WO2021109914A1 - Dimeric immune fusion protein, pharmaceutical composition and use - Google Patents

Dimeric immune fusion protein, pharmaceutical composition and use Download PDF

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WO2021109914A1
WO2021109914A1 PCT/CN2020/131581 CN2020131581W WO2021109914A1 WO 2021109914 A1 WO2021109914 A1 WO 2021109914A1 CN 2020131581 W CN2020131581 W CN 2020131581W WO 2021109914 A1 WO2021109914 A1 WO 2021109914A1
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seq
tlr4
lps
polypeptide chain
tlr2
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Chinese (zh)
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傅文燕
丁敏
胡适
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沣潮医药科技（上海）有限公司
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6813—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin the drug being a peptidic cytokine, e.g. an interleukin or interferon
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    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • A61P31/04—Antibacterial agents
    • 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
    • A61P31/10—Antimycotics
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P33/00—Antiparasitic agents
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    • A61P33/00—Antiparasitic agents
    • A61P33/10—Anthelmintics
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/7056—Lectin superfamily, e.g. CD23, CD72
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention relates to the technical field of biomedical engineering, in particular to a dimer immune fusion protein, a pharmaceutical composition using it as an active component and its medical use, especially its use for diseases related to inflammatory mediators.
  • an inflammatory response occurs when cells or tissues are damaged by bacteria, trauma, toxins, physical or chemical factors (which can be collectively referred to as "inflammatory agents").
  • the pathophysiological characteristics of the inflammatory response are regulated by the complex interaction of multiple pro-inflammatory or anti-inflammatory stimulants or mediators synthesized and released by cells.
  • pro-inflammatory and anti-inflammatory stimulants or mediators include cytokines, nitrous oxide, thromboxane, autolanene, phospholipid platelet activating factor, prostaglandins, kinetic skin, complement factors, and clotting factors , Superantigens, monocytes, chemokines, interferons, free radicals, proteases, arachidonic acid metabolites, cyclic prostaglandins, ⁇ -endorphins, myocardial depressant factor, anadamide, 2-peanut 2-arachidonoylglycerol (2-arachidonoylglycerol), tetrahydrobiological butterfly ridge, cell debris and chemical substances (including histamine, bradykinin and serotonin) and so on.
  • the nature and intensity of the inflammatory response are different according to the location of the attack, the nature of the inflammatory substance, and the interaction of the pro-inflammatory or anti-inflammatory stimulants or mediators involved.
  • an inflammatory response is beneficial.
  • the inflammatory response can cause significant tissue damage and even death.
  • the cytokines involved in inflammation are mainly a type of protein produced by macrophages, monocytes, neutrophils and lymphocytes. These cells are usually stimulated by viral, bacterial, fungal or parasitic infections, and in the immune response. Stimulated release of T cells. Other cells can also release inflammatory cytokines, such as stromal cells such as fibroblasts, endothelial cells, and smooth muscle cells, as well as epithelial cells, keratinocytes, and hepatocytes. Cytokines are usually present in blood or tissues in low concentrations.
  • cytokines The structure and activity of cytokines are a hot topic in immunology. Current research believes that cytokines have a wide range of immune and non-immune activities, which can affect a variety of physiological functions, such as cell growth, differentiation, homeostasis, and pathophysiology. At the same time, cytokines have a variety of biological activities and are involved in multiple activities. A biological regulation process; in addition, cytokines can promote their own synthesis and the production of other cytokines. These phenomena are called "cytokine cascades.” The cytokine cascade is usually associated with systemic changes caused by infection and tissue damage. In this case, the entire cytokine cascade network produces very complex cellular and biological effects, such as interleukins (IL) and interferons. A variety of cytokines such as (IF) and tumor necrosis factor (TNF) can be produced in immune and inflammatory responses.
  • IF interleukins
  • TNF tumor necrosis factor
  • the cytokine cascade mediates the normal host defense response, cell regulation, and cell differentiation.
  • the function of cytokine production may become disordered. This disorder can lead to the appearance of cytokines in excess of the normal concentration.
  • the impact on the body is two-sided: on the one hand, it resists invaders, but on the other hand, if it is too strong or lacks regulation, it can damage the body.
  • the present invention is to solve the above-mentioned problems. It provides a soluble dimer immune fusion protein, and describes the specific structure, preparation method and application of the soluble dimer immune fusion protein, that is, it provides Dimer immune fusion protein, its preparation method and application.
  • the first aspect of the present invention provides a soluble dimer-dimer immune fusion protein, comprising a dimerized first polypeptide chain and a second polypeptide chain, and the structural formula of the first polypeptide chain is Z1-Z2,
  • the general structural formula of the second polypeptide chain is Y1-Y2.
  • Z1 is (i) the extracellular domain of the first pattern recognition receptor or its functional variant or fragment, or (ii) the first co-receptor or its functional variant or fragment;
  • Z2 is a dimerization structure Domain or its functional variant or fragment,
  • Y1 is (i) the extracellular domain of the second pattern recognition receptor or its functional variant or fragment, or (ii) the second co-receptor or its functional variant or fragment ;
  • Y2 is a dimerization domain or a functional variant or fragment thereof.
  • Pattern recognition receptor is an immunological concept.
  • Pattern recognition receptor PRR
  • PRR is a type of recognition molecule that is mainly expressed on the surface of innate immune cells, is non-clonal and can recognize one or more PAMPs. . It is a representative of immune receptors in innate immunity. It is encoded by a limited number of germline genes and is evolutionarily conservative. It also shows that such receptors are extremely important for the survival of organisms. Its mutual recognition and interaction with pathogen-associated molecular pattern (PAMP) on the surface of pathogenic organisms is the key to initiating the innate immune response. Compared with lymphocyte receptors in adaptive immunity, PRR has four characteristics. In addition to being all encoded by germline genes, three other characteristics are: constitutive expression, rapid response and the ability to identify various pathogens. Any pattern recognition receptor is suitable for the fusion protein structure scheme of the present invention.
  • the first pattern recognition receptor and the second pattern recognition receptor are selected from: TLR1 (Gene ID: 7096), TLR2 (Gene ID: 7097), TLR3 (Gene ID: 7098), TLR4 (Gene ID: 7099), TLR5 (Gene ID: 7100), TLR6 (Gene ID: 7100) :10333), TLR7 (Gene ID: 51284), TLR8 (Gene ID: 51311), TLR9 (Gene ID: 54106), TLR10 (Gene ID: 81793), Dectin-1 (Gene ID: 64581), Dectin-2( Gene ID: 93978), Mincle (Gene ID: 26253), CLEC2 (Gene ID: 51266), CLEC5A (Gene ID: 23601), CLEC12A (Gene ID: 160364), DCIR (Gene ID:
  • the first and second pattern recognition receptors may be the same or different.
  • the first co-receptors and the second co-receptors are respectively selected from CD14 (Gene ID: 929) , MD-2 (Gene ID: 23643), LBP (Gene ID: 3929), CD36 (Gene ID: 948).
  • the first and second co-receptors may be the same or different.
  • the dimerization domain Z2 or Y 2 includes the constant region of an immunoglobulin heavy chain.
  • the dimerization domains Z2 and Y2 are Fc fragments of IgG, such as human immunoglobulin ⁇ 1 Fc fragments.
  • the dimerization domains Z2 and Y2 can be engineered to increase the formation of specific heterodimerization.
  • Z2 and Y2 are Fc fragments of IgG or Fc mutations that change their biological activity.
  • the dimerization domains Z2 and Y2 may be active variants of the Fc fragment of human immunoglobulin, such as using the Fc domain of IgG2, IgG3, or IgG4.
  • Fc mutants can be further used to reduce the biological activities of immunoglobulins such as ADCC, complement fixation, etc., such as LALA-PG mutants, L235E, E318A, K320A, K322A mutants, and the like.
  • the dimerization domains Z2 and Y2 also contain a peptide linker consisting of 15-32 amino acid residues, of which 1-8 (for example, 2) of these residues are cysteine Acid residues.
  • Z2 and Y2 comprise an immunoglobulin hinge region or variants thereof, for example, in a specific embodiment, Z2 and Y2 comprise an immunoglobulin hinge variant (for example, a human immunoglobulin ⁇ 1 hinge variant) , Where the cysteine residue corresponding to 220 of the Fc fragment is replaced by serine.
  • Particularly suitable peptide linkers used in accordance with the aforementioned dimerization domains Z2 and Y2 include peptide linkers that comprise a plurality of glycine residues, and optionally at least one serine residue.
  • the amino acid sequences of Z1 and Y1 include any one of the amino acids shown in SEQ ID NO. 1-18 with at least 60%, preferably at least 65%, preferably at least 70%, and more. Preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity.
  • the names of sequences 1-18 are as follows:
  • sequence name sequence name 1 Amino acids 25-475 of TLR1 10 Amino acids 20-576 of TLR10 2 Amino acids 27-506 of TLR2 11 Dectin-1 Amino acids 66-247 3 Amino acids 22-703 of TLR3 12 Dectin-2 Amino acids 64-209 4 Amino acids 27-631 of TLR4 13 Mincle 74-219 amino acids
  • TLR5 21-639 amino acids 14 Amino acids 96-221 of CLEC2 6 TLR6 Amino acids 32-586 15 CLEC5A Amino acids 70-187 7 Amino acids 27-839 of TLR7 16 CLEC12A Amino acids 65-265 8 Amino acids 27-827 of TLR8 17 DCIR 106-237 amino acids 9 Amino acids 26-818 of TLR9 18 CLECSF8 61-215 amino acids
  • the amino acid sequences of Z1 and Y1 include the amino acids shown in SEQ ID NO. 19-22. Any one of the sequences has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, It is preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity.
  • the names of sequences 19-22 are as follows:
  • sequence name sequence name 19 CD14 26-355 amino acids twenty one Amino acids 26-481 of LBP 20 MD-2 17-160 amino acids twenty two CD36 Amino acids 1-472
  • the amino acid sequences of the dimerization domains Z2 and Y2 include SEQ ID NO. 23-28. Any one of the amino acid sequences has at least 60%, preferably at least 65%, preferably at least 70%, and more preferably at least 75%. %, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity.
  • the names of sequences 23-29 are as follows:
  • both Z1 and Y1 are extracellular domains of TLR1 or functional variants or fragments thereof.
  • each of the Z1-Z2 polypeptide chain and Y1-Y2 polypeptide chain contains a sequence identical to the TLR1-IgG1-Fc amino acid sequence shown in SEQ ID NO. 30 below or has at least 60%, preferably at least 65 %, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity.
  • both Z1 and Y1 are extracellular domains of TLR1 or functional variants or fragments thereof.
  • each of the Z1-Z2 polypeptide chain and Y1-Y2 polypeptide chain contains a sequence consistent with the amino acid sequence of TLR1-IgG1-Fc-LALAPG shown in SEQ ID NO. 31 below or has at least 60%, preferably At least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity.
  • both Z1 and Y1 are extracellular domains of TLR2 or functional variants or fragments thereof.
  • each of the Z1-Z2 polypeptide chain and Y1-Y2 polypeptide chain contains a sequence identical to the TLR2-IgG1-Fc amino acid sequence shown in SEQ ID NO. 32 below or has at least 60%, preferably at least 65 %, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity.
  • both Z1 and Y1 are extracellular domains of TLR2 or functional variants or fragments thereof.
  • each of the Z1-Z2 polypeptide chain and Y1-Y2 polypeptide chain contains a sequence consistent with the amino acid sequence of TLR2-IgG1-Fc-LALA shown in SEQ ID NO. 33 below or has at least 60%, preferably At least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity.
  • both Z1 and Y1 are the extracellular domain of TLR4 or functional variants or fragments thereof.
  • each of the Z1-Z2 polypeptide chain and Y1-Y2 polypeptide chain contains a sequence identical to the TLR4-IgG1-Fc amino acid sequence shown in SEQ ID NO. 34 below or has at least 60%, preferably at least 65 %, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity.
  • both Z1 and Y1 are the extracellular domain of TLR4 or functional variants or fragments thereof.
  • each of the Z1-Z2 polypeptide chain and Y1-Y2 polypeptide chain contains a sequence identical to the amino acid sequence of TLR4-IgG1-Fc-LALAGP shown in SEQ ID NO. 35 below or has at least 60%, preferably At least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity.
  • both Z1 and Y1 are extracellular domains of TLR6 or functional variants or fragments thereof.
  • each of the Z1-Z2 polypeptide chain and Y1-Y2 polypeptide chain contains a sequence identical to the TLR6-IgG1-Fc amino acid sequence shown in SEQ ID NO. 36 below or has at least 60%, preferably at least 65 %, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity.
  • both Z1 and Y1 are extracellular domains of TLR6 or functional variants or fragments thereof.
  • each of the Z1-Z2 polypeptide chain and Y1-Y2 polypeptide chain contains a sequence identical to the amino acid sequence of TLR6-IgG1-Fc-LALAGP shown in SEQ ID NO. 37 below or has at least 60%, preferably At least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity.
  • Z1 is the extracellular domain of TLR1 or a functional variant or fragment thereof.
  • Y1 is the extracellular domain of TLR2 or a functional variant or fragment thereof.
  • the Z1-Z2 polypeptide chain includes a sequence identical to the TLR1-Fc-Knob amino acid sequence shown in SEQ ID NO.
  • the Y1-Y2 polypeptide chain comprises the following The TLR2-Fc-Hole amino acid sequence shown in SEQ ID NO. 39 is identical or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85 %, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity;
  • Z1 is the extracellular domain of TLR1 or a functional variant or fragment thereof.
  • Y1 is the extracellular domain of TLR6 or a functional variant or fragment thereof.
  • the Z1-Z2 polypeptide chain includes a sequence identical to the TLR1-Fc-Knob amino acid sequence shown in SEQ ID NO.
  • the Y1-Y2 polypeptide chain comprises the following The TLR6-Fc-hole amino acid sequence shown in SEQ ID NO. 40 is identical or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85 %, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity;
  • Z1 is the extracellular domain of TLR2 or a functional variant or fragment thereof.
  • Y1 is the extracellular domain of TLR4 or a functional variant or fragment thereof.
  • the Z1-Z2 polypeptide chain includes a sequence identical to the TLR2-Fc-Hole amino acid sequence shown in SEQ ID NO.
  • the Y1-Y2 polypeptide chain comprises the following The TLR4-IgG-Knob amino acid sequence shown in SEQ ID NO. 41 is identical or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85 %, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity;
  • Z1 is the extracellular domain of TLR2 or a functional variant or fragment thereof.
  • Y1 is the extracellular domain of TLR6 or a functional variant or fragment thereof.
  • the Z1-Z2 polypeptide chain includes a sequence identical to the TLR2-Fc-Hole amino acid sequence shown in SEQ ID NO.
  • the Y1-Y2 polypeptide chain comprises the following The TLR6-Fc-knob amino acid sequence shown in SEQ ID NO. 42 is identical or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85 %, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity;
  • Z1 is the extracellular domain of TLR4 or a functional variant or fragment thereof.
  • Y1 is the extracellular domain of TLR6 or a functional variant or fragment thereof.
  • the Z1-Z2 polypeptide chain includes a sequence identical to the TLR4-IgG-Knob amino acid sequence shown in SEQ ID NO.
  • the Y1-Y2 polypeptide chain comprises the following The TLR6-Fc-hole amino acid sequence shown in SEQ ID NO. 40 is identical or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85 %, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity;
  • Z1 is the extracellular domain of TLR4 or a functional variant or fragment thereof.
  • Y1 is LBP or a functional variant or fragment thereof.
  • the Z1-Z2 polypeptide chain includes a sequence identical to the TLR4-IgG-Knob amino acid sequence shown in SEQ ID NO. 41 below or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably At least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity; the Y1-Y2 polypeptide chain comprises the following The LBD-Fc-hole amino acid sequence shown in SEQ ID NO.
  • 43 has the same sequence or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85 %, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity;
  • Z1 is the extracellular domain of TLR4 or a functional variant or fragment thereof.
  • Y1 is the extracellular domain of CD14 or a functional variant or fragment thereof.
  • the Z1-Z2 polypeptide chain includes a sequence identical to the TLR4-IgG-Knob amino acid sequence shown in SEQ ID NO.
  • the Y1-Y2 polypeptide chain comprises the following The amino acid sequence of CD14 Fc hole shown in SEQ ID NO. 44 is identical or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, Even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity;
  • Z1 is the extracellular domain of TLR4 or a functional variant or fragment thereof.
  • Y1 is MD-2 or a functional variant or fragment thereof.
  • the Z1-Z2 polypeptide chain includes a sequence identical to the TLR4-IgG-Knob amino acid sequence shown in SEQ ID NO. 41 below or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably At least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity;
  • the Y1-Y2 polypeptide chain comprises the following The sequence of the MD-2 Fc hole shown in SEQ ID NO.
  • 45 is consistent with the amino acid sequence or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85 %, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity;
  • Z1 is the extracellular domain of CD14 or a functional variant or fragment thereof.
  • Y1 is MD-2 or a functional variant or fragment thereof.
  • the Z1-Z2 polypeptide chain comprises a sequence identical to the amino acid sequence of CD14 Fc knob shown in SEQ ID NO. 46 below, or has at least 60%, preferably at least 65%, preferably at least 70%, and more preferably at least 75%.
  • the Y1-Y2 polypeptide chain comprises the following SEQ ID
  • the MD-2 Fc hole amino acid sequence shown in NO.45 has the same sequence or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, Even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity;
  • Z1 is the extracellular domain of TLR4 or a functional variant or fragment thereof.
  • Y1 is the extracellular domain of CD36 or a functional variant or fragment thereof.
  • the Z1-Z2 polypeptide chain includes a sequence identical to the TLR4-IgG-Knob amino acid sequence shown in SEQ ID NO.
  • the Y1-Y2 polypeptide chain comprises the following The amino acid sequence of CD36 Fc hole shown in SEQ ID NO. 47 is identical or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, Even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity;
  • Z1 is the extracellular domain of TLR6 or a functional variant or fragment thereof.
  • Y1 is the extracellular domain of CD36 or a functional variant or fragment thereof.
  • the Z1-Z2 polypeptide chain comprises a sequence identical to the TLR6-Fc-knob amino acid sequence shown in SEQ ID NO. 42 below or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably At least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity;
  • the Y1-Y2 polypeptide chain comprises the following The amino acid sequence of CD36 Fc hole shown in SEQ ID NO.
  • 47 is identical or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, Even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity;
  • both Z1 and Y1 are the extracellular domain of Dectin-1 or functional variants or fragments thereof.
  • each of the Z1-Z2 polypeptide chain and the Y1-Y2 polypeptide chain contains a sequence consistent with the Dectin-1 IgG Fc amino acid sequence shown in SEQ ID NO. 48 below or has at least 60%, preferably at least 65 %, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity.
  • both Z1 and Y1 are the extracellular domain of Dectin-2 or functional variants or fragments thereof.
  • each of the Z1-Z2 polypeptide chain and the Y1-Y2 polypeptide chain contains a sequence consistent with the Dectin-2 IgG Fc amino acid sequence shown in SEQ ID NO. 49 below or has at least 60%, preferably at least 65 %, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity.
  • Z1 is the extracellular domain of Dectin-1 or a functional variant or fragment thereof.
  • Y1 is the extracellular domain of Mincle or a functional variant or fragment thereof.
  • the Z1-Z2 polypeptide chain includes a sequence consistent with the Dectin-1 IgG Fc-knob amino acid sequence shown in SEQ ID NO.
  • the Y1-Y2 polypeptide chain comprises the following The amino acid sequence of Mincle IgG Fc-hole shown in SEQ ID NO. 51 is identical or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably At least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity;
  • Z1 is the extracellular domain of Dectin-2 or a functional variant or fragment thereof.
  • Y1 is the extracellular domain of CLEC2 or a functional variant or fragment thereof.
  • the Z1-Z2 polypeptide chain includes a sequence consistent with the Dectin-2 IgG Fc-knob amino acid sequence shown in SEQ ID NO.
  • the Y1-Y2 polypeptide chain comprises the following
  • the CLEC2-Fc-hole amino acid sequence shown in SEQ ID NO.53 has the same sequence or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably At least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity;
  • Z1 is the extracellular domain of Dectin-1 or a functional variant or fragment thereof.
  • Y1 is the extracellular domain of CLEC5A or a functional variant or fragment thereof.
  • the Z1-Z2 polypeptide chain includes a sequence consistent with the Dectin-1 IgG Fc-knob amino acid sequence shown in SEQ ID NO.
  • the Y1-Y2 polypeptide chain comprises the following The CLEC5A-Fc-hole amino acid sequence shown in SEQ ID NO. 54 is identical or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably At least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity;
  • Z1 is the extracellular domain of Dectin-2 or a functional variant or fragment thereof.
  • Y1 is the extracellular domain of CLEC12A or a functional variant or fragment thereof.
  • the Z1-Z2 polypeptide chain includes a sequence consistent with the Dectin-2 IgG Fc-knob amino acid sequence shown in SEQ ID NO.
  • the Y1-Y2 polypeptide chain comprises the following
  • the CLEC12A Fc hole amino acid sequence shown in SEQ ID NO. 55 has the same sequence or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85 %, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity;
  • Z1 is the extracellular domain of Dectin-1 or a functional variant or fragment thereof.
  • Y1 is the extracellular domain of DCIR or a functional variant or fragment thereof.
  • the Z1-Z2 polypeptide chain includes a sequence consistent with the Dectin-1 IgG Fc-knob amino acid sequence shown in SEQ ID NO.
  • the Y1-Y2 polypeptide chain comprises the following The sequence of the DCIR Fc hole shown in SEQ ID NO.56 is identical or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85 %, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity;
  • Z1 is the extracellular domain of Dectin-2 or a functional variant or fragment thereof.
  • Y1 is the extracellular domain of CLECSF8 or a functional variant or fragment thereof.
  • the Z1-Z2 polypeptide chain includes a sequence consistent with the Dectin-2 IgG Fc-knob amino acid sequence shown in SEQ ID NO.
  • the Y1-Y2 polypeptide chain comprises the following
  • the CLECSF8 Fc hole amino acid sequence shown in SEQ ID NO. 57 has the same sequence or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85 %, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity;
  • both Z1 and Y1 are the extracellular domain of Dectin-1 or functional variants or fragments thereof.
  • each of the Z1-Z2 polypeptide chain and the Y1-Y2 polypeptide chain contains a sequence consistent with the Dectin-1 IgG4 Fc amino acid sequence shown in SEQ ID NO. 58 below or has at least 60%, preferably at least 65 %, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity.
  • Z1 is the extracellular domain of TLR2 or a functional variant or fragment thereof.
  • Y1 is the extracellular domain of Dectin-1 or a functional variant or fragment thereof.
  • the Z1-Z2 polypeptide chain includes a sequence consistent with the amino acid sequence shown in SEQ ID NO.
  • the Y1-Y2 polypeptide chain comprises the following SEQ ID NO.50
  • the shown Dectin-1 IgG Fc-knob amino acid sequence is consistent or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, or even More preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity.
  • the second aspect of the present invention provides a polynucleotide encoding the dimeric immune fusion protein, a vector for carrying the nucleotide, and a cell containing the vector.
  • the expression vector provided by the present invention includes the following operably linked elements: a transcription promoter, a DNA region encoding the dimeric immune fusion protein, and a transcription terminator.
  • culturing a cell containing a vector for the production of the polypeptide or dimeric protein as disclosed above including: (i) culturing a cell containing the expression vector as disclosed above, wherein the cell expresses the dimer immunofusion encoded by the DNA segment Protein and produce the encoded dimer immune fusion protein; (ii) recover the soluble dimer immune fusion protein.
  • the method for preparing a dimeric protein includes: (i) culturing a cell containing the expression vector disclosed above, wherein the cell expresses the dimeric immune fusion protein encoded by the DNA segment, and producing the encoded dimeric immune The fusion protein is used as a dimeric protein; and (ii) the dimeric protein is recovered.
  • the third aspect of the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the above-mentioned soluble dimer immune fusion protein and at least one pharmaceutically acceptable carrier.
  • the pharmaceutical composition uses a soluble dimer immune fusion protein as the main or only active ingredient, and the auxiliary materials can ensure the conformational integrity of the amino acid core sequence of the dimer immune fusion protein disclosed in the present invention, and at the same time protect the multifunctional group of the protein , To prevent its degradation (including but not limited to agglomeration, deamination or oxidation), so as to more stably exert its curative effect.
  • the drug in the form of the drug, it can be a suspension, water injection, freeze-dried preparation commonly used in the pharmaceutical field, and preferably a water injection or freeze-dried preparation.
  • Liquid formulations can be stored at 2°C-8°C for at least one year, and lyophilized formulations can be stored at 30°C for at least six months.
  • pharmaceutically acceptable excipients include one or a combination of surfactants, solution stabilizers, isotonic regulators, and buffers.
  • surfactants include non-ionic surfactants such as polyoxyethylene sorbitol fatty acid esters (Tween 20 or 80); poloxamer (such as poloxamer 188); Triton; sodium dodecyl sulfate (SDS); lauric sulfuric acid Sodium; tetradecyl, linoleyl or octadecyl sarcosine; Pluronics; MONAQUATTM, etc.
