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WO2023011575A1 - Médicament à base de protéine de fusion multifonctionnelle pour cibler et détruire des agents pathogènes et/ou des cellules tumorales - Google Patents

Médicament à base de protéine de fusion multifonctionnelle pour cibler et détruire des agents pathogènes et/ou des cellules tumorales Download PDF

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WO2023011575A1
WO2023011575A1 PCT/CN2022/110178 CN2022110178W WO2023011575A1 WO 2023011575 A1 WO2023011575 A1 WO 2023011575A1 CN 2022110178 W CN2022110178 W CN 2022110178W WO 2023011575 A1 WO2023011575 A1 WO 2023011575A1
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fusion protein
cells
domain
protein
protein according
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Chinese (zh)
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林欣
袁建龙
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Tsinghua University
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Tsinghua University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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 invention relates to the technical field of drug development, in particular to a multifunctional fusion protein drug targeting and killing pathogens and/or tumor cells.
  • Microbial infections including pathogenic viruses, bacteria, fungi and some protists cause a large number of deaths every year.
  • an immune system in the body that can completely or partially resist the infection of these pathogenic microorganisms, when the human body’s immunity declines, it cannot effectively clear the infection of the pathogen, causing the pathogen to multiply in the body, destroying healthy tissues and organs in various parts of the body, and directly life threatening.
  • some pathogenic bacteria are covered with capsules, which can avoid the recognition and attack of immune cells.
  • Cryptococcosis is a subacute or chronic infectious disease, caused by Cryptococcus neoformans, mainly invading the central nervous system.
  • Cryptococcus neoformans mainly invading the central nervous system.
  • fungal meningitis, brain abscess and granuloma have become rare.
  • Intracranial diseases are confused and delayed treatment, so the mortality rate is high.
  • the disease can also involve the lungs, skin, subcutaneous tissue, epiphysis, joints, and other internal organs and tissues, and can occur at any age.
  • the human body is often exposed to environments containing various cryptococci, and the human body's immunity to cryptococci includes cellular immunity and humoral immunity. Macrophages, neutrophils, lymphocytes, natural killer cells play an important role.
  • Humoral immunity includes: anti-capsular polysaccharide antibodies and complement participate in opsonophagocytosis, assisting phagocytes to phagocytose Cryptococcus. Only when the body's resistance is reduced, pathogenic bacteria can easily invade the human body and cause disease.
  • Amoeba infectious disease is an infectious disease caused by amoeba protozoa that grows in water and can erode the human brain. Amoeba will enter the human body through the nose, drill into the skull, and erode brain tissue, resulting in fatality. It is also due to the decline of the body's immunity that it is easy to make people fall ill.
  • pathogens After pathogens enter the body, they will induce specific immunity including cellular immunity and humoral immunity. Some pathogenic bacteria are not easy to be recognized by the human body, so they cannot be attacked. The recognition and killing effect will be greatly reduced, so a drug is needed to enhance this recognition and killing effect. For example, cryptococcal infection, amoeba infection and other pathogen infection diseases currently have no effective drug treatment. Therefore, it is particularly important to invent a new drug to combat the infection of pathogens.
  • the pathogen and/or tumor cell recognition functional domain the immune cell recruitment functional domain, the immune cell activation functional domain and the auxiliary killing functional domain are fused together to form a multi-functional domain.
  • the functional fusion protein also has the function of recognizing pathogens, tumor cells and/or B cell receptors and mobilizing the immune system to kill pathogens and tumor cells.
  • the first aspect of the present invention provides a fusion protein comprising the following functional domains:
  • the fusion protein further comprises:
  • the recognition functional domain includes proteins that recognize pathogens, tumor cells and/or B cell receptors, further preferably, the proteins include but are not limited to proteins that recognize pathogens, tumor cells and/or B cell receptors Antibody variable regions or cell membranes can recognize pathogens, tumor cells and/or natural receptors of B cells.
