WO2021227937A1 - 通过与改变的Fc片段形成融合蛋白增强蛋白/肽抗原免疫原性的方法 - Google Patents

通过与改变的Fc片段形成融合蛋白增强蛋白/肽抗原免疫原性的方法 Download PDF

Info

Publication number
WO2021227937A1
WO2021227937A1 PCT/CN2021/092013 CN2021092013W WO2021227937A1 WO 2021227937 A1 WO2021227937 A1 WO 2021227937A1 CN 2021092013 W CN2021092013 W CN 2021092013W WO 2021227937 A1 WO2021227937 A1 WO 2021227937A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
antigen
cancer
peptide antigen
antibody
Prior art date
Application number
PCT/CN2021/092013
Other languages
English (en)
French (fr)
Inventor
谢良志
Original Assignee
神州细胞工程有限公司
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 神州细胞工程有限公司 filed Critical 神州细胞工程有限公司
Priority to CN202180029479.XA priority Critical patent/CN115461078A/zh
Publication of WO2021227937A1 publication Critical patent/WO2021227937A1/zh

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
    • A61K39/12—Viral antigens
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention belongs to the field of immunology. Specifically, it relates to a method for enhancing the immunogenicity of a protein/peptide antigen, wherein the protein/peptide antigen forms a fusion protein with a modified Fc fragment of an antibody, and the Fc fragment is due to its amino acid sequence and/or sugar
  • the modified form has an improved binding capacity to the Fc receptor and/or complement protein C1q compared to its natural form.
  • the SARS-CoV-2 vaccine prepared by using the fusion protein of the present invention as an immunogen has high binding capacity to Fc receptors, and can maintain long-term humoral and cellular immune responses, and immunized animals can produce higher titer Neutralizing antibodies.
  • the vaccine can be used to prevent SARS-CoV-2 infection-related diseases.
  • B cell-mediated humoral immunity is one of the body's protective mechanisms mediated by vaccines.
  • FcRs Fc receptors
  • CR complement receptors
  • Fc binds to receptors on follicular dendritic cells (fDC) (mediated by FcR or CR) and is displayed on the surface of fDC. This part of the antigen is essential for maintaining the long-term existence of the antigen and maintaining the survival of antigen-specific B cells. Very important [7].
  • cytotoxic T lymphocytes play an important role in resisting virus infection and removing virus-infected cells.
  • CTL cytotoxic T lymphocytes
  • the antigen cross-presentation mechanism allows exogenous antigens to enter the cell's endogenous processing and presentation mechanism, so that the exogenous antigen peptides are displayed on MHC class I molecules to be recognized by T cells and initiate CTL cell responses.
  • the cross-presentation of foreign antigens is of great significance for effectively activating CTLs and triggering antiviral immune responses. Therefore, enhancing the cross-presentation of subunit vaccines is one of the effective strategies to improve the immune effect of vaccines.
  • Dendritic cells are currently known as the most powerful professional antigen-presenting cells, and they are also the main cells for cross-presentation.
  • phagocytosis endocytosis
  • pinocytosis receptor-mediated endocytosis.
  • endocytic receptors related to the cross-presentation of foreign antigens, including C-type lectin receptor (CLR), Fc receptors (FcRs) that recognize immune complex IgG, and scavengers that recognize apoptotic cells Receptors, chemokine receptors, etc.
  • CLR C-type lectin receptor
  • FcRs Fc receptors
  • scavengers that recognize apoptotic cells Receptors, chemokine receptors, etc.
  • these receptors mediate the endocytosis of antigens and enter specific endosomes before they can bind to MHC class I molecules, thereby activating CD8 + T cells.
  • Antigen-antibody complexes can be recognized by the FcRs of DC cells, and the resulting cross-linking can internalize the antigen, and cross-process and present the antigen to specifically activate the CTL response [8-10]. This cross-presentation of antigens mediated by FcRs has been shown to induce strong CTL responses [11].
  • Fc receptors that bind to IgG in the human body mainly include Fc ⁇ RI (CD64), Fc ⁇ RIIA (CD32a), Fc ⁇ RIIB (CD32B), Fc ⁇ RIII (CD16), etc. [12].
  • Fc ⁇ RIIB is an inhibitory receptor, which is mainly expressed on B cells, macrophages and mast cells [13]. It can be divided into Fc ⁇ RIIB-1 and Fc ⁇ RIIB-2.
  • Fc ⁇ RIIB-1 is only expressed on B cells, and controls the excessive activation of B cells and the recognition of self-antigens during the development of B cells. It transmits B cell apoptosis through intracellular receptor tyrosine inhibitory motifs (ITIM).
  • the death signal realizes the negative selection process of B cells and regulates the development process of B cells.
  • Fc ⁇ RIIB-2 is expressed on other immune cells except NK and T cells, and the cross-linking of the receptor can effectively induce the phagocytosis of the antigen-antibody complex [14,15].
  • the other Fc receptors are all activating receptors.
  • CD64 is mainly expressed on monocytes, macrophages, DCs and other cells
  • CD32a is mainly expressed on neutrophils
  • CD16a is mainly It is expressed in NK, monocytes, and macrophages [15].
  • the intracellular receptor tyrosine activation motif determines the antigen-presenting cell APC to initiate the uptake of antigen and the antigen-presenting function exercised by MHC molecules after the uptake [ 16].
  • Fc fusion protein refers to a new type of recombinant protein produced by fusing a certain biologically active functional protein with an Fc fragment using genetic engineering and other technologies. It not only retains the biological activity of the functional protein molecule, but also has some antibody properties, such as FcRs binding and related biological functions mediated.
  • Antigen-Fc fusion protein can be used as an antigen delivery vehicle, with the help of Fc fragments to target and bind antigen-presenting cells, shorten the free time of antigen in plasma, increase the half-life of antigen, and enhance the reaction of antigen presentation and antigen cross-presentation.
  • the Fc fragment is modified to enhance the binding of FcRs to obtain Fe4-Fc modified molecules, which can increase the binding of Fc to complement protein C1q and Fc receptors CD16, CD32, CD64, etc. [17-21], there are It may further enhance Fc and its receptor-mediated antigen capture and presentation, enhance B cell maturation and high-affinity antibody production, maintain long-term humoral immunity, enhance CTL immune response mediated by antigen cross-presentation, and enhance Fc The immune effect of fusion protein vaccines.
  • YIC can increase the maturation (high expression of CD83), antigen recognition and presentation (expression of HLA-II, CD86, CD80, CD40 markers) of DC cells in patients infected with cHBV, and secrete more inflammation Sex factor (IL-12).
  • the patient’s DC-PBMC mixed cells produced more T lymphocyte cytokines (Th1 cells: IL-2, IFN ⁇ ) under YIC stimulation than HBsAg antigen alone (Th2 cells: IL-5, IL-10) [23] .
  • Th1 cells IL-2, IFN ⁇
  • Th2 cells IL-5, IL-10)
  • the excessive stimulation of YIC may cause the body's immune fatigue, thereby reducing the cellular immune response [24]. Therefore, in order to achieve a better immune effect, a suitable immunization program is also particularly important.
  • Some other viral antigen-Fc fusion protein vaccines have also been tried in animal models to achieve effective immune system activation, and the antibodies produced can reduce the level of viral antigens in the serum [25- 28].
  • AE Phase IIb clinical side effects
  • the present invention provides a method for enhancing the immunogenicity of a protein/peptide antigen, the method comprising fusing the protein/peptide antigen with an engineered antibody Fc fragment, which has improved Its ability to bind to Fc receptors and/or complement protein C1q can simulate antigen-antibody complexes to enhance the phagocytosis of DC/B antigen-presenting cells and enhance antigen immunity.
  • the protein/peptide antigen in the method is a pathogen-related protein/peptide antigen or a tumor-related protein/peptide antigen.
  • the pathogen in the method is selected from:
  • Coronavirus human immunodeficiency virus HIV-1, human herpes simplex virus, cytomegalovirus, rotavirus, Epstein-Barr virus, varicella-zoster virus, hepatitis virus, respiratory syncytial virus, parainfluenza virus, measles virus, epidemic Mumps virus, human papilloma virus, flavivirus or influenza virus, Neisseria, Moraxella, Bordetella, Mycobacterium, including Mycobacterium tuberculosis; Escherichia , Including enterotoxin Escherichia coli; Salmonella, Listeria, Helicobacter, Staphylococcus, including Staphylococcus aureus, Staphylococcus epidermidis; Borrelia, Chlamydia, including Chlamydia trachomatis, Chlamydia pneumoniae; Plasmodium , Including Plasmodium falciparum; Toxoplasma gondii, Candida;
  • the tumor is selected from:
  • the protein/peptide protein antigen in the method is selected from a secreted protein or a full-length membrane protein, or a functional domain, a mutein, a truncated protein, or one or more antigen polypeptides.
  • the Fc fragment in the method is derived from the heavy chain constant region of a human antibody, murine antibody, rabbit antibody or other animal antibody.
  • the Fc fragment in the method is derived from an IgG, IgM or IgA subtype antibody of a human antibody,
  • antibodies of IgG1, IgG2, IgG3 or IgG4 subtype are preferred from antibodies of IgG1, IgG2, IgG3 or IgG4 subtype;
  • it is an IgG1 modified Fc fragment with amino acid sequence mutations and/or glycosylation changes for the purpose of improving the function of binding to Fc receptors and C1q complement.
  • the Fc receptor of the method is selected from CD16, CD32a, CD32b, or CD64.
  • the protein/peptide antigen in the method is the ACE2 receptor binding domain (RBD) of the coronavirus spike protein.
  • the modified antibody Fc fragments in the method are Fc receptors CD32a, CD32b, and CD64 binding enhancing fragments/complement C1q binding enhancing fragments;
  • the modified antibody Fc fragments are Fc receptors CD16a, CD32a, CD32b and CD64 binding enhancing fragments/complement C1q binding enhancing fragments;
  • amino acid sequence is shown in SEQ ID NO: 30, and it is produced by Fucose knockout mammalian cells.
  • the mammalian cell is fut8 knockout HEK-293 cell.
  • the Fc fragment in the method is derived from murine antibodies IgG, IgM, and IgA subtype antibodies,
  • it is from an antibody of IgG1, IgG2a, IgG2b or IgG3 subtype.
  • the antigen described in the method is preferably conjugated to other macromolecules through a linker.
  • the other macromolecules are polysaccharides, peptides/proteins.
  • the present invention provides a protein/peptide antigen with enhanced immunogenicity, wherein
  • the protein/peptide antigen is fused with the modified Fc fragment of the antibody.
  • the Fc fragment has improved binding ability to Fc receptor and or complement protein C1q, which can mimic the antigen-antibody complex and enhance the DC/B antigen Phagocytosis of presenting cells enhances the antigen immune effect.
  • the antigen in the protein/peptide antigen is a pathogen-related protein/peptide antigen and a tumor-related protein/peptide antigen.
  • the pathogen in the protein/peptide antigen is selected from:
  • Coronavirus human immunodeficiency virus HIV-1, human herpes simplex virus, cytomegalovirus, rotavirus, Epstein-Barr virus, varicella-zoster virus, hepatitis virus, respiratory syncytial virus, parainfluenza virus, measles virus, epidemic Mumps virus, human papilloma virus, flavivirus or influenza virus, Neisseria, Moraxella, Bordetella, Mycobacterium, including Mycobacterium tuberculosis; Escherichia , Including enterotoxin Escherichia coli; Salmonella, Listeria, Helicobacter, Staphylococcus, including Staphylococcus aureus, Staphylococcus epidermidis; Borrelia, Chlamydia, including Chlamydia trachomatis, Chlamydia pneumoniae; Plasmodium , Including Plasmodium falciparum; Toxoplasma gondii, Candida;
  • the tumor is selected from:
  • the protein antigen in the protein/peptide antigen is selected from a secreted protein or a full-length membrane protein, or a functional domain, a mutein, a truncated protein, or one or more antigen polypeptides.
  • the Fc fragment in the protein/peptide antigen is derived from the heavy chain constant region of a human antibody, murine antibody, rabbit antibody or other animal antibody.
  • the Fc fragment in the protein/peptide antigen is an IgG, IgM or IgA subtype antibody of a human antibody,
  • antibodies of IgG1, IgG2, IgG3 or IgG4 subtype are preferred from antibodies of IgG1, IgG2, IgG3 or IgG4 subtype;
  • it is an IgG1 modified Fc fragment with amino acid sequence mutation and/or glycosylation change for the purpose of improving the function of binding to Fc receptor and/or C1q complement.
  • the Fc receptor in the protein/peptide antigen is selected from CD16, CD32a, CD32b or CD64.
  • the antigen in the protein/peptide antigen is the ACE2 receptor binding domain (RBD) of the coronavirus spike protein.
  • the protein/peptide antigen in one embodiment, the protein/peptide antigen
  • the modified antibody Fc fragments are Fc receptors CD32a, CD32b and CD64 binding enhancing fragments/complement C1q binding enhancing fragments;
  • the protein/peptide antigen in one embodiment, the protein/peptide antigen
  • the modified antibody Fc fragments are Fc receptors CD16a, CD32a, CD32b and CD64 binding enhancing fragments/complement C1q binding enhancing fragments;
  • amino acid sequence is shown in SEQ ID NO: 30, and it is produced by mammalian cells knocked out by Fucose,
  • the mammalian cell is a fut8 gene knockout HEK-293 cell.
  • the Fc fragment in the protein/peptide antigen is derived from murine antibodies IgG, IgM, and IgA subtype antibodies,
  • it is from an antibody of IgG1, IgG2a, IgG2b or IgG3 subtype.
  • the antigen in the protein/peptide antigen is preferably conjugated to other macromolecules through a linker.
  • the other macromolecules are polysaccharides, peptides/proteins.
  • the present invention provides a conjugate comprising, preferably, the protein/peptide antigen of the present invention and other macromolecules connected by a linker, preferably, the other macromolecules are polysaccharides, peptides/proteins .
  • the present invention provides a nucleic acid, which encodes the protein/peptide antigen of the present invention, which is mRNA and/or DNA.
  • the nucleic acid sequence is shown in SEQ ID NO: 31.
  • the present invention provides an expression vector comprising the nucleic acid of the present invention.
  • the present invention provides a host cell comprising the nucleic acid of the present invention or the expression vector of the present invention.
  • the present invention provides a method for producing the protein/peptide antigen of the present invention, which comprises culturing the host cell of the present invention under conditions suitable for the expression of the aforementioned protein molecule, and extracting from the culture medium The expressed product is recovered in the process.
  • the present invention provides an immune composition comprising
  • a pharmaceutically acceptable carrier, excipient or stabilizer preferably
  • a pharmaceutically acceptable carrier, excipient or stabilizer in the form of a lyophilized formulation or an aqueous solution is provided.
  • the adjuvant in the immune composition may be selected from at least one of aluminum adjuvant, MF59, QS-21 or MPL.
  • the present invention provides the protein/peptide antigen of the present invention, the conjugate of the present invention, the nucleic acid of the present invention, the expression vector of the present invention, or the immune combination of the present invention It is used to prevent pathogens, preferably coronaviruses, more preferably SARS-CoV-2 caused diseases ⁇ tumors.
  • pathogens preferably coronaviruses, more preferably SARS-CoV-2 caused diseases ⁇ tumors.
  • the present invention provides the protein/peptide antigen of the present invention, the conjugate of the present invention, the nucleic acid of the present invention, the expression vector of the present invention, or the immune combination of the present invention It is used to prepare vaccines for preventing pathogens, preferably coronaviruses, and more preferably diseases/tumors caused by SARS-CoV-2.
  • the present invention provides an immune combination comprising
  • the protein/peptide antigen of the present invention the conjugate of the present invention, the nucleic acid of the present invention, the expression vector of the present invention, or the immune composition of the present invention.
  • One or more additional immunogenic agents are provided.
  • the present invention provides a kit comprising
  • the protein/peptide antigen of the present invention the conjugate of the present invention, the nucleic acid of the present invention, the expression vector of the present invention, or the immune composition of the present invention;
  • the present invention provides a method for preventing a pathogen, preferably a coronavirus, more preferably a disease caused by SARS-CoV-2 ⁇ preventing a tumor, which comprises administering the protein/peptide antigen of the present invention, the present invention to a subject
  • a pathogen preferably a coronavirus, more preferably a disease caused by SARS-CoV-2 ⁇ preventing a tumor
  • administering the protein/peptide antigen of the present invention, the present invention to a subject
  • the conjugate of the invention, the nucleic acid of the invention, the expression vector of the invention, or the immune composition of the invention, the vaccine combination of the invention or the kit of the invention preferably a pathogen, preferably a coronavirus, more preferably a disease caused by SARS-CoV-2 ⁇ preventing a tumor.
  • the present invention provides a method for immunizing an animal, which comprises administering to the animal the protein/peptide antigen of the present invention, the conjugate of the present invention, the nucleic acid of the present invention, and the nucleic acid of the present invention.
  • Figure 1 Binding of RBD-mFc to murine Fc receptors.
  • Figure 2 The binding of different RBD-Fc fusion proteins to human Fc receptors and C1q.
  • Figure 3 MDM phagocytosis test of different RBD-Fc fusion proteins.
  • the inventors pioneered the discovery that by fusing a protein/peptide antigen with a modified antibody Fc fragment, its immunogenicity is enhanced.
  • the above-mentioned Fc fragment has increased interaction with Fc receptors due to changes in its amino acid sequence and/or glycosylation form. And/or the binding ability of complement protein C1q.
  • the inventors applied this discovery to the new coronavirus (Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2) vaccine.
  • SARS-CoV-2 and SARS-CoV share a common host cell receptor protein, angiotensin converting enzyme 2 (ACE2) [29].
  • ACE2 angiotensin converting enzyme 2
  • the trimeric S protein of the virus binds to the ACE2 receptor and is cleaved by the host protease into the S1 polypeptide containing the receptor binding domain (RBD) and the S2 polypeptide responsible for mediating the fusion of the virus with the cell membrane [30].
  • RBD receptor binding domain
  • the specific interaction between S1 and ACE2 triggers the conformational change of the S2 subunit, which leads to the fusion of the viral envelope and cell membrane or lysosomal membrane and release of viral nucleic acid into the cytoplasm [31].
  • RBD-specific T cells are the most widely distributed. After two weeks of recovery, the level of cellular immunity in the follow-up patients was significantly reduced. RBD can not only cause humoral immunity and produce neutralizing antibodies, but also induce T cell immune response. Therefore, RBD protein is an effective target of SARS-CoV-2 vaccine.
  • a particularly preferred solution of the invention is the RBD-Fe4-Fc fusion protein, which compares to the greatest extent the pharmacodynamic advantages of the antigen-antibody complex vaccine.
  • the Fc fusion protein vaccine has Security and controllability.
  • RBD receptor binding domain
  • SARS-CoV-2 RBD coronavirus spike protein
  • ACE2 coronavirus spike protein
  • SARS-CoV-2 RBD coronavirus spike protein
  • ACE2 angiotensin converting enzyme 2
  • the trimeric S protein of the virus binds to the ACE2 receptor and is cleaved by the host protease into the S1 polypeptide containing the receptor binding domain (RBD) and the S2 polypeptide responsible for mediating the fusion of the virus with the cell membrane.
  • antigen refers to a foreign substance recognized (specifically bound) by an antibody or T cell receptor, but it cannot definitively induce an immune response. Exogenous substances that induce specific immunity are called “immune antigens” or “immunogens.”
  • a "hapten” refers to an antigen that cannot elicit an immune response by itself (although a combination of several molecules of hapten, or a combination of a hapten and a macromolecular carrier can elicit an immune response).
  • polypeptide encompass chains of amino acids of any length, wherein relatively short (for example, shorter than 100 amino acids) amino acid chains are commonly referred to as peptides.
  • the chain may be straight or branched, it may contain modified amino acids, and/or non-amino acids may be intervened.
  • antibody means an immunoglobulin molecule, and refers to any form of antibody that exhibits the desired biological activity. Including but not limited to monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and multispecific antibodies (such as bispecific antibodies), and even antibody fragments.
