient tumor antigen coverage. Early cancer vaccines often used tumor-associated antigens. However, since high-affinity T cells targeting tumor-associated antigens are eliminated early in immune system development, the immune response to these antigens is theoretically weak. Furthermore, the various tumor-specific antigens discovered so far are not universally applicable due to the inherent MHC restriction of T cells. Furthermore, the number of individual antigens present is less than 1 / 100th or even 1 / 1000th of the numerous mutated antigens in a tumor, making them ineffective in activating a polyclonal anti-tumor immune response. 2) The immunosuppressive environment created by the tumor limits the immune activation capacity of the vaccine.

[0005] Therefore, in the means of tumor prevention and / or treatment, new tumor vaccine strategies need to be developed to overcome the obstacles of insufficient tumor antigen coverage and low immune activation ability of existing tumor vaccines.

[0006] Summary of the Invention

[0007] The present application provides a tumor vaccine, which contains and / or expresses antigen molecules, co-stimulatory signal molecules, cytokines, and antigen presentation enhancing molecules, such as NLRC5 and / or CIITA. By activating NLRC5 and CIITA, it can highly express antigen presenting molecules MHC-I, MHC-II and HLA-I, HLA-II and other molecules related to presentation, thereby obtaining antigen presentation capabilities similar to those of dendritic cells, combined with continuously expressed co-stimulatory signal molecules, such as CD80, 4-1BBL, etc., and third cytokine signals, such as IL-2, IL-7, IL-9, IL-15, IL-21, etc., tumor vaccines can provide three signals for T cell activation without relying on other auxiliary conditions, thereby promoting T cell activation and producing corresponding functions. The tumor vaccine of the present application can activate all mutations and related antigens of the tumor, obtain polyclonal anti-tumor immunity, and can be designed for the prevention and / or treatment of various tumors, with a broad spectrum. At the same time, the tumor vaccine provided in this application can also be administered in combination with other therapies (for example, antibody therapy, cell therapy, gene therapy, etc.) to achieve a stronger tumor treatment goal.

[0008] In one aspect, the present application provides a tumor vaccine comprising and / or expressing an antigen molecule, a co-stimulatory signal molecule, a cytokine, and an NLRC5 molecule.

[0009] In certain embodiments, the NLRC5 molecule comprises a functionally active fragment of a NLRC5 molecule.

[0010] In certain embodiments, the NLRC5 molecule comprises the amino acid sequence shown in SEQ ID NO:2.

[0011] In certain embodiments, the tumor vaccine further comprises and / or expresses a CIITA molecule.

[0012] In certain embodiments, the CIITA molecule comprises a functionally active fragment of a CIITA molecule.

[0013] In certain embodiments, the CIITA molecule comprises the amino acid sequence shown in SEQ ID NO:1.

[0014] In certain embodiments, the NLRC5 molecule or a functionally active fragment thereof and the CIITA molecule or a functionally active fragment thereof are expressed as a fusion protein.

[0015] In certain embodiments, the NLRC5 molecule and / or CIITA molecule is capable of enhancing antigen presentation.

[0016] In certain embodiments, the antigenic molecule comprises a tumor-associated antigen.

[0017] In certain embodiments, the antigenic molecule comprises a tumor-specific antigen.

[0018] In certain embodiments, the antigen molecules include endogenous antigens and exogenous antigens.

[0019] In certain embodiments, the antigen molecule is selected from the group consisting of HA, E7, NY-ESO, MAGE-A3, MART, EGFR-VIII, PSMA, GPC-3, Mesothelin, PSA, and CLDN18.2.

[0020] In certain embodiments, the co-stimulatory signal molecule provides a second signal for T cell activation.

[0021] In certain embodiments, the co-stimulatory signaling molecule is selected from the group consisting of CD80, CD86, 4-1BBL, anti-CD28, OX40L, CD40 ligand, ICOS ligand, GITR ligand, 4-1BB ligand, OX40 ligand, TL1A, CD30 ligand, CD27, and Flt3 ligand.

[0022] In certain embodiments, the co-stimulatory signaling molecule is CD80.

[0023] In certain embodiments, the antigen molecule, co-stimulatory signaling molecule, cytokine, NLRC5 molecule and / or CIITA molecule is expressed by a method selected from the group consisting of viral vector expression, plasmid expression, liposome delivery and RNA molecule encoding.

[0024] In certain embodiments, the cytokine is capable of promoting T cell activation and / or proliferation.

[0025] In certain embodiments, the cytokine is selected from the group consisting of IL-2, IL-7, IL-9, IL-15, and IL-21.

[0026] In certain embodiments, the cytokine is selected from the group consisting of a mutant of IL-2, IL-7, IL-9, IL-15, and IL-21.

[0027] In certain embodiments, the cytokine is IL-2.

[0028] In certain embodiments, the tumor vaccine comprises and / or expresses antigenic molecules, NLRC5 and CIITA, CD80, and IL-2.

[0029] In certain embodiments, the tumor vaccine is a protein vaccine, a polypeptide vaccine, a microbial vector vaccine, or a cell vaccine.

[0030] In certain embodiments, the tumor vaccine has independent CD4 and CD8 T cell antigen presentation capabilities that are independent of host immune cells.

[0031] In certain embodiments, the host immune cells include dendritic cells, B cells, and / or macrophages.

[0032] In another aspect, the present application provides a pharmaceutical composition comprising the tumor vaccine and optionally a pharmaceutically acceptable carrier.

[0033] On the other hand, the present application provides a pharmaceutical combination comprising the tumor vaccine and an immunomodulator.

[0034] In certain embodiments, the immunomodulatory agent comprises a therapeutic antibody, a therapeutic cell, a therapeutic nucleic acid, and / or a therapeutic gene.

[0035] In certain embodiments, the therapeutic cells comprise immune cells.

[0036] In certain embodiments, the immune cells are selected from T cells, NK cells, NKT cells and TIL cells.

[0037] In certain embodiments, the immune cell is a modified immune cell.

[0038] In certain embodiments, the modified immune cell comprises an immune cell expressing a chimeric antigen receptor (CAR).

[0039] In certain embodiments, the modified immune cell comprises an immune cell that expresses a T cell receptor (TCR).

[0040] On the other hand, the present application provides a method for preparing the tumor vaccine, the pharmaceutical composition or the drug combination.

[0041] On the other hand, the present application provides uses of the tumor vaccine, the pharmaceutical composition, and the drug combination in preparing a drug for preventing and / or treating tumors.

[0042] In certain embodiments, the tumor is a solid tumor and / or a hematological tumor.

[0043] In certain embodiments, the tumor is selected from the group consisting of pancreatic cancer, gastric cancer, lung cancer, prostate cancer, melanoma, breast cancer, kidney cancer, liver cancer, and ovarian cancer.

[0044] On the other hand, the present application provides a method for preventing and / or treating tumors, which comprises administering the tumor vaccine, the pharmaceutical composition or the drug combination to a subject in need thereof.

[0045] In certain embodiments, the tumor is a solid tumor and / or a hematological tumor.

[0046] In certain embodiments, the tumor is selected from the group consisting of pancreatic cancer, gastric cancer, lung cancer, prostate cancer, melanoma, breast cancer, kidney cancer, liver cancer, and ovarian cancer.

[0047] On the other hand, the present application provides the tumor vaccine, the pharmaceutical composition, and the drug combination, which are used to prevent and / or treat tumors.

[0048] In certain embodiments, the tumor is a solid tumor and / or a hematological tumor.

[0049] In certain embodiments, the tumor is selected from the group consisting of pancreatic cancer, gastric cancer, lung cancer, prostate cancer, melanoma, breast cancer, kidney cancer, liver cancer, and ovarian cancer.

[0050] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] The specific features of the inventions of this application are set forth in the appended claims. The features and advantages of the inventions of this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0052] Figure 1 shows a schematic diagram of the plasmid structures of CIITA, NLRC5, CD80, 4-1BBL, IL-2, IL-21, anti-CD28, and HA-E7.

[0053] FIG2 shows the results of the effects of CIITA, CIITA, and NLRC5 on the expression of HLA-I and HLA-II in the H82 cell line.

[0054] FIG3 shows the results of the activation effect of CIITA on HA-Jurkat T cells.

[0055] FIG4 shows the results of the activation effect of CIITA on HA-CD4 T cells.

[0056] FIG5 shows the results of the activation effect of NLRC5 on E7-Jurkat T cells.

[0057] FIG6 shows the results of NLRC5 activation of E7-CD8 T cells.

[0058] FIG7 shows the results of activation of E7-Jurkat T cells and HA-Jurkat T cells by CIITA and NLRC5.

[0059] FIG8 shows the effect of IL-2 on tumor vaccine-enhanced T cell proliferation.

[0060] FIG9 shows the results of the activation of E7-Jurkat T cells and HA-Jurkat T cells by H82 cells expressing CD80.

[0061] FIG10 shows the results of the activation of E7-Jurkat T cells and HA-Jurkat T cells by CD80 expressed by CFPAC-1 cells.

[0062] FIG11 shows the results of the activation of E7-Jurkat T cells and HA-Jurkat T cells by anti-CD28 expressed on CFPAC-1 cells.

[0063] FIG12 shows the results of the induction effect of the tumor vaccine synergistically induced by NLRC5, CIITA, CD80, and IL-2 on the proliferation of HA-CD4 T cells and E7-CD8 T cells.

[0064] Figure 13 shows the in vivo anti-tumor effect of CAR-T induced by the synergistic tumor vaccine of NLRC5, CIITA, CD80, and IL-2.