  • the amount added should minimize the tendency of bifunctional bispecific antibody protein to granulate
  • solution stabilizers can be sugars, Including reducing sugars and non-reducing sugars, amino acids include monosodium glutamate or histidine, alcohols include triols, higher sugar alcohols, propylene
  • the dimeric immune fusion protein of the present invention and its composition as an active ingredient have the following uses: 1) Combining with pathogenic microorganism surface molecules, cell walls or cell surface components, as described in Example 1; 2) Directly killing pathogens Microorganisms, restrict the growth of pathogenic microorganisms, and improve the resistance of immune cells to pathogenic microorganism invasion, as described in Examples 2-9; 3) Inhibit and reduce overexpression and secretion of inflammatory mediators and/or cytokines, such as HMGB1, TNF ⁇ , IFN- ⁇ , IL-6, COX-2, etc.
  • inflammatory mediators and/or cytokines such as HMGB1, TNF ⁇ , IFN- ⁇ , IL-6, COX-2, etc.
  • Example 10-11 Reduce the over-expression and release of organ inflammatory mediators, reduce organ inflammation damage, enhance organ anti-stress ability, resist acute and chronic organ damage, resist hypertoxicity, etc., as in Example 12 -13; 5) Reduce chronic inflammatory mediator damage and reduce organ inflammation and fibrosis, as in Examples 14-15; 6) Inhibit local immune tolerance disorders, such as immune infertility as described in Example 16. 7) Inhibition of excessive inflammatory mediator diseases mediated by abnormal autoimmune tolerance, such as autoimmune diseases such as lupus, as in Example 17.
  • the dimeric immune fusion protein of the present invention and the composition thereof as an active ingredient have the following uses: including prevention, diagnosis and treatment of drugs, reagents, and drugs related to diseases that require the removal of inflammatory mediators. Any one or a combination of at least two of the kit uses.
  • the inflammatory mediators include pathogenic microorganisms such as viruses, bacteria or parasites, as well as enzymes, cytokines, prostaglandins, eicosanoids, Leukotrienes, Kinins, complements, coagulation factors, toxins, endotoxins, enterotoxins, lipopolysaccharides, substances that induce apoptosis, corrosive substances, bile salts, fatty acids, phospholipids, oxidation by-products, reactive oxygen species, oxygen Any one or a combination of free radicals, surfactants, ions, irritating substances, cell debris, interferons, and immunomodulatory antibodies, biologics, and drugs.
  • pathogenic microorganisms such as viruses, bacteria or parasites, as well as enzymes, cytokines, prostaglandins, eicosanoids, Leukotrienes, Kinins, complements, coagulation factors, toxins, endotoxins, enterotoxins, lipopolysaccharides, substances
  • the inflammatory mediator is present in the physiological fluid or carrier fluid of the subject, and the physiological fluid includes the following fluids: the physiological fluid includes the following fluids: nasopharyngeal, oral cavity, esophagus, stomach, Pancreas, liver, pleura, pericardium, peritoneum, intestine, prostate, semen, vaginal secretions, tears, saliva, mucus, bile, blood, lymph, plasma, serum, synovial fluid, cerebrospinal fluid, urine, as well as spaces, intracellular and cellular Fluid outside.
  • the physiological fluid includes the following fluids: nasopharyngeal, oral cavity, esophagus, stomach, Pancreas, liver, pleura, pericardium, peritoneum, intestine, prostate, semen, vaginal secretions, tears, saliva, mucus, bile, blood, lymph, plasma, serum, synovial fluid, cerebrospinal fluid, urine, as well as spaces, intracellular and cellular
  • the inflammatory mediator-related diseases include: systemic inflammatory response syndrome (SIRS) or sepsis (for example, derived from viral, bacterial, fungal or parasitic infection), autoimmune diseases, surgery, cytotoxic chemotherapy, Bone marrow manipulation, large tissue injury or trauma, mesenteric hypoperfusion, intestinal mucosal injury, malaria, gastrointestinal inflammatory disease, intestinal infection, uterine infection, influenza, acute pneumonia such as acute respiratory distress syndrome or acute lung Injury, pulmonary embolism, pancreatitis, autoimmune and collagen vascular diseases, blood transfusion-related diseases, burns, smoke or inhalation lung injury, graft versus host disease, ischemia or infarction, reperfusion injury, hemorrhage, allergic reactions, drug overdose, Radiation damage or chemical damage.
  • SIRS systemic inflammatory response syndrome
  • sepsis for example, derived from viral, bacterial, fungal or parasitic infection
  • autoimmune diseases surgery, cytotoxic chemotherapy, Bone marrow manipulation, large tissue injury or trauma, mes
  • inflammatory mediators are produced by diseases caused by pathogens, toxins, or agents of biological warfare, such as viral hemorrhagic fever, jellyfish toxin, hantavirus cardiopulmonary syndrome (hantavirus), cholera toxin, botulinum Toxins, hemp toxins, Q fever [Coxiella burnetii], Rickettsia prowaszekii, or psittacosis [Chlamydia psittaci].
  • diseases caused by pathogens, toxins, or agents of biological warfare such as viral hemorrhagic fever, jellyfish toxin, hantavirus cardiopulmonary syndrome (hantavirus), cholera toxin, botulinum Toxins, hemp toxins, Q fever [Coxiella burnetii], Rickettsia prowaszekii, or psittacosis [Chlamydia psittaci].
  • Inflammatory media-related diseases also include: transplantation, immune infertility and other diseases that require the removal of target immune factors.
  • the dimer immune fusion protein, pharmaceutical composition and use provided by the present invention have simple construction and expression processes.
  • the dimer immune fusion protein can directly kill pathogenic microorganisms on the one hand and limit the invasion of foreign pathogens, on the other hand, it can inhibit Excessively produced inflammatory factors can reduce tissue damage and have a good therapeutic effect on inflammatory mediator-related diseases.
  • it can effectively prevent and/or treat inflammatory mediator-related diseases. It has broad prospects for clinical application.
  • Figure 1 is a schematic diagram of the structure of a dimeric immune fusion protein.
  • Example 1 Construction, expression and characterization of soluble dimer immune fusion protein
  • the soluble dimer immune fusion protein is a dimer with an antibody Fc.
  • the method of constructing and expressing the dimer immune fusion protein itself is a conventional experimental technique in the field, which is briefly described as follows:
  • the ELISA method was used to detect the binding ability of the dimer immune fusion protein to specific ligands, as shown in Table 2.
  • Example 2 The effect of dimer immune fusion protein on Staphylococcus aureus
  • Staphylococcus aureus that expresses green fluorescent protein comes from the collection of the Institute of Microbiology, Chinese Academy of Sciences, and the multiplicity of infection is 1:10.
  • Peripheral blood samples of healthy volunteers were collected to separate peripheral mononuclear cells (PBMC, separated by Biyuntian Lymphocyte Separation Kit).
  • PBMC peripheral mononuclear cells
  • the newly isolated cells were stable in RPMI1640 medium containing 10% fetal bovine serum for 2h (37.5°C, 5% CO 2 ).
  • Staphylococcus aureus collect the bacteria by centrifugation at 10000g for 30 seconds in a benchtop centrifuge, and resuspend them to a bacterial density of about 10 8 CFU/ml. Take the bacterial suspension to make a gradient dilution plate count to determine the accurate bacterial density.
  • PBMC cells of Staphylococcus aureus bacteria was added (about 106 bacteria) was resuspended in 10 microliters of PBS after replacing the medium, mix gently shaken and incubated at 37 °C 2 hours.
  • TLR2/TLR4-Fc 84.89 7.21 p ⁇ 0.05 TLR4/TLR6-Fc 96.60 10.39 p ⁇ 0.05 TLR4/MD-2-Fc 88.31 11.60 p ⁇ 0.05 TLR4/CD36-Fc 96.49 5.91 p ⁇ 0.05 TLR2/Dectin-1-Fc 76.19 4.60 p ⁇ 0.05
  • mice BALB/c mice, SPF grade, female, 6-8 weeks old, weight 18-20g, international standard strain MRSA-252, purchased from American Tissue Culture Collection (ATCC).
  • a mouse model was established, and 0.1 mL of the washed bacterial solution was injected through the tail vein (the concentration of the bacterial solution was 1 ⁇ 10 9 CFU/mL).
  • the mice in the blank group were injected with the same amount of sterile normal saline through the tail vein. The mice were then divided into a control group and a treatment group, each with 10 mice.
  • control IgG The control group was given control IgG, and the treatment group was given the representative of the dimer immune fusion protein of the present invention, at a dose of 10 mg/kg, intravenously
  • the injection was once a day, and the observation was continued for 10 days. If the mouse died or all the mice were killed at the end of the experiment on the last day, immediately take the blood to plate and count the bacteria under aseptic conditions. At the same time, take the whole organs of kidney, spleen and liver aseptically, remove part of the tissues and grind them with a glass homogenizer and plate the plates for counting. Bacteria, while performing pathological examination. The results are shown in Table 5 to Table 9:
  • mice 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 Model group 100 20 0 0 0 0 0 Control IgG 100 80 70 70 70 70 70
  • TLR1-Fc 100 70 70 70 70 70 70 TLR2-Fc 100 70 70 70 70 70 70 TLR4-Fc 100 60 60 60 50 50 TLR2/TLR4-Fc 100 70 70 70 70 70 70 TLR4/TLR6-Fc 100 70 70 70 70 70 TLR4/MD-2-Fc 100 80 80 80 80 80 80 80 TLR4/CD36-Fc 100 70 70 70 70 TLR2/Dectin-1-Fc 100 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50
  • Table 6 The relative number of colonies in the blood of each group of mice before death
  • Table 7 The relative number of colonies in the liver of each group of mice before death
  • Table 8 The relative number of colonies in the spleen of each group of mice before death
  • the dimeric immune fusion protein of the present invention has strong microbial killing effect, anti-infection effect, reducing the number of organ colonies, and effectively resisting methicillin-resistant golden yellow staphylococcus aureus.
  • mice Female C57BL/6 mice (about 20g) were selected as experimental animals, and 0.1ml (5 ⁇ 10 5 CFU/ml) of Cryptococcus neoformans at a concentration of 5 ⁇ 10 6 CFU/ml was administered via tail vein injection, resulting in a systemic fungal infection model .
  • mice were divided into groups, each group of 10 mice, the treatment group was administered 10 mg/kg of the dimer immune fusion protein of the present invention via the vein, once a day, and the control group was given control IgG for a total of 5 days, on the 5th day
  • the mice were sacrificed, the brains were taken, the brain tissues were homogenized, the homogenate was diluted to a certain multiple and added to the peptone agar-based coating plate, the colonies on the medium were counted, and the amount of fungi in the brain of the mice was calculated.
  • Table 10 shows.
  • Example 5 Therapeutic study of dimer immune fusion protein treatment on guinea pig model of Trichophyton mentagrophytes infection
  • Trichophyton mentagrophytes was selected as the pathogenic bacteria.
  • ATCC Trichophyton mentagrophytes
  • SDA sandcastle agar
  • the animal model of Trichophyton mentagrophytes infection in guinea pigs was made, it was divided into groups of 10 each.
  • the treatment group was administered 10 mg/kg of the dimer immune fusion protein of the present invention via the veins, once every 2 days, and the control group was given a control IgG, administered for 5 days.
  • the cure is that the skin lesions have subsided, and the fungal microscopy is negative for 2 consecutive times and the fungal culture is negative; invalid is that the skin lesions have not subsided and the fungal microscopy is positive. Record the number of recovered animals in each group and calculate the cure rate, and observe the recurrence of the cured animals after stopping the drug.
  • Table 11 Comparison of the curative effect of Trichophyton mentagrophytes in each group in guinea pigs
  • TLR1-Fc 90 100 100 TLR2-Fc 100 100 100 TLR4-Fc 80 100 100 TLR2/TLR4-Fc 100 100 100 TLR4/TLR6-Fc 100 100 100 TLR4/MD-2-Fc 100 100 100 TLR4/CD36-Fc 100 100 100 TLR2/Dectin-1-Fc 100 100 100 100
  • the dimeric immune fusion protein effectively inhibits the growth of fungi on the superficial skin and achieves an obvious curative effect in killing fungi.
  • Example 6 Therapeutic effect of dimer immune fusion protein on Vibrio vulnificus
  • TLR4/TLR6-Fc 77.54 ⁇ 6.96 87.64 ⁇ 9.02 74.54 ⁇ 5.34 80.88 ⁇ 9.70
  • TLR4/MD-2-Fc 26.20 ⁇ 3.91 88.66 ⁇ 6.48 92.03 ⁇ 7.19 45.15 ⁇ 3.62
  • TLR4/CD36-Fc 7.02 ⁇ 0.81 48.61 ⁇ 2.90 64.55 ⁇ 8.64 72.01 ⁇ 3.76
  • TLR2/Dectin-1-Fc 85.51 ⁇ 7.14 52.39 ⁇ 6.65 60.53 ⁇ 7.92 30.66 ⁇ 2.24
  • the control group was given IgG, and each treatment group was given the dimer immune fusion protein of the present invention, 10 mg/kg, once every 2 days, intraperitoneally injected. Observe and record the incidence and death of suckling mice for 3 weeks, and calculate the survival rate of mice. The results are shown in Table 13:
  • the dimer immune fusion protein effectively inhibits the growth of virulent microorganisms represented by Vibrio vulnificus, and achieves a significant effect of killing microorganisms.
  • Example 7 Dimer immune fusion protein against dengue virus infection
  • Dengue 1 virus 128 strains (Gen Bank FJ176780), Dengue 4 virus 43 strains (GeneBank AF119661), Dengue 3 virus 80-2 strains (Gen Bank AF317645), Dengue 4 virus B5 strains (Gen Bank AF289029), C6/36 cells and BHK21 cells: Both are provided by the Virus Room of the Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences; C6/36 cells are used for dengue virus culture. When the cells grow to a single layer, discard the culture medium, add different virus suspensions, and culture at 37°C to observe the cytopathic changes.
  • the virus stock solution was diluted 10-fold with a cell maintenance solution containing 2% FCS, and then the virus solution of different dilutions was added to the cell monolayer and incubated at 37°C for 1 hour. The supernatant was discarded, DMEM cell maintenance solution containing 1% low melting point agarose was added, and the culture was continued for 5 days.
  • the plaque reduction and neutralization test is carried out by using the method of diluting antibodies to fix the virus.
  • 100PFU of dengue type 4 virus suspension was mixed in equal amounts and treated in a water bath at 37°C for 1 hour. Add the mixed solution to BHK21 cells cultured in a 6-well plate, incubate at 37°C for 1 hour, discard the mixed solution, and wash the cells with PBS buffer. Add nutrient agar cover, continue to culture for 5 days, fix staining, and count the number of plaques.
  • the content of each drug in the treatment group was 50 ⁇ g/ml, and the control group was given blank IgG and the neutralization rate of each administration group was calculated. The neutralization rate was (1-treatment group/blank control) ⁇ 100%. The results are shown in Table 14. .
  • Blank control 100 100 100 100 - Control IgG 100 10 0 0 - TLR1-Fc 100 80 80 80 80 To TLR2-Fc 100 80 70 70 p ⁇ 0.05 TLR4-Fc 100 70 70 p ⁇ 0.05 TLR2/TLR4-Fc 100 60 60 50 p ⁇ 0.05 TLR4/TLR6-Fc 100 50 50 p ⁇ 0.05
  • TLR4/MD-2-Fc 100 50 50 50 50 p ⁇ 0.05
  • TLR4/CD36-Fc 100 90 90 70 p ⁇ 0.05
  • TLR2/Dectin-1-Fc 100 80 80 80 p ⁇ 0.05
  • the dimeric immune fusion protein effectively inhibits the growth of potent microorganisms represented by dengue virus, and achieves a significant effect of killing microorganisms.
  • Example 8 Study on the killing effect of dimer immune fusion protein on parasites represented by Schistosoma
  • New Zealand white rabbits (male, 2.5-3.0Kg) were purchased from Shanghai Slack Laboratory Animal Co., Ltd.; 5-week-old BALB/c mice (male) were purchased from Shanghai Jiesjie Laboratory Animal Co., Ltd.; New Zealand white rabbits passed through the abdomen Infect 1000 ⁇ 5 cercariae of Schistosoma japonicum by skin patch, and they were dissected on the 14th day after infection.
  • the worms of Schistosoma japonicum were collected from the hepatic portal vein by aortic perfusion method, and the worms were washed thoroughly with RPMI1640 medium. body.
  • PBMCs per well were cultured in RPMI1640 medium containing 10% fetal calf blood.
  • the treatment group was given the representative of the dimer immune fusion protein of the present invention, the control group was given control IgG, and the control group was given control IgG. No drugs are given.
  • the administration concentration is 1mg/ml, and 10 (14d) schistosome japonicums with good vigor are added to each well, and then they are cultured in a 37°C, 5% CO 2 incubator (change the medium every 24h). Set three multiple holes.
  • the inverted microscope was used to observe the activity and morphological changes of different groups of Schistosoma japonicum at 24h, 48h, 72h and 96h of culture, and calculate the survival rate of Schistosoma japonicum at the corresponding culture time.
  • the death of Schistosoma japonicum is defined as: continuous observation of the body for 2 minutes is regarded as the death of the body. The results are shown in Table 16.
  • the dimer immune fusion protein effectively inhibits the growth of parasitic pathogenic microorganisms represented by schistosomiasis, and achieves an obvious effect of killing microorganisms.
  • Example 9 Examples of administration of dimer fusion proteins to patients exposed to unknown pathogens
  • the exposure mode is one of many different ways, such as food or water intake, aerosol inhalation, or skin contact.
  • the pathogen is one of many, such as Bacillus anthracis (anthracnose), influenza virus, smallpox virus, Yersinia pestis (plague), Ebola virus or Marburg virus, Tula Francis (hare disease), Han Tan virus, dengue virus, cholera toxin, botulinum toxin, ricin, salmonella, Escherichia coli such as E.coli 0157:H7, Shigella, Listeria, etc.
  • the immune dimer of the present invention can then be used to quickly neutralize inflammatory mediators, for example, as a preventive measure or treatment method for patients who have symptoms of infection and signs of inflammation (fever, chills, etc.), intravenously administer the two of the present invention to the patient.
  • a polymer-dimer immune fusion protein such as a pharmaceutical composition containing an active ingredient of 10 mg/kg TLR2-Fc, a pharmaceutical composition containing an active ingredient of 10 mg/kg TLR2/TLR4-Fc, and an active ingredient of 10 mg/kg TLR4 /TLR6-Fc pharmaceutical composition and other pharmaceutical compositions of the active ingredient of the dimer immune fusion protein in Example 1.
  • the dimers are immune to fusion and isolate the inflammatory mediators in the blood introduced by exogenous or locally produced or introduced through physiological fluids such as bile into the digestive tract. This occurs in these Inflammatory mediators may cause further inflammation or toxicity, or cause worsening inflammation before infection, endotoxemia, and sepsis.
  • the dimeric immune fusion protein reduces the triggers of additional systemic inflammation in the patient's body and reduces the production of systemic inflammatory mediators (such as cytokines), thereby preventing or limiting the induction of cytokines or other inflammatory mediators The occurrence of cell death, organ damage, multiple organ failure and potential death.
  • Raw 264.7 macrophages (Cell Bank of Chinese Academy of Sciences) were cultured in DMEM medium containing 10% fetal bovine serum (FBS; Gibco Laboratories) under the conditions of 37°C and 5% CO2.
  • Raw 264.7 cells were seeded into 96-well plates at a density of 1 ⁇ 10 6 cells/mL and cultured overnight. The next day, the above medium was replaced with fresh DMEM medium, and the 5 ⁇ g/mL dimer immune fusion proteins described in Example 1 were added to the cells, and the control group was added with control human IgG (Sigma). After incubating the cells with the protein for 30 minutes, the medium was added with LPS (final concentration 1 ⁇ g/mL), and the cells were incubated for another 24 hours before the detection experiment was performed.
  • FBS fetal bovine serum
  • the Griess reagent system (Promega, USA) was used to measure the level of nitric oxide (NO) in the raw 264.7 cell culture medium. Add 50 ⁇ L of medium to a 96-well plate, then add the same amount of Griess reagent I (NED) solution and Griess reagent II (para-aminobenzene sulfonamide solution), incubate for 10 minutes, then use a microplate reader (Molecular Devices) , USA) Measure the optical density at 540nm within 30 minutes. Use the sodium nitrite standard curve (0-100 ⁇ M) to calculate the concentration of NO.
  • stimulating cells with LPS increased the expression of NO, but when treated with LPS and the dimeric immune fusion protein of the present invention, the expression level of NO was reduced. Support the dimer immune fusion protein to reduce the effect of macrophage self-inflammatory exudation.
  • the supernatant sample containing the cell culture medium was collected, and the level of cytokine was analyzed using HMGB1, TNF ⁇ , IFN- ⁇ and IL-6 ELISA kit (eBioscience, San Diego). Coat a 96-well plate with 100 ⁇ L of capture antibody (diluted in the coating buffer to the concentration recommended by the manufacturer's operating procedures) at 4°C overnight. Then, after washing the plate 5 times, 200 ⁇ L of the assay diluent was added to each well and incubated at room temperature for 1 hour for blocking. After washing each well with washing buffer 5 times, the cell culture sample or each cytokine standard protein sample was diluted, and 100 ⁇ L of each sample was added to each well.
  • the plate containing the sample was incubated overnight at 4°C. Next, after washing the plate 5 times with a washing buffer, 100 ⁇ L of avidin-conjugated secondary antibody was added and incubated at room temperature for 1 hour. After incubation with the secondary antibody, the plate was washed 5 times and incubated with 100 ⁇ L of avidin-HRP (BD Bioscience) for 30 minutes at room temperature. After washing the plate 7 times, 100 ⁇ L of TMB solution (Pierce) was added and incubated at room temperature for 15 minutes. Add 50 ⁇ l of sulfuric acid to each well to stop the reaction. A microplate reader was used to measure the optical density at 450 nm. The SPSS program's ANOVA operation was used to perform analysis of variance to perform statistical analysis, and Duncan's multivariate domain test was used to verify the significance between the analyses. The test results are shown in Tables 19-22:
  • cytokine protein The cells of the control group and the treatment group were lysed, and the expression of cytokine mRNA in the cells was analyzed by qPCR method as shown in the following Tables 23 to 26. Stimulating the cells with LPS increased the expression of cytokines (HMGB1, TNF- ⁇ , IL -6, COX-2). However, if the cells are treated with LPS and the dimeric immune fusion protein described in the present invention at the same time, the expression level of the above-mentioned pro-inflammatory cytokines is significantly reduced. These results strongly suggest and support the anti-inflammatory effect of the dimeric immune fusion protein of the present invention.
  • TLR4/MD-2-Fc+LPS 16.89 1.15 p ⁇ 0.05 TLR4/CD36-Fc+LPS 23.83 2.91 p ⁇ 0.05 TLR2/Dectin-1-Fc 36.76 3.75 p ⁇ 0.05
  • the dimeric immune fusion protein effectively inhibits the inflammatory exudation of immune cells represented by macrophages mediated by foreign stimuli.
  • Example 11 Anti-inflammatory activity of dimeric immune fusion protein to peripheral monocytes
  • Biocoll Separating Solution Biochrom AG, Berlin, Germany was used to separate PBMC (peripheral blood mononuclear cells) from blood samples (50ml) collected from healthy subjects.
  • PBMC peripheral blood mononuclear cells
  • the cells were processed according to the method of Example 2 and the secretion levels of cytokines (TNF ⁇ and IL-6) in the culture medium and the mRNA levels of intracellular cytokines (HMGB1, TNF ⁇ ) were detected.
  • cytokines TNF ⁇ and IL-6
  • HMGB1, TNF ⁇ intracellular cytokines
  • the dimeric immune fusion protein effectively inhibits the inflammatory exudation of immune cells represented by peripheral mononuclear cells mediated by foreign stimuli.
  • Example 12 Treatment of acute lung injury with dimeric immune fusion protein
  • the mice were injected into the tail vein at a dosage of 10 mg/kg, once a day for 3 consecutive days, and the model was started 1 hour after the last administration.
  • the mice were anesthetized by intraperitoneal injection of 2% isopentobarbital sodium, and the mice were fixed on their back at 37. °C Constant temperature operating table. Refer to the literature method to make the model.
  • the main steps of the model are as follows: carefully shave the middle hair of the neck, disinfect with alcohol, cut the neck skin about 2cm in the middle, expose and separate the trachea, use an insulin syringe to slowly instill LPS5mg/kg into the trachea (0.5mL/kg).
  • LPS5mg/kg 0.5mL/kg
  • the same amount of normal saline was instilled into the trachea, iodophor disinfected the wound and sutured the skin to establish a mouse model of acute lung injury.
  • mice After 24 hours of modeling, the eyeballs were taken and blood was collected. After standing in a refrigerator at 4°C for 3 hours, centrifuged at 3500 r/min for 15 minutes, the serum was separated, and stored in liquid nitrogen for testing. Each group of mice carefully cut the neck skin and separated the trachea, and intubated the trachea. The chest was opened, the right bronchus was ligated, and the left lung was lavaged with phosphate buffer solution for a total of 3 times, 2 mL/time. The bronchoalveolar lavage fluid was collected and centrifuged (4°C, 1300r/min, 5min).