  • the full-length antibody structure preferably comprises 4 polypeptide chains, usually 2 heavy (H) chains and 2 light (L) chains connected to each other by disulfide bonds. Each heavy chain contains a heavy chain variable region and a heavy chain constant region. Each light chain contains a light chain variable region and a light chain constant region. In addition to this typical full-length antibody structure, its structure also includes other derivative forms.
  • complete antibodies can be classified into five classes of antibodies: IgA, IgD, IgE, IgG, and IgM, among which IgG and IgA can be further divided into subclasses (isotypes), such as IgG1, IgG2 , IgG3, IgG4, IgA1 and IgA2.
  • the heavy chains of the five types of antibodies are classified into ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ chains, respectively.
  • the amino acid sequence of the constant region of its light chain the light chain of an antibody can be classified into ⁇ and ⁇ .
  • variable region refers to the domain in the heavy or light chain of an antibody that is involved in the binding of the antibody to the antigen.
  • constant region refers to such amino acid sequences on the light chain and heavy chain of an antibody that do not directly participate in the binding of the antibody to the antigen, but exhibit a variety of effector functions, such as antibody-dependent cytotoxicity.
  • Fc region is used to define the C-terminal region of an immunoglobulin heavy chain.
  • the "Fc region” can be a native sequence Fc region or a variant Fc region.
  • the Fc region of a human IgG heavy chain is generally defined as extending from the amino acid residue at position 226 of Cys or from Pro230 to its carboxy terminus.
  • the numbering of residues in the Fc region is like the EU index in Kabat. Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991
  • the Fc region of IgG usually has two constant regions, CH 2 and CH 3 .
  • Fc receptor refers to a receptor that binds to the Fc region of an antibody. Natural sequence human FcR is preferred, and receptors ( ⁇ receptors) that bind to IgG antibodies are preferred, which include Fc ⁇ RI, Fc ⁇ RII and Fc ⁇ RIII subtypes, and variants of these receptors. Other FcRs are included in the term “FcR”.
  • the term also includes the neonatal receptor (FcRn), which is responsible for the transport of maternal IgG to the fetus (Guyer et al., Journal of Immunology 117:587 (1976) and Kim et al., Journal of Immunology 24:249 (1994)).
  • FcRn neonatal Fc receptor
  • the neonatal Fc receptor (FcRn) plays an important role in the metabolic fate of IgG antibodies in the body. FcRn functions to rescue IgG from the lysosomal degradation pathway, thereby reducing its clearance in serum and increasing its half-life. Therefore, the in vitro FcRn binding properties/characteristics of IgG indicate its in vivo pharmacokinetic properties in the blood circulation.
  • engineered Fc polypeptide engineered Fc region
  • engineered Fc engineered Fc
  • Fc fusion protein refers to a new type of recombinant protein produced by fusing a certain biologically active functional protein with an Fc fragment using genetic engineering and other technologies. It not only retains the biological activity of the functional protein molecule, but also has some antibodies. Properties, such as the binding of FcRs and related biological functions mediated.
  • effector function refers to those biological activities attributable to the Fc region of an antibody, which vary with antibody isotype.
  • antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor (such as CD16, CD32, CD64) binding, antibody-dependent cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP) , Cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, down-regulation of cell surface receptors (such as B cell receptors) and B cell activation.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • cytotoxic cells such as NK cells, neutrophils, and macrophages.
  • Ig on the Fc ⁇ receptor enables these cytotoxic effector cells to specifically bind to the target cell carrying the antigen, and then kill the target cell using, for example, a cytotoxin.
  • an in vitro ADCC assay can be performed, such as the in vitro ADCC assay described in U.S. Patent No. 5,500,362 or 5,821,337 or U.S. Patent No. 6,737,056 (Presta), and the method described in the Examples of this application .
  • Useful effector cells for such assays include PBMC and NK cells.
  • “Complement dependent cytotoxicity” or “CDC” refers to the lysis of target cells in the presence of complement.
  • the activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (of the appropriate subclass), wherein the antibody binds to its corresponding antigen.
  • C1q the first component of the complement system
  • CDC assays can be performed, such as the CDC assays described in Gazzano-Santoro et al., J. Immunol Methods 202:163 (1996), such as the methods described in the examples of this application, such as The method described in U.S. Patent No. 6,194,551B1 and WO1999/51642, in which polypeptide variants with altered Fc region amino acid sequence (polypeptides with variant Fc region) and polypeptide variants with enhanced or reduced C1q binding are described .
  • Human immune response is an antibody-mediated immune response and involves the introduction and production of antibodies that recognize and bind to the antigen in the immunogenic composition of the present invention with a certain affinity.
  • Cell-mediated immune response is composed of T cells and / Or other leukocyte-mediated immune responses.
  • Cell-mediated immune response is induced by providing epitopes associated with major histocompatibility complex (MHC) class I or class II molecules, CD1 or other atypical MHC-like molecules.
  • MHC major histocompatibility complex
  • conjugate refers to a protein/peptide covalently conjugated to other molecules.
  • immunogenic composition refers to any pharmaceutical composition containing an antigen such as a microorganism or its components, which composition can be used to induce an immune response in an individual.
  • Immunogenicity means an antigen (or epitope of an antigen) such as the coronavirus spike protein receptor binding region or a glycoconjugate or immunogenic composition containing the antigen in a host (e.g., lactating The ability of animals) to induce humoral or cell-mediated immune responses or both.
  • a “protective” immune response refers to the ability of an immunogenic composition to induce a humoral or cell-mediated immune response, or both, used to protect an individual from infection.
  • the protection provided does not have to be absolute, that is, it does not have to completely prevent or eradicate the infection, as long as there is a statistically significant improvement relative to a control population of individuals (for example, infected animals not administered a vaccine or immunogenic composition) . Protection can be limited to alleviating the severity of infection symptoms or rapid onset.
  • Immunogenic amount and “immune effective amount” are used interchangeably herein, and refer to an antigen or immunogenic composition sufficient to elicit an immune response (cell (T cell) or humoral (B cell or antibody) response or two Or, as measured by standard determinations known to those skilled in the art).
  • the effectiveness of an antigen as an immunogen can be measured by a proliferation assay, by a cytolysis assay, or by measuring the level of B cell activity.
  • the present invention is a technological invention.
  • the inventors have discovered that the immunogenicity of the protein/peptide antigen is enhanced by fusion of the modified antibody Fc fragment.
  • the above-mentioned Fc fragment is changed due to its amino acid sequence and/or glycosylation form. It has improved binding ability to Fc receptor and/or complement protein C1q.
  • immunogenicity-enhanced protein/peptide antigen of the present invention is immunogenicity-enhanced protein/peptide antigen of the present invention.
  • the starting protein/peptide antigen is the ACE2 receptor binding domain (RBD) of the coronavirus spike protein.
  • RBD ACE2 receptor binding domain
  • the Fc fragment is the Fc receptor CD32a, CD32b, and CD64 binding enhancement fragment/complement C1q binding enhancement fragment; its amino acid sequence is shown in SEQ ID NO: 30.
  • the modified antibody Fc fragments are Fc receptors CD16a, CD32a, CD32b and CD64 binding enhancing fragments/complement C1q binding enhancing fragments; its amino acid sequence is shown in SEQ ID NO: 30, and Fucose is used. Produced by knockout CHO cells.
  • Coronaviruses mediate virus invasion through the binding of spike protein (S protein) to host cell receptors, and determine the tissue or host tropism of the virus.
  • the host cell receptor protein of SARS-CoV-2 is angiotensin converting enzyme 2 (ACE2).
  • ACE2 angiotensin converting enzyme 2
  • the trimeric spike protein (S protein) of the virus binds to the ACE2 receptor and is cleaved by the host protease into the S1 polypeptide containing the receptor binding domain (SARS-COV-2 RBD) and is responsible for mediating the virus with the cell membrane
  • SARS-COV-2 RBD receptor binding domain
  • the immunogenic composition of the present invention further comprises at least one of adjuvants, buffers, cryoprotectants, salts, divalent cations, non-ionic detergents, free radical oxidation inhibitors, diluents or carriers A sort of.
  • An adjuvant is a substance that enhances the immune response when administered with an immunogen or antigen.
  • the immunogenic composition of the present invention may or may not contain a vaccine adjuvant.
  • Adjuvants that can be used in the composition of the present invention include, but are not limited to: at least one of MF59, QS-21 or MPL.
  • the adjuvant in the immunogenic composition of the present invention is an aluminum-based adjuvant.
  • the adjuvant used will depend on the individual to whom the immunogenic composition is administered, the prescribed route of injection, and the number of injections.
  • the immunogenic composition may optionally include a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier includes the carriers used in the pharmacopoeias of various countries for animals (including humans and non-human mammals).
  • the term carrier can be used to refer to diluents, adjuvants, excipients or vehicles with which the pharmaceutical composition is administered. Water, saline solutions, and aqueous dextrose and glycerol solutions can be used as liquid carriers especially for injection solutions.
  • the immunogenic composition of the present invention may also contain one or more additional immunogenic agents.
  • the immunogenic composition of the present invention used for treatment or prophylactic treatment can be administered to the oral cavity/esophagus, respiratory tract, urogenital tract by intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection; or via mucosal administration. Intranasal administration of vaccines is preferred for the treatment of certain diseases, such as pneumonia or otitis media.
  • the vaccine of the present invention can be administered in a single dose, its components can also be co-administered at the same time or in time sharing. In addition to a single route of administration, two different routes of administration can be used.
  • the optimal amount of the components for a particular immunogenic composition can be determined by standard studies involving observation of the appropriate immune response in the individual. After the initial vaccination, the individual can receive one or several well-spaced booster immunizations.
  • the protein/peptide antigen and immune complex of the present invention can prevent or treat diseases caused by pathogens, especially coronaviruses, and more especially diseases caused by SARS-CoV-2 virus.
  • the protein/peptide antigen and immune complex of the present invention can prevent or treat tumor diseases. It can also be used to immunize animals to produce neutralizing antibodies.
  • Example 1 SARS-CoV-2 RBD and RBD-Fc fusion protein expression vector construction and protein production
  • the SARS-CoV-2-Spike-RBD sequence (SEQ ID NO: 5) was obtained by PCR amplification (the PCR amplification template was from Beijing Yiqiao Shenzhou Technology Co., Ltd., the same below), including the signal peptide sequence (SEQ ID NO: 3) and SARS-CoV-2-Spike-RBD sequence (SEQ ID NO:1), inserted into Hind III+Xba I (source: Fermentas, the same below) digested pSE vector (source: Shenzhou) by in-fusion method
  • the pSE-CoV-2-RBD expression vector (SEQ ID NO: 5) was obtained from Cell Engineering Co., Ltd., the same below).
  • the pSE-CoV-2-RBD plasmid was extracted, transfected into HEK-293 cells (source: Invitrogen, the same below), cultured and expressed for 7 days, and purified to obtain high-purity SARS-CoV-2 RBD protein.
  • the SARS-CoV-2-Spike-RBD sequence was amplified by PCR and inserted into Afe I digested FastAP dephosphorized by in-fusion method, including signal peptide (SEQ ID NO: 3), linker (SEQ ID NO: 7) and The pSE-mFc expression vector (SEQ ID NO: 11) was obtained from the pSE-mFc vector (source: Shenzhou Cell Engineering Co., Ltd.) of the mouse IgG1 constant region sequence (SEQ ID NO: 9).
  • the pSE-CoV-2-RBD-mFc plasmid was extracted, transfected into HEK-293 cells, cultured and expressed for 7 days, and purified by a protein A purification column to obtain high-purity RBD-mFc protein.
  • the SARS-CoV-2-Spike-RBD sequence was amplified by PCR and inserted into Afe I digested FastAP dephosphorized by in-fusion method, including signal peptide (SEQ ID NO: 3), linker (SEQ ID NO: 7) and The pSE-CoV-2-RBD-Fc expression vector (SEQ ID NO: 15) was obtained from the pSTEP2-Fc vector (source: Shenzhou Cell Engineering Co., Ltd.) of the human IgG1 constant region sequence (SEQ ID NO: 13).
  • the pSE-CoV-2-RBD-Fc plasmid was extracted, transfected into HEK-293 cells, cultured and expressed for 7 days, and purified by a protein A purification column to obtain high-purity RBD-Fc protein.
  • nucleotide mutations in the constant region of the IgG1 subtype were carried out with reference to the literature [36, 37] to obtain the genetically engineered heavy chain IgG1 constant region nucleotide sequence (Fc-Ce3, SEQ ID NO: 17).
  • SARS-CoV-2-Spike-RBD-Ce3-Fc sequence (SEQ ID NO: 19) was amplified by PCR, and inserted into the pSE vector digested with HindIII+Xba I by in-fusion method to obtain pSE-nCoV-2- RBD-Fc-Ce3 expression vector (SEQ ID NO: 19).
  • Amplification primers :
  • the pSE-nCoV-2-RBD-Fc-Ce3 plasmid was extracted, transfected into HEK-293 cells, cultured and expressed for 7 days, and purified by a protein A purification column to obtain high-purity RBD-Fc-Ce3.
  • the pSE-nCoV-2-RBD-Fc-Ce3 plasmid was extracted and transfected into HEK-293 cells with fut8 gene knockout (source: Shenzhou Cell Engineering Co., Ltd.) for culture and expression 7 Today, a protein A purification column was used to obtain defucosylated high-purity RBD-Fc-Fe4.
  • nucleotide mutations in the constant region of the IgG4 subtype were carried out with reference to the literature [38] to obtain the genetically engineered heavy chain IgG4 constant region nucleotide sequence (Fd11-IgG4, SEQ ID NO:21).
  • SARS-CoV-2-Spike-RBD-Fd11-IgG4 sequence (SEQ ID NO: 23) was amplified by splicing PCR, and inserted into the pSE vector digested with Hind III+Xba I by in-fusion method to obtain pSE-CoV- 2-RBD-Fc-Fd11-IgG4 expression vector (SEQ ID NO: 23).
  • the pSE-CoV-2-RBD-Fc-Fd11-IgG4 plasmid was extracted, transfected into HEK-293 cells, cultured and expressed for 7 days, and purified by a protein A purification column to obtain high-purity RBD-Fc-Fd11-IgG4 protein.
  • Example 2 SARS-CoV-2 RBD and RBD-Fc fusion protein Fc receptor binding test
  • Avidin protein (source: Thermo Fisher, the same below) at a concentration of 10 ⁇ g/mL was coated on a 96-well plate, 100 ⁇ L per well, and coated overnight at 2-8°C. The plate was washed the next day and sealed at room temperature for 1 hour, and then 100 ⁇ L of different biotin-labeled Fc receptor proteins (all from Beijing Yiqiao Shenzhou Technology Co., Ltd.): 5 ⁇ g/mL mCD16a-AVI-His(V158)+BirA, 5 ⁇ g/ mL of mCD32b-AVI-His+BirA or 0.5 ⁇ g/mL of mCD64-AVI-His+BirA, and no protein as a blank control.
  • biotin-labeled Fc receptor proteins all from Beijing Yiqiao Shenzhou Technology Co., Ltd.
  • the protein concentration when the detection is combined with mCD16a and mCD32b is 40 ⁇ g/mL, 10 ⁇ g/mL, 2.5 ⁇ g/mL, 0.6250 ⁇ g/mL, 0.1563 ⁇ g/mL, 0.0391 ⁇ g/mL, 0.0098 ⁇ g/mL and 0.0024 ⁇ g/ml; the detection is the same
  • the protein concentration of mCD64 binding is 10 ⁇ g/mL, 2.5 ⁇ g/mL, 0.6250 ⁇ g/mL, 0.1563 ⁇ g/mL, 0.0391 ⁇ g/mL, 0.0098 ⁇ g/mL and 0.0024 ⁇ g/ml.
  • the plate After incubating for 1 hour at room temperature, the plate was washed to remove unbound proteins, and 100 ⁇ L of horseradish peroxidase labeled CoV-2-RBD antibody CoV-2-HB27-Fd11-IgG4 without Fc receptor binding function at a concentration of 0.1 ⁇ g/mL was added. (Source: Shenzhou Cell Engineering Co., Ltd., as a detection antibody). After incubating for 1 hour, the plate was washed repeatedly, and the substrate color developing solution was added for color development. After termination, the OD 450 was read by the microplate reader, and the data of the sample OD 450 -blank control OD 450 was used for analysis.
  • the protein concentration when detecting binding to CD16a is 40 ⁇ g/mL, 10 ⁇ g/mL, 2.5 ⁇ g/mL, 0.625 ⁇ g/mL, 0.156 ⁇ g/mL, 0.039 ⁇ g/mL, 0.01 ⁇ g/mL and 0.002 ⁇ g/ml; the detection is the same as CD32a and
  • the protein concentration when CD32b is bound is 40 ⁇ g/mL, 10 ⁇ g/mL, 2.5 ⁇ g/mL, 0.625 ⁇ g/mL and 0.156 ⁇ g/mL; the protein concentration when detecting binding with CD64 is 10 ⁇ g/mL, 2.5 ⁇ g/mL, 0.625 ⁇ g/mL , 0.156 ⁇ g/mL, 0.039 ⁇ g/mL, 0.01 ⁇ g/mL and 0.002 ⁇ g/mL.
  • the plate After incubating for 1 hour at room temperature, the plate was washed to remove unbound proteins, and 100 ⁇ L of horseradish peroxidase labeled CoV-2-RBD antibody CoV-2-HB27-Fd11-IgG4 without Fc receptor binding function at a concentration of 0.1 ⁇ g/mL was added. . After incubating for 1 hour, the plate was washed repeatedly, and the substrate color developing solution was added for color development. After termination, the OD 450 was read by the microplate reader, and the data of the sample OD 450 -blank control OD 450 was used for analysis.
  • RBD-Fc-Fe4 has the strongest binding ability to CD16, CD32a and CD32b, which is better than RBD-Fc and RBD-Fc-Ce3, RBD-Fc- Fd11-IgG4 and RBD protein hardly bind to CD16, CD32a and CD32b.
  • RBD-Fc-Fe4, RBD-Fc-Ce3 and RBD-Fc have high and similar binding capacity to CD64, while RBD-Fc-Fd11-IgG4 protein binds weakly to CD64.
  • RBD protein is presumed to be non-specific at high concentrations Combine.
  • RBD-Fc-Fe4 and RBD-Fc-Ce3 The binding of RBD-Fc-Fe4 and RBD-Fc-Ce3 to C1q is slightly stronger than that of RBD-Fc, while RBD and RBD-Fc-Fd11-IgG4 have no binding to C1q.
  • Example 3 SARS-CoV-2 RBD and RBD-Fc fusion protein cell phagocytosis test
  • Monocyte-derived macrophages (MDM, monocyte-derived macrophages) are obtained from PBMCs isolated from fresh human peripheral blood through cell attachment methods, and then induced by inducing media for 7 days. By labeling the FITC fluorescent dye on the antigen protein and co-incubating with macrophages, the phagocytosis of the cells was observed under a confocal microscope.
  • 3 ⁇ 10 4 /well was seeded with MDM cells (source: Shenzhou Cell Engineering Co., Ltd. induced), 50 ⁇ L/well, cultured overnight in 1640+10% FBS+1 ⁇ PS medium (source: Shenzhou Cell Engineering Co., Ltd.), Then 50 ⁇ L/well was added with 20 ⁇ g/mL FITC-labeled different antigen proteins. Incubate the 96-well plate in an incubator at 37°C and 5% CO 2 in the dark for 2.5 hours. After the effect is over, the 96-well plate is cleared away, the unbound antigen protein is washed with PBS, and 4% paraformaldehyde (source: Sigma-aldrich) is added to 50 ⁇ L/well. Fix for 15min at 4°C, discard the fixative, wash with PBS, add 50 ⁇ L/well to PBS, use the confocal microscope FITC channel fluorescence to take pictures, use Image pro plus software to analyze the fluorescence intensity.
  • MDM cells source: Shenzhou Cell Engineering
  • Aluminum-containing adjuvant antigen preparation RBD-his (source: Beijing Yiqiao Shenzhou Technology Co., Ltd.) and RBD-mFc antigen were diluted with PBS to 0.06mg/mL, aluminum adjuvant (source: Shenzhou Cell Engineering Co., Ltd., the same below ) Dilute with PBS to 1mg/mL. The diluted antigen and aluminum adjuvant are mixed in equal volumes. Obtained 0.03 mg/mL of aluminum-containing adjuvant antigen.
  • RBD, RBD-Fc, RBD-Fc-Ce3, RBD-Fc-Fe4 antigens containing MF59 (source: Shenzhou Cell Engineering Co., Ltd., the same below) and aluminum adjuvant mixed adjuvant: Take 1.5 mL aluminum adjuvant and add 1.5 mL MF59 adjuvant is added with 0.18 mL of antigen protein with a concentration of 0.53 mg/mL to obtain a protein concentration of 0.03 mg/mL, and the adjuvant is the antigen of the mixed adjuvant.
  • mice were selected from Balb/c mice for 4-6 weeks (source: China Institute for Food and Drug Control), and intraperitoneally injected 0.1 mL of aluminum-containing adjuvant antigen or antigen with mixed adjuvants, which contained 3 ⁇ g of antigen, respectively on the 14th day And boost immunization on the 28th day.
  • the RBD-mFc protein at a concentration of 5 ⁇ g/mL was coated on a 96-well plate, 100 ⁇ L/well, and CD155(D1)-mFc (source: Shenzhou Cell Engineering Co., Ltd.) was used as an irrelevant control with the same label. After incubating for 2 hours at room temperature, the plate was washed and blocked with 2% BSA (bovine serum albumin), and incubated for 1 hour at room temperature.
  • BSA bovine serum albumin
  • the immune serum titer and neutralizing titer of different RBD-Fc fusion proteins are better than those of RBD protein, among which RBD-Fc-Ce3
  • the serum neutralization titer of RBD-Fc-Fe4 is higher, indicating that the RBD-Fc fusion protein can activate a stronger humoral immune response (Figure 5).

