WO2021227937A1 - Procédé d'amélioration de l'immunogénicité d'un antigène de protéine/peptide au moyen de la formation d'une protéine de fusion avec un fragment fc modifié - Google Patents

Procédé d'amélioration de l'immunogénicité d'un antigène de protéine/peptide au moyen de la formation d'une protéine de fusion avec un fragment fc modifié Download PDF

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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
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protein
antigen
cancer
peptide antigen
antibody
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PCT/CN2021/092013
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Chinese (zh)
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谢良志
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神州细胞工程有限公司
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    • 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).

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Abstract

La présente invention concerne un procédé d'amélioration de l'immunogénicité d'un antigène de protéine/peptide, l'antigène de protéine/peptide formant une protéine de fusion avec un fragment Fc d'anticorps modifié, et le fragment Fc présente une capacité de liaison améliorée à un récepteur Fc et/ou à une protéine complémentaire C1q par rapport à la forme naturelle de celui-ci due à des changements de la séquence d'acides aminés et/ou de la forme de glycosylation de celui-ci. Le vaccin contre le SARS-CoV-2 préparé au moyen de l'utilisation de la protéine de fusion en tant qu'immunogène présente une capacité de liaison élevée au récepteur Fc, et peut maintenir des réponses immunitaires humorales et cellulaires à long terme, et des animaux immunisés peuvent produire des titres supérieurs d'anticorps neutralisants. Le vaccin à base de protéine recombinante peut être utilisé pour prévenir des maladies liées à l'infection par le SARS-CoV-2.
PCT/CN2021/092013 2020-05-11 2021-05-07 Procédé d'amélioration de l'immunogénicité d'un antigène de protéine/peptide au moyen de la formation d'une protéine de fusion avec un fragment fc modifié WO2021227937A1 (fr)

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EP4331571A1 (fr) 2022-09-02 2024-03-06 Formycon AG Formulations de protéines de fusion ace2-igm

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EP4331571A1 (fr) 2022-09-02 2024-03-06 Formycon AG Formulations de protéines de fusion ace2-igm

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