Immune-enhancing polypeptides and uses thereof

Abstract

This invention relates to an immune-enhancing polypeptide and its applications, specifically providing an immune-enhancing polypeptide, wherein the polypeptide is the N-terminal amino acid sequence of pyroptosis-associated protein GSDMD from position 1 to 500 or a truncated sequence thereof. The immune-enhancing polypeptide described herein can induce a stronger immune response and can simultaneously satisfy multiple antigen loading formats.

Description

[0001] This application claims priority to Chinese Patent Application No. 202411966900.1, filed on December 30, 2024, entitled "An Immune-Enhancing Polypeptide and Its Application Thereof," the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of tumor immunotherapy, specifically relating to an immune-enhancing polypeptide and its applications. Background Technology

[0003] Tumor vaccines work by introducing tumor antigens to activate or enhance the body's own immunity to kill tumor cells and achieve the effect of treating tumors. Due to their high safety, high specificity and long duration of maintaining immunity, they have made great progress in clinical treatment.

[0004] Existing technologies for enhancing the immunogenicity of tumor vaccines mainly fall into two categories: ① improving antigen presentation efficiency; ② promoting immune cell activation. For example, BioNTech has used strategies to enhance antigen presentation in its BNT-111 and BNT-113 pipelines by adjusting the subcellular localization of antigen proteins and MHC co-localization.

[0005] Pyroptosis is a form of autonomous programmed cell death that triggers a strong immune response in the body. The pyroptosis-associated protein GSDMD causes cell rupture by creating pores in the cell membrane, releasing inflammatory signals and other contents that activate the body's immune response. Summary of the Invention

[0006] This invention selects an amino acid fragment at the N-terminus of the GSDMD protein and links it to a tumor antigen. Through pyroptosis, immune cells recognize tumor cells that highly express the relevant tumor antigen, thereby achieving the purpose of treating tumors.

[0007] One objective of this invention is to provide a protein with immune-enhancing effects, wherein the protein is the N-terminal amino acid 1-500 of the pyroptosis-associated protein GSDMD or a truncated sequence thereof, preferably the N-terminal amino acid 1-275 of the GSDMD protein, and more preferably, has the amino acid sequence shown in SEQ ID NO:1.

[0008] A second objective of this invention is to provide a recombinant antigen with a self-adjuvant effect, comprising the immune-enhancing polypeptide and antigen described in objective one.

[0009] In some embodiments, the antigen is selected from viral antigens, bacterial antigens, fungal antigens, autoimmune disease-related antigens, and / or tumor antigens.

[0010] In some embodiments, the viral antigen includes one or more selected from the group consisting of: Epstein-Barr virus (EBV), adenovirus, cytomegalovirus, influenza virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes simplex virus, human immunodeficiency virus, human papillomavirus, Japanese encephalitis virus, measles virus, poliovirus, respiratory syncytial virus, rubella virus, smallpox virus, varicella-zoster virus, West Nile virus, polyomavirus, or Zika virus.

[0011] In other embodiments, the bacterial antigen includes one or more selected from the group consisting of: anthrax antigen, Gram-negative bacilli, chlamydia, diphtheria, Haemophilus influenzae, Helicobacter pylori, malaria, Mycobacterium tuberculosis, pertussis toxin, pneumococcus, rickettsia, Staphylococcus, Streptococcus, or Clostridium tetani; and the fungal antigen is selected from Candida, Coccidioides, Cryptococcus, Histoplasma capsulatum, Leishmania, Plasmodium, protozoa, parasites, Schistosoma, tinea fungi, Toxoplasma gondii, or Trypanosoma krusei.

[0012] In other embodiments, the fungal antigen includes one or more selected from the group consisting of: Candida, Coccidioides, Cryptococcus, Histoplasma capsulatum, Leishmania, Plasmodium, protozoa, parasites, Schistosoma, tinea fungi, Toxoplasma gondii, or Trypanosoma krusei.

[0013] In other embodiments, the autoimmune disease-associated antigens include one or more selected from the group consisting of: acute necrotizing hemorrhagic encephalopathy, allergic asthma, anemia, aphthous ulcers, arthritis, asthma, autoimmune thyroiditis, conjunctivitis, Crohn's disease, cutaneous lupus erythematosus, atopic dermatitis, eczematous dermatitis, diabetes, erythema nodosum leprosy, keratoconjunctivitis, multiple sclerosis, myasthenia gravis, psoriasis, scleroderma, systemic lupus erythematosus, ulcerative colitis, or vaginitis.

[0014] In other embodiments, the tumor antigen includes one or more selected from the group consisting of: A33, BAGE, Bcl-2, B cell maturation antigen, BCR-ABL, β-catenin, testicular cancer antigen, CA125, CA19-9, CA50, CA27.29, CA15-3, CD5, CD19, CD20, CD21, CD22, CD33, CD37, CD45, CD123, CEA, c-Met, CS-1, cyclin B1, DAGE, EBNA, EGFR, ELA2, ephrin B2, estrogen receptor, FAP, ferritin, folate-binding protein, GAGE, G250 / CA. IX, GD-2, GM2, gp75, gp100, HA-1, HA-2, HER-2 / neu, HM1.24, Survivin, KRAS, hTERT, Ki-67, LRP, mesothelin, mucin-like cancer-associated antigen, MUC1, p53, PR1, PRAME, PRTN3, RHAMM, or WT-1.

[0015] In some specific embodiments, the tumor antigen is selected from WT1; preferably, the amino acid sequence of WT1 is shown in SEQ ID NO:2.

[0016] A third objective of this invention is to provide a polynucleotide, wherein the polynucleotide includes a polynucleotide encoding the immune-enhancing polypeptide described in objective one or a polynucleotide encoding the recombinant antigen described in objective two.

[0017] In some specific implementations, the polynucleotide encoding the immune-enhancing polypeptide is covalently linked to the polynucleotide encoding the antigen.

[0018] A fourth objective of this invention is to provide a recombinant nucleic acid vaccine comprising the polynucleotides described in objective three.

[0019] In some embodiments, the vaccine further contains a delivery vector molecule, wherein the polynucleotide is delivered after being encapsulated by the delivery vector molecule.

[0020] The fifth objective of this invention is to provide a recombinant vector comprising the polynucleotide and backbone vector described in objective three.

[0021] In some implementations, the backbone vector includes a plasmid vector or a viral vector.

[0022] In some specific implementations, the skeleton carrier is selected from p2023.

[0023] The sixth objective of this invention is to provide an engineered host cell comprising the recombinant vector as described in objective five, and / or capable of expressing and / or secreting recombinant antigens.

[0024] In some implementations, the host cell is selected from antigen-presenting cells.

[0025] In some specific implementations, the host cell is selected from macrophages, B cells, or dendritic cells.

[0026] The seventh objective of this invention is to provide a method for preparing a recombinant RNA vaccine, comprising the following steps:

[0027] Provide the recombinant vector described in objective five;

[0028] The recombinant vector was transcribed into RNA in vitro.

[0029] In some embodiments, the preparation process of the recombinant vector includes: artificially synthesizing a polynucleotide sequence of the recombinant antigen and linking it to a backbone vector;

[0030] The ligated product was transfected into host cells and cultured to obtain a positive clone recombinant expression vector.

[0031] In some specific implementations, the skeleton carrier is p2023.

[0032] The eighth objective of this invention is to provide a method for preparing engineered host cells, comprising loading the host cells with the recombinant antigen described in objective two or introducing the polynucleotide described in objective three; preferably, loading the host cells with the recombinant antigen or polynucleotide comprises: (1) contacting the antigen-presenting cell with the recombinant antigen or with the polynucleotide;

[0033] In some specific implementations, the host cell is selected from antigen-presenting cells.

[0034] In some specific implementations, the host cell is selected from macrophages, B cells, or dendritic cells.

[0035] In some specific implementations, the antigen-presenting cells are contacted with the mature composition before or after step (1).