Abstract

一种增强蛋白/肽抗原免疫原性的方法，其中该蛋白/肽抗原与经过改造的抗体Fc片段形成融合蛋白，该Fc片段因其氨基酸序列和/或糖基化形式改变，与其天然形式相比，具有提高的与Fc受体和/或补体蛋白C1q的结合能力。以所述融合蛋白作为免疫原制备的SARS-CoV-2疫苗同Fc受体具有较高的结合能力，可维持长时程的体液免疫和细胞免疫反应，免疫动物可产生较高滴度的中和抗体。所述重组蛋白疫苗可用于预防SARS-CoV-2感染相关疾病。

Description

通过与改变的Fc片段形成融合蛋白增强蛋白/肽抗原免疫原性的方法
相关申请的交叉引用
本申请要求2020年05月11日提交的中国专利申请202010394463.6的权益，该申请的内容通过引用被合并于本文。
技术领域
本发明属于免疫学领域，具体地，涉及增强蛋白/肽抗原免疫原性的方法，其中该蛋白/肽抗原与经过改造的抗体Fc片段形成融合蛋白，该Fc片段因其氨基酸序列和/或糖基化形式改变，与其天然形式相比，具有提高的与Fc受体和/或补体蛋白C1q的结合能力。以本发明的融合蛋白作为免疫原制备的SARS-CoV-2疫苗同Fc受体具有较高的结合能力，可维持长时程的体液免疫和细胞免疫反应，免疫动物可产生较高滴度的中和抗体。所述疫苗可用于预防SARS-CoV-2感染相关疾病。
背景技术
1.Fc受体融合蛋白疫苗以及FcRs结合增强Fc片段融合蛋白疫苗
B细胞介导的体液免疫是疫苗介导的机体保护机制之一。已有研究表明，Fc与Fc受体(FcRs)及补体受体(CR)相互作用可介导更优的抗原捕获及呈递、促进B细胞亲和力成熟及高亲和力抗体的产生[1-6]。此外，Fc与滤泡树突细胞(fDC)上的受体结合(通过FcR或CR介导)后，展示于fDC表面，这部分抗原对于维持抗原的长期存在、维持抗原特异性B细胞的存活极为重要[7]。
此外，细胞毒性T淋巴细胞(CTL)对抵抗病毒感染，清除病毒感染细胞具有重要的作用。抗原交叉递呈机制可使外源性抗原进入细胞内源性的加工和递呈机制，从而使外源抗原肽展示在MHC I类分子上被T细胞识别，启动CTL细胞应答。外源抗原的交叉递呈对有效激活CTL从而引发抗病毒免疫反应有着重要意义，因此增强亚单位疫苗的交叉递呈是提升疫苗免疫效果的有效策略之一。树突状细胞(DC)是目前已知功能最强的专职抗原递呈细胞，也是主要进行交叉递呈的细胞。外源抗原进入DC的方式主要有三种:吞噬作用(胞吞)、胞饮和受体介导的内吞作用。目前已知的与外源抗原交叉递呈有关的内吞受体，包括C-型凝集素受体(CLR)、识别免疫复合物IgG的Fc受体(FcRs)、识别凋亡细胞的清道夫受体、趋化因子受体等，这些受体介导抗原内吞后进入特定的内涵体才能与MHCⅠ类分子结合，从而激活CD8 +T细胞。抗原抗体复合物(免疫复合物)可被DC细胞的FcRs识别，由此引发的交联可将抗原内化，并将抗原进行交叉加工递呈，特异性激活CTL反应[8-10]。这种由FcRs介导的抗原交叉递呈已证实可诱导强CTL反应[11]。
人体中与IgG结合的Fc受体(FcγR)主要包含FcγRI(CD64)、FcγRIIA(CD32a)、FcγRIIB(CD32B)、FcγRIII(CD16)等[12]。其中FcγRIIB为抑制性受体，主要表达在B细胞、巨噬及肥大细胞上[13]。其又可分为FcγRIIB-1、FcγRIIB-2。FcγRIIB-1仅表达在B细胞上，在B细胞发育过程中控制着B细胞的过度激活以及对自身抗原的识别，其通过胞内的受体酪氨酸抑制基序(ITIM)传递B细胞凋亡信号实现B细胞的阴性选择过程，调控B细胞的发育过程。FcγRIIB-2表达在除NK及T细胞的其它免疫细胞上，通过受体的交联可以有效的诱导抗原抗体复合物的吞噬作用[14,15]。其余几种Fc受体均为激活性受体，其中CD64主要表达在单核、巨噬、DC等细胞上，CD32a主要表达在中性粒、单核、巨噬、DC等细胞上，CD16a主要表达在NK、单核、巨噬细胞[15]。激活型受体在识别抗原抗体复合物后，胞内受体酪氨酸激活基序(ITAM)决定着抗原提呈细胞APC启动抗原的摄取以及摄取后经MHC类分子行使的抗原提呈功能[16]。
Fc融合蛋白是指利用基因工程等技术将某种具有生物活性的功能蛋白与Fc片段融合而 产生的新型重组蛋白，其不仅保留了功能蛋白分子的生物学活性，还具有一些抗体的性质，如同FcRs的结合及介导的相关生物学功能等。抗原-Fc融合蛋白可作为抗原运载工具，借助Fc片段靶向结合抗原递呈细胞，缩短抗原在血浆中的游离时间，提高抗原半衰期，从而加强抗原递呈和抗原交叉递呈反应。在此基础上对Fc片段进行增强FcRs结合的改造，获得Fe4-Fc改造分子，可实现增加Fc与补体蛋白C1q及Fc受体CD16、CD32、CD64等结合的作用[17-21]，则有可能进一步提升Fc及其受体介导的抗原捕获及呈递、提升B细胞成熟及高亲和力抗体的产生、维持长时程的体液免疫、提升抗原交叉递呈介导的CTL免疫反应，进而增强Fc融合蛋白类疫苗的免疫效果。
2.现有的Fc融合蛋白类疫苗临床案例
目前在临床上，开展的Fc融合蛋白类疫苗的相对较少。一项IIb期临床中，慢性乙型cHBV感染者，按照60μg/4周给予一种YIC免疫原性复合物(由酵母表达的乙肝表面抗原HBsAg与HBsAg免疫的人血清中和抗体HBIG以特定比例混合孵育，并加入铝佐剂)治疗，与仅给予铝佐剂的对照组相比，其血清乙肝病毒E抗原(HBeAg)转阴率有明显提升(21.8％vs 9％)，病毒滴度显著下降，并且伴随抗E抗原抗体的产生[22]。于此同时体外条件下，YIC可增加cHBV感染病人DC细胞的成熟(CD83高表达)、抗原识别与提呈(HLA-II、CD86、CD80、CD40的标志物的表达)，并分泌更多炎性因子(IL-12)。病人的DC-PBMC混合细胞在YIC刺激下比单独HBsAg抗原刺激产生更多的T淋巴细胞细胞因子(Th1细胞：IL-2，IFNγ)(Th2细胞:IL-5,IL-10)[23]。然而，YIC的过度刺激可能会引发机体的免疫疲劳，从而降低细胞免疫反应[24]。因此，为了实现更好的免疫效果，合适的免疫方案也尤为重要。
其它一些病毒抗原-Fc融合蛋白的疫苗(RSV，HBV，DENV，TB)也在动物模型中展开了尝试，实现了有效的免疫***激活，同时产生的抗体可降低血清中病毒抗原水平[25-28]。
Fc融合蛋白类疫苗对免疫***的激活所引发的安全性风险也是值得考虑的议题。上文中提到的慢性乙肝cHBV感染者，给予YIC治疗后一段时间内，少量病人出现丙氨酸转移酶(ALT)的瞬时上升。与仅给予铝佐剂的对照组相比，发生ALT上升病人的比例相近。转氨酶水平的上升在一定程度上反映着肝损伤，但随后的观察显示，ALT的可恢复到正常水平[24]。其IIa期临床也观察到HBeAg抗原血清转阴的病人中部分观察到ALT升高的现象[23]。从YIC的IIb期临床副反应(AE)数据可见，YIC 30μg剂量组，YIC 60μg剂量组，铝佐剂对照组发生严重AE的比例相近(3.6％vs 5％vs 5.1％)，YIC组最常见的AE为注射点相关反应，包括皮疹、肿胀、瘙痒等与炎性相关反应，其他***性的AE如发烧、头疼、恶心等与对照组均无明显差异[22]，因此包含Fc的免疫复合物在临床中的安全性相对较好，无因过度激活免疫***产生的相关严重副反应发生。
发明内容
在一个方面，本发明提供了一种增强蛋白/肽抗原的免疫原性的方法，该方法包含将蛋白/肽抗原与经过改造的抗体Fc片段融合，该Fc片段与其天然形式相比，具有提高的与Fc受体和/或补体蛋白C1q的结合能力，可模拟抗原抗体复合物增强DC/B抗原呈递细胞的吞噬，增强抗原免疫效果。
在一个实施方式中，所述方法中所述的蛋白/肽抗原为病原体相关的蛋白/肽抗原或肿瘤相关蛋白/肽抗原。
在一个实施方式中，所述方法中所述的病原体选自：
冠状病毒，人免疫缺陷性病毒HIV-1，人单纯疱疹病毒，巨细胞病毒，轮状病毒，EB病毒，水痘带状疱疹病毒，肝炎病毒，呼吸道合胞体病毒，副流感病毒，麻疹病毒，流行性腮腺炎病毒，人***瘤病毒，黄病毒或流感病毒，奈瑟氏菌属，莫拉氏菌属，博代氏杆菌属，分枝杆菌属，包括结核分枝杆菌；埃希氏菌属，包括肠毒素大肠埃希菌；沙门氏菌属，李斯特氏菌属，螺杆菌属，葡萄球菌属，包括金黄葡萄球菌，表皮葡萄球菌；疏螺旋体属，衣原体，包括砂眼衣原体，肺炎衣原体；疟原虫，包括恶性疟原虫；弓形虫，念珠菌；
其中的肿瘤选自：
弥漫性大B细胞淋巴瘤、滤泡性淋巴瘤、其他淋巴瘤、白血病、多发性骨髓瘤、间皮瘤、胃癌、恶性横纹肌瘤、肝细胞癌、***癌，乳腺癌、胆管癌和胆囊癌、膀胱癌、脑肿瘤包括神经母细胞瘤、神经鞘瘤、胶质瘤、胶质母细胞瘤和星形细胞瘤、***、结肠癌、黑色素瘤、子宫内膜癌、食管癌、头颈癌、肺癌、鼻咽癌、卵巢癌、胰腺癌、肾细胞癌、直肠癌、甲状腺癌、甲状旁腺肿瘤、子宫肿瘤和软组织肉瘤。
在一个实施方式中，所述方法中的蛋白/肽蛋白抗原选自分泌蛋白或全长膜蛋白，或其功能结构域、突变蛋白、截短蛋白，或由其1个或多个抗原多肽表位拼接组成的改造蛋白。
在一个实施方式中，所述方法中的Fc片段来自人抗体、鼠源抗体、兔源抗体或其他动物抗体的重链恒定区。
在一个实施方式中，所述方法中的Fc片段来自人抗体的IgG、IgM或IgA亚型抗体，
优选地，来自IgG1、IgG2、IgG3或IgG4亚型抗体；
更优选地，是为提高与Fc受体、C1q补体结合功能之目的，而进行氨基酸序列突变和/或糖基化形式改变的IgG1改造Fc片段。
在一个实施方式中，所述方法的Fc受体选自CD16、CD32a、CD32b或CD64。
在一个实施方式中，所述方法中的蛋白/肽抗原为冠状病毒棘突蛋白的ACE2受体结合结构域(RBD)。
在一个实施方式中，所述方法中经过改造的抗体Fc片段为Fc受体CD32a、CD32b和CD64结合增强片段/补体C1q结合增强片段；
其氨基酸序列如SEQ ID NO：30所示。
在一个实施方式中，所述方法中
经过改造的抗体Fc片段为Fc受体CD16a、CD32a、CD32b和CD64结合增强片段/补体C1q结合增强片段；
其氨基酸序列如SEQ ID NO：30所示，且用Fucose敲除的哺乳细胞生产，优选地，哺乳动物细胞为fut8基因敲除的HEK-293细胞。
在一个实施方式中，所述方法中的Fc片段来自鼠抗体IgG，IgM，IgA亚型抗体，
优选地，来自IgG1、IgG2a、IgG2b或IgG3亚型抗体。
在一个实施方式中，所述方法中所述的抗原优选地通过接头和其他大分子缀合，优选地，其他大分子为多糖、肽/蛋白。
在另一个方面，本发明提供一种免疫原性增强的蛋白/肽抗原，其中
该蛋白/肽抗原与经过改造的抗体Fc片段融合，该Fc片段与其天然形式相比，具有提高的与Fc受体和或补体蛋白C1q的结合能力,可模拟抗原抗体复合物增强DC/B抗原呈递细胞的吞噬，增强抗原免疫效果。
在一个实施方式中，所述蛋白/肽抗原中所述的抗原为病原体相关的蛋白/肽抗原、肿瘤相关蛋白/肽抗原。
在一个实施方式中，所述蛋白/肽抗原中所述的病原体选自：
冠状病毒，人免疫缺陷性病毒HIV-1，人单纯疱疹病毒，巨细胞病毒，轮状病毒，EB病毒，水痘带状疱疹病毒，肝炎病毒，呼吸道合胞体病毒，副流感病毒，麻疹病毒，流行性腮腺炎病毒，人***瘤病毒，黄病毒或流感病毒，奈瑟氏菌属，莫拉氏菌属，博代氏杆菌属，分枝杆菌属，包括结核分枝杆菌；埃希氏菌属，包括肠毒素大肠埃希菌；沙门氏菌属，李斯特氏菌属，螺杆菌属，葡萄球菌属，包括金黄葡萄球菌，表皮葡萄球菌；疏螺旋体属，衣原体，包括砂眼衣原体，肺炎衣原体；疟原虫，包括恶性疟原虫；弓形虫，念珠菌；
其中的肿瘤选自：
弥漫性大B细胞淋巴瘤、滤泡性淋巴瘤、其他淋巴瘤、白血病、多发性骨髓瘤、间皮瘤、胃癌、恶性横纹肌瘤、肝细胞癌、***癌，乳腺癌、胆管癌和胆囊癌、膀胱癌、脑肿瘤包括神经母细胞瘤、神经鞘瘤、胶质瘤、胶质母细胞瘤和星形细胞瘤、***、结肠癌、黑色 素瘤、子宫内膜癌、食管癌、头颈癌、肺癌、鼻咽癌、卵巢癌、胰腺癌、肾细胞癌、直肠癌、甲状腺癌、甲状旁腺肿瘤、子宫肿瘤和软组织肉瘤。
在一个实施方式中，所述蛋白/肽抗原中的蛋白抗原选自分泌蛋白或全长膜蛋白，或其功能结构域、突变蛋白、截短蛋白，或由其1个或多个抗原多肽表位拼接组成的改造蛋白。
在一个实施方式中，所述蛋白/肽抗原中的Fc片段来自人抗体、鼠源抗体、兔源抗体或其他动物抗体的重链恒定区。
在一个实施方式中，所述蛋白/肽抗原中的Fc片段来人抗体的IgG、IgM或IgA亚型抗体，
优选地，来自IgG1、IgG2、IgG3或IgG4亚型抗体；
更优选地，是为提高与Fc受体和/或C1q补体结合功能之目的，而进行氨基酸序列突变和/或糖基化形式改变的IgG1改造Fc片段。
在一个实施方式中，所述蛋白/肽抗原中的Fc受体选自CD16、CD32a、CD32b或CD64。
在一个实施方式中，所述蛋白/肽抗原中的抗原为冠状病毒棘突蛋白的ACE2受体结合结构域(RBD)。
在一个实施方式中，所述的蛋白/肽抗原其中
经过改造的抗体Fc片段为Fc受体CD32a、CD32b和CD64结合增强片段/补体C1q结合增强片段；
其氨基酸序列如SEQ ID NO：30所示。
在一个实施方式中，所述的蛋白/肽抗原其中
经过改造的抗体Fc片段为Fc受体CD16a、CD32a、CD32b和CD64结合增强片段/补体C1q结合增强片段；
其氨基酸序列如SEQ ID NO：30所示，且用Fucose敲除的哺乳动物细胞生产，
优选地，哺乳动物细胞为fut8基因敲除的HEK-293细胞。
在一个实施方式中，所述蛋白/肽抗原中的Fc片段来自鼠抗体IgG，IgM，IgA亚型抗体，
优选地，来自IgG1、IgG2a、IgG2b或IgG3亚型抗体。
在一个实施方式中，所述蛋白/肽抗原中所述的抗原优选地通过接头和其他大分子缀合，优选地，其他大分子为多糖、肽/蛋白。
在又一个方面，本发明提供一种缀合物，其包含，优选地通过接头连接的如本发明所述的蛋白/肽抗原和其他大分子，优选地，其他大分子为多糖、肽/蛋白。
在又一个方面，本发明提供一种核酸，其编码本发明所述的蛋白/肽抗原，其为mRNA和/或DNA。
在一个实施方式中，所述核酸序列如SEQ ID NO:31所示。
在又一个方面，本发明提供一种表达载体，其包含本发明所述的核酸。
在又一个方面，本发明提供一种宿主细胞，其包含本发明所述的核酸或本发明所述的表达载体。
在又一个方面，本发明提供一种用于生产本发明所述的蛋白/肽抗原的方法，其包括在适合于前述蛋白质分子表达的条件下培养本发明所述的宿主细胞，和从培养基中回收表达的产物。
在又一个方面，本发明提供一种免疫组合物，其包含
a)本发明所述的蛋白/肽抗原、本发明所述的缀合物、本发明所述的核酸或本发明所述的表达载体；和/或
b)佐剂；和
