[go: up one dir, main page]
More Web Proxy on the site http://driver.im/

WO2023125976A1 - Fusion protein vaccine - Google Patents

Fusion protein vaccine Download PDF

Info

Publication number
WO2023125976A1
WO2023125976A1 PCT/CN2022/144163 CN2022144163W WO2023125976A1 WO 2023125976 A1 WO2023125976 A1 WO 2023125976A1 CN 2022144163 W CN2022144163 W CN 2022144163W WO 2023125976 A1 WO2023125976 A1 WO 2023125976A1
Authority
WO
WIPO (PCT)
Prior art keywords
antigen
domain
linker
pan
csf2
Prior art date
Application number
PCT/CN2022/144163
Other languages
French (fr)
Chinese (zh)
Inventor
彭华
曹学智
王修业
Original Assignee
广州国家实验室
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 广州国家实验室 filed Critical 广州国家实验室
Publication of WO2023125976A1 publication Critical patent/WO2023125976A1/en

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the invention belongs to the field of vaccines, in particular to protein vaccines and applications thereof.
  • a subunit vaccine is a vaccine that contains a purified portion of a pathogen's antigen, or the portion necessary to elicit a protective immune response.
  • Subunit vaccines do not contain the entire pathogen like live attenuated or inactivated vaccines, but only antigenic parts such as proteins, polysaccharides or peptides. Since the vaccine does not contain "live" components of the pathogen, there is no risk of introducing the disease and it is safer and more stable than vaccines containing the whole pathogen. Other advantages of subunit vaccines include proven technology and suitability for immunocompromised individuals. Protein vaccines are a type of subunit vaccine that contain proteins isolated from pathogens (viruses or bacteria).
  • Recombinant protein vaccine is to integrate a key fragment of the pathogen into bacteria, yeast, animal or insect cells, cultivate it in large quantities in vitro, then collect and purify it and add an adjuvant to make a vaccine.
  • the recombinant protein subunit vaccine only has the main surface protein of the pathogen, which avoids the production of antibodies induced by many irrelevant antigens, thereby reducing the side effects of the vaccine and related diseases caused by the vaccine.
  • recombinant protein vaccines are their low immunogenicity.
  • Specific antigens used in recombinant protein vaccines may lack pathogen-associated molecular structures common to pathogens. These molecular structures are used by immune cells to recognize danger, so without them, the immune response is weaker.
  • the second disadvantage of recombinant protein vaccines is the lack of cellular immune response.
  • Recombinant protein subunit vaccines will not infect cells, and the immune response induced by it is mainly antibody-mediated humoral immune response, but lacks important cellular immune response, which is not conducive to effective pathogen removal and long-term immune protection.
  • subunit vaccines need to be used with adjuvants. This not only increases the dependence of recombinant protein vaccines on adjuvants, but also increases the complexity of vaccine immunization procedures and the commercial cost of vaccines.
  • the invention provides a fusion protein vaccine with enhanced immune effect, and the fusion protein vaccine comprises an antigen structural domain and an immune cell targeting structural domain.
  • the fusion protein vaccine with enhanced immune effect of the present invention has enhanced immune effect, enhances the immune response induced by the recombinant protein vaccine, and reduces the dependence of the recombinant protein vaccine on the adjuvant.
  • the present invention provides a fusion protein comprising an antigen domain and an immune cell targeting domain.
  • the immune cell targeting domain is one or more domains selected from the following:
  • Domain A an antibody or polypeptide or an active fragment thereof capable of binding to an immune cell surface protein
  • Domain B cytokines or their active fragments capable of activating immune cells
  • Domain D Immunoglobulin Fc capable of binding immune cells.
  • the fusion protein comprises an antigenic domain and domain A; a fusion protein of an antigenic domain with domain A and domain B; a fusion protein of an antigenic domain with domain A, domain B, and domain C Fusion protein; fusion protein of antigenic domain with domain A, domain B, domain C and domain D; fusion protein of antigenic domain with domain B; fusion of antigenic domain with domain B and domain C Protein; the fusion protein of antigenic domain and structural domain B, structural domain C and structural domain D; the fusion protein of antigenic domain and structural domain C; the fusion protein of antigenic domain and structural domain C and structural domain D; Or antigenic structure A fusion protein of domain and domain D.
  • the fusion protein can be any arrangement of the antigen domain and domain A and/or domain B and/or domain C and/or domain D, for example, the antigen domain and/or structure in the fusion protein Domain A and/or domain B and/or domain C and/or domain D are C-terminal or N-terminal relative to each other.
  • the domain A includes, but is not limited to, antibodies against CD274 (PDL1), PDCD1LG2 (PDL2), CLEC9A, LY75 (DEC205), CD40, TNFSF9 (4-1BB-L) and/or TNFSF4 (OX4OL), or a partner thereof active fragments of the body.
  • the domain B includes but not limited to interleukin (interleukin, IL) and/or colony-stimulating factor (Colony-stimulating factor, CSF), or active fragments thereof.
  • interleukin interleukin
  • CSF colony-stimulating factor
  • the interleukins include IL2, IL12, IL15 and/or IL21, or active fragments thereof.
  • the colony-stimulating factors include CSF1, CSF2 and/or CSF3, or active fragments thereof.
  • the domain C has the amino acid sequence shown as AKFVAAWTLKAAA.
  • Said domain D may be Fc from IgG, IgM, IgA, IgE or IgD or a mutant thereof, said Fc being mutant to form a heterodimeric protein.
  • the Fc is a modified Fc, for example, the two Fc domains of the dimer have Fc knob modification and Fc Hole modification respectively.
  • the antigenic domain may be an immunogenic protein or an immunogenic fragment thereof capable of inducing an immune response against pathogenic microorganisms.
  • Described pathogenic microorganism can be SARS-Cov-2, SARS, cytomegalovirus CMV, herpes virus, respiratory syncytial virus RSV, influenza virus, Ebola virus, Epstein-Barr virus EBV, dengue fever virus, Zike virus, HIV virus , Rabies virus, Plasmodium gametophyte, herpes zoster virus HZV, hepatitis B virus HBV, hepatitis C virus HCV, hepatitis D virus HDV, HPV, Mycobacterium tuberculosis, Helicobacter pylori, etc.
  • the antigenic domain may be an immunogenic protein capable of inducing an immune response against cancer cells, or an immunogenic fragment thereof.
  • the antigenic domain may be a tumor antigenic domain, such as MelanA/MART1, cancer-germline antigen, gp100, tyrosinase, CEA, PSA, Her-2/neu, survivin, terminal Granzyme, or an immunogenic fragment thereof.
  • a tumor antigenic domain such as MelanA/MART1, cancer-germline antigen, gp100, tyrosinase, CEA, PSA, Her-2/neu, survivin, terminal Granzyme, or an immunogenic fragment thereof.
  • the antigen domain and the immune cell targeting domain, and/or the immune cell targeting domain can be connected through a connecting fragment.
  • the junctional fragment can be a flexible junctional fragment, a rigid junctional fragment, or an in vivo cleavage junctional fragment.
  • the amino acid sequence of the flexible linking fragment can be (G) N , (GS) N , (GGS) N , (GGGS) N , or (GGGGS) N .
  • the fusion protein is a homodimer or a heterodimer.
  • the fusion protein comprises an antigenic domain and domain D
  • the fusion protein constitutes a homodimer or heterodimer similar to an antibody form.
  • the fusion protein consists of a homodimer or a heterodimer formed by disulfide bonds of two Fc domains.
  • the fusion protein includes a first polypeptide chain and a second polypeptide chain, the first polypeptide chain includes an antigenic domain and domain D, and the second polypeptide chain includes an antigenic domain and a domain d.
  • the first polypeptide chain and the second polypeptide chain are connected by a disulfide bond between the domain D of the first polypeptide chain and the domain D of the second polypeptide chain to form a homodimer or heterodimer source dimer.
  • the first polypeptide chain further comprises domain B and/or domain C
  • the second polypeptide chain further comprises domain B and/or domain C.
  • the first polypeptide chain further includes domain B
  • the second polypeptide chain further includes domain B, thereby forming a homodimer.
  • the first polypeptide chain further includes domain B
  • the second polypeptide chain further includes domain C, thereby forming a heterodimer, and vice versa.
  • the antigen domain and/or domain B and/or domain C may be linked to the C-terminus or N-terminus of domain D.
  • the first polypeptide chain further includes domain B and domain C
  • the second polypeptide chain further includes domain B and domain C, thereby forming a homodimer.
  • the first polypeptide chain includes domain D, antigen domain, and domain A from C-terminus to N-terminus
  • the second polypeptide chain includes domain D, antigen domain, from C-terminus to N-terminus, Domain A, thereby forming a homodimer, and vice versa.
  • the first polypeptide chain includes domain D, an antigen domain, and domain B from the C-terminus to the N-terminus
  • the second polypeptide chain includes domain D, an antigen domain, from the C-terminus to the N-terminus, Domain B, thereby forming a homodimer, and vice versa.
  • the first polypeptide chain includes domain D, antigen domain, domain C, and domain A from C-terminus to N-terminus
  • the second polypeptide chain includes domain D from C-terminus to N-terminus, The antigenic domain, domain C, domain A, thereby constituting a homodimer and vice versa.
  • the first polypeptide chain includes domain D, antigen domain, domain C, and domain B from C-terminus to N-terminus
  • the second polypeptide chain includes domain D from C-terminus to N-terminus
  • the antigenic domain, domain C, domain B thus constitutes a homodimer and vice versa.
  • the first polypeptide chain includes domain B, domain D, antigen domain, and domain A from C-terminus to N-terminus
  • the second polypeptide chain includes domain B from C-terminus to N-terminus, Domain D, antigenic domain, and domain A, thus forming a homodimer.
  • the first polypeptide chain includes domain B, domain D, antigen domain, domain C, and domain A from the C-terminus to the N-terminus
  • the second polypeptide chain includes from the C-terminus to the N-terminus Domain B, domain D, antigenic domain, domain C, domain A, thereby forming a homodimer and vice versa.
  • the first polypeptide chain includes domain D, antigen domain, and domain A from C-terminus to N-terminus
  • the second polypeptide chain includes domain D, antigen domain, from C-terminus to N-terminus, Domain C, domain B, thereby forming a heterodimer and vice versa.
  • the first polypeptide chain includes domain D, antigen domain, domain C, and domain A from C-terminus to N-terminus
  • the second polypeptide chain includes domain D from C-terminus to N-terminus
  • the antigenic domain, domain C, domain B thus constitutes a heterodimer and vice versa.
  • domain D has Fc knob and Fc Hole modifications, respectively.
  • the antigen domain and the immune cell targeting domain can be arranged and combined in any form.
  • the antigen domain and the immune cell targeting domain A, domain B, domain C and domain D can be arranged and combined in any form.
  • the antigen and immune cell targeting molecule A can be arranged and combined in any form.
  • the antigen and immune cell targeting molecule B can be arranged and combined in any form.
  • the antigen and immune cell targeting molecule C can be arranged and combined in any form.
  • the fusion protein has a structure selected from:
  • the antigenic domain is the S protein of SARS-Cov-2 or a fragment thereof.
  • the S protein is a pre-fusion stable S protein.
  • the prefusion stabilized S protein comprises a double proline (S2P) mutation or a hexaproline (S6P) mutation.
  • the antigenic domain is the RBD domain of the S protein of SARS-Cov-2.
  • the fusion protein of the present invention can be used as a recombinant protein vaccine.
  • the present invention provides nucleic acid molecules for encoding the fusion protein of the first aspect.
  • the present invention provides a vector for expressing the fusion protein of the first aspect or comprising the nucleic acid molecule of the second aspect.
  • the vector can be a plasmid vector, an adenoviral vector or a lentiviral vector.
  • the present invention provides a host cell for expressing the fusion protein of the first aspect or comprising the nucleic acid molecule of the second aspect, or the vector of the third aspect.
  • the host cells can be, for example, Chinese hamster ovary cells (CHO cells), young hamster kidney cells (BHK cells), COS cells, mouse NSO thymoma cells, mouse myeloma cells (SP2/0 cells), human embryonic kidney cells Cells HEK293 cells.
  • CHO cells Chinese hamster ovary cells
  • BHK cells young hamster kidney cells
  • COS cells mouse NSO thymoma cells
  • mouse myeloma cells SP2/0 cells
  • human embryonic kidney cells Cells HEK293 cells human embryonic kidney cells
  • the present invention provides a composition containing the fusion protein of the first aspect, or the nucleic acid molecule of the second aspect, or the vector of the third aspect, or the host cell of the fourth aspect.
  • the composition does not contain a pharmaceutically acceptable adjuvant.
  • the composition further comprises a pharmaceutically acceptable adjuvant.
  • the pharmaceutically acceptable adjuvants include, but are not limited to, aluminum adjuvants, such as aluminum hydroxide, aluminum phosphate or aluminum sulfate, or CpG adjuvants.
  • the present invention provides a method for preventing or treating infection with pathogenic microorganisms or tumors, the method comprising administering to a subject the fusion protein of the first aspect, the nucleic acid molecule of the second aspect, and the carrier of the third aspect, The host cell of the fourth aspect or the step of the composition of the fifth aspect.
  • the subject is a human or an animal.
  • the animals include but are not limited to cattle, sheep, cats, dogs, horses, rabbits, monkeys, mice, rats, alpacas, camels and the like.
  • the subject is an immunocompromised human or animal.
  • the subject has a chronic lung disease, such as chronic obstructive pulmonary disease or asthma.
  • the patient has an underlying disease, such as heart disease, diabetes, or lung disease.
  • an underlying disease such as heart disease, diabetes, or lung disease.
  • the present invention provides the fusion protein of the first aspect, the nucleic acid molecule of the second aspect, the vector of the third aspect, the host cell of the fourth aspect or the composition of the fifth aspect in preparation for preventing or treating an object with The application of pathogenic microorganism infection or tumor-related drugs.
  • the subject is a human or an animal.
  • the animals include, but are not limited to, cows, sheep, cats, dogs, horses, rabbits, monkeys, mice, rats, alpacas, camels, chickens, ducks, geese, and the like.
  • the subject is an immunocompromised human or animal.
  • the subject has a chronic lung disease, such as chronic obstructive pulmonary disease or asthma.
  • the patient has an underlying disease, such as heart disease, diabetes, or lung disease.
  • an underlying disease such as heart disease, diabetes, or lung disease.
  • the cancer may be prostate cancer, non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, brain cancer, melanoma, acute myeloid leukemia, pancreatic cancer, colorectal cancer, squamous cell carcinoma of the head and neck, squamous cell carcinoma of the skin Cystoid cell carcinoma, adenoid cystic carcinoma, glioblastoma, breast cancer, mesothelioma, ovarian cancer, glioma, bladder cancer, liver cancer, bone cancer, bone marrow cancer, stomach cancer, thyroid cancer, lymphoma, Cervical cancer, endometrial cancer, laryngeal cancer, acute lymphoblastic leukemia, etc.
  • the antigen is a tumor antigen, including but not limited to 5T4, AIM2, AKAP4 2, Art-4, AuraA1 (AURKA), Aura B1 (AURKB), BAGE, BCAN, B-cycle BSG, CCND1, CD133, CDC45L, CDCA1 (TTK), CEA, CHI3L2 (chitinase 3-like 2), CSPG4, EpCAM4, Epha2, EPHX1, Ezh2, FABP7, Fosl1 (Fra-1), GAGE, Galt -3, G250(CA9), gBK, glast, GnT-V, gp100, HB-EGF, HER2, HNPRL, HO-1, hTERT, IGF2BP3, IL13-Ra2, IMP-3, IQGAP1, ITGAV, KIF1C, KIF20A, KIF21B, KIFC3, KK-LC-1, LAGE-1, Lck, LRRC8A, MAGE
  • the antigens include, but are not limited to, antigens associated with any tumor/cancer, such as lung cancer (MTFR2D326Y, CHTF18L769V, MYADM R30W, HERC1P3278S, FAM3C K193E, CSMD1G3446E, SLC26A7R117Q, PGAP1Y903F, HELB P987S, ANKRD K60 3T); Melanoma (TMEM48F169L, TKT R438W, SEC24A P469L, AKAP13Q285K, EXOC8Q656P, PABPC1R520Q, MRPS5P59L, ABCC2S1342F, SEC23A P52L, SYTL4S363F, MAP3K9E689K, AKAP6M14 82I,RPBMP42L,HCAPG2P333L,H3F3C T4I,GABPA E161K,
  • lung cancer
  • the diseases related to the pathogenic microorganisms include but are not limited to: Acquired Immunodeficiency Syndrome (AIDS) (Human Immunodeficiency Virus (HIV)); Argentine Teagan fever (Argentine Teagan fever) (Junin virus); Astrovirus infection (Astrovirus) Viridae); BK virus infection (BK virus); Venezuelan hemorrhagic fever (Machupo virus); Brazilian hemorrhagic fever (Sabiá virus); chickenpox (varicella zoster virus (VZV)); A virus); Colorado tick fever (CTF) (Colorado tick fever virus (CTFV)); common cold, acute viral nasopharyngitis, acute rhinitis (usually rhinovirus and coronavirus); cytomegalovirus infection (CMV); dengue fever (Dengue virus (DEN-1, DEN-2, DEN-3 and DEN-4) and other flaviviruses, including but not limited to West Nile virus (West Nile fever), yellow fever virus (
  • the recombinant protein vaccine of the invention has improved immunogenicity and enhanced immune response, and at the same time reduces the dependence of the recombinant protein vaccine on the adjuvant.
  • antigen fusion expression in this protein vaccine can also be applied to other types of vaccines, including but not limited to nucleic acid vaccines (such as mRNA vaccines, circular RNA vaccines and DNA vaccines, etc.), viral vector vaccines (such as adenovirus vector vaccines and influenza Viral vector vaccines, etc.), and nanoparticle vaccines, etc.
  • nucleic acid vaccines such as mRNA vaccines, circular RNA vaccines and DNA vaccines, etc.
  • viral vector vaccines such as adenovirus vector vaccines and influenza Viral vector vaccines, etc.
  • nanoparticle vaccines etc.
  • the present invention shows:
  • the immunogenicity of the free RBD protein is weak.
  • the Fc part is added to the RBD, its immunogenicity is improved.
  • the cytokines IL12 and Pan are added to the RBD-Fc, its immune
  • the cytokines IL21 and Pan are added on the basis of RBD-Fc
  • its immunogenicity has been further improved
  • the cytokines CSF2 and Pan are added on the basis of RBD-Fc
  • Its immunogenicity has been further improved, and its immunogenicity has been further improved when the ⁇ PDL1 antibody and Pan that can bind to the immune cell surface protein PDL1 are added on the basis of RBD-Fc;
  • the immunogenicity of the free RBD protein is weak, and the antibody titer can be increased when the Fc part is added to the RBD, and the antibody titer can be further increased when the cytokine CSF2 is added to the RBD-Fc , when the ⁇ PDL1 antibody that can bind to the immune cell surface protein PDL1 is added on the basis of RBD-Fc, the antibody titer can be further increased;
  • the immunogenicity of the free gp350 protein vaccine is weak.
  • the antibody titer can be increased.
  • the antibody titer can be improved when the ⁇ PDL1 antibody of PDL1 is added, and the antibody titer can be increased when the cytokines CSF2 and Pan are added on the basis of gp350-Fc, and the antibody titer can be increased when the gp350-Fc is added on the basis of the ability to bind to the immune cell surface protein PDL1 ⁇ PDL1 antibody and Pan can increase the antibody titer.
  • Figure 1 shows a schematic diagram of a fusion protein vaccine in the form of a homodimer. Among them, antigen-Fc and immune cell targeting molecule A are arranged and combined in the order of A-antigen-Fc.
  • Figure 2 shows a schematic diagram of a fusion protein vaccine in the form of a homodimer. Wherein, the antigen-Fc and the immune cell targeting molecule B are arranged and combined in the order of B-antigen-Fc.
  • Figure 3 shows a schematic diagram of a fusion protein vaccine in the form of a homodimer. Wherein, antigen-Fc and immune cell targeting molecules A and C are arranged and combined in the order of A-C-antigen-Fc.
  • Figure 4 shows a schematic diagram of a fusion protein vaccine in the form of a homodimer. Wherein, antigen-Fc and immune cell targeting molecules B and C are arranged and combined in the order of B-C-antigen-Fc.
  • Figure 5 shows a schematic diagram of a fusion protein vaccine in the form of a homodimer. Wherein, antigen-Fc and immune cell targeting molecules A and B are arranged and combined in the order of A-antigen-Fc-B.
  • Figure 6 shows a schematic diagram of a fusion protein vaccine in the form of a homodimer.
  • antigen-Fc and immune cell targeting molecules A, B and C are arranged and combined in the order of A-C-antigen-Fc-B.
  • Figure 7 shows a schematic diagram of a fusion protein vaccine in the form of a heterodimer.
  • the antigen-Fcknob and the immune cell targeting molecule A are arranged and combined in the order of A-antigen-Fcknob
  • the antigen-Fchole and the immune cell targeting molecule B are arranged and combined in the order of B-antigen-Fchole.
  • Figure 8 shows a schematic diagram of a fusion protein vaccine in the form of a heterodimer.
  • antigen-Fcknob and immune cell targeting molecules A and C are arranged and combined in the order of A-C-antigen-Fcknob
  • antigen-Fchole and immune cell targeting molecules B and C are arranged and combined in the order of B-C-antigen-Fcknob.
  • Figure 9 shows the polyacrylamide gel (SDS-PAGE) electrophoresis identification diagram of the SARS-CoV-2 spike protein RBD-related immune cell targeting fusion protein.
  • RBD, RBD-Fc, IL4-Pan-RBD-Fc, IL10-Pan-RBD-Fc, IL12-Pan-RBD-Fc, IL21-Pan-RBD-Fc, CSF2-Pan were identified on SDS-PAGE - RBD-Fc, PD1-Pan-RBD-Fc, PDL1-Pan-RBD-Fc corresponding proteins.
  • Figure 10 shows the protein production of SARS-CoV-2 spike protein RBD-related immune cell targeting fusion protein vaccine transiently expressed in eukaryotic cell 293F.
  • Figure 11 shows the results of antibody responses against the SARS-CoV-2 spike protein RBD induced by immune cell targeting fusion protein vaccines.
  • Figure 12 shows the SDS-PAGE electrophoresis identification diagram of the fusion protein related to the RBD of the novel coronavirus SARS-CoV-2 spike protein.
  • Figure 13 shows the expression yield of the fusion protein related to the SARS-CoV-2 spike protein RBD of the new coronavirus.
  • Figure 14 shows that the RBD-related protein vaccine targeting the new coronavirus SARS-CoV-2 spike protein by immune cells can elicit a stronger antibody response in mice than the RBD protein vaccine alone.
  • Figure 15 shows the SDS-PAGE electrophoresis identification diagram of the fusion protein related to the membrane protein gp350 of Epstein-Barr virus (EBV).
  • EBV Epstein-Barr virus
  • Figure 16 shows the expression yield of Epstein-Barr virus (Epstein-Barr virus, EBV) membrane protein gp350-related fusion protein.
  • Figure 17 shows that immune cells targeting EBV membrane protein gp350-associated protein vaccine can elicit a stronger antibody response in mice than pure gp350 protein vaccine.
  • Embodiment 1 Design of fusion protein vaccine platform
  • Immune cell-targeting fusion protein vaccines are obtained by fusing immune cell-targeting molecules to antigens.
  • Immune cell targeting molecules can include the following four components:
  • Pan epitopes capable of activating immune cells PADRE
  • the antigen can be combined with two, three or four of these four components in any form to form a fusion protein. Its representative form is as follows:
  • antigen-Fc and immune cell targeting molecule A are arranged and combined in the order of A-antigen-Fc, as shown in Figure 1.
  • Antigen-Fc and immune cell targeting molecule A can be arranged and combined in any form. Specifically include: ⁇ PDL1-Antigen-Fc, CLEC9A binding peptide-Antigen-Fc, ⁇ DEC205-Antigen-Fc, Antigen-Fc-CLEC9A binding peptide.
  • antigen-Fc and immune cell targeting molecule B are arranged and combined in the order of B-antigen-Fc, as shown in FIG. 2 .
  • Antigen-Fc and immune cell targeting molecule B can be arranged and combined in any form. Specifically include: IL2-Antigen-Fc, IL12-Antigen-Fc, IL15-Antigen-Fc, IL21-Antigen-Fc, CSF2-Antigen-Fc, Antigen-Fc-CSF2.
  • antigen-Fc and immune cell targeting molecules A and C are arranged and combined in the order of A-C-antigen-Fc, as shown in FIG. 3 .
  • Antigen-Fc and immune cell targeting molecules A and C can be arranged and combined in any form. Specifically include: ⁇ PDL1-Pan-Antigen-Fc, CLEC9A binding peptide-Pan-Antigen-Fc, ⁇ DEC205-Pan-Antigen-Fc.
  • antigen-Fc and immune cell targeting molecules B and C are arranged and combined in the order of B-C-antigen-Fc, as shown in FIG. 4 .
  • Antigen-Fc and immune cell targeting molecules B and C can be arranged and combined in any form. Specifically include: IL2-Pan-Antigen-Fc, IL12-Pan-Antigen-Fc, IL15-Pan-Antigen-Fc, IL21-Pan-Antigen-Fc, CSF2-Pan-Antigen-Fc, Pan-Antigen-Fc-CSF2 .
  • antigen-Fc and immune cell targeting molecules A and B are arranged and combined in the order of A-antigen-Fc-B, as shown in FIG. 5 .
  • Antigen-Fc and immune cell targeting molecules A and B can be arranged and combined in any form. Specifically include: ⁇ PDL1-Antigen-Fc-IL12, ⁇ PDL1-Antigen-Fc-IL21, ⁇ PDL1-Antigen-Fc-CSF2, CLEC9A binding peptide-Antigen-Fc-IL12, CLEC9A binding peptide-Antigen-Fc-IL21, CLEC9A binding peptide -Antigen-Fc-CSF2.
  • antigen-Fc and immune cell targeting molecules A, B and C are arranged and combined in the order of A-C-antigen-Fc-B, as shown in FIG. 6 .
  • Antigen-Fc and immune cell targeting molecules A, B and C can be arranged and combined in any form.
  • ⁇ PDL1-Pan-Antigen-Fc-IL12 ⁇ PDL1-Pan-Antigen-Fc-IL21, ⁇ PDL1-Pan-Antigen-Fc-CSF2, CLEC9A binding peptide-Pan-Antigen-Fc-IL12, CLEC9A binding peptide-Pan -Antigen-Fc-IL21, CLEC9A binding peptide-Pan-Antigen-Fc-CSF2, PDL1-CSF2-Pan-Antigen-Fc.
  • antigen-Fc knob and immune cell targeting molecule A are arranged in the order of A-antigen-Fc knob, and antigen-Fc hole and immune cell targeting molecule B are in the order of B-antigen-Fc hole Arrange and combine, as shown in Figure 7.
  • Antigen-Fc knob and immune cell targeting molecule A can be arranged and combined in any form.
  • Antigen-Fc hole and immune cell targeting molecule B can be arranged and combined in any form. Specifically include: ⁇ PDL1-Antigen-Fc knob/CSF2-Antigen-Fc hole, CLEC9A binding peptide-Antigen-Fc knob/CSF2-Antigen-Fc hole.
  • the antigen-Fc hole and immune cell targeting molecules A and C are arranged in the order of A-C-antigen-Fc knob, and the antigen-Fc hole and immune cell targeting molecules B and C are arranged in the order of B-C-antigen -Fc hole sequence arrangement and combination, as shown in Figure 8.
  • Antigen-Fc knob and immune cell targeting molecules A and C can be arranged and combined in any form.
  • Antigen-Fc hole and immune cell targeting molecules B and C can be arranged and combined in any form.
  • ⁇ PDL1-Pan-Antigen-Fc knob/CSF2-Pan-Antigen-Fc hole Specifically include: ⁇ PDL1-Pan-Antigen-Fc knob/CSF2-Pan-Antigen-Fc hole, CLEC9A binding peptide-Pan-Antigen-Fc knob/CSF2-Pan-Antigen-Fc hole.
  • the expression and production of the vaccine platform is described by taking the homodimer form of the spike protein RBD protein of the novel coronavirus SARS-CoV-2 as an example.
  • antigens and immune cell targeting molecules are constructed on the vector by means of molecular cloning, so as to obtain plasmids that can express fusion proteins.
  • KOZAK is the Kozak sequence
  • SP signal peptide
  • Freestyle 293F cells were frozen in Freestyle 293 Expression Medium (10% DMSO) at a concentration of 3 ⁇ 10 7 cells/ml. After taking it out from the liquid nitrogen, dissolve it quickly in a 37°C water bath, wash with SMM 293-TII medium, and resuspend the cells in 50ml of SMM 293-TII medium at 37°C, 8% CO 2 , 120rpm.
  • Freestyle 293F cells were frozen in Freestyle 293 Expression Medium (10% DMSO) at a concentration of 3 ⁇ 10 7 cells/ml. After taking it out from the liquid nitrogen, dissolve it quickly in a 37°C water bath, wash with SMM 293-TII medium, and resuspend the cells in 50ml of SMM 293-TII medium at 37°C, 8% CO 2 , 120rpm.
  • the plasmid/PEI mixture was added to the cell suspension, and placed in an incubator at 37° C., 8% CO 2 , 85 rpm.
  • the proteins were loaded onto polyacrylamide gels with reducing buffer (R) and non-reducing buffer (N-R), respectively. After electrophoresis, the gel was stained with Coomassie brilliant blue, and then the gel was stained with Imager imaging to check the integrity and purity of the fusion protein.
  • the SDS-PAGE electrophoresis identification diagram of the fusion protein is shown in FIG. 9 .
  • the expression yield of the fusion protein is shown in FIG. 10 .
  • SARS-CoV-2 spike protein RBD immune cell-targeting fusion protein vaccine can cause a stronger antibody response in mice than a simple RBD protein vaccine.
  • C57BL/6 male mice (6-8) weeks were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.; horseradish peroxidase (HRP)-labeled goat anti-mouse IgG was purchased from Jiangsu Kangwei Century Biotechnology Co., Ltd.
  • HRP horseradish peroxidase
  • HRP horseradish peroxidase
  • IgG1, IgG2b and IgG2c horseradish peroxidase
  • 96-well ELISA assay plate was purchased from Bioland Company
  • ELISA chromogenic solution Purchased from Shanghai Biyuntian Biotechnology Co., Ltd.
  • ELISA stop solution was purchased from Beijing Suolaibao Technology Co., Ltd.
  • microplate reader Multiskan FC was purchased from Thermo Fisher Scientific.
  • mice were immunized with immune cell-targeted fusion protein vaccine. After diluting the fusion protein vaccine in PBS, each mouse was inoculated with 0.5 ⁇ g of RBD protein or other fusion proteins of the same molar amount, and each mouse was injected intramuscularly with 50 ⁇ l. Mice were immunized on day 0 and day 21 using two immunization procedures. On the 14th day after each immunization, mouse serum was collected by cheek blood collection for antibody detection.
  • the immunogenicity of the free RBD protein was weak, and its immunogenicity was improved when the Fc part was added on the basis of RBD, and its immunogenicity was improved when the cytokines IL12 and Pan were added on the basis of RBD-Fc.
  • FIG. 11(b) IL12 or CSF2 together with Pan induced higher levels of IgG2b and IgG2c representing Th1 immune responses on the basis of RBD-Fc, as shown in Figure 11(c) and Figure 11(d).
  • the ratio of (IgG2b+IgG2c)/IgG1 showed that IL12-Pan-RBD-Fc induced relatively balanced Th1 and Th2 immune responses, while RBD-Fc, CSF2-Pan-RBD-Fc and aPDL1-Pan-RBD-Fc induced Th2-biased immune responses, as shown in Figure 11(e).
  • antigens and immune cell targeting molecules are constructed on the vector by means of molecular cloning, so as to obtain plasmids that can express fusion proteins.
  • KOZAK is the Kozak sequence
  • SP signal peptide
  • Freestyle 293F cells were frozen in Freestyle 293 Expression Medium (10% DMSO) at a concentration of 3 ⁇ 10 7 cells/ml. After taking it out from the liquid nitrogen, dissolve it quickly in a 37°C water bath, wash with SMM 293-TII medium, and resuspend the cells in 50ml of SMM 293-TII medium at 37°C, 8% CO 2 , 120rpm.
  • Freestyle 293F cells were frozen in Freestyle 293 Expression Medium (10% DMSO) at a concentration of 3 ⁇ 10 7 cells/ml. After taking it out from the liquid nitrogen, dissolve it quickly in a 37°C water bath, wash with SMM 293-TII medium, and resuspend the cells in 50ml of SMM 293-TII medium at 37°C, 8% CO 2 , 120rpm.
  • the plasmid/PEI mixture was added to the cell suspension, and placed in an incubator at 37° C., 8% CO 2 , 85 rpm.
  • the proteins were loaded onto polyacrylamide gels with reducing buffer (R) and non-reducing buffer (N-R), respectively. After electrophoresis, the gel was stained with Coomassie brilliant blue, and then the gel was stained with Imager imaging to check the integrity and purity of the fusion protein.
  • the SDS-PAGE electrophoresis identification diagram of the fusion protein is shown in FIG. 12 .
  • the expression yield of the fusion protein is shown in FIG. 13 .
  • Example 5 Immune cells targeting the new coronavirus SARS-CoV-2 spike protein RBD-related protein vaccine can cause a stronger antibody response in mice than a simple RBD protein vaccine
  • C57BL/6 male mice (6-8) weeks were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.; horseradish peroxidase (HRP)-labeled goat anti-mouse IgG was purchased from Jiangsu Kangwei Century Biotechnology Co., Ltd. Co., Ltd.; 96-well ELISA assay plate was purchased from Bioland; ELISA chromogenic solution was purchased from Shanghai Biyuntian Biotechnology Co., Ltd.; ELISA stop solution was purchased from Beijing Solaibao Technology Co., Ltd.; Microplate reader Multiskan FC was purchased from Thermo Fisher Scientific company.
  • HRP horseradish peroxidase
  • mice were immunized with immune cell-targeted fusion protein vaccine. After diluting the fusion protein vaccine in PBS, each mouse was inoculated with 0.5 ⁇ g of RBD protein or other fusion proteins of the same molar number, and each mouse was injected intramuscularly with 50 ⁇ l. Mice were immunized on day 0 and day 21 using two immunization procedures. On the 14th day after each immunization, mouse serum was collected by cheek blood collection for antibody detection.
  • the immunogenicity of free RBD protein is weak, and the antibody titer can be increased when the Fc part is added on the basis of RBD, and the antibody titer can be further improved when the cytokine CSF2 is added on the basis of RBD-Fc.
  • Adding the ⁇ PDL1 antibody capable of binding to the immune cell surface protein PDL1 on the basis of RBD-Fc can also further increase the antibody titer, as shown in FIG. 14 .
  • Epstein-Barr virus (Epstein-Barr virus, EBV) membrane protein gp350-related protein vaccine, production and identification
  • EBV also known as human herpesvirus 4
  • human herpesvirus 4 is one of nine known types of human herpesviruses in the herpes family and one of the most common viruses in humans. Best known as the cause of infectious mononucleosis, EBV is also associated with a variety of non-malignant, precancerous, and malignant EBV-associated lymphoproliferative disorders, with approximately 200,000 cancer cases worldwide each year considered probable Attributed to EBV.
  • antigens and immune cell targeting molecules are constructed on the vector by means of molecular cloning, so as to obtain plasmids that can express fusion proteins.
  • KOZAK is the Kozak sequence
  • SP signal peptide
  • Freestyle 293F cells were frozen in Freestyle 293 Expression Medium (10% DMSO) at a concentration of 3 ⁇ 10 7 cells/ml. After taking it out from the liquid nitrogen, dissolve it quickly in a 37°C water bath, wash with SMM 293-TII medium, and resuspend the cells in 50ml of SMM 293-TII medium at 37°C, 8% CO 2 , 120rpm.
  • Freestyle 293F cells were frozen in Freestyle 293 Expression Medium (10% DMSO) at a concentration of 3 ⁇ 10 7 cells/ml. After taking it out from the liquid nitrogen, dissolve it quickly in a 37°C water bath, wash with SMM 293-TII medium, and resuspend the cells in 50ml of SMM 293-TII medium at 37°C, 8% CO 2 , 120rpm.
  • the plasmid/PEI mixture was added to the cell suspension, and placed in an incubator at 37° C., 8% CO 2 , 85 rpm.
  • the proteins were loaded onto polyacrylamide gels with reducing buffer (R) and non-reducing buffer (N-R), respectively. After electrophoresis, the gel was stained with Coomassie brilliant blue, and then the gel was stained with Imager imaging to check the integrity and purity of the fusion protein.
  • the SDS-PAGE electrophoresis identification diagram of the fusion protein is shown in FIG. 15 .
  • the expression yield of the fusion protein is shown in FIG. 16 .
  • Example 7 Immune cells targeting EBV membrane protein gp350-related protein vaccines can elicit stronger antibody responses in mice than pure gp350 protein vaccines
  • C57BL/6 male mice (6-8) weeks were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.; horseradish peroxidase (HRP)-labeled goat anti-mouse IgG was purchased from Jiangsu Kangwei Century Biotechnology Co., Ltd. Co., Ltd.; 96-well ELISA assay plate was purchased from Bioland; ELISA chromogenic solution was purchased from Shanghai Biyuntian Biotechnology Co., Ltd.; ELISA stop solution was purchased from Beijing Solaibao Technology Co., Ltd.; Microplate reader Multiskan FC was purchased from Thermo Fisher Scientific company.
  • HRP horseradish peroxidase
  • mice were immunized with immune cell-targeted fusion protein vaccine. After diluting the fusion protein vaccine in PBS, each mouse was inoculated with 0.5 ⁇ g gp350 protein or other fusion proteins of the same molar amount, and each mouse was injected intramuscularly with 50 ⁇ l. Mice were immunized on day 0 and day 21 using two immunization procedures. On the 14th day after each immunization, mouse serum was collected by cheek blood collection for antibody detection.
  • the immunogenicity of the free gp350 protein vaccine is weak.
  • the antibody titer is not improved.
  • the cytokine CSF2 is added on the basis of gp350-Fc
  • the antibody titer can be improved.
  • the antibody titer can be increased when the ⁇ PDL1 antibody that can bind to the immune cell surface protein PDL1 is added.
  • the cytokines CSF2 and Pan are added on the basis of gp350-Fc, the antibody titer can be increased.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Epidemiology (AREA)
  • Immunology (AREA)
  • Mycology (AREA)
  • Zoology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Oncology (AREA)
  • Hematology (AREA)
  • Cell Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Virology (AREA)
  • Communicable Diseases (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