  • BCA protein quantification kit (Biyuntian) was used to detect the protein content in bronchoalveolar lavage fluid, and the experimental operations were carried out in accordance with the kit instructions.
  • the level of white blood cell in bronchoalveolar lavage fluid 800 ⁇ L 0.01mol/L (pH 7.4) PBS buffer is used to resuspend the bronchoalveolar lavage fluid sediment, after pipetting evenly, take 400 ⁇ L in the blood analyzer to detect the number of white blood cells.
  • W/D Lung wet-to-dry mass ratio
  • Lung tissue pathological morphology score take the lower lobe of the right lung, Neutral formaldehyde immersion and fixation for 24h, running water for 12h, conventional paraffin embedding, sectioning, hematoxylin and eosin staining, mounting, and pathological observation under a microscope. Different visual fields were selected for pathological scoring according to the standard pneumonia score. The scoring method refers to the literature [Zhu Shan, Pan Linghui, Lin Fei, et al.
  • the dimeric immune fusion protein described in the present invention can reduce acute inflammation exudation, inhibit leukocyte exudation, reduce tissue edema, reduce tissue damage, enhance SOD activity, reduce serum MDA content, and inhibit the expression of Smad2 and TGF ⁇ 1, It has strong anti-inflammatory and anti-LPS effects. It can treat acute organ inflammation damage.
  • Example 13 Overview of the protocol for administering the dimer immune fusion protein in a mouse model of cecal ligation and perforation poisoning
  • mice For C57 male mice, the operation is as follows: Use a short-acting isoflurane for anesthesia to minimize the harmful effects of anesthesia on cardiovascular function.
  • the surgical procedure included a 5cm midline laparotomy starting 2cm below the chest plate. Isolate the cecum on a sterile gauze outside the abdominal cavity to avoid vascular damage. Afterwards, a 2-0 Vicryl line (vicry1) was used to ligate immediately below the ileocecal valve and maintain the continuity of the intestine. Then squeeze the contents of the cecum to one end of the cecum. Use a 20-gauge needle to puncture the cecum 3 times, and then squeeze a single drop of excretion from each puncture site.
  • mice in the sham-operated group did not undergo cecal ligation and perforation, and the rest of the surgical procedures were exactly the same.
  • the survival of the mice was observed and recorded every 12 hours until 7 days after the operation.
  • a mouse blood sample was collected 48 hours after the operation, and the level of TNF ⁇ in the plasma of each group was detected with an ELISA kit (CST). The results are shown in Tables 39 and 40:
  • TLR2-Fc 100 90 80 80 70 70 70 70 TLR4-Fc 100 90 80 80 60 60 60 TLR2/TLR4-Fc 100 100 70 70 60 60 60 50 TLR4/TLR6-Fc 100 100 80 60 60 60 60 50 TLR4/MD-2-Fc 100 100 80 70 60 60 60 50 TLR4/CD36-Fc 100 100 60 60 60 60 60 50 TLR2/Dectin-1-Fc 100 100 60 60 60 60 60 60 50
  • dimeric immune fusion protein described in the present invention can reduce septic cytokines and improve the survival rate of patients against sepsis. It can treat acute organ inflammation damage.
  • Example 14 The effect of dimeric immune fusion protein on schistosoma infection and liver fibrosis caused by infection
  • mice choose Balb/c mice, weighing 20-25g.
  • Oncomelania snails infected by Schistosoma japonicum were provided by Jiangsu Institute of Schistosomiasis Control.
  • the experimental animals were randomly divided into groups with 10 mice in each group, namely the healthy control group (blank group), the infection control group (model group), and the control IgG combined dimer immune fusion protein treatment groups. Except for the blank group, each mouse in the other groups was infected with (30 ⁇ 2) cercariae by abdominal patch method. Starting from 42 days after infection, the drug was continuously administered for 30 days.
  • the dose of dimer immune fusion protein and control IgG was 10 mg/kg, once every 3 days, 24 hours after the last administration, the animals were sacrificed under anesthesia with an inhaled isoflurane anesthesia machine, blood samples were collected, and the serum was separated for testing. After the mouse liver was taken out, it was washed twice in pre-cooled normal saline to remove residual blood stains. Each liver is cut into several parts immediately after being weighed, one part is fixed in 4% neutral paraformaldehyde solution for subsequent histopathological examination, and the other part is quick-frozen in liquid nitrogen and stored in a -80°C refrigerator For subsequent molecular biology testing.
  • the content detection is performed according to the instructions of the detection kit, and the results are shown in Tables 41 to 49.
  • TGF ⁇ % SD p value Blank control 1.65 0.19 To Model group 99.47 9.40 To Control IgG 100 10.55 To TLR1-Fc 13.54 1.34 p ⁇ 0.05 TLR2-Fc 5.42 0.36 p ⁇ 0.05 TLR4-Fc 10.32 2.02 p ⁇ 0.05 TLR2/TLR4-Fc 8.72 1.11 p ⁇ 0.05 TLR4/TLR6-Fc 2.94 0.32 p ⁇ 0.05 TLR4/MD-2-Fc 16.22 2.22 p ⁇ 0.05 TLR4/CD36-Fc 19.99 1.06 p ⁇ 0.05 TLR2/Dectin-1-Fc 19.05 1.16 p ⁇ 0.05
  • dimeric immune fusion protein described in the present invention can inhibit schistosomiasis liver colonization, reduce liver fibrosis, reduce organ inflammatory damage, and can be used as a product against organ fibrosis.
  • mice Eight-week-old female mice were divided into groups, each with 10 mice, and the double (infection + mechanical) injury method was used to construct an endometrial injury model, that is, after the mice were anesthetized, the median length of the abdomen was removed and the longitudinal length was about 2 cm.
  • the incision is made into the abdomen, and a 0.5cm longitudinal incision is made at the middle and lower 1/3 of the uterus, and the upper and middle uterine cavity is scraped with an endometrial spatula.
  • the spatula enters and exits the uterus, the unevenness disappears and the four walls feel rough, stop curettage. Lipopolysaccharide cotton thread was left in the uterine cavity after curettage, the abdominal incision was sutured, and the lipopolysaccharide cotton thread was taken out 48 hours later.
  • a blank control group (sham operation group), a saline injection only (model) group, and a treatment group are set up.
  • the treatment group was administered 10 mg/kg representative of the dimer immune fusion protein of the present invention via the vein, once every 3 days, for a total of 3 administrations.
  • the mice were mated with male mice after 3 cycles of estrus. One month later, the specimens were collected for HE staining and Masson staining to evaluate the function of the endometrium. Three months later, the mouse pregnancy results were evaluated.
  • Results 1 month after operation, the histological function evaluation showed that compared with each control group, the degree of fibrosis in each group treated with immunodimer was significantly reduced (Table 50); compared with each control group, the exosomal glands The numbers are higher than those of each control group. The evaluation of pregnancy results showed that the pregnancy rate of each group treated with immunodimer was higher than that of each control group. The results are shown in Table 51.
  • the dimeric immune fusion protein described in the present invention can inhibit organ inflammatory damage, inhibit chronic inflammation of organs, especially endometrial inflammation and fibrosis, and can be used as a product to combat organ fibrosis. Improve the treatment of infertility diseases.
  • Example 16 Therapeutic effect of dimeric immune fusion protein on spontaneous abortion model
  • CBA/J female mice and DBA/2J male mice were used to establish a stress abortion model.
  • This abortion model is a classic research model of maternal-fetal immune tolerance.
  • the establishment methods, experimental methods and observation time points are equivalent to the literature (Blois S M ,et al.. Nature Medicine, 2007,13(12):1450-1457.).
  • CBA/J female mice were divided into negative control group, stress pressure group, and treatment group before being caged.
  • the treatment group was administered 10 mg/kg of the dimer immune fusion protein of the present invention via the vein, once every 3 days, for a total of 3 administrations.
  • the cages were closed 3 days after the first application.
  • mice in the control group, the stress pressure group, and the treatment group were further separated, and the level of Foxp3-positive T helper lymphocytes in them was detected.
  • the level of TNF ⁇ in the tissue is detected; the method of separation and detection is the same as that in the literature (Kim B J, et al.. Proceedings of the National Academy of Sciences, 2015, 112(5): 1559-1564. Results Shows that dimer immune fusion protein treatment can effectively increase the level of Foxp3-positive T helper lymphocytes and reduce the level of tissue TNF ⁇ (Table 53).
  • Example 17 Therapeutic effect of dimeric immune fusion protein on lupus mouse model
  • lupus nephritis As a representative disease of the immune system, lupus nephritis has an incidence of about 50/100,000, which accounts for about 0.7 ⁇ of the population in my country. More than 90% of lupus nephritis is seen in women, mainly young and middle-aged women. It is generally believed that people under 30 have a high renal involvement rate. About 70% of patients have clinical manifestations of renal damage to varying degrees, with varying degrees of proteinuria, Hematuria under microscope is more common, often accompanied by tubular urine and renal function damage, which seriously affects the normal life of patients.
  • Lupus mouse models are mostly produced by crossing NZB female mice and NZW male mice.
  • the first-generation (NZB ⁇ NZW) F1 hybridization can produce typical lupus symptoms including lupus nephritis. It is currently recognized as an animal model for studying lupus nephritis. one.
  • the establishment of the model refers to the non-patent literature Brinks et al. Circ Res (2010) 107:1140-1149.
  • the administration dose of the dimer immune fusion protein is: 10 mg/kg, twice a week by tail vein injection for ten consecutive weeks.
  • the control group was injected with control IgG at the same dose as the treatment group.
  • Blank group 100 To Control IgG 100 To TLR1-Fc 40 p ⁇ 0.05 TLR2-Fc 30 p ⁇ 0.05 TLR4-Fc 30 p ⁇ 0.05 TLR2/TLR4-Fc 30 p ⁇ 0.05 TLR4/TLR6-Fc 20 p ⁇ 0.05 TLR4/MD-2-Fc 30 p ⁇ 0.05 TLR4/CD36-Fc 30 p ⁇ 0.05 TLR2/Dectin-1-Fc 30 p ⁇ 0.05
  • Group Pathology score SD p value Blank group (model group) 3.20 0.26 To Control IgG 3.30 0.26 To TLR1-Fc 0.85 0.47 p ⁇ 0.05 TLR2-Fc 0.80 0.35 p ⁇ 0.05 TLR4-Fc 0.75 0.26 p ⁇ 0.05 TLR2/TLR4-Fc 1.05 0.44 p ⁇ 0.05 TLR4/TLR6-Fc 1.10 0.70 p ⁇ 0.05 TLR4/MD-2-Fc 1.10 0.66 p ⁇ 0.05 TLR4/CD36-Fc 0.80 0.35 p ⁇ 0.05 TLR2/Dectin-1-Fc 0.95 0.44 p ⁇ 0.05
  • the immune dimer fusion protein of the present invention can reduce autoimmune antibodies, reduce pathological inflammatory exudation of organs, and has a good therapeutic effect on immune system diseases, which is beneficial to follow-up The development of clinical trials.

Abstract

Provided is a soluble dimeric immune fusion protein, comprising a first dimerized polypeptide chain and a second dimerized polypeptide chain. The general structural formula of the first polypeptide chain is Z1-Z2, and the general structural formula of the second polypeptide chain is Y1-Y2, wherein Z1 is (i) an extracellular domain of a first pattern recognition receptor or a functional variant or fragment thereof, or (ii) a first co-receptor or a functional variant or fragment thereof; Z2 is a dimerized domain or a functional variant or fragment thereof; Y1 is (i) an extracellular domain of a second pattern recognition receptor or a functional variant or fragment thereof, or (ii) a second co-receptor or a functional variant or fragment thereof; and Y2 is a dimerized domain or a functional variant or fragment thereof. The dimeric protein can block pathogen invasion and limit generation and expansion of inflammatory mediators, and can be used for preparing products for preventing or treating inflammatory mediator-related diseases.

Description

二聚体免疫融合蛋白、药物组合物和用途Dimer immune fusion protein, pharmaceutical composition and use 技术领域Technical field
本发明涉及生物医药工程技术领域，具体涉及一种二聚体免疫融合蛋白、以其作为活性组分的药物组合物和其医药用途，尤其是用于炎症介质相关疾病的用途。The present invention relates to the technical field of biomedical engineering, in particular to a dimer immune fusion protein, a pharmaceutical composition using it as an active component and its medical use, especially its use for diseases related to inflammatory mediators.
背景技术Background technique
在动物中，当细胞或组织受到细菌、外伤、毒素、物理或化学因素(其可以统称为“炎性物质，inflammatory agent”)损伤时，会发生炎性应答。炎症应答的病理生理特征受到由细胞合成并释放的多种促炎或抗炎剌激物或介质的复杂相互作用的调节。一些己知种类的促炎、抗炎剌激物或介质包括细胞因子、氧化亚氮、血栓皖(thromboxane)、自兰烯、磷脂样血小板活化因子、前列腺素、激肤、补体因子、凝血因子、超抗原、单核因子、趋化因子、干扰素、自由基、蛋白酶、花生四烯酸代谢物、环前列腺素、β内啡肽、心肌抑制因子(myocardial depressant factor)、anadamide，2一花生酷甘油(2-arachidonoylglycerol)、四氢生物蝶岭、细胞碎片以及化学物质(包括组胺、缓激肽和血清素)等。In animals, an inflammatory response occurs when cells or tissues are damaged by bacteria, trauma, toxins, physical or chemical factors (which can be collectively referred to as "inflammatory agents"). The pathophysiological characteristics of the inflammatory response are regulated by the complex interaction of multiple pro-inflammatory or anti-inflammatory stimulants or mediators synthesized and released by cells. Some known types of pro-inflammatory and anti-inflammatory stimulants or mediators include cytokines, nitrous oxide, thromboxane, autolanene, phospholipid platelet activating factor, prostaglandins, kinetic skin, complement factors, and clotting factors , Superantigens, monocytes, chemokines, interferons, free radicals, proteases, arachidonic acid metabolites, cyclic prostaglandins, β-endorphins, myocardial depressant factor, anadamide, 2-peanut 2-arachidonoylglycerol (2-arachidonoylglycerol), tetrahydrobiological butterfly ridge, cell debris and chemical substances (including histamine, bradykinin and serotonin) and so on.
根据被侵袭的位置、炎性物质的性质以及所涉及的促炎或抗炎剌激物或介质的相互作用，炎性应答的性质和强度不同。当受到调节并且为局限性时，炎性应答是有益的。但是，如果不受调节并且广泛化时，炎性应答可造成显著的组织损伤甚至死亡。The nature and intensity of the inflammatory response are different according to the location of the attack, the nature of the inflammatory substance, and the interaction of the pro-inflammatory or anti-inflammatory stimulants or mediators involved. When regulated and limited, an inflammatory response is beneficial. However, if unregulated and generalized, the inflammatory response can cause significant tissue damage and even death.
近年来，高度耐药的微生物感染成为临床常见的棘手问题。由于患者救治时间延长，微生物在体内进一步进化，产生如耐甲氧西林金黃色葡萄球菌(methicillin-resist-ant Staphylococcus aureus，MRSA)等“超级耐药菌”，耐药微生物在体内持续存在，使得参与炎症的细胞因子网络调控紊乱，因此开发能抑制整个微生物-免疫系统作用的药物成为前沿热点。利用超剂量的抗生素可以杀灭微生物，但死亡微生物可以进一步介导炎症因子风暴，且大剂量抗生素造成患者肝肾损害，依然无法对抗疾病。In recent years, highly drug-resistant microbial infections have become a common and difficult clinical problem. Due to the prolonged treatment time of patients, microorganisms have further evolved in the body to produce "super drug-resistant bacteria" such as methicillin-resist-ant Staphylococcus aureus (MRSA). Drug-resistant microorganisms continue to exist in the body, making The cytokine network involved in inflammation is regulated in disorder, so the development of drugs that can inhibit the action of the entire microbe-immune system has become a frontier hot spot. Overdose antibiotics can kill microorganisms, but dead microorganisms can further mediate inflammatory factor storms, and large doses of antibiotics cause liver and kidney damage in patients, which still cannot fight the disease.
参与炎症的细胞因子主要由巨噬细胞、单核细胞、中性粒细胞和淋巴细胞产生的一类蛋白，通常由上述细胞受到病毒、细菌、真菌或寄生虫感染刺激，以及在免疫应答中由T细胞剌激释放。其他一些细胞也可以释放炎性细胞因子，例如基质细胞如成纤维细胞、内皮细胞和平滑肌细胞，以及上皮细胞、角质形成细胞和肝细胞。细胞因子通常以低浓度存在于血液或组织中。The cytokines involved in inflammation are mainly a type of protein produced by macrophages, monocytes, neutrophils and lymphocytes. These cells are usually stimulated by viral, bacterial, fungal or parasitic infections, and in the immune response. Stimulated release of T cells. Other cells can also release inflammatory cytokines, such as stromal cells such as fibroblasts, endothelial cells, and smooth muscle cells, as well as epithelial cells, keratinocytes, and hepatocytes. Cytokines are usually present in blood or tissues in low concentrations.
细胞因子的结构和活性是免疫学研究热点。目前研究认为，细胞因子拥有广泛的免疫和非免疫活性，可以影响多种生理功能，例如细胞生长、分化、内稳态和病理生理等；同时，细胞因子具有多种生物活性，并且与参与多种生物调控过程；此外，细胞因子可以促进其自身的合成，以及来其他细胞因子的产生，这些现象被称作“细胞因子级联”。细胞因子级联通常与由感染和组织损伤所造成的全身性变化相关，在这种情况下，整个细胞因子级联网络产生非常复杂的细胞和生物效应，例如，白介素(IL)类、干扰素(IF)类和肿 瘤坏死因子(TNF)类的多种细胞因子在免疫和炎性应答中可以产生。The structure and activity of cytokines are a hot topic in immunology. Current research believes that cytokines have a wide range of immune and non-immune activities, which can affect a variety of physiological functions, such as cell growth, differentiation, homeostasis, and pathophysiology. At the same time, cytokines have a variety of biological activities and are involved in multiple activities. A biological regulation process; in addition, cytokines can promote their own synthesis and the production of other cytokines. These phenomena are called "cytokine cascades." The cytokine cascade is usually associated with systemic changes caused by infection and tissue damage. In this case, the entire cytokine cascade network produces very complex cellular and biological effects, such as interleukins (IL) and interferons. A variety of cytokines such as (IF) and tumor necrosis factor (TNF) can be produced in immune and inflammatory responses.
通常，细胞因子级联介导正常的宿主防御应答、细胞调节和细胞分化。在级联环境下，细胞因子生产的功能可能变得紊乱。该紊乱可导致出现超过正常浓度的细胞因子，此时，对机体的影响是双面的：一方面对抗入侵物，但另一方面如果过度强烈或缺乏调节，即可以损伤机体。Generally, the cytokine cascade mediates the normal host defense response, cell regulation, and cell differentiation. In a cascade environment, the function of cytokine production may become disordered. This disorder can lead to the appearance of cytokines in excess of the normal concentration. At this time, the impact on the body is two-sided: on the one hand, it resists invaders, but on the other hand, if it is too strong or lacks regulation, it can damage the body.
当外源性感染物、内源性的免疫刺激物介导细胞因子级联紊乱，就可以产生全身性炎性应答综合征(SIRS)、脓毒症(以及被称作重症脓毒症(具有器官功能障碍的脓毒症)，甚至脓毒症休克。另一方面，感染物的持续存在和慢性炎症、纤维化性炎症等密切相关。When exogenous infectious agents and endogenous immune stimulants mediate cytokine cascade disorders, systemic inflammatory response syndrome (SIRS), sepsis (and what is called severe sepsis (with Organ dysfunction, sepsis), or even septic shock. On the other hand, the persistence of infectious agents is closely related to chronic inflammation and fibrotic inflammation.
综上所述，能够清除入侵物，又能抑制引起体内过度产生的细胞因子的药物是亟待开发的，目前发明人未见任何报道有类似药物。In summary, drugs that can eliminate invaders and inhibit the excessive production of cytokines in the body are in urgent need of development. At present, the inventor has not seen any reports of similar drugs.
发明内容Summary of the invention
本发明是为解决上述问题而进行的，提供了一种可溶性二聚体免疫融合蛋白，并对该可溶性二聚二聚体免疫融合蛋白的具体结构、制备方法和用途进行了描述，即提供了二聚体免疫融合蛋白、其制备方法和用途。The present invention is to solve the above-mentioned problems. It provides a soluble dimer immune fusion protein, and describes the specific structure, preparation method and application of the soluble dimer immune fusion protein, that is, it provides Dimer immune fusion protein, its preparation method and application.
本发明的第一方面，提供了可溶性二聚二聚体免疫融合蛋白，包含二聚化的第一条多肽链和第二条多肽链，第一条多肽链的结构通式为Z1-Z2，第二条多肽链的结构通式为Y1-Y2。其中，Z1是(i)第一种模式识别受体的细胞外结构域或其功能变体或片段，或(ii)第一共受体或其功能变体或片段；Z2是二聚化结构域或其功能变体或片段，Y1是(i)第二种模式识别受体的细胞外结构域或其功能变体或片段，或(ii)第二共受体或其功能变体或片段；Y2是二聚化结构域或其功能变体或片段。The first aspect of the present invention provides a soluble dimer-dimer immune fusion protein, comprising a dimerized first polypeptide chain and a second polypeptide chain, and the structural formula of the first polypeptide chain is Z1-Z2, The general structural formula of the second polypeptide chain is Y1-Y2. Among them, Z1 is (i) the extracellular domain of the first pattern recognition receptor or its functional variant or fragment, or (ii) the first co-receptor or its functional variant or fragment; Z2 is a dimerization structure Domain or its functional variant or fragment, Y1 is (i) the extracellular domain of the second pattern recognition receptor or its functional variant or fragment, or (ii) the second co-receptor or its functional variant or fragment ; Y2 is a dimerization domain or a functional variant or fragment thereof.
术语“模式识别受体”是免疫学概念，模式识别受体(pattern recognition receptor，PRR)是一类主要表达于固有免疫细胞表面、非克隆性分布、可识别一种或多种PAMP的识别分子。是固有免疫中免疫受体的代表，由有限数量的胚系基因编码，进化上十分保守，也表明此类受体对生物体的生存极为重要。其与病原生物表面的病原体相关分子模式(pathogen-associated molecular pattern，PAMP)的相互识别和作用是启动固有免疫应答的关键。和适应性免疫中淋巴细胞受体相比较，PRR有四个特点。除了全部由胚系基因编码外，另外三个特点是：组成性地表达、引起快速应答和能够识别各种病原体。任何模式识别受体均适用于本发明所述的融合蛋白结构方案。The term "pattern recognition receptor" is an immunological concept. Pattern recognition receptor (PRR) is a type of recognition molecule that is mainly expressed on the surface of innate immune cells, is non-clonal and can recognize one or more PAMPs. . It is a representative of immune receptors in innate immunity. It is encoded by a limited number of germline genes and is evolutionarily conservative. It also shows that such receptors are extremely important for the survival of organisms. Its mutual recognition and interaction with pathogen-associated molecular pattern (PAMP) on the surface of pathogenic organisms is the key to initiating the innate immune response. Compared with lymphocyte receptors in adaptive immunity, PRR has four characteristics. In addition to being all encoded by germline genes, three other characteristics are: constitutive expression, rapid response and the ability to identify various pathogens. Any pattern recognition receptor is suitable for the fusion protein structure scheme of the present invention.
在本发明的某些优选的实施方案中，当Z1和Y1均为模式识别受体的细胞外结构域或其功能变体或片段时，该第一种模式识别受体和第二种模式识别受体分别选自：TLR1(Gene ID:7096)，TLR2(Gene ID:7097)，TLR3(Gene ID:7098)，TLR4(Gene ID:7099)，TLR5(Gene ID:7100)，TLR6(Gene ID:10333)，TLR7(Gene ID:51284)，TLR8(Gene ID:51311)，TLR9(Gene ID:54106)，TLR10(Gene ID:81793)，Dectin-1(Gene ID:64581)，Dectin-2(Gene ID:93978)，Mincle(Gene ID:26253)，CLEC2(Gene ID:51266)，CLEC5A(Gene ID:23601)，CLEC12A(Gene ID:160364)，DCIR(Gene ID:50856)， CLECSF8(Gene ID:338339)中的任一个。In certain preferred embodiments of the present invention, when both Z1 and Y1 are the extracellular domains of pattern recognition receptors or functional variants or fragments thereof, the first pattern recognition receptor and the second pattern recognition receptor The receptors are selected from: TLR1 (Gene ID: 7096), TLR2 (Gene ID: 7097), TLR3 (Gene ID: 7098), TLR4 (Gene ID: 7099), TLR5 (Gene ID: 7100), TLR6 (Gene ID: 7100) :10333), TLR7 (Gene ID: 51284), TLR8 (Gene ID: 51311), TLR9 (Gene ID: 54106), TLR10 (Gene ID: 81793), Dectin-1 (Gene ID: 64581), Dectin-2( Gene ID: 93978), Mincle (Gene ID: 26253), CLEC2 (Gene ID: 51266), CLEC5A (Gene ID: 23601), CLEC12A (Gene ID: 160364), DCIR (Gene ID: 50856), CLECSF8 (Gene ID) :338339).