c)药学上可接受的载体、赋形剂或稳定剂，优选为
冻干制剂或水溶液形式的药学上可接受的载体、赋形剂或稳定剂。
在一个实施方式中，所述免疫组合物中的佐剂可选自铝佐剂、MF59、QS-21或MPL的至少一种。
在又一个方面，本发明提供本发明所述的蛋白/肽抗原、本发明所述的缀合物、本发明所述的核酸、本发明所述的表达载体、或本发明所述的免疫组合物，其用于预防病原体，优选冠状病毒、更优选SARS-CoV-2引起的疾病\肿瘤的应用。
在又一个方面，本发明提供本发明所述的蛋白/肽抗原、本发明所述的缀合物、本发明所述的核酸、本发明所述的表达载体、或本发明所述的免疫组合物，其用于制备预防病原体，优选冠状病毒、更优选SARS-CoV-2引起的疾病\肿瘤的疫苗中的应用。
在又一个方面，本发明提供一种免疫组合，其包含
本发明所述的蛋白/肽抗原、本发明所述的缀合物、本发明所述的核酸、本发明所述的表达载体、或本发明所述的免疫组合物；以及
一种或多种另外的免疫原性剂。
在又一个方面，本发明提供一种试剂盒，其包含
本发明所述的蛋白/肽抗原、本发明所述的缀合物、本发明所述的核酸、本发明所述的表达载体、或本发明所述的免疫组合物；
优选地，
还进一步包含给予疫苗的装置。
在又一方面，本发明提供一种预防病原体，优选冠状病毒、更优选SARS-CoV-2引起的疾病\预防肿瘤的方法，其包含给予受治疗者本发明所述的蛋白/肽抗原、本发明所述的缀合物、本发明所述的核酸、本发明所述的表达载体、或本发明所述的免疫组合物、本发明所述的疫苗组合或本发明所述的试剂盒。
在又一方面，本发明提供一种免疫动物的方法，其包含给予动物本发明所述的蛋白/肽抗原、本发明所述的缀合物、本发明所述的核酸、本发明所述的表达载体、或本发明所述的免疫组合物、本发明所述的疫苗组合或本发明所述的试剂盒，以产生中和抗体。
附图说明
图1：RBD-mFc同鼠Fc受体的结合。
图2：不同RBD-Fc融合蛋白同人Fc受体及C1q的结合。
图3：不同RBD-Fc融合蛋白MDM吞噬试验。
图4：RBD-his和RBD-mFc免疫血清抗体滴度及中和滴度比较(Mean±SEM，n＝5)。
图5：RBD和不同RBD-Fc融合蛋白免疫血清抗体滴度及中和滴度比较(Mean±SEM，n＝5)。
具体实施方式
发明人开创性地发现通过将蛋白/肽抗原与经过改造的抗体Fc片段融合，其免疫原性得以增强，上述Fc片段由于其氨基酸序列和/或糖基化形式改变具有提高的与Fc受体和/或补体蛋白C1q的结合能力。
发明人将此发现用于新型冠状病毒(Severe Acute Respiratory Syndrome Coronavirus 2，SARS-CoV-2)疫苗。
SARS-CoV-2和SARS-CoV具有共同的宿主细胞受体蛋白，即血管紧张素转化酶2(ACE2)[29]。病毒的三聚体S蛋白同ACE2受体结合后被宿主蛋白酶切割为包含受体结合域(Receptor binding domain,RBD)的S1多肽和负责介导病毒同细胞膜融合的S2多肽[30]。S1和ACE2之间的特异性相互作用会触发S2亚基的构象变化，从而导致病毒包膜和细胞膜或溶酶体膜融合并释放病毒核酸进入细胞质[31]。数据表明COVID-19患者，尤其是在重症患者中，伴随肺炎症状，淋巴细胞显著降低，血浆促炎因子显著增加，提示了免疫***在疾病进程中发挥着重要的作用[32-34]。对23例COVID-19患者出现症状后血清抗体的分析表明，大多数患者在出现症状10天后发生针对RBD蛋白的抗体反应 [35]。发病早期血清中RBD蛋白抗体阳性患者比例高于N蛋白抗体阳性患者比例，说明机体可能先产生具有中和作用的抗 体，以抑制病毒通过RBD侵入细胞。对细胞免疫的分析表明，刚出院患者针对不同抗原的特异性T细胞与未感染者的T细胞有显著差异，其中RBD特异性T细胞分布最广。康复两周后的随访患者细胞免疫水平则明显降低。RBD不仅可引起体液免疫，产生中和抗体，而且还可诱导T细胞免疫应答，因此RBD蛋白是SARS-CoV-2疫苗的有效靶标。
发明的一个特别优选的方案是RBD-Fe4-Fc融合蛋白，最大程度上比拟了抗原抗体复合物类疫苗的药效优势，通过对抗原抗体复合物的临床AE评估，推测Fc融合蛋白类疫苗具有安全可控性。
定义
除非另有说明，本文使用的所有技术和科学术语具有本发明所属的技术领域的普通技术人员通常理解的含义。为了本发明的目的，进一步定义以下术语。
当用于本文和所附权利要求书中时，单数形式“一”、“一种”、“另一”和“所述”包括复数指代对象，除非上下文明确地另有指示。
术语“包括”、“包含”是指包括具体成分而不排除任何其他的成分。诸如“基本上由……组成”允许包括不损害本发明的新颖或基本特征的其他成分或步骤，即，它们排除损害本发明的新颖或基本的特征的其他未列举的成分或步骤。术语“由……组成”是指包括具体成分或成分组并且排除所有其他成分。
术语“RBD受体结合域(Receptor binding domain,RBD)”在本说明书和所附的权利要求书中特指“冠状病毒棘突蛋白的ACE2受体结合结构域(SARS-CoV-2 RBD)”，以上术语互换使用。SARS-CoV-2和SARS-CoV具有共同的宿主细胞受体蛋白，即血管紧张素转化酶2(ACE2)。病毒的三聚体S蛋白同ACE2受体结合后被宿主蛋白酶切割为包含受体结合域(Receptor binding domain,RBD)的S1多肽和负责介导病毒同细胞膜融合的S2多肽。
术语“抗原”是指一种由抗体或T细胞受体所识别(特异性结合)的外源物质，但是其不能确定性地诱导免疫应答。诱导特异性免疫的外源性物质称为“免疫性抗原”或“免疫原”。“半抗原”是指一种本身不能引发免疫应答(尽管几个分子半抗原的结合物，或半抗原与大分子载体的结合物可引发免疫应答)的抗原。
术语“多肽”、“寡肽”、“肽”和“蛋白质”，涵盖任何长度的氨基酸的链，其中相对短(例如，短于100个氨基酸)氨基酸链通常称为肽。该链可以是直链或支链的，其可包含修饰氨基酸，和/或可间插非氨基酸。
术语“抗体”意指免疫球蛋白分子，是指表现所需生物学活性的抗体的任何形式。包括但不限于单克隆抗体(包括全长单克隆抗体)、多克隆抗体和多特异性抗体(例如双特异性抗体),甚至包括抗体片段。典型地，全长抗体结构优选包含4条多肽链，通常通过二硫键相互连接的2条重(H)链和2条轻(L)链。每条重链包含重链可变区和重链恒定区。每条轻链包含轻链可变区和轻链恒定区。在此典型全长抗体结构外,其结构还包括其他衍生形式。
根据其重链恒定区的氨基酸序列，完整的抗体可归属于IgA、IgD、IgE、IgG和IgM五类抗体，其中IgG和IgA还可进一步分为亚类(同种型)，例如IgG1、IgG2、IgG3、IgG4、IgA1和IgA2。相应地，五类抗体的重链分别归入α、δ、ε、γ和μ链。根据其轻链恒定区的氨基酸序列，抗体的轻链可归入κ和λ。
术语“可变区”指抗体重链或轻链中涉及抗体结合抗原的域。
术语“恒定区”是指抗体的轻链和重链上的这样一些氨基酸序列，不直接参与抗体与抗原的结合，但展现出多种效应子功能，例如抗体依赖性细胞毒性。
术语“Fc区”用于定义免疫球蛋白重链的C-末端区。“Fc区”可以是天然序列Fc区或变体Fc区。尽管免疫球蛋白重链的Fc区的边界可变化，但通常将人IgG重链Fc区定义为从Cys226位处的氨基酸残基或从Pro230延伸至其羧基末端。Fc区中残基的编号如Kabat中的EU索引。Kabat等人,Sequences of Proteins of Immunological Interest,5thEd.Public Health Service,National Institutes of Health,Bethesda,Md.,1991 IgG的Fc区通常具有两个恒定区，CH 2和CH 3。
术语“Fc受体”或“FcR”指与抗体Fc区结合的受体。优选天然序列的人FcR，且优选与IgG抗体结合的受体(γ受体)，其包括FcγRI，FcγRII和FcγRIII亚型，以及这些受体的变体。其它FcR均被包含在术语“FcR”中。该术语也包括新生儿受体(FcRn)，其负责将母体的IgG转运至胎儿(Guyer等，免疫学杂志117：587(1976)和Kim等，免疫学杂志24：249(1994))。
术语“新生儿Fc受体”、简称“FcRn”，其结合IgG抗体Fc区。新生儿Fc受体(FcRn)在体内IgG类抗体的代谢命运中起重要作用。FcRn行使功能以从溶酶体降解途径营救IgG，从而降低其在血清中的清除率并加长半衰期。因此，IgG体外FcRn结合性质/特征指示它在血液循环中的体内药代动力学性质。
术语“改造的Fc多肽”、“改造的Fc区”和“改造的Fc”在本文中可互换使用，意指包含至少一个氨基酸改变的、或其糖基化修饰改变的Fc多肽或其部分。
术语“Fc融合蛋白”是指利用基因工程等技术将某种具有生物活性的功能蛋白与Fc片段融合而产生的新型重组蛋白，其不仅保留了功能蛋白分子的生物学活性，还具有一些抗体的性质，如同FcRs的结合及介导的相关生物学功能等。
术语“效应子功能”指可归因于抗体的Fc区的那些生物学活性，其随抗体同种型而不同。抗体效应子功能的实例包括：C1q结合和依赖补体的细胞毒性(CDC)、Fc受体(如CD16、CD32、CD64)结合、依赖抗体的细胞毒性(ADCC)、依赖抗体的吞噬作用(ADCP)、细胞因子分泌、免疫复合物介导的抗原呈递细胞对抗原的摄取、细胞表面受体(例如B细胞受体)的下调和B细胞激活。
“抗体依赖性细胞介导的细胞毒性”或“ADCC”是指一种细胞毒性形式，其中结合到在某些细胞毒性细胞(例如NK细胞、嗜中性粒细胞和巨噬细胞)上存在的Fcγ受体上的分泌Ig使得这些细胞毒性效应细胞能够特异性结合至承载抗原的靶细胞，随后使用例如细胞毒素杀死所述靶细胞。为了评估目的抗体的ADCC活性，可进行体外ADCC测定法，例如记载于美国专利No.5,500,362或5,821,337或美国专利No.6,737,056(Presta)中的体外ADCC测定法、本申请的实施例中记载的方法。用于这类测定法的有用效应细胞包括PBMC和NK细胞。
“补体依赖性细胞毒性”或“CDC”是指在补体的存在下靶细胞的裂解。经典补体途径的活化由补体***的第一组分(C1q)与(适当亚类的)抗体结合起始，其中该抗体与其相应抗原结合。为了评估补体活化，可进行CDC测定法，例如记载于Gazzano-Santoro et al.,J.Immunol Methods 202:163(1996)中的CDC测定法、例如本申请的实施例中记载的方法、例如在美国专利No.6,194,551Bl和WO1999/51642中记载的方法，其中描述了具有改变的Fc区氨基酸序列的多肽变体(具有变体Fc区的多肽)和具有增强或降低的C1q结合的多肽变体。
“体液免疫应答”是抗体介导的免疫应答并且涉及引入和生成以一定亲和力识别和结合本发明的免疫原性组合物中的抗原的抗体，“细胞介导的免疫应答”是由T细胞和/或其他白细胞介导的免疫应答。“细胞介导的免疫应答”是通过提供与主要组织相容性复合物(MHC)的I类或II类分子、CD1或其他非典型MHC样分子相关的抗原表位而诱发的。
术语“缀合物”是指与其他分子共价缀合的蛋白/肽。
术语“免疫原性组合物”是指含有抗原如微生物或其组分的任何药物组合物，该组合物可用于在个体中诱发免疫应答。
如本文所使用的“免疫原性”意指抗原(或抗原的表位)例如冠状病毒棘突蛋白受体结合区或包含该抗原的糖缀合物或免疫原性组合物在宿主(例如哺乳动物)中诱发体液或细胞介导的免疫应答或二者的能力。
“保护性”免疫应答是指免疫原性组合物诱发用于保护个体免于感染的体液或细胞介导的免疫应答或两者的能力。所提供的保护不必是绝对的，即，不必完全阻止或根除感染，只要相对于对照个体群体(例如未给药疫苗或免疫原性组合物的受感染动物)存在统计学上显著的改进即可。保护可限于缓和感染症状的严重性或发作快速性。
“免疫原性量”和“免疫有效量”二者在本文可交换使用，是指抗原或免疫原性组合物足以引发免疫应答(细胞(T细胞)或体液(B细胞或抗体)应答或二者，如通过本领域技术人员已知的 标准测定所测量的)的量。
抗原作为免疫原的有效性可通过增殖测定、通过细胞溶解测定、或通过测量B细胞活性水平来测量。
本发明的提高蛋白/肽抗原免疫原性的方法
本发明是一个开创性的发明，发明人发现包括通过将蛋白/肽抗原与经过改造的抗体Fc片段融合，其免疫原性得以增强，上述Fc片段由于其氨基酸序列和/或糖基化形式改变具有提高的与Fc受体和/或补体蛋白C1q的结合能力。
在此发明之前，没有研究报道蛋白/肽抗原与具有提高的与Fc受体和/或补体蛋白C1q的结合能力的Fc片段形成融合蛋白后，其蛋白/肽的免疫原性的提高。
本发明的免疫原性增强的蛋白/肽抗原
本发明的一个实例中，出发蛋白/肽抗原是冠状病毒棘突蛋白的ACE2受体结合结构域(RBD)，其免疫原性通过与提高的Fc受体和/或补体蛋白C1q的结合能力的Fc片段形成融合蛋白得以提高。在一个特别优选的方案中，Fc片段是Fc受体CD32a、CD32b和CD64结合增强片段/补体C1q结合增强片段；其氨基酸序列SEQ ID NO：30所示。在一个最优选的方案中，经过改造的抗体Fc片段为Fc受体CD16a、CD32a、CD32b和CD64结合增强片段/补体C1q结合增强片段；其氨基酸序列如SEQ ID NO：30所示，且用Fucose敲除的CHO细胞生产。
冠状病毒主要通过棘突蛋白(S蛋白)与宿主细胞受体结合来介导病毒的入侵，并决定病毒的组织或宿主嗜性。SARS-CoV-2的宿主细胞受体蛋白为血管紧张素转化酶2(ACE2)。病毒的三聚体棘突蛋白(S蛋白)同ACE2受体结合后被宿主蛋白酶切割为包含受体结合域(Receptor binding domain,SARS-COV-2 RBD)的S1多肽和负责介导病毒同细胞膜融合的S2多肽，进而侵入体内。
本发明的免疫原性组合物
在一个实施方案中，本发明的免疫原性组合物还包含佐剂、缓冲剂、冷冻保护剂、盐、二价阳离子、非离子清洁剂、自由基氧化抑制剂、稀释剂或载体中的至少一种。
佐剂是当与免疫原或抗原一起给药时增强免疫应答的物质。本发明的免疫原性组合物可含有或不含有疫苗佐剂。可用于本发明组合物的佐剂包括但不限于：MF59、QS-21或MPL的至少一种。
在一个实施方案中，本发明的免疫原性组合物中的佐剂是铝系佐剂。所用的佐剂会取决于被给药所述免疫原性组合物的个体、规定的注射途径及注射次数。
所述免疫原性组合物可任选地包含药学上可接受的载体。所述药学上可接受的载体包括各国药典用于动物(包括人类以及非人类哺乳动物)的载体。术语载体可用于指与药物组合物一起给药的稀释剂、佐剂、赋形剂或媒介物。可采用水、盐水溶液以及含水的右旋糖和甘油溶液作为尤其用于注射溶液剂的液体载体。