A fusion protein, comprising an antigen domain and an immune cell targeting domain.

Description

融合蛋白疫苗fusion protein vaccine 技术领域technical field

本发明属于疫苗领域，具体地涉及蛋白疫苗及其应用。The invention belongs to the field of vaccines, in particular to protein vaccines and applications thereof.

背景技术Background technique

亚单位疫苗是一种含有纯化的病原体抗原部分，或引起保护性免疫反应所必需的部分的疫苗。亚单位疫苗不像减毒活疫苗或灭活疫苗那样包含全部病原体，而只包含抗原部分，如蛋白质、多糖或肽。由于该疫苗不包含病原体的“活”成分，因此不存在引入该疾病的风险，而且比含有完整病原体的疫苗更安全、更稳定。亚单位疫苗的其他优势包括成熟的技术和适用于免疫功能低下的个体。蛋白疫苗是亚单位疫苗中的一种，含有从病原体(病毒或细菌)中分离出来的蛋白质。重组蛋白疫苗，是将病原体的某个关键片段，整合到细菌、酵母、动物或昆虫细胞中，在体外大量培养，然后收集纯化后添加佐剂制成疫苗。重组蛋白亚单位疫苗仅有病原体主要表面蛋白质，避免产生许多无关抗原诱发的抗体，从而减少疫苗的副反应和疫苗引起的相关疾病。A subunit vaccine is a vaccine that contains a purified portion of a pathogen's antigen, or the portion necessary to elicit a protective immune response. Subunit vaccines do not contain the entire pathogen like live attenuated or inactivated vaccines, but only antigenic parts such as proteins, polysaccharides or peptides. Since the vaccine does not contain "live" components of the pathogen, there is no risk of introducing the disease and it is safer and more stable than vaccines containing the whole pathogen. Other advantages of subunit vaccines include proven technology and suitability for immunocompromised individuals. Protein vaccines are a type of subunit vaccine that contain proteins isolated from pathogens (viruses or bacteria). Recombinant protein vaccine is to integrate a key fragment of the pathogen into bacteria, yeast, animal or insect cells, cultivate it in large quantities in vitro, then collect and purify it and add an adjuvant to make a vaccine. The recombinant protein subunit vaccine only has the main surface protein of the pathogen, which avoids the production of antibodies induced by many irrelevant antigens, thereby reducing the side effects of the vaccine and related diseases caused by the vaccine.

重组蛋白疫苗的缺点之一是免疫原性较低。重组蛋白疫苗中使用的特定抗原可能缺乏病原体常见的病原体相关的分子结构。这些分子结构可以被免疫细胞用来识别危险，所以没有它们，免疫反应会更弱。重组蛋白疫苗的缺点之二是缺乏细胞免疫应答。重组蛋白亚单位疫苗不会感染细胞，其诱导的免疫反应主要是抗体介导的体液免疫应答，而缺乏重要的细胞免疫应答，不利于有效的清除病原体和长期的免疫保护。为了增强重组蛋白疫苗的免疫原性，亚单位疫苗需要配合佐剂使用。这不仅增加了重组蛋白疫苗对佐剂的依赖，同时也提高了疫苗免疫程序的复杂性以及疫苗的商用成本。One of the disadvantages of recombinant protein vaccines is their low immunogenicity. Specific antigens used in recombinant protein vaccines may lack pathogen-associated molecular structures common to pathogens. These molecular structures are used by immune cells to recognize danger, so without them, the immune response is weaker. The second disadvantage of recombinant protein vaccines is the lack of cellular immune response. Recombinant protein subunit vaccines will not infect cells, and the immune response induced by it is mainly antibody-mediated humoral immune response, but lacks important cellular immune response, which is not conducive to effective pathogen removal and long-term immune protection. In order to enhance the immunogenicity of recombinant protein vaccines, subunit vaccines need to be used with adjuvants. This not only increases the dependence of recombinant protein vaccines on adjuvants, but also increases the complexity of vaccine immunization procedures and the commercial cost of vaccines.

发明内容Contents of the invention

本发明提供了免疫效果增强的融合蛋白疫苗，所述融合蛋白疫苗包含抗原结构域和免疫细胞靶向结构域。本发明的免疫效果增强的融合蛋白疫苗具有增强的免疫效果，增强了重组蛋白疫苗诱导的免疫应答，降低重组蛋白疫苗对佐剂的依赖性。The invention provides a fusion protein vaccine with enhanced immune effect, and the fusion protein vaccine comprises an antigen structural domain and an immune cell targeting structural domain. The fusion protein vaccine with enhanced immune effect of the present invention has enhanced immune effect, enhances the immune response induced by the recombinant protein vaccine, and reduces the dependence of the recombinant protein vaccine on the adjuvant.

第一方面，本发明提供了含有抗原结构域和免疫细胞靶向结构域的融合蛋白。In a first aspect, the present invention provides a fusion protein comprising an antigen domain and an immune cell targeting domain.

所述免疫细胞靶向结构域为选自如下的一种或多种结构域：The immune cell targeting domain is one or more domains selected from the following:

结构域A：能够结合免疫细胞表面蛋白的抗体或多肽或其活性片段；Domain A: an antibody or polypeptide or an active fragment thereof capable of binding to an immune cell surface protein;

结构域B：能够激活免疫细胞的细胞因子或其活性片段；Domain B: cytokines or their active fragments capable of activating immune cells;

结构域C：能够激活免疫细胞的Pan表位(PADRE)或其活性片段；Domain C: Pan epitope (PADRE) or its active fragments capable of activating immune cells;

结构域D：能够结合免疫细胞的免疫球蛋白Fc。Domain D: Immunoglobulin Fc capable of binding immune cells.

在一些实施方案中，所述融合蛋白包含抗原结构域和结构域A；抗原结构域与结构域A和结构域B的融合蛋白；抗原结构域与结构域A，结构域B和结构域C的融合蛋白；抗原结构域与结构域A，结构域B，结构域C和结构域D的融合蛋白；抗原结构域与结构域B的融合蛋白；抗原结构域与结构域B和结构域C的融合蛋白；抗原结构域与结构域B，结构域C和结构域D的融合蛋白；抗原结构域与结构域C的融合蛋白；抗原结构域与结构域C和结构域D的融合蛋白；或抗原结构域与结构域D的融合蛋白。In some embodiments, the fusion protein comprises an antigenic domain and domain A; a fusion protein of an antigenic domain with domain A and domain B; a fusion protein of an antigenic domain with domain A, domain B, and domain C Fusion protein; fusion protein of antigenic domain with domain A, domain B, domain C and domain D; fusion protein of antigenic domain with domain B; fusion of antigenic domain with domain B and domain C Protein; the fusion protein of antigenic domain and structural domain B, structural domain C and structural domain D; the fusion protein of antigenic domain and structural domain C; the fusion protein of antigenic domain and structural domain C and structural domain D; Or antigenic structure A fusion protein of domain and domain D.

所述融合蛋白可以是抗原结构域与结构域A和/或结构域B和/或结构域C和/或结构域D的任意排列方式，例如，所述融合蛋白中抗原结构域和/或结构域A和/或结构域B和/或结构域C和/或结构域D相对于彼此位于C端或者N端。The fusion protein can be any arrangement of the antigen domain and domain A and/or domain B and/or domain C and/or domain D, for example, the antigen domain and/or structure in the fusion protein Domain A and/or domain B and/or domain C and/or domain D are C-terminal or N-terminal relative to each other.

所述结构域A包括但不限于针对CD274(PDL1)，PDCD1LG2(PDL2)，CLEC9A，LY75(DEC205)，CD40，TNFSF9(4-1BB-L)和/或TNFSF4(OX4OL)的抗体，或其配体的活性片段。The domain A includes, but is not limited to, antibodies against CD274 (PDL1), PDCD1LG2 (PDL2), CLEC9A, LY75 (DEC205), CD40, TNFSF9 (4-1BB-L) and/or TNFSF4 (OX4OL), or a partner thereof active fragments of the body.

所述结构域B包括但不限于白介素(interleukin，IL)和/或集落刺激因子(Colony-stimulating factor，CSF)，或其活性片段。The domain B includes but not limited to interleukin (interleukin, IL) and/or colony-stimulating factor (Colony-stimulating factor, CSF), or active fragments thereof.

优选地，所述白介素包括IL2，IL12，IL15和/或IL21，或其活性片段。Preferably, the interleukins include IL2, IL12, IL15 and/or IL21, or active fragments thereof.

优选地，所述集落刺激因子包括CSF1，CSF2和/或CSF3，或其活性片段。Preferably, the colony-stimulating factors include CSF1, CSF2 and/or CSF3, or active fragments thereof.

优选地，所述结构域C具有AKFVAAWTLKAAA所示的氨基酸序列。Preferably, the domain C has the amino acid sequence shown as AKFVAAWTLKAAA.

所述结构域D可以为来自IgG，IgM，IgA，IgE或IgD的Fc或其突变体，所述Fc为突变体以形成异源二聚体蛋白。在一些实施方案中，所述Fc是经修饰的Fc，例如二聚体的两个Fc结构域分别具有Fc knob修饰和Fc Hole修饰。Said domain D may be Fc from IgG, IgM, IgA, IgE or IgD or a mutant thereof, said Fc being mutant to form a heterodimeric protein. In some embodiments, the Fc is a modified Fc, for example, the two Fc domains of the dimer have Fc knob modification and Fc Hole modification respectively.

在一些实施方案中，所述抗原结构域(Antigen)可以为能够诱导针对病原微生物的免疫反应的免疫原性蛋白或其免疫原性片段。In some embodiments, the antigenic domain (Antigen) may be an immunogenic protein or an immunogenic fragment thereof capable of inducing an immune response against pathogenic microorganisms.

所述病原微生物可以为SARS-Cov-2，SARS，巨细胞病毒CMV，疱疹病毒，呼吸道合胞病毒RSV，流感病毒，Ebola病毒，爱泼斯坦-巴尔病毒EBV，登革热 病毒，Zike病毒，HIV病毒，狂犬病毒，疟原虫配子体，带状疱疹病毒HZV，乙肝病毒HBV，丙型肝炎病毒HCV，丁肝病毒HDV，HPV，结核分枝杆菌，幽门螺旋杆菌等。Described pathogenic microorganism can be SARS-Cov-2, SARS, cytomegalovirus CMV, herpes virus, respiratory syncytial virus RSV, influenza virus, Ebola virus, Epstein-Barr virus EBV, dengue fever virus, Zike virus, HIV virus , Rabies virus, Plasmodium gametophyte, herpes zoster virus HZV, hepatitis B virus HBV, hepatitis C virus HCV, hepatitis D virus HDV, HPV, Mycobacterium tuberculosis, Helicobacter pylori, etc.

所述抗原结构域可以为能够诱导针对癌症细胞的免疫反应的免疫原性蛋白，或其免疫原性片段。The antigenic domain may be an immunogenic protein capable of inducing an immune response against cancer cells, or an immunogenic fragment thereof.

在一些实施方案中，所述抗原结构域可以为肿瘤抗原结构域，例如MelanA/MART1、癌-种系抗原、gp100、酪氨酸酶、CEA、PSA、Her-2/neu、存活蛋白、端粒酶，或其免疫原性片段。In some embodiments, the antigenic domain may be a tumor antigenic domain, such as MelanA/MART1, cancer-germline antigen, gp100, tyrosinase, CEA, PSA, Her-2/neu, survivin, terminal Granzyme, or an immunogenic fragment thereof.

在一些实施方案中，所述抗原结构域与免疫细胞靶向结构域之间，和/或免疫细胞靶向结构域之间可以通过连接片段进行连接。In some embodiments, the antigen domain and the immune cell targeting domain, and/or the immune cell targeting domain can be connected through a connecting fragment.

在一些实施方案中，所述连接片段可以是柔性连接片段、刚性连接片段或体内剪切连接片段。其中柔性连接片段的氨基酸序列可以是(G) N，(GS) N，(GGS) N，(GGGS) N，或(GGGGS) N。 In some embodiments, the junctional fragment can be a flexible junctional fragment, a rigid junctional fragment, or an in vivo cleavage junctional fragment. Wherein the amino acid sequence of the flexible linking fragment can be (G) N , (GS) N , (GGS) N , (GGGS) N , or (GGGGS) N .

在一些实施方案中，所述融合蛋白是同源二聚体或异源二聚体。In some embodiments, the fusion protein is a homodimer or a heterodimer.