[0065] Figure 14 shows the in vivo anti-tumor effect of TCR-T induced by tumor vaccine synergistically induced by NLRC5, CIITA, CD80, and IL-2.

[0066] FIG15 shows the activation results of endogenous and exogenous antigen-specific T cells induced by tumor vaccines synergistically induced by NLRC5, CIITA, CD80, and IL-2.

[0067] FIG16 shows the co-stimulatory effect of the tumor vaccine synergistically administered with NLRC5, CIITA, and OX40L on T cells.

[0068] FIG17 shows the results of the promotion of T cell proliferation by tumor vaccines synergized by NLRC5, CIITA, and IL-7 and tumor vaccines synergized by NLRC5, CIITA, and IL-15. DETAILED DESCRIPTION

[0069] The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0070] Definition of terms

[0071] In this application, the term "antigen" generally refers to any substance that elicits an immune response. For example, the immune response may involve the activation of specific immunocompetent cells or an immune reaction such as antibody production. In this application, the antigen may include a protein, peptide, or a nucleic acid molecule encoding the protein or peptide. In this application, the antigen may include a portion thereof that is capable of eliciting the desired immune response. For example, the portion that can elicit an immune response may include the full-length antigen or a fragment of the antigen. For example, a fragment of the antigen may retain the antigenic properties of a natural or synthetic antigen polypeptide and may elicit an immune response against the natural or synthetic antigen polypeptide. In this application, the antigen may include tumor-specific antigens and tumor-associated antigens. In this application, the term "tumor-specific antigen," also referred to as a mutated antigen, can be generated by mutation in normal cells and is typically expressed on tumor cells. In this application, the term "tumor-associated antigen" generally refers to an antigen that is also expressed in normal tissues and can be highly expressed in tumors. In this application, the antigen may include both endogenous and exogenous antigens. In this application, the term "endogenous antigen" generally refers to tumor antigens synthesized in tumor cells. The endogenous antigen peptides produced by proteasome degradation enter the endoplasmic reticulum and bind to class I molecules. For example, the endogenous antigen can be presented to CD8 T cells in the form of an antigen peptide-MHC-I class molecule complex. In this application, the term "exogenous antigen" generally refers to tumor antigens taken up from outside the cell by antigen-presenting cells and present in the cell capsule system. For example, after being degraded into short peptides by lysosomes, exogenous antigens can be presented to CD4 T cells for recognition through MHC-II class molecules, inducing an immune response involving CD4 T cells. In this application, for example, the antigens can be HA, E7, NY-ESO, MAGE-A3, MART, EGFR-VIII, PSMA, GPC-3, Mesothelin, PSA and CLDN18.2. In this application, the antigen can be a full-length protein and its variants, homologs, derivatives, analogs, functionally active fragments or fusion proteins. In the present application, the HA may be Influenza HA, or its variants, homologs, derivatives, analogs, functionally active fragments, or fusion proteins. For example, the HA may form a fusion protein with E7. In the present application, the E7 may be HPV E7, or its variants, homologs, derivatives, analogs, functionally active fragments, or fusion proteins. For example, the E7 may form a fusion protein with HA. For example, the antigen may be HA-E7.

[0072] In this application, the terms "costimulatory signal molecule" and "costimulatory molecule" can be used interchangeably, generally referring to a cell surface molecule or its ligand that specifically binds to a costimulatory ligand, provides a costimulatory signal for T cell activation, and mediates the costimulatory response of T cells. For example, the costimulatory signal molecule may include a costimulatory molecule or its ligand. For example, the costimulatory signal molecule may be a TNF / TNFR superfamily, an immunoglobulin superfamily, a Tim family or a SLAM family or its ligand. For example, exemplary costimulatory signal molecules may be selected from CD80, CD86, 4-1BBL, OX40L, CD40 ligand, ICOS ligand, GITR ligand, 4-1BB ligand, OX40 ligand, TL1A, CD30 ligand, CD27 and Flt3 ligand.

[0073] In this application, the term "cytokine" generally refers to a class of small molecule proteins with a wide range of biological activities that are synthesized and secreted by immune cells or certain non-immune cells upon stimulation. In this application, the cytokines may include cytokines that can enhance T cell function. For example, the cytokines may induce T cell maturation, proliferation, growth, and / or activation. In this application, the cytokines used to construct the tumor vaccine may include exogenous cytokines. In this application, cytokines may include interleukins (ILs), tumor necrosis factor, and other polypeptide factors. For example, the cytokines may include IL-2, IL-7, IL-9, IL-15, and IL-21. The cytokines in this application may include functionally active fragments thereof, proteins derived from natural sources or from recombinant cell culture, and biologically active equivalents of native sequence cytokines. The cytokines in this application may also include modified / optimized cytokines that have undergone one or more mutations. For example, the cytokines may include mutants of IL-2, IL-7, IL-9, IL-15, and IL-21. In the present application, the mutant may retain its function of causing T cell maturation, proliferation, growth and / or activation.

[0074] In this application, the term "NLRC5 (NLR family CARD domain containing 5)" generally refers to a key transcriptional regulator of MHC class I gene expression, a member of the NOD-like protein family, that can regulate the activity of immune cells. In this application, the NLRC5 may include NLRC5 from any species. For example, the NLRC5 may be human-derived NLRC5. In this application, the NLRC5 may also include full-length NLRC5 or a truncated NLRC5 that retains its function, for example, the NLRC5 may be a functionally active fragment of NLRC5. In this application, the NLRC5 may also include homologs, analogs, etc. of NLRC5. For example, the NLRC5 can be a protein or polypeptide having an amino acid sequence with at least about 70% (e.g., at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more) sequence identity. In the present application, the NLRC5 can include modified / artificially engineered NLRC5. For example, the NLRC5 can be linked to another protein to form a fusion protein. For example, the NLRC5 can be linked to CIITA to form a fusion protein.

[0075] In this application, the term "CIITA" can be used interchangeably with "type II transactivator protein", which generally refers to a regulatory factor for the transcription of MHC-II class molecule genes. It is a member of the NOD-like receptor family and can play a regulatory role in the constitutive expression of MHC-II class genes and IFN-γ induced expression. In this application, the CIITA can include CIITA of any species. For example, the CIITA can be CIITA derived from humans. In this application, the CIITA can also include full-length CIITA or truncated CIITA that retains its function, for example, the CIITA can be a functionally active fragment of CIITA. In this application, the CIITA can also include homologues, analogs, etc. of CIITA. For example, the CIITA can be a protein or polypeptide having an amino acid sequence with at least about 70% sequence identity (e.g., at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more). In the present application, the CIITA can include modified / artificially engineered CIITA. For example, the CIITA can be combined with other proteins to form a fusion protein. For example, the CIITA can form a fusion protein with NLRC5.

[0076] The proteins and / or amino acid sequences referred to in this application should also be understood to include at least the following scope: variants, homologs, derivatives, analogs, functionally active fragments, or fusion proteins that have the same or similar functions as the protein. In this application, the variant can be a protein or polypeptide in which one or more amino acids are substituted, deleted, or added in the amino acid sequence of the protein and / or polypeptide (e.g., NLRC5 or CIITA described in this application). For example, the functional variant can include a protein or polypeptide that has amino acid changes through at least one, such as 1-30, 1-20, or 1-10, or for example 1, 2, 3, 4, or 5 amino acid substitutions, deletions, and / or insertions. The functional variant can substantially retain the biological properties of the protein or polypeptide before the change (e.g., substitution, deletion, or addition). For example, the functional variant can retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., enhanced antigen presentation ability) of the protein or polypeptide before the change. For example, the substitution can be a conservative substitution.

[0077] In the present application, the homolog can be a protein or polypeptide having at least about 70% (e.g., at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence identity with the amino acid sequence of the protein and / or the polypeptide (e.g., NLRC5 or CIITA described herein).

[0078] In the present application, the derivative may be a protein or polypeptide (e.g., NLRC5 or CIITA described in the present application) whose amino acid sequence is modified, such as by coupling or complexing with other chemical substances or by modification techniques known in the art, derived from the basic sequence.

[0079] As used herein, the term "functionally active fragment" generally refers to a fragment that has a partial region of a full-length protein (e.g., NLRC5 or CIITA as described herein) but retains or partially retains the biological activity or function of the full-length protein. For example, a functionally active fragment of NLRC5 or CIITA may retain or partially retain the ability of the full-length protein to enhance antigen presentation.

[0080] In the present application, the fusion protein can be a protein composed of two or more polypeptides, which are generally not bound in their native state, but are bound together by peptide bonds at the amino and carboxyl termini to form a continuous polypeptide. It is understood that the binding refers to direct or indirect binding of the two or more polypeptide components. In the present application, the indirect binding can be through a peptide linker or spacer.

[0081] In the present application, the term "second signal of T cell activation" generally refers to a "costimulatory signal", which refers to the costimulatory signal required for T cell maturation, proliferation, growth and / or activation as an effector T cell process. For example, a costimulatory signal can be generated by the mutual binding of a costimulatory molecule and its ligand. For example, a costimulatory signal can be generated by the mutual binding of a costimulatory molecule and its ligand expressed on the surface of an immune cell. In the present application, the costimulatory signal molecule may include a costimulatory molecule or its ligand. For example, the costimulatory signal can be produced by the TNF / TNFR superfamily, the immunoglobulin superfamily, the Tim family or the SLAM family or its ligand. For example, the costimulatory signal of the present application can be produced by CD80, CD86, 4-1BBL, OX40L, CD40 ligand, ICOS ligand, GITR ligand, 4-1BB ligand, OX40 ligand, TL1A, CD30 ligand, CD27 and Flt3 ligand.