本发明的免疫原性组合物还可包含一种或多种额外的免疫原性剂。
本发明的免疫原性组合物的给药形式
用于治疗或预防性治疗的本发明免疫原性组合物可以通过肌内注射、腹膜内注射、皮内注射或皮下注射；或者经由粘膜给药至口腔/食道、呼吸道、泌尿生殖道。鼻内给予疫苗对于治疗某些疾病，例如肺炎或中耳炎是优选的。虽然本发明的疫苗可单剂量给予，但是其组分也可同时或分时共同给予。除了单一给药途径以外，可以使用两种不同的给药途径。
用于特定免疫原性组合物的组分的最佳量可通过涉及在个体中观察适当免疫应答的标准研究来确定。在进行初始疫苗接种后，个体可接受一次或若干次充分间隔的加强免疫。
本发明的免疫原性组合物的用途
本发明的蛋白/肽抗原及免疫复合物可以预防或治疗病原体引起的疾病，尤其是冠状病毒，更尤其是SARS-CoV-2病毒引起的的疾病。本发明的蛋白/肽抗原及免疫复合物可以预防或***疾病。亦可以用于免疫动物，以产生中和抗体。
实施例
实施例1：SARS-CoV-2 RBD及RBD-Fc融合蛋白表达载体的构建及蛋白生产
1.1 SARS-CoV-2 RBD表达载体的构建及蛋白生产
通过PCR扩增获得SARS-CoV-2-Spike-RBD序列(SEQ ID NO:5)(PCR扩增模板来源于北京义翘神州科技有限公司，下文同)，包含信号肽序列(SEQ ID NO:3)和SARS-CoV-2-Spike-RBD序列(SEQ ID NO:1)，通过in-fusion方法***到Hind III+Xba I(来源：Fermentas，下文同)酶切的pSE载体(来源：神州细胞工程有限公司，下文同)中获得pSE-CoV-2-RBD表达载体(SEQ ID NO:5)。
扩增引物：
Figure PCTCN2021092013-appb-000001
提取pSE-CoV-2-RBD质粒，转染HEK-293细胞(来源：Invitrogen，下文同)进行培养表达7天，纯化获得高纯度SARS-CoV-2 RBD蛋白。
1.2 SARS-CoV-2 RBD-mFc表达载体的构建及蛋白生产
通过PCR扩增SARS-CoV-2-Spike-RBD序列，通过in-fusion方法***到Afe I酶切FastAP去磷的包含信号肽(SEQ ID NO:3)、linker(SEQ ID NO:7)和鼠IgG1恒定区序列(SEQ ID NO:9)的pSE-mFc载体(来源：神州细胞工程有限公司)中获得pSE-CoV-2-RBD-mFc表达载体(SEQ ID NO:11)。
扩增引物：
Figure PCTCN2021092013-appb-000002
提取pSE-CoV-2-RBD-mFc质粒，转染HEK-293细胞进行培养表达7天，采用蛋白A纯化柱纯化获得高纯度RBD-mFc蛋白。
1.3 SARS-CoV-2 RBD-Fc表达载体的构建及蛋白生产
通过PCR扩增SARS-CoV-2-Spike-RBD序列，通过in-fusion方法***到Afe I酶切FastAP去磷的包含信号肽(SEQ ID NO:3)、linker(SEQ ID NO:7)和人IgG1恒定区序列(SEQ ID NO:13)的pSTEP2-Fc载体(来源：神州细胞工程有限公司)中获得pSE-CoV-2-RBD-Fc表达载体(SEQ ID NO:15)。
扩增引物：
Figure PCTCN2021092013-appb-000003
提取pSE-CoV-2-RBD-Fc质粒，转染HEK-293细胞进行培养表达7天，采用蛋白A纯化柱纯化获得高纯度RBD-Fc蛋白。
1.4 SARS-CoV-2 RBD-Fc-Ce3表达载体的构建及蛋白生产
为增强抗体Fc片段介导的CDC功能，参照文献对IgG1亚型的恒定区进行核苷酸突变[36,37]，得到基因工程改造的重链IgG1恒定区核苷酸序列(Fc-Ce3，SEQ ID NO:17)。
通过PCR扩增SARS-CoV-2-Spike-RBD-Ce3-Fc序列(SEQ ID NO:19)，通过in-fusion方法***到HindIII+Xba I酶切的pSE载体中获得pSE-nCoV-2-RBD-Fc-Ce3表达载体(SEQ  ID NO:19)。
扩增引物:
Figure PCTCN2021092013-appb-000004
提取pSE-nCoV-2-RBD-Fc-Ce3质粒，转染HEK-293细胞进行培养表达7天，采用蛋白A纯化柱纯化获得高纯度RBD-Fc-Ce3。
1.5 SARS-CoV-2 RBD-Fc-Fe4表达载体的构建及蛋白生产
为进一步增强抗体Fc片段介导的免疫功能，提取pSE-nCoV-2-RBD-Fc-Ce3质粒，转染fut8基因敲除的HEK-293细胞(来源：神州细胞工程有限公司)进行培养表达7天，采用蛋白A纯化柱纯化获得去岩藻糖基化的高纯度RBD-Fc-Fe4。
1.6 SARS-CoV-2 RBD-Fc-Fd11-IgG4表达载体的构建及蛋白生产
为降低抗体Fc片段介导的免疫功能，参照文献对IgG4亚型的恒定区进行核苷酸突变[38]，得到基因工程改造的重链IgG4恒定区核苷酸序列(Fd11-IgG4，SEQ ID NO:21)。
通过拼接PCR扩增SARS-CoV-2-Spike-RBD-Fd11-IgG4序列(SEQ ID NO:23)，通过in-fusion方法***到Hind III+Xba I酶切的pSE载体中获得pSE-CoV-2-RBD-Fc-Fd11-IgG4表达载体(SEQ ID NO:23)。
扩增引物：
Figure PCTCN2021092013-appb-000005
提取pSE-CoV-2-RBD-Fc-Fd11-IgG4质粒，转染HEK-293细胞进行培养表达7天，采用蛋白A纯化柱纯化获得高纯度RBD-Fc-Fd11-IgG4蛋白。
实施例2：SARS-CoV-2 RBD及RBD-Fc融合蛋白Fc受体结合试验
2.1 RBD-mFc增加同鼠Fc受体的结合
将浓度为10μg/mL的Avidin蛋白(来源：Thermo Fisher，下文同)包被于96孔板上，每孔100μL，2-8℃包被过夜。次日洗板，室温封闭1h后，加入100μL不同生物素标记Fc受体蛋白(均来源于北京义翘神州科技有限公司):5μg/mL的mCD16a-AVI-His(V158)+BirA、5μg/mL的mCD32b-AVI-His+BirA或0.5μg/mL的mCD64-AVI-His+BirA，同时以无蛋白为空白对照。室温孵育1h后洗板，加入100μL不同浓度的RBD-mFc蛋白或RBD蛋白。检测同mCD16a和mCD32b结合时蛋白浓度为40μg/mL、10μg/mL、2.5μg/mL、0.6250μg/mL、0.1563μg/mL、0.0391μg/mL、0.0098μg/mL和0.0024μg/ml；检测同mCD64结合时蛋白浓度为10μg/mL、2.5μg/mL、0.6250μg/mL、0.1563μg/mL、0.0391μg/mL、0.0098μg/mL和0.0024μg/ml。室温孵育1h后洗板去除未结合蛋白，加入100μL浓度为0.1μg/mL的辣根过氧化物酶标记的无Fc受体结合功能的CoV-2-RBD抗体CoV-2-HB27-Fd11-IgG4(来源：神州细胞工程有限公司，作为检测抗体)。孵育1h后重复洗板，加入底物显色液进行显色，终止后酶标仪读取OD 450，以样品OD 450–空白对照OD 450的数据进行分析。
结果如图1所示，RBD-mFc同mCD16a和mCD32b的结合EC 50分别为0.19μg/mL和0.27μg/mL，而RBD蛋白同mCD16a和mCD32b均无结合。RBD-mFc和RBD同mCD64蛋白无结合。
2.2 RBD-Fc-Fe4增加同人Fc受体及C1q的结合
检测同Fc受体结合时，将浓度为10μg/mL的Avidin蛋白包被于96孔板上，每孔100μL，2-8℃包被过夜。次日洗板，室温封闭1h后，加入100μL不同生物素标记Fc受体蛋白(均来源于北京义翘神州科技有限公司):5μg/mL的CD16a-AVI-His(V158)+BirA、5μg/mL的CD32a-AVI-His(R131)+BirA、5μg/mL的CD32b-AVI-His(R131)+BirA、或0.5μg/mL的CD64-AVI-His(V158)+BirA，同时以无蛋白为空白对照。室温孵育1h后洗板，加入100μL不同的RBD-Fc融合蛋白或RBD蛋白。检测同CD16a结合时蛋白浓度为40μg/mL、10μg/mL、2.5μg/mL、0.625μg/mL、0.156μg/mL、0.039μg/mL、0.01μg/mL和0.002μg/ml；检测同CD32a和CD32b结合时蛋白浓度为40μg/mL、10μg/mL、2.5μg/mL、0.625μg/mL和0.156μg/mL；检测同CD64结合时蛋白浓度为10μg/mL、2.5μg/mL、0.625μg/mL、0.156μg/mL、0.039μg/mL、0.01μg/mL和0.002μg/mL。室温孵育1h后洗板去除未结合蛋白，加入100μL浓度为0.1μg/mL的辣根过氧化物酶标记的无Fc受体结合功能的CoV-2-RBD抗体CoV-2-HB27-Fd11-IgG4。孵育1h后重复洗板，加入底物显色液进行显色，终止后酶标仪读取OD 450，以样品OD 450–空白对照OD 450的数据进行分析。
检测同C1q结合时，将不同浓度的RBD蛋白或不同RBD-Fc融合蛋白分别包被于96孔板上，100μL/孔，4℃包被过夜，蛋白包被浓度为200μg/mL、40μg/mL、8.0μg/mL、1.6μg/mL、0.32μg/mL和0.064μg/mL。次日洗板，室温封闭1h后，加入5μg/mL的C1q补体蛋白(来源：北京义翘神州科技有限公司)，100μg/孔，孵育1h。洗板去除未结合的蛋白，加入0.5μg/mL anti-C1q/HRP(来源：Abcam)孵育后重复洗板，最后加入底物显色液进行显色，终止后检测OD 450。
结果如图2所示，不同RBD-Fc融合蛋白中，RBD-Fc-Fe4同CD16、CD32a和CD32b均有最强的结合能力，优于RBD-Fc及RBD-Fc-Ce3，RBD-Fc-Fd11-IgG4及RBD蛋白同CD16、CD32a和CD32b几乎无结合。RBD-Fc-Fe4、RBD-Fc-Ce3及RBD-Fc同CD64具有较高且相似的结合能力，而RBD-Fc-Fd11-IgG4蛋白同CD64结合较弱，RBD蛋白在高浓度推测有非特异性结合。RBD-Fc-Fe4、RBD-Fc-Ce3同C1q的结合略强于RBD-Fc，RBD及RBD-Fc-Fd11-IgG4同C1q无结合。
实施例3：SARS-CoV-2 RBD及RBD-Fc融合蛋白细胞吞噬试验
单核来源的巨噬细胞(MDM,monocyte-derived macrophages)是将从新鲜人外周血分离的PBMC通过细胞贴壁的方法获得单核细胞，再经过诱导培养基诱导培养7天得到。通过在抗原蛋白上标记FITC荧光染料，与巨噬细胞共孵育后在共聚焦显微镜观察细胞吞噬作用。
3×10 4/孔接种MDM细胞(来源：神州细胞工程有限公司诱导获得)，50μL/孔，在1640+10％FBS+1×PS培养基(来源：神州细胞工程有限公司)中培养过夜，然后50μL/孔加入20μg/mL FITC标记的不同抗原蛋白。将96孔板置37℃、5％CO 2条件下的培养箱内避光孵育2.5h。作用结束后，将96孔板上清弃去，使用PBS洗去未结合的抗原蛋白，50μL/孔加入4％多聚甲醛(来源：Sigma-aldrich)。4℃固定15min,弃去固定液，PBS洗一遍，50μL/孔加入PBS，利用共聚焦显微镜FITC通道荧光拍照，使用Image pro plus软件分析荧光强度。
结果如图3所示，RBD-Fc-Fe4-FITC可介导更强的吞噬作用。
实施例4：小鼠免疫及免疫效果评价
4.1小鼠免疫
含铝佐剂抗原制备：RBD-his(来源：北京义翘神州科技有限公司)和RBD-mFc抗原分别用PBS稀释成0.06mg/mL，铝佐剂(来源：神州细胞工程有限公司，下文同)用PBS稀释成1mg/mL。稀释后的抗原和铝佐剂等体积混合。得到0.03mg/mL的含铝佐剂的抗原。
含MF59(来源：神州细胞工程有限公司，下文同)和铝佐剂混合佐剂的RBD、RBD-Fc、RBD-Fc-Ce3、RBD-Fc-Fe4抗原制备：取1.5mL铝佐剂加1.5mL MF59佐剂再加入0.18mL浓度为0.53mg/mL的抗原蛋白，得到蛋白浓度为0.03mg/mL，佐剂为混合佐剂的抗原。
小鼠选用4-6周Balb/c小鼠(来源：中国食品药品检定研究院)，腹腔注射0.1mL含铝佐剂抗原或含混合佐剂的抗原，其中含抗原3μg，分别于第14天和第28天加强免疫。
4.2小鼠血清抗体滴度及中和滴度的测定
将浓度为5μg/mL的RBD-mFc蛋白包被于96孔板，100μL/孔，以CD155(D1)-mFc(来源：神州细胞工程有限公司)作为相同标签的无关对照。室温孵育2h后，洗板加2％BSA(牛血清白蛋白)封闭，室温孵育1h。使用0.1％BSA的TBST缓冲液作为样品稀释剂将小鼠铝佐剂免疫原血清稀释8000×，混合佐剂免疫原血清稀释1000×，以无关免疫靶点的小鼠血清为阴性对照，同时加入待测血清、阴性对照血清和80ng/mL检测二抗兔抗鼠IgG F(ab)2/HRP(来源：Jackson Immuno Research)，各100μL/孔，共同孵育2h后洗板5遍，加入底物显色液进行显色，终止后酶标仪读取OD 450，测定小鼠血清抗体滴度。
将小鼠铝佐剂免疫原血清稀释500×，混合佐剂免疫原血清稀释10×，与SARS-CoV-2假病毒(来源：中国食品药品检定研究院)等体积混合，37℃孵育1h后，加入293FT-ACE2细胞(来源：神州细胞工程有限公司)。以加假病毒、不加抗体组作为阳性对照，不加假病毒和抗体组为阴性对照。侵染后细胞在37℃，5％CO 2条件下培养20-28h，在微孔板式发光检测仪上检测荧光信号值(RLU)，并计算中和抑制率。中和抑制率％＝(阳性对照RLUs–样品RLUs)/(阳性对照RLUs–阴性对照RLUs)×100％。
结果表明，含铝佐剂的抗原第三次免疫小鼠7天后，RBD-mFc蛋白的免疫血清效价及中和滴度效价均优于RBD-his，说明RBD-mFc可激活更强的体液免疫反应(图4)。
含铝佐剂和MF59混合佐剂的抗原第一次免疫小鼠7天后，不同RBD-Fc融合蛋白的免疫血清效价及中和滴度效价均优于RBD蛋白，其中RBD-Fc-Ce3和RBD-Fc-Fe4的血清中和滴度较高，说明RBD-Fc融合蛋白可激活更强的体液免疫反应(图5)。
参考文献