在一些实施方案中，所述融合蛋白包含抗原结构域和结构域D，所述融合蛋白构成类似抗体形式的同源二聚体或异源二聚体。所述融合蛋白由两个Fc结构域的二硫键构成同源二聚体或异源二聚体。优选地，所述融合蛋白包括第一多肽链和第二多肽链，所述第一多肽链包括抗原结构域和结构域D，所述第二多肽链包括抗原结构域和结构域D。所述第一多肽链与所述第二多肽链经第一多肽链的结构域D和所述第二多肽链的结构域D的二硫键连接形成同源二聚体或异源二聚体。优选地，所述第一多肽链还包括结构域B和/或结构域C，所述第二多肽链还包括结构域B和/或结构域C。例如，所述第一多肽链还包括结构域B，所述第二多肽链还包括结构域B，从而构成同源二聚体。或者，所述第一多肽链还包括结构域B，所述第二多肽链还包括结构域C，从而构成异源二聚体，反之亦然。所述抗原结构域和/或结构域B和/或结构域C可以连接在结构域D的C端或N端。例如，所述第一多肽链还包括结构域B和结构域C，所述第二多肽链还包括结构域B和结构域C，从而构成同源二聚体。In some embodiments, the fusion protein comprises an antigenic domain and domain D, the fusion protein constitutes a homodimer or heterodimer similar to an antibody form. The fusion protein consists of a homodimer or a heterodimer formed by disulfide bonds of two Fc domains. Preferably, the fusion protein includes a first polypeptide chain and a second polypeptide chain, the first polypeptide chain includes an antigenic domain and domain D, and the second polypeptide chain includes an antigenic domain and a domain d. The first polypeptide chain and the second polypeptide chain are connected by a disulfide bond between the domain D of the first polypeptide chain and the domain D of the second polypeptide chain to form a homodimer or heterodimer source dimer. Preferably, the first polypeptide chain further comprises domain B and/or domain C, and the second polypeptide chain further comprises domain B and/or domain C. For example, the first polypeptide chain further includes domain B, and the second polypeptide chain further includes domain B, thereby forming a homodimer. Alternatively, the first polypeptide chain further includes domain B, and the second polypeptide chain further includes domain C, thereby forming a heterodimer, and vice versa. The antigen domain and/or domain B and/or domain C may be linked to the C-terminus or N-terminus of domain D. For example, the first polypeptide chain further includes domain B and domain C, and the second polypeptide chain further includes domain B and domain C, thereby forming a homodimer.

或者，所述第一多肽链从C端至N端包括结构域D，抗原结构域，结构域A，所述第二多肽链从C端至N端包括结构域D，抗原结构域，结构域A，从而构成同 源二聚体，反之亦然。Alternatively, the first polypeptide chain includes domain D, antigen domain, and domain A from C-terminus to N-terminus, and the second polypeptide chain includes domain D, antigen domain, from C-terminus to N-terminus, Domain A, thereby forming a homodimer, and vice versa.

或者，所述第一多肽链从C端至N端包括结构域D，抗原结构域，结构域B，所述第二多肽链从C端至N端包括结构域D，抗原结构域，结构域B，从而构成同源二聚体，反之亦然。Alternatively, the first polypeptide chain includes domain D, an antigen domain, and domain B from the C-terminus to the N-terminus, and the second polypeptide chain includes domain D, an antigen domain, from the C-terminus to the N-terminus, Domain B, thereby forming a homodimer, and vice versa.

或者，所述第一多肽链从C端至N端包括结构域D，抗原结构域，结构域C，结构域A，所述第二多肽链从C端至N端包括结构域D，抗原结构域，结构域C，结构域A，从而构成同源二聚体，反之亦然。Alternatively, the first polypeptide chain includes domain D, antigen domain, domain C, and domain A from C-terminus to N-terminus, and the second polypeptide chain includes domain D from C-terminus to N-terminus, The antigenic domain, domain C, domain A, thereby constituting a homodimer and vice versa.

或者，所述第一多肽链从C端至N端包括结构域D，抗原结构域，结构域C，结构域B，所述第二多肽链从C端至N端包括结构域D，抗原结构域，结构域C，结构域B，从而构成同源二聚体，反之亦然。Alternatively, the first polypeptide chain includes domain D, antigen domain, domain C, and domain B from C-terminus to N-terminus, and the second polypeptide chain includes domain D from C-terminus to N-terminus, The antigenic domain, domain C, domain B, thus constitutes a homodimer and vice versa.

或者，所述第一多肽链从C端至N端包括结构域B，结构域D，抗原结构域，结构域A，所述第二多肽链从C端至N端包括结构域B，结构域D，抗原结构域，结构域A，从而构成同源二聚体。Alternatively, the first polypeptide chain includes domain B, domain D, antigen domain, and domain A from C-terminus to N-terminus, and the second polypeptide chain includes domain B from C-terminus to N-terminus, Domain D, antigenic domain, and domain A, thus forming a homodimer.

或者，所述第一多肽链从C端至N端包括结构域B，结构域D，抗原结构域，结构域C，结构域A，所述第二多肽链从C端至N端包括结构域B，结构域D，抗原结构域，结构域C，结构域A，从而构成同源二聚体，反之亦然。Alternatively, the first polypeptide chain includes domain B, domain D, antigen domain, domain C, and domain A from the C-terminus to the N-terminus, and the second polypeptide chain includes from the C-terminus to the N-terminus Domain B, domain D, antigenic domain, domain C, domain A, thereby forming a homodimer and vice versa.

或者，所述第一多肽链从C端至N端包括结构域D，抗原结构域，结构域A，所述第二多肽链从C端至N端包括结构域D，抗原结构域，结构域C，结构域B，从而构成异源二聚体，反之亦然。Alternatively, the first polypeptide chain includes domain D, antigen domain, and domain A from C-terminus to N-terminus, and the second polypeptide chain includes domain D, antigen domain, from C-terminus to N-terminus, Domain C, domain B, thereby forming a heterodimer and vice versa.

或者，所述第一多肽链从C端至N端包括结构域D，抗原结构域，结构域C，结构域A，所述第二多肽链从C端至N端包括结构域D，抗原结构域，结构域C，结构域B，从而构成异源二聚体，反之亦然。Alternatively, the first polypeptide chain includes domain D, antigen domain, domain C, and domain A from C-terminus to N-terminus, and the second polypeptide chain includes domain D from C-terminus to N-terminus, The antigenic domain, domain C, domain B, thus constitutes a heterodimer and vice versa.

优选地，在异源二聚体的情况下，结构域D分别具有Fc knob修饰和Fc Hole修饰。Preferably, in case of a heterodimer, domain D has Fc knob and Fc Hole modifications, respectively.

所述抗原结构域与所述免疫细胞靶向结构域可以用任意形式排列组合。所述抗原结构域与所述免疫细胞靶向结构域A，结构域B，结构域C，结构域D可以任意形式排列组合。The antigen domain and the immune cell targeting domain can be arranged and combined in any form. The antigen domain and the immune cell targeting domain A, domain B, domain C and domain D can be arranged and combined in any form.

抗原与免疫细胞靶向分子A可以任意形式排列组合。抗原与免疫细胞靶向分子B可以用任意形式排列组合。抗原与免疫细胞靶向分子C可以用任意形式 排列组合。The antigen and immune cell targeting molecule A can be arranged and combined in any form. The antigen and immune cell targeting molecule B can be arranged and combined in any form. The antigen and immune cell targeting molecule C can be arranged and combined in any form.

在一些实施方案，所述融合蛋白具有选自如下的结构：In some embodiments, the fusion protein has a structure selected from:

αPDL1-Antigen-Fc；αPDL1-Antigen-Fc;

CLEC9A binding peptide-Antigen-Fc；CLEC9A binding peptide-Antigen-Fc;

αDEC205-Antigen-Fc；αDEC205-Antigen-Fc;

Antigen-Fc-CLEC9A binding peptide；Antigen-Fc-CLEC9A binding peptide;

αPDL1-linker-Antigen-linker-Fc；αPDL1-linker-Antigen-linker-Fc;

αPDL1-(GGGGS) 3-Antigen-(G) 3-Fc； αPDL1-(GGGGS) 3 -Antigen-(G) 3 -Fc;

CLEC9A binding peptide-linker-Antigen-linker-Fc；CLEC9A binding peptide-linker-Antigen-linker-Fc;

CLEC9A binding peptide-(GGGGS) 3-Antigen-(G) 3-Fc； CLEC9A binding peptide-(GGGGS) 3 -Antigen-(G) 3 -Fc;

αDEC205-linker-Antigen-linker-Fc；αDEC205-linker-Antigen-linker-Fc;

αDEC205-(GGGGS) 3-Antigen-(G) 3-Fc； αDEC205-(GGGGS) 3 -Antigen-(G) 3 -Fc;

Antigen-linker-Fc-linker-CLEC9A binding peptide；Antigen-linker-Fc-linker-CLEC9A binding peptide;

Antigen-(G) 3-Fc-(GS) 3-CLEC9A binding peptide； Antigen-(G) 3 -Fc-(GS) 3 -CLEC9A binding peptide;

IL2-Antigen-Fc；IL2-Antigen-Fc;

IL12-Antigen-Fc；IL12-Antigen-Fc;

IL15-Antigen-Fc；IL15-Antigen-Fc;

IL21-Antigen-Fc；IL21-Antigen-Fc;

CSF2-Antigen-Fc；CSF2-Antigen-Fc;

Antigen-Fc-CSF2；Antigen-Fc-CSF2;

IL2-linker-Antigen-linker-Fc；IL2-linker-Antigen-linker-Fc;

IL2-(GGGGS) 3-Antigen-(G) 3-Fc； IL2-(GGGGS) 3 -Antigen-(G) 3 -Fc;

IL12-linker-Antigen-linker-Fc；IL12-linker-Antigen-linker-Fc;

IL12-(GGGGS) 3-Antigen-(G) 3-Fc； IL12-(GGGGS) 3 -Antigen-(G) 3 -Fc;

IL15-linker-Antigen-linker-Fc；IL15-linker-Antigen-linker-Fc;

IL15-(GGGGS) 3-Antigen-(G) 3-Fc； IL15-(GGGGS) 3 -Antigen-(G) 3 -Fc;

IL21-linker-Antigen-linker-Fc；IL21-linker-Antigen-linker-Fc;

IL21-(GGGGS) 3-Antigen-(G) 3-Fc； IL21-(GGGGS) 3 -Antigen-(G) 3 -Fc;

CSF2-linker-Antigen-linker-Fc；CSF2-linker-Antigen-linker-Fc;

CSF2-(GGGGS) 3-Antigen-(G) 3-Fc； CSF2-(GGGGS) 3 -Antigen-(G) 3 -Fc;

Antigen-linker-Fc-linker-CSF2；Antigen-linker-Fc-linker-CSF2;

Antigen-(GGGGS) 3-Fc-(G) 3-CSF2； Antigen-(GGGGS) 3 -Fc-(G) 3 -CSF2;

αPDL1-Pan-Antigen-Fc；αPDL1-Pan-Antigen-Fc;

CLEC9A binding peptide-Pan-Antigen-Fc；CLEC9A binding peptide-Pan-Antigen-Fc;

αDEC205-Pan-Antigen-Fc；αDEC205-Pan-Antigen-Fc;

αPDL1-linker-Pan-Antigen-linker-Fc；αPDL1-linker-Pan-Antigen-linker-Fc;

αPDL1-(GGGGS) 3-Pan-Antigen-(G) 3-Fc； αPDL1-(GGGGS) 3 -Pan-Antigen-(G) 3 -Fc;

CLEC9A binding peptide-linker-Pan-Antigen-linker-Fc；CLEC9A binding peptide-linker-Pan-Antigen-linker-Fc;

CLEC9A binding peptide-(GGGGS) 3-Pan-Antigen-(G) 3-Fc； CLEC9A binding peptide-(GGGGS) 3 -Pan-Antigen-(G) 3 -Fc;

αDEC205-linker-Pan-Antigen-linker-Fc；αDEC205-linker-Pan-Antigen-linker-Fc;

αDEC205-(GGGGS) 3-Pan-Antigen-(G) 3-Fc； αDEC205-(GGGGS) 3 -Pan-Antigen-(G) 3 -Fc;

IL2-Pan-Antigen-Fc；IL2-Pan-Antigen-Fc;

IL12-Pan-Antigen-Fc；IL12-Pan-Antigen-Fc;

IL15-Pan-Antigen-Fc；IL15-Pan-Antigen-Fc;

IL21-Pan-Antigen-Fc；IL21-Pan-Antigen-Fc;

CSF2-Pan-Antigen-Fc；CSF2-Pan-Antigen-Fc;

Pan-Antigen-Fc-CSF2；Pan-Antigen-Fc-CSF2;

IL2-linker-Pan-linker-Antigen-(G) 3-Fc； IL2-linker-Pan-linker-Antigen-(G) 3 -Fc;

IL2-(GGGGS) 3-Pan-(GS) 3-Antigen-linker-Fc； IL2-(GGGGS) 3 -Pan-(GS) 3 -Antigen-linker-Fc;

IL12-linker-Pan-linker-Antigen-linker-Fc；IL12-linker-Pan-linker-Antigen-linker-Fc;

IL12-(GGGGS) 3-Pan-(GS) 3-Antigen-(G) 3-Fc； IL12-(GGGGS) 3 -Pan-(GS) 3 -Antigen-(G) 3 -Fc;

IL15-linker-Pan-linker-Antigen-linker-Fc；IL15-linker-Pan-linker-Antigen-linker-Fc;

IL15-(GGGGS) 3-Pan-(GS) 3-Antigen-(G) 3-Fc； IL15-(GGGGS) 3 -Pan-(GS) 3 -Antigen-(G) 3 -Fc;

IL21-linker-Pan-linker-Antigen-linker-Fc；IL21-linker-Pan-linker-Antigen-linker-Fc;

IL21-(GGGGS) 3-Pan-(GS) 3-Antigen-(G) 3-Fc； IL21-(GGGGS) 3 -Pan-(GS) 3 -Antigen-(G) 3 -Fc;

CSF2-linker-Pan-linker-Antigen-linker-Fc；CSF2-linker-Pan-linker-Antigen-linker-Fc;

CSF2-(GGGGS) 3-Pan-(GS) 3-Antigen-(G) 3-Fc； CSF2-(GGGGS) 3 -Pan-(GS) 3- Antigen-(G) 3 -Fc;

Pan-linker-Antigen-linker-Fc-linker-CSF2；Pan-linker-Antigen-linker-Fc-linker-CSF2;

Pan-(GGGGS) 3-Antigen-(G) 3-Fc-(GS) 3-CSF2； Pan-(GGGGS) 3 -Antigen-(G) 3 -Fc-(GS) 3 -CSF2;

αPDL1-Antigen-Fc-IL12；αPDL1-Antigen-Fc-IL12;

αPDL1-Antigen-Fc-IL21；αPDL1-Antigen-Fc-IL21;

αPDL1-Antigen-Fc-CSF2；αPDL1-Antigen-Fc-CSF2;

CLEC9A binding peptide-Antigen-Fc-IL12；CLEC9A binding peptide-Antigen-Fc-IL12;

CLEC9A binding peptide-Antigen-Fc-IL21；CLEC9A binding peptide-Antigen-Fc-IL21;

CLEC9A binding peptide-Antigen-Fc-CSF2；CLEC9A binding peptide-Antigen-Fc-CSF2;

αPDL1-linker-Antigen-linker-Fc-linker-IL12；αPDL1-linker-Antigen-linker-Fc-linker-IL12;

αPDL1-(GGGGS) 3-Antigen-(G) 3-Fc-(GS) 3-IL12； αPDL1-(GGGGS) 3 -Antigen-(G) 3 -Fc-(GS) 3 -IL12;

αPDL1-linker-Antigen-linker-Fc-linker-IL21；αPDL1-linker-Antigen-linker-Fc-linker-IL21;

αPDL1-(GGGGS) 3-Antigen-(G) 3-Fc-(GS) 3-IL21； αPDL1-(GGGGS) 3 -Antigen-(G) 3 -Fc-(GS) 3 -IL21;

αPDL1-linker-Antigen-linker-Fc-linker-CSF2；αPDL1-linker-Antigen-linker-Fc-linker-CSF2;

αPDL1-(GGGGS) 3-Antigen-(G) 3-Fc-(GS) 3-CSF2； αPDL1-(GGGGS) 3 -Antigen-(G) 3 -Fc-(GS) 3 -CSF2;

CLEC9A binding peptide-linker-Antigen-linker-Fc-linker-IL12；CLEC9A binding peptide-linker-Antigen-linker-Fc-linker-IL12;

CLEC9A binding peptide-(GGGGS) 3-Antigen-(G) 3-Fc-(GS) 3-IL12； CLEC9A binding peptide-(GGGGS) 3 -Antigen-(G) 3 -Fc-(GS) 3 -IL12;

CLEC9A binding peptide-linker-Antigen-linker-Fc-linker-IL21；CLEC9A binding peptide-linker-Antigen-linker-Fc-linker-IL21;

CLEC9A binding peptide-(GGGGS) 3-Antigen-(G) 3-Fc-(GS) 3-IL21； CLEC9A binding peptide-(GGGGS) 3 -Antigen-(G) 3 -Fc-(GS) 3 -IL21;

CLEC9A binding peptide-linker-Antigen-linker-Fc-linker-CSF2；CLEC9A binding peptide-linker-Antigen-linker-Fc-linker-CSF2;

CLEC9A binding peptide-(GGGGS) 3-Antigen-(G) 3-Fc-(GS) 3-CSF2； CLEC9A binding peptide-(GGGGS) 3 -Antigen-(G) 3 -Fc-(GS) 3 -CSF2;

αPDL1-Pan-Antigen-Fc-IL12；αPDL1-Pan-Antigen-Fc-IL12;

αPDL1-Pan-Antigen-Fc-IL21；αPDL1-Pan-Antigen-Fc-IL21;

αPDL1-Pan-Antigen-Fc-CSF2；αPDL1-Pan-Antigen-Fc-CSF2;

CLEC9A binding peptide-Pan-Antigen-Fc-IL12；CLEC9A binding peptide-Pan-Antigen-Fc-IL12;

CLEC9A binding peptide-Pan-Antigen-Fc-IL21；CLEC9A binding peptide-Pan-Antigen-Fc-IL21;

CLEC9A binding peptide-Pan-Antigen-Fc-CSF2；CLEC9A binding peptide-Pan-Antigen-Fc-CSF2;

αPDL1-CSF2-Pan-Antigen-Fc；αPDL1-CSF2-Pan-Antigen-Fc;

αPDL1-linker-Pan-linker-Antigen-linker-Fc-linker-IL12；αPDL1-linker-Pan-linker-Antigen-linker-Fc-linker-IL12;

αPDL1-(GGGGS) 3-Pan-(GS) 3-Antigen-(G) 3-Fc-(GS) 3-IL12； αPDL1-(GGGGS) 3 -Pan-(GS) 3 -Antigen-(G) 3 -Fc-(GS) 3 -IL12;

αPDL1-linker-Pan-linker-Antigen-linker-Fc-linker-IL21；αPDL1-linker-Pan-linker-Antigen-linker-Fc-linker-IL21;

αPDL1-(GGGGS) 3-Pan-(GS) 3-Antigen-(G) 3-Fc-(GS) 3-IL21； αPDL1-(GGGGS) 3 -Pan-(GS) 3 -Antigen-(G) 3 -Fc-(GS) 3 -IL21;

αPDL1-linker-Pan-linker-Antigen-linker-Fc-linker-CSF2；αPDL1-linker-Pan-linker-Antigen-linker-Fc-linker-CSF2;

αPDL1-(GGGGS) 3-Pan-(GS) 3-Antigen-(G) 3-Fc-(GS) 3-CSF2； αPDL1-(GGGGS) 3 -Pan-(GS) 3 -Antigen-(G) 3 -Fc-(GS) 3 -CSF2;

CLEC9A binding peptide-linker-Pan-linker-Antigen-linker-Fc-linker-IL12；CLEC9A binding peptide-linker-Pan-linker-Antigen-linker-Fc-linker-IL12;

CLEC9A binding peptide-(GGGGS) 3-Pan-(GS) 3-Antigen-(G) 3-Fc-(GS) 3-IL12； CLEC9A binding peptide-(GGGGS) 3 -Pan-(GS) 3 -Antigen-(G) 3 -Fc-(GS) 3 -IL12;

CLEC9A binding peptide-linker-Pan-linker-Antigen-linker-Fc-linker-IL21；CLEC9A binding peptide-linker-Pan-linker-Antigen-linker-Fc-linker-IL21;

CLEC9A binding peptide-(GGGGS) 3-Pan-(GS) 3-Antigen-(G) 3-Fc-(GS) 3-IL21； CLEC9A binding peptide-(GGGGS) 3 -Pan-(GS) 3 -Antigen-(G) 3 -Fc-(GS) 3 -IL21;

CLEC9A binding peptide-linker-Pan-linker-Antigen-linker-Fc-linker-CSF2；CLEC9A binding peptide-linker-Pan-linker-Antigen-linker-Fc-linker-CSF2;

CLEC9A binding peptide-(GGGGS) 3-Pan-(GS) 3-Antigen-(G) 3-Fc-(GS) 3-CSF2； CLEC9A binding peptide-(GGGGS) 3 -Pan-(GS) 3 -Antigen-(G) 3 -Fc-(GS) 3 -CSF2;

αPDL1-linker‐CSF2-linker-Pan-linker-Antigen-linker‐Fc；αPDL1-linker-CSF2-linker-Pan-linker-Antigen-linker-Fc;

αPDL1-(GGGGS) 3‐CSF2-(GS) 3‐Pan-(GS) 3‐Antigen-(G) 3‐Fc； αPDL1-(GGGGS) 3 ‐CSF2-(GS) 3 ‐Pan-(GS) 3 ‐Antigen-(G) 3 ‐Fc;

αPDL1-Antigen-Fc knob/CSF2-Antigen-Fc hole；αPDL1-Antigen-Fc knob/CSF2-Antigen-Fc hole;

CLEC9A binding peptide-Antigen-Fc knob/CSF2-Antigen-Fc hole；CLEC9A binding peptide-Antigen-Fc knob/CSF2-Antigen-Fc hole;

αPDL1-linker-Antigen-linker-Fc knob/CSF2-linker-Antigen-linker-Fc hole；αPDL1-linker-Antigen-linker-Fc knob/CSF2-linker-Antigen-linker-Fc hole;

αPDL1-(GGGGS) 3-Antigen-(G) 3-Fc knob/CSF2-(GGGGS) 3-Antigen-(G) 3-Fchole； αPDL1-(GGGGS) 3 -Antigen-(G) 3 -Fc knob/CSF2-(GGGGS) 3 -Antigen-(G) 3 -Fchole;

CLEC9A binding peptide-linker-Antigen-linker-Fc knob/CSF2-linker-Antigen-linker-Fc hole；CLEC9A binding peptide-linker-Antigen-linker-Fc knob/CSF2-linker-Antigen-linker-Fc hole;

CLEC9A binding peptide-(GGGGS) 3-Antigen-(G) 3-Fc knob/CSF2-(GGGGS) 3-Antigen-(G) 3-Fc hole； CLEC9A binding peptide-(GGGGS) 3 -Antigen-(G) 3 -Fc knob/CSF2-(GGGGS) 3 -Antigen-(G) 3 -Fc hole;

αPDL1-Pan-Antigen-Fc knob/CSF2-Pan-Antigen-Fc hole；αPDL1-Pan-Antigen-Fc knob/CSF2-Pan-Antigen-Fc hole;

CLEC9A binding peptide-Pan-Antigen-Fc knob/CSF2-Pan-Antigen-Fc hole；CLEC9A binding peptide-Pan-Antigen-Fc knob/CSF2-Pan-Antigen-Fc hole;

αPDL1-linker-Pan-linker-Antigen-linker-Fc knob/CSF2-linker-Pan-linker-Antigen-linker-Fc hole；αPDL1-linker-Pan-linker-Antigen-linker-Fc knob/CSF2-linker-Pan-linker-Antigen-linker-Fc hole;

αPDL1-(GGGGS) 3-Pan-(GS) 3-Antigen-(G) 3-Fc knob/CSF2-(GGGGS) 3-Pan-(GS) 3-Antigen-(G) 3-Fc hole； αPDL1-(GGGGS) 3 -Pan-(GS) 3 -Antigen-(G) 3 -Fc knob/CSF2-(GGGGS) 3 -Pan-(GS) 3 -Antigen-(G) 3 -Fc hole;

CLEC9A binding peptide-linker-Pan-linker-Antigen-linker-Fc knob/CSF2-linker-Pan-linker-Antigen-linker-Fc hole；CLEC9A binding peptide-linker-Pan-linker-Antigen-linker-Fc knob/CSF2-linker-Pan-linker-Antigen-linker-Fc hole;

CLEC9A binding peptide-(GGGGS) 3-Pan-(GS) 3-Antigen-(G) 3-Fc knob/ CSF2-(GGGGS) 3-Pan-(GS) 3-Antigen-(G) 3-Fc hole。 CLEC9A binding peptide-(GGGGS) 3 -Pan-(GS) 3 -Antigen-(G) 3 -Fc knob/ CSF2-(GGGGS) 3 -Pan-(GS) 3 -Antigen-(G) 3 -Fc hole.

在一些实施方案中，所述抗原结构域为SARS-Cov-2的S蛋白或其片段。优选地，所述S蛋白为预融合稳定的S蛋白。在一些实施方案中，所述预融合稳定的S蛋白包含双脯氨酸(S2P)突变或六脯氨酸(S6P)突变。In some embodiments, the antigenic domain is the S protein of SARS-Cov-2 or a fragment thereof. Preferably, the S protein is a pre-fusion stable S protein. In some embodiments, the prefusion stabilized S protein comprises a double proline (S2P) mutation or a hexaproline (S6P) mutation.

在一些实施方案中，所述抗原结构域为SARS-Cov-2的S蛋白的RBD结构域。In some embodiments, the antigenic domain is the RBD domain of the S protein of SARS-Cov-2.

本发明的融合蛋白可以作为重组蛋白疫苗。The fusion protein of the present invention can be used as a recombinant protein vaccine.

第二方面，本发明提供了用于编码第一方面的融合蛋白的核酸分子。In a second aspect, the present invention provides nucleic acid molecules for encoding the fusion protein of the first aspect.

第三方面，本发明提供了用于表达第一方面的融合蛋白或包含第二方面的核酸分子的载体。In a third aspect, the present invention provides a vector for expressing the fusion protein of the first aspect or comprising the nucleic acid molecule of the second aspect.

所述载体可以为质粒载体，腺病毒载体或慢病毒载体。The vector can be a plasmid vector, an adenoviral vector or a lentiviral vector.

第四方面，本发明提供了用于表达第一方面的融合蛋白或包含第二方面的核酸分子，或第三方面的载体的宿主细胞。In a fourth aspect, the present invention provides a host cell for expressing the fusion protein of the first aspect or comprising the nucleic acid molecule of the second aspect, or the vector of the third aspect.