[0036] The ninth objective of this invention is to provide a method for in vitro activation of immune-active cells derived from a patient with a tumor, comprising:

[0037] Immune-active cells were obtained from the patient.

[0038] Engineered antigen-presenting cells are generated by the method described in objective eight; and

[0039] The various immune-active cells are co-cultured with the engineered antigen-presenting cells for a sufficient period of time to activate the immune-active cells, thereby obtaining activated immune-active cells.

[0040] The tenth objective of this invention is to provide an activated immune-active cell, obtained by the method described in objective nine.

[0041] The eleventh objective of this invention is to provide a pharmaceutical composition comprising the recombinant antigen described in objective two, the polynucleotide described in objective three, the recombinant nucleic acid vaccine described in objective four, the recombinant vector described in objective five, the host cell described in objective six, the recombinant RNA vaccine prepared by the method described in objective seven, the engineered host cell prepared by the method described in objective eight, or the immune active cell described in objective ten, and pharmaceutically acceptable excipients.

[0042] The twelfth objective of this invention is to provide the use of the immune-enhancing polypeptide of objective one, the recombinant antigen of objective two, the polynucleotide of objective three, the recombinant nucleic acid vaccine of objective four, the recombinant vector of objective five, the host cell of objective six, the recombinant RNA vaccine prepared by the method of objective seven, the engineered host cell prepared by the method of objective eight, the immune-active cell of objective ten, and / or the pharmaceutical composition of objective eleven in the preparation of a medicament for the prevention or treatment of antigen-related diseases.

[0043] For example, WT1-related diseases include, but are not limited to: hematologic malignancies such as acute myeloid leukemia and multiple myeloma; solid tumors such as lung cancer, pancreatic cancer, liver cancer, breast cancer, and glioma; and others such as stomach cancer, kidney cancer, and skin cancer.

[0044] The beneficial effects of the technical solution of this invention are as follows:

[0045] The immune-enhancing peptides described in this invention are applicable to various vaccines. They are translated from mRNA into proteins, inducing pyroptosis and triggering an immune response. The immune-enhancing peptides are covalently linked to the antigen sequence in the form of a nucleic acid chain drug. In vitro, they are delivered via dendritic cells (DCs) as a carrier and directly injected intramuscularly into tissues. By inducing pyroptosis in cells or tissues, the released danger signals trigger an immune response. Compared to traditional adjuvants, they elicit a stronger immune response and can simultaneously support multiple antigen loading formats, such as DC-mRNA vaccines, DNA vaccines, and LNP-mRNA vaccines. Attached Figure Description

[0046] Figure 1 This is the 275-WT1 plasmid map.

[0047] Figure 2 Electrophoresis image of 275-WT1 mRNA at 5200 nm.

[0048] Figure 3The results of Western blot analysis for 275-WT1 mRNA were as follows.

[0049] Figure 4 The result for 275-WT1 mRNA LDH is...

[0050] Figure 5(AB) shows the effect of T cell co-culture. Figure 5-A Indicates the number of ELISpot spots. Figure 5-B This represents the data for IFN-γ. Detailed Implementation

[0051] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used herein are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0052] In this invention, the term "pyroptosis" refers to programmed cell death, also known as inflammatory necrosis, which is characterized by the continuous swelling of cells until the cell membrane ruptures, leading to the release of cell contents and activating a strong inflammatory response. Pyroptosis is a programmed cell death mediated by the gasdermin protein.

[0053] The invention provides an immune-enhancing polypeptide, wherein the protein is the N-terminal amino acid 1-500 of the pyroptosis-associated protein GSDMD or a truncated sequence thereof, preferably the N-terminal amino acid 1-275 of the GSDMD protein.

[0054] The term "immune enhancement" refers to strengthening the body's immune system, enabling it to more effectively combat the invasion of external pathogens. This includes increasing the activity of immune cells, increasing antibody production, or improving the overall function of the immune system.

[0055] In some specific embodiments, the immune-enhancing polypeptide has an amino acid sequence as shown in SEQ ID NO:1.

[0056] Without substantially affecting activity, those skilled in the art can modify the sequence of the present invention by one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) to obtain variants of the recombinant antigen or its functional fragment sequence. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically up to 20, preferably up to 10, more preferably up to 5) at the C-terminus and / or N-terminus. In the art, conservative substitutions with amino acids of similar or comparable properties generally do not alter the function of the protein. Similarly, the addition of one or more amino acids at the C-terminus and / or N-terminus generally does not alter the function of the protein. These are all considered to be included within the scope of protection of the present invention.

[0057] In some embodiments, the sequence of the variant described in this invention may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with its source sequence. This sequence identity can be measured using sequence analysis software, such as the computer program BLAST with default parameters, particularly BLASTP or TBLASTN.

[0058] A second objective of this invention is to provide a recombinant antigen with a self-adjuvant effect, wherein the recombinant antigen comprises the protein sequence and antigen sequence described in objective one.

[0059] Self-adjuvant is a novel vaccine design strategy in which recombinant antigens can simultaneously act as adjuvants, thereby enhancing the immune response to the vaccine. The immune-enhancing peptides described in this invention are applicable to various vaccines. They are translated from mRNA into proteins, inducing pyroptosis and triggering an immune response, exhibiting a "self-adjuvant effect."

[0060] The term "antigen" has its conventional meaning and refers to a molecule capable of inducing an immune response. In the context of this invention, an antigen can be a protein or a fragment thereof, such as a (poly)peptide that presents an epitope of said protein. However, the antigen used may also be an artificial peptide or a peptide mimic.

[0061] For example, antigens can be intact cells (e.g., live or dead cells), cell fractions (e.g., lysed cells), cellular antigens (e.g., cell surface antigens), protein extracts, purified proteins, or synthetic peptides. For example, one or more antigens in some embodiments of the present invention include malignancy-associated antigens (e.g., tumor antigens) and viral antigens (i.e., viral antigens).

[0062] In some embodiments, the antigen includes infectious organisms (e.g., viruses, bacteria, fungi) that typically affect the immunity of a subject (e.g., a transplant patient). Examples of infectious organisms that can affect a patient's immunity include, but are not limited to, viruses, bacteria, fungi, etc.

[0063] In some implementations, the antigen is a viral antigen, such as, but not limited to, Epstein-Barr virus (EBV), adenovirus (Adv), cytomegalovirus (CMV), influenza virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes simplex virus, human immunodeficiency virus (HIV), human papillomavirus (HPV), Japanese encephalitis virus, measles virus, poliovirus, respiratory syncytial virus, rubella virus, smallpox virus, varicella-zoster virus, West Nile virus, polyomavirus (e.g., BK virus), or Zika virus.

[0064] In some embodiments, the antigen is a bacterial antigen, such as, but not limited to, anthrax antigen, Gram-negative bacilli, chlamydia, diphtheria, Haemophilus influenzae, Helicobacter pylori, malaria, Mycobacterium tuberculosis, pertussis toxin, pneumococcus, rickettsia, staphylococcus, streptococcus, or tetanus.

[0065] In some embodiments, the antigen is a fungal antigen, such as, but not limited to, Candida, Coccidioides, Cryptococcus, Histoplasma capsulatum, Leishmania, Plasmodium, protozoa, parasites, schistosomiasis, tinea fungi, Toxoplasma gondii, or Trypanosoma krusei.

[0066] In some implementations, the antigen is an antigen expressed by cells associated with an undesirable autoimmune or allergic condition. Exemplary autoimmune conditions include, but are not limited to, acute necrotizing hemorrhagic encephalopathy, allergic asthma, anemia, aphthous ulcers, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), asthma, autoimmune thyroiditis, conjunctivitis, Crohn's disease, cutaneous lupus erythematosus, dermatitis (including atopic dermatitis and eczematous dermatitis), diabetes mellitus, erythema nodosum leprosy, keratoconjunctivitis, multiple sclerosis, myasthenia gravis, psoriasis, scleroderma, Sjogren's syndrome, including dry keratoconjunctivitis secondary to Sjogren's syndrome, Stevens-Johnson syndrome, systemic lupus erythematosus, ulcerative colitis, vaginitis, or Wegener's granulomatosis.