在Z1和Y1都是模式识别受体的细胞外结构域或其功能变体或片段的情况下，第一和第二种模式识别受体可以是相同的，也可以不同。In the case where both Z1 and Y1 are the extracellular domains of pattern recognition receptors or functional variants or fragments thereof, the first and second pattern recognition receptors may be the same or different.
在本发明的一些优选的实施方案中，当Z1和Y1均为共受体或其功能变体或片段时，第一共受体和第二共受体分别选自CD14(Gene ID:929)，MD-2(Gene ID:23643)，LBP(Gene ID:3929)，CD36(Gene ID:948)中的任一个。In some preferred embodiments of the present invention, when both Z1 and Y1 are co-receptors or functional variants or fragments thereof, the first co-receptors and the second co-receptors are respectively selected from CD14 (Gene ID: 929) , MD-2 (Gene ID: 23643), LBP (Gene ID: 3929), CD36 (Gene ID: 948).
在Z1和Y1都是共受体或其功能变体或片段的情况下，第一种和第二种共受体可以是相同的或不同的。In the case where both Z1 and Y1 are co-receptors or functional variants or fragments thereof, the first and second co-receptors may be the same or different.
二聚化结构域Z2或Y 2包括免疫球蛋白重链恒定区。例如，在具体的变化中，二聚化结构域Z2和Y2是IgG的Fc片段，诸如人免疫球蛋白γ1 Fc片段。当Z1与Y1不同时，二聚化结构域Z2和Y2可以采用工程化的手段以增加特异性的异源二聚化形成，如Z2和Y2为IgG的Fc片段或改变其生物活性的Fc突变体，或利用Knob-in-hole技术、改变电荷极性的ART-Ig技术或BiMab技术构建的异源二聚IgG-Fc片段(综述文献Brinkmann U,Kontermann R E.mAbs,2017,9(2):182-212.)。The dimerization domain Z2 or Y 2 includes the constant region of an immunoglobulin heavy chain. For example, in a specific variation, the dimerization domains Z2 and Y2 are Fc fragments of IgG, such as human immunoglobulin γ1 Fc fragments. When Z1 and Y1 are different, the dimerization domains Z2 and Y2 can be engineered to increase the formation of specific heterodimerization. For example, Z2 and Y2 are Fc fragments of IgG or Fc mutations that change their biological activity. Or a heterodimeric IgG-Fc fragment constructed by Knob-in-hole technology, ART-Ig technology that changes the charge polarity, or BiMab technology (review literature Brinkmann U, Kontermann R E. mAbs, 2017, 9(2) ):182-212.).
关于改变其生物活性的Fc突变体，如二聚化结构域Z2和Y2可以是人免疫球蛋白Fc片段的活性变体，如采用IgG2、IgG3或IgG4的Fc结构域。在某些实施方案中，可以进一步采用Fc的突变体以降低免疫球蛋白诸如ADCC、补体结合等生物活性，如LALA-PG突变体，L235E、E318A、K320A、K322A突变体等。Regarding Fc mutants that change their biological activity, for example, the dimerization domains Z2 and Y2 may be active variants of the Fc fragment of human immunoglobulin, such as using the Fc domain of IgG2, IgG3, or IgG4. In certain embodiments, Fc mutants can be further used to reduce the biological activities of immunoglobulins such as ADCC, complement fixation, etc., such as LALA-PG mutants, L235E, E318A, K320A, K322A mutants, and the like.
此外，二聚化结构域Z2和Y2还包含有肽接头，所述肽接头由15-32个氨基酸残基组成，其中这些残基中的1-8个(例如，2个)是半胱氨酸残基。在具体变化中，Z2和Y2包含免疫球蛋白铰链区或其变体，例如，在一个具体实施方案中，Z2和Y2包含免疫球蛋白铰链变体(例如，人免疫球蛋白γ1铰链变体)，其中Fc片段的220相对应的半胱氨酸残基被丝氨酸替代。根据上述二聚化结构域Z2和Y2使用的特别合适的肽接头包括这样的肽接头：所述接头包含多个甘氨酸残基，且任选地包含至少一个丝氨酸残基。In addition, the dimerization domains Z2 and Y2 also contain a peptide linker consisting of 15-32 amino acid residues, of which 1-8 (for example, 2) of these residues are cysteine Acid residues. In specific variations, Z2 and Y2 comprise an immunoglobulin hinge region or variants thereof, for example, in a specific embodiment, Z2 and Y2 comprise an immunoglobulin hinge variant (for example, a human immunoglobulin γ1 hinge variant) , Where the cysteine residue corresponding to 220 of the Fc fragment is replaced by serine. Particularly suitable peptide linkers used in accordance with the aforementioned dimerization domains Z2 and Y2 include peptide linkers that comprise a plurality of glycine residues, and optionally at least one serine residue.
关于Z1和Y1的具体结构，通过下述描述进行具体说明：Regarding the specific structures of Z1 and Y1, the following descriptions are provided for specific explanations:
(1)当Z1和Y1中的每一种都是相同的一种模式识别受体的细胞外结构域或其功能变体或片段时：在具有上述的通式Z1-Z2和Y1-Y2的可溶性二聚体免疫融合蛋白具体变化中，Z1和Y1的氨基酸序列包括SEQ ID NO.1-18所示的氨基酸中的任一个序列具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性。序列1～18的名称如下：(1) When each of Z1 and Y1 is the same extracellular domain of a pattern recognition receptor or a functional variant or fragment thereof: in the case of the above-mentioned general formulas Z1-Z2 and Y1-Y2 In the specific changes of the soluble dimer immune fusion protein, the amino acid sequences of Z1 and Y1 include any one of the amino acids shown in SEQ ID NO. 1-18 with at least 60%, preferably at least 65%, preferably at least 70%, and more. Preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity. The names of sequences 1-18 are as follows:
序列sequence 名称name 序列sequence 名称name
11 TLR1 25-475位氨基酸Amino acids 25-475 of TLR1 1010 TLR10 20-576位氨基酸Amino acids 20-576 of TLR10
22 TLR2 27-506位氨基酸Amino acids 27-506 of TLR2 1111 Dectin-1 66-247位氨基酸Dectin-1 Amino acids 66-247
33 TLR3 22-703位氨基酸Amino acids 22-703 of TLR3 1212 Dectin-2 64-209位氨基酸Dectin-2 Amino acids 64-209
44 TLR4 27-631位氨基酸Amino acids 27-631 of TLR4 1313 Mincle 74-219位氨基酸Mincle 74-219 amino acids
55 TLR5 21-639位氨基酸TLR5 21-639 amino acids 1414 CLEC2 96-221位氨基酸Amino acids 96-221 of CLEC2
66 TLR6 32-586位氨基酸TLR6 Amino acids 32-586 1515 CLEC5A 70-187位氨基酸CLEC5A Amino acids 70-187
77 TLR7 27-839位氨基酸Amino acids 27-839 of TLR7 1616 CLEC12A 65-265位氨基酸CLEC12A Amino acids 65-265
88 TLR8 27-827位氨基酸Amino acids 27-827 of TLR8 1717 DCIR 106-237位氨基酸DCIR 106-237 amino acids
99 TLR9 26-818位氨基酸Amino acids 26-818 of TLR9 1818 CLECSF8 61-215位氨基酸CLECSF8 61-215 amino acids
(2)当Z1和Y1中的每一种都是相同的一种共受体或其功能变体或片段时：在具有上述的通式Z1-Z2和Y1-Y2的可溶性二聚体免疫融合蛋白具体变化中，Z1和Y1的氨基酸序列包括SEQ ID NO.19-22所示的氨基酸中的任一个序列具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性。序列19～22的名称如下：(2) When each of Z1 and Y1 is the same co-receptor or its functional variants or fragments: immune fusion of soluble dimers with the above-mentioned general formulas Z1-Z2 and Y1-Y2 In the specific changes of the protein, the amino acid sequences of Z1 and Y1 include the amino acids shown in SEQ ID NO. 19-22. Any one of the sequences has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, It is preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity. The names of sequences 19-22 are as follows:
序列sequence 名称name 序列sequence 名称name
1919 CD14 26-355位氨基酸CD14 26-355 amino acids 21twenty one LBP 26-481位氨基酸Amino acids 26-481 of LBP
2020 MD-2 17-160位氨基酸MD-2 17-160 amino acids 22twenty two CD36 1-472位氨基酸CD36 Amino acids 1-472
(3)二聚化结构域Z2和Y2的氨基酸序列包括SEQ ID NO.23-28所示的氨基酸中的任一个序列具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性。序列23～29的名称如下：(3) The amino acid sequences of the dimerization domains Z2 and Y2 include SEQ ID NO. 23-28. Any one of the amino acid sequences has at least 60%, preferably at least 65%, preferably at least 70%, and more preferably at least 75%. %, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity. The names of sequences 23-29 are as follows:
序列sequence 名称name 序列sequence 名称name
23twenty three IgG-FcIgG-Fc 2727 IgG4-FcIgG4-Fc
24twenty four IgG-Fc-LALAGPIgG-Fc-LALAGP 2828 人IgG4 FC-hole突变体Human IgG4 FC-hole mutant
2525 IgG-Fc-holeIgG-Fc-hole 2929 人IgG4 FC-knob突变体Human IgG4 FC-knob mutant
2626 IgG-Fc-knobIgG-Fc-knob  To  To
根据Z1和Y1的来源，Z1-Z2多肽链和Y1-Y2多肽链的具体序列举例如下：According to the source of Z1 and Y1, the specific sequences of Z1-Z2 polypeptide chain and Y1-Y2 polypeptide chain are as follows:
在本发明的一些具体的优选实施例中，Z1和Y1都是TLR1的细胞外结构域或其功能变体或片段。例如，所述Z1-Z2多肽链和Y1-Y2多肽链的每一条包含与下述的SEQ ID NO.30所示的TLR1-IgG1-Fc氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性。In some specific preferred embodiments of the present invention, both Z1 and Y1 are extracellular domains of TLR1 or functional variants or fragments thereof. For example, each of the Z1-Z2 polypeptide chain and Y1-Y2 polypeptide chain contains a sequence identical to the TLR1-IgG1-Fc amino acid sequence shown in SEQ ID NO. 30 below or has at least 60%, preferably at least 65 %, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity.
在本发明的一些具体的优选实施例中，Z1和Y1都是TLR1的细胞外结构域或其功能变体或片段。例如，所述Z1-Z2多肽链和Y1-Y2多肽链的每一条包含与下述的SEQ ID NO.31所示的TLR1-IgG1-Fc-LALAPG氨基酸序列一致的序列或具有至少60％、优选至 少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性。In some specific preferred embodiments of the present invention, both Z1 and Y1 are extracellular domains of TLR1 or functional variants or fragments thereof. For example, each of the Z1-Z2 polypeptide chain and Y1-Y2 polypeptide chain contains a sequence consistent with the amino acid sequence of TLR1-IgG1-Fc-LALAPG shown in SEQ ID NO. 31 below or has at least 60%, preferably At least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity.
在本发明的一些具体的优选实施例中，Z1和Y1都是TLR2的细胞外结构域或其功能变体或片段。例如，所述Z1-Z2多肽链和Y1-Y2多肽链的每一条包含与下述的SEQ ID NO.32所示的TLR2-IgG1-Fc氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性。In some specific preferred embodiments of the present invention, both Z1 and Y1 are extracellular domains of TLR2 or functional variants or fragments thereof. For example, each of the Z1-Z2 polypeptide chain and Y1-Y2 polypeptide chain contains a sequence identical to the TLR2-IgG1-Fc amino acid sequence shown in SEQ ID NO. 32 below or has at least 60%, preferably at least 65 %, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity.
在本发明的一些个具体的优选实施例中，Z1和Y1都是TLR2的细胞外结构域或其功能变体或片段。例如，所述Z1-Z2多肽链和Y1-Y2多肽链的每一条包含与下述的SEQ ID NO.33所示的TLR2-IgG1-Fc-LALA氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性。In some specific preferred embodiments of the present invention, both Z1 and Y1 are extracellular domains of TLR2 or functional variants or fragments thereof. For example, each of the Z1-Z2 polypeptide chain and Y1-Y2 polypeptide chain contains a sequence consistent with the amino acid sequence of TLR2-IgG1-Fc-LALA shown in SEQ ID NO. 33 below or has at least 60%, preferably At least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity.
在本发明的一些具体的优选实施例中，Z1和Y1都是TLR4的细胞外结构域或其功能变体或片段。例如，所述Z1-Z2多肽链和Y1-Y2多肽链的每一条包含与下述的SEQ ID NO.34所示的TLR4-IgG1-Fc氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性。In some specific preferred embodiments of the present invention, both Z1 and Y1 are the extracellular domain of TLR4 or functional variants or fragments thereof. For example, each of the Z1-Z2 polypeptide chain and Y1-Y2 polypeptide chain contains a sequence identical to the TLR4-IgG1-Fc amino acid sequence shown in SEQ ID NO. 34 below or has at least 60%, preferably at least 65 %, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity.
在本发明的一些具体的优选实施例中，Z1和Y1都是TLR4的细胞外结构域或其功能变体或片段。例如，所述Z1-Z2多肽链和Y1-Y2多肽链的每一条包含与下述的SEQ ID NO.35所示的TLR4-IgG1-Fc-LALAGP氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性。In some specific preferred embodiments of the present invention, both Z1 and Y1 are the extracellular domain of TLR4 or functional variants or fragments thereof. For example, each of the Z1-Z2 polypeptide chain and Y1-Y2 polypeptide chain contains a sequence identical to the amino acid sequence of TLR4-IgG1-Fc-LALAGP shown in SEQ ID NO. 35 below or has at least 60%, preferably At least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity.
在本发明的一些具体的优选实施例中，Z1和Y1都是TLR6的细胞外结构域或其功能变体或片段。例如，所述Z1-Z2多肽链和Y1-Y2多肽链的每一条包含与下述的SEQ ID NO.36所示的TLR6-IgG1-Fc氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性。In some specific preferred embodiments of the present invention, both Z1 and Y1 are extracellular domains of TLR6 or functional variants or fragments thereof. For example, each of the Z1-Z2 polypeptide chain and Y1-Y2 polypeptide chain contains a sequence identical to the TLR6-IgG1-Fc amino acid sequence shown in SEQ ID NO. 36 below or has at least 60%, preferably at least 65 %, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity.
在本发明的一些具体的优选实施例中，Z1和Y1都是TLR6的细胞外结构域或其功能变体或片段。例如，所述Z1-Z2多肽链和Y1-Y2多肽链的每一条包含与下述的SEQ ID NO.37所示的TLR6-IgG1-Fc-LALAGP氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性。In some specific preferred embodiments of the present invention, both Z1 and Y1 are extracellular domains of TLR6 or functional variants or fragments thereof. For example, each of the Z1-Z2 polypeptide chain and Y1-Y2 polypeptide chain contains a sequence identical to the amino acid sequence of TLR6-IgG1-Fc-LALAGP shown in SEQ ID NO. 37 below or has at least 60%, preferably At least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity.
在本发明的一些具体的优选实施例中，Z1是TLR1的细胞外结构域或其功能变体或片段。Y1是TLR2的细胞外结构域或其功能变体或片段。例如，所述Z1-Z2多肽链包含与下述的SEQ ID NO.38所示的TLR1-Fc-Knob氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、 甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性；所述Y1-Y2多肽链包含与下述的SEQ ID NO.39所示的TLR2-Fc-Hole氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性；In some specific preferred embodiments of the present invention, Z1 is the extracellular domain of TLR1 or a functional variant or fragment thereof. Y1 is the extracellular domain of TLR2 or a functional variant or fragment thereof. For example, the Z1-Z2 polypeptide chain includes a sequence identical to the TLR1-Fc-Knob amino acid sequence shown in SEQ ID NO. 38 below or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably At least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity; the Y1-Y2 polypeptide chain comprises the following The TLR2-Fc-Hole amino acid sequence shown in SEQ ID NO. 39 is identical or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85 %, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity;
在本发明的一些具体的优选实施例中，Z1是TLR1的细胞外结构域或其功能变体或片段。Y1是TLR6的细胞外结构域或其功能变体或片段。例如，所述Z1-Z2多肽链包含与下述的SEQ ID NO.38所示的TLR1-Fc-Knob氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性；所述Y1-Y2多肽链包含与下述的SEQ ID NO.40所示的TLR6-Fc-hole氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性；In some specific preferred embodiments of the present invention, Z1 is the extracellular domain of TLR1 or a functional variant or fragment thereof. Y1 is the extracellular domain of TLR6 or a functional variant or fragment thereof. For example, the Z1-Z2 polypeptide chain includes a sequence identical to the TLR1-Fc-Knob amino acid sequence shown in SEQ ID NO. 38 below or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably At least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity; the Y1-Y2 polypeptide chain comprises the following The TLR6-Fc-hole amino acid sequence shown in SEQ ID NO. 40 is identical or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85 %, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity;
在本发明的一些具体的优选实施例中，Z1是TLR2的细胞外结构域或其功能变体或片段。Y1是TLR4的细胞外结构域或其功能变体或片段。例如，所述Z1-Z2多肽链包含与下述的SEQ ID NO.39所示的TLR2-Fc-Hole氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性；所述Y1-Y2多肽链包含与下述的SEQ ID NO.41所示的TLR4-IgG-Knob氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性；In some specific preferred embodiments of the present invention, Z1 is the extracellular domain of TLR2 or a functional variant or fragment thereof. Y1 is the extracellular domain of TLR4 or a functional variant or fragment thereof. For example, the Z1-Z2 polypeptide chain includes a sequence identical to the TLR2-Fc-Hole amino acid sequence shown in SEQ ID NO. 39 below or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably At least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity; the Y1-Y2 polypeptide chain comprises the following The TLR4-IgG-Knob amino acid sequence shown in SEQ ID NO. 41 is identical or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85 %, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity;
在本发明的一些具体的优选实施例中，Z1是TLR2的细胞外结构域或其功能变体或片段。Y1是TLR6的细胞外结构域或其功能变体或片段。例如，所述Z1-Z2多肽链包含与下述的SEQ ID NO.39所示的TLR2-Fc-Hole氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性；所述Y1-Y2多肽链包含与下述的SEQ ID NO.42所示的TLR6-Fc-knob氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性；In some specific preferred embodiments of the present invention, Z1 is the extracellular domain of TLR2 or a functional variant or fragment thereof. Y1 is the extracellular domain of TLR6 or a functional variant or fragment thereof. For example, the Z1-Z2 polypeptide chain includes a sequence identical to the TLR2-Fc-Hole amino acid sequence shown in SEQ ID NO. 39 below or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably At least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity; the Y1-Y2 polypeptide chain comprises the following The TLR6-Fc-knob amino acid sequence shown in SEQ ID NO. 42 is identical or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85 %, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity;
在本发明的一些具体的优选实施例中，Z1是TLR4的细胞外结构域或其功能变体或片段。Y1是TLR6的细胞外结构域或其功能变体或片段。例如，所述Z1-Z2多肽链包含与下述的SEQ ID NO.41所示的TLR4-IgG-Knob氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性；所述Y1-Y2多肽链包含与下述的SEQ ID NO.40所示的TLR6-Fc-hole氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至 少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性；In some specific preferred embodiments of the present invention, Z1 is the extracellular domain of TLR4 or a functional variant or fragment thereof. Y1 is the extracellular domain of TLR6 or a functional variant or fragment thereof. For example, the Z1-Z2 polypeptide chain includes a sequence identical to the TLR4-IgG-Knob amino acid sequence shown in SEQ ID NO. 41 below or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably At least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity; the Y1-Y2 polypeptide chain comprises the following The TLR6-Fc-hole amino acid sequence shown in SEQ ID NO. 40 is identical or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85 %, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity;
在本发明的一些具体的优选实施例中，Z1是TLR4的细胞外结构域或其功能变体或片段。Y1是LBP或其功能变体或片段。例如，所述Z1-Z2多肽链包含与下述的SEQ ID NO.41所示的TLR4-IgG-Knob氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性；所述Y1-Y2多肽链包含与下述的SEQ ID NO.43所示的LBD-Fc-hole氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性；In some specific preferred embodiments of the present invention, Z1 is the extracellular domain of TLR4 or a functional variant or fragment thereof. Y1 is LBP or a functional variant or fragment thereof. For example, the Z1-Z2 polypeptide chain includes a sequence identical to the TLR4-IgG-Knob amino acid sequence shown in SEQ ID NO. 41 below or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably At least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity; the Y1-Y2 polypeptide chain comprises the following The LBD-Fc-hole amino acid sequence shown in SEQ ID NO. 43 has the same sequence or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85 %, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity;
在本发明的一些具体的优选实施例中，Z1是TLR4的细胞外结构域或其功能变体或片段。Y1是CD14的细胞外结构域或其功能变体或片段。例如，所述Z1-Z2多肽链包含与下述的SEQ ID NO.41所示的TLR4-IgG-Knob氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性；所述Y1-Y2多肽链包含与下述的SEQ ID NO.44所示的CD14 Fc hole氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性；In some specific preferred embodiments of the present invention, Z1 is the extracellular domain of TLR4 or a functional variant or fragment thereof. Y1 is the extracellular domain of CD14 or a functional variant or fragment thereof. For example, the Z1-Z2 polypeptide chain includes a sequence identical to the TLR4-IgG-Knob amino acid sequence shown in SEQ ID NO. 41 below or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably At least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity; the Y1-Y2 polypeptide chain comprises the following The amino acid sequence of CD14 Fc hole shown in SEQ ID NO. 44 is identical or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, Even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity;
在本发明的一些具体的优选实施例中，Z1是TLR4的细胞外结构域或其功能变体或片段。Y1是MD-2或其功能变体或片段。例如，所述Z1-Z2多肽链包含与下述的SEQ ID NO.41所示的TLR4-IgG-Knob氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性；所述Y1-Y2多肽链包含与下述的SEQ ID NO.45所示的MD-2 Fc hole氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性；In some specific preferred embodiments of the present invention, Z1 is the extracellular domain of TLR4 or a functional variant or fragment thereof. Y1 is MD-2 or a functional variant or fragment thereof. For example, the Z1-Z2 polypeptide chain includes a sequence identical to the TLR4-IgG-Knob amino acid sequence shown in SEQ ID NO. 41 below or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably At least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity; the Y1-Y2 polypeptide chain comprises the following The sequence of the MD-2 Fc hole shown in SEQ ID NO. 45 is consistent with the amino acid sequence or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85 %, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity;
在本发明的一些具体的优选实施例中，Z1是CD14的细胞外结构域或其功能变体或片段。Y1是MD-2或其功能变体或片段。例如，所述Z1-Z2多肽链包含与下述的SEQ ID NO.46所示的CD14 Fc knob氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性；所述Y1-Y2多肽链包含与下述的SEQ ID NO.45所示的MD-2 Fc hole氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性；In some specific preferred embodiments of the present invention, Z1 is the extracellular domain of CD14 or a functional variant or fragment thereof. Y1 is MD-2 or a functional variant or fragment thereof. For example, the Z1-Z2 polypeptide chain comprises a sequence identical to the amino acid sequence of CD14 Fc knob shown in SEQ ID NO. 46 below, or has at least 60%, preferably at least 65%, preferably at least 70%, and more preferably at least 75%. %, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity; the Y1-Y2 polypeptide chain comprises the following SEQ ID The MD-2 Fc hole amino acid sequence shown in NO.45 has the same sequence or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, Even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity;
在本发明的一些具体的优选实施例中，Z1是TLR4的细胞外结构域或其功能变体或片段。Y1是CD36的细胞外结构域或其功能变体或片段。例如，所述Z1-Z2多肽链包含 与下述的SEQ ID NO.41所示的TLR4-IgG-Knob氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性；所述Y1-Y2多肽链包含与下述的SEQ ID NO.47所示的CD36 Fc hole氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性；In some specific preferred embodiments of the present invention, Z1 is the extracellular domain of TLR4 or a functional variant or fragment thereof. Y1 is the extracellular domain of CD36 or a functional variant or fragment thereof. For example, the Z1-Z2 polypeptide chain includes a sequence identical to the TLR4-IgG-Knob amino acid sequence shown in SEQ ID NO. 41 below or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably At least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity; the Y1-Y2 polypeptide chain comprises the following The amino acid sequence of CD36 Fc hole shown in SEQ ID NO. 47 is identical or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, Even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity;
在本发明的一些具体的优选实施例中，Z1是TLR6的细胞外结构域或其功能变体或片段。Y1是CD36的细胞外结构域或其功能变体或片段。例如，所述Z1-Z2多肽链包含与下述的SEQ ID NO.42所示的TLR6-Fc-knob氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性；所述Y1-Y2多肽链包含与下述的SEQ ID NO.47所示的CD36 Fc hole氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性；In some specific preferred embodiments of the present invention, Z1 is the extracellular domain of TLR6 or a functional variant or fragment thereof. Y1 is the extracellular domain of CD36 or a functional variant or fragment thereof. For example, the Z1-Z2 polypeptide chain comprises a sequence identical to the TLR6-Fc-knob amino acid sequence shown in SEQ ID NO. 42 below or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably At least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity; the Y1-Y2 polypeptide chain comprises the following The amino acid sequence of CD36 Fc hole shown in SEQ ID NO. 47 is identical or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, Even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity;
在本发明的一些具体的优选实施例中，Z1和Y1都是Dectin-1的细胞外结构域或其功能变体或片段。例如，所述Z1-Z2多肽链和Y1-Y2多肽链的每一条包含与下述的SEQ ID NO.48所示的Dectin-1 IgG Fc氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性。In some specific preferred embodiments of the present invention, both Z1 and Y1 are the extracellular domain of Dectin-1 or functional variants or fragments thereof. For example, each of the Z1-Z2 polypeptide chain and the Y1-Y2 polypeptide chain contains a sequence consistent with the Dectin-1 IgG Fc amino acid sequence shown in SEQ ID NO. 48 below or has at least 60%, preferably at least 65 %, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity.