  • the natural receptors include but are not limited to Dectin1, DC-205 (dendritic cell receptor), MR (mannose receptor), DNGR-1 (dendritic cell natural killer cell lectin family receptor body 1), DNGR-2 (dendritic cell natural killer cell lectin family receptor 2), Mincle (macrophage-inducible C-type lectin receptor), CLECSF8, CLEC5A (C-type lectin 5A), DCIR One or more of (dendritic cell immune receptor), DC-SIGN (dendritic cell-specific intercellular adhesion molecule 3-binding non-integrin).
  • the recognition functional domain includes antibody scFv and/or antigens that specifically recognize pathogens, tumor cells and/or B cell receptors.
  • the recognition domain also includes a tag protein
  • the tag protein includes but not limited to streptavidin-binding peptide tag, polyarginine, polyhistidine, calmodulin Binding polypeptide (CBP), c-myc protein, glutathione S-transferase (GST), FLAG polypeptide, staphylococcal protein A, maltose binding protein, thioredox protein, small molecule ubiquitin-like modifying protein (SUMO), HA protein, AviTag protein, enhanced green fluorescent protein (eGFP), enhanced yellow-green fluorescent protein (eCFP), enhanced yellow-green fluorescent protein (eYFP), monomeric red fluorescent protein (mCherry).
  • streptavidin-binding peptide tag polyarginine, polyhistidine, calmodulin Binding polypeptide (CBP), c-myc protein, glutathione S-transferase (GST), FLAG polypeptide, staphylococcal protein A, maltose binding protein, thior
  • the tagged protein is a small molecule tagged protein, preferably, the tagged protein is 8aa.
  • the pathogen includes but not limited to parasite, fungi, bacteria or virus.
  • the parasite includes but is not limited to one of egg antigen, amoeba, helminth, malaria or leishmania or Various.
  • the fungi include, but are not limited to, Cryptococcus, Malaria furfur, Trichomonas, Microsporum, Epidermophyton flocculus, colored fungi, Sporotrichum, Candida, Aspergillus, Mucor or One or more species of Pneumocystis carinii.
  • the bacteria include but are not limited to Streptococcus (Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus viridans, Streptococcus agalactiae, Streptococcus equi, Streptococcus bovis), staphylococcus (Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus), Mycoplasma (Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma genitalium, Mycoplasma penetrating, Ureaplasma urealyticum), Actinomycetes, Nocardia, Mycobacterium, Escherichia, Salmonella, Shigella, Spirochetes (Leptospira, Borrelia burgdorferi, Borrelia relapsing fever, Treponema pallidum, Treponema fragilis), Chlamydia
  • the viruses include but are not limited to hepatitis virus (type A, type B, type C, type D, type E), norovirus, rotavirus, coxsackie virus, echovirus, star Virus, measles virus, varicella-zoster virus, rubella virus, Epstein-Barr virus, mumps virus, herpes virus, influenza virus, avian influenza virus or common coronavirus.
  • the pathogen includes cryptococcus or amoeba.
  • the recruitment domain includes a domain of a protein that has a recruitment function and/or regulates the function of endogenously recruiting immune cells.
  • the domain of a protein includes but is not limited to chemokines, other cellular Domains of factors or antibodies.
  • the chemokine is selected from CC chemokine subfamily, CXC chemokine subfamily, XC chemokine subfamily or CX3C chemokine subfamily.
  • the chemokines include but are not limited to CXCL8, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CCL11, CCL7, CCL8, CCL13, CCL5, CCL3, CCL4, CCL2, CCL20, CCL19, CCL21, CXCL12, CXCL13 , XCL1, CX 3 CL1, CCL17, CCL22, CXCL9, CXCL10, CXCL11, CCL1, CCL25, CCL27, CCL28, CXCL16.
  • the antibody is selected from full-length antibodies, chimeric antibodies, Fab fragments, Fab' fragments, Fd fragments, Fd' fragments, Fv fragments, dAb fragments, isolated CDR regions, F(ab') 2 fragments, Single-domain antibody (DAB), single-chain antibody molecule (such as single-chain Fv; scFv), diabody, triabody, linear antibody or a combination of two or more.
  • DAB Single-domain antibody
  • single-chain antibody molecule such as single-chain Fv; scFv
  • the recruitment domain can also activate immune cells.