[0082] In this application, the term "viral vector expression" generally refers to the expression of a target protein molecule via viral vector infection. For example, the viral vector expression can be the delivery of a nucleic acid to a target cell via a viral vector, causing it to express the target protein. In this application, the viral vector can be a virus for delivering nucleic acids. For example, the viral vector can be a retrovirus, adenovirus, adeno-associated virus, herpes virus, poxvirus, baculovirus, papillomavirus, or papovavirus. For example, the viral vector can be a lentivirus.

[0083] In this application, the term "plasmid expression" generally refers to the expression of a target protein via a DNA or RNA molecule capable of autonomous replication. In this application, the plasmid can be used as a vector for transferring genetic material into cells, has the ability to autonomously replicate, can maintain a constant copy number in daughter cells, and can express the genetic information it carries. For example, the plasmid can be a DNA plasmid or an RNA plasmid. For example, the plasmid can be a linear plasmid or a circular plasmid.

[0084] In this application, the term "liposomal delivery" generally refers to the delivery of a target protein molecule to a target cell via a vesicle having an interior space that is isolated from the external medium by one or more bilayer membranes. In this application, the bilayer membrane of the liposome can be formed by amphiphilic molecules, such as synthetic or naturally derived lipids containing spatially isolated hydrophilic and hydrophobic domains, or by amphiphilic polymers and surfactants. For example, the liposome can be a unilamellar liposome, a multilamellar liposome, or a multivesicular liposome. For example, the liposome can be a neutral liposome, a negatively charged liposome, or a positively charged liposome.

[0085] In the present application, the term "RNA molecule encoding" generally refers to the introduction of modified nucleosides into an RNA molecule sequence, which is delivered into a cell and encodes a target protein. In the present application, the RNA molecule can be an RNA molecule with a smaller molecular weight. For example, the RNA can be siRNA, antisense oligonucleotides (ASOs), and oligonucleotides. In the present application, the RNA molecule can be mRNA.

[0086] In this application, the term "tumor vaccine" generally refers to vaccines that induce an immune response to tumor antigens. For example, the tumor antigen can be a tumor-specific antigen or a tumor-associated antigen. In this application, the tumor vaccine can activate the body's immune system and alleviate and / or control the occurrence and progression of tumors. In this application, tumor vaccines can be protein vaccines, peptide vaccines, microbial vector vaccines, or cell vaccines. In this application, the term "protein vaccine" generally refers to vaccines produced using genetic engineering, where DNA encoding the antigen is inserted into a system such as Escherichia coli / yeast, insect-baculovirus, or mammalian cells for expression and purification to produce the vaccine. In this application, the term "peptide vaccine" generally refers to vaccines prepared by chemical synthesis techniques using amino acid sequence determination of antigenic peptides eluted from the surface of tumor cells or proteins abnormally expressed within tumor cells. In this application, the term "microbial vector vaccine" generally refers to vaccines that use attenuated or harmless microorganisms (such as adenovirus) to deliver a portion of the antigen to stimulate an immune response. In this application, the term "cell vaccine" generally refers to tumor cell vaccines or dendritic cell vaccines. In this application, the cell vaccine is typically prepared by in vitro cultivation of autologous or allogeneic tumor cells or host dendritic cells loaded with tumor antigens. In this application, the tumor vaccine can be a preventive tumor vaccine or a therapeutic tumor vaccine. For example, the tumor vaccine can be a preventive tumor vaccine that can be used to prevent tumors associated with specific viruses. For example, the tumor vaccine can be a therapeutic tumor vaccine that can treat existing tumors by stimulating the immune system.

[0087] In this application, the term "pharmaceutically acceptable carrier" generally refers to a carrier that is compatible with the other ingredients of the formulation and is generally non-toxic to cells or mammals exposed thereto at the doses and concentrations employed. In this application, the pharmaceutically acceptable carrier can be a pharmaceutically acceptable carrier, excipient, or stabilizer. For example, the carrier can be water, salt, protein, polysaccharide, lipid, or inactive viral particles.

[0088] In the present application, the term "drug combination" generally refers to a mixture or combination of more than one active ingredients. For example, the drug combination may be a combination of a tumor vaccine and a drug related to the immune response. For example, the drug combination may be a combination of a tumor vaccine and an immunomodulator. In the present application, the immunomodulator generally refers to a drug that regulates the host immune system. For example, the immunomodulator may be an immunosuppressant and an immunostimulator. For example, the immunomodulator may inhibit or reduce the immune system response of the subject. For example, the immunomodulator may activate or enhance the immune system response of the subject. In the present application, the immunomodulator may be a small molecule, peptide, polypeptide, protein, gene, nucleic acid, antibody, synthetic or natural inorganic molecule, synthetic or natural organic molecule and cell. For example, the immunomodulator may be a therapeutic antibody, therapeutic cell, therapeutic nucleic acid and / or therapeutic gene.

[0089] In the present application, the pharmaceutical combination can be administered separately, simultaneously, or sequentially. In the present application, the pharmaceutical combination can be administered in the same or different dosages or modes of administration. For example, the components of the pharmaceutical combination can be administered to the patient simultaneously as a single entity or dosage. For example, the components of the pharmaceutical combination can be administered to the patient simultaneously, jointly, or sequentially as separate entities. For example, the active ingredients of the pharmaceutical combination can be administered to the patient as separate entities in the same or different dosages or modes of administration.

[0090] In this application, the term "therapeutic antibody" generally refers to an antibody that is administered to treat a disease. In this application, the therapeutic antibody as an immunomodulator can be an agonist antibody or an inhibitory antibody. In this application, the agonist antibody can activate the body's immune response by changing signal transduction within immune cells, thereby prompting immune cells to have stronger anti-cancer activity. In this application, the inhibitory antibody can inhibit a specific immune response by targeting receptors on immune cells. In this application, the therapeutic antibody can be selected from the full-length sequence of an antibody or a fragment thereof with antigen-binding function. For example, the therapeutic antibody can be a full-length sequence, Fab', F(ab')2, Fab or Fv.

[0091] In this application, the term "therapeutic cells" generally refers to cells for the purpose of treatment. For example, the therapeutic cells may be cells participating in an immune response. For example, the therapeutic cells may be natural immune cells or modified immune cells. In this application, the natural immune cells generally refer to naturally occurring cells that participate in or are associated with an immune response. For example, natural immune cells may be T cells, NK cells, NKT cells, and TIL cells. In this application, the modified immune cells, also known as engineered immune cells, generally refer to immune cells that are genetically modified by transcribing additional genetic material in the form of DNA or RNA. In this application, the modification may be that the immune cells express chimeric antigen receptors (CARs) and T cell receptors (TCRs). For example, the modified immune cells may be CAR-T cells and TCR-T cells.

[0092] In this application, the term "therapeutic nucleic acid" generally refers to RNA or DNA with different functions, which can act on pathogenic target genes or target mRNA to treat diseases. In this application, the therapeutic nucleic acid can be DNA, RNA or its derivatives. For example, the therapeutic nucleic acid can be an antisense nucleic acid (ASO), small interfering RNA (siRNA), microRNA (miRNA), small activating RNA (saRNA), messenger RNA (mRNA) aptamer, ribozyme (ribozyme) and antibody nucleic acid conjugate drug (ARC).

[0093] In this application, the term "therapeutic gene" generally refers to a gene that is introduced into a patient's body using biological methods to treat a disease or enhance disease resistance. In this application, the therapeutic gene can be a gene that corrects, replaces, or adds a gene to a target cell. In this application, the therapeutic gene can be a gene that enhances or inactivates a gene in a target cell. In this application, any technique known in the art can be used to deliver the therapeutic gene.

[0094] In this application, the term "prevention and / or treatment" generally refers to the prevention and / or treatment of a disease. For example, the prevention and / or treatment may include preventing the onset of the disease, slowing or reversing the progression of the disease, preventing or slowing the onset of one or more symptoms associated with the disease, reducing or alleviating one or more symptoms associated with the disease, reducing the severity and duration of the disease and any symptoms associated therewith, or preventing further increase in the severity of the disease and any symptoms associated therewith. In this application, the disease may be a neoplastic disease. For example, the onset of one or more symptoms associated with a tumor may be prevented or alleviated, and the severity and duration of a tumor and symptoms associated therewith may be reduced.

[0095] In this application, the term "tumor" generally refers to any new pathological tissue proliferation in which tumor antigens are recognized by the immune system. In this application, the tumor may include benign or malignant tumors (cancer). In this application, the cancer may be metastatic cancer or non-metastatic cancer. In this application, the tumor may include solid tumors and hematologic tumors. In this application, the solid tumor generally refers to a tangible tumor that can be detected by clinical examination means. For example, the solid tumor may include a growth or solid lesion formed by abnormal cell growth. In this application, the hematologic tumor generally refers to a class of hematopoietic system diseases. In this application, the hematologic tumor may include various types of leukemia, multiple myeloma or malignant lymphoma. For example, the tumor may be pancreatic cancer, gastric cancer, lung cancer, prostate cancer, melanoma, breast cancer, kidney cancer, liver cancer and ovarian cancer.