[0067] In some embodiments, the antigen is an antigen (or a portion thereof, such as an epitope) expressed by tumor cells, and the antigen (or a portion thereof) is derived from a protein expressed in hematopoietic tissue (e.g., hematologic malignancies, such as leukemia antigen) or in solid tumors (e.g., melanoma, pancreatic cancer, liver cancer, gastrointestinal cancer).

[0068] Examples of tumor antigens include, but are not limited to, A33, BAGE, Bcl-2, B cell maturation antigen (BCMA), BCR-ABL, β-catenin, testicular cancer antigens (CTA, such as MAGE-1, MAGE-A2 / A3, and NY-ESO-1), CA125, CA19-9, CA50, CA27.29 (BR 27.29), CA15-3, CD5, CD19, CD20, CD21, CD22, CD33, CD37, CD45, CD123, CEA, c-Met, CS-1, cyclin B1, DAGE, EBNA, EGFR, ELA2, ephrin B2, estrogen receptor, FAP, ferritin, folate-binding protein, GAGE, G250 / CA IX, GD-2, GM2, gp75, gp100 (Pmel 17) HA-1, HA-2, HER-2 / neu, HM1.24, Survivin, KRAS, hTERT, Ki-67, LRP, mesothelin, mucin-like cancer-associated antigen (MCA), MUC1, p53, PR1, PRAME, PRTN3, RHAMM (CD168), WT-1.

[0069] The antigen can be one or more in combination; for example, in some specific embodiments, the tumor antigen is selected from WT1.

[0070] In this article, "multiple" or "various types" refers to more than one or more kinds, that is, two or more types.

[0071] The term "WT1" stands for Wilms' Tumor gene 1, a zinc finger gene located in the 11p13 region of chromosome 1. It is approximately 12,000 base pairs long and includes multiple exons and introns. The WT1 gene is widely expressed in the human body and has a wide range of functions, playing a crucial role in maintaining normal embryonic development, hematopoiesis, and the immune system.

[0072] In some specific implementations, the amino acid sequence of WT1 is shown in SEQ ID NO:2.

[0073] A third objective of this invention is to provide a polynucleotide, wherein the polynucleotide includes a polynucleotide encoding the recombinant antigen described in objective two.

[0074] The coding sequence of a recombinant antigen can be DNA or RNA, such as mRNA.

[0075] The polynucleotide encoding the immune-enhancing polypeptide and the polynucleotide encoding the antigen can exist independently or in tandem. The tandem connection can be achieved through a specific linker or through covalent chemical bonding.

[0076] In some specific implementations, the polynucleotide encoding the immune-enhancing polypeptide is covalently linked to the polynucleotide encoding the antigen.

[0077] A fourth objective of this invention is to provide a recombinant nucleic acid vaccine comprising the polynucleotides described in objective three.

[0078] In some embodiments, the vaccine further contains a delivery vector molecule, wherein the polynucleotide is delivered after being encapsulated by the delivery vector molecule.

[0079] The delivery medium molecule includes any one of lipids, liposomes, cationic liposomes, lipid nanoparticles (LNPs), polymeric compounds, peptides, proteins, cells, nanoparticle mimics, nanotubes, or conjugates, preferably liposomes, cationic liposomes, or lipid nanoparticles.

[0080] Lipid nanoparticles (LNPs) are nanocarriers for delivering nucleic acid substances to cells, containing ionizable lipids, auxiliary lipid molecules, cholesterol or cholesterol derivatives, or polymer-conjugated lipid molecules. Examples include those described in PCT / CN2022 / 118198. The full text of PCT / CN2022 / 118198 is incorporated herein by reference.

[0081] Cationic liposomes typically comprise (1) cationic lipids, and (2) one or both of accessory lipids and cholesterol. Optionally, cationic liposomes may also comprise polymer-conjugated lipid molecules.

[0082] In some embodiments, cationic liposomes contain DOTMA, DOPE, and cholesterol, as described, for example, as in CN109331176B. The cationic (positively charged) liposomes and the anionic (negatively charged) nucleic acid form a "lipoplex (LPX)," which can deliver the nucleic acid into the cell.

[0083] The fifth objective of this invention is to provide a recombinant vector containing a backbone vector and the polynucleotide described in objective three.

[0084] In some implementations, the backbone vector includes a plasmid vector or a viral vector.

[0085] In some specific implementations, the skeleton carrier is selected from p2023.

[0086] The sixth objective of this invention is to provide an engineered host cell comprising the recombinant vector as described in objective five, and / or capable of expressing and / or secreting recombinant antigens.

[0087] In some implementations, the host cell is selected from antigen-presenting cells.

[0088] In some specific implementations, the host cell is selected from macrophages, B cells, or dendritic cells.

[0089] The term "engineering" and its grammatical equivalents can refer to one or more human-designed alterations to nucleic acids (e.g., nucleic acids within an organism's genome). In another embodiment, engineering can refer to alterations, additions, and / or deletions of genes.

[0090] In some implementations, the term "engineered host cell" is selected from "engineered antigen-presenting cells," which refers to immune cells with added, deleted, and / or altered genes. As used herein, the terms "immune cell" or "engineered antigen-presenting cell" and their grammatical equivalents may refer to immune cells of human or non-human animal origin.

[0091] Antigen-presenting cells are cells that can transmit antigen information to lymphocytes (such as T cells) to trigger an immune response, including macrophages, B cells, and dendritic cells (DC cells or DCs).

[0092] Recombinant antigens or their coding sequences can be introduced into antigen-presenting cells in the form of proteins, RNA, or DNA. For example, DNA vectors expressing recombinant antigens can be constructed and transformed into antigen-presenting cells.

[0093] Therefore, the present invention also includes polynucleotides (in DNA or RNA form) encoding the immune-enhancing peptides or recombinant antigens described herein, and nucleic acid constructs (e.g., expression vectors and integration vectors) containing these polynucleotides. The vectors described herein typically contain sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences. These sequences (collectively referred to as “flanking sequences” in some embodiments) typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splicing sites, a leader sequence encoding a peptide secretion site, a ribosome binding site, a polyadenylated sequence, a multi-linker region for inserting a nucleic acid encoding an antibody to be expressed, and optional marker elements.

[0094] The polynucleotides introduced into antigen-presenting cells according to the present invention can be in the form of DNA or RNA (e.g., mRNA or saRNA). The inventors have found that mRNA offers advantages in safety compared to traditional vaccines, such as not introducing gene mutations, being degraded by normal cells, and having its half-life altered by regulating sequence modifications and delivery vectors. Methods for introducing mRNA into antigen-presenting cells (e.g., dendritic cells) are well known in the art, for example, by electroporation.

[0095] The seventh objective of this invention is to provide a method for preparing a recombinant RNA vaccine, comprising the following steps:

[0096] Provide the recombinant vector described in objective five;

[0097] The recombinant vector was transcribed into RNA in vitro.

[0098] In some embodiments, the preparation process of the recombinant vector includes: artificially synthesizing a polynucleotide sequence of the recombinant antigen and linking it to a backbone vector;

[0099] The ligated product was transfected into host cells and cultured to obtain a positive clone recombinant expression vector.

[0100] In some specific implementations, the skeleton carrier is p2023.

[0101] The eighth objective of this invention is to provide a method for preparing engineered host cells, comprising loading the host cells with the recombinant antigen described in objective two or introducing the polynucleotide described in objective three.

[0102] In some implementations, loading host cells with recombinant antigens or polynucleotides includes: (1) contacting antigen-presenting cells with recombinant antigens or polynucleotides;

[0103] In some specific implementations, the host cell is selected from antigen-presenting cells.

[0104] In some specific implementations, the host cell is selected from macrophages, B cells, or dendritic cells (DCs).

[0105] In some specific implementations, the antigen-presenting cells are contacted with the mature composition before or after step (1).