  • the recognition functional domain is a protein domain that can recognize pathogenic bacteria such as scFv, nanobody or cell surface receptors (such as Dectin1, etc.), and the chemokine binding domain behind the recognition functional domain (including strepTactin, antibody ScFv, leucine zipper, etc.), can bind chemokines with small tags such as strep, Flag, HA, etc., and then recruit immune cells.
  • pathogenic bacteria such as scFv, nanobody or cell surface receptors (such as Dectin1, etc.)
  • chemokine binding domain behind the recognition functional domain including strepTactin, antibody ScFv, leucine zipper, etc.
  • small tags such as strep, Flag, HA, etc.
  • the activation domain includes a domain of a protein that activates immune cells or regulates a protein that activates immune cells. More preferably, the protein domain includes a cytokine or antibody domain.
  • the cytokines include but not limited to interferon or interleukin, further preferably, the cytokines include INF- ⁇ , IL-4 or IL-13.
  • the antibody is selected from full-length antibodies, chimeric antibodies, Fab fragments, Fab' fragments, Fd fragments, Fd' fragments, Fv fragments, dAb fragments, isolated CDR regions, F(ab') 2 fragments, Single-domain antibody (DAB), single-chain antibody molecule (such as single-chain Fv; scFv), diabody, triabody, linear antibody or a combination of two or more.
  • DAB Single-domain antibody
  • single-chain antibody molecule such as single-chain Fv; scFv
  • the activation domain also includes a protein domain that regulates endogenous cytokines.
  • the activation domain is the domain of anti-trem2.
  • the immune cells described in the present invention include but are not limited to neutrophil (neutrophils), basophil (basophils), eosinophil (eosinophils), monocyte (monocytes), immature DC (not Mature dendritic cells), mature DC (mature dendritic cells), B cell (B cells), Tfh (follicular helper T cells), NK cell (NK cells), Naive T (initial T cells), Tcm (central memory T cell), Treg (regulatory T cell), Th1 cell, Th2 cell, Th17 cell, ⁇ 4 ⁇ 7 + T cell, CLA + T cell, colon T (colon T cell), CD8 + T cell, NKT cell (NKT cells).
  • neutrophil neutrophil
  • basophil basophil
  • eosinophil eosinophils
  • monocyte monocyte
  • immature DC not Mature dendritic cells
  • mature DC mature dendritic cells
  • B cell B cells
  • Tfh follicular helper T cells
  • the helper killing domain includes, but is not limited to, antibody crystallizable fragments or antigen receptor fragments of immune cells.
  • the crystallizable fragment is an antibody constant region, further preferably, the constant region includes a KIH structure.
  • the crystallizable fragment includes interaction sites with Fc ⁇ R and C1q.
  • the crystallizable fragment includes interaction sites with FcRn, SpA, and SpG.
  • the effect of the helper killing domain includes a domain that induces antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) or complement-dependent cytotoxicity (CDC) .
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • CDC complement-dependent cytotoxicity
  • the antigen receptor fragment is TCR ⁇ or ⁇ chain.
  • the recognition domain, recruitment domain, activation domain or auxiliary killing domain is derived from human.
  • the second aspect of the present invention provides a nucleic acid encoding the above fusion protein.
  • the third aspect of the present invention provides an expression vector, said expression vector comprising the above-mentioned nucleic acid.
  • the expression vector can be expressed in vivo or in vitro or in vitro. Further preferably, the expression vector is continuously expressed at a high level in cells in vivo.
  • the expression vector may be a prokaryotic expression vector or a retrovirus vector.
  • the prokaryotic expression vector is Escherichia coli series.
  • Rous sarcoma virus (RSV), lentivirus, human immunodeficiency virus (HIV), murine leukemia virus (MLV), equine infectious anemia virus (EIAV), mouse breast cancer virus (MMTV), Fujinami Sarcoma virus (FuSV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine leukemia virus (Mo-MLV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV ), avian myeloproliferative virus 29 (MC29) or avian erythroblastosis virus (AEV), etc.