[0096] Detailed Description of the Invention

[0097] Tumor vaccines

[0098] The present application provides a tumor vaccine, which contains and / or expresses antigen molecules, co-stimulatory signal molecules, cytokines, and antigen presentation enhancing molecules. For example, the antigen presentation enhancing molecule can be an NLRC5 molecule and / or a CⅡTA molecule. The antigen molecule can be recognized by the immune system after presentation by the antigen presenting molecule, and binds to the TCR to produce a TCR activation signal. By activating the NLRC5 molecule and / or the CⅡTA molecule, it can be made to highly express the antigen presenting molecules MHC-I, MHC-II and HLA-I, HLA-II and other molecules related to presentation, thereby obtaining an antigen presentation ability similar to that of the strongest antigen presenting cells (dendritic cells). At the same time, combined with the continuously expressed co-stimulatory cells and cytokine signals, it is possible to activate all mutations and related antigens of the tumor and enhance the anti-tumor immunity. The tumor vaccine designed in the present application is not limited by the type of tumor antigen, is generally applicable to the design of tumor vaccines for various tumor types, and has universal applicability.

[0099] In one aspect, the present application provides a tumor vaccine comprising and / or expressing an antigen molecule, a co-stimulatory signaling molecule, a cytokine, and an NLRC5 molecule. Activation of the NLRC5 molecule can enhance MHC-I class antigen presentation, enhance HLA-I expression, and activate CD8 T cells.

[0100] In the present application, the NLRC5 molecule may comprise a full-length NLRC5 or a truncated NLRC5 that retains its functional activity. For example, the NLRC5 molecule may be a functionally active fragment that retains the ability of the full-length NLRC5 molecule to enhance MHC class I antigen presentation. For example, the NLRC5 molecule may be a domain that retains the antigen presentation enhancing function of NLRC5.

[0101] In the present application, the NLRC5 molecule may include homologs, analogs, etc. of NLRC5. For example, the NLRC5 may be a protein or polypeptide having an amino acid sequence having at least about 70% (e.g., at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence identity.

[0102] In the present application, the NLRC5 molecule may include NLRC5 from any species. For example, the NLRC5 may be human-derived NLRC5.

[0103] In the present application, the NLRC5 molecule may comprise a modified / artificially engineered NLRC5. For example, the NLRC5 molecule may have one or more amino acid mutations compared to a wild-type NLRC5 molecule. The amino acid mutations do not affect the antigen presentation-enhancing function of the NLRC5 molecule. For example, the NLRC5 molecule may be directly or indirectly linked to another protein to form a fusion protein. For example, the NLRC5 molecule may be linked to CIITA to form a fusion protein.

[0104] In certain embodiments, the NLRC5 molecule may comprise the amino acid sequence set forth in SEQ ID NO: 2. In certain embodiments, the NLRC5 molecule may comprise an amino acid sequence having at least about 70% (e.g., at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence identity to the amino acid sequence set forth in SEQ ID NO: 2.

[0105] In the present application, the tumor vaccine may further comprise and / or express CIITA molecules. Activated CIITA can enhance MHC-II class antigen presentation, enhance HLA-II expression, and activate CD4 T cells.

[0106] In this application, the tumor vaccine comprises and / or expresses antigen molecules, co-stimulatory signaling molecules, cytokines, NLRC5 molecules, and CⅡTA molecules. The expression of these two molecules can promote the high expression of antigen-presenting molecules MHC-I and MHC-II molecules, enabling antigen-presenting cells to acquire antigen-presenting capabilities similar to those of dendritic cells.

[0107] In the present application, the CⅡTA molecule may comprise a full-length CⅡTA or a truncated CⅡTA that retains its function. For example, the CⅡTA molecule may be a functionally active fragment that retains the ability of the full-length protein to enhance MHC-II class antigen presentation.

[0108] In the present application, the CIITA molecule may include homologs, analogs, etc. of CIITA. For example, the CIITA may be a protein or polypeptide having an amino acid sequence having at least about 70% (e.g., at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence identity.

[0109] In certain embodiments, the CIITA molecule may comprise CIITA of any species, for example, the CIITA may be human-derived CIITA.

[0110] In certain embodiments, the CIITA molecule may comprise a modified / artificially engineered CIITA. For example, the CIITA molecule may have one or more amino acid mutations compared to a wild-type CIITA molecule. These amino acid mutations do not affect the antigen presentation-enhancing function of the CIITA molecule. For example, the CIITA may be directly or indirectly linked to another protein to form a fusion protein. For example, the CIITA may be linked to CIITA and NLRC5 to form a fusion protein.

[0111] In the present application, the CIITA and NLRC5 forming the fusion protein may be full-length CIITA and NLRC5, or functionally active fragments of CIITA and NLRC5.

[0112] In certain embodiments, the CIITA molecule may comprise the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the CIITA molecule may comprise an amino acid sequence having at least about 70% (e.g., at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence identity to the amino acid sequence set forth in SEQ ID NO: 1.

[0113] In the present application, the tumor vaccine may have independent CD4 and / or CD8 T cell antigen presentation capabilities independent of host immune cells. For example, the host immune cells may be dendritic cells, B cells, and / or macrophages.

[0114] In the present application, the antigen molecule can be any substance that causes an immune response. For example, the antigen can include a protein, a peptide, or a nucleic acid molecule encoding the protein or peptide. In the present application, the antigen can include a portion thereof that can cause the desired immune response, for example, the antigen can include a full-length antigen, or a fragment of the antigen.

[0115] In the present application, the antigen can include any type of antigen. For example, the antigen can include any type of tumor antigen. For example, the antigen can include tumor-specific antigens and tumor-associated antigens. For example, the tumor-specific antigens and tumor-associated antigens can cover any existing mutant antigens. The tumor vaccine described in the present application is not restricted by the MHC of the T cells themselves.

[0116] In the present application, the antigens may include endogenous antigens and exogenous antigens. For example, the endogenous antigens may be presented to CD8 T cells in the form of antigen peptide-MHC class I molecule complexes. For example, exogenous antigens, after being degraded into short peptides by lysosomes, may be presented to CD4 T cells via MHC class II molecules for recognition, thereby inducing an immune response involving CD4 T cells.

[0117] In the present application, exemplary antigens may include, but are not limited to, HA, E7, NY-ESO, MAGE-A3, MART, EGFR-VIII, PSMA, GPC-3, Mesothelin, PSA, and CLDN18.2. In the present application, the antigen may be a full-length protein and its variants, homologs, derivatives, analogs, functionally active fragments, or fusion proteins. In the present application, the HA may be Influenza HA and its variants, homologs, derivatives, analogs, functionally active fragments, or fusion proteins. For example, the HA may form a fusion protein with E7. In the present application, the E7 may be HPV E7 and its variants, homologs, derivatives, analogs, functionally active fragments, or fusion proteins. For example, the E7 may form a fusion protein with HA. For example, the antigen molecule may be HA-E7. For example, the HA-E7 may comprise the amino acid sequence shown in SEQ ID NO: 8.

[0118] In the present application, the costimulatory signal molecule can provide a second signal for T cell activation - a costimulatory signal. The costimulatory signal is generated by the interaction between the costimulatory signal molecule and the corresponding T cell receptor, which can enhance the TCR signal. For example, the costimulatory signal molecule provides a signal that mediates a T cell response, including but not limited to proliferation, activation, differentiation, etc.

[0119] In the present application, the costimulatory signal molecule may include a costimulatory molecule or a ligand thereof.

[0120] For example, the co-stimulatory signal molecule can be a TNF / TNFR superfamily, an immunoglobulin superfamily, a Tim family, or a SLAM family, or a ligand thereof.

[0121] For example, exemplary costimulatory signal molecules can be CD80, CD86, 4-1BBL, anti-CD28, OX40L, CD40 ligand, ICOS ligand, GITR ligand, 4-1BB ligand, OX40 ligand, TL1A, CD30 ligand, CD27 and Flt3 ligand. For example, the costimulatory signal molecule can be CD80. For example, the CD80 costimulatory signal molecule can comprise the amino acid sequence shown in SEQ ID NO:3. For example, the costimulatory signal molecule can be 4-1BBL. For example, the 4-1BBL can comprise the amino acid sequence shown in SEQ ID NO:4. For example, the costimulatory signal molecule can be 4-1BBL. For example, the 4-1BBL can comprise the amino acid sequence shown in SEQ ID NO:4. For example, the costimulatory signal molecule can be anti-CD28. For example, the anti-CD28 can comprise the amino acid sequence shown in SEQ ID NO:7.

[0122] In the present application, the tumor vaccine may comprise and / or express antigen molecules, cytokines, NLRC5 molecules and CD80.

[0123] In the present application, the tumor vaccine may comprise and / or express antigen molecules, cytokines, NLRC5 molecules, CⅡTA molecules and CD80.

[0124] In the present application, the cytokine can provide a third signal for T cell activation. For example, the cytokine can enhance the function of T cells. For example, the cytokine can promote the maturation, proliferation, growth and / or activation of T cells.

[0125] In the present application, the cytokines may be interleukins (ILs), tumor necrosis factors and other polypeptide factors.

[0126] In the present application, the cytokine may comprise a full-length cytokine or a truncated cytokine that retains its function, for example, the cytokine may be a functionally active fragment that retains the ability of the full-length protein to enhance T cell function.

[0127] In the present application, the cytokine can be IL-2, IL-7, IL-9, IL-15 and IL-21. For example, IL-2 can play an important role in T cell development and amplification, can stimulate activated T cell proliferation, and produce cytotoxic T cells. For example, IL-7 can play a key role in the generation of naive T cells and memory T cell subsets. For example, IL-9 can promote the survival and activation of T cells and stimulate cell proliferation. For example, IL-15 can stimulate the proliferation and activation of T cells. For example, IL-21 can promote CD8+ T cell proliferation and cytotoxicity and participate in tumor immunity.