1.Gonzalez,S.F.,etal.,Trafficking of B cell antigen in lymph nodes.Annu Rev Immunol,2011.29:p.215-33.
2.Bergtold,A.,et al.,Cell Surface Recycling of Internalized Antigen Permits Dendritic Cell Priming of B Cells.Immunity,2005.23(5):p.503-514.
3.Batista,F.D.and N.E.Harwood,The who,how and where of antigen presentation to B cells.Nature Reviews Immunology,2009.9(1):p.15-27.
4.Gonzalez,S.F.,et al.,Complement-dependent transport of antigen into B cell follicles.Journal of immunology(Baltimore,Md.:1950),2010.185(5):p.2659-2664.
5.Heesters,B.A.,et al.,Endocytosis and recycling of immune complexes by follicular dendritic cells enhances B cell antigen binding and activation.Immunity,2013.38(6):p.1164-1175.
6.Harwood,N.E.and F.D.Batista,The Antigen Expressway:Follicular Conduits Carry Antigen to B Cells.Immunity,2009.30(2):p.177-179.
7.Mandels,T.E.,et al.,The Follicular Dendritic Cell:Long Term Antigen Retention During Immunity.ImmunologicalReviews,1980.53(1):p.29-59.
8.Regnault,A.,et al.,Fcgamma receptor-mediated induction of dendritic cell maturation and major histocompatibility complex class I-restricted antigen presentation after immune complex internalization.J Exp Med,1999.189(2):p.371-80.
9.Rodriguez,A.,et al.,Selective transport of internalized antigens to the cytosol for MHC class I presentation in dendritic cells.Nat Cell Biol,1999.1(6):p.362-8.
10.Machy,P.,K.Serre,and L.Leserman,Class I-restricted presentation of exogenous antigen acquired by Fcgamma receptor-mediated endocytosis is regulated in dendritic cells.Eur J Immunol,2000.30(3):p.848-57.
11.Kotsias,F.,I.Cebrian,and A.Alloatti,Antigen processing and presentation.Int Rev Cell Mol Biol,2019.348:p.69-121.
12.Nimmerjahn,F.and J.V.Ravetch,Fcγ receptors as regulators of immune responses.Nature Reviews Immunology,2008.8(1):p.34-47.
13.Ben Mkaddem,S.,M.Benhamou,and R.C.Monteiro,Understanding Fc Receptor Involvement in Inflammatory Diseases:From Mechanisms to New Therapeutic Tools.Frontiers in Immunology,2019.10(811).
14.Takai,T.,Roles of Fc receptors in autoimmunity.Nature Reviews Immunology,2002.2(8):p.580-592.
15.Bournazos,S.and J.V.Ravetch,Fcγ Receptor Function and the Design of Vaccination Strategies.Immunity,2017.47(2):p.224-233.
16.Guilliams,M.,et al.,The function of Fcγ receptors in dendritic cells and macrophages.Nature Reviews Immunology,2014.14(2):p.94-108.
17.Idusogie,E.E.,et al.,Mapping of the C1q Binding Site on Rituxan,a Chimeric Antibody with a Human IgG1 Fc.Journal of Immunology,2000.164(8):p.4178-4184.
18.Idusogie,E.E.,et al.,Engineered Antibodies with Increased Activity to Recruit Complement.Journal of Immunology,2001.166(4):p.2571-2575.
19.Steurer,W.,et al.,Ex vivo coating of islet cell allografts with murine CTLA4/Fc promotes graft tolerance.Journal of immunology(Baltimore,Md.:1950),1995.155:p.1165-74.
20.Lazar,G.A.,et al.,Engineered antibody Fc variants with enhanced effector function.Proceedings of the National Academy of Sciences of the United States of America,2006.103(11):p.4005.
21.Ryan,M.C.,et al.,Antibody targeting of B-cell maturation antigen on malignant plasma cells.(1535-7163(Print)).
22.Xu,D.-Z.,et al.,A Randomized Controlled Phase IIb Trial of Antigen-Antibody Immunogenic Complex Therapeutic Vaccine in Chronic Hepatitis B Patients.PLOS ONE,2008.3(7):p.e2565.
23.Yao,X.,et al.,Therapeutic effect of hepatitis B surface antigen–antibody complex is associated with cytolytic and non-cytolytic immune responses in hepatitis B patients.Vaccine,2007.25(10):p.1771-1779.
24.Xu,D.-Z.,et al.,Results of a phase III clinical trial with an HBsAg-HBIG immunogenic complex therapeutic vaccine for chronic hepatitis B patients:Experiences and findings.Journal of Hepatology,2013.59(3):p.450-456.
25.Zhang,Y.,et al.,A novel RSV F-Fcfusion protein vaccine reduces lung injury induced by respiratory syncytial virus infection.Antiviral Research,2019.165:p.11-22.
26.Meng,Z.,et al.,Immunization with HBsAg-Fc fusion protein induces a predominant production of Th1 cytokines and reduces HBsAg level in transgenic mice.Chinese medical journal,2012.125:p.3266-72.
27.Kebriaei,A.,et al.,Construction and immunogenicity of a new Fc-based subunit vaccine candidate against Mycobacterium tuberculosis.Molecular Biology Reports,2016.43(9):p.911-922.
28.Kim,M.-Y.,et al.,Novel vaccination approach for dengue infection based on recombinant immune complex universal platform.Vaccine,2015.33.
29.Zhou,P.,et al.,Discovery of a novel coronavirus associated with the recentpneumonia outbreak in humans and its potential bat origin.BioRxiv,2020.
30.Wan,Y.,et al.,Receptor recognition by novel coronavirus from Wuhan:An analysis based on decade-long structural studies of SARS.Journal of virology,2020.
31.Bosch,B.J.,et al.,Severe acute respiratory syndrome coronavirus(SARS-CoV)infection inhibition using spike protein heptad repeat-derived peptides.Proceedings of the National Academy of Sciences of the United States of America,2004.101(22):p.8455-8460.
32.Huang,C.,et al.,Clinical features of patients infected with 2019 novel coronavirus in Wuhan,China.Lancet,2020.395(10223):p.497-506.
33.Zhou,F.,et al.,Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan,China:a retrospective cohort study.The Lancet,2020.
34.Dong,C.,et al.,Characterization of anti-viral immunity in recovered individuals infected by SARS-CoV-2.medRxiv,2020.
35.To,K.K.,et al.,Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2:an observational cohort study.Lancet Infect Dis,2020.
36.de Jong,R.N.,et al.,A novel platform for the potentiation of therapeutic antibodies based on antigen-dependent formation of IgG hexamers at the cell surface.PLoS biology,2016.14(1).
37.Parren,P.,et al.,Antibody variants and uses thereof.2014,Google Patents.
38.叶鑫,etal.,Cd47 antibody,antigen-binding fragment and medical use thereof.2018,Google Patents.
本发明涉及的主要序列
Figure PCTCN2021092013-appb-000006
Figure PCTCN2021092013-appb-000007
Figure PCTCN2021092013-appb-000008
Figure PCTCN2021092013-appb-000009
Figure PCTCN2021092013-appb-000010
Figure PCTCN2021092013-appb-000011
Figure PCTCN2021092013-appb-000012
Figure PCTCN2021092013-appb-000013
Figure PCTCN2021092013-appb-000014
Figure PCTCN2021092013-appb-000015
Figure PCTCN2021092013-appb-000016
Figure PCTCN2021092013-appb-000017