所述宿主细胞可以为例如中国仓鼠卵巢细胞(CHO细胞)，幼年仓鼠肾细胞(BHK细胞)，COS细胞，小鼠NSO胸腺瘤细胞，小鼠骨髓瘤细胞(SP2/0细胞)，人胚胎肾细胞HEK293细胞。The host cells can be, for example, Chinese hamster ovary cells (CHO cells), young hamster kidney cells (BHK cells), COS cells, mouse NSO thymoma cells, mouse myeloma cells (SP2/0 cells), human embryonic kidney cells Cells HEK293 cells.

第五方面，本发明提供了含有第一方面的融合蛋白，或第二方面的核酸分子，或第三方面的载体，或第四方面的宿主细胞的组合物。In the fifth aspect, the present invention provides a composition containing the fusion protein of the first aspect, or the nucleic acid molecule of the second aspect, or the vector of the third aspect, or the host cell of the fourth aspect.

在一些实施方案中，所述组合物不包含可药用佐剂。In some embodiments, the composition does not contain a pharmaceutically acceptable adjuvant.

在一些实施方案中，所述组合物还包含可药用佐剂。所述可药用佐剂包括但不限于铝佐剂，例如氢氧化铝、磷酸铝或硫酸铝，或CpG佐剂。In some embodiments, the composition further comprises a pharmaceutically acceptable adjuvant. The pharmaceutically acceptable adjuvants include, but are not limited to, aluminum adjuvants, such as aluminum hydroxide, aluminum phosphate or aluminum sulfate, or CpG adjuvants.

第六方面，本发明提供了用于预防或治疗与病原微生物感染或肿瘤的方法，所述方法包括向对象给药第一方面的融合蛋白，第二方面的核酸分子，第三方面的载体，第四方面的宿主细胞或第五方面的组合物的步骤。In a sixth aspect, the present invention provides a method for preventing or treating infection with pathogenic microorganisms or tumors, the method comprising administering to a subject the fusion protein of the first aspect, the nucleic acid molecule of the second aspect, and the carrier of the third aspect, The host cell of the fourth aspect or the step of the composition of the fifth aspect.

所述对象为人类或动物。所述动物包括但不限于牛，羊，猫，犬，马，兔，猴，小鼠，大鼠，羊驼，骆驼等。The subject is a human or an animal. The animals include but are not limited to cattle, sheep, cats, dogs, horses, rabbits, monkeys, mice, rats, alpacas, camels and the like.

在一些实施方案中，所述对象为免疫功能低下的人或动物。In some embodiments, the subject is an immunocompromised human or animal.

在一些实施方案中，所述对象患有慢性肺病，例如慢性阻塞性肺疾病或哮喘。In some embodiments, the subject has a chronic lung disease, such as chronic obstructive pulmonary disease or asthma.

在一些实施方案中，所述患者患有基础疾病，例如心脏病，糖尿病或肺病。In some embodiments, the patient has an underlying disease, such as heart disease, diabetes, or lung disease.

第七方面，本发明提供了第一方面的融合蛋白，第二方面的核酸分子，第三 方面的载体，第四方面的宿主细胞或第五方面的组合物在制备用于预防或治疗对象与病原微生物感染或肿瘤相关的药物中的应用。In the seventh aspect, the present invention provides the fusion protein of the first aspect, the nucleic acid molecule of the second aspect, the vector of the third aspect, the host cell of the fourth aspect or the composition of the fifth aspect in preparation for preventing or treating an object with The application of pathogenic microorganism infection or tumor-related drugs.

所述对象为人类或动物。所述动物包括但不限于牛，羊，猫，犬，马，兔，猴，小鼠，大鼠，羊驼，骆驼，鸡，鸭，鹅，等。The subject is a human or an animal. The animals include, but are not limited to, cows, sheep, cats, dogs, horses, rabbits, monkeys, mice, rats, alpacas, camels, chickens, ducks, geese, and the like.

在一些实施方案中，所述对象为免疫功能低下的人或动物。In some embodiments, the subject is an immunocompromised human or animal.

在一些实施方案中，所述对象患有慢性肺病，例如慢性阻塞性肺疾病或哮喘。In some embodiments, the subject has a chronic lung disease, such as chronic obstructive pulmonary disease or asthma.

在一些实施方案中，所述患者患有基础疾病，例如心脏病，糖尿病或肺病。In some embodiments, the patient has an underlying disease, such as heart disease, diabetes, or lung disease.

所述癌症可以为前列腺癌，非小细胞肺癌，小细胞肺癌，肾细胞癌，脑癌，黑色素瘤，急性髓细胞性白血病，胰腺癌，结直肠癌，头颈部鳞状细胞癌，皮肤鳞状细胞癌，腺样囊性癌，胶质母细胞瘤，乳腺癌，间皮瘤，卵巢癌，神经胶质瘤，膀胱癌，肝癌，骨癌，骨髓癌，胃癌，甲状腺癌，淋巴癌，宫颈癌，子宫内膜癌，喉癌，急性淋巴细胞性白血病等。The cancer may be prostate cancer, non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, brain cancer, melanoma, acute myeloid leukemia, pancreatic cancer, colorectal cancer, squamous cell carcinoma of the head and neck, squamous cell carcinoma of the skin Cystoid cell carcinoma, adenoid cystic carcinoma, glioblastoma, breast cancer, mesothelioma, ovarian cancer, glioma, bladder cancer, liver cancer, bone cancer, bone marrow cancer, stomach cancer, thyroid cancer, lymphoma, Cervical cancer, endometrial cancer, laryngeal cancer, acute lymphoblastic leukemia, etc.

在一些实施方案中，所述抗原为肿瘤抗原，所述肿瘤抗原包括但不限于5T4,AIM2,AKAP4 2,Art-4,AuraA1(AURKA),Aura B1(AURKB),BAGE,BCAN,B-周期素,BSG,CCND1,CD133,CDC45L,CDCA1(TTK),CEA,CHI3L2(几丁质酶3样2),CSPG4,EpCAM4,Epha2,EPHX1,Ezh2,FABP7,Fosl1(Fra-1),GAGE,Galt-3,G250(CA9),gBK,glast,GnT-V,gp100,HB-EGF,HER2,HNPRL,HO-1,hTERT,IGF2BP3,IL13-Ra2,IMP-3,IQGAP1,ITGAV,KIF1C,KIF20A,KIF21B,KIFC3,KK-LC-1,LAGE-1,Lck,LRRC8A,MAGE-1(MAGEA1),MAGE-2(MAGEA2B),MAGE-3,MAGE-4,MAGE-6,MAGE-10,MAGE-12,MAGE-C1(CT7),MAGE-C2,MAGE-C3,Mart-1,MELK,MRP3,MUC1,NAPSA,NLGN4X,Nrcam,NY-ESO-1(CTAG1B),NY-SAR-35,OFA/iLRP,PCNA,PIK3R1,Prame,PRKDC,PTH-rP,PTPRZ1,PTTG1 2,PRKDC,RAN,RGS1,RGS5,RHAMM(RHAMM-3R),RPL19,Sart-1,Sart-2,Sart-3,SEC61G,SGT-1,SOX2,Sox10,Sox11,SP17,SPANX-B,SQSTM1,S.S.X-2,STAT1,STAT3,生存素,TARA,TNC,Trag-3,TRP-1,TRP2,酪氨酸酶,URLC10(LY6K),Ube2V,WT1,XAGE-1b(GAGED2a),YKL-40(CHI3L1),ACRBP,SCP-1,S.S.X-1,S.S.X-4,NY-TLU-57,CAIX,Brachyury,NY-BR-1,ErbB,间皮素,EGFRvIII,IL-13Ra2,MSLN,GPC3,FR,PSMA,GD2,L1-CAM,VEGFR1,VEGFR2,KOC1,OFA,SL-701,突变体P53,DEPDC1,MPHOSPH1,ONT-10,GD2L,GD3L,TF,PAP,BRCA1DLC1,XPO1,HIF1A, ADAM2,CALR3,SAGE1,SCP-1,ppMAPkkk,WHSC,突变体Ras,COX1,COX2,FOXP3,IDO1,IDO2,TDO,PDL1,PDL2和PGE2。In some embodiments, the antigen is a tumor antigen, including but not limited to 5T4, AIM2, AKAP4 2, Art-4, AuraA1 (AURKA), Aura B1 (AURKB), BAGE, BCAN, B-cycle BSG, CCND1, CD133, CDC45L, CDCA1 (TTK), CEA, CHI3L2 (chitinase 3-like 2), CSPG4, EpCAM4, Epha2, EPHX1, Ezh2, FABP7, Fosl1 (Fra-1), GAGE, Galt -3, G250(CA9), gBK, glast, GnT-V, gp100, HB-EGF, HER2, HNPRL, HO-1, hTERT, IGF2BP3, IL13-Ra2, IMP-3, IQGAP1, ITGAV, KIF1C, KIF20A, KIF21B, KIFC3, KK-LC-1, LAGE-1, Lck, LRRC8A, MAGE-1(MAGEA1), MAGE-2(MAGEA2B), MAGE-3, MAGE-4, MAGE-6, MAGE-10, MAGE- 12, MAGE-C1 (CT7), MAGE-C2, MAGE-C3, Mart-1, MELK, MRP3, MUC1, NAPSA, NLGN4X, Nrcam, NY-ESO-1 (CTAG1B), NY-SAR-35, OFA/ iLRP, PCNA, PIK3R1, Prame, PRKDC, PTH-rP, PTPRZ1, PTTG1 2, PRKDC, RAN, RGS1, RGS5, RHAMM (RHAMM-3R), RPL19, Sart-1, Sart-2, Sart-3, SEC61G, SGT-1, SOX2, Sox10, Sox11, SP17, SPANX-B, SQSTM1, S.S.X-2, STAT1, STAT3, Survivin, TARA, TNC, Trag-3, TRP-1, TRP2, Tyrosinase, URLC10( LY6K), Ube2V, WT1, XAGE-1b (GAGED2a), YKL-40 (CHI3L1), ACRBP, SCP-1, S.S.X-1, S.S.X-4, NY-TLU-57, CAIX, Brachyury, NY-BR-1 , ErbB, Mesothelin, EGFRvIII, IL-13Ra2, MSLN, GPC3, FR, PSMA, GD2, L1-CAM, VEGFR1, VEGFR2, KOC1, OFA, SL-701, Mutant P53, DEPDC1, MPHOSPH1, ONT-10 , GD2L, GD3L, TF, PAP, BRCA1DLC1, XPO1, HIF1A, ADAM2, CALR3, SAGE1, SCP-1, ppMAPkkk, WHSC, mutant Ras, COX1, COX2, FOXP3, IDO1, IDO2, TDO, PDL1, PDL2 and PGE2 .

在一些实施方案中，所述抗原包括但不限于与任何肿瘤/癌症有关的抗原，例如肺癌(MTFR2D326Y,CHTF18L769V,MYADM R30W,HERC1P3278S,FAM3C K193E,CSMD1G3446E,SLC26A7R117Q,PGAP1Y903F,HELB P987S,ANKRD K603T)；黑色素瘤(TMEM48F169L,TKT R438W,SEC24A P469L,AKAP13Q285K,EXOC8Q656P,PABPC1R520Q,MRPS5P59L,ABCC2S1342F,SEC23A P52L,SYTL4S363F,MAP3K9E689K,AKAP6M1482I,RPBMP42L,HCAPG2P333L,H3F3C T4I,GABPA E161K,SEPT2Q125R,SRPX P55L,WDR46T300I,PRDX3P101L,HELZ2D614N,GCN1L1P769L,AFMID A52V,PLSCR4R247C,CENPL P79L,TPX2H458Y,SEC22C H218Y,POLA2L420F,SLC24A5mut)；间皮瘤(NOTCH2G703D,PDE4DIPL288M,BAP1V523fs,ATP10B E210K,NSD1K2482T)；胶质瘤/胶质母细胞瘤(IDH1R132H,POLE L424V)；乳腺癌(mPALB2,mROBO3,mZDHHC16,mPTPRS,RBPJ H204L)；胆管癌((ERBB2IPE805G)；和宫颈癌(MAPK1E322K,PIK3CA E545K,PIK3CA E542K,EP300D1399N,ERBB2S310F,ERBB3V104M,KRAS G12D)。In some embodiments, the antigens include, but are not limited to, antigens associated with any tumor/cancer, such as lung cancer (MTFR2D326Y, CHTF18L769V, MYADM R30W, HERC1P3278S, FAM3C K193E, CSMD1G3446E, SLC26A7R117Q, PGAP1Y903F, HELB P987S, ANKRD K60 3T); Melanoma (TMEM48F169L, TKT R438W, SEC24A P469L, AKAP13Q285K, EXOC8Q656P, PABPC1R520Q, MRPS5P59L, ABCC2S1342F, SEC23A P52L, SYTL4S363F, MAP3K9E689K, AKAP6M14 82I,RPBMP42L,HCAPG2P333L,H3F3C T4I,GABPA E161K,SEPT2Q125R,SRPX P55L,WDR46T300I,PRDX3P101L,HELZ2D614N , GCN1L1P769L, AFMID A52V, PLSCR4R247C, CENPL P79L, TPX2H458Y, SEC22C H218Y, POLA2L420F, SLC24A5mut); mesothelioma (NOTCH2G703D, PDE4DIPL288M, BAP1V523fs, ATP10B E 210K, NSD1K2482T); glioma/glioblastoma (IDH1R132H, POLE L424V); breast cancer (mPALB2, mROBO3, mZDHHC16, mPTPRS, RBPJ H204L); cholangiocarcinoma ((ERBB2IPE805G); and cervical cancer (MAPK1E322K, PIK3CA E545K, PIK3CA E542K, EP300D1399N, ERBB2S310F, ERBB3V1 04M, KRAS G12D).

所述病原微生物相关的疾病包括但不限于：获得性免疫缺陷综合症(AIDS)(人类免疫缺陷病毒(HIV))；阿根廷提根热(ArgentineTeagan fever)(Junin病毒)；星状病毒感染(星状病毒科)；BK病毒感染(BK病毒)；玻利维亚出血热(Machupo病毒)；巴西出血热(Sabiá病毒)；水痘(水痘带状疱疹病毒(VZV))；基孔肯雅热(Chikungunya)(甲病毒)；科罗拉多蜱传热(Colorado tick fever)(CTF)(科罗拉多蜱传热病毒(Colorado tick fever virus)(CTFV))；普通感冒，急性病毒性鼻咽炎，急性鼻炎(通常是鼻病毒和冠状病毒)；巨细胞病毒感染(巨细胞病毒)；登革热(登革热病毒(DEN-1，DEN-2，DEN-3和DEN-4)和其他黄病毒，包括但不限于西尼罗河病毒(西尼罗河热)，黄热病病毒(黄热病)；寨卡病毒(寨卡热)和蜱传脑炎病毒；埃博拉出血热(埃博拉病毒(EBOV))；肠病毒感染(肠病毒种)；感染性红斑病(第五种病)(细小病毒B19)；幼儿急疹(Exanthemsubitum)(第六种病)(人类疱疹病毒6(HHV-6)和人类疱疹病毒7(HHV-7))；手足口病(HFMD)(肠病毒，主要是柯萨奇A病毒和肠病毒71(EV71))；汉坦病毒肺综合征(HantavirusPulmonary Syndrome， HPS)(Sin Nombre病毒)；甲型肝炎(甲型肝炎病毒)；乙型肝炎(乙型肝炎病毒)；丙型肝炎(丙型肝炎病毒)；丁型肝炎(丁型肝炎病毒)；戊型肝炎(戊型肝炎病毒)；单纯疱疹(单纯疱疹病毒1和2(HSV-1和HSV-2))；人博卡病毒感染(人博卡病毒(HBoV))；人间质性肺炎病毒感染(人间质性肺炎病毒(hMPV))；人乳头瘤病毒(HPV)感染(人乳头瘤病毒(HPV))；人副流感病毒感染(人副流感病毒(HPIV))；爱泼斯坦-巴尔病毒传染性单核细胞增多症(Mono)(爱泼斯坦-巴尔病毒(EBV))；人流感病毒(甲型流感，包括但不限于H1N1，H2N2，H3N2，H5N1，H7N9，乙型流感和正黏病毒科的其他成员)；拉沙热(拉沙病毒)；淋巴细胞性脉络丛脑膜炎(淋巴细胞性脉络丛脑膜炎病毒(LCMV))；马尔堡出血热(MHF)(马尔堡病毒)；麻疹(麻疹病毒)；中东呼吸综合征(MERS)(中东呼吸综合征冠状病毒)；传染性软疣(MC)(传染性软疣病毒(MCV))；猴痘(猴痘病毒)；流行性腮腺炎(流行性腮腺炎病毒)；诺如病毒(Norovirus)(儿童和婴儿)(诺如病毒)；脊髓灰质炎(脊髓灰质炎病毒)；进行性多灶性白质脑病(JC病毒)；狂犬病(狂犬病病毒)；呼吸道合胞病毒感染(呼吸道合胞病毒(RSV))；鼻病毒感染(鼻病毒)；裂谷热(RVF)(裂谷热病毒)；轮状病毒感染(轮状病毒)；风疹(风疹病毒)；带状疱疹(Shingles)(带状疱疹(Herpes zoster))(水痘带状疱疹病毒(VZV))；天花(Smallpox)(天花(Variola))(大天花或小天花)；亚急性硬化性全脑炎(麻疹病毒)；委内瑞拉马脑炎(委内瑞拉马脑炎病毒)；委内瑞拉出血热(瓜纳里托病毒)；病毒性肺炎。The diseases related to the pathogenic microorganisms include but are not limited to: Acquired Immunodeficiency Syndrome (AIDS) (Human Immunodeficiency Virus (HIV)); Argentine Teagan fever (Argentine Teagan fever) (Junin virus); Astrovirus infection (Astrovirus) Viridae); BK virus infection (BK virus); Bolivian hemorrhagic fever (Machupo virus); Brazilian hemorrhagic fever (Sabiá virus); chickenpox (varicella zoster virus (VZV)); A virus); Colorado tick fever (CTF) (Colorado tick fever virus (CTFV)); common cold, acute viral nasopharyngitis, acute rhinitis (usually rhinovirus and coronavirus); cytomegalovirus infection (CMV); dengue fever (Dengue virus (DEN-1, DEN-2, DEN-3 and DEN-4) and other flaviviruses, including but not limited to West Nile virus (West Nile fever), yellow fever virus (Yellow fever); Zika virus (Zika fever) and tick-borne encephalitis virus; Ebola hemorrhagic fever (Ebola virus (EBOV)); enterovirus infection (Enterovirus species ); erythema infectious disease (fifth disease) (parvovirus B19); exanthemsubitum (sixth disease) (human herpesvirus 6 (HHV-6) and human herpesvirus 7 (HHV-7) ); hand, foot, and mouth disease (HFMD) (enteroviruses, mainly Coxsackie virus A and enterovirus 71 (EV71)); Hantavirus pulmonary syndrome (HPS) (Sin Nombre virus); hepatitis A ( hepatitis A virus); hepatitis B (hepatitis B virus); hepatitis C (hepatitis C virus); hepatitis D (hepatitis D virus); hepatitis E (hepatitis E virus); herpesvirus 1 and 2 (HSV-1 and HSV-2)); human bocavirus infection (human bocavirus (HBoV)); human metapneumovirus infection (human interstitial pneumovirus (hMPV)); Human papillomavirus (HPV) infection (human papillomavirus (HPV)); human parainfluenza virus infection (human parainfluenza virus (HPIV)); Epstein-Barr virus infectious mononucleosis (Mono) ( Epstein-Barr virus (EBV)); human influenza viruses (influenza A, including but not limited to H1N1, H2N2, H3N2, H5N1, H7N9, influenza B and other members of the Orthomyxoviridae family); Lassa fever (Lassa arenavirus); lymphocytic choriomeningitis (lymphocytic choriomeningitis virus (LCMV)); Marburg hemorrhagic fever (MHF) (Marburg virus); measles (measles virus); Middle East respiratory syndrome (MERS ) (MERS-CoV); Molluscum contagiosum (MC) (Molluscum contagiosum virus (MCV)); Monkeypox (Monkeypox virus); Mumps (Mumps virus); Norovirus Norovirus (children and infants) (Norovirus); Poliomyelitis (poliomyelitis virus); Progressive multifocal leukoencephalopathy (JC virus); Rabies (Rabies virus); Syncytial virus (RSV)); Rhinovirus infection (Rhinovirus); Rift Valley fever (RVF) (Rift Valley fever virus); Rotavirus infection (Rotavirus); Rubella (Rubella virus); Herpes zoster (Shingles ) (Herpes zoster) (varicella-zoster virus (VZV)); smallpox (Variola) (variola major or minor); subacute sclerosing panencephalitis (measles virus) ; Venezuelan equine encephalitis (Venezuelan equine encephalitis virus); Venezuelan hemorrhagic fever (Guararito virus); Viral pneumonia.

本发明的重组蛋白疫苗具有提高的免疫原性，增强的免疫应答，同时降低了重组蛋白疫苗对佐剂的依赖性。The recombinant protein vaccine of the invention has improved immunogenicity and enhanced immune response, and at the same time reduces the dependence of the recombinant protein vaccine on the adjuvant.

该蛋白疫苗中抗原融合表达的设计同样可以应用于其它类型的疫苗，包括但不限于核酸疫苗(如mRNA疫苗、环状RNA疫苗和DNA疫苗等)，病毒载体疫苗(如腺病毒载体疫苗和流感病毒载体疫苗等)，以及纳米颗粒疫苗等。The design of antigen fusion expression in this protein vaccine can also be applied to other types of vaccines, including but not limited to nucleic acid vaccines (such as mRNA vaccines, circular RNA vaccines and DNA vaccines, etc.), viral vector vaccines (such as adenovirus vector vaccines and influenza Viral vector vaccines, etc.), and nanoparticle vaccines, etc.

本发明显示了：The present invention shows:

(1)游离的RBD蛋白免疫原性较弱，当在RBD的基础上加上Fc部分时其免疫原性得到了提升，当在RBD-Fc的基础上加上细胞因子IL12和Pan时其免疫原性得到了进一步的提升，当在RBD-Fc的基础上加上细胞因子IL21和Pan时其免疫原性得到了进一步的提升，当在RBD-Fc的基础上加上细胞因子CSF2和Pan时其免疫原性得到了进一步的提升，当在RBD-Fc的基础上加上能够结合免疫细胞表面 蛋白PDL1的αPDL1抗体和Pan时其免疫原性得到了进一步的提升；(1) The immunogenicity of the free RBD protein is weak. When the Fc part is added to the RBD, its immunogenicity is improved. When the cytokines IL12 and Pan are added to the RBD-Fc, its immune The originality has been further improved, when the cytokines IL21 and Pan are added on the basis of RBD-Fc, its immunogenicity has been further improved, and when the cytokines CSF2 and Pan are added on the basis of RBD-Fc Its immunogenicity has been further improved, and its immunogenicity has been further improved when the αPDL1 antibody and Pan that can bind to the immune cell surface protein PDL1 are added on the basis of RBD-Fc;

(2)游离的RBD蛋白免疫原性较弱，当在RBD的基础上加上Fc部分时可提高抗体滴度，当在RBD-Fc的基础上加上细胞因子CSF2时可进一步提高抗体滴度，当在RBD-Fc的基础上加上能够结合免疫细胞表面蛋白PDL1的αPDL1抗体时也可进一步提高抗体滴度；(2) The immunogenicity of the free RBD protein is weak, and the antibody titer can be increased when the Fc part is added to the RBD, and the antibody titer can be further increased when the cytokine CSF2 is added to the RBD-Fc , when the αPDL1 antibody that can bind to the immune cell surface protein PDL1 is added on the basis of RBD-Fc, the antibody titer can be further increased;

(3)游离的gp350蛋白疫苗免疫原性较弱，当在gp350-Fc的基础上加上细胞因子CSF2时可提高抗体滴度，当在gp350-Fc的基础上加上能够结合免疫细胞表面蛋白PDL1的αPDL1抗体时可提高抗体滴度，当在gp350-Fc的基础上加上细胞因子CSF2和Pan时可提高抗体滴度，当在gp350-Fc的基础上加上能够结合免疫细胞表面蛋白PDL1的αPDL1抗体和Pan时可提高抗体滴度。(3) The immunogenicity of the free gp350 protein vaccine is weak. When the cytokine CSF2 is added on the basis of gp350-Fc, the antibody titer can be increased. The antibody titer can be improved when the αPDL1 antibody of PDL1 is added, and the antibody titer can be increased when the cytokines CSF2 and Pan are added on the basis of gp350-Fc, and the antibody titer can be increased when the gp350-Fc is added on the basis of the ability to bind to the immune cell surface protein PDL1 αPDL1 antibody and Pan can increase the antibody titer.

附图说明Description of drawings

图1示出了一种同源二聚体形式的融合蛋白疫苗的示意图。其中，抗原‐Fc与免疫细胞靶向分子A按照A‐抗原‐Fc顺序排列组合。Figure 1 shows a schematic diagram of a fusion protein vaccine in the form of a homodimer. Among them, antigen-Fc and immune cell targeting molecule A are arranged and combined in the order of A-antigen-Fc.

图2示出了一种同源二聚体形式的融合蛋白疫苗的示意图。其中，抗原-Fc与免疫细胞靶向分子B按照B-抗原-Fc顺序排列组合。Figure 2 shows a schematic diagram of a fusion protein vaccine in the form of a homodimer. Wherein, the antigen-Fc and the immune cell targeting molecule B are arranged and combined in the order of B-antigen-Fc.

图3示出了一种同源二聚体形式的融合蛋白疫苗的示意图。其中，抗原-Fc与免疫细胞靶向分子A和C按照A-C-抗原-Fc顺序排列组合。Figure 3 shows a schematic diagram of a fusion protein vaccine in the form of a homodimer. Wherein, antigen-Fc and immune cell targeting molecules A and C are arranged and combined in the order of A-C-antigen-Fc.

图4示出了一种同源二聚体形式的融合蛋白疫苗的示意图。其中，抗原-Fc与免疫细胞靶向分子B和C按照B-C-抗原-Fc顺序排列组合。Figure 4 shows a schematic diagram of a fusion protein vaccine in the form of a homodimer. Wherein, antigen-Fc and immune cell targeting molecules B and C are arranged and combined in the order of B-C-antigen-Fc.