[0106] In this study, DCs can be derived from DC precursor cells isolated from the subject's own blood, such as CD34+ hematopoietic precursor cells derived from umbilical cord blood, or differentiated from CD14+ monocytes derived from peripheral blood. DCs were obtained after isolation, culture, expansion, and differentiation from the subject.

[0107] The method for culturing DC precursor cells to differentiate into DCs can be any method known in the art or any other method capable of differentiating DC precursor cells into DCs, such as adding cytokines GM-CSF and IL-4 to the culture medium for differentiation culture. In other embodiments, the DCs can be cells obtained by in vitro expansion and culture followed by differentiation culture of an immortalized DC precursor cell line. The immortalized DC precursor cell line can be a cell line known in the art or publicly reported, such as the MUTZ3 cell line, or an immortalized DC precursor cell line prepared by the method described in CN201810368646.3. The immortalized DC precursor cell line can be expanded in large quantities in vitro and then differentiated into DCs by the aforementioned method.

[0108] The term "load" as used herein refers to enabling antigen-presenting cells to contain (capture) tumor antigens in a certain way, thereby processing the antigens and presenting them to other immune cells. Taking dendritic cells (DCs) as an example, loading can be achieved through various methods of contacting the antigen or its coding sequence, such as incubation with recombinant, synthetic, or purified tumor antigen peptides or proteins, incubation with tumor cell lysates, incubation with apoptotic or necrotic tumor cells, or enabling the cells to express the antigen. Enabling cells to express antigens can be achieved by contacting the cells with nucleic acids (DNA or RNA) encoding the tumor antigen (e.g., co-incubation) or by introducing the nucleic acids into the cells (e.g., via electroporation). Introducing a DNA-coding sequence into cells typically involves a nucleic acid (DNA) construct, such as an expression vector and an integration vector, containing the DNA sequence along with a suitable promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins. Alternatively, the RNA-coding sequence of the antigen (e.g., mRNA) can be directly introduced into the cells to express the antigen. Depending on the tumor to be targeted, the antigen can be contacted with and loaded with the corresponding tumor antigen or its encoding nucleic acid (e.g., mRNA). Methods of loading antigens are known in the art, such as incubation, cell transformation (e.g., electroporation of DNA or mRNA), etc.

[0109] Antigen-presenting cells (e.g., dendritic cells) are induced to mature by contact with a maturation composition (maturation cocktail). The maturation composition used exemplary herein comprises one or more selected from IFN-γ, PolyI:C, R848, and PGE2. The dendritic cells are contacted with the maturation composition for at least 10 hours, at least 20 hours, at least 30 hours, or at least 40 hours.

[0110] There are generally no particular restrictions on the order of engineering and maturation of antigen-presenting cells; that is, cells can be loaded with antigens before being exposed to the cytokine composition, or antigens can be exposed to the cytokine composition before being loaded with antigens. This is within the knowledge of those skilled in the art.

[0111] The method for preparing the DC cells provided by the present invention includes enabling dendritic cells to secrete multispecific nanobodies and loading them with tumor antigens. The method may include the steps of: (1) loading the tumor antigen or its coding sequence onto dendritic cells capable of secreting multispecific nanobodies; or (2) enabling the tumor antigen-loaded dendritic cells to secrete multispecific nanobodies; or (3) contacting the dendritic cells with the tumor antigen or its coding sequence and the coding sequence of the multispecific nanobodies. The method of loading the antigen is as described herein, for example by contacting the cells with the antigen or its coding nucleic acid (DNA or RNA) (e.g., co-incubation) or introducing the antigen or its coding nucleic acid into the cells (e.g., by electroporation, or by electroporation of RNA).

[0112] The RNA coding sequence of the recombinant antigen can be synthesized by a gene company or obtained through in vitro transcription. Those skilled in the art are familiar with methods for preparing RNA sequences through in vitro transcription. Exemplary in vitro transcription methods include the steps of constructing a transcription template DNA vector and incubating it in a transcription system. Transcription systems and incubation conditions are well known in the art; transcription systems include, for example, transcription buffers (containing, but not limited to, Tris-HCl, MgCl2, DTT, spermidine), NTPs, RNase inhibitors, RNA polymerase, etc.; incubation conditions include, for example, 37°C for at least 2 hours.

[0113] Furthermore, in embodiments expressing antigens and immune-enhancing peptides, the coding sequences of the antigens and immune-enhancing peptides can be introduced into cells separately or simultaneously. Similarly, the coding sequences of the antigens and immune-enhancing peptides can be located on separate nucleic acid constructs or combined in a suitable manner on the same nucleic acid construct.

[0114] Specific steps for preparing dendritic cells (DCs) include, for example, antigen-presenting cells (APCs) such as DCs being obtained from subjects, such as patients with cancer or at risk of developing cancer, via apheresis. Purified dendritic cells are cultured in the presence of a maturation composition to obtain mature DCs. Mature DCs are loaded with antigens (e.g., P53, Survivin, MUC1, hTERT, and KRAS), for example, by electroporation of the mRNA encoding the antigen, thereby obtaining mature DCs containing the antigen and a DC vaccine. Before, during, or after antigen loading, the DCs may be introduced with the multispecific nanobody encoding sequences described herein, for example, by electroporation of the mRNA, saRNA, or DNA of the multispecific nanobody. The resulting DCs are then given to the patient. An exemplary treatment procedure involves administering DCs three times over a 4-week period.

[0115] The DC cells used in this invention are either freshly prepared or obtained by cryopreservation followed by thawing, such as DC cells obtained by thawing and culturing after one month of cryopreservation. The reagents, conditions, etc. required for cryopreservation and thawing can be obtained using conventional methods in the art.

[0116] In this article, the culture medium and culture conditions required for preparing DC cells can be the same as those for conventional DC cell culture. Exemplary culture media and culture conditions are shown in the examples.

[0117] The ninth objective of this invention is to provide a method for in vitro activation of immune-active cells derived from a patient with a tumor, comprising:

[0118] Immune-active cells were obtained from the patient.

[0119] Engineered antigen-presenting cells are generated by the method described in objective eight; and

[0120] The various immune-active cells are co-cultured with the engineered antigen-presenting cells for a sufficient period of time to activate the immune-active cells, thereby obtaining activated immune-active cells.

[0121] The tenth objective of this invention is to provide an activated immune-active cell, obtained by the method described in objective nine.

[0122] The eleventh objective of this invention is to provide a pharmaceutical composition comprising the recombinant antigen described in objective two, the polynucleotide described in objective three, the recombinant nucleic acid vaccine described in objective four, the recombinant vector described in objective five, the host cell described in objective six, the recombinant RNA vaccine prepared by the method described in objective seven, the engineered host cell prepared by the method described in objective eight, or the immune active cell described in objective ten, and pharmaceutically acceptable excipients.

[0123] Pharmaceutically acceptable excipients include, but are not limited to, diluents, carriers, solubilizers, emulsifiers, and / or preservatives and adjuvants. The excipients are preferably non-toxic to the recipient at the dosage and concentration used. Such excipients include, but are not limited to, saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. In some embodiments, the pharmaceutical composition may contain substances for improving, maintaining, or retaining, for example, the composition's pH, permeability, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption, or permeation. These substances are known in the art. The optimal pharmaceutical composition can be determined based on the intended route of administration, delivery method, and required dosage.