[0128] In the present application, the cytokine can be a mutant of IL-2, IL-7, IL-9, IL-15 and IL-21. For example, the mutant of IL-2, IL-7, IL-9, IL-15 and IL-21 can retain its function of causing T cell maturation, proliferation, growth and / or activation.

[0129] In a specific embodiment, the cytokine may be IL-2. For example, the IL-2 may comprise the amino acid sequence set forth in SEQ ID NO: 5. In a specific embodiment, the cytokine may be IL-21. For example, the IL-21 may comprise the amino acid sequence set forth in SEQ ID NO: 6.

[0130] In certain embodiments, the tumor vaccine comprises and / or expresses an antigen molecule, a co-stimulatory signaling molecule, an NLRC5 molecule, and IL-2.

[0131] In certain embodiments, the tumor vaccine comprises and / or expresses an antigen molecule, a co-stimulatory signaling molecule, an NLRC5 molecule, a CⅡTA molecule, and IL-2.

[0132] In the present application, the tumor vaccine can provide the three signals required for T cell activation, can activate tumor mutations and related antigens, stimulate immune responses, and obtain polyclonal anti-tumor immunity.

[0133] In certain embodiments, the tumor vaccine comprises and / or expresses antigenic molecules, NLRC5 and CIITA, CD80 and IL-2.

[0134] In the present application, the antigen molecules, co-stimulatory signaling molecules, cytokines, NLRC5 molecules, and / or CIITA molecules of the tumor vaccine can be expressed in target cells by delivering nucleic acids encoding the target proteins into target cells. For example, the protein molecules can be expressed via viral vectors, plasmids, liposomes, or RNA molecules.

[0135] In certain embodiments, the tumor vaccine can be expressed by viral vector infection of the target protein molecule. For example, the viral vector expression can be the delivery of nucleic acid to target cells via a viral vector, causing them to express the target protein. In the present application, the viral vector can be a virus for delivering nucleic acid. For example, the viral vector can be a retrovirus, adenovirus, adeno-associated virus, herpes virus, poxvirus, baculovirus, papillomavirus, or papovavirus. For example, the viral vector can be a lentivirus.

[0136] In certain embodiments, the tumor vaccine can be delivered to cells using a plasmid as a vector for transferring genetic material and expressing the tumor vaccine. For example, the plasmid can be a DNA plasmid or an RNA plasmid. For example, the plasmid can be a linear plasmid or a circular plasmid.

[0137] In certain embodiments, the tumor vaccine can be delivered to target cells via one or more liposomes. For example, the liposomes can be unilamellar liposomes, multilamellar liposomes, or multivesicular liposomes. For example, the liposomes can be neutral liposomes, negatively charged liposomes, or positively charged liposomes.

[0138] In certain embodiments, the tumor vaccine can encode the target protein by introducing modified nucleosides into the RNA molecule sequence, encapsulating the RNA with a carrier and delivering it into the cell. For example, the RNA molecule can be an RNA molecule with a smaller molecular weight. For example, the RNA can be siRNA, antisense oligonucleotide (ASO), and oligonucleotide. In the present application, the RNA molecule can be mRNA.

[0139] In the present application, the tumor vaccine can be prepared as a preparation to induce an immune response against tumor-specific antigens or tumor-associated antigens.

[0140] In the present application, the tumor vaccine can be in the form of any tumor vaccine known in the art. For example, the tumor vaccine preparation can be a protein vaccine, a polypeptide vaccine, a microbial vector vaccine, a genetically engineered vaccine, and / or a cell vaccine. For example, the tumor vaccine can be a protein vaccine, in which the DNA encoding the antigen is inserted into a system such as Escherichia coli / yeast, insect-baculovirus, or mammalian cells for expression using genetic engineering, and the expressed molecule is then purified and prepared into a vaccine. For example, the tumor vaccine can be a polypeptide vaccine, in which the amino acid sequence of the antigen polypeptide eluted from the surface of the tumor cell or the protein abnormally expressed inside the tumor cell is determined, and the vaccine is prepared by chemical synthesis technology. For example, the tumor vaccine is a microbial vector vaccine, which uses an attenuated or harmless microorganism (such as an adenovirus) to transport a portion of the antigen to stimulate an immune response, and can induce effective cell-mediated immunity. For example, the tumor vaccine can be a genetically engineered vaccine, in which the gene encoding the tumor antigen and other genes encoding the target molecule can be loaded onto a recombinant viral vector or plasmid DNA using genetic engineering technology. For example, the tumor vaccine can be a cell vaccine, in which autologous or allogeneic tumor cells are selected to prepare the vaccine, so that the cells release multiple tumor antigens and express target molecules at the same time to enhance the anti-tumor effect of the tumor vaccine.

[0141] On the other hand, the present application provides one or more pharmaceutical compositions, which may include the tumor vaccine of the present application. In certain embodiments, the pharmaceutical composition may further include an optional pharmaceutically acceptable carrier.

[0142] For example, the pharmaceutical composition may also include one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or preservatives and other suitable formulations. The acceptable ingredients of the composition are preferably non-toxic to the recipient at the dosage and concentration used. The pharmaceutical composition of the present invention may include liquid, frozen and lyophilized compositions.

[0143] In certain embodiments, the pharmaceutically acceptable adjuvant may include any and all solvents, dispersion media, coatings, isotonic agents, and absorption delaying agents that are compatible with pharmaceutical administration and are generally safe, non-toxic, and neither biologically nor otherwise undesirable.

[0144] In certain embodiments, the pharmaceutical composition can comprise parenteral, transdermal, intracavitary, intraarterial, intrathecal and / or intranasal administration or direct injection into a tissue. For example, the pharmaceutical composition can be administered to a patient or subject by infusion or injection. In certain embodiments, the administration of the pharmaceutical composition can be carried out in different ways, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

[0145] Drug combinations

[0146] On the other hand, the present application provides one or more drug combinations, which may include the tumor vaccine of the present application. The drug combination may also include one or more active ingredients other than the tumor vaccine.

[0147] In certain embodiments, the drug combination may further comprise a drug associated with immune response.

[0148] In certain embodiments, the drug combination may be composed of a tumor vaccine and an immunomodulator.

[0149] On the other hand, the present application also provides a scheme for combining the tumor vaccine with one or more other active ingredients. For example, the tumor vaccine described in the present application can be administered in combination with other immunomodulators.

[0150] In certain embodiments, the immunomodulator can be an immunosuppressant or an immunostimulator. For example, the immunomodulator can suppress or reduce the immune system response of the subject. For example, the immunomodulator can activate or enhance the immune system response of the subject.

[0151] In certain embodiments, the immunomodulatory agent may be an anti-tumor agent.

[0152] In certain embodiments, the immunomodulator can be a small molecule, peptide, polypeptide, protein, gene, nucleic acid, antibody, synthetic or natural inorganic molecule, synthetic or natural organic molecule or cell. For example, the immunomodulator can be a therapeutic antibody, therapeutic cell, therapeutic nucleic acid and / or therapeutic gene.

[0153] In certain embodiments, the immunomodulator can be a therapeutic antibody. For example, the therapeutic antibody can be an agonist antibody or an inhibitory antibody as an immunomodulator. In the present application, the therapeutic antibody can be selected from a full-length antibody sequence or a fragment thereof having antigen-binding function. For example, the therapeutic antibody can be a full-length sequence, Fab', F(ab')2, Fab, or Fv.

[0154] In certain embodiments, the immunomodulator can be a therapeutic cell. For example, the therapeutic cell can be a natural immune cell or a modified immune cell. In the present application, the therapeutic cell can be a natural immune cell. For example, the natural immune cell can be a T cell, a NK cell, a NKT cell or a TIL cell. In the present application, the therapeutic cell can be a modified immune cell. In the present application, the modification can be an immune cell expressing a chimeric antigen receptor (CAR) or a T cell receptor (TCR). For example, the modified immune cell can be a CAR-T cell or a TCR-T cell.

[0155] In certain embodiments, the immunomodulator can be a therapeutic nucleic acid. In the present application, the therapeutic nucleic acid can be DNA, RNA, or a derivative thereof. For example, the therapeutic nucleic acid can be an antisense nucleic acid (ASO), a small interfering RNA (siRNA), a microRNA (miRNA), a small activating RNA (saRNA), a messenger RNA (mRNA) aptamer, a ribozyme, or an antibody nucleic acid conjugate (ARC).

[0156] In certain embodiments, the immunomodulator can be a therapeutic gene. In this application, the therapeutic gene can be a gene that corrects, replaces, or adds a gene in the target cell. In this application, the therapeutic gene can be a gene that enhances or inactivates the gene in the target cell.

[0157] In the present application, the drug combination can be administered separately, simultaneously or sequentially. In the present application, the drug combination can be administered in the same or different dosages or modes of administration. For example, the components of the drug combination can be administered to the patient simultaneously in the form of a single entity or dosage. For example, the components of the drug combination can be administered to the patient simultaneously, jointly or sequentially as separate entities. For example, the active ingredients in the drug combination can be administered to the patient as separate entities in the same / different dosages or modes of administration. The specific mode of administration can be determined based on the type of active ingredient. The specific dosage can be adjusted based on the severity of the subject's condition, the subject's physical condition, etc.