图5示出了一种同源二聚体形式的融合蛋白疫苗的示意图。其中，抗原-Fc与免疫细胞靶向分子A和B按照A-抗原-Fc-B顺序排列组合。Figure 5 shows a schematic diagram of a fusion protein vaccine in the form of a homodimer. Wherein, antigen-Fc and immune cell targeting molecules A and B are arranged and combined in the order of A-antigen-Fc-B.

图6示出了一种同源二聚体形式的融合蛋白疫苗的示意图。其中，抗原-Fc与免疫细胞靶向分子A，B和C按照A-C-抗原-Fc-B顺序排列组合。Figure 6 shows a schematic diagram of a fusion protein vaccine in the form of a homodimer. Wherein, antigen-Fc and immune cell targeting molecules A, B and C are arranged and combined in the order of A-C-antigen-Fc-B.

图7示出了一种异源二聚体形式的融合蛋白疫苗的示意图。其中，抗原-Fcknob与免疫细胞靶向分子A按照A-抗原-Fcknob顺序排列组合，抗原-Fchole与免疫细胞靶向分子B按照B-抗原-Fchole顺序排列组合。Figure 7 shows a schematic diagram of a fusion protein vaccine in the form of a heterodimer. Among them, the antigen-Fcknob and the immune cell targeting molecule A are arranged and combined in the order of A-antigen-Fcknob, and the antigen-Fchole and the immune cell targeting molecule B are arranged and combined in the order of B-antigen-Fchole.

图8示出了一种异源二聚体形式的融合蛋白疫苗的示意图。其中，抗原-Fcknob与免疫细胞靶向分子A和C按照A-C-抗原-Fcknob顺序排列组合，抗原-Fchole与免疫细胞靶向分子B和C按照B-C-抗原-Fchole顺序排列组合。Figure 8 shows a schematic diagram of a fusion protein vaccine in the form of a heterodimer. Among them, antigen-Fcknob and immune cell targeting molecules A and C are arranged and combined in the order of A-C-antigen-Fcknob, and antigen-Fchole and immune cell targeting molecules B and C are arranged and combined in the order of B-C-antigen-Fcknob.

图9示出了SARS-CoV-2刺突蛋白RBD相关免疫细胞靶向融合蛋白的聚丙烯酰胺 凝胶(SDS-PAGE)电泳鉴定图。其中分别在SDS-PAGE上鉴定了RBD，RBD-Fc，IL4-Pan-RBD-Fc，IL10-Pan-RBD-Fc，IL12-Pan-RBD-Fc，IL21-Pan-RBD-Fc，CSF2-Pan-RBD-Fc，PD1-Pan-RBD-Fc，PDL1-Pan-RBD-Fc对应的蛋白。Figure 9 shows the polyacrylamide gel (SDS-PAGE) electrophoresis identification diagram of the SARS-CoV-2 spike protein RBD-related immune cell targeting fusion protein. Among them, RBD, RBD-Fc, IL4-Pan-RBD-Fc, IL10-Pan-RBD-Fc, IL12-Pan-RBD-Fc, IL21-Pan-RBD-Fc, CSF2-Pan were identified on SDS-PAGE - RBD-Fc, PD1-Pan-RBD-Fc, PDL1-Pan-RBD-Fc corresponding proteins.

图10示出了SARS-CoV-2刺突蛋白RBD相关免疫细胞靶向融合蛋白疫苗在真核细胞293F中瞬时表达的蛋白产量。Figure 10 shows the protein production of SARS-CoV-2 spike protein RBD-related immune cell targeting fusion protein vaccine transiently expressed in eukaryotic cell 293F.

图11示出了免疫细胞靶向融合蛋白疫苗诱导的针对SARS-CoV-2刺突蛋白RBD的抗体反应结果。Figure 11 shows the results of antibody responses against the SARS-CoV-2 spike protein RBD induced by immune cell targeting fusion protein vaccines.

图12示出了新冠病毒SARS-CoV-2刺突蛋白RBD相关融合蛋白SDS-PAGE电泳鉴定图。Figure 12 shows the SDS-PAGE electrophoresis identification diagram of the fusion protein related to the RBD of the novel coronavirus SARS-CoV-2 spike protein.

图13示出了新冠病毒SARS-CoV-2刺突蛋白RBD相关融合蛋白的表达产量。Figure 13 shows the expression yield of the fusion protein related to the SARS-CoV-2 spike protein RBD of the new coronavirus.

图14示出了免疫细胞靶向新冠病毒SARS-CoV-2刺突蛋白RBD相关蛋白疫苗相较于单纯的RBD蛋白疫苗在小鼠体内可以引起更强的抗体反应。Figure 14 shows that the RBD-related protein vaccine targeting the new coronavirus SARS-CoV-2 spike protein by immune cells can elicit a stronger antibody response in mice than the RBD protein vaccine alone.

图15示出了EB病毒(Epstein-Barr virus，EBV)膜蛋白gp350相关融合蛋白SDS-PAGE电泳鉴定图。Figure 15 shows the SDS-PAGE electrophoresis identification diagram of the fusion protein related to the membrane protein gp350 of Epstein-Barr virus (EBV).

图16示出了EB病毒(Epstein-Barr virus，EBV)膜蛋白gp350相关融合蛋白的表达产量。Figure 16 shows the expression yield of Epstein-Barr virus (Epstein-Barr virus, EBV) membrane protein gp350-related fusion protein.

图17示出了免疫细胞靶向EBV膜蛋白gp350相关蛋白疫苗相较于单纯的gp350蛋白疫苗在小鼠体内可以引起更强的抗体反应。Figure 17 shows that immune cells targeting EBV membrane protein gp350-associated protein vaccine can elicit a stronger antibody response in mice than pure gp350 protein vaccine.

序列：sequence:

Figure PCTCN2022144163-appb-000001
Figure PCTCN2022144163-appb-000001

Figure PCTCN2022144163-appb-000002
Figure PCTCN2022144163-appb-000002

Figure PCTCN2022144163-appb-000003
Figure PCTCN2022144163-appb-000003

Figure PCTCN2022144163-appb-000004
Figure PCTCN2022144163-appb-000004

具体实施方式Detailed ways

以下所述的是本发明的优选实施方式，本发明所保护的不限于以下优选实施方式。应当指出，对于本领域的技术人员来说在此发明创造构思的基础上，做出的若干变形和改进，都属于本发明的保护范围。所用试剂未注明生产商者，均为可以通过市购获得的常规产品。What is described below is the preferred implementation of the present invention, and the protection of the present invention is not limited to the following preferred implementation. It should be pointed out that for those skilled in the art, some modifications and improvements made on the basis of this inventive concept all belong to the protection scope of the present invention. The reagents used were not indicated by the manufacturer, but were commercially available conventional products.

实施例Example

实施例1.融合蛋白疫苗平台的设计Embodiment 1. Design of fusion protein vaccine platform

免疫细胞靶向融合蛋白疫苗通过在抗原上融合免疫细胞靶向分子来获得。免疫细胞靶向分子可以包括以下四种成分：Immune cell-targeting fusion protein vaccines are obtained by fusing immune cell-targeting molecules to antigens. Immune cell targeting molecules can include the following four components:

A.能够结合免疫细胞表面蛋白的抗体或多肽；A. Antibodies or polypeptides capable of binding to immune cell surface proteins;

B.能够激活免疫细胞的细胞因子；B. Cytokines capable of activating immune cells;

C.能够激活免疫细胞的Pan表位(PADRE)；C. Pan epitopes capable of activating immune cells (PADRE);

D.能够结合免疫细胞的免疫球蛋白Fc。D. Immunoglobulin Fc capable of binding immune cells.

抗原可以和这四种成分中的两种、三种或四种，以任意形式排列组合形成融合蛋白。其代表形式如下：The antigen can be combined with two, three or four of these four components in any form to form a fusion protein. Its representative form is as follows:

为同源二聚体的形式，抗原-Fc与免疫细胞靶向分子A按照A-抗原-Fc的顺序排列组合，如图1所示。抗原-Fc与免疫细胞靶向分子A可以任意形式排列组合。具体包括：αPDL1-Antigen-Fc，CLEC9A binding peptide-Antigen-Fc，αDEC205-Antigen-Fc，Antigen-Fc-CLEC9A binding peptide。In the form of a homodimer, antigen-Fc and immune cell targeting molecule A are arranged and combined in the order of A-antigen-Fc, as shown in Figure 1. Antigen-Fc and immune cell targeting molecule A can be arranged and combined in any form. Specifically include: αPDL1-Antigen-Fc, CLEC9A binding peptide-Antigen-Fc, αDEC205-Antigen-Fc, Antigen-Fc-CLEC9A binding peptide.

为同源二聚体的形式，抗原-Fc与免疫细胞靶向分子B按照B-抗原-Fc顺序排列组合，如图2所示。抗原-Fc与免疫细胞靶向分子B可以任意形式排列组合。具体包括：IL2-Antigen-Fc，IL12-Antigen-Fc，IL15-Antigen-Fc，IL21-Antigen-Fc， CSF2-Antigen-Fc，Antigen-Fc-CSF2。In the form of a homodimer, antigen-Fc and immune cell targeting molecule B are arranged and combined in the order of B-antigen-Fc, as shown in FIG. 2 . Antigen-Fc and immune cell targeting molecule B can be arranged and combined in any form. Specifically include: IL2-Antigen-Fc, IL12-Antigen-Fc, IL15-Antigen-Fc, IL21-Antigen-Fc, CSF2-Antigen-Fc, Antigen-Fc-CSF2.

为同源二聚体的形式，抗原-Fc与免疫细胞靶向分子A和C按照A-C-抗原-Fc顺序排列组合，如图3所示。抗原-Fc与免疫细胞靶向分子A和C可以任意形式排列组合。具体包括：αPDL1-Pan-Antigen-Fc，CLEC9A binding peptide-Pan-Antigen-Fc，αDEC205-Pan-Antigen-Fc。In the form of a homodimer, antigen-Fc and immune cell targeting molecules A and C are arranged and combined in the order of A-C-antigen-Fc, as shown in FIG. 3 . Antigen-Fc and immune cell targeting molecules A and C can be arranged and combined in any form. Specifically include: αPDL1-Pan-Antigen-Fc, CLEC9A binding peptide-Pan-Antigen-Fc, αDEC205-Pan-Antigen-Fc.

为同源二聚体的形式，抗原-Fc与免疫细胞靶向分子B和C按照B-C-抗原-Fc顺序排列组合，如图4所示。抗原-Fc与免疫细胞靶向分子B和C可以任意形式排列组合。具体包括：IL2-Pan-Antigen-Fc，IL12-Pan-Antigen-Fc，IL15-Pan-Antigen-Fc，IL21-Pan-Antigen-Fc，CSF2-Pan-Antigen-Fc，Pan-Antigen-Fc-CSF2。In the form of a homodimer, antigen-Fc and immune cell targeting molecules B and C are arranged and combined in the order of B-C-antigen-Fc, as shown in FIG. 4 . Antigen-Fc and immune cell targeting molecules B and C can be arranged and combined in any form. Specifically include: IL2-Pan-Antigen-Fc, IL12-Pan-Antigen-Fc, IL15-Pan-Antigen-Fc, IL21-Pan-Antigen-Fc, CSF2-Pan-Antigen-Fc, Pan-Antigen-Fc-CSF2 .

为同源二聚体的形式，抗原-Fc与免疫细胞靶向分子A和B按照A-抗原-Fc-B顺序排列组合，如图5所示。抗原-Fc与免疫细胞靶向分子A和B可以任意形式排列组合。具体包括：αPDL1-Antigen-Fc-IL12，αPDL1-Antigen-Fc-IL21，αPDL1-Antigen-Fc-CSF2，CLEC9A binding peptide-Antigen-Fc-IL12，CLEC9A binding peptide-Antigen-Fc-IL21，CLEC9A binding peptide-Antigen-Fc-CSF2。In the form of a homodimer, antigen-Fc and immune cell targeting molecules A and B are arranged and combined in the order of A-antigen-Fc-B, as shown in FIG. 5 . Antigen-Fc and immune cell targeting molecules A and B can be arranged and combined in any form. Specifically include: αPDL1-Antigen-Fc-IL12, αPDL1-Antigen-Fc-IL21, αPDL1-Antigen-Fc-CSF2, CLEC9A binding peptide-Antigen-Fc-IL12, CLEC9A binding peptide-Antigen-Fc-IL21, CLEC9A binding peptide -Antigen-Fc-CSF2.

为同源二聚体的形式，抗原-Fc与免疫细胞靶向分子A，B和C按照A-C-抗原-Fc-B顺序排列组合，如图6所示。抗原-Fc与免疫细胞靶向分子A，B和C可以任意形式排列组合。具体包括：αPDL1-Pan-Antigen-Fc-IL12，αPDL1-Pan-Antigen-Fc-IL21，αPDL1-Pan-Antigen-Fc-CSF2，CLEC9A binding peptide-Pan-Antigen-Fc-IL12，CLEC9A binding peptide-Pan-Antigen-Fc-IL21，CLEC9A binding peptide-Pan-Antigen-Fc-CSF2，PDL1-CSF2-Pan-Antigen-Fc。In the form of a homodimer, antigen-Fc and immune cell targeting molecules A, B and C are arranged and combined in the order of A-C-antigen-Fc-B, as shown in FIG. 6 . Antigen-Fc and immune cell targeting molecules A, B and C can be arranged and combined in any form. Specifically include: αPDL1-Pan-Antigen-Fc-IL12, αPDL1-Pan-Antigen-Fc-IL21, αPDL1-Pan-Antigen-Fc-CSF2, CLEC9A binding peptide-Pan-Antigen-Fc-IL12, CLEC9A binding peptide-Pan -Antigen-Fc-IL21, CLEC9A binding peptide-Pan-Antigen-Fc-CSF2, PDL1-CSF2-Pan-Antigen-Fc.

为异源二聚体的形式，抗原-Fc knob与免疫细胞靶向分子A按照A-抗原-Fc knob顺序排列组合，抗原-Fc hole与免疫细胞靶向分子B按照B-抗原-Fc hole顺序排列组合，如图7所示。抗原-Fc knob与免疫细胞靶向分子A可以任意形式排列组合。抗原-Fc hole与免疫细胞靶向分子B可以任意形式排列组合。具体包括：αPDL1-Antigen-Fc knob/CSF2-Antigen-Fc hole，CLEC9A binding peptide-Antigen-Fc knob/CSF2-Antigen-Fc hole。In the form of heterodimers, antigen-Fc knob and immune cell targeting molecule A are arranged in the order of A-antigen-Fc knob, and antigen-Fc hole and immune cell targeting molecule B are in the order of B-antigen-Fc hole Arrange and combine, as shown in Figure 7. Antigen-Fc knob and immune cell targeting molecule A can be arranged and combined in any form. Antigen-Fc hole and immune cell targeting molecule B can be arranged and combined in any form. Specifically include: αPDL1-Antigen-Fc knob/CSF2-Antigen-Fc hole, CLEC9A binding peptide-Antigen-Fc knob/CSF2-Antigen-Fc hole.

为异源二聚体的形式，抗原-Fc knob与免疫细胞靶向分子A和C按照A-C-抗原-Fc knob顺序排列组合，抗原-Fc hole与免疫细胞靶向分子B和C按照B-C- 抗原-Fc hole顺序排列组合，如图8所示。抗原-Fc knob与免疫细胞靶向分子A和C可以任意形式排列组合。抗原-Fc hole与免疫细胞靶向分子B和C可以任意形式排列组合。具体包括：αPDL1-Pan-Antigen-Fc knob/CSF2-Pan-Antigen-Fc hole，CLEC9A binding peptide-Pan-Antigen-Fc knob/CSF2-Pan-Antigen-Fc hole。In the form of heterodimers, the antigen-Fc hole and immune cell targeting molecules A and C are arranged in the order of A-C-antigen-Fc knob, and the antigen-Fc hole and immune cell targeting molecules B and C are arranged in the order of B-C-antigen -Fc hole sequence arrangement and combination, as shown in Figure 8. Antigen-Fc knob and immune cell targeting molecules A and C can be arranged and combined in any form. Antigen-Fc hole and immune cell targeting molecules B and C can be arranged and combined in any form. Specifically include: αPDL1-Pan-Antigen-Fc knob/CSF2-Pan-Antigen-Fc hole, CLEC9A binding peptide-Pan-Antigen-Fc knob/CSF2-Pan-Antigen-Fc hole.

实施例2.疫苗平台的构建纯化和生产Example 2. Construction, purification and production of the vaccine platform

本实施例中，以新型冠状病毒SARS-CoV-2的刺突蛋白RBD蛋白同源二聚体形式为例，对该疫苗平台的表达生产进行了描述。In this example, the expression and production of the vaccine platform is described by taking the homodimer form of the spike protein RBD protein of the novel coronavirus SARS-CoV-2 as an example.

1、载体的构建1. Construction of the carrier

以PEE6.4为载体，通过分子克隆的方式，将抗原和免疫细胞靶向分子构建到该载体上，从而得到可以表达融合蛋白的质粒。其中KOZAK为科扎克序列，SP(signal peptide)为信号肽。Using PEE6.4 as a vector, antigens and immune cell targeting molecules are constructed on the vector by means of molecular cloning, so as to obtain plasmids that can express fusion proteins. Among them, KOZAK is the Kozak sequence, and SP (signal peptide) is the signal peptide.

(1)RBD：(1) RBD:

PEE6.4-HindIII-KOZAK-Start-SP-XbaI-RBD-XhoI-6His-EcoRIPEE6.4-HindIII-KOZAK-Start-SP-XbaI-RBD-XhoI-6His-EcoRI

(2)RBD-Fc：(2) RBD-Fc:

PEE6.4-HindIII-KOZAK-Start-SP-BsiwI-RBD-XhoI-G3-Fc-EcoRIPEE6.4-HindIII-KOZAK-Start-SP-BsiwI-RBD-XhoI-G3-Fc-EcoRI

(3)IL4-Pan-RBD-Fc：(3) IL4-Pan-RBD-Fc:

PEE6.4-HindIII-KOZAK-Start-SP-BsiwI-IL4-SfuI-(G4S) 3-Pan-(GS) 3-XbaI-RBD-XhoI-G 3-Fc-EcoRI PEE6.4-HindIII-KOZAK-Start-SP-BsiwI-IL4-SfuI-(G4S) 3 -Pan-(GS) 3 -XbaI-RBD-XhoI-G 3 -Fc-EcoRI

(4)IL10-Pan-RBD-Fc：(4) IL10-Pan-RBD-Fc:

PEE6.4-HindIII-KOZAK-Start-SP-BsiwI-IL10-SfuI-(G4S) 3-Pan-(GS) 3-XbaI-RBD-XhoI-G 3-Fc-EcoRI PEE6.4-HindIII-KOZAK-Start-SP-BsiwI-IL10-SfuI-(G4S) 3 -Pan-(GS) 3 -XbaI-RBD-XhoI-G 3 -Fc-EcoRI

(5)IL12-Pan-RBD-Fc：(5) IL12-Pan-RBD-Fc:

PEE6.4-HindIII-KOZAK-Start-SP-BsiwI-IL12-SfuI-(G4S) 3-Pan-(GS) 3-XbaI-RBD-XhoI-G 3-Fc-EcoRI PEE6.4-HindIII-KOZAK-Start-SP-BsiwI-IL12-SfuI-(G4S) 3 -Pan-(GS) 3 -XbaI-RBD-XhoI-G 3 -Fc-EcoRI

(6)IL21-Pan-RBD-Fc：(6) IL21-Pan-RBD-Fc:

PEE6.4-HindIII-KOZAK-Start-SP-BsiwI-IL21-SfuI-(G4S) 3-Pan-(GS) 3-XbaI-RBD-XhoI-G 3-Fc-EcoRI PEE6.4-HindIII-KOZAK-Start-SP-BsiwI-IL21-SfuI-(G4S) 3 -Pan-(GS) 3 -XbaI-RBD-XhoI-G 3 -Fc-EcoRI

(7)CSF2-Pan-RBD-Fc：(7) CSF2-Pan-RBD-Fc:

PEE6.4-HindIII-KOZAK-Start-SP-BsiwI-CSF2-SfuI-(G4S) 3-Pan-(GS) 3-XbaI-RBD-XhoI-G 3-Fc-EcoRI PEE6.4-HindIII-KOZAK-Start-SP-BsiwI-CSF2-SfuI-(G4S) 3 -Pan-(GS) 3 -XbaI-RBD-XhoI-G 3 -Fc-EcoRI

(8)PD1-Pan-RBD-Fc：(8) PD1-Pan-RBD-Fc:

PEE6.4-HindIII-KOZAK-Start-SP-BsiwI-PD1-SfuI-(G4S) 3-Pan-(GS) 3-XbaI-RBD-XhoI-G 3-Fc-EcoRI PEE6.4-HindIII-KOZAK-Start-SP-BsiwI-PD1-SfuI-(G4S) 3 -Pan-(GS) 3 -XbaI-RBD-XhoI-G 3 -Fc-EcoRI

(9)αPDL1-Pan-RBD-Fc：(9) αPDL1-Pan-RBD-Fc:

PEE6.4-HindIII-KOZAK-Start-SP-BsiwI-aPDL1-SfuI-(G4S) 3-Pan-(GS) 3-XbaI-RBD-XhoI-G 3-Fc-EcoRI。 PEE6.4-HindIII-KOZAK-Start-SP-BsiwI-aPDL1-SfuI-(G4S) 3 -Pan-(GS) 3- XbaI-RBD-XhoI- G3- Fc-EcoRI.

2、瞬时转染表达融合蛋白2. Transient transfection to express fusion protein

(1)细胞复苏：Freestyle 293F细胞以3×10 7个细胞/ml的浓度，于Freestyle 293 Expression Medium(10％DMSO)中冻存。从液氮中取出后，在37℃水浴锅中快速溶化，用SMM 293-TII培养基洗过后，将细胞重悬于50ml SMM 293-TII培养基中，37℃，8％CO 2，120rpm。 (1) Cell recovery: Freestyle 293F cells were frozen in Freestyle 293 Expression Medium (10% DMSO) at a concentration of 3×10 7 cells/ml. After taking it out from the liquid nitrogen, dissolve it quickly in a 37°C water bath, wash with SMM 293-TII medium, and resuspend the cells in 50ml of SMM 293-TII medium at 37°C, 8% CO 2 , 120rpm.

(2)用100ml Freestyle 293培养基重新悬浮293F细胞，细胞密度3×10 6cells/ml。 (2) 293F cells were resuspended with 100ml Freestyle 293 medium, and the cell density was 3×10 6 cells/ml.

(3)用2ml Freestyle 293培养基稀释100μg质粒，用2ml Freestyle 293培养基稀释400μg PEI。随后立即将2mL质粒和2mL的PEI混匀，室温静置5分钟。(3) Dilute 100μg plasmid with 2ml Freestyle 293 medium, and dilute 400μg PEI with 2ml Freestyle 293 medium. Immediately thereafter, 2 mL of the plasmid and 2 mL of PEI were mixed and allowed to stand at room temperature for 5 minutes.

(4)将质粒/PEI混合物加入细胞悬液中，放置在37℃，8％CO 2，85rpm培养箱中培养。 (4) The plasmid/PEI mixture was added to the cell suspension, and placed in an incubator at 37° C., 8% CO 2 , 85 rpm.

(5)4小时后，补加100ml EX-CELL293培养基，将转速调至120rpm继续培养。(5) After 4 hours, add 100ml EX-CELL293 medium, and adjust the rotation speed to 120rpm to continue culturing.

(6)24小时后，加入3.8mM细胞增殖抑制剂VPA，转染6天后收集上清液进行纯化。(6) After 24 hours, 3.8 mM cell proliferation inhibitor VPA was added, and the supernatant was collected 6 days after transfection for purification.

3、融合蛋白的纯化3. Purification of fusion protein

(1)将悬浮细胞培养液上清离心，弃沉淀，经0.45μM滤器过滤除去杂质。(1) Centrifuge the supernatant of the suspended cell culture solution, discard the precipitate, and filter through a 0.45 μM filter to remove impurities.

(2)取适量Protein A琼脂糖珠加入层析柱中，用10倍层析柱体积的蒸馏水和PBS分别冲洗和平衡层析柱。(2) Take an appropriate amount of Protein A agarose beads and add them to the chromatography column, wash and equilibrate the chromatography column with distilled water and PBS of 10 times the volume of the chromatography column respectively.

(3)利用恒流泵进行上样，流速为0.2mL/min。(3) A constant flow pump was used to load the sample at a flow rate of 0.2 mL/min.

(4)用10倍层析柱体积以上的PBS冲洗柱子后，用洗脱缓冲液(0.1M甘氨酸,pH 2.7)进行洗脱，洗脱蛋白加入适量的1M Tris，pH 9.0进行中和。(4) After washing the column with PBS more than 10 times the volume of the chromatography column, elution was performed with elution buffer (0.1M glycine, pH 2.7), and the eluted protein was neutralized by adding an appropriate amount of 1M Tris, pH 9.0.

(5)利用浓缩离心柱浓缩蛋白，利用Zeba脱盐离心柱将蛋白溶液置换到所需要的缓冲液中，利用Nano-500测定蛋白浓度，并计算蛋白表达产量。(5) Use concentrated spin column to concentrate protein, use Zeba desalting spin column to replace protein solution into required buffer, use Nano-500 to measure protein concentration, and calculate protein expression yield.

4、融合蛋白的纯度鉴定4. Purity identification of fusion protein

分别用还原性上样缓冲液(R)和非还原性上样缓冲液(N-R)将蛋白上样到聚丙烯酰胺凝胶上，电泳后用考马斯亮蓝对凝胶进行染色，然后用凝胶成像仪成像，查看融合蛋白的完整性和纯度。所述融合蛋白SDS-PAGE电泳鉴定图如图9所示。所述融合蛋白表达产量如图10所示。The proteins were loaded onto polyacrylamide gels with reducing buffer (R) and non-reducing buffer (N-R), respectively. After electrophoresis, the gel was stained with Coomassie brilliant blue, and then the gel was stained with Imager imaging to check the integrity and purity of the fusion protein. The SDS-PAGE electrophoresis identification diagram of the fusion protein is shown in FIG. 9 . The expression yield of the fusion protein is shown in FIG. 10 .