[0124] The excipients in the pharmaceutical composition also include vaccine adjuvants. The adjuvants can be small molecules, biomolecules, compositions, complexes, or extracts of compounds known in the art that can enhance immune responses. In one or more embodiments, the adjuvants include those selected from aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete and incomplete Freund's adjuvants), prostaglandin E2, alpha-interferon, Corynebacterium breviculae, lipopolysaccharides, cytokines, oil-in-water emulsions, water-in-oil emulsions, nanoemulsions, microparticle delivery systems, liposomes, microspheres, biodegradable microspheres, plaque virions, protein liposomes, proteasomes, immune-enhancing complexes (ISCOMs, ISCOMATRIX), microparticles, nanoparticles, biodegradable nanoparticles, silicon nanoparticles, polymeric micro / nanoparticles, polymeric sheet substrate particles (PLSP), microparticle resins, nanoliposome polymeric gels, synthetic / biodegradable and biocompatible semi-synthetic or natural polymers or dendritic polymers (e.g., PLG, PL...). GA, PLA, polycaprolactone, silicone polymers, polyesters, polydimethylsiloxane, sodium polystyrene sulfonate, polystyrene benzyltrimethylammonium chloride, polystyrene divinylbenzene resin, polyphosphazene, poly-[di-(carboxyacetylphenoxy)phosphazene (PCPP), poly-(methyl methacrylate), dextran, polyvinylpyrrolidone, hyaluronic acid and its derivatives, chitosan and its derivatives, polysaccharides, δ-inulin polysaccharide, glycolipids (synthetic or natural), lipopolysaccharides, one or more polycationic compounds (such as polyamino acids, poly-(γ-glutamic acid), poly-arginine-HCl, poly-L-lysine, polypeptides, biopolymers), cationic dimethyl di(octadecyl)ammonium (DDA), α-galactoside ceramide and its derivatives, archaeal lipids and their derivatives, lactams, gallons, glycerides, phospholipids, and spirochetes.

[0125] Pharmaceutical compositions for internal administration are typically provided in sterile formulations. Sterilization is achieved by filtration through a sterile filter membrane. When the composition is lyophilized, sterilization can be performed using this method before or after lyophilization and rehydration. The pharmaceutical compositions of the present invention may be used for parenteral delivery. Compositions for parenteral administration may be lyophilized or stored in solution. They are prepared, for example, by conventional methods using physiological saline or aqueous solutions containing glucose and other excipients. Parenteral compositions are typically placed in containers with sterile access openings, such as intravenous solution bands or vials with stoppers puncturable by a hypodermic needle. Alternatively, the compositions may be used for inhalation or delivery via the digestive tract (e.g., orally). The preparation of the pharmaceutically acceptable compositions is within the scope of the art. Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations containing antibodies in sustained or controlled-release delivery formulations. Techniques for formulating a variety of other sustained or controlled delivery methods (such as liposome carriers, bioeasily perishable microparticles or porous beads, and accumulation injection) are also known to those skilled in the art.

[0126] Once formulated, the pharmaceutical composition is stored in sterile vials as a solution, suspension, gel, emulsion, solid, crystal, or as a dehydrated or lyophilized powder. The formulation may be stored in a ready-to-use form or rehydrated before administration (e.g., lyophilized). The present invention also provides kits for generating single-dose administration units. The kits of the present invention may each contain a first container with dried protein and a second container with an aqueous formulation. In some embodiments of the invention, kits containing single-lumen and multi-lumen pre-filled syringes (e.g., liquid syringes and lyophilized syringes) are provided.

[0127] The twelfth objective of this invention is to provide the use of the immune-enhancing polypeptide of objective one, the recombinant antigen of objective two, the polynucleotide of objective three, the recombinant nucleic acid vaccine of objective four, the recombinant vector of objective five, the host cell of objective six, the recombinant RNA vaccine prepared by the method of objective seven, the engineered host cell prepared by the method of objective eight, the immune-active cell of objective ten, and / or the pharmaceutical composition of objective eleven in the preparation of a medicament for the prevention or treatment of antigen-related diseases.

[0128] For example, tumor antigens can trigger conditions and diseases such as the occurrence, growth, and / or metastasis of tumors (cancer), including but not limited to: lung cancer, non-small cell lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma, kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematologic malignancies, head and neck cancer, glioma, mesothelioma, colorectal cancer, stomach cancer, nasopharyngeal carcinoma, laryngeal cancer, cervical cancer, uterine fibroids and osteosarcoma, bone cancer, pancreatic cancer, renal cell carcinoma, skin cancer, prostate cancer, malignant melanoma of the skin or eye, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small bowel cancer, endocrine system cancers, and bile duct cancer. Thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, urothelial carcinoma, penile cancer, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia), childhood solid tumors, lymphocytic lymphoma, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, Hodgkin's lymphoma, epidermal carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, including asbestos-induced cancers, and various types of leukemia and lymphoma, as well as various precancerous lesions.

[0129] For example, WT1-related diseases include, but are not limited to: hematologic malignancies such as acute myeloid leukemia and multiple myeloma; solid tumors such as lung cancer, pancreatic cancer, liver cancer, breast cancer, and glioma; and others such as stomach cancer, kidney cancer, and skin cancer.

[0130] This invention also provides a method for treating patients (especially those with mesothelin-related diseases) by administering the immune-enhancing peptides described in Objective 1, the recombinant antigens described in Objective 2, the polynucleotides described in Objective 3, the recombinant nucleic acid vaccines described in Objective 4, the recombinant vectors described in Objective 5, the host cells described in Objective 6, the recombinant RNA vaccines prepared by the method described in Objective 7, the engineered host cells prepared by the method described in Objective 8, the immune-active cells described in Objective 10, and / or the pharmaceutical compositions described in Objective 11. In this document, the terms “patient,” “subject,” “individual,” and “object” are used interchangeably and include any living organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit, etc.), and most preferably a human. “Treatment” refers to a subject receiving the treatment regimen described herein to achieve at least one positive therapeutic effect (e.g., a reduction in the number of cancer cells, a reduction in tumor volume, a decrease in the rate of cancer cell invasion into surrounding organs, or a decrease in the rate of tumor metastasis or tumor growth). “Prevention” refers to a subject at risk receiving the treatment regimen described herein to achieve at least one effect of preventing the occurrence of disease or symptoms. Effective treatment or prevention regimens for patients can vary depending on various factors (e.g., the patient's disease state, age, weight, and the ability of the therapy to elicit an anti-cancer response in the subject).

[0131] The term "effective dose" refers to a dose that can achieve therapeutic, preventive, alleviating and / or relieving disease or condition as described in this invention in a subject.

[0132] The term "disease and / or symptom" refers to a physical condition of the subject that is related to the disease and / or symptom described in this invention.

[0133] The term "subject" or "patient" may refer to a patient or other animal, particularly a mammal, such as a human, dog, monkey, cow, horse, etc., that receives the pharmaceutical composition of the present invention to treat, prevent, reduce and / or alleviate the disease or condition described in the present invention.

[0134] The therapeutically effective dose of the drug composition comprising the immune-enhancing peptide described in Objective 1, the recombinant antigen described in Objective 2, the polynucleotide described in Objective 3, the recombinant nucleic acid vaccine described in Objective 4, the recombinant vector described in Objective 5, the host cell described in Objective 6, the recombinant RNA vaccine prepared by the method described in Objective 7, the engineered host cell prepared by the method described in Objective 8, the immune-active cells described in Objective 10, and / or the pharmaceutical composition described in Objective 11 will depend, for example, on the extent of treatment and the target. Those skilled in the art will understand that the appropriate dose level for treatment will vary in part depending on the delivered molecule, indication, route of administration, and the patient's size (weight, body surface or organ size) and / or condition (age and general health status). In some embodiments, clinicians may titrate the dose and change the route of administration to obtain optimal therapeutic effect. For example, approximately 10 micrograms / kg body weight to approximately 50 milligrams / kg body weight per day.

[0135] The frequency of administration will depend on the pharmacokinetic parameters of the bound molecules in the formulation used. Clinicians typically administer the composition until a dose is reached to achieve the desired effect. The composition can therefore be administered as a single dose, or over time as two or more doses (with or without the same amount of the desired molecule), or via implanted device or catheter as a continuous infusion.

[0136] The administration routes of the above-mentioned technical solutions are based on known methods, such as oral, intravenous, intraperitoneal, intracerebral (within brain parenchyma), intraventricular, intramuscular, intraocular, intraarterial, portal vein, or intralesional injection; via sustained-release systems or implantable devices. In one or more embodiments, the pharmaceutical composition as a vaccine can be administered to the inguinal region via intrasegmental injection. Optionally, depending on the vaccine target, the vaccine can be administered subcutaneously or intradermally to the hands and feet of a cancer patient receiving treatment. Other routes of administration, such as intramuscular injection or blood injection, may also be employed.