  • RSV Rous sarcoma virus
  • HAV human immunodeficiency virus
  • MMV murine leukemia virus
  • EIAV equine infectious anemia virus
  • mouse breast cancer virus MMTV
  • Fujinami Sarcoma virus FuSV
  • FBR murine osteosarcoma virus FBR MSV
  • the fourth aspect of the present invention provides a host cell, said host cell comprising the above-mentioned nucleic acid or expression vector.
  • said host cell can be eukaryotic or prokaryotic.
  • the host cells include but not limited to yeast cells, animal cells (such as 293 cells, CHO cells), insect cells, Escherichia coli, Bacillus subtilis, Streptomyces and the like.
  • the fifth aspect of the present invention provides a method for preparing the above-mentioned fusion protein, comprising the following steps:
  • step (2) transforming or transfecting the expression vector obtained in step (1) into a host cell, and then inducing its expression;
  • the fusion protein is obtained after purification.
  • the sixth aspect of the present invention provides a pharmaceutical composition, which includes the above-mentioned fusion protein and pharmaceutically acceptable excipients.
  • the pharmaceutical composition can also be used together with other therapeutic agents.
  • the therapeutic agent may be an immunomodulator.
  • the seventh aspect of the present invention provides the application of the above-mentioned fusion protein, the above-mentioned expression vector, the above-mentioned host cell or the fusion protein prepared by the method for preparing the above-mentioned fusion protein in the preparation of a pharmaceutical composition for treating pathogenic infection or tumor.
  • the pathogen includes but not limited to parasite, fungi, bacteria or virus.
  • the parasite includes but is not limited to one of egg antigen, amoeba, helminth, malaria or leishmania or Various.
  • the fungi include, but are not limited to, Cryptococcus, Malaria furfur, Trichomonas, Microsporum, Epidermophyton flocculus, colored fungi, Sporotrichum, Candida, Aspergillus, Mucor or One or more species of Pneumocystis carinii.
  • the bacteria include but are not limited to Streptococcus (Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus viridans, Streptococcus agalactiae, Streptococcus equi, Streptococcus bovis), staphylococcus (Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus), Mycoplasma (Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma genitalium, Mycoplasma penetrating, Ureaplasma urealyticum), Actinomycetes, Nocardia, Mycobacterium, Escherichia, Salmonella, Shigella, Spirochetes (Leptospira, Borrelia burgdorferi, Borrelia relapsing fever, Treponema pallidum, Treponema fragilis), Chlamydia
  • the viruses include but are not limited to hepatitis virus (type A, type B, type C, type D, type E), norovirus, rotavirus, coxsackie virus, echovirus, star Virus, measles virus, varicella-zoster virus, rubella virus, Epstein-Barr virus, mumps virus, herpes virus, influenza virus, avian influenza virus or common coronavirus.
  • the pathogenic infection includes cryptococcal infection or amoeba infection.
  • the eighth aspect of the present invention provides a method for treating pathogen infection or tumor disease, the method comprising administering to an individual an effective amount of the fusion protein of the present invention, the expression vector, the host cell, the fusion protein The fusion protein or the pharmaceutical composition prepared by the preparation method.
  • the pathogen includes but not limited to parasite, fungi, bacteria or virus.
  • the parasite includes but is not limited to one of egg antigen, amoeba, helminth, malaria or leishmania or Various.
  • the fungi include, but are not limited to, Cryptococcus, Malaria furfur, Trichomonas, Microsporum, Epidermophyton flocculus, colored fungi, Sporotrichum, Candida, Aspergillus, Mucor or One or more species of Pneumocystis carinii.
  • the bacteria include but are not limited to Streptococcus (Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus viridans, Streptococcus agalactiae, Streptococcus equi, Streptococcus bovis), staphylococcus (Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus), Mycoplasma (Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma genitalium, Mycoplasma penetrating, Ureaplasma urealyticum), Actinomycetes, Nocardia, Mycobacterium, Escherichia, Salmonella, Shigella, Spirochetes (Leptospira, Borrelia burgdorferi, Borrelia relapsing fever, Treponema pallidum, Treponema fragilis), Chlamydia
  • the viruses include but are not limited to hepatitis virus (type A, type B, type C, type D, type E), norovirus, rotavirus, coxsackie virus, echovirus, star Virus, measles virus, varicella-zoster virus, rubella virus, Epstein-Barr virus, mumps virus, herpes virus, influenza virus, avian influenza virus or common coronavirus.