实施例3.SARS-CoV-2刺突蛋白RBD免疫细胞靶向融合蛋白疫苗相较于单纯的RBD蛋白疫苗在小鼠体内可以引起更强的抗体反应。Example 3. The SARS-CoV-2 spike protein RBD immune cell-targeting fusion protein vaccine can cause a stronger antibody response in mice than a simple RBD protein vaccine.

1、材料1. Materials

C57BL/6雄性小鼠(6-8)周购买于江苏集萃药康生物科技股份有限公司；辣根过氧化物氧化酶(HRP)标记的山羊抗小鼠IgG购自江苏康为世纪生物科技股份有限公司；辣根过氧化物氧化酶(HRP)标记的山羊抗小鼠IgG1，IgG2b和IgG2c购自江苏康为世纪生物科技股份有限公司；96孔ELISA测定板购自Bioland公司；ELISA显色液购自上海碧云天生物技术有限公司；ELISA终止液购自北京索莱宝科技有限公司；酶标仪Multiskan FC购自Thermo Fisher Scientific公司。C57BL/6 male mice (6-8) weeks were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.; horseradish peroxidase (HRP)-labeled goat anti-mouse IgG was purchased from Jiangsu Kangwei Century Biotechnology Co., Ltd. Co., Ltd.; horseradish peroxidase (HRP)-labeled goat anti-mouse IgG1, IgG2b and IgG2c were purchased from Jiangsu Kangwei Century Biotechnology Co., Ltd.; 96-well ELISA assay plate was purchased from Bioland Company; ELISA chromogenic solution Purchased from Shanghai Biyuntian Biotechnology Co., Ltd.; ELISA stop solution was purchased from Beijing Suolaibao Technology Co., Ltd.; microplate reader Multiskan FC was purchased from Thermo Fisher Scientific.

2、方法2. Method

(1)免疫细胞靶向融合蛋白疫苗免疫小鼠。将融合蛋白疫苗在PBS中稀释后，每只小鼠接种0.5μg RBD蛋白或者相同摩尔数的其它融合蛋白，每只小鼠肌肉注射50μl。采用两次免疫程序在第0天与21天免疫小鼠。每次免疫后第14天通过脸颊取血的方式收集小鼠血清，进行抗体检测。(1) Mice were immunized with immune cell-targeted fusion protein vaccine. After diluting the fusion protein vaccine in PBS, each mouse was inoculated with 0.5 μg of RBD protein or other fusion proteins of the same molar amount, and each mouse was injected intramuscularly with 50 μl. Mice were immunized on day 0 and day 21 using two immunization procedures. On the 14th day after each immunization, mouse serum was collected by cheek blood collection for antibody detection.

(2)ELISA检测血清中RBD特异性抗体。将RBD(2μg/ml)包被液以每孔100μl的体系加入到Elisa板中，4℃包被过夜。5％的封闭液(5％FBS的PBS)37℃封闭1小时。PBST洗3次，血清样品按照10倍梯度稀释，每孔加100μl到封闭好的ELISA板中，37℃孵育1小时。PBST洗3次，每孔加入100μl酶标二抗(1:5000)，37℃孵育1小时。用PBST洗5次，加底物TMB 100μl/孔，室温避光孵育，等待底物显色；每孔加50μl ELISA终止液终止显色，酶标仪读板，OD450-620。(2) ELISA detection of RBD-specific antibodies in serum. RBD (2 μg/ml) coating solution was added to the Elisa plate at 100 μl per well, and coated overnight at 4°C. 5% blocking solution (5% FBS in PBS) was used to block for 1 hour at 37°C. Wash 3 times with PBST, serum samples were diluted according to 10-fold gradient, and 100 μl was added to each well of a well-blocked ELISA plate, and incubated at 37°C for 1 hour. Wash 3 times with PBST, add 100 μl enzyme-labeled secondary antibody (1:5000) to each well, and incubate at 37°C for 1 hour. Wash 5 times with PBST, add substrate TMB 100 μl/well, incubate at room temperature in the dark, wait for substrate color development; add 50 μl ELISA stop solution to each well to stop color development, read the plate with a microplate reader, OD450-620.

3、结果3. Results

游离的RBD蛋白免疫原性较弱，当在RBD的基础上加上Fc部分时其免疫原性得到了提升，当在RBD-Fc的基础上加上细胞因子IL12和Pan时其免疫原性得到了进一步的提升，当在RBD-Fc的基础上加上细胞因子IL21和Pan时其免疫原性得到了进一步的提升，当在RBD-Fc的基础上加上细胞因子CSF2和Pan时 其免疫原性得到了进一步的提升，当在RBD-Fc的基础上加上能够结合免疫细胞表面蛋白PDL1的αPDL1抗体和Pan时其免疫原性得到了进一步的提升，二次免疫后抗体的水平普遍高于一次免疫后的抗体水平，如图11(a)所示。CSF2或αPDL1与Pan一起在RBD-Fc基础上诱导了更高水平的代表Th2免疫反应的IgG1，如图11(b)所示。IL12或CSF2与Pan一起在RBD-Fc基础上诱导了更高水平的代表Th1免疫反应的IgG2b和IgG2c，如图11(c)和图11(d)所示。(IgG2b+IgG2c)/IgG1的比例显示IL12-Pan-RBD-Fc诱导了比较平衡的Th1和Th2免疫反应，而RBD-Fc，CSF2-Pan-RBD-Fc和aPDL1-Pan-RBD-Fc诱导了偏向Th2的免疫反应，如图11(e)所示。The immunogenicity of the free RBD protein was weak, and its immunogenicity was improved when the Fc part was added on the basis of RBD, and its immunogenicity was improved when the cytokines IL12 and Pan were added on the basis of RBD-Fc. When the cytokines IL21 and Pan were added on the basis of RBD-Fc, its immunogenicity was further improved, and when the cytokines CSF2 and Pan were added on the basis of RBD-Fc, its immunogenicity The immunogenicity has been further improved, and the immunogenicity has been further improved when the αPDL1 antibody and Pan that can bind to the immune cell surface protein PDL1 are added on the basis of RBD-Fc, and the level of antibodies after the second immunization is generally higher than that of Antibody levels after one immunization are shown in Figure 11(a). CSF2 or αPDL1 together with Pan induced a higher level of IgG1 representing Th2 immune response on the basis of RBD-Fc, as shown in Fig. 11(b). IL12 or CSF2 together with Pan induced higher levels of IgG2b and IgG2c representing Th1 immune responses on the basis of RBD-Fc, as shown in Figure 11(c) and Figure 11(d). The ratio of (IgG2b+IgG2c)/IgG1 showed that IL12-Pan-RBD-Fc induced relatively balanced Th1 and Th2 immune responses, while RBD-Fc, CSF2-Pan-RBD-Fc and aPDL1-Pan-RBD-Fc induced Th2-biased immune responses, as shown in Figure 11(e).

实施例4.新冠病毒SARS-CoV-2刺突蛋白RBD相关蛋白疫苗的构建，生产和鉴定Example 4. Construction, production and identification of novel coronavirus SARS-CoV-2 spike protein RBD-related protein vaccine

1、载体的构建1. Construction of the carrier

以PEE6.4为载体，通过分子克隆的方式，将抗原和免疫细胞靶向分子构建到该载体上，从而得到可以表达融合蛋白的质粒。其中KOZAK为科扎克序列，SP(signal peptide)为信号肽。Using PEE6.4 as a vector, antigens and immune cell targeting molecules are constructed on the vector by means of molecular cloning, so as to obtain plasmids that can express fusion proteins. Among them, KOZAK is the Kozak sequence, and SP (signal peptide) is the signal peptide.

(1)CSF2-RBD-Fc：(1) CSF2-RBD-Fc:

PEE6.4-HindIII-KOZAK-Start-SP-BsiwI-CSF2-SfuI-(G4S)3-XbaI-RBD-XhoI-G3-Fc-EcoRIPEE6.4-HindIII-KOZAK-Start-SP-BsiwI-CSF2-SfuI-(G4S)3-XbaI-RBD-XhoI-G3-Fc-EcoRI

(2)αPDL1-RBD-Fc：(2) αPDL1-RBD-Fc:

PEE6.4-HindIII-KOZAK-Start-SP-BsiwI-αPDL1-SfuI-(G4S)3-XbaI-RBD-XhoI-G3-Fc-EcoRI。PEE6.4-HindIII-KOZAK-Start-SP-BsiwI-αPDL1-SfuI-(G4S)3-XbaI-RBD-XhoI-G3-Fc-EcoRI.

2、瞬时转染表达融合蛋白2. Transient transfection to express fusion protein

(1)细胞复苏：Freestyle 293F细胞以3×10 7个细胞/ml的浓度，于Freestyle 293 Expression Medium(10％DMSO)中冻存。从液氮中取出后，在37℃水浴锅中快速溶化，用SMM 293-TII培养基洗过后，将细胞重悬于50ml SMM 293-TII培养基中，37℃，8％CO 2，120rpm。 (1) Cell recovery: Freestyle 293F cells were frozen in Freestyle 293 Expression Medium (10% DMSO) at a concentration of 3×10 7 cells/ml. After taking it out from the liquid nitrogen, dissolve it quickly in a 37°C water bath, wash with SMM 293-TII medium, and resuspend the cells in 50ml of SMM 293-TII medium at 37°C, 8% CO 2 , 120rpm.

(2)用100ml Freestyle 293培养基重新悬浮293F细胞，细胞密度3×10 6cells/ml。 (2) 293F cells were resuspended with 100ml Freestyle 293 medium, and the cell density was 3×10 6 cells/ml.

(3)用2ml Freestyle 293培养基稀释100μg质粒，用2ml Freestyle 293培养基稀释400μg PEI。随后立即将2mL质粒和2mL的PEI混匀，室温静置5分钟。(3) Dilute 100μg plasmid with 2ml Freestyle 293 medium, and dilute 400μg PEI with 2ml Freestyle 293 medium. Immediately thereafter, 2 mL of the plasmid and 2 mL of PEI were mixed and allowed to stand at room temperature for 5 minutes.

(4)将质粒/PEI混合物加入细胞悬液中，放置在37℃，8％CO 2，85rpm培养箱中培养。 (4) The plasmid/PEI mixture was added to the cell suspension, and placed in an incubator at 37° C., 8% CO 2 , 85 rpm.

(5)4小时后，补加100ml EX-CELL293培养基，将转速调至120rpm继续培养。(5) After 4 hours, add 100ml EX-CELL293 medium, and adjust the rotation speed to 120rpm to continue culturing.

(6)24小时后，加入3.8mM细胞增殖抑制剂VPA，转染6天后收集上清液进行纯化。(6) After 24 hours, 3.8 mM cell proliferation inhibitor VPA was added, and the supernatant was collected 6 days after transfection for purification.

3、融合蛋白的纯化3. Purification of fusion protein

(1)将悬浮细胞培养液上清离心，弃沉淀，经0.45μM滤器过滤除去杂质。(1) Centrifuge the supernatant of the suspended cell culture solution, discard the precipitate, and filter through a 0.45 μM filter to remove impurities.

(2)取适量Protein A琼脂糖珠加入层析柱中，用10倍层析柱体积的蒸馏水和PBS分别冲洗和平衡层析柱。(2) Take an appropriate amount of Protein A agarose beads and add them to the chromatography column, wash and equilibrate the chromatography column with distilled water and PBS of 10 times the volume of the chromatography column respectively.

(3)利用恒流泵进行上样，流速为0.2mL/min。(3) A constant flow pump was used to load the sample at a flow rate of 0.2 mL/min.

(4)用10倍层析柱体积以上的PBS冲洗柱子后，用洗脱缓冲液(0.1M甘氨酸,pH 2.7)进行洗脱，洗脱蛋白加入适量的1M Tris，pH 9.0进行中和。(4) After washing the column with PBS more than 10 times the volume of the chromatography column, elution was performed with elution buffer (0.1M glycine, pH 2.7), and the eluted protein was neutralized by adding an appropriate amount of 1M Tris, pH 9.0.

(5)利用浓缩离心柱浓缩蛋白，利用Zeba脱盐离心柱将蛋白溶液置换到所需要的缓冲液中，利用Nano-500测定蛋白浓度，并计算蛋白表达产量。(5) Use concentrated spin column to concentrate protein, use Zeba desalting spin column to replace protein solution into required buffer, use Nano-500 to measure protein concentration, and calculate protein expression yield.

4、融合蛋白的纯度鉴定4. Purity identification of fusion protein

分别用还原性上样缓冲液(R)和非还原性上样缓冲液(N-R)将蛋白上样到聚丙烯酰胺凝胶上，电泳后用考马斯亮蓝对凝胶进行染色，然后用凝胶成像仪成像，查看融合蛋白的完整性和纯度。所述融合蛋白SDS-PAGE电泳鉴定图如图12所示。所述融合蛋白表达产量如图13所示。The proteins were loaded onto polyacrylamide gels with reducing buffer (R) and non-reducing buffer (N-R), respectively. After electrophoresis, the gel was stained with Coomassie brilliant blue, and then the gel was stained with Imager imaging to check the integrity and purity of the fusion protein. The SDS-PAGE electrophoresis identification diagram of the fusion protein is shown in FIG. 12 . The expression yield of the fusion protein is shown in FIG. 13 .

实施例5.免疫细胞靶向新冠病毒SARS-CoV-2刺突蛋白RBD相关蛋白疫苗相较于单纯的RBD蛋白疫苗在小鼠体内可以引起更强的抗体反应Example 5. Immune cells targeting the new coronavirus SARS-CoV-2 spike protein RBD-related protein vaccine can cause a stronger antibody response in mice than a simple RBD protein vaccine

1、材料1. Materials

C57BL/6雄性小鼠(6-8)周购买于江苏集萃药康生物科技股份有限公司；辣根过氧化物氧化酶(HRP)标记的山羊抗小鼠IgG购自江苏康为世纪生物科技股份有限公司；96孔ELISA测定板购自Bioland公司；ELISA显色液购自上海碧云天生物技术有限公司；ELISA终止液购自北京索莱宝科技有限公司；酶标仪Multiskan FC购自Thermo Fisher Scientific公司。C57BL/6 male mice (6-8) weeks were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.; horseradish peroxidase (HRP)-labeled goat anti-mouse IgG was purchased from Jiangsu Kangwei Century Biotechnology Co., Ltd. Co., Ltd.; 96-well ELISA assay plate was purchased from Bioland; ELISA chromogenic solution was purchased from Shanghai Biyuntian Biotechnology Co., Ltd.; ELISA stop solution was purchased from Beijing Solaibao Technology Co., Ltd.; Microplate reader Multiskan FC was purchased from Thermo Fisher Scientific company.

2、方法2. Method

(1)免疫细胞靶向融合蛋白疫苗免疫小鼠。将融合蛋白疫苗在PBS中稀释后， 每只小鼠接种0.5μg RBD蛋白或者相同摩尔数的其它融合蛋白，每只小鼠肌肉注射50μl。采用两次免疫程序在第0天与21天免疫小鼠。每次免疫后第14天通过脸颊取血的方式收集小鼠血清，进行抗体检测。(1) Mice were immunized with immune cell-targeted fusion protein vaccine. After diluting the fusion protein vaccine in PBS, each mouse was inoculated with 0.5 μg of RBD protein or other fusion proteins of the same molar number, and each mouse was injected intramuscularly with 50 μl. Mice were immunized on day 0 and day 21 using two immunization procedures. On the 14th day after each immunization, mouse serum was collected by cheek blood collection for antibody detection.

(2)ELISA检测血清中RBD特异性抗体。将RBD(2μg/ml)包被液以每孔100μl的体系加入到Elisa板中，4℃包被过夜。5％的封闭液(5％FBS的PBS)37℃封闭1小时。PBST洗3次，血清样品按照10倍梯度稀释，每孔加100μl到封闭好的ELISA板中，37℃孵育1小时。PBST洗3次，每孔加入100μl酶标二抗(1:5000)，37℃孵育1小时。用PBST洗5次，加底物TMB 100μl/孔，室温避光孵育，等待底物显色；每孔加50μl ELISA终止液终止显色，酶标仪读板，OD450-620。(2) ELISA detection of RBD-specific antibodies in serum. RBD (2 μg/ml) coating solution was added to the Elisa plate at 100 μl per well, and coated overnight at 4°C. 5% blocking solution (5% FBS in PBS) was used to block for 1 hour at 37°C. Wash 3 times with PBST, serum samples were diluted according to 10-fold gradient, and 100 μl was added to each well of a well-blocked ELISA plate, and incubated at 37°C for 1 hour. Wash 3 times with PBST, add 100 μl enzyme-labeled secondary antibody (1:5000) to each well, and incubate at 37°C for 1 hour. Wash 5 times with PBST, add substrate TMB 100 μl/well, incubate at room temperature in the dark, wait for substrate color development; add 50 μl ELISA stop solution to each well to stop color development, read the plate with a microplate reader, OD450-620.

3、结果3. Results

游离的RBD蛋白免疫原性较弱，当在RBD的基础上加上Fc部分时可提高抗体滴度，当在RBD-Fc的基础上加上细胞因子CSF2时可进一步提高抗体滴度，当在RBD-Fc的基础上加上能够结合免疫细胞表面蛋白PDL1的αPDL1抗体时也可进一步提高抗体滴度，如图14所示。这些结果说明CSF2-RBD-Fc的免疫原性高于RBD和RBD-Fc，αPDL1-RBD-Fc的免疫原性高于RBD和RBD-Fc。The immunogenicity of free RBD protein is weak, and the antibody titer can be increased when the Fc part is added on the basis of RBD, and the antibody titer can be further improved when the cytokine CSF2 is added on the basis of RBD-Fc. Adding the αPDL1 antibody capable of binding to the immune cell surface protein PDL1 on the basis of RBD-Fc can also further increase the antibody titer, as shown in FIG. 14 . These results indicated that the immunogenicity of CSF2-RBD-Fc was higher than that of RBD and RBD-Fc, and that of αPDL1-RBD-Fc was higher than that of RBD and RBD-Fc.

实施例6.EB病毒(Epstein-Barr virus，EBV)膜蛋白gp350相关蛋白疫苗的构建，生产和鉴定The construction of embodiment 6.Epstein-Barr virus (Epstein-Barr virus, EBV) membrane protein gp350-related protein vaccine, production and identification

EBV也称为人类疱疹病毒4，是疱疹家族中已知的九种人类疱疹病毒类型之一，也是人类最常见的病毒之一。EBV最出名的是传染性单核细胞增多症的原因，EBV还与各种非恶性、癌前病变和恶性EB病毒相关的淋巴组织增生性疾病有关，全球每年约有200,000例癌症病例被认为可归因于EBV。EBV, also known as human herpesvirus 4, is one of nine known types of human herpesviruses in the herpes family and one of the most common viruses in humans. Best known as the cause of infectious mononucleosis, EBV is also associated with a variety of non-malignant, precancerous, and malignant EBV-associated lymphoproliferative disorders, with approximately 200,000 cancer cases worldwide each year considered probable Attributed to EBV.

1、载体的构建1. Construction of the carrier

以PEE6.4为载体，通过分子克隆的方式，将抗原和免疫细胞靶向分子构建到该载体上，从而得到可以表达融合蛋白的质粒。其中KOZAK为科扎克序列，SP(signal peptide)为信号肽。Using PEE6.4 as a vector, antigens and immune cell targeting molecules are constructed on the vector by means of molecular cloning, so as to obtain plasmids that can express fusion proteins. Among them, KOZAK is the Kozak sequence, and SP (signal peptide) is the signal peptide.

(1)gp350：(1) gp350:

PEE6.4-HindIII-KOZAK-Start-SP-XbaI-gp350-XhoI-6His-EcoRIPEE6.4-HindIII-KOZAK-Start-SP-XbaI-gp350-XhoI-6His-EcoRI

(2)gp350-Fc：(2) gp350-Fc:

PEE6.4-HindIII-KOZAK-Start-SP-BsiwI-gp350-XhoI-G 3-Fc-EcoRI PEE6.4-HindIII-KOZAK-Start-SP-BsiwI-gp350-XhoI- G3 -Fc-EcoRI

(3)CSF2-gp350-Fc：(3) CSF2-gp350-Fc:

PEE6.4-HindIII-KOZAK-Start-SP-BsiwI-CSF2-SfuI-(G 4S) 3-XbaI-gp350-XhoI-G 3-Fc-EcoRI PEE6.4-HindIII-KOZAK-Start-SP-BsiwI-CSF2-SfuI-(G 4 S) 3 -XbaI-gp350-XhoI-G 3 -Fc-EcoRI

(4)αPDL1-gp350-Fc：(4) αPDL1-gp350-Fc:

PEE6.4-HindIII-KOZAK-Start-SP-BsiwI-αPDL1-SfuI-(G 4S) 3-XbaI-gp350-XhoI-G 3-Fc-EcoRI PEE6.4-HindIII-KOZAK-Start-SP-BsiwI-αPDL1-SfuI-(G 4 S) 3 -XbaI-gp350-XhoI-G 3 -Fc-EcoRI

(5)CSF2-Pan-gp350-Fc：(5) CSF2-Pan-gp350-Fc:

PEE6.4-HindIII-KOZAK-Start-SP-BsiwI-CSF2-SfuI-(G 4S) 3-Pan-(GS) 3-XbaI-gp350-XhoI-G 3-Fc-EcoRI PEE6.4-HindIII-KOZAK-Start-SP-BsiwI-CSF2-SfuI-(G 4 S) 3 -Pan-(GS) 3 -XbaI-gp350-XhoI-G 3 -Fc-EcoRI

(6)αPDL1-Pan-gp350-Fc：(6) αPDL1-Pan-gp350-Fc:

PEE6.4-HindIII-KOZAK-Start-SP-BsiwI-αPDL1-SfuI-(G 4S) 3-Pan-(GS) 3-XbaI-gp350-XhoI-G 3-Fc-EcoRI PEE6.4-HindIII-KOZAK-Start-SP-BsiwI-αPDL1-SfuI-(G 4 S) 3 -Pan-(GS) 3 -XbaI-gp350-XhoI-G 3 -Fc-EcoRI

2、瞬时转染表达融合蛋白2. Transient transfection to express fusion protein

(1)细胞复苏：Freestyle 293F细胞以3×10 7个细胞/ml的浓度，于Freestyle 293 Expression Medium(10％DMSO)中冻存。从液氮中取出后，在37℃水浴锅中快速溶化，用SMM 293-TII培养基洗过后，将细胞重悬于50ml SMM 293-TII培养基中，37℃，8％CO 2，120rpm。 (1) Cell recovery: Freestyle 293F cells were frozen in Freestyle 293 Expression Medium (10% DMSO) at a concentration of 3×10 7 cells/ml. After taking it out from the liquid nitrogen, dissolve it quickly in a 37°C water bath, wash with SMM 293-TII medium, and resuspend the cells in 50ml of SMM 293-TII medium at 37°C, 8% CO 2 , 120rpm.

(2)用100ml Freestyle 293培养基重新悬浮293F细胞，细胞密度3×10 6cells/ml。 (2) 293F cells were resuspended with 100ml Freestyle 293 medium, and the cell density was 3×10 6 cells/ml.

(3)用2ml Freestyle 293培养基稀释100μg质粒，用2ml Freestyle 293培养基稀释400μg PEI。随后立即将2mL质粒和2mL的PEI混匀，室温静置5分钟。(3) Dilute 100μg plasmid with 2ml Freestyle 293 medium, and dilute 400μg PEI with 2ml Freestyle 293 medium. Immediately thereafter, 2 mL of the plasmid and 2 mL of PEI were mixed and allowed to stand at room temperature for 5 minutes.

(4)将质粒/PEI混合物加入细胞悬液中，放置在37℃，8％CO 2，85rpm培养箱中培养。 (4) The plasmid/PEI mixture was added to the cell suspension, and placed in an incubator at 37° C., 8% CO 2 , 85 rpm.

(5)4小时后，补加100ml EX-CELL293培养基，将转速调至120rpm继续培养。(5) After 4 hours, add 100ml EX-CELL293 medium, and adjust the rotation speed to 120rpm to continue culturing.

(6)24小时后，加入3.8mM细胞增殖抑制剂VPA，转染6天后收集上清液进行纯化。(6) After 24 hours, 3.8 mM cell proliferation inhibitor VPA was added, and the supernatant was collected 6 days after transfection for purification.

3、融合蛋白的纯化3. Purification of fusion protein

(1)将悬浮细胞培养液上清离心，弃沉淀，经0.45μM滤器过滤除去杂质。(1) Centrifuge the supernatant of the suspended cell culture solution, discard the precipitate, and filter through a 0.45 μM filter to remove impurities.

(2)取适量Protein A琼脂糖珠加入层析柱中，用10倍层析柱体积的蒸馏水和PBS分别冲洗和平衡层析柱。(2) Take an appropriate amount of Protein A agarose beads and add them to the chromatography column, wash and equilibrate the chromatography column with distilled water and PBS of 10 times the volume of the chromatography column respectively.

(3)利用恒流泵进行上样，流速为0.2mL/min。(3) A constant flow pump was used to load the sample at a flow rate of 0.2 mL/min.

(4)用10倍层析柱体积以上的PBS冲洗柱子后，用洗脱缓冲液(0.1M甘氨酸,pH 2.7)进行洗脱，洗脱蛋白加入适量的1M Tris，pH 9.0进行中和。(4) After washing the column with PBS more than 10 times the volume of the chromatography column, elution was performed with elution buffer (0.1M glycine, pH 2.7), and the eluted protein was neutralized by adding an appropriate amount of 1M Tris, pH 9.0.

(5)利用浓缩离心柱浓缩蛋白，利用Zeba脱盐离心柱将蛋白溶液置换到所需要的缓冲液中，利用Nano-500测定蛋白浓度，并计算蛋白表达产量。(5) Use concentrated spin column to concentrate protein, use Zeba desalting spin column to replace protein solution into required buffer, use Nano-500 to measure protein concentration, and calculate protein expression yield.