在本发明的一些具体的优选实施例中，Z1和Y1都是Dectin-2的细胞外结构域或其功能变体或片段。例如，所述Z1-Z2多肽链和Y1-Y2多肽链的每一条包含与下述的SEQ ID NO.49所示的Dectin-2 IgG Fc氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性。In some specific preferred embodiments of the present invention, both Z1 and Y1 are the extracellular domain of Dectin-2 or functional variants or fragments thereof. For example, each of the Z1-Z2 polypeptide chain and the Y1-Y2 polypeptide chain contains a sequence consistent with the Dectin-2 IgG Fc amino acid sequence shown in SEQ ID NO. 49 below or has at least 60%, preferably at least 65 %, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity.
在本发明的一些具体的优选实施例中，Z1是Dectin-1的细胞外结构域或其功能变体或片段。Y1是Mincle的细胞外结构域或其功能变体或片段。例如，所述Z1-Z2多肽链包含与下述的SEQ ID NO.50所示的Dectin-1 IgG Fc-knob氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性；所述Y1-Y2多肽链包含与下述的SEQ ID NO.51所示的Mincle IgG Fc-hole氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性；In some specific preferred embodiments of the present invention, Z1 is the extracellular domain of Dectin-1 or a functional variant or fragment thereof. Y1 is the extracellular domain of Mincle or a functional variant or fragment thereof. For example, the Z1-Z2 polypeptide chain includes a sequence consistent with the Dectin-1 IgG Fc-knob amino acid sequence shown in SEQ ID NO. 50 below or has at least 60%, preferably at least 65%, preferably at least 70%, More preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity; the Y1-Y2 polypeptide chain comprises the following The amino acid sequence of Mincle IgG Fc-hole shown in SEQ ID NO. 51 is identical or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably At least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity;
在本发明的一些具体的优选实施例中，Z1是Dectin-2的细胞外结构域或其功能变体或片段。Y1是CLEC2的细胞外结构域或其功能变体或片段。例如，所述Z1-Z2多肽链 包含与下述的SEQ ID NO.52所示的Dectin-2 IgG Fc-knob氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性；所述Y1-Y2多肽链包含与下述的SEQ ID NO.53所示的CLEC2-Fc-hole氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性；In some specific preferred embodiments of the present invention, Z1 is the extracellular domain of Dectin-2 or a functional variant or fragment thereof. Y1 is the extracellular domain of CLEC2 or a functional variant or fragment thereof. For example, the Z1-Z2 polypeptide chain includes a sequence consistent with the Dectin-2 IgG Fc-knob amino acid sequence shown in SEQ ID NO. 52 below or has at least 60%, preferably at least 65%, preferably at least 70%, More preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity; the Y1-Y2 polypeptide chain comprises the following The CLEC2-Fc-hole amino acid sequence shown in SEQ ID NO.53 has the same sequence or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably At least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity;
在本发明的一些具体的优选实施例中，Z1是Dectin-1的细胞外结构域或其功能变体或片段。Y1是CLEC5A的细胞外结构域或其功能变体或片段。例如，所述Z1-Z2多肽链包含与下述的SEQ ID NO.50所示的Dectin-1 IgG Fc-knob氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性；所述Y1-Y2多肽链包含与下述的SEQ ID NO.54所示的CLEC5A-Fc-hole氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性；In some specific preferred embodiments of the present invention, Z1 is the extracellular domain of Dectin-1 or a functional variant or fragment thereof. Y1 is the extracellular domain of CLEC5A or a functional variant or fragment thereof. For example, the Z1-Z2 polypeptide chain includes a sequence consistent with the Dectin-1 IgG Fc-knob amino acid sequence shown in SEQ ID NO. 50 below or has at least 60%, preferably at least 65%, preferably at least 70%, More preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity; the Y1-Y2 polypeptide chain comprises the following The CLEC5A-Fc-hole amino acid sequence shown in SEQ ID NO. 54 is identical or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably At least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity;
在本发明的一些具体的优选实施例中，Z1是Dectin-2的细胞外结构域或其功能变体或片段。Y1是CLEC12A的细胞外结构域或其功能变体或片段。例如，所述Z1-Z2多肽链包含与下述的SEQ ID NO.52所示的Dectin-2 IgG Fc-knob氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性；所述Y1-Y2多肽链包含与下述的SEQ ID NO.55所示的CLEC12A Fc hole氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性；In some specific preferred embodiments of the present invention, Z1 is the extracellular domain of Dectin-2 or a functional variant or fragment thereof. Y1 is the extracellular domain of CLEC12A or a functional variant or fragment thereof. For example, the Z1-Z2 polypeptide chain includes a sequence consistent with the Dectin-2 IgG Fc-knob amino acid sequence shown in SEQ ID NO. 52 below or has at least 60%, preferably at least 65%, preferably at least 70%, More preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity; the Y1-Y2 polypeptide chain comprises the following The CLEC12A Fc hole amino acid sequence shown in SEQ ID NO. 55 has the same sequence or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85 %, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity;
在本发明的一些具体的优选实施例中，Z1是Dectin-1的细胞外结构域或其功能变体或片段。Y1是DCIR的细胞外结构域或其功能变体或片段。例如，所述Z1-Z2多肽链包含与下述的SEQ ID NO.50所示的Dectin-1 IgG Fc-knob氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性；所述Y1-Y2多肽链包含与下述的SEQ ID NO.56所示的DCIR Fc hole氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性；In some specific preferred embodiments of the present invention, Z1 is the extracellular domain of Dectin-1 or a functional variant or fragment thereof. Y1 is the extracellular domain of DCIR or a functional variant or fragment thereof. For example, the Z1-Z2 polypeptide chain includes a sequence consistent with the Dectin-1 IgG Fc-knob amino acid sequence shown in SEQ ID NO. 50 below or has at least 60%, preferably at least 65%, preferably at least 70%, More preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity; the Y1-Y2 polypeptide chain comprises the following The sequence of the DCIR Fc hole shown in SEQ ID NO.56 is identical or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85 %, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity;
在本发明的一些具体的优选实施例中，Z1是Dectin-2的细胞外结构域或其功能变体或片段。Y1是CLECSF8的细胞外结构域或其功能变体或片段。例如，所述Z1-Z2多肽链包含与下述的SEQ ID NO.52所示的Dectin-2 IgG Fc-knob氨基酸序列一致的序列或具 有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性；所述Y1-Y2多肽链包含与下述的SEQ ID NO.57所示的CLECSF8 Fc hole氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性；In some specific preferred embodiments of the present invention, Z1 is the extracellular domain of Dectin-2 or a functional variant or fragment thereof. Y1 is the extracellular domain of CLECSF8 or a functional variant or fragment thereof. For example, the Z1-Z2 polypeptide chain includes a sequence consistent with the Dectin-2 IgG Fc-knob amino acid sequence shown in SEQ ID NO. 52 below or has at least 60%, preferably at least 65%, preferably at least 70%, More preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity; the Y1-Y2 polypeptide chain comprises the following The CLECSF8 Fc hole amino acid sequence shown in SEQ ID NO. 57 has the same sequence or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85 %, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity;
在本发明的一些具体的优选实施例中，Z1和Y1都是Dectin-1的细胞外结构域或其功能变体或片段。例如，所述Z1-Z2多肽链和Y1-Y2多肽链的每一条包含与下述的SEQ ID NO.58所示的Dectin-1 IgG4 Fc氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性。In some specific preferred embodiments of the present invention, both Z1 and Y1 are the extracellular domain of Dectin-1 or functional variants or fragments thereof. For example, each of the Z1-Z2 polypeptide chain and the Y1-Y2 polypeptide chain contains a sequence consistent with the Dectin-1 IgG4 Fc amino acid sequence shown in SEQ ID NO. 58 below or has at least 60%, preferably at least 65 %, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity.
本发明的一些具体的优选实施例中，Z1是TLR2的细胞外结构域或其功能变体或片段。Y1是Dectin-1的细胞外结构域或其功能变体或片段。例如，所述Z1-Z2多肽链包含与下述的SEQ ID NO.39所示的氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性；所述Y1-Y2多肽链包含与下述的SEQ ID NO.50所示的Dectin-1 IgG Fc-knob氨基酸序列一致的序列或具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性。In some specific preferred embodiments of the present invention, Z1 is the extracellular domain of TLR2 or a functional variant or fragment thereof. Y1 is the extracellular domain of Dectin-1 or a functional variant or fragment thereof. For example, the Z1-Z2 polypeptide chain includes a sequence consistent with the amino acid sequence shown in SEQ ID NO. 39 below or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more Preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity; the Y1-Y2 polypeptide chain comprises the following SEQ ID NO.50 The shown Dectin-1 IgG Fc-knob amino acid sequence is consistent or has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, or even More preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity.
本发明的第二方面，提供了编码上述的二聚体免疫融合蛋白的多核苷酸、运载该核苷酸的载体以及包含这种载体的细胞。The second aspect of the present invention provides a polynucleotide encoding the dimeric immune fusion protein, a vector for carrying the nucleotide, and a cell containing the vector.
本发明提供的表达载体包含下述可操作地连接的元件：转录启动子、编码上述二聚体免疫融合蛋白的DNA区和转录终止子。The expression vector provided by the present invention includes the following operably linked elements: a transcription promoter, a DNA region encoding the dimeric immune fusion protein, and a transcription terminator.
通过培养包含载体的细胞，用于生产如上公开的多肽或二聚蛋白，包括：(i)培养包含如上公开的表达载体的细胞，其中细胞表达由所述DNA区段编码的二聚体免疫融合蛋白，并生产编码的二聚体免疫融合蛋白；(ii)回收可溶性二聚体免疫融合蛋白。By culturing a cell containing a vector for the production of the polypeptide or dimeric protein as disclosed above, including: (i) culturing a cell containing the expression vector as disclosed above, wherein the cell expresses the dimer immunofusion encoded by the DNA segment Protein and produce the encoded dimer immune fusion protein; (ii) recover the soluble dimer immune fusion protein.
类似地，制备二聚蛋白的方法包括：(i)培养包含如上公开的表达载体的细胞，其中细胞表达由所述DNA区段编码的二聚体免疫融合蛋白，并生产编码的二聚体免疫融合蛋白作为二聚蛋白；和(ii)回收二聚蛋白。Similarly, the method for preparing a dimeric protein includes: (i) culturing a cell containing the expression vector disclosed above, wherein the cell expresses the dimeric immune fusion protein encoded by the DNA segment, and producing the encoded dimeric immune The fusion protein is used as a dimeric protein; and (ii) the dimeric protein is recovered.
本发明的第三方面，提供了一种药物组合物，该药物组合物包含上述可溶性二聚体免疫融合蛋白和至少一种药学上可接受的载体。该药物组合物以可溶性二聚体免疫粘融合蛋白为主要或唯一活性成分，辅料可以保证本发明公开的二聚体免疫融合蛋白氨基酸核心序列的构像完整性，同时还要保护蛋白质的多官能团，防止其降解(包括但不限于凝聚、脱氨或氧化)，从而更稳定地发挥疗效。The third aspect of the present invention provides a pharmaceutical composition comprising the above-mentioned soluble dimer immune fusion protein and at least one pharmaceutically acceptable carrier. The pharmaceutical composition uses a soluble dimer immune fusion protein as the main or only active ingredient, and the auxiliary materials can ensure the conformational integrity of the amino acid core sequence of the dimer immune fusion protein disclosed in the present invention, and at the same time protect the multifunctional group of the protein , To prevent its degradation (including but not limited to agglomeration, deamination or oxidation), so as to more stably exert its curative effect.
在药物形式上，可为制药领域常用的混悬、水针、冻干等制剂，优选水针或冻干制剂。液体制剂可以在2℃-8℃条件下保存至少稳定一年，冻干制剂在30℃至少六个月保 持稳定。In the form of the drug, it can be a suspension, water injection, freeze-dried preparation commonly used in the pharmaceutical field, and preferably a water injection or freeze-dried preparation. Liquid formulations can be stored at 2°C-8°C for at least one year, and lyophilized formulations can be stored at 30°C for at least six months.
对于本发明公开的上述二聚体免疫融合蛋白的水针或冻干制剂，药学上可以接受的辅料包括表面活性剂、溶液稳定剂、等渗调节剂和缓冲液之一或其组合。其中，表面活性剂包括非离子型表面活性剂如聚氧乙烯山梨醇脂肪酸酯(吐温20或80)；poloxamer(如poloxamer 188)；Triton；十二烷基硫酸钠(SDS)；月桂硫酸钠；十四烷基、亚油基或十八烷基肌氨酸；Pluronics；MONAQUATTM等，其加入量应使双功能双特异性抗体蛋白的颗粒化趋势最小；溶液稳定剂可以为糖类，包括还原性糖和非还原性糖，氨基酸类包括谷氨酸单钠或组氨酸，醇类包括三元醇、高级糖醇、丙二醇、聚乙二醇之一或其组合，溶液稳定剂的加入量应该使最后形成的制剂在本领域的技术人员认为达到稳定的时间内保持稳定状态；等渗调节剂可以为氯化钠、甘露醇之一；缓冲液可以为TRIS、组氨酸缓冲液、磷酸盐缓冲液之一。For the water injection or lyophilized preparation of the dimeric immune fusion protein disclosed in the present invention, pharmaceutically acceptable excipients include one or a combination of surfactants, solution stabilizers, isotonic regulators, and buffers. Among them, surfactants include non-ionic surfactants such as polyoxyethylene sorbitol fatty acid esters (Tween 20 or 80); poloxamer (such as poloxamer 188); Triton; sodium dodecyl sulfate (SDS); lauric sulfuric acid Sodium; tetradecyl, linoleyl or octadecyl sarcosine; Pluronics; MONAQUATTM, etc., the amount added should minimize the tendency of bifunctional bispecific antibody protein to granulate; solution stabilizers can be sugars, Including reducing sugars and non-reducing sugars, amino acids include monosodium glutamate or histidine, alcohols include triols, higher sugar alcohols, propylene glycol, polyethylene glycol, one or a combination of them, solution stabilizers The amount added should enable the final formulation to remain stable within the time considered by those skilled in the art to reach a stable state; the isotonicity regulator can be one of sodium chloride and mannitol; the buffer can be TRIS, histidine buffer , One of the phosphate buffer.
本发明所述的二聚体免疫融合蛋白及其作为活性成分的组合物具有如下的用途：1)结合病原微生物表面分子、细胞壁或细胞表面成分，如实施例1所述；2)直接杀伤病原微生物、限制病原微生物生长、提高免疫细胞对病原微生物入侵的抵抗作用，如实施例2-9所述；3)抑制和减少过度表达、分泌的炎性介质和/或细胞因子，如HMGB1、TNFα、IFN-γ、IL-6、COX-2等。如实施例10-11；4)减少脏器炎症介质过度表达释放、减轻脏器炎症损伤、增强脏器抗应激能力、抵抗急慢性脏器损伤、抵抗浓毒血症等，如实施例12-13；5)减少慢性炎性介质损伤、减轻脏器炎症纤维化的作用，如实施例14-15；6)抑制局部的免疫耐受紊乱，如实施例16所述的免疫不孕不育；7)抑制自身免疫耐受异常介导的炎性介质过度疾病，如狼疮等自身免疫病，如实施例17。The dimeric immune fusion protein of the present invention and its composition as an active ingredient have the following uses: 1) Combining with pathogenic microorganism surface molecules, cell walls or cell surface components, as described in Example 1; 2) Directly killing pathogens Microorganisms, restrict the growth of pathogenic microorganisms, and improve the resistance of immune cells to pathogenic microorganism invasion, as described in Examples 2-9; 3) Inhibit and reduce overexpression and secretion of inflammatory mediators and/or cytokines, such as HMGB1, TNFα , IFN-γ, IL-6, COX-2, etc. As in Examples 10-11; 4) Reduce the over-expression and release of organ inflammatory mediators, reduce organ inflammation damage, enhance organ anti-stress ability, resist acute and chronic organ damage, resist hypertoxicity, etc., as in Example 12 -13; 5) Reduce chronic inflammatory mediator damage and reduce organ inflammation and fibrosis, as in Examples 14-15; 6) Inhibit local immune tolerance disorders, such as immune infertility as described in Example 16. 7) Inhibition of excessive inflammatory mediator diseases mediated by abnormal autoimmune tolerance, such as autoimmune diseases such as lupus, as in Example 17.
因此，在本发明的一些方面，本发明所述的二聚体免疫融合蛋白及其作为活性成分的组合物具有如下的用途：包括预防、诊断和治疗需要去除炎性介质相关疾病药物、试剂、试剂盒用途中的任意一种或至少两种的组合。Therefore, in some aspects of the present invention, the dimeric immune fusion protein of the present invention and the composition thereof as an active ingredient have the following uses: including prevention, diagnosis and treatment of drugs, reagents, and drugs related to diseases that require the removal of inflammatory mediators. Any one or a combination of at least two of the kit uses.
在一些方法中，所述炎性介质包括：病毒、细菌或寄生虫等病原微生物，还包括酶、细胞因子、前列腺素(prostaglandins)、类花生酸(eicosanoids)、自三烯类(Leukotrienes)、激肽类(kinins)、补体、凝血因子、毒素、内毒素、肠毒素、脂多糖、诱导细胞凋亡的物质、腐蚀性物质、胆汁盐、脂肪酸、磷脂、氧化副产物、活性氧簇、氧自由基、表面活性剂、离子、刺激性物质、细胞碎片、干扰素、以及免疫调节性抗体、生物制品(biologics)、药物中的任一种或至少两种的组合。In some methods, the inflammatory mediators include pathogenic microorganisms such as viruses, bacteria or parasites, as well as enzymes, cytokines, prostaglandins, eicosanoids, Leukotrienes, Kinins, complements, coagulation factors, toxins, endotoxins, enterotoxins, lipopolysaccharides, substances that induce apoptosis, corrosive substances, bile salts, fatty acids, phospholipids, oxidation by-products, reactive oxygen species, oxygen Any one or a combination of free radicals, surfactants, ions, irritating substances, cell debris, interferons, and immunomodulatory antibodies, biologics, and drugs.
在一些方面，所述炎性介质存在于受试者的生理性流体或载体流体中，所述生理性流体包括以下的流体:生理性流体包括以下的流体:鼻咽、口腔、食道、胃、胰腺、肝、胸膜、心包、腹膜、肠、前列腺、精液、阴道分泌物、眼泪、唾液、粘液、胆汁、血液、淋巴、血浆、血清、滑液、脑脊液、尿，以及间隙、细胞内和细胞外的流体。In some aspects, the inflammatory mediator is present in the physiological fluid or carrier fluid of the subject, and the physiological fluid includes the following fluids: the physiological fluid includes the following fluids: nasopharyngeal, oral cavity, esophagus, stomach, Pancreas, liver, pleura, pericardium, peritoneum, intestine, prostate, semen, vaginal secretions, tears, saliva, mucus, bile, blood, lymph, plasma, serum, synovial fluid, cerebrospinal fluid, urine, as well as spaces, intracellular and cellular Fluid outside.
所述炎性介质相关性疾病包括：全身性炎性应答综合征(SIRS)或脓毒症(例如源自病毒、细菌、真菌或寄生虫感染)、自身免疫病、外科手术、细胞毒性化疗、骨髓操作、大的组织损伤或外伤、肠系膜灌注不足、肠粘膜损伤、疟疾、胃肠道炎性疾病、肠道感 染、宫腔感染、流行性感冒、急性肺炎如急性呼吸窘迫综合症或急性肺损伤、肺栓塞、胰腺炎、自身免疫和胶原血管病、输血相关疾病、烧伤、烟或吸入肺损伤、移植物抗宿主病、缺血或梗死、再灌注损伤、出血、过敏反应、药物过量、辐射损伤或化学损伤。在一些实施方案中，炎性介质由生物战的病原体、毒素或制剂导致的疾病产生，例如病毒性出血热、水母毒素、汉坦病毒心肺综合征(汉坦病毒)、霍乱毒素、肉毒杆菌毒素、草麻毒素、Q热[博纳特氏立克次氏体(Coxiella burnetii)]、斑痊伤寒症(Rickettsia prowaszekii)或鹦鹉热[鹦鹉热衣原体(Chlamydia psittaci)]。The inflammatory mediator-related diseases include: systemic inflammatory response syndrome (SIRS) or sepsis (for example, derived from viral, bacterial, fungal or parasitic infection), autoimmune diseases, surgery, cytotoxic chemotherapy, Bone marrow manipulation, large tissue injury or trauma, mesenteric hypoperfusion, intestinal mucosal injury, malaria, gastrointestinal inflammatory disease, intestinal infection, uterine infection, influenza, acute pneumonia such as acute respiratory distress syndrome or acute lung Injury, pulmonary embolism, pancreatitis, autoimmune and collagen vascular diseases, blood transfusion-related diseases, burns, smoke or inhalation lung injury, graft versus host disease, ischemia or infarction, reperfusion injury, hemorrhage, allergic reactions, drug overdose, Radiation damage or chemical damage. In some embodiments, inflammatory mediators are produced by diseases caused by pathogens, toxins, or agents of biological warfare, such as viral hemorrhagic fever, jellyfish toxin, hantavirus cardiopulmonary syndrome (hantavirus), cholera toxin, botulinum Toxins, hemp toxins, Q fever [Coxiella burnetii], Rickettsia prowaszekii, or psittacosis [Chlamydia psittaci].
炎性介质相关性疾病还包括：接受移植，免疫不孕等需要去除目的免疫因素的疾病。Inflammatory media-related diseases also include: transplantation, immune infertility and other diseases that require the removal of target immune factors.
本发明的有益保障及效果：The beneficial guarantees and effects of the present invention:
本发明提供的二聚体免疫融合蛋白、药物组合物和用途，构建和表达过程简单，通过实验证实二聚体免疫融合蛋白一方面可以直接杀伤病原微生物，限制外来病原体的入侵，另一方面抑制过度产生的炎症因子，减轻组织损伤，对炎性介质相关性疾病等具有良好的治疗作用，通过单独应用或与其他相关病症药物联用，能有效预防和/或治疗炎性介质相关性疾病，具备广阔的临床应用前景。The dimer immune fusion protein, pharmaceutical composition and use provided by the present invention have simple construction and expression processes. Experiments have proved that the dimer immune fusion protein can directly kill pathogenic microorganisms on the one hand and limit the invasion of foreign pathogens, on the other hand, it can inhibit Excessively produced inflammatory factors can reduce tissue damage and have a good therapeutic effect on inflammatory mediator-related diseases. By using alone or in combination with other related disease drugs, it can effectively prevent and/or treat inflammatory mediator-related diseases. It has broad prospects for clinical application.
附图说明Description of the drawings
图1为二聚体免疫融合蛋白的结构示意图。Figure 1 is a schematic diagram of the structure of a dimeric immune fusion protein.
具体实施方式Detailed ways
以下实施例、实验例对本发明进行进一步的说明，不应理解为对本发明的限制。实施例不包括对传统方法的详细描述，如那些用于构建载体和质拉的方法，将编码蛋白的基因插入到这样的载体和质拉的方法或将质粒引入宿主细胞的方法。这样的方法对于本领域中具有普通技术的人员是众所周知的，并且在许多出版物中都有所描述，包括Sambrook,J.,Fritsch,E.F.and Maniais，T.(1989)Molecular Cloning：A Laboratory Manual，2 ndedition，Cold spring Harbor Laboratory Press。 The following examples and experimental examples further illustrate the present invention, and should not be construed as limiting the present invention. The examples do not include detailed descriptions of traditional methods, such as those used to construct vectors and plasmids, methods of inserting genes encoding proteins into such vectors and plasmids, or methods of introducing plasmids into host cells. Such methods are well known to those of ordinary skill in the art and are described in many publications, including Sambrook, J., Fritsch, EF and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2 nd edition, Cold spring Harbor Laboratory Press.
实施例1.可溶性二聚体免疫融合蛋白的构建、表达、表征Example 1. Construction, expression and characterization of soluble dimer immune fusion protein
如图1所示，可溶性二聚体免疫融合蛋白是一种带有抗体Fc的二聚体，二聚体免疫融合蛋白本身的构建和表达的方法为领域内的常规实验技术，简单描述如下：As shown in Figure 1, the soluble dimer immune fusion protein is a dimer with an antibody Fc. The method of constructing and expressing the dimer immune fusion protein itself is a conventional experimental technique in the field, which is briefly described as follows:
(1)委托基因合成商(苏州金维智公司)针对本实施例需要的二聚体免疫融合蛋白的氨基酸序列进行编码核苷酸密码子优化和全基因合成，优化后的核苷酸序列直接装载到PCDNA3.4载体上，所有载体编码后的氨基酸序列描述见表1。(1) Entrust a gene synthesizer (Suzhou Jinweizhi Company) to optimize the coding nucleotide codon and synthesize the whole gene for the amino acid sequence of the dimer immune fusion protein required in this example, and load the optimized nucleotide sequence directly into On the PCDNA3.4 vector, the amino acid sequences encoded by all vectors are described in Table 1.