  • the pathogenic infection includes cryptococcal infection or amoeba infection.
  • the method for treating diseases also includes other synergistic treatment means, preferably one or a combination of two or more of chemotherapy, surgery, radiotherapy, gene therapy, hormone therapy, and immunotherapy.
  • other synergistic treatment means preferably one or a combination of two or more of chemotherapy, surgery, radiotherapy, gene therapy, hormone therapy, and immunotherapy.
  • the "tumor” mentioned in the present invention includes but not limited to lymphoma, non-small cell lung cancer, cervical cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer , glioma, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophagus cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma Cytoma, melanoma, myelodysplastic syndrome, and sarcoma.
  • the leukemia is selected from acute lymphocytic (lymphoblastic) leukemia, acute myelogenous leukemia, myelogenous leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myelogenous leukemia;
  • the lymphoma is selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma , T-cell lymphoma, and Waldenstrom macroglobulinemia;
  • said sarcoma is selected from the group consisting of osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma
  • treating means slowing, interrupting, arresting, controlling, stopping, alleviating, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease after the disease has begun to develop, but not necessarily The complete elimination of all disease-related signs, symptoms, conditions, or disorders is contemplated.
  • the "expression vector” in the present invention can be any vector in the prior art that can carry nucleic acid and stably replicate and express in the host. It contains an origin of replication, a promoter, marker genes and translational control elements. Preferably, they may be bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian viruses, and the like.
  • the "antibody” of the present invention includes, but is not limited to: Fab fragments, which have VL, CL, VH and CH1 domains; Fab' fragments, which have one or more cysteine residues at the C-terminal of the CH1 domain Fab fragment; Fd fragment, which has VH and CH1 domains; Fd' fragment, which has VH and CH1 domains and one or more cysteine residues at the C-terminus of CH1 domain; Fv fragment, which has a single antibody
  • a bivalent fragment of a single chain antibody molecule e.g.
  • scFv single chain Fv
  • scFv single chain Fv
  • a "diabody” with two antigen binding sites comprising a heavy chain linked to a light chain variable domain (VL) in the same polypeptide chain a variable domain (VH); a "linear antibody” comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) which together with a complementary light chain polypeptide form a pair of antigen-binding regions; and any of the foregoing
  • Ig immunoglobulin
  • TCR T cell antigen receptor
  • the immunoglobulin can be an antibody, and the CDRs correspond to the complementarity determining regions in the variable sequence of the antibody.
  • the system described by Kabat et al (Kabat et al, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides unambiguous residue numbering applicable to antibody variable regions system, but also provides residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs.
  • Each CDR may contain amino acid residues from a "complementarity determining region" as defined by Kabat. Chothia et al. ( Chothia & Lesk, J. Mol.
  • the residue boundary of the CDR in the TCR is the same as above.
  • the "antibody variable region” refers to the light chain and the heavy chain of the antibody molecule comprising a complementarity determining region (CDR) and a framework region (FR) part of the amino acid sequence.
  • VH refers to the variable domain of the heavy chain.
  • VL refers to the variable domain of the light chain.
  • a fusion protein by constructing a fusion protein, the pathogen and/or tumor cell recognition functional domain, the immune cell recruitment functional domain, the immune cell activation functional domain and the auxiliary killing functional domain are fused together to form a multifunctional protein, which has the ability to detect pathogens, tumor cells and /or the recognition of B cell receptors and the function of inducing the immune system to kill pathogens and tumor cells.
  • the fusion protein in the present application can effectively eliminate pathogenic infection and/or tumor cells, and can effectively identify and attack even pathogenic bacteria covered with capsules on the surface. Compared with ordinary antibody drugs, the fusion protein drug has a more effective ability to kill pathogens and/or tumor cells.