4、融合蛋白的纯度鉴定4. Purity identification of fusion protein

分别用还原性上样缓冲液(R)和非还原性上样缓冲液(N-R)将蛋白上样到聚丙烯酰胺凝胶上，电泳后用考马斯亮蓝对凝胶进行染色，然后用凝胶成像仪成像，查看融合蛋白的完整性和纯度。所述融合蛋白SDS-PAGE电泳鉴定图如图15所示。所述融合蛋白表达产量如图16所示。The proteins were loaded onto polyacrylamide gels with reducing buffer (R) and non-reducing buffer (N-R), respectively. After electrophoresis, the gel was stained with Coomassie brilliant blue, and then the gel was stained with Imager imaging to check the integrity and purity of the fusion protein. The SDS-PAGE electrophoresis identification diagram of the fusion protein is shown in FIG. 15 . The expression yield of the fusion protein is shown in FIG. 16 .

实施例7.免疫细胞靶向EBV膜蛋白gp350相关蛋白疫苗相较于单纯的gp350蛋白疫苗在小鼠体内可以引起更强的抗体反应Example 7. Immune cells targeting EBV membrane protein gp350-related protein vaccines can elicit stronger antibody responses in mice than pure gp350 protein vaccines

1、材料1. Materials

C57BL/6雄性小鼠(6-8)周购买于江苏集萃药康生物科技股份有限公司；辣根过氧化物氧化酶(HRP)标记的山羊抗小鼠IgG购自江苏康为世纪生物科技股份有限公司；96孔ELISA测定板购自Bioland公司；ELISA显色液购自上海碧云天生物技术有限公司；ELISA终止液购自北京索莱宝科技有限公司；酶标仪Multiskan FC购自Thermo Fisher Scientific公司。C57BL/6 male mice (6-8) weeks were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.; horseradish peroxidase (HRP)-labeled goat anti-mouse IgG was purchased from Jiangsu Kangwei Century Biotechnology Co., Ltd. Co., Ltd.; 96-well ELISA assay plate was purchased from Bioland; ELISA chromogenic solution was purchased from Shanghai Biyuntian Biotechnology Co., Ltd.; ELISA stop solution was purchased from Beijing Solaibao Technology Co., Ltd.; Microplate reader Multiskan FC was purchased from Thermo Fisher Scientific company.

2、方法2. Method

(1)免疫细胞靶向融合蛋白疫苗免疫小鼠。将融合蛋白疫苗在PBS中稀释后，每只小鼠接种0.5μg gp350蛋白或者相同摩尔数的其它融合蛋白，每只小鼠肌肉注射50μl。采用两次免疫程序在第0天与21天免疫小鼠。每次免疫后第14天通过脸颊取血的方式收集小鼠血清，进行抗体检测。(1) Mice were immunized with immune cell-targeted fusion protein vaccine. After diluting the fusion protein vaccine in PBS, each mouse was inoculated with 0.5 μg gp350 protein or other fusion proteins of the same molar amount, and each mouse was injected intramuscularly with 50 μl. Mice were immunized on day 0 and day 21 using two immunization procedures. On the 14th day after each immunization, mouse serum was collected by cheek blood collection for antibody detection.

(2)ELISA检测血清中gp350特异性抗体。将gp350(2μg/ml)包被液以每孔100μl的体系加入到Elisa板中，4℃包被过夜。5％的封闭液(5％FBS的PBS)37℃封闭1小时。PBST洗3次，血清样品按照10倍梯度稀释，每孔加100μl到封闭好的ELISA板中，37℃孵育1小时。PBST洗3次，每孔加入100μl酶标二抗(1:5000)，37℃孵育1小时。用PBST洗5次，加底物TMB 100μl/孔，室温避光孵育，等待底物显色；每孔加50μl ELISA终止液终止显色，酶标仪读板， OD450-620。(2) Detection of gp350-specific antibody in serum by ELISA. The gp350 (2 μg/ml) coating solution was added to the Elisa plate at 100 μl per well, and coated overnight at 4°C. 5% blocking solution (5% FBS in PBS) was used to block for 1 hour at 37°C. Wash 3 times with PBST, serum samples were diluted according to 10-fold gradient, and 100 μl was added to each well of a well-blocked ELISA plate, and incubated at 37°C for 1 hour. Wash 3 times with PBST, add 100 μl enzyme-labeled secondary antibody (1:5000) to each well, and incubate at 37°C for 1 hour. Wash 5 times with PBST, add substrate TMB 100 μl/well, incubate at room temperature in the dark, wait for substrate color development; add 50 μl ELISA stop solution to each well to stop color development, read the plate with a microplate reader, OD450-620.

3、结果3. Results

游离的gp350蛋白疫苗免疫原性较弱，当在gp350的基础上加上Fc部分时没有提高抗体滴度，当在gp350-Fc的基础上加上细胞因子CSF2时可提高抗体滴度，当在gp350-Fc的基础上加上能够结合免疫细胞表面蛋白PDL1的αPDL1抗体时可提高抗体滴度，当在gp350-Fc的基础上加上细胞因子CSF2和Pan时可提高抗体滴度，当在gp350-Fc的基础上加上能够结合免疫细胞表面蛋白PDL1的αPDL1抗体和Pan时可提高抗体滴度，如图17所示。这些结果说明CSF2-gp350-Fc的免疫原性高于gp350和gp350-Fc，αPDL1-gp350-Fc的免疫原性高于gp350和gp350-Fc，CSF2-Pan-gp350-Fc的免疫原性高于gp350和gp350-Fc，αPDL1-Pan-gp350-Fc的免疫原性高于gp350和gp350-Fc。The immunogenicity of the free gp350 protein vaccine is weak. When the Fc part is added on the basis of gp350, the antibody titer is not improved. When the cytokine CSF2 is added on the basis of gp350-Fc, the antibody titer can be improved. On the basis of gp350-Fc, the antibody titer can be increased when the αPDL1 antibody that can bind to the immune cell surface protein PDL1 is added. When the cytokines CSF2 and Pan are added on the basis of gp350-Fc, the antibody titer can be increased. When gp350 The addition of αPDL1 antibody capable of binding to the immune cell surface protein PDL1 and Pan on the basis of -Fc can increase the antibody titer, as shown in Figure 17. These results indicate that the immunogenicity of CSF2-gp350-Fc is higher than that of gp350 and gp350-Fc, the immunogenicity of αPDL1-gp350-Fc is higher than that of gp350 and gp350-Fc, and the immunogenicity of CSF2-Pan-gp350-Fc is higher than that of The immunogenicity of gp350 and gp350-Fc, αPDL1-Pan-gp350-Fc was higher than that of gp350 and gp350-Fc.

Claims (16)

含有抗原结构域和免疫细胞靶向结构域的融合蛋白，所述免疫细胞靶向结构域为选自如下的一种或多种结构域：A fusion protein containing an antigen domain and an immune cell targeting domain, wherein the immune cell targeting domain is one or more domains selected from the following: 结构域A：能够结合免疫细胞表面蛋白的抗体或多肽或其活性片段；Domain A: an antibody or polypeptide or an active fragment thereof capable of binding to an immune cell surface protein; 结构域B：能够激活免疫细胞的细胞因子或其活性片段；Domain B: cytokines or their active fragments capable of activating immune cells; 结构域C：能够激活免疫细胞的Pan表位(PADRE)或其活性片段；Domain C: Pan epitope (PADRE) or its active fragments capable of activating immune cells; 结构域D：能够结合免疫细胞的免疫球蛋白Fc。Domain D: Immunoglobulin Fc capable of binding immune cells. 根据权利要求1所述的融合蛋白，所述融合蛋白包含抗原结构域和结构域A；抗原结构域与结构域A和结构域B的融合蛋白；抗原结构域与结构域A，结构域B和结构域C的融合蛋白；抗原结构域与结构域A，结构域B，结构域C和结构域D的融合蛋白；抗原结构域与结构域A和结构域C的融合蛋白；抗原结构域与结构域A和结构域D的融合蛋白；抗原结构域与结构域A，结构域C和结构域D的融合蛋白；抗原结构域与结构域B的融合蛋白；抗原结构域与结构域B和结构域C的融合蛋白；抗原结构域与结构域B和结构域D的融合蛋白；抗原结构域与结构域B，结构域C和结构域D的融合蛋白；抗原结构域与结构域C的融合蛋白；抗原结构域与结构域C和结构域D的融合蛋白；抗原结构域与结构域C的融合蛋白；抗原结构域与结构域C和结构域D的融合蛋白；或抗原结构域与结构域D的融合蛋白；The fusion protein according to claim 1, said fusion protein comprising antigen domain and domain A; fusion protein of antigen domain and domain A and domain B; antigen domain and domain A, domain B and The fusion protein of structural domain C; the fusion protein of antigenic domain and structural domain A, structural domain B, structural domain C and structural domain D; the fusion protein of antigenic domain and structural domain A and structural domain C; antigenic domain and structural Fusion protein of domain A and domain D; fusion protein of antigenic domain with domain A, domain C and domain D; fusion protein of antigenic domain with domain B; antigenic domain with domain B and domain A fusion protein of C; a fusion protein of an antigen domain and domain B and domain D; a fusion protein of an antigen domain and domain B, domain C and domain D; a fusion protein of an antigen domain and domain C; A fusion protein of an antigenic domain with domain C and domain D; a fusion protein of an antigenic domain with domain C; a fusion protein of an antigenic domain with domain C and domain D; or a fusion protein of an antigenic domain with domain D fusion protein; 所述结构域A选自针对CD274(PDL1)，PDCD1LG2(PDL2)，CLEC9A，LY75(DEC205)，CD40，TNFSF9(4-1BB-L)和/或TNFSF4(OX4OL)的抗体，或其配体的活性片段；The domain A is selected from antibodies against CD274 (PDL1), PDCD1LG2 (PDL2), CLEC9A, LY75 (DEC205), CD40, TNFSF9 (4-1BB-L) and/or TNFSF4 (OX4OL), or ligands thereof active fragment; 所述结构域B选自白介素(interleukin，IL)，集落刺激因子(Colony-stimulating factor，CSF)，或其活性片段，优选地，所述白介素选自IL2，IL12，IL15和/或IL21，或其活性片段；优选地，所述集落刺激因子选自CSF1，CSF2和/或CSF3，或其活性片段；The domain B is selected from interleukin (interleukin, IL), colony-stimulating factor (Colony-stimulating factor, CSF), or its active fragment, preferably, the interleukin is selected from IL2, IL12, IL15 and/or IL21, or an active fragment thereof; preferably, the colony-stimulating factor is selected from CSF1, CSF2 and/or CSF3, or an active fragment thereof; 优选地，所述结构域C具有AKFVAAWTLKAAA所示的氨基酸序列；Preferably, the domain C has the amino acid sequence shown as AKFVAAWTLKAAA; 优选地，所述结构域D选自IgG、IgM、IgA、IgE或IgD的Fc或其突变体；Preferably, the domain D is selected from Fc of IgG, IgM, IgA, IgE or IgD or mutants thereof; 优选地，所述Fc是经修饰的Fc，更优选地，所述Fc具有Fc knob修饰和Fc Hole修饰；Preferably, the Fc is a modified Fc, more preferably, the Fc has Fc knob modification and Fc Hole modification; 优选地，所述融合蛋白是抗原结构域与结构域A和/或结构域B和/或结构域C 和/或结构域D的任意排列方式；或者，Preferably, the fusion protein is any arrangement of antigenic domains and domain A and/or domain B and/or domain C and/or domain D; or, 所述融合蛋白中抗原结构域和/或结构域A和/或结构域B和/或结构域C和/或结构域D相对于彼此位于C端或者N端。In the fusion protein, the antigen domain and/or domain A and/or domain B and/or domain C and/or domain D are located at the C-terminal or N-terminal relative to each other. 根据权利要求1-2任一项所述的融合蛋白，所述融合蛋白是同源二聚体或异源二聚体；优选地，所述融合蛋白包含抗原结构域和结构域D，所述融合蛋白构成类似抗体形式的同源二聚体或异源二聚体；According to the fusion protein according to any one of claims 1-2, the fusion protein is a homodimer or a heterodimer; preferably, the fusion protein comprises an antigen domain and a domain D, the The fusion protein constitutes a homodimer or heterodimer similar to the form of an antibody; 优选地，所述融合蛋白由两个Fc结构域的二硫键构成同源二聚体或异源二聚体；Preferably, the fusion protein consists of a homodimer or a heterodimer formed by disulfide bonds of two Fc domains; 优选地，所述融合蛋白包括第一多肽链和第二多肽链，所述第一多肽链包括抗原结构域和结构域D，所述第二多肽链包括抗原结构域和结构域D；或者Preferably, the fusion protein includes a first polypeptide chain and a second polypeptide chain, the first polypeptide chain includes an antigenic domain and domain D, and the second polypeptide chain includes an antigenic domain and a domain D; or 优选地，所述第一多肽链还包括结构域B和/或结构域C，所述第二多肽链还包括结构域B和/或结构域C；或者，Preferably, the first polypeptide chain further comprises domain B and/or domain C, and the second polypeptide chain further comprises domain B and/or domain C; or, 优选地，所述第一多肽链还包括结构域B，所述第二多肽链还包括结构域C，从而构成异源二聚体；或者Preferably, said first polypeptide chain further comprises domain B and said second polypeptide chain further comprises domain C, thereby forming a heterodimer; or 所述第一多肽链从C端至N端包括结构域D，抗原结构域，结构域A，所述第二多肽链从C端至N端包括结构域D，抗原结构域，结构域A，从而构成同源二聚体，反之亦然；或者The first polypeptide chain includes domain D, antigen domain, and domain A from C-terminus to N-terminus, and the second polypeptide chain includes domain D, antigen domain, and domain A from C-terminus to N-terminus A, thereby forming a homodimer and vice versa; or 所述第一多肽链从C端至N端包括结构域D，抗原结构域，结构域B，所述第二多肽链从C端至N端包括结构域D，抗原结构域，结构域B，从而构成同源二聚体，反之亦然；或者The first polypeptide chain includes domain D, antigen domain, domain B from C-terminus to N-terminus, and the second polypeptide chain includes domain D, antigen domain, domain B from C-terminus to N-terminus B, thereby forming a homodimer, or vice versa; or 所述第一多肽链从C端至N端包括结构域D，抗原结构域，结构域C，结构域A，所述第二多肽链从C端至N端包括结构域D，抗原结构域，结构域C，结构域A，从而构成同源二聚体，反之亦然；或者The first polypeptide chain includes domain D from C-terminus to N-terminus, antigen domain, domain C, domain A, and the second polypeptide chain includes domain D from C-terminus to N-terminus, antigen structure domain, domain C, domain A, thereby constituting a homodimer, or vice versa; or 所述第一多肽链从C端至N端包括结构域D，抗原结构域，结构域C，结构域B，所述第二多肽链从C端至N端包括结构域D，抗原结构域，结构域C，结构域B，从而构成同源二聚体，反之亦然；或者The first polypeptide chain includes domain D from C-terminus to N-terminus, antigen domain, domain C, domain B, and the second polypeptide chain includes domain D from C-terminus to N-terminus, antigen structure domain, domain C, domain B, thereby constituting a homodimer, or vice versa; or 所述第一多肽链从C端至N端包括结构域B，结构域D，抗原结构域，结构域A，所述第二多肽链从C端至N端包括结构域B，结构域D，抗原结构域，结构域A，从而构成同源二聚体；或者The first polypeptide chain includes domain B, domain D, antigen domain, and domain A from C-terminus to N-terminus, and the second polypeptide chain includes domain B, domain A from C-terminus to N-terminus D, the antigenic domain, domain A, thereby constituting a homodimer; or 所述第一多肽链从C端至N端包括结构域B，结构域D，抗原结构域，结构域C，结构域A，所述第二多肽链从C端至N端包括结构域B，结构域D，抗原结构域，结构域C，结构域A，从而构成同源二聚体，反之亦然；或者The first polypeptide chain includes domain B, domain D, antigen domain, domain C, and domain A from C-terminus to N-terminus, and the second polypeptide chain includes domain A from C-terminus to N-terminus B, domain D, antigenic domain, domain C, domain A, thereby constituting a homodimer, or vice versa; or 所述第一多肽链从C端至N端包括结构域D，抗原结构域，结构域A，所述第二多肽链从C端至N端包括结构域D，抗原结构域，结构域C，结构域B，从而构成异源二聚体，反之亦然；或者The first polypeptide chain includes domain D, antigen domain, and domain A from C-terminus to N-terminus, and the second polypeptide chain includes domain D, antigen domain, and domain A from C-terminus to N-terminus C, domain B, thereby constituting a heterodimer and vice versa; or 所述第一多肽链从C端至N端包括结构域D，抗原结构域，结构域C，结构域A，所述第二多肽链从C端至N端包括结构域D，抗原结构域，结构域C，结构域B，从而构成异源二聚体，反之亦然；The first polypeptide chain includes domain D from C-terminus to N-terminus, antigen domain, domain C, domain A, and the second polypeptide chain includes domain D from C-terminus to N-terminus, antigen structure domain, domain C, domain B, thereby forming a heterodimer, and vice versa; 优选地，所述抗原结构域与所述免疫细胞靶向结构域A，结构域B，结构域C，结构域D可以任意形式排列组合。Preferably, the antigen domain and the immune cell targeting domain A, domain B, domain C and domain D can be arranged and combined in any form. 根据权利要求1-3任一项所述的融合蛋白，所述融合蛋白抗原与免疫细胞靶向分子之间，和/或免疫细胞靶向分子之间通过连接片段进行连接；According to the fusion protein according to any one of claims 1-3, the fusion protein antigen is connected to the immune cell targeting molecule, and/or the immune cell targeting molecule is connected through a linking fragment; 优选地，所述连接片段是柔性连接片段、刚性连接片段或体内剪切连接片段，更优选地，所述柔性连接片段的氨基酸序列是(G) N，(GS) N，(GGS) N，(GGGS) N，或(GGGGS) N。 Preferably, the connecting fragment is a flexible connecting fragment, a rigid connecting fragment or an in vivo shear connecting fragment, more preferably, the amino acid sequence of the flexible connecting fragment is (G) N , (GS) N , (GGS) N , (GGGS) N , or (GGGGS) N . 根据权利要求1-4任一项所述的融合蛋白，所述融合蛋白具有选自如下的结构：The fusion protein according to any one of claims 1-4, which has a structure selected from the group consisting of: αPDL1-Antigen-Fc；αPDL1-Antigen-Fc; CLEC9A binding peptide-Antigen-Fc；CLEC9A binding peptide-Antigen-Fc; αDEC205-Antigen-Fc；αDEC205-Antigen-Fc; Antigen-Fc-CLEC9A binding peptide；Antigen-Fc-CLEC9A binding peptide; αPDL1-linker-Antigen-linker-Fc；αPDL1-linker-Antigen-linker-Fc; αPDL1-(GGGGS) 3-Antigen-(G) 3-Fc； αPDL1-(GGGGS) 3 -Antigen-(G) 3 -Fc; CLEC9A binding peptide-linker-Antigen-linker-Fc；CLEC9A binding peptide-linker-Antigen-linker-Fc; CLEC9A binding peptide-(GGGGS) 3-Antigen-(G) 3-Fc； CLEC9A binding peptide-(GGGGS) 3 -Antigen-(G) 3 -Fc; αDEC205-linker-Antigen-linker-Fc；αDEC205-linker-Antigen-linker-Fc; αDEC205-(GGGGS) 3-Antigen-(G) 3-Fc； αDEC205-(GGGGS) 3 -Antigen-(G) 3 -Fc; Antigen-linker-Fc-linker-CLEC9A binding peptide；Antigen-linker-Fc-linker-CLEC9A binding peptide; Antigen-(G) 3-Fc-(GS) 3-CLEC9A binding peptide； Antigen-(G) 3 -Fc-(GS) 3 -CLEC9A binding peptide; IL2-Antigen-Fc；IL2-Antigen-Fc; IL12-Antigen-Fc；IL12-Antigen-Fc; IL15-Antigen-Fc；IL15-Antigen-Fc; IL21-Antigen-Fc；IL21-Antigen-Fc; CSF2-Antigen-Fc；CSF2-Antigen-Fc; Antigen-Fc-CSF2；Antigen-Fc-CSF2; IL2-linker-Antigen-linker-Fc；IL2-linker-Antigen-linker-Fc; IL2-(GGGGS) 3-Antigen-(G) 3-Fc； IL2-(GGGGS) 3 -Antigen-(G) 3 -Fc; IL12-linker-Antigen-linker-Fc；IL12-linker-Antigen-linker-Fc; IL12-(GGGGS) 3-Antigen-(G) 3-Fc； IL12-(GGGGS) 3 -Antigen-(G) 3 -Fc; IL15-linker-Antigen-linker-Fc；IL15-linker-Antigen-linker-Fc; IL15-(GGGGS) 3-Antigen-(G) 3-Fc； IL15-(GGGGS) 3 -Antigen-(G) 3 -Fc; IL21-linker-Antigen-linker-Fc；IL21-linker-Antigen-linker-Fc; IL21-(GGGGS) 3-Antigen-(G) 3-Fc； IL21-(GGGGS) 3 -Antigen-(G) 3 -Fc; CSF2-linker-Antigen-linker-Fc；CSF2-linker-Antigen-linker-Fc; CSF2-(GGGGS) 3-Antigen-(G) 3-Fc； CSF2-(GGGGS) 3 -Antigen-(G) 3 -Fc; Antigen-linker-Fc-linker-CSF2；Antigen-linker-Fc-linker-CSF2; Antigen-(GGGGS) 3-Fc-(G) 3-CSF2； Antigen-(GGGGS) 3 -Fc-(G) 3 -CSF2; αPDL1-Pan-Antigen-Fc；αPDL1-Pan-Antigen-Fc; CLEC9A binding peptide-Pan-Antigen-Fc；CLEC9A binding peptide-Pan-Antigen-Fc; αDEC205-Pan-Antigen-Fc；αDEC205-Pan-Antigen-Fc; αPDL1-linker-Pan-Antigen-linker-Fc；αPDL1-linker-Pan-Antigen-linker-Fc; αPDL1-(GGGGS) 3-Pan-Antigen-(G) 3-Fc； αPDL1-(GGGGS) 3 -Pan-Antigen-(G) 3 -Fc; CLEC9A binding peptide-linker-Pan-Antigen-linker-Fc；CLEC9A binding peptide-linker-Pan-Antigen-linker-Fc; CLEC9A binding peptide-(GGGGS) 3-Pan-Antigen-(G) 3-Fc； CLEC9A binding peptide-(GGGGS) 3 -Pan-Antigen-(G) 3 -Fc; αDEC205-linker-Pan-Antigen-linker-Fc；αDEC205-linker-Pan-Antigen-linker-Fc; αDEC205-(GGGGS) 3-Pan-Antigen-(G) 3-Fc；IL2-Pan-Antigen-Fc； αDEC205-(GGGGS) 3 -Pan-Antigen-(G) 3 -Fc; IL2-Pan-Antigen-Fc; IL12-Pan-Antigen-Fc；IL12-Pan-Antigen-Fc; IL15-Pan-Antigen-Fc；IL15-Pan-Antigen-Fc; IL21-Pan-Antigen-Fc；IL21-Pan-Antigen-Fc; CSF2-Pan-Antigen-Fc；CSF2-Pan-Antigen-Fc; Pan-Antigen-Fc-CSF2；Pan-Antigen-Fc-CSF2; IL2-linker-Pan-linker-Antigen-(G) 3-Fc； IL2-linker-Pan-linker-Antigen-(G) 3 -Fc; IL2-(GGGGS) 3-Pan-(GS) 3-Antigen-linker-Fc； IL2-(GGGGS) 3 -Pan-(GS) 3 -Antigen-linker-Fc; IL12-linker-Pan-linker-Antigen-linker-Fc；IL12-linker-Pan-linker-Antigen-linker-Fc; IL12-(GGGGS) 3-Pan-(GS) 3-Antigen-(G) 3-Fc； IL12-(GGGGS) 3 -Pan-(GS) 3 -Antigen-(G) 3 -Fc; IL15-linker-Pan-linker-Antigen-linker-Fc；IL15-linker-Pan-linker-Antigen-linker-Fc; IL15-(GGGGS) 3-Pan-(GS) 3-Antigen-(G) 3-Fc； IL15-(GGGGS) 3 -Pan-(GS) 3 -Antigen-(G) 3 -Fc; IL21-linker-Pan-linker-Antigen-linker-Fc；IL21-linker-Pan-linker-Antigen-linker-Fc; IL21-(GGGGS) 3-Pan-(GS) 3-Antigen-(G) 3-Fc； IL21-(GGGGS) 3 -Pan-(GS) 3 -Antigen-(G) 3 -Fc; CSF2-linker-Pan-linker-Antigen-linker-Fc；CSF2-linker-Pan-linker-Antigen-linker-Fc; CSF2-(GGGGS) 3-Pan-(GS) 3-Antigen-(G) 3-Fc； CSF2-(GGGGS) 3 -Pan-(GS) 3- Antigen-(G) 3 -Fc; Pan-linker-Antigen-linker-Fc-linker-CSF2；Pan-linker-Antigen-linker-Fc-linker-CSF2; Pan-(GGGGS) 3-Antigen-(G) 3-Fc-(GS) 3-CSF2； Pan-(GGGGS) 3 -Antigen-(G) 3 -Fc-(GS) 3 -CSF2; αPDL1-Antigen-Fc-IL12；αPDL1-Antigen-Fc-IL12; αPDL1-Antigen-Fc-IL21；αPDL1-Antigen-Fc-IL21; αPDL1-Antigen-Fc-CSF2；αPDL1-Antigen-Fc-CSF2; CLEC9A binding peptide-Antigen-Fc-IL12；CLEC9A binding peptide-Antigen-Fc-IL12; CLEC9A binding peptide-Antigen-Fc-IL21；CLEC9A binding peptide-Antigen-Fc-IL21; CLEC9A binding peptide-Antigen-Fc-CSF2；CLEC9A binding peptide-Antigen-Fc-CSF2; αPDL1-linker-Antigen-linker-Fc-linker-IL12；αPDL1-linker-Antigen-linker-Fc-linker-IL12; αPDL1-(GGGGS) 3-Antigen-(G) 3-Fc-(GS) 3-IL12； αPDL1-(GGGGS) 3 -Antigen-(G) 3 -Fc-(GS) 3 -IL12; αPDL1-linker-Antigen-linker-Fc-linker-IL21；αPDL1-linker-Antigen-linker-Fc-linker-IL21; αPDL1-(GGGGS) 3-Antigen-(G) 3-Fc-(GS) 3-IL21； αPDL1-(GGGGS) 3 -Antigen-(G) 3 -Fc-(GS) 3 -IL21; αPDL1-linker-Antigen-linker-Fc-linker-CSF2；αPDL1-linker-Antigen-linker-Fc-linker-CSF2; αPDL1-(GGGGS) 3-Antigen-(G) 3-Fc-(GS) 3-CSF2； αPDL1-(GGGGS) 3 -Antigen-(G) 3 -Fc-(GS) 3 -CSF2; CLEC9A binding peptide-linker-Antigen-linker-Fc-linker-IL12；CLEC9A binding peptide-linker-Antigen-linker-Fc-linker-IL12; CLEC9A binding peptide-(GGGGS) 3-Antigen-(G) 3-Fc-(GS) 3-IL12； CLEC9A binding peptide-(GGGGS) 3 -Antigen-(G) 3 -Fc-(GS) 3 -IL12; CLEC9A binding peptide-linker-Antigen-linker-Fc-linker-IL21；CLEC9A binding peptide-linker-Antigen-linker-Fc-linker-IL21; CLEC9A binding peptide-(GGGGS) 3-Antigen-(G) 3-Fc-(GS) 3-IL21； CLEC9A binding peptide-(GGGGS) 3 -Antigen-(G) 3 -Fc-(GS) 3 -IL21; CLEC9A binding peptide-linker-Antigen-linker-Fc-linker-CSF2；CLEC9A binding peptide-linker-Antigen-linker-Fc-linker-CSF2; CLEC9A binding peptide-(GGGGS) 3-Antigen-(G) 3-Fc-(GS) 3-CSF2； CLEC9A binding peptide-(GGGGS) 3 -Antigen-(G) 3 -Fc-(GS) 3 -CSF2; αPDL1-Pan-Antigen-Fc-IL12；αPDL1-Pan-Antigen-Fc-IL12; αPDL1-Pan-Antigen-Fc-IL21；αPDL1-Pan-Antigen-Fc-IL21; αPDL1-Pan-Antigen-Fc-CSF2；αPDL1-Pan-Antigen-Fc-CSF2; CLEC9A binding peptide-Pan-Antigen-Fc-IL12；CLEC9A binding peptide-Pan-Antigen-Fc-IL12; CLEC9A binding peptide-Pan-Antigen-Fc-IL21；CLEC9A binding peptide-Pan-Antigen-Fc-IL21; CLEC9A binding peptide-Pan-Antigen-Fc-CSF2；CLEC9A binding peptide-Pan-Antigen-Fc-CSF2; αPDL1‐CSF2‐Pan‐Antigen‐Fc；αPDL1‐CSF2‐Pan‐Antigen‐Fc; αPDL1-linker-Pan-linker-Antigen-linker-Fc-linker-IL12；αPDL1-linker-Pan-linker-Antigen-linker-Fc-linker-IL12; αPDL1-(GGGGS) 3-Pan-(GS) 3-Antigen-(G) 3-Fc-(GS) 3-IL12； αPDL1-(GGGGS) 3 -Pan-(GS) 3 -Antigen-(G) 3 -Fc-(GS) 3 -IL12; αPDL1-linker-Pan-linker-Antigen-linker-Fc-linker-IL21；αPDL1-linker-Pan-linker-Antigen-linker-Fc-linker-IL21; αPDL1-(GGGGS) 3-Pan-(GS) 3-Antigen-(G) 3-Fc-(GS) 3-IL21； αPDL1-(GGGGS) 3 -Pan-(GS) 3 -Antigen-(G) 3 -Fc-(GS) 3 -IL21; αPDL1-linker-Pan-linker-Antigen-linker-Fc-linker-CSF2；αPDL1-linker-Pan-linker-Antigen-linker-Fc-linker-CSF2; αPDL1-(GGGGS) 3-Pan-(GS) 3-Antigen-(G) 3-Fc-(GS) 3-CSF2； αPDL1-(GGGGS) 3 -Pan-(GS) 3 -Antigen-(G) 3 -Fc-(GS) 3 -CSF2; CLEC9A binding peptide-linker-Pan-linker-Antigen-linker-Fc-linker-IL12；CLEC9A binding peptide-linker-Pan-linker-Antigen-linker-Fc-linker-IL12; CLEC9A binding peptide-(GGGGS) 3-Pan-(GS) 3-Antigen-(G) 3-Fc-(GS) 3-IL12； CLEC9A binding peptide-(GGGGS) 3 -Pan-(GS) 3 -Antigen-(G) 3 -Fc-(GS) 3 -IL12; CLEC9A binding peptide-linker-Pan-linker-Antigen-linker-Fc-linker-IL21；CLEC9A binding peptide-linker-Pan-linker-Antigen-linker-Fc-linker-IL21; CLEC9A binding peptide-(GGGGS) 3-Pan-(GS) 3-Antigen-(G) 3-Fc-(GS) 3-IL21； CLEC9A binding peptide-(GGGGS) 3 -Pan-(GS) 3 -Antigen-(G) 3 -Fc-(GS) 3 -IL21; CLEC9A binding peptide-linker-Pan-linker-Antigen-linker-Fc-linker-CSF2；CLEC9A binding peptide-linker-Pan-linker-Antigen-linker-Fc-linker-CSF2; CLEC9A binding peptide-(GGGGS) 3-Pan-(GS) 3-Antigen-(G) 3-Fc-(GS) 3-CSF2； CLEC9A binding peptide-(GGGGS) 3 -Pan-(GS) 3 -Antigen-(G) 3 -Fc-(GS) 3 -CSF2; αPDL1-linker‐CSF2-linker‐Pan-linker‐Antigen-linker‐Fc；αPDL1-linker‐CSF2-linker‐Pan-linker‐Antigen-linker‐Fc; αPDL1-(GGGGS) 3‐CSF2-(GS) 3‐Pan-(GS) 3‐Antigen-(G) 3‐Fc； αPDL1-(GGGGS) 3 ‐CSF2-(GS) 3 ‐Pan-(GS) 3 ‐Antigen-(G) 3 ‐Fc; αPDL1-Antigen-Fc knob/CSF2-Antigen-Fc hole；αPDL1-Antigen-Fc knob/CSF2-Antigen-Fc hole; CLEC9A binding peptide-Antigen-Fc knob/CSF2-Antigen-Fc hole；CLEC9A binding peptide-Antigen-Fc knob/CSF2-Antigen-Fc hole; αPDL1-linker-Antigen-linker-Fc knob/CSF2-linker-Antigen-linker-Fc hole；αPDL1-linker-Antigen-linker-Fc knob/CSF2-linker-Antigen-linker-Fc hole; αPDL1-(GGGGS) 3-Antigen-(G) 3-Fc knob/ αPDL1-(GGGGS) 3 -Antigen-(G) 3 -Fc knob/ CSF2-(GGGGS) 3-Antigen-(G) 3-Fc hole； CSF2-(GGGGS) 3 -Antigen-(G) 3 -Fc hole; CLEC9A binding peptide-linker-Antigen-linker-Fc knob/CLEC9A binding peptide-linker-Antigen-linker-Fc knob/ CSF2-linker-Antigen-linker-Fc hole；CSF2-linker-Antigen-linker-Fc hole; CLEC9A binding peptide-(GGGGS) 3-Antigen-(G) 3-Fc knob/ CLEC9A binding peptide-(GGGGS) 3 -Antigen-(G) 3 -Fc knob/ CSF2-(GGGGS) 3-Antigen-(G) 3-Fc hole； CSF2-(GGGGS) 3 -Antigen-(G) 3 -Fc hole; αPDL1-Pan-Antigen-Fc knob/CSF2-Pan-Antigen-Fc hole；αPDL1-Pan-Antigen-Fc knob/CSF2-Pan-Antigen-Fc hole; CLEC9A binding peptide-Pan-Antigen-Fc knob/CSF2-Pan-Antigen-Fc hole；CLEC9A binding peptide-Pan-Antigen-Fc knob/CSF2-Pan-Antigen-Fc hole; αPDL1-linker-Pan-linker-Antigen-linker-Fc knob/αPDL1-linker-Pan-linker-Antigen-linker-Fc knob/ CSF2-linker-Pan-linker-Antigen-linker-Fc hole；CSF2-linker-Pan-linker-Antigen-linker-Fc hole; αPDL1-(GGGGS) 3-Pan-(GS) 3-Antigen-(G) 3-Fc knob/ αPDL1-(GGGGS) 3 -Pan-(GS) 3 -Antigen-(G) 3 -Fc knob/ CSF2-(GGGGS) 3-Pan-(GS) 3-Antigen-(G) 3-Fc hole； CSF2-(GGGGS) 3 -Pan-(GS) 3 -Antigen-(G) 3 -Fc hole; αPDL1-linker-Pan-linker-Antigen-linker-Fc knob/αPDL1-linker-Pan-linker-Antigen-linker-Fc knob/ CLEC9A binding peptide-linker-Pan-linker-Antigen-linker-Fc knob/CLEC9A binding peptide-linker-Pan-linker-Antigen-linker-Fc knob/ CSF2-linker-Pan-linker-Antigen-linker-Fc hole；CSF2-linker-Pan-linker-Antigen-linker-Fc hole; CLEC9A binding peptide-(GGGGS) 3-Pan-(GS) 3-Antigen-(G) 3-Fc knob/ CLEC9A binding peptide-(GGGGS) 3 -Pan-(GS) 3 -Antigen-(G) 3 -Fc knob/ CSF2-(GGGGS) 3-Pan-(GS) 3-Antigen-(G) 3-Fc hole。 CSF2-(GGGGS) 3 -Pan-(GS) 3 -Antigen-(G) 3 -Fc hole. 根据权利要求1-5任一项所述的融合蛋白，所述抗原结构域为能够诱导针对病原微生物的免疫反应的免疫原性蛋白或其免疫原性片段；According to the fusion protein according to any one of claims 1-5, the antigenic domain is an immunogenic protein or an immunogenic fragment thereof capable of inducing an immune response against pathogenic microorganisms; 优选地所述病原微生物为SARS-Cov-2，SARS，巨细胞病毒CMV，疱疹病毒，呼吸道合胞病毒RSV，流感病毒，Ebola病毒，爱泼斯坦-巴尔病毒EBV，登革热病毒，Zike病毒，HIV病毒，狂犬病毒，疟原虫配子体，带状疱疹病毒HZV，乙肝病毒HBV，丙型肝炎病毒HCV，丁肝病毒HDV，HPV，结核分枝杆菌，或幽门螺旋杆菌。Preferably, the pathogenic microorganism is SARS-Cov-2, SARS, cytomegalovirus CMV, herpes virus, respiratory syncytial virus RSV, influenza virus, Ebola virus, Epstein-Barr virus EBV, dengue fever virus, Zike virus, HIV Viruses, rabies virus, Plasmodium gametophyte, herpes zoster virus HZV, hepatitis B virus HBV, hepatitis C virus HCV, hepatitis D virus HDV, HPV, Mycobacterium tuberculosis, or Helicobacter pylori. 根据权利要求6所述的融合蛋白，所述抗原结构域为SARS-Cov-2的S蛋白或其片段，优选地，所述S蛋白为预融合稳定的S蛋白，更优选地，所述预融合稳定的S蛋白包含双脯氨酸(S2P)突变或六脯氨酸(S6P)突变；或者The fusion protein according to claim 6, the antigen domain is the S protein or fragment thereof of SARS-Cov-2, preferably, the S protein is a pre-fusion stable S protein, more preferably, the pre-fused S protein The fusion-stabilized S protein contains a double proline (S2P) mutation or a hexaproline (S6P) mutation; or 所述抗原为SARS-Cov-2的S蛋白的RBD结构域。The antigen is the RBD domain of the S protein of SARS-Cov-2. 根据权利要求1-5任一项所述的融合蛋白，所述抗原结构域为能够诱导针对癌症细胞的免疫反应的免疫原性蛋白，或其免疫原性片段；The fusion protein according to any one of claims 1-5, the antigen domain is an immunogenic protein capable of inducing an immune response against cancer cells, or an immunogenic fragment thereof; 优选地，所述抗原结构域为选自如下的肿瘤抗原或其免疫原性片段：MelanA/MART1、癌-种系抗原、gp100、酪氨酸酶、CEA、PSA、Her-2/neu、存活蛋白、端粒酶，或其免疫原性片段；Preferably, the antigenic domain is a tumor antigen or an immunogenic fragment thereof selected from the group consisting of: MelanA/MART1, cancer-germline antigen, gp100, tyrosinase, CEA, PSA, Her-2/neu, survival protein, telomerase, or immunogenic fragments thereof; 优选地，所述癌症可以为前列腺癌，非小细胞肺癌，小细胞肺癌，肾细胞癌，脑癌，黑色素瘤，急性髓细胞性白血病，胰腺癌，结直肠癌，头颈部鳞状细胞癌，皮肤鳞状细胞癌，腺样囊性癌，胶质母细胞瘤，乳腺癌，间皮瘤，卵巢癌，神经胶质瘤，膀胱癌，肝癌，骨癌，骨髓癌，胃癌，甲状腺癌，淋巴癌，宫颈癌，子宫内膜癌，喉癌，急性淋巴细胞性白血病。Preferably, the cancer may be prostate cancer, non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, brain cancer, melanoma, acute myeloid leukemia, pancreatic cancer, colorectal cancer, squamous cell carcinoma of the head and neck , squamous cell carcinoma of the skin, adenoid cystic carcinoma, glioblastoma, breast cancer, mesothelioma, ovarian cancer, glioma, bladder cancer, liver cancer, bone cancer, bone marrow cancer, stomach cancer, thyroid cancer, Lymphoma, cervical cancer, endometrial cancer, laryngeal cancer, acute lymphoblastic leukemia. 编码权利要求1-8任一项的融合蛋白的核酸分子。A nucleic acid molecule encoding the fusion protein of any one of claims 1-8. 载体，所述载体表达权利要求1-9任一项的融合蛋白或包含权利要求9的核酸分子。A vector expressing the fusion protein of any one of claims 1-9 or comprising the nucleic acid molecule of claim 9. 权利要求10所述的载体，所述载体为质粒载体，腺病毒载体或慢病毒载体。The carrier according to claim 10, which is a plasmid vector, an adenoviral vector or a lentiviral vector. 用于表达权利要求1-8任一项所述的融合蛋白或包含权利要求9所述的核酸分子，或权利要求10或11所述载体的宿主细胞，A host cell for expressing the fusion protein according to any one of claims 1-8 or comprising the nucleic acid molecule according to claim 9, or the vector according to claim 10 or 11, 优选地，所述宿主细胞为中国仓鼠卵巢细胞(CHO细胞)，幼年仓鼠肾细胞(BHK细胞)，COS细胞，小鼠NSO胸腺瘤细胞，小鼠骨髓瘤细胞(SP2/0细胞)，人胚胎肾细胞HEK293细胞。Preferably, the host cells are Chinese hamster ovary cells (CHO cells), young hamster kidney cells (BHK cells), COS cells, mouse NSO thymoma cells, mouse myeloma cells (SP2/0 cells), human embryo Kidney cells HEK293 cells. 一种组合物，其包含权利要求1-8任一项的融合蛋白或权利要求9所述的核酸分子，或权利要求10或11所述的载体，或权利要求12所述的宿主细胞。A composition comprising the fusion protein of any one of claims 1-8 or the nucleic acid molecule of claim 9, or the vector of claim 10 or 11, or the host cell of claim 12. 根据权利要求13所述的组合物，所述组合物不包含药用佐剂；或者所述组合物还包含药用佐剂，优选地，所述药用佐剂包括但不限于铝佐剂或CpG佐剂，优选地，所述铝佐剂为氢氧化铝、磷酸铝或硫酸铝。The composition according to claim 13, which does not comprise a pharmaceutical adjuvant; or the composition further comprises a pharmaceutical adjuvant, preferably, the pharmaceutical adjuvant includes but not limited to aluminum adjuvant or CpG adjuvant, preferably, the aluminum adjuvant is aluminum hydroxide, aluminum phosphate or aluminum sulfate. 用于预防或治疗与病原微生物感染或肿瘤的方法，所述方法包括向对象给药权利要求1-8任一项的融合蛋白或权利要求9所述的核酸分子，或权利要求10或11所述的载体，权利要求12的宿主细胞，或权利要求13或14所述的组合物的步骤，A method for preventing or treating infection with pathogenic microorganisms or tumors, the method comprising administering to a subject the fusion protein of any one of claims 1-8 or the nucleic acid molecule of claim 9, or the nucleic acid molecule of claim 10 or 11. described carrier, the host cell of claim 12, or the step of the composition described in claim 13 or 14, 优选地，所述对象为人类或动物，Preferably, the subject is a human or an animal, 优选地，所述动物为牛，羊，猫，犬，马，兔，猴，小鼠，大鼠，羊驼或骆 驼；Preferably, the animal is a cow, a sheep, a cat, a dog, a horse, a rabbit, a monkey, a mouse, a rat, an alpaca or a camel; 优选地，所述对象为免疫功能低下的人或动物；Preferably, the subject is an immunocompromised human or animal; 优选地，所述对象患有慢性肺病，慢性阻塞性肺疾病或哮喘；Preferably, the subject suffers from chronic lung disease, chronic obstructive pulmonary disease or asthma; 优选地，所述患者患有选自心脏病，糖尿病或肺病的基础疾病。Preferably, said patient suffers from an underlying disease selected from heart disease, diabetes or lung disease. 权利要求1-8任一项的融合蛋白或权利要求9的核酸分子，或权利要求10或11的载体，权利要求12的宿主细胞，或权利要求13或14的组合物在制备用于预防或治疗对象与病原微生物感染或肿瘤相关的药物或试剂盒中的应用，The fusion protein of any one of claims 1-8 or the nucleic acid molecule of claim 9, or the carrier of claim 10 or 11, the host cell of claim 12, or the composition of claim 13 or 14 in the preparation for preventing or The application of drugs or kits related to pathogenic microorganism infection or tumor in the treatment object, 所述对象为人类或动物，said subject is a human or an animal, 优选地，所述动物为牛，羊，猫，犬，马，兔，猴，小鼠，大鼠，羊驼或骆驼；Preferably, the animal is cow, sheep, cat, dog, horse, rabbit, monkey, mouse, rat, alpaca or camel; 优选地，所述对象为免疫功能低下的人或动物；Preferably, the subject is an immunocompromised human or animal; 优选地，所述对象患有慢性肺病，慢性阻塞性肺疾病或哮喘；Preferably, the subject suffers from chronic lung disease, chronic obstructive pulmonary disease or asthma; 优选地，所述患者患有选自心脏病，糖尿病或肺病的基础疾病。Preferably, said patient suffers from an underlying disease selected from heart disease, diabetes or lung disease.
PCT/CN2022/144163 2021-12-31 2022-12-30 Fusion protein vaccine WO2023125976A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202111683236 2021-12-31
CN202111683236.6 2021-12-31