[0137] Depending on the type of the intended technical solution (e.g., a vaccine) being prepared, the production scale of the solution can be expanded if necessary by culturing cells in a bioreactor, fermenter, or similar container and apparatus suitable for mass cell growth. In one or more embodiments, according to the invention, a device or composition comprising the produced or recycled vaccine or antigen is adapted for continuous or intermittent release and can be implanted in the body or administered locally at a corresponding location in the body to achieve the effect of slow and timed release of these materials into the body.

[0138] The present invention also provides a method for treating and / or preventing cancer, the method comprising administering to a subject an effective dose of one or more of the following: the immune-enhancing polypeptide of objective one, the recombinant antigen of objective two, the polynucleotide of objective three, the recombinant nucleic acid vaccine of objective four, the recombinant vector of objective five, the host cell of objective six, the recombinant RNA vaccine prepared by the method of objective seven, the engineered host cell prepared by the method of objective eight, the immune-active cell of objective ten, and / or the pharmaceutical composition of objective eleven. The method comprises effects of at least one of both treatment and prevention. In one or more embodiments, the method of the present invention is for preventative purposes, and one or more of the objective measures of the present invention are administered to the subject before the occurrence of cancer or precancerous lesions. In some cases, the objective measures are administered to the subject after the onset of one or more of the aforementioned cancers, aiming to prevent the occurrence of further symptoms or further deterioration of existing symptoms. Prophylactic administration of one or more of the objective measures of the present invention aims to prevent or alleviate any subsequent symptoms. In one or more embodiments, the method of the present invention is for therapeutic purposes, and one or more of the objective measures of the present invention are administered to the subject at the onset of cancer or after the onset of cancer, aiming to alleviate the symptoms of existing cancer.

[0139] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0140] Experimental steps:

[0141] 1. DC cell culture and induction

[0142] 1.1 Separation of PBMC

[0143] (1) Use a syringe to draw apheresis blood from the blood bag into a 50ml centrifuge tube, wash the blood bag with an equal volume of PBS (HyClone brand) and mix it with the extracted apheresis blood (1:1 dilution);

[0144] (2) Take a 50ml centrifuge tube (containing 15ml Ficoll lymphocyte separation solution (brand name: GE)) and slowly add the blood and PBS mixture from step 1 along the tube wall. Then centrifuge at 800g for 20min with an acceleration of 1 and a deceleration of 0.

[0145] (3) Carefully aspirate the white cell layer into another 50ml centrifuge tube, add PBS, centrifuge and wash at 400g / min for 10min, with acceleration and deceleration at 9.

[0146] (4) Discard the waste liquid, but do not discard it completely. Add DPBS, wash again, and centrifuge (same as step 3).

[0147] (5) Drain the waste liquid, add AIM-V culture medium (gibco brand) to suspend the cells, and add them to the culture flask. Let them adhere to the wall overnight.

[0148] 1.2 DC cell generation

[0149] Monocyte-derived dendritic cells (DCs) were generated from peripheral blood mononuclear cells (PBMCs) via standard Ficoll density centrifugation to isolate PBMCs from patient leukocyte removal samples. PBMCs were seeded in serum-free AIM-V medium and allowed to adhere to culture flasks with 0.22 μm filter caps. After 2 hours, non-adherent cells were removed, and adherent monocytes were subsequently cultured for 6 days in AIM-V medium containing 50 ng / ml rhIL-4 and 100 ng / ml rhGM-CSF. On day 3, half of the medium was replaced with fresh medium containing GM-CSF and IL-4. A maturation mixture consisting of 100 IU / ml IFN-γ, 30 μg / ml poly(I:C), 5 μg / ml R848, and 1 μg / ml PGE2 was used to induce DC maturation for 24 hours.

[0150] 1.3 DC cell transformation

[0151] Observe cell morphology under a microscope, collect DCs into centrifuge tubes, and centrifuge at 300g for 10 min at room temperature; discard the supernatant, add an appropriate amount of DPBS to resuspend, and count the cells; take the number of cells for electroporation, and centrifuge at 300g for 10 min at room temperature. Take the electroporation kit (from Lonza), add 100 μl of electroporation reagent according to the 4D electroporation kit instructions, and then add the electroporation plasmid or mRNA; resuspend the centrifuged cell pellet, gently mix, transfer the mixture to an electroporation cuvette, and select the desired program for electroporation; use the micropipettes in the kit to transfer the electroporated cell suspension to a 6-well or 12-well plate (containing DC induction medium), mix well, and incubate at 37℃ in a 5% CO2 incubator.

[0152] 2. T cell sorting

[0153] Prepare sorting buffer (PBS solution containing 0.5% HSA and 2mM EDTA), preheating it to 37°C. Take PBMC cells and resuspend them in AIM-V complete medium containing SR, IL-7, and IL-15 to a final volume of 1×10⁻⁶. 7 Cells / mL. Based on cell quantity, administer per 1×10⁻⁶ cells. 7 Add 20 μL of CD4 and CD8 magnetic beads to the cells, adding the beads as needed, mix well, and incubate in the refrigerator in the dark for 15 min. After incubation with the magnetic beads, add 1 × 10⁻⁶ beads per cell. 7 Add 1-2 mL of sorting buffer to the cells, mix well, wash once, centrifuge at 300 x g for 10 min at room temperature, discard the supernatant, and repeat the process with 1 × 10⁻⁶ cells per centrifuge.8 Add 500 μL of sorting buffer to the cells, resuspend them, and thoroughly disperse the cells.

[0154] Based on the cell volume, select and install an MS / LS sorting column (Miltenyi). Rinse the column with 3 mL of sorting buffer. When the liquid in the column is almost completely drained, slowly add the cell suspension into the column. After the solution has completely passed through the column, add 3 mL of sorting buffer to wash the column, repeating once. Remove the sorting column from the separator and place it on a suitable collection tube. Add 5 mL of sorting buffer and quickly push the plunger into the column to rapidly wash the target cells. After counting a small number of cells, centrifuge at 300 g for 10 min at room temperature.

[0155] Example 1: Construction of plasmids containing recombinant antigens

[0156] The sequence of the immune-enhancing peptide (hereinafter referred to as adjuvant 275) is shown in SEQ ID NO:1, and the amino acid sequence of WT1 is shown in SEQ ID NO:2.

[0157] We commissioned Genewiz to synthesize DNA fragment 275-WT1 containing adjuvant 275 and WT1, respectively; the fusion fragment 275-WT1 nucleotide fragment was cloned into the backbone vector p2023 (amino acid sequence as shown in SEQ ID NO:3) by NcoI and XhoI restriction enzyme digestion and ligation, thus obtaining p2023-275-WT1; the results of p2023-275-WT1 in vitro transcription template plasmid construction are as follows. Figure 1 The 275-WT1 sequence is shown in SEQ ID NO:4, and the sequencing results show that the sequence is correct.

[0158] Example 2: In vitro transcription of plasmids containing recombinant antigens

[0159] 2.1 Linearization of plasmid digestion

[0160] Take a 1.5 mL sterile centrifuge tube and, at room temperature, add the following enzyme digestion reagents in sequence (RNase-free water, 10× Buffer, template plasmid, and restriction enzyme) for digestion. The template plasmid concentration in the digestion system is 1 mg / mL, the BsaI addition is 1 μL, and the enzyme is digested at 37°C for 1 hour to digest 1 μg of plasmid template. Linearize according to the required dosage and gently shake the centrifuge tube to mix.

[0161] 2.2 Recycling:

[0162] (1) Add 1 volume of isopropanol to the enzyme digestion system and vortex to mix.