[0158] In the present application, the different active ingredients in the pharmaceutical combination can be mixed or placed separately, and can be placed in the same container or in different containers.

[0159] Methods and uses

[0160] In another aspect, the present application provides a method for preparing the tumor vaccine, the pharmaceutical composition, or the pharmaceutical combination. The tumor vaccine described herein can be prepared using any method known in the art.

[0161] On the other hand, the present application provides uses of the tumor vaccine, the pharmaceutical composition, and the drug combination in preparing drugs, which can be used to prevent and / or treat tumors.

[0162] On the other hand, the present application provides a method for preventing and / or treating tumors, which comprises administering the tumor vaccine, the pharmaceutical composition or the drug combination to a subject in need thereof.

[0163] On the other hand, the present application provides the tumor vaccine, the pharmaceutical composition, and the drug combination, which are used to prevent and / or treat tumors.

[0164] In the present application, the prevention and / or treatment of tumors may be the prevention of the occurrence or development of tumors. For example, the prevention and / or treatment of tumors may be the slowing down or reversing of tumor progression, the prevention or slowing down of the onset of one or more symptoms associated with the tumor, the reduction or alleviation of one or more symptoms associated with the tumor, the reduction of the severity and duration of the tumor and any symptoms associated therewith, or the prevention of further increase in the severity of the tumor and any symptoms associated therewith.

[0165] In the present application, the tumor can be any new pathological tissue proliferation, and there are tumor antigens that can be recognized by the immune system. In the present application, the tumor can include benign or malignant tumors (cancer). In the present application, the cancer can be metastatic cancer or non-metastatic cancer. In the present application, the tumor can include solid tumors or hematologic tumors. In the present application, the hematologic tumor can include various types of leukemia, multiple myeloma or malignant lymphoma.

[0166] In the present application, the tumor type targeted by the tumor vaccine is related to the antigen expressed in the tumor vaccine. For example, the tumor can be pancreatic cancer, gastric cancer, lung cancer, prostate cancer, melanoma, breast cancer, kidney cancer, liver cancer and ovarian cancer.

[0167] In the present application, the tumor vaccine can be used alone or in combination with other treatment modalities, such as surgery, drug therapy, or radiotherapy.

[0168] Without intending to be bound by any theory, the following examples are merely intended to illustrate the tumor vaccine, preparation method, and use of the present application, and are not intended to limit the scope of the present invention.

[0169] Example

[0170] Example 1 Construction of CIITA, NLRC5, CD80, 4-1BBL, IL-2, IL-21, anti-CD28, HA-E7, HA-CD4 TCR, E7-CD8 TCR, and HLA-DRB101 plasmids

[0171] To construct the plasmid described in the present application, the antigen presentation enhancing molecules CIITA (SEQ ID NO: 1), NLRC5 (SEQ ID NO: 2); T cell activation enhancing molecules CD80 (SEQ ID NO: 3), 4-1BBL (SEQ ID NO: 4), IL-2 (SEQ ID NO: 5), IL-21 (SEQ ID NO: 6), anti-CD28 (SEQ ID NO: 7), OX40L (SEQ ID NO: 14), IL-7 (SEQ ID NO: 16), IL-15 (SEQ ID NO: 17); antigen expression molecules HA-E7 (SEQ ID NO: 8), exogenous antigen recognized by JM22 TCR (SEQ ID NO: 13); antigen recognition TCR molecules HA-CD4 TCR (SEQ ID NO: 9), E7-CD8 TCR (SEQ ID NO: 10), JM22-CD8 TCR (SEQ ID NO: 12), E7-OX40 TCR (SEQ ID NO: 13); The nucleic acid fragments of the HLA molecule HLA-DRB101 (SEQ ID NO: 15) and HLA-DRB101 (SEQ ID NO: 11) were synthesized at GenScript, with XbaI and BamHI restriction sites added at both ends, and cloned into the pCDH-MSCVEF vector. The structure is shown in Figure 1. Correctly sequenced clones were extracted using the NucleoBond Xtra Midi Plus EF kit without endotoxin and used for subsequent viral packaging.

[0172] Example 2 Production of CIITA, NLRC5, CD80, 4-1BBL, IL-2, IL-21, anti-CD28, OX40L, IL-7, IL-15, HA-E7, HA-CD4 TCR, E7-CD8 TCR, JM22-CD8 TCR, E7-OX40 TCR, HLA-DRB101 Lentivirus and Preparation of Corresponding Cells

[0173] 2.1 Production of CIITA, NLRC5, CD80, 4-1BBL, IL-2, IL-21, anti-CD28, OX40L, IL-7, IL-15, HA-E7, HA-CD4 TCR, E7-CD8 TCR, JM22-CD8 TCR, E7-OX40 TCR, and HLA-DRB101 Viruses

[0174] 293X cells were co-transfected with CIITA, NLRC5, CD80, 4-1BBL, IL-2, IL-21, anti-CD28, OX40L, IL-7, IL-15, HA-E7, HA-CD4 TCR, E7-CD8 TCR, JM22-CD8 TCR, E7-OX40 TCR, and HLA-DRB101 viral plasmids constructed in Example 1, and lentiviral packaging plasmids (VSV-g, pMD Gag / Pol or RSV-REV). The cells were cultured at 37° C., 5% CO 2 for 48 hours, and the supernatant was collected and used directly or concentrated after 0.45 μM filtration. Use a Beckman ultracentrifuge and SW28 rotor at 25,000 RPM for 2 hours to concentrate the virus, which is the corresponding CIITA, NLRC5, CD80, 4-1BBL, IL-2, IL-21, anti-CD28, OX40L, IL-7, IL-15, HA-E7, HA-CD4 TCR, E7-CD8 TCR, JM22-CD8 TCR, E7-OX40 TCR, HLA-DRB101 lentivirus for subsequent tumor vaccine, cell line and T cell production.

[0175] Preparation of tumor vaccine cell lines containing CIITA, NLRC5, CD80, 4-1BBL, IL-2, IL-21, anti-CD28, OX40L, IL-7, IL-15, HA-E7, HA-CD4 TCR, E7-CD8 TCR, JM22-CD8 TCR, E7-OX40 TCR, and HLA-DRB101

[0176] The HLA-DRB101 and HA-E7 lentiviruses constructed in Example 2.1 were used to infect CFPAC-1 and H82 tumor cells to obtain CFPAC-1-HE and H82-HE expressing HA and E7 antigens; CFPAC-1-HE and H82-HE were infected with CIITA and NLRC5 alone or in combination to obtain CFPAC-1-HE-CIITA, CFPAC-1-HE-NLRC5, CFPAC-1-HE-CIITA-NLRC5, H82-HE-CIITA, H82-HE, which express CIITA and NLRC5 alone or in combination. -NLRC5, H82-HE-CIITA-NLRC5; based on CFPAC-1-HE-CIITA-NLRC5 and H82-HE-CIITA-NLRC5, they were infected with CD80, 4-1BBL, IL-2, IL-21, anti-CD28, OX40L, IL-7, and IL-15 alone or in combination to obtain CFPAC-1-HE-CIITA-NLRC5-IL2, CFPAC-1-HE-CIITA-NLRC5-IL21, and CFPAC-1-HE-CIITA-NLRC5-CD 80, CFPAC-1-HE-CIITA-NLRC5-4-1BBL, CFPAC-1-HE-CIITA-NLRC5-anti-CD28, H82-HE-CIITA-NLRC5-IL2, H82-HE-CIITA-NLRC5- IL21, H82-HE-CIITA-NLRC5-CD80, H82-HE-CIITA-NLRC5-4-1BBL, H82-HE-CIITA-NLRC5-anti-CD28, CFPAC-1-HE-CIITA-NLRC5-O The expression levels of HLA-I and HLA-II in X40L, CFPAC-1-HE-CIITA-NLRC5-IL7, CFPAC-1-HE-CIITA-NLRC5-IL15 tumor vaccine cell lines and co-expressing tumor cell lines CFPAC-1-HE-CIITA-NLRC5-CD80-IL2 and H82-HE-CIITA-NLRC5-CD80-IL2 were detected by flow cytometry. The results are shown in Figure 2. NLRC5 and CIITA in H82 cells can enhance the expression of HLA-I and HLA-II, respectively.

[0177] 2.3 Preparation of HA-CD4 TCR, E7-CD8 TCR Primary T Cells and Jurkat T Cells

[0178] First, sgRNA (sequence) targeting the constant regions of TCRa and TCRb was designed and synthesized at GenScript. It formed an RNP complex with Cas9 at a 1:2 molar ratio and was electroporated into Jurkat cells. TCRa and b knockout Jurkat cell lines were obtained by flow cytometry sorting. The E7-CD8 TCR, HA-CD4 TCR, and JM22-CD8 TCR constructed in Example 2.1 were used to infect TCRa and b knockout Jurkat cell lines, obtaining E7-Jurkat, HA-Jurkat, and JM22-Jurkat cell lines that reacted with the exogenous antigens recognized by the E7 polypeptide, HA polypeptide, and JM22 TCR.

[0179] First, primary T cells were activated using anti-CD3 / CD28. Two days later, sgRNAs for the TCRα and TCRb constant regions and the Cas9 RNP complex were used to electroporate the primary T cells. One day later, the E7-CD8 TCR and HA-CD4 TCR constructed in Example 2.1 were used to infect the above T cells to obtain E7-CD8 T cells, HA-CD4 T cells, and E7-OX40 T cells that react with E7 and HA polypeptides.