Claims (38)

  1. 一种增强蛋白/肽抗原的免疫原性的方法，该方法包含将蛋白/肽抗原与经过改造的抗体Fc片段融合，该Fc片段与其天然形式相比，具有提高的与Fc受体和/或补体蛋白C1q的结合能力，可模拟抗原抗体复合物增强DC/B抗原呈递细胞的吞噬，增强抗原免疫效果。
  2. 如权利要求1所述的方法，其中所述的蛋白/肽抗原为病原体相关的蛋白/肽抗原或肿瘤相关蛋白/肽抗原。
  3. 如权利要求2所述的方法，
    其中所述的病原体选自：
    冠状病毒，人免疫缺陷性病毒HIV-1，人单纯疱疹病毒，巨细胞病毒，轮状病毒，EB病毒，水痘带状疱疹病毒，肝炎病毒，呼吸道合胞体病毒，副流感病毒，麻疹病毒，流行性腮腺炎病毒，人***瘤病毒，黄病毒或流感病毒，奈瑟氏菌属，莫拉氏菌属，博代氏杆菌属，分枝杆菌属，包括结核分枝杆菌；埃希氏菌属，包括肠毒素大肠埃希菌；沙门氏菌属，李斯特氏菌属，螺杆菌属，葡萄球菌属，包括金黄葡萄球菌，表皮葡萄球菌；疏螺旋体属，衣原体，包括砂眼衣原体，肺炎衣原体；疟原虫，包括恶性疟原虫；弓形虫，念珠菌；
    其中的肿瘤选自：
    弥漫性大B细胞淋巴瘤、滤泡性淋巴瘤、其他淋巴瘤、白血病、多发性骨髓瘤、间皮瘤、胃癌、恶性横纹肌瘤、肝细胞癌、***癌，乳腺癌、胆管癌和胆囊癌、膀胱癌、脑肿瘤包括神经母细胞瘤、神经鞘瘤、胶质瘤、胶质母细胞瘤和星形细胞瘤、***、结肠癌、黑色素瘤、子宫内膜癌、食管癌、头颈癌、肺癌、鼻咽癌、卵巢癌、胰腺癌、肾细胞癌、直肠癌、甲状腺癌、甲状旁腺肿瘤、子宫肿瘤和软组织肉瘤。
  4. 如权利要求1-3所述的方法，其中蛋白/肽蛋白抗原选自分泌蛋白或全长膜蛋白，或其功能结构域、突变蛋白、截短蛋白，或由其1个或多个抗原多肽表位拼接组成的改造蛋白。
  5. 如权利要求1-4之任一所述的方法，其中Fc片段来自人抗体、鼠源抗体、兔源抗体或其他动物抗体的重链恒定区。
  6. 如权利要求5所述的方法，其中Fc片段来自人抗体的IgG、IgM或IgA亚型抗体，
    优选地，来自IgG1、IgG2、IgG3或IgG4亚型抗体；
    更优选地，是为提高与Fc受体和/或C1q补体结合功能之目的，而进行氨基酸序列突变和/或糖基化形式改变的IgG1改造Fc片段。
  7. 如权利要求1-6之任一所述的方法，Fc受体选自CD16、CD32a、CD32b或CD64。
  8. 如权利要求1-7之任一所述的方法，其中
    蛋白/肽抗原为冠状病毒棘突蛋白的ACE2受体结合结构域(RBD)。
  9. 如权利要求7所述的方法，其中
    经过改造的抗体Fc片段为Fc受体CD32a、CD32b和CD64结合增强片段/补体C1q结合增强片段；
    其氨基酸序列如SEQ ID NO：30所示。
  10. 如权利要求7所述的方法，其中
    经过改造的抗体Fc片段为Fc受体CD16a、CD32a、CD32b和CD64结合增强片段/补体C1q结合增强片段；
    其氨基酸序列如SEQ ID NO：30所示，且用Fucose敲除的哺乳细胞生产，优选地，哺乳动物细胞为fut8基因敲除的HEK-293细胞。
  11. 如权利要求4所述的方法，其中Fc片段来自鼠抗体IgG，IgM，IgA亚型抗体，
    优选地，来自IgG1、IgG2a、IgG2b或IgG3亚型抗体。
  12. 如权利要求1-11之任一所述的方法，其中所述抗原优选地通过接头和其他大分子缀合，
    优选地，其他大分子为多糖、肽/蛋白。
  13. 一种免疫原性增强的蛋白/肽抗原，其中
    该蛋白/肽抗原与经过改造的抗体Fc片段融合，该Fc片段与其天然形式相比，具有提高的与Fc受体和/或补体蛋白C1q的结合能力,可模拟抗原抗体复合物增强DC/B抗原呈递细胞的吞噬，增强抗原免疫效果。
  14. 如权利要求13所述蛋白/肽抗原，其中所述的抗原为病原体相关的蛋白/肽抗原、肿瘤相关蛋白/肽抗原。
  15. 如权利要求14所述蛋白/肽抗原，
    其中所述的病原体选自：
    冠状病毒，人免疫缺陷性病毒HIV-1，人单纯疱疹病毒，巨细胞病毒，轮状病毒，EB病毒，水痘带状疱疹病毒，肝炎病毒，呼吸道合胞体病毒，副流感病毒，麻疹病毒，流行性腮腺炎病毒，人***瘤病毒，黄病毒或流感病毒，奈瑟氏菌属，莫拉氏菌属，博代氏杆菌属，分枝杆菌属，包括结核分枝杆菌；埃希氏菌属，包括肠毒素大肠埃希菌；沙门氏菌属，李斯特氏菌属，螺杆菌属，葡萄球菌属，包括金黄葡萄球菌，表皮葡萄球菌；疏螺旋体属，衣原体，包括砂眼衣原体，肺炎衣原体；疟原虫，包括恶性疟原虫；弓形虫，念珠菌；
    其中的肿瘤选自：弥漫性大B细胞淋巴瘤、滤泡性淋巴瘤、其他淋巴瘤、白血病、多发性骨髓瘤、间皮瘤、胃癌、恶性横纹肌瘤、肝细胞癌、***癌，乳腺癌、胆管癌和胆囊癌、膀胱癌、脑肿瘤包括神经母细胞瘤、神经鞘瘤、胶质瘤、胶质母细胞瘤和星形细胞瘤、***、结肠癌、黑色素瘤、子宫内膜癌、食管癌、头颈癌、肺癌、鼻咽癌、卵巢癌、胰腺癌、肾细胞癌、直肠癌、甲状腺癌、甲状旁腺肿瘤、子宫肿瘤和软组织肉瘤。
  16. 如权利要求13-15之任一所述的蛋白/肽抗原，其中蛋白抗原选自分泌蛋白或全长膜蛋白，或其功能结构域、突变蛋白、截短蛋白，或由其1个或多个抗原多肽表位拼接组成的改造蛋白。
  17. 如权利要求13-16之任一所述的蛋白/肽抗原，其中Fc片段来自人抗体、鼠源抗体、兔源抗体或其他动物抗体的重链恒定区。
  18. 如权利要求17所述的蛋白/肽抗原，其中Fc片段来人抗体的IgG、IgM或IgA亚型抗体，
    优选地，来自IgG1、IgG2、IgG3或IgG4亚型抗体；
    更优选地，是为提高与Fc受体、C1q补体结合功能之目的，而进行氨基酸序列突变和/或糖基化形式改变的IgG1改造Fc片段。
  19. 如权利要求13-18之任一所述的蛋白/肽抗原，其中Fc受体选自CD16、CD32a、CD32b 或CD64。
  20. 如权利要求13-19之任一所述的蛋白/肽抗原，其中
    抗原为冠状病毒棘突蛋白的ACE2受体结合结构域(RBD)。
  21. 如权利要求20所述的蛋白/肽抗原，其中
    经过改造的抗体Fc片段为Fc受体CD32a、CD32b和CD64结合增强片段/补体C1q结合增强片段；
    其氨基酸序列如SEQ ID NO：30所示。
  22. 如权利要求20所述的蛋白/肽抗原，其中
    经过改造的抗体Fc片段为Fc受体CD16a、CD32a、CD32b和CD64结合增强片段/补体C1q结合增强片段；
    其氨基酸序列如SEQ ID NO：30所示，且用Fucose敲除的哺乳动物细胞生产，
    优选地，哺乳动物细胞为fut8基因敲除的HEK-293细胞。
  23. 如权利要求17所述的蛋白/肽抗原，其中Fc片段来自鼠抗体IgG，IgM，IgA亚型抗体，优选地，来自IgG1、IgG2a、IgG2b或IgG3亚型抗体。
  24. 如权利要求13-23所述的蛋白/肽抗原，其中所述抗原优选地通过接头和其他大分子缀合，优选地，其他大分子为多糖、肽/蛋白。
  25. 一种缀合物，其包含，优选地通过接头连接的权利要求13-23任一项所述的蛋白/肽抗原和其他大分子，优选地，其他大分子为多糖、肽/蛋白。
  26. 一种核酸，其编码权利要求13-24任一项所述的蛋白/肽抗原，其为mRNA和/或DNA。
  27. 权利要求26所述的核酸，其序列如SEQ ID NO:31所示。
  28. 一种表达载体，其包含权利要求26或27所述的核酸。
  29. 一种宿主细胞，其包含权利要求26或27所述的核酸或权利要求28所述的表达载体。
  30. 一种用于生产权利要求13-24任一所述的蛋白/肽抗原的方法，其包括在适合于前述蛋白质分子表达的条件下培养权利要求29所述的宿主细胞，和从培养基中回收表达的产物。
  31. 一种免疫组合物，其包含
    a)权利要求13-24任一所述的蛋白/肽抗原、权利要求25所述的缀合物、权利要求26-27任一项所述的核酸或权利要求28所述的表达载体；和/或
    b)佐剂；和
    c)药学上可接受的载体、赋形剂或稳定剂，优选为
    冻干制剂或水溶液形式的药学上可接受的载体、赋形剂或稳定剂。
  32. 权利要求31所述的免疫组合物，其中佐剂可选自铝佐剂、MF59、QS-21或MPL的至少一种。
  33. 权利要求13-24任一所述的蛋白/肽抗原、权利要求25所述的缀合物、权利要求26-27任一项所述的核酸、权利要求28所述的表达载体、或权利要求31或32所述的免疫组合物，其用于预防病原体，优选冠状病毒、更优选SARS-CoV-2引起的疾病\肿瘤的应用。
  34. 权利要求13-24任一所述的蛋白/肽抗原、权利要求25所述的缀合物、权利要求26-27任一项所述的核酸、权利要求28所述的表达载体、或权利要求31或32所述的免疫组合物，其用于制备预防病原体，优选冠状病毒、更优选SARS-CoV-2引起的疾病\肿瘤的疫苗中的应用。
  35. 一种免疫组合，其包含
    权利要求13-24任一所述的蛋白/肽抗原、权利要求25所述的缀合物、权利要求26-27任一项所述的核酸、权利要求28所述的表达载体、或权利要求31或32所述的免疫组合物；以及一种或多种另外的免疫原性剂。
  36. 一种试剂盒，其包含
    权利要求13-24任一所述的蛋白/肽抗原、权利要求25所述的缀合物、权利要求26-27任一项所述的核酸、权利要求28所述的表达载体、或权利要求31或32所述的免疫组合物；
    优选地，
    还进一步包含给予疫苗的装置。
  37. 一种预防病原体，优选冠状病毒、更优选SARS-CoV-2引起的疾病\预防肿瘤的方法，其包含给予受治疗者权利要求13-24任一所述的蛋白/肽抗原、权利要求25所述的缀合物、权利要求26-27任一项所述的核酸、权利要求28所述的表达载体、或权利要求31或32所述的免疫组合物、权利要求35的疫苗组合或权利要求36的试剂盒。
  38. 一种免疫动物的方法，其包含给予动物权利要求13-24任一所述的蛋白/肽抗原、权利要求25所述的缀合物、权利要求26-27任一项所述的核酸、权利要求28所述的表达载体、或权利要求31或32所述的免疫组合物、权利要求35的疫苗组合或权利要求36的试剂盒，以产生中和抗体。
PCT/CN2021/092013 2020-05-11 2021-05-07 通过与改变的Fc片段形成融合蛋白增强蛋白/肽抗原免疫原性的方法 WO2021227937A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202180029479.XA CN115461078A (zh) 2020-05-11 2021-05-07 通过与改变的Fc片段形成融合蛋白增强蛋白/肽抗原免疫原性的方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010394463.6 2020-05-11
CN202010394463 2020-05-11