[0163] (2) After mixing, centrifuge at 12000 rpm for 5 min to collect the precipitate and remove the supernatant;

[0164] (3) Add 1 ml of 75% ethanol to wash the precipitate, centrifuge at 12000 rpm for 5 min, remove the supernatant and collect the precipitate;

[0165] (4) Repeat the washing operation once;

[0166] (5) After opening, air dry at room temperature;

[0167] (6) After drying, add 200μL TE buffer to reconstitute the precipitate, and take samples to detect the concentration, A260 / 280, etc.

[0168] 2.3 mRNA transcription and synthesis

[0169] (1) Under room temperature conditions, add the following reaction components in the order shown in the table below to the reaction vessel: 10×IVTBuffer, 100mM ATP-Na2, 100mM CTP-Na2, 100mM GTP-Na2, 100mM UTP-Na2, linearized template plasmid, MgCl2 (1M), T7-M5 RNA polymerase, RNase inhibitor, and water for injection. The final concentration of NTP is 10mM, the template plasmid is 30mM, the final concentration of Mg2+ is 30mM, and the T7 RNA polymerase is added. Gently shake the centrifuge tube to mix well, then place the centrifuge tube in a metal bath and set the temperature of the metal bath to 37℃. The transcription time is 2 hours.

[0170] (2) Template digestion: Take 20 μL of the sample after transcription and add 10×DNase I buffer, 0.5 μL / ug template DNase I, and 5 μL / mL RNase inhibitor in sequence to expand the system to 5 times. Incubate at 37℃ for 30 min.

[0171] (3) LiCl precipitation (used to estimate transcription yield): After template digestion, LiCl precipitation was performed at -20 degrees Celsius for 30 min.

[0172] (4) Sample retention (for purity testing): After precipitation, centrifuge at 13000 rpm for 5 min, wash twice with 75% ethanol, add pure water to dissolve overnight, and retain 20 μL of sample from the reaction system.

[0173] 2.4 Adding a cap

[0174] (1) After the precipitate has completely dissolved on the second day, add the other components of the capping system in sequence. 20 μL of the system corresponds to 10 μg of mRNA, with a final concentration of 3 mM for GTP, 3.2 mM for SAM, and 0.1 μL for VCE mix.

[0175] Mix by gentle shaking with a pressure of 0.5 μL, and then cap at 37°C for 1.5 h.

[0176] 2.5 Purity test results

[0177] The purity of mRNA was controlled using an Agilent 5200 capillary electrophoresis system.

[0178] The results of in vitro transcription of 275-WT1 mRNA are shown below. Figure 2 The 5200 electrophoresis results showed that the 275-WT1 mRNA was the correct size, had no impurities, and was of good purity.

[0179] Example 3: Transfection and Expression of Recombinant Antigen

[0180] 3.1 Cell Seeding

[0181] (1) Approximately 24 hours before transfection, 293T cells (purchased from ATCC) were plated at a seeding density of approximately 0.5-1×10⁻⁶ cells / cm². 6 cells / six-well plate;

[0182] (2) Transfection should be performed when the cell confluence reaches 70%-90%;

[0183] 3.2 Cell transfection

[0184] Prepare a mixture of transfection reagent and mRNA, and perform cell transfection according to the Novizan Lipo3000 transfection reagent instructions. The specific steps are as follows:

[0185] (1) Add 125 μl of opti-MEM medium and 1 μl of transfection reagent (Novazia Lipo3000) to a 1.5 ml sterile centrifuge tube and mix gently with a pipette;

[0186] (2) Add 125 μl opti-MEM and 0.5 μg mRNA to a 1.5 ml sterile centrifuge tube and mix gently.

[0187] (3) Transfection steps: Mix the solutions prepared in steps (1) and (2) and gently mix with a pipette. After standing at room temperature for 10 minutes, it can be used for transfection. Other transfection doses can be calculated by using 2 μl of transfection reagent for 1 μg mRNA.

[0188] 3.3 Experimental Results

[0189] (1) Transfection dose was 1 μg or 2 μg / 1×10 6 Cells were analyzed at 2h and 4h for antigen expression. Western blotting results showed that antigen expression was detected at approximately 90kD in cells and supernatant samples at 2h and 4h. Figure 3 .

[0190] 90kD indicates the size of the target band, which can be detected in both cells and supernatant, while 36kD GAPDH indicates the size of the internal reference gene.

[0191] (2) Transfection doses were 0.5ug, 1ug, 2ug, 3ug, and 4ug / 5×10 5 Cells were selected for expression over a 24-hour period; LDH levels in the cell supernatant were measured using the Yisheng LDH40209 kit. Results of 275-WT1 mRNA expression validation in cells are shown below. Figure 4 LDH results showed that 275 mRNA could induce cell death, and it was significantly different from the control group EGFP at different doses. The LDH results of 275 showed that the degree of cell death was not significantly related to the transfection dose, indicating that the pyroptosis induced by 275 was not dose-dependent.

[0192] Example 4: T cell co-culture

[0193] 4.1 Experimental Grouping

[0194]

[0195] 275-WT1 mRNA carrying an immunostimulatory peptide causes cell death via pyroptosis after translation. Therefore, electroporation of DCs with mRNA will also cause DC cell death, thus causing the electroporated DCs to lose their original function. In order to simulate the tumor environment in vivo, the experimental group was a mixture of DCs transfected with 275-WT1 mRNA and DCs transfected with WT1 mRNA (without adjuvant, only WT1, simulating DCs that have recognized and expressed WT1 tumors in vivo).

[0196] 4.2 Experimental Procedure

[0197] 4.2.1 Preparation of DCs: Cytokines were added to PBMCs according to Method 1 to induce DCs and prepare mature DCs.

[0198] 4.2.2 Co-culture and primary immunization steps

[0199] (1) Prepare co-culture medium AIM-V+50IUIL-2+10%FBS, and preheat the water bath for later use;

[0200] (2) DC cells loaded with mRNA-TAA (TAA) by electroporation are called TAA-DC: After TAA-DC (first batch) is incubated for 24 hours, the delivered DC cells are collected and grouped. After centrifugation, the supernatant of the G4 group is collected into a centrifuge tube (the supernatant is recorded as solution A). The supernatant of the other groups is discarded after centrifugation. All groups are resuspended in 1 ml DPBS for counting.

[0201] (3) Take 5×10 from each group. 5Cells were centrifuged, and the supernatant (DPBS) was discarded. Group G4 was resuspended in 1 ml of solution A, and the other groups were resuspended in 1 ml of co-culture medium. The cells were then seeded into 6-well plates according to the experimental protocol.

[0202] (4) Resuscitate PBMC cells, centrifuge at 400g for 5 min, discard the supernatant; resuspend in co-culture medium, count, take a portion of cells, and adjust the cell density to 2.5×10⁻⁶. 6 Add cells / ml to the culture plate according to the experimental group, 2ml / well, i.e., 5×10⁶ cells / ml. 6 cells / well. Final DC:PBMC ratio = 1:10;

[0203] (5) Mix gently and incubate in a 37°C, 5% CO2 incubator;

[0204] (6) Co-culture for 7 days. On the third day (D3), observe the cells and add 1 ml of co-culture medium or change the medium. At the same time, prepare TAA-DC (second batch) for re-immunization;

[0205] 4.2.3 Re-immunization after 7 days of co-culture (ELISpot plate preparation)

[0206] (1) Prepare ELISpot culture medium AIM-V + 10% FBS (IL-2-free), and preheat the water bath for later use;

[0207] (2) Take out the 96-well plate for IFN-γELISpot detection, remove enough strips according to the experimental group requirements, assemble the required number of strips, wash 4 times with sterile DPBS (200μl / well), and rinse.

[0208] (3) Culture plates (200 μl / well) in a medium containing 10% serum (the same concentration as the cell suspension). Incubate at room temperature for at least 30 minutes. AIM-V + 10% FBS medium is used here.