[0180] Example 3 CIITA enhances the activation of CD4 T cells by tumor vaccines

[0181] 3.1 CIITA enhances the activation of HA-Jurkat T cells by tumor vaccines

[0182] CFPAC-1-HE, CFPAC-1-HE-CIITA, H82-HE, H82-HE-CIITA, and HA-Jurkat T cells were prepared according to Example 2, and CFPAC-1-HE, CFPAC-1-HE-CIITA, H82-HE, and H82-HE-CIITA tumor vaccines were mixed with HA-Jurkat T cells at a ratio of 1:1, 10 5 Each cell type was co-cultured in a 96-well plate, and the expression of T cell activation molecules CD25 and CD69 was detected 24 hours later (Figure 3). The results showed that the tumor vaccine CFPAC-1-HE-CIITA was better able to activate HA-Jurkat T cells than CFPAC-1-HE, enhancing CD25 expression by 2.7 times and CD69 expression by 2 times. The tumor vaccine H82-HE-CIITA was better able to activate HA-Jurkat cells than H82-HE, enhancing CD25 expression by 3.6 times and CD69 expression by 24.7 times.

[0183] 3.2 CIITA enhances the activation of primary T cells by tumor vaccines

[0184] H82-HE, H82-HE-CIITA and HA-CD4 T cells were prepared according to Example 2, and H82-HE and H82-HE-CIITA tumor vaccines were mixed with HA-CD4 T cells at a ratio of 1:1, 10 5 Each cell type was co-cultured in a 96-well plate, and the expression of T cell activation cytokines IFNg and TNFa in the culture supernatant was detected 6 hours later (Figure 4). Compared with H82-HE, the tumor vaccine H82-HE-CIITA was able to better activate HA-CD4 T cells and significantly enhance the expression of IFNg and TNFa.

[0185] Example 4 NLRC5 enhances the activation of CD8 T cells by tumor vaccines

[0186] 4.1 NLRC5 enhances the activation of E7-Jurkat T cells by tumor vaccines

[0187] CFPAC-1-HE, CFPAC-1-HE-NLRC5, H82-HE, H82-HE-NLRC5, and E7-Jurkat T cells were prepared according to Example 2, and CFPAC-1-HE, CFPAC-1-HE-NLRC5, H82-HE, and H82-HE-NLRC5 tumor vaccines were mixed with E7-Jurkat T cells at a ratio of 1:1, 10 5 Each cell type was co-cultured in a 96-well plate, and the expression of the T cell activation molecules CD25 and CD69 was measured 24 hours later (Figure 5). The results showed that due to the high baseline HLA-I expression of CFPAC1, the tumor vaccine CFPAC-1-HE-NLRC5 activated E7-Jurkat T cells to the same degree as CFPAC-1-HE. However, the tumor vaccine H82-HE-NLRC5 was able to better activate E7-Jurkat T cells than H82-HE, with a 2-fold increase in CD25 expression and a 2-fold increase in CD69 expression.

[0188] 4.2 NLRC5 enhances the activation of primary T cells by tumor vaccines

[0189] H82-HE, H82-HE-NLRC5, and E7-CD8 T cells were prepared according to Example 2, and H82-HE and H82-HE-NLRC5 tumor vaccines were mixed with E7-CD8 T cells at a ratio of 1:1, 10 5Each cell type was co-cultured in a 96-well plate, and the expression of T cell activation cytokines IFNg and TNFa in the culture supernatant was detected 48 hours later (Figure 6). The results showed that the tumor vaccine H82-HE-NLRC5 was able to better activate E7-CD8 T cells than H82-HE, and could enhance IFNg expression by 1.25 times and TNFa expression by 1.875 times.

[0190] Example 5 CIITA and NLRC5 synergistically enhance the activation of CD4 and CD8 T cells

[0191] H82-HE, H82-HE-NLRC5, H82-HE-CIITA, H82-HE-CIITA-NLRC5, E7-Jurkat T, and HA-Jurkat T cells were prepared according to Example 2. H82-HE, H82-HE-NLRC5, H82-HE-CIITA, and H82-HE-CIITA-NLRC5 tumor vaccines were mixed with E7-Jurkat T and HA-Jurkat T cells at a ratio of 1:1, 10 5 Each cell type was co-cultured in a 96-well plate, and the expression of T cell activation molecules CD25 and CD69 was detected after 24 hours (Figure 7). The simultaneous expression of CIITA and NLRC5 can simultaneously activate the expression of CD25 and CD69 in CD4 and CD8 T cells, and has the ability to simultaneously enhance the activation of CD4 and CD8 T cells.

[0192] Example 6 IL-2 Enhances the Effect of Tumor Vaccine on T Cell Proliferation

[0193] The CFPAC-1-HE-CIITA-NLRC5 and CFPAC-1-HE-CIITA-NLRC5-CD80-IL2 stable cell lines prepared in Example 2 were collected, and the cell culture supernatant was collected. The IL2 secreted by them can activate STAT5 in Jurkat-T cells (Figure 8). CFPAC-1-HE and CFPAC-1-HE-CIITA-NLRC5-IL2 were co-cultured with HA-CD4 T or E7-CD8 T cells, and cell proliferation was detected by flow cytometry every 2 days (Figure 8). The results showed that CFPAC-1-HE-CIITA-NLRC5-IL2 increased the proliferation of T cells in vitro by about 8-9 times, and had the ability to promote the proliferation of T cells.

[0194] Example 7 CD80 and anti-CD28 enhance the co-stimulatory effect of tumor vaccines on T cells

[0195] The stable cell lines CFPAC-1-HE-CIITA-NLRC5, CFPAC-1-HE-CIITA-NLRC5-CD80, CFPAC-1-HE-CIITA-NLRC5-anti-CD28, H82-HE-CIITA-NLRC5, and H82-HE-CIITA-NLRC5-CD80 were obtained according to Example 2. The obtained cells were mixed with Jurkat T cells at a ratio of 1:1, 10 5 Each cell type was co-cultured in a 96-well plate, and the expression of activation markers CD25 and CD69 on the T cell surface was measured 24 hours later. The results showed that H82-HE-CIITA-NLRC5-CD80 enhanced the expression of activation markers CD69 and CD25 (Figure 9), and CFPAC-1-HE-CIITA-NLRC5-CD80 enhanced the expression of activation markers CD69 and CD25 (Figure 10). CFPAC-1-HE-CIITA-NLRC5-anti-CD28 enhanced the expression of activation markers CD69 and CD25 (Figure 11), indicating the ability to costimulate T cells.

[0196] Example 8 Tumor vaccines co-expressing NLRC5, CIITA, CD80, and IL-2 induce stronger T cell proliferation in vivo

[0197] HA-CD4 T cells and E7-CD8 T cells, and CFPAC-1-HE and CFPAC-1-HE-CIITA-NLRC5-CD80-IL2 tumor vaccines were constructed according to Example 2 and subcutaneously inoculated into NSG mice for 10 6 The tumor vaccines CFPAC-1-HE-CIITA-NLRC5 and CFPAC-1-HE-CIITA-NLRC5-CD80-IL2 were used. After 10 days, 5×10 6 HA-CD4 T and E7-CD8 T cells were intravenously injected into NSG mice, and the number of HA-CD4 T and E7-CD8 T cells in the peripheral blood was detected 10 days later (Figure 12). As can be seen from the figure, CFPAC-1-HE-CIITA-NLRC5-CD80-IL2 can better activate CD4 and CD8 T cells than CFPAC-1-HE tumor vaccine. CFPAC-1-HE-CIITA-NLRC5-CD80-IL2 can enhance the proliferation of HA-CD4 and E7-CD8 T cells by 4 times and 19 times, and has the ability to induce T cell proliferation in vivo.

[0198] Example 9 Tumor vaccines synergistically combining NLRC5, CIITA, CD80, and IL-2 induce stronger CAR-T anti-tumor activity

[0199] CFPAC-1-HE and CFPAC-1-HE-CIITA-NLRC5-CD80-IL2 tumor vaccines were constructed according to Example 2, and 1×10 6 CFPAC-1-HE, CFPAC-1-HE-CIITA-NLRC5-CD80-IL2 tumor vaccine, 3×10 6 CLDN18.2-targeted CAR-T cells were injected intravenously into NSG mice, and tumor burden was measured. Results showed that compared to the control CFPAC-1-HE, CFPAC-1-HE-CIITA-NLRC5-CD80-IL2 cell line, tumor size was reduced by 46% after treatment, and the proportion of CAR-T cells in the tumor was significantly increased (Figure 13), demonstrating the ability to induce CAR-T anti-tumor activity.

[0200] Example 10 Tumor vaccines synergistically combining NLRC5, CIITA, CD80, and IL-2 induce stronger TCR-T anti-tumor activity

[0201] CFPAC-1-HE and CFPAC-1-HE-CIITA-NLRC5-CD80-IL2 tumor vaccines were prepared according to Example 2, and 1×10 6 CFPAC-1-HE, CFPAC-1-HE-CIITA-NLRC5-CD80-IL2 tumors, 2.5 × 10 6 E7-CD8 TCR-T or HA-CD4 TCR-T was intravenously injected into NSG mice, and tumor burden was detected. The results showed that compared with the control CFPAC-1-HE, CFPAC-1-HE-CIITA-NLRC5-CD80-IL2 reduced tumor size by 93% on day 32 after E7-CD8 TCR-T treatment and by 88% on day 58 after HA-CD4 TCR-T treatment (Figure 14), demonstrating the ability to induce TCR-T anti-tumor activity.