  • the recognition domain of the present invention can recognize pathogens and/or tumor cells that cannot be recognized by the human immune system.
  • the combination of the recognition domain and the recruitment domain of the present invention can be used as a multiple Attractant for immune cells, its small molecular structure may increase permeability, while a small tag (8aa) at the C-terminus of chemokines maintains its activity and facilitates the combination of different scFvs and chemokines.
  • pathogens and/or tumor cells can be recognized and killed when the human immunity is insufficient, the recognition and killing effects will be greatly reduced. This drug can enhance the killing performance of the immune system.
  • the fusion protein of the present invention also includes a recruitment domain, which can recruit and mobilize the innate and adaptive immune systems in the body, thereby further improving the killing effect on pathogens and/or tumor cells.
  • the functional domain of the fusion protein of the present invention can use the domain of the protein in the human body to avoid the immunogenicity of other protein drugs and the immune rejection caused by the immunogenicity.
  • the preparation method of the fusion protein of the present invention is simple, and various functional domains can be easily replaced to realize the recognition of various diseases and the recruitment and killing of various corresponding immune cells.
  • the fusion protein or pharmaceutical composition of the present invention because the recognition functional domain can recognize multiple pathogens or tumor cells, can treat multiple diseases at the same time, especially tumor patients, who are more likely to be infected due to chemotherapy and other reasons , the pharmaceutical composition of the present invention can not only treat tumors but also treat pathogens, simplify the method of administration, and realize the all-in-one administration mode.
  • Figure 1 Schematic diagram of fusion protein drug
  • FIG. 1 Schematic diagram of the recruitment functional domain
  • Figure 3 Schematic diagram of the interaction between the recognition domain and the recruitment domain
  • Figure 4 Flow diagram of the recognition function detection of fusion protein drugs on pathogens (taking Candida albicans as an example).
  • the nucleic acid of the fusion protein is connected to the carrier backbone to obtain the expression vector; the obtained expression vector is transformed into a host cell, and then its expression is induced; after purification, the fusion protein is obtained, and the structure of the fusion protein and the schematic diagram of the recruitment functional domain are shown in Figure 1 and As shown in FIG. 2 , in the constructed fusion protein, the interaction mechanism between the recognition functional domain and the recruitment functional domain is shown in FIG. 3 .
  • the effectiveness of the drug is tested by constructing a mouse pathogen (Candida albicans, Cryptococcus or amoeba) infection model or a tumor model.
  • a mouse pathogen Candida albicans, Cryptococcus or amoeba
  • group A/B/C/D Take 4 groups of 10 male and female C57BL/6 mice aged 6-8 weeks, named group A/B/C/D, and inject 5 ⁇ 10 5 CFU white rosary beads into group A/B/C at the same time Bacteria, group D was not treated;
  • group A was injected with 100 microliters of PBS; group B was injected with 1 microgram of the fusion protein prepared in Example 1; group C/D was injected with 10 micrograms of the fusion protein prepared in example 1;
  • mice injected with protein drugs was significantly longer than that of mice injected with PBS.

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Abstract

L'invention concerne une protéine de fusion contenant un domaine à fonction de reconnaissance, un domaine à fonction de recrutement, un domaine à fonction d'activation, et un domaine à fonction de destruction auxiliaire, son procédé de construction et son utilisation dans le domaine des biomédicaments. La protéine de fusion peut reconnaître des agents pathogènes et mobiliser un système immunitaire pour détruire les agents pathogènes, et présente une valeur d'application importante pour de nouvelles recherches et développement de médicament pour traiter une infection pathogène ou des cellules tumorales.
PCT/CN2022/110178 2021-08-04 2022-08-04 Médicament à base de protéine de fusion multifonctionnelle pour cibler et détruire des agents pathogènes et/ou des cellules tumorales Ceased WO2023011575A1 (fr)

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CN202280062222.9A CN117957257A (zh) 2021-08-04 2022-08-04 一种靶向并杀伤病原体和/或肿瘤细胞的多功能融合蛋白药物

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