(2)委托蛋白质制备商(义翘神州公司)针对本实施例需要二聚体免疫融合蛋白进行表达纯化。采用文献Finck B K.Science,265.；Mihara M et al..Journal of Clinical Investigation.2000；106:91-101；Yu X,et al.Nature Immunology.2009；10:48-57.Liu S,et al.Clin Immunol.2019 Jun；203:72-80.)方法，利用293F系统进行瞬时转染表达技术进行蛋白表达，然后利用 protein A和离子交换的方法获取大量的可溶性二聚体免疫融合蛋白，SDS-PAGE、western blot、质谱证实目的蛋白。(2) Entrust a protein preparation company (Yiqiao Shenzhou Company) to express and purify the dimer immune fusion protein for this embodiment. Using the literature Finck B K. Science, 265.; Mihara M et al.. Journal of Clinical Investigation. 2000; 106: 91-101; Yu X, et al. Nature Immunology. 2009; 10: 48-57. Liu S, et al. Clin Immunol. 2019 Jun; 203:72-80.) method, using the 293F system for transient transfection expression technology for protein expression, and then using protein A and ion exchange methods to obtain a large number of soluble dimeric immune fusion proteins , SDS-PAGE, western blot, and mass spectrometry confirmed the target protein.
(3)测定二聚体免疫融合蛋白对配体结合能力(3) Determine the binding ability of the dimer immune fusion protein to the ligand
利用ELISA方法检测二聚体免疫融合蛋白对特定配体的结合能力，如表2所示。The ELISA method was used to detect the binding ability of the dimer immune fusion protein to specific ligands, as shown in Table 2.
表1可溶性二聚体免疫融合蛋白信息Table 1 Soluble dimer immune fusion protein information
Figure PCTCN2020131581-appb-000001
Figure PCTCN2020131581-appb-000001
Figure PCTCN2020131581-appb-000002
Figure PCTCN2020131581-appb-000002
表2二聚体免疫融合蛋白结合能力检测Table 2 Detection of binding ability of dimer immune fusion protein
Figure PCTCN2020131581-appb-000003
Figure PCTCN2020131581-appb-000003
Figure PCTCN2020131581-appb-000004
Figure PCTCN2020131581-appb-000004
*+++：结合力达PM级别；++结合力达NM级别。*+++: The binding force reaches PM level; ++The binding force reaches NM level.
实施例2二聚体免疫融合蛋白对金葡菌作用Example 2 The effect of dimer immune fusion protein on Staphylococcus aureus
表达绿色荧光蛋白的金黄色葡萄球菌来自中国科学院微生物所保藏，感染复数采用1:10的感染比。采集健康志愿者外周血样分离外周单核细胞(PBMC，采用碧云天淋巴细胞分离试剂盒分离)。新分离的细胞在含有10％胎牛血清的RPMI1640培养基中稳定2h(37.5℃，5％CO 2)。 Staphylococcus aureus that expresses green fluorescent protein comes from the collection of the Institute of Microbiology, Chinese Academy of Sciences, and the multiplicity of infection is 1:10. Peripheral blood samples of healthy volunteers were collected to separate peripheral mononuclear cells (PBMC, separated by Biyuntian Lymphocyte Separation Kit). The newly isolated cells were stable in RPMI1640 medium containing 10% fetal bovine serum for 2h (37.5°C, 5% CO 2 ).
按实验需求培养金黄色葡萄球菌，在台式离心机中以10000g离心30秒收集细菌，并用重悬到10 8CFU/ml左右的细菌密度。取该细菌悬液作梯度稀释涂板计数以确定准确细菌密度。PBMC细胞更换培养基后加入10微升PBS重悬的金黄色葡萄球菌菌液(约10 6左右细菌)，轻轻振荡混匀，于37℃孵育2小时。吸取培养基上清，用1mL冰PBS清洗细胞三次，合并上清培养基和清洗液，梯度稀释涂板计数，计算未被吞噬的金黄色葡萄球菌数。 Cultivate Staphylococcus aureus according to the experimental requirements, collect the bacteria by centrifugation at 10000g for 30 seconds in a benchtop centrifuge, and resuspend them to a bacterial density of about 10 8 CFU/ml. Take the bacterial suspension to make a gradient dilution plate count to determine the accurate bacterial density. PBMC cells of Staphylococcus aureus bacteria was added (about 106 bacteria) was resuspended in 10 microliters of PBS after replacing the medium, mix gently shaken and incubated at 37 ℃ 2 hours. Aspirate the medium supernatant, wash the cells three times with 1 mL of ice PBS, combine the supernatant medium and the washing solution, and count the plates with gradient dilution, and calculate the number of Staphylococcus aureus that has not been swallowed.
加入新鲜培养基，添加10μg/ml红霉素培养12小时以彻底清除PBMC细胞外残余细菌，12小时更换无抗生素的新鲜培养基。分别取吞噬实验起始后12小时、24小时、36小时、小时和48小时的细胞，清洗后加入胰蛋白酶水溶液,用枪头彻底吹吸重悬细胞,该过程同时造成巨噬细胞的溶胀并破裂。将该裂解液取梯度稀释涂板计数,得到被吞噬后在巨噬细胞内存活的细菌数量，计算金葡菌的裂解率，结果如表3和表4所示。Add fresh medium, add 10μg/ml erythromycin and culture for 12 hours to completely remove residual bacteria outside the PBMC cells, and replace with fresh medium without antibiotics for 12 hours. Respectively take the cells 12 hours, 24 hours, 36 hours, hours and 48 hours after the start of the phagocytosis experiment. After washing, add trypsin aqueous solution, and thoroughly suck and resuspend the cells with a pipette tip. This process also causes the swelling of macrophages and rupture. The lysate was taken on a gradient dilution plate and counted to obtain the number of bacteria that survived in the macrophages after being swallowed, and the lysis rate of Staphylococcus aureus was calculated. The results are shown in Table 3 and Table 4.
表3：金葡菌吞噬率Table 3: Phagocytosis rate of Staphylococcus aureus
组别Group 吞噬率(％)Phagocytosis rate (%) SDSD p值p value
空白对照Blank control 44.9644.96 3.563.56  To
对照IgGControl IgG 43.6343.63 4.714.71  To
TLR1-FcTLR1-Fc 88.4288.42 8.578.57 p＜0.05p＜0.05
TLR2-FcTLR2-Fc 81.2481.24 9.139.13 p＜0.05p＜0.05
TLR4-FcTLR4-Fc 91.5791.57 12.7712.77 p＜0.05p＜0.05
TLR2/TLR4-FcTLR2/TLR4-Fc 84.8984.89 7.217.21 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 96.6096.60 10.3910.39 p＜0.05p＜0.05
TLR4/MD-2-FcTLR4/MD-2-Fc 88.3188.31 11.6011.60 p＜0.05p＜0.05
TLR4/CD36-FcTLR4/CD36-Fc 96.4996.49 5.915.91 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 76.1976.19 4.604.60 p＜0.05p＜0.05
表4：金葡菌裂解率Table 4: Lysis rate of Staphylococcus aureus
组别Group 吞噬率(％已吞噬细菌)Phagocytosis rate (% swallowed bacteria) SDSD p值p value
空白对照Blank control 22.4522.45 2.472.47  To
对照IgGControl IgG 24.6224.62 1.391.39  To
TLR1-FcTLR1-Fc 99.1599.15 5.725.72 p＜0.05p＜0.05
TLR2-FcTLR2-Fc 95.6195.61 5.725.72 p＜0.05p＜0.05
TLR4-FcTLR4-Fc 70.7370.73 9.639.63 p＜0.05p＜0.05
TLR2/TLR4-FcTLR2/TLR4-Fc 96.6096.60 10.3910.39 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 88.3188.31 11.6011.60 p＜0.05p＜0.05
TLR4/MD-2-FcTLR4/MD-2-Fc 75.4675.46 9.839.83 p＜0.05p＜0.05
TLR4/CD36-FcTLR4/CD36-Fc 81.8981.89 8.688.68 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 94.6594.65 8.668.66 p＜0.05p＜0.05
这些实验可以证实，二聚体免疫融合蛋白可以有效增强巨噬细胞吞噬、裂解能力，证实二聚体免疫融合蛋白可以作为对抗感染的产品。These experiments can confirm that the dimer immune fusion protein can effectively enhance the phagocytosis and lysis ability of macrophages, and confirm that the dimer immune fusion protein can be used as a product against infection.
实施例3免疫二聚体对耐甲氧西林金黄色葡萄球菌作用Example 3 The effect of immunodimer on methicillin-resistant Staphylococcus aureus
BALB/c小鼠，SPF级，雌性，6～8周龄，体重18～20g，国际标准株MRSA-252，购自美国组织培养库(American Tissue Culture Collection，ATCC)。建立小鼠模型，经尾静脉注射0.1mL洗涤后的菌液(菌液浓度为1×10 9CFU/mL)，空白组小鼠经尾静脉注射等量无菌生理盐水。然后将小鼠分为对照组和处理组，每组10只，对照组给与对照IgG，处理组给与本发明所述的二聚体免疫融合蛋白的代表物，剂量为10mg/kg，静脉注射一天一次，连续观察10d。如有小鼠死亡或最后一天实验结束处死全部老鼠，立即无菌条件下取血液铺板计数细菌，同时无菌取肾脏、脾脏、肝脏全器官，去部分组织用玻璃匀浆器磨碎后铺板计数细菌，同时进行病理检查。结果如表5～表9所示： BALB/c mice, SPF grade, female, 6-8 weeks old, weight 18-20g, international standard strain MRSA-252, purchased from American Tissue Culture Collection (ATCC). A mouse model was established, and 0.1 mL of the washed bacterial solution was injected through the tail vein (the concentration of the bacterial solution was 1×10 9 CFU/mL). The mice in the blank group were injected with the same amount of sterile normal saline through the tail vein. The mice were then divided into a control group and a treatment group, each with 10 mice. The control group was given control IgG, and the treatment group was given the representative of the dimer immune fusion protein of the present invention, at a dose of 10 mg/kg, intravenously The injection was once a day, and the observation was continued for 10 days. If the mouse died or all the mice were killed at the end of the experiment on the last day, immediately take the blood to plate and count the bacteria under aseptic conditions. At the same time, take the whole organs of kidney, spleen and liver aseptically, remove part of the tissues and grind them with a glass homogenizer and plate the plates for counting. Bacteria, while performing pathological examination. The results are shown in Table 5 to Table 9:
表5：小鼠生存率％Table 5: Survival rate of mice%
组别Group 0d0d 2d2d 4d4d 6d6d 8d8d 10d10d
正常小鼠Normal mice 100100 100100 100100 100100 100100 100100
模型组Model group 100100 2020 00 00 00 00
对照IgGControl IgG 100100 8080 7070 7070 7070 7070
TLR1-FcTLR1-Fc 100100 7070 7070 7070 7070 7070
TLR2-FcTLR2-Fc 100100 7070 7070 7070 7070 7070
TLR4-FcTLR4-Fc 100100 6060 6060 6060 5050 5050
TLR2/TLR4-FcTLR2/TLR4-Fc 100100 7070 7070 7070 7070 7070
TLR4/TLR6-FcTLR4/TLR6-Fc 100100 7070 7070 7070 7070 7070
TLR4/MD-2-FcTLR4/MD-2-Fc 100100 8080 8080 8080 8080 8080
TLR4/CD36-FcTLR4/CD36-Fc 100100 7070 7070 7070 7070 7070
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 100100 5050 5050 5050 5050 5050
表6：各组小鼠死亡前血液相对菌落数Table 6: The relative number of colonies in the blood of each group of mice before death
组别Group 相对菌落数％Relative number of colonies% SDSD p值p value
空白对照Blank control 00 00  To
模型组Model group 94.6594.65 8.668.66  To
对照IgGControl IgG 100100 7.887.88  To
TLR1-FcTLR1-Fc 14.9414.94 1.311.31 p＜0.05p＜0.05
TLR2-FcTLR2-Fc 21.0321.03 3.103.10 p＜0.05p＜0.05
TLR4-FcTLR4-Fc 13.9013.90 0.710.71 p＜0.05p＜0.05
TLR2/TLR4-FcTLR2/TLR4-Fc 13.4113.41 1.291.29 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 23.3123.31 1.341.34 p＜0.05p＜0.05
TLR4/MD-2-FcTLR4/MD-2-Fc 14.7514.75 1.931.93 p＜0.05p＜0.05
TLR4/CD36-FcTLR4/CD36-Fc 13.0313.03 1.171.17 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 23.1723.17 2.382.38 p＜0.05p＜0.05
表7：各组小鼠死亡前肝脏相对菌落数Table 7: The relative number of colonies in the liver of each group of mice before death
组别Group 相对菌落数％Relative number of colonies% SDSD p值p value
空白对照Blank control 00 00  To
模型组Model group 100.54100.54 10.5610.56  To
对照IgGControl IgG 100100 5.655.65  To
TLR1-FcTLR1-Fc 14.0014.00 1.511.51 p＜0.05p＜0.05
TLR2-FcTLR2-Fc 15.6115.61 1.971.97 p＜0.05p＜0.05
TLR4-FcTLR4-Fc 5.655.65 0.450.45 p＜0.05p＜0.05
TLR2/TLR4-FcTLR2/TLR4-Fc 8.928.92 1.311.31 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 1.351.35 0.180.18 p＜0.05p＜0.05
TLR4/MD-2-FcTLR4/MD-2-Fc 5.675.67 0.650.65 p＜0.05p＜0.05
TLR4/CD36-FcTLR4/CD36-Fc 0.920.92 0.060.06 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 15.5315.53 0.790.79 p＜0.05p＜0.05
表8：各组小鼠死亡前脾脏相对菌落数Table 8: The relative number of colonies in the spleen of each group of mice before death
组别Group 相对菌落数％Relative number of colonies% SDSD p值p value
空白对照Blank control 00 00  To
模型组Model group 107.97107.97 11.5711.57  To
对照IgGControl IgG 100100 6.976.97  To
TLR1-FcTLR1-Fc 5.515.51 0.370.37 p＜0.05p＜0.05
TLR2-FcTLR2-Fc 18.7018.70 1.531.53 p＜0.05p＜0.05
TLR4-FcTLR4-Fc 8.298.29 0.610.61 p＜0.05p＜0.05
TLR2/TLR4-FcTLR2/TLR4-Fc 2.402.40 0.190.19 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 11.3211.32 1.121.12 p＜0.05p＜0.05
TLR4/MD-2-FcTLR4/MD-2-Fc 1.801.80 0.250.25 p＜0.05p＜0.05
TLR4/CD36-FcTLR4/CD36-Fc 14.2714.27 1.231.23 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 17.3617.36 2.402.40 p＜0.05p＜0.05
表9：各组小鼠死亡前肾脏相对菌落数Table 9: The relative number of colonies in the kidneys of each group of mice before death
组别Group 相对菌落数％Relative number of colonies% SDSD p值p value
空白对照Blank control 00 00  To
模型组Model group 108.35108.35 12.4212.42  To
对照IgGControl IgG 100100 8.158.15  To
TLR1-FcTLR1-Fc 5.285.28 0.290.29 p＜0.05p＜0.05
TLR2-FcTLR2-Fc 10.1210.12 0.550.55 p＜0.05p＜0.05
TLR4-FcTLR4-Fc 14.2714.27 2.072.07 p＜0.05p＜0.05
TLR2/TLR4-FcTLR2/TLR4-Fc 2.222.22 0.010.01 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 19.1519.15 1.471.47 p＜0.05p＜0.05
TLR4/MD-2-FcTLR4/MD-2-Fc 16.1616.16 0.890.89 p＜0.05p＜0.05
TLR4/CD36-FcTLR4/CD36-Fc 8.008.00 0.480.48 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 15.9015.90 2.362.36 p＜0.05p＜0.05
这些结果表明，本发明所述的二聚体免疫融合蛋白具有较强的微生物杀灭作用、抗感染作用、减少脏器菌落数量，有效对抗耐甲氧西林金黄色葡萄球。These results show that the dimeric immune fusion protein of the present invention has strong microbial killing effect, anti-infection effect, reducing the number of organ colonies, and effectively resisting methicillin-resistant golden yellow staphylococcus aureus.
实施例4二聚体免疫融合蛋白治疗的动物系统性真菌感染实验Example 4 Animal Systemic Fungal Infection Experiments Treated by Dimer Immune Fusion Protein
选用雌性C57BL/6小鼠作为实验动物(20g左右)，经尾静脉注射给予5×10 6CFU/ml浓度的新生隐球菌0.1ml(5×10 5CFU/ml)，造成系统性真菌感染模型。然后进行分组， 每组10只小鼠，处理组分别经脉施用10mg/kg本发明所述二聚体免疫融合蛋白，每日一次，对照组给予对照IgG，给药共5天，在第5天将小鼠处死、取脑、将脑组织匀浆均匀，将匀浆液稀释一定倍数后加入到蛋白胨琼脂基涂板，将培养基上的菌落计数，计算小鼠脑部真菌荷菌量，结果如表10所示。 Female C57BL/6 mice (about 20g) were selected as experimental animals, and 0.1ml (5×10 5 CFU/ml) of Cryptococcus neoformans at a concentration of 5×10 6 CFU/ml was administered via tail vein injection, resulting in a systemic fungal infection model . Then they were divided into groups, each group of 10 mice, the treatment group was administered 10 mg/kg of the dimer immune fusion protein of the present invention via the vein, once a day, and the control group was given control IgG for a total of 5 days, on the 5th day The mice were sacrificed, the brains were taken, the brain tissues were homogenized, the homogenate was diluted to a certain multiple and added to the peptone agar-based coating plate, the colonies on the medium were counted, and the amount of fungi in the brain of the mice was calculated. The results are as follows Table 10 shows.
表10：脑组织来源菌落计数Table 10: Colony counts from brain tissues
组别Group 相对菌落(％)Relative colony (%) SDSD p值p value
正常小鼠Normal mice 00 00  To
模型组Model group 95.9595.95 4.824.82  To
对照IgGControl IgG 100100 6.686.68  To
TLR1-FcTLR1-Fc 17.2717.27 1.361.36 p＜0.05p＜0.05
TLR2-FcTLR2-Fc 27.8827.88 3.843.84 p＜0.05p＜0.05
TLR4-FcTLR4-Fc 13.4013.40 1.291.29 p＜0.05p＜0.05
TLR2/TLR4-FcTLR2/TLR4-Fc 28.4828.48 4.244.24 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 21.6921.69 2.802.80 p＜0.05p＜0.05
TLR4/MD-2-FcTLR4/MD-2-Fc 12.5512.55 0.690.69 p＜0.05p＜0.05
TLR4/CD36-FcTLR4/CD36-Fc 29.8229.82 3.813.81 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 27.3727.37 2.292.29 p＜0.05p＜0.05
通过表10可以看出，二聚体免疫融合蛋白系统性给药可以有效的抑制脑组织内的真菌生长，达到真菌杀灭效果疗效明显。It can be seen from Table 10 that the systemic administration of the dimer immune fusion protein can effectively inhibit the growth of fungi in the brain tissue, and achieve a significant curative effect in killing fungi.
实施例5二聚体免疫融合蛋白治疗对须癣毛癣菌感染豚鼠模型的治疗试验研究Example 5 Therapeutic study of dimer immune fusion protein treatment on guinea pig model of Trichophyton mentagrophytes infection
选取须癣毛癣菌(ATCC)作为致病菌，实验前恢复其致病力并接种于沙堡琼脂(SDA)斜面试管，26℃培养，7～10d后小心刮取菌落，用生理盐水制成感染真菌混悬液备用。SDA由4％葡萄糖、1％蛋白胨和2％琼脂组成。Trichophyton mentagrophytes (ATCC) was selected as the pathogenic bacteria. Before the experiment, the pathogenicity was restored and inoculated into sandcastle agar (SDA) oblique interview tubes, cultured at 26℃, 7-10 days later, the colonies were carefully scraped and prepared with physiological saline. Into a suspension of infected fungus for later use. SDA is composed of 4% glucose, 1% peptone and 2% agar.
健康白色豚鼠48只，雌雄兼用，体重250～350g。将所有白色豚鼠的腹背部的长毛剪短，然后用刮须刀脱去毛做出8cm×10cm去毛区，并在脱毛区涂适量甘油以防皮肤干裂，再用砂纸反复磨擦去毛区皮肤，损伤面约3cm×5cm，以皮肤有渗出液但又不大出血为准，将制备的须癣毛癣菌混悬液涂擦于损伤皮肤，感染真菌后，于第10天，观察白色豚鼠的感染区皮肤的病变程度，刮取皮疹、鳞屑或痂皮，镜检出真菌的菌丝或孢子。证明磨砂创伤感染法感染豚鼠动物模型成功。48 healthy white guinea pigs, both male and female, weighing 250-350g. Cut all the long hairs on the abdomen and back of the white guinea pigs short, then use a shaver to remove the hair to make an 8cm×10cm depilated area, and apply a proper amount of glycerin to the depilated area to prevent the skin from drying out, and then repeatedly rub the depilated area with sandpaper The skin, the damaged surface is about 3cm×5cm, and the prepared T. mentagrophytes suspension is rubbed on the damaged skin based on the exudation of the skin but no major bleeding. After infection with the fungus, observe the whiteness on the 10th day The extent of skin lesions in the infected area of guinea pigs, rashes, scales or crusts were scraped, and fungal hyphae or spores were detected under a microscope. Prove that the scrub wound infection method infects the guinea pig animal model successfully.
须癣毛癣菌感染豚鼠动物模型制成后，然后进行分组，每组10只，处理组分别经脉施用10mg/kg本发明所述二聚体免疫融合蛋白，每2日一次，对照组给予对照IgG，给药共5天。治愈为皮损消退，真菌镜检连续2次阴性及真菌培养阴性；无效为皮损未消退，真菌镜检阳性。记录各组动物的痊愈数并计算治愈率，停药后观察治愈动物的复发情况。After the animal model of Trichophyton mentagrophytes infection in guinea pigs was made, it was divided into groups of 10 each. The treatment group was administered 10 mg/kg of the dimer immune fusion protein of the present invention via the veins, once every 2 days, and the control group was given a control IgG, administered for 5 days. The cure is that the skin lesions have subsided, and the fungal microscopy is negative for 2 consecutive times and the fungal culture is negative; invalid is that the skin lesions have not subsided and the fungal microscopy is positive. Record the number of recovered animals in each group and calculate the cure rate, and observe the recurrence of the cured animals after stopping the drug.
各组分别给药克霉唑乳膏、茯茶提取物外用膜剂、对照外用膜剂和外用空白膜剂，连续治疗观察14d，其中检查7、l0、14d的治愈率如表11所示。由表11的试验统计结果可以看出，本发明所述二聚体免疫融合蛋白对治疗豚鼠须癣毛癣菌感染模型具有较好的疗效。Each group was given clotrimazole cream, Fucha extract topical membrane, control topical membrane, and topical blank membrane. Continuous treatment was observed for 14 days. The cure rates of 7, 10, and 14 days are shown in Table 11. It can be seen from the experimental statistical results in Table 11 that the dimeric immune fusion protein of the present invention has a good curative effect on the treatment of Trichophyton mentagrophytes infection model in guinea pigs.
表11：各组须癣毛癣菌感染豚鼠的疗效比较Table 11: Comparison of the curative effect of Trichophyton mentagrophytes in each group in guinea pigs
 To 7d治愈率(％)7d cure rate (%) 10d治愈率(％)10d cure rate (%) 14d治愈率(％)14d cure rate (%)
模型组Model group 00 00 00
对照IgGControl IgG 00 00 00
TLR1-FcTLR1-Fc 9090 100100 100100
TLR2-FcTLR2-Fc 100100 100100 100100
TLR4-FcTLR4-Fc 8080 100100 100100
TLR2/TLR4-FcTLR2/TLR4-Fc 100100 100100 100100
TLR4/TLR6-FcTLR4/TLR6-Fc 100100 100100 100100
TLR4/MD-2-FcTLR4/MD-2-Fc 100100 100100 100100
TLR4/CD36-FcTLR4/CD36-Fc 100100 100100 100100
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 100100 100100 100100
通过结果可以看出，二聚体免疫融合蛋白有效的抑制皮肤浅表真菌生长，达到真菌杀灭效果疗效明显。It can be seen from the results that the dimeric immune fusion protein effectively inhibits the growth of fungi on the superficial skin and achieves an obvious curative effect in killing fungi.