[0202] Example 11 Tumor vaccines expressed by NLRC5 and CIITA have the ability to induce activation of endogenous and exogenous antigen-specific T cells

[0203] E7-Jurkat T and JM22-Jurkat T cells, and CFPAC-1-HE-CIITA-NLRC5-CD80-IL2 tumor vaccines were constructed according to Example 2, wherein E7 is equivalent to the endogenous antigen of the tumor. 5Each cell type was co-cultured in a 96-well plate. An exogenous polypeptide recognized by the JM22 TCR (SEQ ID NO: 13, GILGFVFTL) was added to the culture system as an exogenous antigen. The expression of T cell activation molecules CD25 and CD69 was detected 24 hours later ( Figure 15 ). The results showed that the CFPAC-1-HE-CIITA-NLRC5-CD80-IL2 tumor vaccine was able to activate both E7-Jurkat T and JM22-Jurkat T cells, indicating that it has the ability to simultaneously induce the activation of endogenous and exogenous antigen-specific T cells.

[0204] Example 12 OX40L enhances the co-stimulatory effect of tumor vaccines on T cells

[0205] CFPAC-1-HE-CIITA-NLRC5, CFPAC-1-HE-CIITA-NLRC5-OX40L tumor vaccines, and E7-OX40 T cells were prepared according to Example 2. The obtained tumor vaccines were mixed with TCR-T cells at a ratio of 1:1. 5 Each cell type was co-cultured in a 96-well plate, and the expression of the T cell activation molecule IFNg was measured 24 hours later. The results showed that CFPAC-1-HE-CIITA-NLRC5-OX40L enhanced IFNg release compared to CFPAC-1-HE-CIITA-NLRC5 (Figure 16), indicating that OX40L can promote T cell co-stimulation. Combined with the ability of CD80 and anti-CD28 to enhance T cell co-stimulation in Example 7, this demonstrates that multiple co-stimulations can enhance T cell activation in tumor vaccines.

[0206] Example 13 IL7 and IL15 enhance the effect of tumor vaccines on T cell proliferation

[0207] CFPAC-1-HE-CIITA-NLRC5, CFPAC-1-HE-CIITA-NLRC5-IL7, and CFPAC-1-HE-CIITA-NLRC5-IL15 tumor vaccines were prepared according to Example 2. CFPAC-1-HE-CIITA-NLRC5, CFPAC-1-HE-CIITA-NLRC5-IL7, and CFPAC-1-HE-CIITA-NLRC5-IL15 were co-cultured with HA-CD4 T or E7-CD8 T cells. Cell proliferation was detected after 6 days ( FIG. 17 ). The results showed that CFPAC-1-HE-CIITA-NLRC5-IL7 and CFPAC-1-HE-CIITA-NLRC5-IL15 increased T cell proliferation in vitro by approximately 5-fold, indicating the ability to promote T cell proliferation.

Claims

1. A tumor vaccine comprising and / or expressing an antigen molecule, a co-stimulatory signal molecule, a cytokine, and a NLRC5 molecule.

2. The tumor vaccine according to claim 1, wherein the NLRC5 molecule comprises a functionally active fragment of the NLRC5 molecule.

3. The tumor vaccine according to any one of claims 1-2, wherein the NLRC5 molecule comprises the amino acid sequence shown in SEQ ID NO:

2.

4. The tumor vaccine according to any one of claims 1 to 3, further comprising and / or expressing a CⅡTA molecule.

5. The tumor vaccine according to claim 4, wherein the CⅡTA molecule comprises a functionally active fragment of the CⅡTA molecule.

6. The tumor vaccine according to any one of claims 4-5, wherein the CⅡTA molecule comprises the amino acid sequence shown in SEQ ID NO:

1.

7. The tumor vaccine according to any one of claims 4 to 6, wherein the NLRC5 molecule or a functionally active fragment thereof and the CⅡTA molecule or a functionally active fragment thereof are expressed in the form of a fusion protein.

8. The tumor vaccine according to any one of claims 4 to 7, wherein the NLRC5 molecule and / or the CⅡTA molecule is capable of enhancing antigen presentation.

9. The tumor vaccine according to any one of claims 1 to 8, wherein the antigen molecule comprises a tumor-associated antigen.

10. The tumor vaccine according to any one of claims 1 to 9, wherein the antigen molecule comprises a tumor-specific antigen.

11. The tumor vaccine according to any one of claims 1 to 10, wherein the antigen molecules include endogenous antigens and exogenous antigens.

12. The tumor vaccine according to any one of claims 1-11, wherein the antigen molecule is selected from the group consisting of HA, E7, NY-ESO, MAGE-A3, MART, EGFR-VIII, PSMA, GPC-3, Mesothelin, PSA and CLDN18.

2.

13. The tumor vaccine according to any one of claims 1 to 12, wherein the co-stimulatory signal molecule provides a second signal for T cell activation.

14. The tumor vaccine according to any one of claims 1-13, wherein the co-stimulatory signal molecule is selected from the group consisting of CD80, CD86, 4-1BBL, anti-CD28, OX40L, CD40 ligand, ICOS ligand, GITR ligand, 4-1BB ligand, OX40 ligand, TL1A, CD30 ligand, CD27 and Flt3 ligand. The tumor vaccine according to claim 14 , wherein the co-stimulatory signal molecule is CD80.

16. The tumor vaccine according to any one of claims 4-15, wherein the antigen molecule, co-stimulatory signal molecule, cytokine, NLRC5 molecule and / or CⅡTA molecule is expressed by a method selected from the group consisting of viral vector expression, plasmid expression, liposome delivery and RNA molecule encoding.

17. The tumor vaccine according to any one of claims 1 to 16, wherein the cytokine is capable of promoting the activation and / or proliferation of T cells.

18. The tumor vaccine according to any one of claims 1 to 17, wherein the cytokine is selected from the group consisting of IL-2, IL-7, IL-9, IL-15 and IL-21 or mutants of these molecules. The tumor vaccine according to claim 18 , wherein the cytokine is IL-2.

20. The tumor vaccine according to any one of claims 1 to 19, comprising and / or expressing antigen molecules, NLRC5 and CIITA, CD80 and IL-2.

21. The tumor vaccine according to any one of claims 1 to 20, which is a protein vaccine, a polypeptide vaccine, a microbial vector vaccine or a cell vaccine.

22. The tumor vaccine according to any one of claims 1-21, which has independent CD4 and CD8 T cell antigen presentation capabilities that are independent of host immune cells.

23. The tumor vaccine according to claim 22, wherein the host immune cells include dendritic cells, B cells, and / or macrophages.

24. A pharmaceutical composition comprising the tumor vaccine according to any one of claims 1 to 23, and optionally a pharmaceutically acceptable carrier.

25. A pharmaceutical combination comprising the tumor vaccine according to any one of claims 1 to 23, and an immunomodulator.

26. The pharmaceutical combination of claim 25, wherein the immunomodulator comprises a therapeutic antibody, a therapeutic cell, a therapeutic nucleic acid and / or a therapeutic gene.

27. The pharmaceutical combination of claim 26, wherein the therapeutic cells comprise immune cells.

28. The pharmaceutical combination according to claim 27, wherein the immune cells are selected from T cells, NK cells, NKT cells and TIL cells.

29. The pharmaceutical combination according to any one of claims 27-28, wherein the immune cells are modified immune cells.

30. The pharmaceutical combination of claim 29, wherein the modified immune cells comprise immune cells expressing a chimeric antigen receptor (CAR).

31. The pharmaceutical combination of claim 29, wherein the modified immune cells comprise immune cells expressing a T cell receptor (TCR).

32. A method for preparing the tumor vaccine of any one of claims 1-23, the pharmaceutical composition of claim 24, or the pharmaceutical combination of any one of claims 25-31.

33. Use of the tumor vaccine according to any one of claims 1 to 23, the pharmaceutical composition according to claim 24, or the pharmaceutical combination according to any one of claims 25 to 31 in the preparation of a drug for preventing and / or treating tumors.

34. The use according to claim 33, wherein the tumor is a solid tumor and / or a hematological tumor.

35. The use according to any one of claims 33-34, wherein the tumor is selected from the group consisting of pancreatic cancer, gastric cancer, lung cancer, prostate cancer, melanoma, breast cancer, kidney cancer, liver cancer and ovarian cancer.

36. A method for preventing and / or treating tumors, comprising administering the tumor vaccine according to any one of claims 1 to 23, the pharmaceutical composition according to claim 42, or the pharmaceutical combination according to any one of claims 25 to 31 to a subject in need thereof.

37. The method of claim 36, wherein the tumor is a solid tumor and / or a hematological tumor.

38. The method of any one of claims 36-37, wherein the tumor is selected from the group consisting of pancreatic cancer, gastric cancer, lung cancer, prostate cancer, melanoma, breast cancer, kidney cancer, liver cancer, and ovarian cancer.

39. The tumor vaccine according to any one of claims 1 to 23, the pharmaceutical composition according to claim 24, or the pharmaceutical combination according to any one of claims 5 to 31, for use in preventing and / or treating tumors.

40. The tumor vaccine, pharmaceutical composition or pharmaceutical combination according to claim 39, wherein the tumor is a solid tumor and / or a blood tumor.

41. The tumor vaccine, pharmaceutical composition or pharmaceutical combination according to any one of claims 39-40, wherein the tumor is selected from the group consisting of pancreatic cancer, gastric cancer, lung cancer, prostate cancer, melanoma, breast cancer, kidney cancer, liver cancer and ovarian cancer.

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