[0209] (4) Take the PBMCs from 4.2.2 after primary immunization, and count them using trypan blue staining to determine their viability. Centrifuge at 400g for 5 minutes, discard the supernatant; resuspend in DPBS, take the required amount of cells, and sort for T cells according to method 2. After separating T cells from each group, centrifuge at 400g for 5 minutes, discard the supernatant; count using trypan blue staining, and adjust the T cell density to 1×10⁻⁶. 6 cells / ml, for later use;

[0210] (5) Collect TAA-DC (second batch), process it as in 4.2.2(2), and adjust the density to 1×10 5 cells / ml for later use (G4 group was resuspended in solution A);

[0211] (6) Take out the pretreated ELISpot culture plate, discard the supernatant, and gently pat the plate dry; set up the plate according to the group, DC:T=1:10 co-culture (re-immunization), the culture system is 200ul.

[0212] 4.3 Experimental Results

[0213] Figure 5 shows the results of T cell activation promoted by 275-WT1 loaded DC cells in vitro:

[0214] (1) Consistent with the conclusion of activation for 20 hours, the number of ELISpot spots in the experimental group was significantly greater than that in the control group;

[0215] (2) The number of ELISpot points of DCs loaded with 275-WT1 after 20h and 5d of activation was small. The number of ELISpot points of the 275+N4.1 group after 5d of activation with supernatant was greater than that of the group without supernatant. It is speculated that the 275-initiated DC pyroptosis adjuvant DC cell subsets promote the presentation of antigen-loaded DC subsets through paracrine pathway.

Claims

1. An immune-enhancing polypeptide, characterized in that, The polypeptide is the N-terminal amino acid 1-500 of the pyroptosis-associated protein GSDMD or a truncated sequence thereof, preferably the N-terminal amino acid 1-275 of the GSDMD protein, and more preferably, has the amino acid sequence shown in SEQ ID NO:

1.

2. A recombinant antigen having a self-adjuvanting effect, characterized in that, The recombinant antigen includes the immune-enhancing polypeptide and antigen protein as described in claim 1.

3. The recombinant antigen of claim 2, wherein, The antigens are selected from viral antigens, bacterial antigens, fungal antigens, autoimmune disease-related antigens, and / or tumor antigens.

4. The recombinant antigen of claim 3, wherein, The viral antigens include one or more selected from the group consisting of: Epstein-Barr virus, adenovirus, cytomegalovirus, influenza virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes simplex virus, human immunodeficiency virus, human papillomavirus, Japanese encephalitis virus, measles virus, poliovirus, respiratory syncytial virus, rubella virus, smallpox virus, varicella-zoster virus, West Nile virus, polyomavirus, or Zika virus; the bacterial antigens include one or more selected from the group consisting of: anthrax antigen, Gram-negative bacilli, chlamydia, diphtheria, Haemophilus influenzae, Helicobacter pylori, malaria, Mycobacterium tuberculosis, pertussis toxin, pneumococcus, rickettsia, Staphylococcus, Streptococcus, or Clostridium tetani; the fungal antigens include one or more selected from the group consisting of: Candida, Coccidioides, Cryptococcus. Histoplasma capsulatum, Leishmania, Plasmodium, protozoa, parasites, schistosomiasis, tinea fungi, Toxoplasma gondii, or Trypanosoma krusei; the autoimmune disease-related antigens include one or more selected from the group consisting of: acute necrotizing hemorrhagic encephalopathy, allergic asthma, anemia, aphthous ulcers, arthritis, asthma, autoimmune thyroiditis, conjunctivitis, Crohn's disease, cutaneous lupus erythematosus, atopic dermatitis, eczematous dermatitis, diabetes mellitus, erythema nodosum leprosy, keratoconjunctivitis, multiple sclerosis, myasthenia gravis, psoriasis, scleroderma, systemic lupus erythematosus, ulcerative colitis, or vaginitis; the tumor antigens include one or more selected from the group consisting of: A33, BAGE, Bcl-2, B cell maturation antigen, BCR-ABL, β-catenin, testicular cancer antigen, CA125, CA19-9, CA 50, CA 27.29, CA 15-3, CD5, CD19, CD20, CD21, CD22, CD33, CD37, CD45, CD123, CEA, c-Met, CS-1, cyclin B1, DAGE, EBNA, EGFR, ELA2, ephrinB2, estrogen receptor, FAP, ferritin, folate-binding protein, GAGE, G250 / CAIX, GD-2, GM2, gp75, gp100, HA-1, HA-2, HER-2 / neu, HM1.24, Survivin, KRAS, hTERT, Ki-67, LRP, mesothelin, mucin-like cancer-associated antigen, MUC1, p53, PR1, PRAME, PRTN3, RHAMM, or WT-1.

5. The recombinant antigen of claim 4, wherein, The tumor antigen is selected from WT1; preferably, the amino acid sequence of WT1 is shown in SEQ ID NO:

2.

6. A polynucleotide comprising a nucleic acid sequence encoding a polypeptide of any one of claims 1-5. The polynucleotide includes a polynucleotide encoding the immune-enhancing polypeptide of claim 1 or the recombinant antigen of any one of claims 2-5; more preferably, the polynucleotide encoding the immune-enhancing polypeptide is covalently linked to the polynucleotide encoding the antigen.

7. A recombinant nucleic acid vaccine, characterized in that, The nucleic acid vaccine comprises the polynucleotide as described in claim 6; more preferably, the vaccine further comprises a delivery medium molecule, wherein the polynucleotide is delivered after being encapsulated by the delivery medium molecule.

8. A recombinant vector, characterized in that, The recombinant vector includes the polynucleotide and backbone vector as described in claim 6; preferably, the backbone vector includes a plasmid vector or a viral vector.

9. An engineered host cell, characterized in that, The engineered host cell contains the recombinant vector as described in claim 8, and / or is capable of expressing and / or secreting recombinant antigens; Preferably, the host cell is selected from antigen-presenting cells; more preferably, it is selected from macrophages, B cells, or dendritic cells.

10. A method for preparing a recombinant RNA vaccine, characterized in that, Includes the following steps: Provide the recombinant vector as described in claim 8, The recombinant vector is transcribed into RNA in vitro; preferably, The preparation process of the recombinant vector includes: artificially synthesizing the polynucleotide sequence of the recombinant antigen and linking it to the backbone vector; The ligated product was transfected into host cells and cultured to obtain a positive clone recombinant expression vector.

11. A method of making an engineered host cell, comprising, This includes loading host cells with the recombinant antigen as described in any one of claims 2-5 or introducing the polynucleotide as described in claim 6; preferably, loading host cells with the recombinant antigen or polynucleotide includes: (1) contacting the antigen-presenting cell with the recombinant antigen or with the polynucleotide; Preferably, the host cell is selected from antigen-presenting cells; more preferably, it is selected from macrophages, B cells, or dendritic cells.

12. The method of claim 11, wherein, Before or after step (1), the antigen-presenting cells are exposed to the mature composition.

13. A method for in vitro activation of immune-active cells derived from a patient with a tumor, comprising: Immune-active cells were obtained from the patient. Engineered antigen-presenting cells are generated by the method of claim 11 or 12; and The various immune-active cells are co-cultured with the engineered antigen-presenting cells for a sufficient time to activate the immune-active cells, thereby obtaining activated immune-active cells.

14. An activated immune-active cell, obtained by the method of claim 13.

15. A pharmaceutical composition comprising the recombinant antigen of any one of claims 2-5, the polynucleotide of claim 6, the recombinant nucleic acid vaccine of claim 7, the recombinant vector of claim 8, the engineered host cell of claim 9, the host cell produced by the method of claim 11 or 12 or the immune-active cell of claim 14, and pharmaceutically acceptable excipients.

16. Use of the immune-enhancing polypeptide of claim 1, the recombinant antigen of any one of claims 2-5, the polynucleotide of claim 6, the recombinant nucleic acid vaccine of claim 7, the recombinant vector of claim 8, the engineered host cell of claim 9, the host cell produced by the method of claim 11 or 12, the immune-active cell of claim 14, and / or the pharmaceutical composition of claim 15 in the preparation of a medicament for the prevention and / or treatment of antigen-related diseases.

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