实施例6二聚体免疫融合蛋白对创伤弧菌的治疗作用Example 6 Therapeutic effect of dimer immune fusion protein on Vibrio vulnificus
1)结合实验1) Combined experiment
分别用灭活的创伤弧菌和鳗弧菌、迟缓爱德华菌、和溶藻弧菌(均来自中科院微生物研究所)进行本实验，利用ELISA方法检测本发明所述的二聚体免疫融合蛋白的代表物跟上述菌体的结合力，检测方法同文献(Goldberg M E,Djavadi-Ohaniance L.Current opinion in immunology,1993,5(2):278-281.)对照组采用对照IgG或TIGIT-Ig，检测结果如表12所示：This experiment was carried out with inactivated Vibrio vulnificus and Vibrio anguillarum, Edwards tarda, and Vibrio alginolyticus (all from the Institute of Microbiology, Chinese Academy of Sciences), and the ELISA method was used to detect the dimer immune fusion protein of the present invention. The binding ability of the representative substance and the above-mentioned bacteria, the detection method is the same as that of the literature (Goldberg M E, Djavadi-Ohaniance L. Current opinion in immunology, 1993, 5(2): 278-281.) The control group uses control IgG or TIGIT-Ig , The test results are shown in Table 12:
表12：菌体结合力(nM)Table 12: Bacterial binding capacity (nM)
组别Group 创伤弧菌Vibrio vulnificus 鳗弧菌Vibrio anguillarum 迟缓爱德华菌Edwardsiella tarda 溶藻弧菌Vibrio alginolyticus
空白对照Blank control -- -- -- --
对照IgGControl IgG -- -- -- --
TIGIT-IgTIGIT-Ig -- -- -- --
TLR1-FcTLR1-Fc 71.36±5.5071.36±5.50 15.59±2.2115.59±2.21 93.47±9.7393.47±9.73 50.30±6.4050.30±6.40
TLR2-FcTLR2-Fc 51.97±2.8651.97±2.86 82.08±9.9282.08±9.92 48.07±5.0448.07±5.04 43.54±3.0943.54±3.09
TLR4-FcTLR4-Fc 22.29±2.7722.29±2.77 40.84±2.7840.84±2.78 39.55±2.8739.55±2.87 28.79±3.7628.79±3.76
TLR2/TLR4-FcTLR2/TLR4-Fc 6.91±0.646.91±0.64 13.64±1.9713.64±1.97 74.14±10.9774.14±10.97 73.64±4.9473.64±4.94
TLR4/TLR6-FcTLR4/TLR6-Fc 77.54±6.9677.54±6.96 87.64±9.0287.64±9.02 74.54±5.3474.54±5.34 80.88±9.7080.88±9.70
TLR4/MD-2-FcTLR4/MD-2-Fc 26.20±3.9126.20±3.91 88.66±6.4888.66±6.48 92.03±7.1992.03±7.19 45.15±3.6245.15±3.62
TLR4/CD36-FcTLR4/CD36-Fc 7.02±0.817.02±0.81 48.61±2.9048.61±2.90 64.55±8.6464.55±8.64 72.01±3.7672.01±3.76
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 85.51±7.1485.51±7.14 52.39±6.6552.39±6.65 60.53±7.9260.53±7.92 30.66±2.2430.66±2.24
2)乳鼠保护实验2) Suckling mouse protection experiment
首先建立动物模型，用腹腔注射法人工感染乳鼠，以3×10 9菌量腹腔注射乳鼠，感染12h后分组，每组n＝10。对照组给与IgG，各处理组给与本发明所述二聚体免疫融合蛋白，10mg/kg,每2d，一次，腹腔注射。观察和记录乳鼠发病和死亡情况共3周，计算小鼠存活率，结果如表13所示： First, establish an animal model, and artificially infect the suckling mice by intraperitoneal injection. The suckling mice were injected intraperitoneally with 3×10 9 bacteria, and they were divided into groups after infection for 12 hours, n=10 in each group. The control group was given IgG, and each treatment group was given the dimer immune fusion protein of the present invention, 10 mg/kg, once every 2 days, intraperitoneally injected. Observe and record the incidence and death of suckling mice for 3 weeks, and calculate the survival rate of mice. The results are shown in Table 13:
表13：创伤弧菌存活率Table 13: Survival rate of Vibrio vulnificus
组别Group 0周存活率％0 week survival rate% 1周存活率％1 week survival rate% 2周存活率2-week survival rate 3周存活率3-week survival rate p值p value
空白对照Blank control 100100 00 00 00 --
对照IgGControl IgG 100100 00 00 00 --
TLR1-FcTLR1-Fc 100100 6060 6060 6060  To
TLR2-FcTLR2-Fc 100100 6060 6060 6060 p＜0.05p＜0.05
TLR4-FcTLR4-Fc 100100 6060 6060 6060 p＜0.05p＜0.05
TLR2/TLR4-FcTLR2/TLR4-Fc 100100 7070 7070 7070 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 100100 7070 7070 7070 p＜0.05p＜0.05
TLR4/MD-2-FcTLR4/MD-2-Fc 100100 7070 7070 7070 p＜0.05p＜0.05
TLR4/CD36-FcTLR4/CD36-Fc 100100 7070 7070 7070 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 100100 7070 7070 7070 p＜0.05p＜0.05
通过结果可以看出，二聚体免疫融合蛋白有效的抑制创伤弧菌为代表的烈性菌微生物生长，达到微生物杀灭效果疗效明显。It can be seen from the results that the dimer immune fusion protein effectively inhibits the growth of virulent microorganisms represented by Vibrio vulnificus, and achieves a significant effect of killing microorganisms.
实施例7二聚体免疫融合蛋白对抗登革病毒感染Example 7 Dimer immune fusion protein against dengue virus infection
登革1型病毒128株(Gen Bank FJ176780)、登革4型病毒43株(GeneBank AF119661)、登革3型病毒80-2株(Gen Bank AF317645)、登革4型病毒B5株(Gen Bank AF289029)，C6/36细胞和BHK21细胞：均为军事医学科学院微生物流行病研究所病毒室提供；登革病毒培养采用C6/36细胞。待细胞长至单层，弃去培养液，加入不同病毒悬液，于37℃培养，观察细胞病变。待细胞病变达到+++时，冻融病毒培养液，于2,000rpm离心5min，收集上清即为病毒原液。分装后于-80℃保存。为了测定不同病毒的滴度，首先用含2％FCS的细胞维持液将病毒原液10倍比稀释，然后将不同稀释度的病毒液加入细胞单层，37℃孵育1h。弃上清，加入含1％低熔点琼脂糖的DMEM细胞维持液，继续培养5天。显微镜下观察，细胞出现病变后，4％甲醛溶液固定细胞30min，弃上层琼 盖，用去离子水清洗后，加入1％结晶紫染色30min，去离子水清洗后计数蚀斑数，病毒滴度用蚀斑形成单位(plaque form unit，PFU/ml)表示。Dengue 1 virus 128 strains (Gen Bank FJ176780), Dengue 4 virus 43 strains (GeneBank AF119661), Dengue 3 virus 80-2 strains (Gen Bank AF317645), Dengue 4 virus B5 strains (Gen Bank AF289029), C6/36 cells and BHK21 cells: Both are provided by the Virus Room of the Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences; C6/36 cells are used for dengue virus culture. When the cells grow to a single layer, discard the culture medium, add different virus suspensions, and culture at 37°C to observe the cytopathic changes. When the cytopathic effect reaches +++, freeze-thaw the virus culture solution, centrifuge at 2,000 rpm for 5 min, and collect the supernatant to obtain the virus stock solution. Store at -80°C after aliquoting. In order to determine the titer of different viruses, the virus stock solution was diluted 10-fold with a cell maintenance solution containing 2% FCS, and then the virus solution of different dilutions was added to the cell monolayer and incubated at 37°C for 1 hour. The supernatant was discarded, DMEM cell maintenance solution containing 1% low melting point agarose was added, and the culture was continued for 5 days. Observed under the microscope, after the cells showed lesions, fix the cells with 4% formaldehyde solution for 30 minutes, discard the upper agar, wash with deionized water, add 1% crystal violet to stain for 30 minutes, and count the number of plaques and virus titer after washing with deionized water It is expressed in plaque form unit (PFU/ml).
1)体外实验1) In vitro experiment
采用固定病毒稀释抗体的方法进行蚀斑减少中和试验：将不同浓度单抗与含The plaque reduction and neutralization test is carried out by using the method of diluting antibodies to fix the virus.
100PFU的登革4型病毒悬液等量混合，37℃水浴作用1h。将混合液加入培养于6孔板的BHK21细胞，37℃孵育1h，弃去混合液，用PBS缓冲液洗细胞。加入营养琼盖，继续培养5d后固定染色，计数蚀斑数。处理组各药物含量为50μg/ml，对照组给与空白IgG并计算各给药组的中和率，中和率为(1－处理组/空白对照)×100％，结果如表14所示。100PFU of dengue type 4 virus suspension was mixed in equal amounts and treated in a water bath at 37°C for 1 hour. Add the mixed solution to BHK21 cells cultured in a 6-well plate, incubate at 37°C for 1 hour, discard the mixed solution, and wash the cells with PBS buffer. Add nutrient agar cover, continue to culture for 5 days, fix staining, and count the number of plaques. The content of each drug in the treatment group was 50μg/ml, and the control group was given blank IgG and the neutralization rate of each administration group was calculated. The neutralization rate was (1-treatment group/blank control)×100%. The results are shown in Table 14. .
表14：登革病毒中和率Table 14: Dengue virus neutralization rate
组别Group 中和率％Neutralization rate% SDSD p值p value
空白对照Blank control 00 4.674.67 --
对照IgGControl IgG 3.823.82 0.570.57 --
TLR1-FcTLR1-Fc 91.6591.65 9.689.68  To
TLR2-FcTLR2-Fc 87.9087.90 11.5811.58 p＜0.05p＜0.05
TLR4-FcTLR4-Fc 88.2188.21 8.768.76 p＜0.05p＜0.05
TLR2/TLR4-FcTLR2/TLR4-Fc 64.8264.82 4.964.96 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 98.1698.16 14.2414.24 p＜0.05p＜0.05
TLR4/MD-2-FcTLR4/MD-2-Fc 91.0991.09 11.4511.45 p＜0.05p＜0.05
TLR4/CD36-FcTLR4/CD36-Fc 84.5284.52 12.6012.60 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 93.8693.86 10.4310.43 p＜0.05p＜0.05
2)体内实验2) In vivo experiment
将与10 5PFU/ml的各型登革病毒等量混匀，37℃孵育1h。将病毒合液颅内接种1日龄昆明种乳鼠，每只约30μL，每组n＝10。感染24h后分组，对照组给与IgG，各处理组给与本发明所述二聚体免疫融合蛋白，10mg/kg，每天一次，腹腔注射。观察和记录乳鼠发病和死亡情况共3周，计算小鼠存活率，如表15所示。 Mix the same amount with each type of dengue virus at 10 5 PFU/ml, and incubate at 37°C for 1 hour. 1 day-old Kunming suckling mice were intracranially inoculated with the virus mixture, each with about 30 μL, n=10 in each group. After 24 hours of infection, they were divided into groups, the control group was given IgG, and each treatment group was given the dimer immune fusion protein of the present invention, 10 mg/kg, once a day, intraperitoneal injection. Observe and record the incidence and death of suckling mice for 3 weeks, and calculate the survival rate of mice, as shown in Table 15.
表15：登革病毒存活率Table 15: Dengue virus survival rate
组别Group 0周存活率％0 week survival rate% 1周存活率％1 week survival rate% 2周存活率2-week survival rate 3周存活率3-week survival rate p值p value
空白对照Blank control 100100 100100 100100 100100 --
对照IgGControl IgG 100100 1010 00 00 --
TLR1-FcTLR1-Fc 100100 8080 8080 8080  To
TLR2-FcTLR2-Fc 100100 8080 7070 7070 p＜0.05p＜0.05
TLR4-FcTLR4-Fc 100100 7070 7070 7070 p＜0.05p＜0.05
TLR2/TLR4-FcTLR2/TLR4-Fc 100100 6060 6060 5050 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 100100 5050 5050 5050 p＜0.05p＜0.05
TLR4/MD-2-FcTLR4/MD-2-Fc 100100 5050 5050 5050 p＜0.05p＜0.05
TLR4/CD36-FcTLR4/CD36-Fc 100100 9090 9090 7070 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 100100 8080 8080 8080 p＜0.05p＜0.05
通过结果可以看出，二聚体免疫融合蛋白有效的抑制以登革病毒为代表的烈性微生物生长，达到微生物杀灭效果疗效明显。From the results, it can be seen that the dimeric immune fusion protein effectively inhibits the growth of potent microorganisms represented by dengue virus, and achieves a significant effect of killing microorganisms.
实施例8二聚体免疫融合蛋白对血吸虫为代表的的寄生虫杀伤作用研究Example 8 Study on the killing effect of dimer immune fusion protein on parasites represented by Schistosoma
新西兰大白兔(雄性,2.5-3.0Kg)购于上海斯莱克实验动物有限责任公司；5周龄的BALB/c小鼠(雄性)购自上海杰思捷实验动物有限公司；新西兰大白兔通过腹部皮肤贴片的方法感染1000±5条日本血吸虫尾蚴，于感染后第14d进行剖杀，以主动脉灌注法从肝门静脉收集相应感染时间的日木血吸虫虫体，然后用RPMI1640培养基充分洗涤虫体。New Zealand white rabbits (male, 2.5-3.0Kg) were purchased from Shanghai Slack Laboratory Animal Co., Ltd.; 5-week-old BALB/c mice (male) were purchased from Shanghai Jiesjie Laboratory Animal Co., Ltd.; New Zealand white rabbits passed through the abdomen Infect 1000±5 cercariae of Schistosoma japonicum by skin patch, and they were dissected on the 14th day after infection. The worms of Schistosoma japonicum were collected from the hepatic portal vein by aortic perfusion method, and the worms were washed thoroughly with RPMI1640 medium. body.
1)体外研究1) In vitro research
将10 5个PBMC每孔培养于含有10％胎牛血淸的RPMI1640培养基中，处理组给与本发明所述的二聚体免疫融合蛋白的代表物，对照组给与对照IgG，空白组不给与任何药物。给药浓度为1mg/ml，每孔分别加入10条(14d)活力较好的日本血吸虫，然后放入37℃，5％CO 2培养箱中培养(每24h更换一次培养基)，每组均设置三个复孔。利用倒置显微镜分别在培养的第24h、48h、72h和96h观察不同组别的日本血吸虫的活动性和形态学变化，并计算相应培养时间时日本血吸虫的存活率。日本血吸虫的死亡定义为:连续观察2min虫体静止不动则视为虫体死亡，结果如表16所示。 10 5 PBMCs per well were cultured in RPMI1640 medium containing 10% fetal calf blood. The treatment group was given the representative of the dimer immune fusion protein of the present invention, the control group was given control IgG, and the control group was given control IgG. No drugs are given. The administration concentration is 1mg/ml, and 10 (14d) schistosome japonicums with good vigor are added to each well, and then they are cultured in a 37°C, 5% CO 2 incubator (change the medium every 24h). Set three multiple holes. The inverted microscope was used to observe the activity and morphological changes of different groups of Schistosoma japonicum at 24h, 48h, 72h and 96h of culture, and calculate the survival rate of Schistosoma japonicum at the corresponding culture time. The death of Schistosoma japonicum is defined as: continuous observation of the body for 2 minutes is regarded as the death of the body. The results are shown in Table 16.
表16：PBMC对14d血吸虫存活率影响Table 16: Effect of PBMC on the survival rate of schistosome at 14 days
Figure PCTCN2020131581-appb-000005
Figure PCTCN2020131581-appb-000005
2)体内研究2) In vivo research
将5周龄雄性的BALB/c小鼠随机分组，分别为健康对照组(空白组)、感染对照组 (模型组)、对照IgG组合二聚体免疫融合蛋白处理各组。除空白组外，通过腹部皮肤贴片的方法每只小鼠感染40±2条日本血吸虫尾蚴，在感染的第14d进行给药，处理各组给与本发明所述的二聚体免疫融合蛋白的代表，对照组给与对照IgG，给药剂量为10mg/kg静脉给药，每3天给药一次。于感染的第42d进行剖杀，收集虫体，并对每组的小鼠体内的日本血吸虫进行计数，计算减虫率。减虫率的计算方法如下：减虫率＝(1-实验组平均虫体数/对照组平均虫体数)×100％，结果如表17所示：Five-week-old male BALB/c mice were randomly divided into healthy control group (blank group), infection control group (model group), and control IgG combined dimer immune fusion protein treatment groups. Except for the blank group, each mouse was infected with 40±2 cercariae of Schistosoma japonicum by the method of abdominal skin patch. The administration was administered on the 14th day of infection, and each group was treated with the dimer immune fusion protein of the present invention. The representative of the control group was given control IgG at a dose of 10 mg/kg intravenously, once every 3 days. On the 42nd day of infection, they were dissected and the parasites were collected, and the Schistosoma japonicum in the mice of each group was counted to calculate the reduction rate. The calculation method of the reduction rate is as follows: reduction rate=(1-average number of worms in the experimental group/average number of worms in the control group)×100%, the results are shown in Table 17:
表17：血吸虫减虫率Table 17: Reduction rate of schistosomiasis
组别Group 减虫率％Insect reduction rate% SDSD p值p value
空白对照(健康小鼠)Blank control (healthy mice) -- -- --
模型组Model group -- --  To
对照IgGControl IgG 00 1.251.25 --
TLR1-FcTLR1-Fc 99.4799.47 9.409.40  To
TLR2-FcTLR2-Fc 78.0878.08 8.928.92 p＜0.05p＜0.05
TLR4-FcTLR4-Fc 91.0291.02 10.0110.01 p＜0.05p＜0.05
TLR2/TLR4-FcTLR2/TLR4-Fc 81.8781.87 11.4111.41 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 78.5578.55 7.017.01 p＜0.05p＜0.05
TLR4/MD-2-FcTLR4/MD-2-Fc 97.6897.68 8.068.06 p＜0.05p＜0.05
TLR4/CD36-FcTLR4/CD36-Fc 90.5090.50 6.236.23 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 92.7092.70 7.827.82 p＜0.05p＜0.05
通过结果可以看出，二聚体免疫融合蛋白有效的抑制以血吸虫为代表的寄生虫类病原微生物生长，达到微生物杀灭效果疗效明显。From the results, it can be seen that the dimer immune fusion protein effectively inhibits the growth of parasitic pathogenic microorganisms represented by schistosomiasis, and achieves an obvious effect of killing microorganisms.
实施例9向暴露于未知病原体的患者施用二聚体融合蛋白举例Example 9 Examples of administration of dimer fusion proteins to patients exposed to unknown pathogens
在突发公共安全或生物恐怖主义袭击中，人们暴露于未知的病原体或毒素。暴露模式为很多不同方式中的一种，例如食物或水摄取、气雾剂吸入或皮肤接触。病原体为众多中的一种，例如炭疽杆菌(炭疽热)、流感病毒、天花病毒、鼠疫耶尔森菌(鼠疫)、埃博拉病毒或马尔堡病毒、土拉弗朗西斯菌(野兔病)、汉坦病毒、登革病毒、霍乱毒素、肉毒杆菌毒素、蓖麻毒素、沙门氏菌、大肠杆菌如E.coli 0157:H7、志贺氏杆菌、李斯特菌等。In sudden public safety or bioterrorism attacks, people are exposed to unknown pathogens or toxins. The exposure mode is one of many different ways, such as food or water intake, aerosol inhalation, or skin contact. The pathogen is one of many, such as Bacillus anthracis (anthracnose), influenza virus, smallpox virus, Yersinia pestis (plague), Ebola virus or Marburg virus, Tula Francis (hare disease), Han Tan virus, dengue virus, cholera toxin, botulinum toxin, ricin, salmonella, Escherichia coli such as E.coli 0157:H7, Shigella, Listeria, etc.
当威胁性的微生物尚未确定时，一些病人已经迅速开始患有相似症状的严重疾病，包括高烧、寒颤、咳嗽、严重疲劳和腹泻。患者可接受标准治疗，例如抗病毒药、抗生素、抗毒素、免疫球蛋白。When the threatening microorganisms have not yet been identified, some patients have quickly begun to suffer from serious illnesses with similar symptoms, including high fever, chills, cough, severe fatigue, and diarrhea. Patients can receive standard treatments such as antiviral drugs, antibiotics, antitoxins, and immunoglobulins.
然后可以利用本发明所述的免疫二聚体快速中和炎症介质，如作为发生感染症状和炎症体征(发烧、寒颤等)的患者的预防措施或治疗手段，向患者静脉施用本发明所述二聚体二聚体免疫融合蛋白，例如包含活性成分为10mg/kg TLR2-Fc的药物组合物、包含活 性成分为10mg/kg TLR2/TLR4-Fc的药物组合物、包含活性成分为10mg/kg TLR4/TLR6-Fc的药物组合物等实施例1中的二聚体免疫融合蛋白活性成分的药物组合物。一旦药物分布到体液(尤其是消化道血液)中，二聚体免疫融合并且隔绝外源引入或局部产生或通过生理性流体如胆汁引入消化道造成的血液中的炎性介质，这发生在这些炎性介质可能造成进一步的炎症或毒性，或造成可导致恶化的炎症感染、内毒素血症和脓毒症之前。通过去除这些炎性介质，二聚体免疫融合蛋白减少患者体内额外全身性炎症的引发物，减少全身性炎性介质(如细胞因子)的产生，从而防止或限制细胞因子或其他炎性介质诱导的细胞死亡、器官损伤、多器官衰竭和潜在死亡的发生。The immune dimer of the present invention can then be used to quickly neutralize inflammatory mediators, for example, as a preventive measure or treatment method for patients who have symptoms of infection and signs of inflammation (fever, chills, etc.), intravenously administer the two of the present invention to the patient. A polymer-dimer immune fusion protein, such as a pharmaceutical composition containing an active ingredient of 10 mg/kg TLR2-Fc, a pharmaceutical composition containing an active ingredient of 10 mg/kg TLR2/TLR4-Fc, and an active ingredient of 10 mg/kg TLR4 /TLR6-Fc pharmaceutical composition and other pharmaceutical compositions of the active ingredient of the dimer immune fusion protein in Example 1. Once the drug is distributed into body fluids (especially the blood of the digestive tract), the dimers are immune to fusion and isolate the inflammatory mediators in the blood introduced by exogenous or locally produced or introduced through physiological fluids such as bile into the digestive tract. This occurs in these Inflammatory mediators may cause further inflammation or toxicity, or cause worsening inflammation before infection, endotoxemia, and sepsis. By removing these inflammatory mediators, the dimeric immune fusion protein reduces the triggers of additional systemic inflammation in the patient's body and reduces the production of systemic inflammatory mediators (such as cytokines), thereby preventing or limiting the induction of cytokines or other inflammatory mediators The occurrence of cell death, organ damage, multiple organ failure and potential death.
实施例10二聚体免疫融合蛋白对巨噬细胞抗炎活性Example 10 Anti-inflammatory activity of dimeric immune fusion protein on macrophages
Raw 264.7巨噬细胞(中科院细胞库)以含有10％的胎牛血清(FBS；Gibco Laboratories)的DMEM培养基培养，培养条件为在37℃和5％CO2。以1×10 6个细胞/mL的密度将Raw 264.7细胞接种至96孔板中并贴壁培养过夜。次日，用新鲜的DMEM培养基替换上述培养基，并将实施例1所述的5μg/mL的多种二聚体免疫融合蛋白加至细胞中，对照组加入对照人IgG(Sigma)。将细胞与蛋白孵育30分钟后，培养基添加LPS(终浓度1μg/mL)，并将细胞再温育24小时后进行检测实验。 Raw 264.7 macrophages (Cell Bank of Chinese Academy of Sciences) were cultured in DMEM medium containing 10% fetal bovine serum (FBS; Gibco Laboratories) under the conditions of 37°C and 5% CO2. Raw 264.7 cells were seeded into 96-well plates at a density of 1×10 6 cells/mL and cultured overnight. The next day, the above medium was replaced with fresh DMEM medium, and the 5 μg/mL dimer immune fusion proteins described in Example 1 were added to the cells, and the control group was added with control human IgG (Sigma). After incubating the cells with the protein for 30 minutes, the medium was added with LPS (final concentration 1 μg/mL), and the cells were incubated for another 24 hours before the detection experiment was performed.
1)NO水平测试1) NO level test
使用Griess试剂系统(Promega，USA)测量上述Raw 264.7细胞培养基中的中的一氧化氮(NO)水平。将50μL培养基加入96孔板，接着加入相同量的Griess试剂I(NED)溶液和Griess试剂II(对氨基苯磺酰胺溶液)，孵育10分钟，之后，使用微孔板读取仪(Molecular Devices，USA)在30分钟内测量540nm下的光密度。使用亚硝酸钠标准曲线(0～100μM)来计算NO的浓度。The Griess reagent system (Promega, USA) was used to measure the level of nitric oxide (NO) in the raw 264.7 cell culture medium. Add 50μL of medium to a 96-well plate, then add the same amount of Griess reagent I (NED) solution and Griess reagent II (para-aminobenzene sulfonamide solution), incubate for 10 minutes, then use a microplate reader (Molecular Devices) , USA) Measure the optical density at 540nm within 30 minutes. Use the sodium nitrite standard curve (0-100μM) to calculate the concentration of NO.
如下表18所示，以LPS刺激细胞增加了NO的表达，但在以LPS与本发明所述的二聚体免疫融合蛋白共同处理时，上述NO表达水平降低了。支持了二聚体免疫融合蛋白的减少巨噬细胞自身炎性渗出的效果。As shown in Table 18 below, stimulating cells with LPS increased the expression of NO, but when treated with LPS and the dimeric immune fusion protein of the present invention, the expression level of NO was reduced. Support the dimer immune fusion protein to reduce the effect of macrophage self-inflammatory exudation.