Publications (1)

Publication Number Publication Date
WO2023125976A1 true WO2023125976A1 (en) 2023-07-06

Family

ID=86966224

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/144163 WO2023125976A1 (en) 2021-12-31 2022-12-30 Fusion protein vaccine

Country Status (2)

Country Link
CN (1) CN116375881A (en)
WO (1) WO2023125976A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117003895A (en) * 2023-08-09 2023-11-07 成都新诺明生物科技有限公司 gE fusion protein containing IL2, fc and PADRE, and preparation method and application thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1374871A (en) * 1999-07-21 2002-10-16 利思进药品公司 FC fusion proteins for enhancing the immunogenicity of protein and piptide antigens
WO2005120565A2 (en) * 2004-06-02 2005-12-22 New York Blood Center Sars vaccines and methods to produce highly potent antibodies
CN102884194A (en) * 2010-03-11 2013-01-16 健康研究股份有限公司 Methods and compositions containing Fc fusiong proteins for enhancing immune responses
CN107286245A (en) * 2016-04-11 2017-10-24 生命序有限公司 PD-L1 and PD-L2 recombinant proteins and application thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1374871A (en) * 1999-07-21 2002-10-16 利思进药品公司 FC fusion proteins for enhancing the immunogenicity of protein and piptide antigens
WO2005120565A2 (en) * 2004-06-02 2005-12-22 New York Blood Center Sars vaccines and methods to produce highly potent antibodies
CN102884194A (en) * 2010-03-11 2013-01-16 健康研究股份有限公司 Methods and compositions containing Fc fusiong proteins for enhancing immune responses
CN107286245A (en) * 2016-04-11 2017-10-24 生命序有限公司 PD-L1 and PD-L2 recombinant proteins and application thereof

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CHEN ELAINE C., GILCHUK PAVLO, ZOST SETH J., SURYADEVARA NAVEENCHANDRA, WINKLER EMMA S., CABEL CARLY R., BINSHTEIN ELAD, CHEN RITA: "Convergent antibody responses to the SARS-CoV-2 spike protein in convalescent and vaccinated individuals", CELL REPORTS, ELSEVIER INC, US, vol. 36, no. 8, 1 August 2021 (2021-08-01), US , pages 109604, XP093076277, ISSN: 2211-1247, DOI: 10.1016/j.celrep.2021.109604 *
SUN, S.Y. ET AL.: "Interferon-armed RBD Dimer Enhances the Immunogenicity of RBD for Sterilizing Immunity Against SARS-CoV-2", CELL RESEARCH, vol. 31, 15 July 2021 (2021-07-15), XP037566854, DOI: 10.1038/s41422-021-00531-8 *
WEI LI: "Synergistic antibody induction by antigen–CD40 ligand fusion protein as improved immunogen", IMMUNOLOGY, vol. 115, no. 2, 31 March 2005 (2005-03-31), GB , pages 215 - 222, XP071275201, ISSN: 0019-2805, DOI: 10.1111/j.1365-2567.2005.02141.x *
XU ZIYANG, NEETHU CHOKKALINGAM, EDGAR TELLO-RUIZ, SUSANNE WALKER, DANIEL W. KULP, DAVID B. WEINER: "Incorporation of a Novel CD4+ Helper Epitope Identified from Aquifex aeolicus Enhances Humoral Responses Induced by DNA and Protein Vaccinations", ISCIENCE, ELSEVIER, vol. 23, no. 8, 21 August 2020 (2020-08-21), pages 101399, XP093076276, DOI: 10.1016/j.isci *
ZHAO GANG, WU NAN-PING, YAO HANG-PING: "Expression of recombinant plasmid of HIV-1 gp120 and IFN-Y in E. coli and its immunoregulatory effect", JOURNAL OF ZHEJIANG UNIVERSITY (MEDICAL SCIENCES), vol. 35, no. 6, 1 January 2006 (2006-01-01), pages 610 - 614, XP093076272 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117003895A (en) * 2023-08-09 2023-11-07 成都新诺明生物科技有限公司 gE fusion protein containing IL2, fc and PADRE, and preparation method and application thereof
CN117003895B (en) * 2023-08-09 2024-05-28 成都新诺明生物科技有限公司 GE fusion protein containing IL2, fc and PADRE, and preparation method and application thereof

Also Published As

Publication number Publication date
CN116375881A (en) 2023-07-04

Similar Documents

Publication Publication Date Title
Sun et al. Interferon-armed RBD dimer enhances the immunogenicity of RBD for sterilizing immunity against SARS-CoV-2
WO2021239147A1 (en) β-CORONAVIRUS ANTIGEN, β-CORONAVIRUS BIVALENT VACCINE, PREPARATION METHODS THEREFOR, AND APPLICATIONS THEREOF
KR101912330B1 (en) Influenza nucleic acid molecules and vaccines made therefrom
CN104039833B (en) For hpv vaccine
ES2848064T3 (en) Therapeutic immunity-enhancing vaccine for HPV and related diseases
WO2021189571A1 (en) Vaccine vector capable of efficiently inducing body humoral immune response as well as preparation method therefor and application thereof
Stepanova et al. Flagellin-fused protein targeting M2e and HA2 induces potent humoral and T-cell responses and protects mice against various influenza viruses a subtypes
Fan et al. Targeting the HA2 subunit of influenza A virus hemagglutinin via CD40L provides universal protection against diverse subtypes
US11185580B2 (en) Compositions, methods and uses for improved human papilloma virus constructs
US20210106667A1 (en) Vaccine compositions and methods for enhanced antigen-specific vaccination
JP2016202185A (en) Homodimeric protein constructs
CN103002909A (en) Pharmaceutical compositions comprising polypeptides containing at least one CXXC motif and heterologous antigens and uses thereof
CN113876938A (en) Construction and application of fusion protein vaccine platform
WO2023125976A1 (en) Fusion protein vaccine
JP2014534807A (en) Vaccibody targeting dendritic cells for cross-presentation
WO2021253172A1 (en) Method for inducing anti-novel coronavirus neutralizing antibody using receptor recognition domain
WO2023023940A1 (en) Immunogen for inducing broad-spectrum anti-coronavirus t cell vaccine and use thereof
Kwak et al. Multivalent human papillomavirus l1 DNA vaccination utilizing electroporation
WO2022135563A1 (en) Method for simultaneously inducing immune response against multiple viruses
Muralidharan et al. Targeting CD40 enhances antibody-and CD8-mediated protection against respiratory syncytial virus infection
CN115137812A (en) Construction and application of fusion protein vaccine platform
Li et al. A recombinant protein containing influenza viral conserved epitopes and superantigen induces broad-spectrum protection
WO2023125974A1 (en) Mrna vaccine
Ivanova et al. Targeting of influenza viral epitopes to antigen-presenting cells by genetically engineered chimeric molecules in a humanized NOD SCID Gamma Transfer Model
Jiang et al. Aggregation by peptide conjugation rescues poor immunogenicity of the HA stem

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22915223

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22915223

Country of ref document: EP

Kind code of ref document: A1