Publications (1)

Publication Number Publication Date
WO2021227937A1 true WO2021227937A1 (zh) 2021-11-18

Family

ID=78526432

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/092013 WO2021227937A1 (zh) 2020-05-11 2021-05-07 通过与改变的Fc片段形成融合蛋白增强蛋白/肽抗原免疫原性的方法

Country Status (2)

Country Link
CN (1) CN115461078A (zh)
WO (1) WO2021227937A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022184854A2 (en) 2021-03-03 2022-09-09 Formycon Ag Formulations of ace2 fc fusion proteins
EP4331571A1 (en) 2022-09-02 2024-03-06 Formycon AG Formulations of ace2-igm fusion proteins

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101098710A (zh) * 2004-06-02 2008-01-02 纽约血液中心 产生高度有效抗体的sars疫苗和方法
WO2013004841A1 (en) * 2011-07-06 2013-01-10 Genmab A/S Modulation of complement-dependent cytotoxicity through modifications of the c-terminus of antibody heavy chains
CN104220093A (zh) * 2011-07-06 2014-12-17 根马布私人有限公司 抗体变体及其用途
CN109705222A (zh) * 2018-12-27 2019-05-03 中国科学院武汉病毒研究所 黄病毒科病毒囊膜蛋白的融合蛋白及其制备方法与应用
US20190328865A1 (en) * 2014-02-28 2019-10-31 New York Blood Center, Inc. Immunogenic composition for mers coronavirus infection

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101098710A (zh) * 2004-06-02 2008-01-02 纽约血液中心 产生高度有效抗体的sars疫苗和方法
WO2013004841A1 (en) * 2011-07-06 2013-01-10 Genmab A/S Modulation of complement-dependent cytotoxicity through modifications of the c-terminus of antibody heavy chains
CN104220093A (zh) * 2011-07-06 2014-12-17 根马布私人有限公司 抗体变体及其用途
US20190328865A1 (en) * 2014-02-28 2019-10-31 New York Blood Center, Inc. Immunogenic composition for mers coronavirus infection
CN109705222A (zh) * 2018-12-27 2019-05-03 中国科学院武汉病毒研究所 黄病毒科病毒囊膜蛋白的融合蛋白及其制备方法与应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
PROCKO ERIK: "The sequence of human ACE2 is suboptimal for binding the S spike protein of SARS coronavirus 2", 17 March 2020 (2020-03-17), pages 1 - 12, XP055864460, Retrieved from the Internet <URL:https://www.biorxiv.org/content/10.1101/2020.03.16.994236v3> DOI: 10.1101/2020.03.16.994236 *
ROB N DE JONG; BEURSKENS FRANK; VERPLOEGEN SANDRA; STRUMANE KRISTIN; VAN KAMPEN MURIEL; VOORHORST MARLEEN; HORSTMAN WENDY; ENGELBE: "A Novel Platform for the Potentiation of Therapeutic Antibodies Based on Antigen-Dependent Formation of IgG Hexamers at the Cell Surface", PLOS BIOLOGY, vol. 14, no. 1, 6 January 2016 (2016-01-06), pages 1 - 24, XP055268752, ISSN: 1544-9173, DOI: 10.1371/journal.pbio.1002344 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022184854A2 (en) 2021-03-03 2022-09-09 Formycon Ag Formulations of ace2 fc fusion proteins
EP4331571A1 (en) 2022-09-02 2024-03-06 Formycon AG Formulations of ace2-igm fusion proteins

Also Published As

Publication number Publication date
CN115461078A (zh) 2022-12-09

Similar Documents

Publication Publication Date Title
US10385120B2 (en) Molecular complex for targeting antigens towards cells comprising antigens and uses thereof for vaccination
WO2021227937A1 (zh) 通过与改变的Fc片段形成融合蛋白增强蛋白/肽抗原免疫原性的方法
WO2021228167A1 (zh) 通过形成Fc片段融合蛋白糖缀合物增强抗原免疫原性的方法
Sterlin et al. When therapeutic IgA antibodies might come of age
ES2961791T3 (es) Anticuerpos que neutralizan el virus de la hepatitis B y usos de los mismos
Grødeland et al. The specificity of targeted vaccines for APC surface molecules influences the immune response phenotype
US11352416B2 (en) Mosaic chimeric viral vaccine particle
TW201725214A (zh) 以抗原呈獻細胞為標的之癌症疫苗
EP1246643A2 (en) Dna vaccines encoding antigen linked to a domain that binds cd40
Ruffini et al. Human chemokine MIP1α increases efficiency of targeted DNA fusion vaccines
Lunde et al. Efficient delivery of T cell epitopes to APC by use of MHC class II-specific Troybodies
EP3226892A1 (en) Dna antibody constructs and method of using same
TWI425952B (zh) 具有抗原呈現細胞結合區及半胱胺酸富含區之免疫蛋白載體
JP2023528017A (ja) 重症急性呼吸器症候群コロナウイルス２（ｓａｒｓ－ｃｏｖ－２）ポリペプチドおよびワクチン目的でのその使用
WO2021198999A1 (en) Epitope-based vaccines for treatment of coronavirus associated diseases
JP2023512709A (ja) 抗原由来ｔ細胞抗原エピトープ又はこれを含むペプチドを細胞表面に提示するための融合抗体、及びこれを含む組成物
WO2022011005A1 (en) Immunostimulatory adjuvants
Lapuente et al. B-cell and antibody responses to SARS-CoV-2: infection, vaccination, and hybrid immunity
US11780924B2 (en) HLA binding vaccine moieties and uses thereof
WO2023137156A2 (en) T cell modulatory polypeptides with conjugation sites and methods of use thereof
TW202245838A (zh) 用於治療b型肝炎病毒感染的組成物及方法
KR20230107260A (ko) Sars-cov-2 스파이크 단백질의 수용체-결합 도메인에 접합되거나 융합된 항체, 및 백신 목적을 위한 이의 용도
EP4204448A2 (en) Anti-golph2 antibodies for macrophage and dendritic cell differentiation
AU2012367204B2 (en) Compositions and methods of modulating an immune response
Herrmann et al. A targeted vaccine against COVID-19: S1-Fc vaccine targeting the antigen-presenting cell compartment elicits protection against SARS-CoV-2 infection

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: 21804747

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: 21804747

Country of ref document: EP

Kind code of ref document: A1