表18相对NO含量Table 18 Relative NO content
组别Group 相对NO表达％Relative NO expression% SDSD p值(vs.对照IgG+LPS)p value (vs. control IgG+LPS)
空白对照(仅培养基)Blank control (medium only) 97.6697.66 4.884.88  To
对照IgGControl IgG 100100 6.356.35  To
对照IgG+LPSControl IgG+LPS 612.27612.27 60.5660.56  To
TLR1-Fc+LPSTLR1-Fc+LPS 163.04163.04 23.2123.21 p＜0.05p＜0.05
TLR2-Fc+LPSTLR2-Fc+LPS 235.52235.52 31.3031.30 p＜0.05p＜0.05
TLR4-Fc+LPSTLR4-Fc+LPS 153.073153.073 17.4517.45 p＜0.05p＜0.05
TLR6-Fc+LPSTLR6-Fc+LPS 126.13126.13 7.537.53 p＜0.05p＜0.05
TLR4-Fc-LALAPG+LPSTLR4-Fc-LALAPG+LPS 131.534131.534 18.2818.28 p＜0.05p＜0.05
TLR1/TLR2-Fc+LPSTLR1/TLR2-Fc+LPS 196.87196.87 12.3812.38 p＜0.05p＜0.05
TLR2/TLR4-Fc+LPSTLR2/TLR4-Fc+LPS 141.86141.86 12.2512.25 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 153.07153.07 17.4517.45 p＜0.05p＜0.05
TLR4/MD-2-Fc+LPSTLR4/MD-2-Fc+LPS 137.16137.16 19.9919.99 p＜0.05p＜0.05
TLR4/CD36-Fc+LPSTLR4/CD36-Fc+LPS 91.4891.48 12.4512.45 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 150.97150.97 16.0816.08 p＜0.05p＜0.05
2)细胞因子检测2) Cytokine detection
收集含有细胞培养基的上清液样品，并使用HMGB1、TNFα、IFN-γ和IL-6ELISA试剂盒(eBioscience，San Diego)分析细胞因子的水平。在4℃下用100μL捕获抗体(在涂布缓冲液中稀释至制造商的操作规程所建议的浓度)涂布96孔板过夜。接着，在洗涤该板5次之后，每孔中加入200μL测定稀释液，并于室温下温育1小时以进行封闭。在用洗涤缓冲液洗涤各孔5次之后，将细胞培养物样品或每个细胞因子标准蛋白样品稀释，并在每孔中加入100μL各样品。在4℃下过夜温育含有样品的板。接着，在用洗涤缓冲液洗涤该板5次之后，加入100μL与抗生物素蛋白偶联的二抗，并在室温下温育1小时。在与二抗温育之后，洗涤该板5次，并在室温下与100μL抗生物素蛋白-HRP(BDBioscience)温育30分钟。在洗涤该板7次之后，加入100μL TMB溶液(Pierce)并在室温下温育15分钟。在各孔中加入50μl硫酸来终止反应。使用微孔板读取仪测量450nm下的光密度。使用SPSS程序的ANOVA操作进行方差分析，从而进行统计学分析，并使用邓肯氏多变域检验法来验证分析之间的显著性。检测结果如表19～22所示：The supernatant sample containing the cell culture medium was collected, and the level of cytokine was analyzed using HMGB1, TNFα, IFN-γ and IL-6 ELISA kit (eBioscience, San Diego). Coat a 96-well plate with 100 μL of capture antibody (diluted in the coating buffer to the concentration recommended by the manufacturer's operating procedures) at 4°C overnight. Then, after washing the plate 5 times, 200 μL of the assay diluent was added to each well and incubated at room temperature for 1 hour for blocking. After washing each well with washing buffer 5 times, the cell culture sample or each cytokine standard protein sample was diluted, and 100 μL of each sample was added to each well. The plate containing the sample was incubated overnight at 4°C. Next, after washing the plate 5 times with a washing buffer, 100 μL of avidin-conjugated secondary antibody was added and incubated at room temperature for 1 hour. After incubation with the secondary antibody, the plate was washed 5 times and incubated with 100 μL of avidin-HRP (BD Bioscience) for 30 minutes at room temperature. After washing the plate 7 times, 100 μL of TMB solution (Pierce) was added and incubated at room temperature for 15 minutes. Add 50 μl of sulfuric acid to each well to stop the reaction. A microplate reader was used to measure the optical density at 450 nm. The SPSS program's ANOVA operation was used to perform analysis of variance to perform statistical analysis, and Duncan's multivariate domain test was used to verify the significance between the analyses. The test results are shown in Tables 19-22:
表19各处理组HMGB1水平Table 19 HMGB1 level of each treatment group
组别Group HMGB1(pg/ml)HMGB1(pg/ml) SDSD p值(vs.对照IgG+LPS)p value (vs. control IgG+LPS)
空白对照(仅培养基)Blank control (medium only) 26.6326.63 3.213.21  To
对照IgGControl IgG 30.4630.46 2.322.32  To
对照IgG+LPSControl IgG+LPS 575.15575.15 32.1832.18  To
TLR1-Fc+LPSTLR1-Fc+LPS 144.23144.23 11.1311.13 p＜0.05p＜0.05
TLR2-Fc+LPSTLR2-Fc+LPS 154.83154.83 19.0119.01 p＜0.05p＜0.05
TLR4-Fc+LPSTLR4-Fc+LPS 155.32155.32 20.3420.34 p＜0.05p＜0.05
TLR6-Fc+LPSTLR6-Fc+LPS 295.83295.83 39.2439.24 p＜0.05p＜0.05
TLR4-Fc-LALAPG+LPSTLR4-Fc-LALAPG+LPS 147.27147.27 15.1715.17 p＜0.05p＜0.05
TLR1/TLR2-Fc+LPSTLR1/TLR2-Fc+LPS 61.9661.96 5.715.71 p＜0.05p＜0.05
TLR2/TLR4-Fc+LPSTLR2/TLR4-Fc+LPS 85.8285.82 8.828.82 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 187.14187.14 11.9711.97 p＜0.05p＜0.05
TLR4/MD-2-Fc+LPSTLR4/MD-2-Fc+LPS 104.50104.50 14.4014.40 p＜0.05p＜0.05
TLR4/CD36-Fc+LPSTLR4/CD36-Fc+LPS 262.44262.44 27.6927.69 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 132.86132.86 10.3010.30 p＜0.05p＜0.05
表20各处理组TNFα水平Table 20 TNFα levels in each treatment group
组别Group TNFα(pg/ml)TNFα(pg/ml) SDSD p值(vs.对照IgG+LPS)p value (vs. control IgG+LPS)
空白对照(仅培养基)Blank control (medium only) 38.8138.81 3.043.04  To
对照IgGControl IgG 26.9726.97 1.551.55  To
对照IgG+LPSControl IgG+LPS 846.19846.19 109.55109.55  To
TLR1-Fc+LPSTLR1-Fc+LPS 222.31222.31 29.6329.63 p＜0.05p＜0.05
TLR2-Fc+LPSTLR2-Fc+LPS 283.73283.73 32.1032.10 p＜0.05p＜0.05
TLR4-Fc+LPSTLR4-Fc+LPS 109.83109.83 12.8412.84 p＜0.05p＜0.05
TLR6-Fc+LPSTLR6-Fc+LPS 280.92280.92 41.0741.07 p＜0.05p＜0.05
TLR4-Fc-LALAPG+LPSTLR4-Fc-LALAPG+LPS 231.82231.82 25.7325.73 p＜0.05p＜0.05
TLR1/TLR2-Fc+LPSTLR1/TLR2-Fc+LPS 156.57156.57 13.7513.75 p＜0.05p＜0.05
TLR2/TLR4-Fc+LPSTLR2/TLR4-Fc+LPS 96.9896.98 10.4010.40 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 212.85212.85 10.7910.79 p＜0.05p＜0.05
TLR4/MD-2-Fc+LPSTLR4/MD-2-Fc+LPS 77.5477.54 5.555.55 p＜0.05p＜0.05
TLR4/CD36-Fc+LPSTLR4/CD36-Fc+LPS 54.1354.13 5.915.91 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 176.33176.33 20.5320.53 p＜0.05p＜0.05
表21各处理组IFN-γ水平Table 21 IFN-γ levels in each treatment group
组别Group IFN-γ(pg/ml)IFN-γ(pg/ml) SDSD p值(vs.对照IgG+LPS)p value (vs. control IgG+LPS)
空白对照(仅培养基)Blank control (medium only) 32.1932.19 3.383.38  To
对照IgGControl IgG 41.4741.47 4.764.76  To
对照IgG+LPSControl IgG+LPS 468.55468.55 26.0726.07  To
TLR1-Fc+LPSTLR1-Fc+LPS 174.12174.12 25.9025.90 p＜0.05p＜0.05
TLR2-Fc+LPSTLR2-Fc+LPS 70.7470.74 9.159.15 p＜0.05p＜0.05
TLR4-Fc+LPSTLR4-Fc+LPS 237.00237.00 28.5828.58 p＜0.05p＜0.05
TLR6-Fc+LPSTLR6-Fc+LPS 174.12174.12 25.9025.90 p＜0.05p＜0.05
TLR4-Fc-LALAPG+LPSTLR4-Fc-LALAPG+LPS 136.35136.35 19.1619.16 p＜0.05p＜0.05
TLR1/TLR2-Fc+LPSTLR1/TLR2-Fc+LPS 206.10206.10 30.4730.47 p＜0.05p＜0.05
TLR2/TLR4-Fc+LPSTLR2/TLR4-Fc+LPS 77.8877.88 6.726.72 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 216.75216.75 28.2828.28 p＜0.05p＜0.05
TLR4/MD-2-Fc+LPSTLR4/MD-2-Fc+LPS 91.3491.34 10.9710.97 p＜0.05p＜0.05
TLR4/CD36-Fc+LPSTLR4/CD36-Fc+LPS 202.65202.65 15.4615.46 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 199.29199.29 23.2123.21 p＜0.05p＜0.05
表22各处理组IL-6水平Table 22 IL-6 levels in each treatment group
组别Group IL-6(pg/ml)IL-6(pg/ml) SDSD p值(vs.对照IgG+LPS)p value (vs. control IgG+LPS)
空白对照(仅培养基)Blank control (medium only) 31.4731.47 4.694.69  To
对照IgGControl IgG 41.0041.00 3.533.53  To
对照IgG+LPSControl IgG+LPS 634.98634.98 71.0671.06  To
TLR1-Fc+LPSTLR1-Fc+LPS 160.10160.10 12.2912.29 p＜0.05p＜0.05
TLR2-Fc+LPSTLR2-Fc+LPS 71.7271.72 7.427.42 p＜0.05p＜0.05
TLR4-Fc+LPSTLR4-Fc+LPS 295.24295.24 34.6234.62 p＜0.05p＜0.05
TLR6-Fc+LPSTLR6-Fc+LPS 157.59157.59 8.618.61 p＜0.05p＜0.05
TLR4-Fc-LALAPG+LPSTLR4-Fc-LALAPG+LPS 211.16211.16 13.2613.26 p＜0.05p＜0.05
TLR1/TLR2-Fc+LPSTLR1/TLR2-Fc+LPS 262.55262.55 21.1021.10 p＜0.05p＜0.05
TLR2/TLR4-Fc+LPSTLR2/TLR4-Fc+LPS 89.2689.26 5.955.95 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 57.3357.33 5.835.83 p＜0.05p＜0.05
TLR4/MD-2-Fc+LPSTLR4/MD-2-Fc+LPS 109.77109.77 8.018.01 p＜0.05p＜0.05
TLR4/CD36-Fc+LPSTLR4/CD36-Fc+LPS 189.63189.63 13.5713.57 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 171.20171.20 21.5121.51 p＜0.05p＜0.05
进一步确定二聚体免疫融合蛋白对细胞因子蛋白表达的抑制效果。将所述的对照组和处理组细胞裂解，利用qPCR方法分析细胞内的细胞因子mRNA的表达如下表23～26所示，以LPS刺激细胞增加了细胞因子的表达(HMGB1、TNF-α、IL-6、COX-2)。然而，若用LPS和本发明所描述的二聚体免疫融合蛋白同时处理细胞，上述促炎细胞因子的表达水平显著降低。这些结果强烈的提示和支持支持本发明所述的二聚体免疫融合蛋白的抗炎效果。To further determine the inhibitory effect of the dimeric immune fusion protein on the expression of cytokine protein. The cells of the control group and the treatment group were lysed, and the expression of cytokine mRNA in the cells was analyzed by qPCR method as shown in the following Tables 23 to 26. Stimulating the cells with LPS increased the expression of cytokines (HMGB1, TNF-α, IL -6, COX-2). However, if the cells are treated with LPS and the dimeric immune fusion protein described in the present invention at the same time, the expression level of the above-mentioned pro-inflammatory cytokines is significantly reduced. These results strongly suggest and support the anti-inflammatory effect of the dimeric immune fusion protein of the present invention.
表23各处理组细胞HMGB1表达水平Table 23 Cell HMGB1 expression level in each treatment group
组别Group HMGB1相对表达水平％Relative expression level of HMGB1% SDSD p值(vs.对照IgG+LPS)p value (vs. control IgG+LPS)
空白对照(仅培养基)Blank control (medium only) 20.0420.04 2.762.76  To
对照IgGControl IgG 17.9817.98 1.511.51  To
对照IgG+LPSControl IgG+LPS 100100 7.557.55  To
TLR1-Fc+LPSTLR1-Fc+LPS 13.2313.23 1.871.87 p＜0.05p＜0.05
TLR2-Fc+LPSTLR2-Fc+LPS 22.8622.86 2.352.35 p＜0.05p＜0.05
TLR4-Fc+LPSTLR4-Fc+LPS 40.5840.58 3.103.10 p＜0.05p＜0.05
TLR6-Fc+LPSTLR6-Fc+LPS 17.3917.39 2.132.13 p＜0.05p＜0.05
TLR4-Fc-LALAPG+LPSTLR4-Fc-LALAPG+LPS 13.6413.64 1.141.14 p＜0.05p＜0.05
TLR1/TLR2-Fc+LPSTLR1/TLR2-Fc+LPS 10.0910.09 1.181.18 p＜0.05p＜0.05
TLR2/TLR4-Fc+LPSTLR2/TLR4-Fc+LPS 33.6133.61 4.414.41 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 14.6914.69 1.401.40 p＜0.05p＜0.05
TLR4/MD-2-Fc+LPSTLR4/MD-2-Fc+LPS 16.8916.89 1.151.15 p＜0.05p＜0.05
TLR4/CD36-Fc+LPSTLR4/CD36-Fc+LPS 23.8323.83 2.912.91 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 36.7636.76 3.753.75 p＜0.05p＜0.05
表24各处理组细胞TNFα表达水平Table 24 Cell TNFα expression levels in each treatment group
组别Group TNFα相对表达水平％Relative expression level of TNFα% SDSD p值(vs.对照IgG+LPS)p value (vs. control IgG+LPS)
空白对照(仅培养基)Blank control (medium only) 10.6710.67 0.980.98  To
对照IgGControl IgG 10.1210.12 0.690.69  To
对照IgG+LPSControl IgG+LPS 100100 7.847.84  To
TLR1-Fc+LPSTLR1-Fc+LPS 17.6317.63 1.991.99 p＜0.05p＜0.05
TLR2-Fc+LPSTLR2-Fc+LPS 23.0423.04 2.932.93 p＜0.05p＜0.05
TLR4-Fc+LPSTLR4-Fc+LPS 14.3314.33 1.041.04 p＜0.05p＜0.05
TLR6-Fc+LPSTLR6-Fc+LPS 18.8718.87 2.132.13 p＜0.05p＜0.05
TLR4-Fc-LALAPG+LPSTLR4-Fc-LALAPG+LPS 24.6524.65 2.802.80 p＜0.05p＜0.05
TLR1/TLR2-Fc+LPSTLR1/TLR2-Fc+LPS 13.4613.46 1.471.47 p＜0.05p＜0.05
TLR2/TLR4-Fc+LPSTLR2/TLR4-Fc+LPS 14.8214.82 0.750.75 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 20.7720.77 2.982.98 p＜0.05p＜0.05
TLR4/MD-2-Fc+LPSTLR4/MD-2-Fc+LPS 35.8635.86 5.385.38 p＜0.05p＜0.05
TLR4/CD36-Fc+LPSTLR4/CD36-Fc+LPS 18.4718.47 2.622.62 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 22.1822.18 2.262.26 p＜0.05p＜0.05
表25各处理组细胞IL-6表达水平Table 25 Cell IL-6 expression level in each treatment group
组别Group IL-6相对表达水平％Relative expression level of IL-6% SDSD p值(vs.对照IgG+LPS)p value (vs. control IgG+LPS)
空白对照(仅培养基)Blank control (medium only) 11.8011.80 0.970.97  To
对照IgGControl IgG 8.888.88 0.530.53  To
对照IgG+LPSControl IgG+LPS 100100 7.657.65  To
TLR1-Fc+LPSTLR1-Fc+LPS 17.5417.54 1.351.35 p＜0.05p＜0.05
TLR2-Fc+LPSTLR2-Fc+LPS 27.1527.15 3.313.31 p＜0.05p＜0.05
TLR4-Fc+LPSTLR4-Fc+LPS 29.1829.18 3.513.51 p＜0.05p＜0.05
TLR6-Fc+LPSTLR6-Fc+LPS 39.8239.82 4.444.44 p＜0.05p＜0.05
TLR4-Fc-LALAPG+LPSTLR4-Fc-LALAPG+LPS 19.7419.74 1.631.63 p＜0.05p＜0.05
TLR1/TLR2-Fc+LPSTLR1/TLR2-Fc+LPS 21.4621.46 1.761.76 p＜0.05p＜0.05
TLR2/TLR4-Fc+LPSTLR2/TLR4-Fc+LPS 30.3230.32 4.064.06 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 25.8825.88 2.592.59 p＜0.05p＜0.05
TLR4/MD-2-Fc+LPSTLR4/MD-2-Fc+LPS 23.8223.82 3.183.18 p＜0.05p＜0.05
TLR4/CD36-Fc+LPSTLR4/CD36-Fc+LPS 26.1526.15 3.673.67 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 25.9425.94 2.932.93 p＜0.05p＜0.05
表26各处理组细胞COX-2表达水平Table 26 COX-2 expression level of cells in each treatment group
组别Group COX-2相对表达水平％Relative expression level of COX-2% SDSD p值(vs.对照IgG+LPS)p value (vs. control IgG+LPS)
空白对照(仅培养基)Blank control (medium only) 22.8622.86 3.163.16  To
对照IgGControl IgG 28.8928.89 1.911.91  To
对照IgG+LPSControl IgG+LPS 100100 8.818.81  To
TLR1-Fc+LPSTLR1-Fc+LPS 30.6930.69 3.443.44 p＜0.05p＜0.05
TLR2-Fc+LPSTLR2-Fc+LPS 26.5026.50 3.033.03 p＜0.05p＜0.05
TLR4-Fc+LPSTLR4-Fc+LPS 29.6529.65 3.213.21 p＜0.05p＜0.05
TLR6-Fc+LPSTLR6-Fc+LPS 34.0334.03 3.443.44 p＜0.05p＜0.05
TLR4-Fc-LALAPG+LPSTLR4-Fc-LALAPG+LPS 28.1528.15 1.651.65 p＜0.05p＜0.05
TLR1/TLR2-Fc+LPSTLR1/TLR2-Fc+LPS 26.3226.32 3.853.85 p＜0.05p＜0.05
TLR2/TLR4-Fc+LPSTLR2/TLR4-Fc+LPS 22.4422.44 2.282.28 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 23.4523.45 2.822.82 p＜0.05p＜0.05
TLR4/MD-2-Fc+LPSTLR4/MD-2-Fc+LPS 21.6421.64 1.421.42 p＜0.05p＜0.05
TLR4/CD36-Fc+LPSTLR4/CD36-Fc+LPS 41.0941.09 3.673.67 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 38.4638.46 3.763.76 p＜0.05p＜0.05
通过结果可以看出，二聚体免疫融合蛋白有效的抑制由于外来刺激物介导的以巨噬细胞为代表的免疫细胞炎性渗出。It can be seen from the results that the dimeric immune fusion protein effectively inhibits the inflammatory exudation of immune cells represented by macrophages mediated by foreign stimuli.
实施例11二聚体免疫融合蛋白对外周单核细胞抗炎活性Example 11 Anti-inflammatory activity of dimeric immune fusion protein to peripheral monocytes
使用Biocoll Separating Solution(Biochrom AG,Berlin,Germany)从收集自健康受试对象的血样(50ml)中分离出PBMC(外周血单个核细胞)。按照实施例2的方法进行细胞处理同时检测培养基中的细胞因子(TNFα和IL-6)分泌水平、细胞内细胞因子(HMGB1、TNFα)的mRNA水平，结果如表27～30所示：Biocoll Separating Solution (Biochrom AG, Berlin, Germany) was used to separate PBMC (peripheral blood mononuclear cells) from blood samples (50ml) collected from healthy subjects. The cells were processed according to the method of Example 2 and the secretion levels of cytokines (TNFα and IL-6) in the culture medium and the mRNA levels of intracellular cytokines (HMGB1, TNFα) were detected. The results are shown in Tables 27-30:
表27各处理组TNFα水平Table 27 TNFα levels in each treatment group
组别Group TNFα(pg/ml)TNFα(pg/ml) SDSD p值(vs.对照IgG+LPS)p value (vs. control IgG+LPS)
空白对照(仅培养基)Blank control (medium only) 33.7833.78 4.384.38  To
对照IgGControl IgG 31.2931.29 1.251.25  To
对照IgG+LPSControl IgG+LPS 950.71950.71 53.6753.67  To
TLR1-Fc+LPSTLR1-Fc+LPS 223.38223.38 18.4018.40 p＜0.05p＜0.05
TLR2-Fc+LPSTLR2-Fc+LPS 128.09128.09 9.369.36 p＜0.05p＜0.05
TLR4-Fc+LPSTLR4-Fc+LPS 280.90280.90 39.4239.42 p＜0.05p＜0.05
TLR6-Fc+LPSTLR6-Fc+LPS 84.7784.77 8.028.02 p＜0.05p＜0.05
TLR4-Fc-LALAPG+LPSTLR4-Fc-LALAPG+LPS 65.1665.16 4.324.32 p＜0.05p＜0.05
TLR1/TLR2-Fc+LPSTLR1/TLR2-Fc+LPS 33.3633.36 1.831.83 p＜0.05p＜0.05
TLR2/TLR4-Fc+LPSTLR2/TLR4-Fc+LPS 59.5659.56 5.065.06 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 37.5737.57 3.413.41 p＜0.05p＜0.05
TLR4/MD-2-Fc+LPSTLR4/MD-2-Fc+LPS 38.5738.57 3.643.64 p＜0.05p＜0.05
TLR4/CD36-Fc+LPSTLR4/CD36-Fc+LPS 54.1754.17 5.385.38 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 48.3848.38 5.225.22 p＜0.05p＜0.05
表28各处理组IL-6水平Table 28 IL-6 levels in each treatment group
组别Group IL-6(pg/ml)IL-6(pg/ml) SDSD p值(vs.对照IgG+LPS)p value (vs. control IgG+LPS)
空白对照(仅培养基)Blank control (medium only) 13.2313.23 1.721.72  To
对照IgGControl IgG 22.6022.60 2.992.99  To
对照IgG+LPSControl IgG+LPS 263.02263.02 32.1032.10  To
TLR1-Fc+LPSTLR1-Fc+LPS 31.1331.13 3.083.08 p＜0.05p＜0.05
TLR2-Fc+LPSTLR2-Fc+LPS 85.9885.98 7.087.08 p＜0.05p＜0.05
TLR4-Fc+LPSTLR4-Fc+LPS 95.7495.74 8.388.38 p＜0.05p＜0.05
TLR6-Fc+LPSTLR6-Fc+LPS 46.9346.93 5.895.89 p＜0.05p＜0.05
TLR4-Fc-LALAPG+LPSTLR4-Fc-LALAPG+LPS 12.6312.63 1.321.32 p＜0.05p＜0.05
TLR1/TLR2-Fc+LPSTLR1/TLR2-Fc+LPS 84.7984.79 5.115.11 p＜0.05p＜0.05
TLR2/TLR4-Fc+LPSTLR2/TLR4-Fc+LPS 15.6015.60 0.960.96 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 45.7645.76 6.016.01 p＜0.05p＜0.05
TLR4/MD-2-Fc+LPSTLR4/MD-2-Fc+LPS 44.2244.22 6.546.54 p＜0.05p＜0.05
TLR4/CD36-Fc+LPSTLR4/CD36-Fc+LPS 19.7519.75 1.161.16 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 97.0197.01 6.276.27 p＜0.05p＜0.05
表29各处理组细胞HMGB1表达水平Table 29 HMGB1 expression level of cells in each treatment group
组别Group HMGB1相对表达水平％Relative expression level of HMGB1% SDSD p值(vs.对照IgG+LPS)p value (vs. control IgG+LPS)
空白对照(仅培养基)Blank control (medium only) 10.6410.64 0.820.82  To
对照IgGControl IgG 15.9315.93 1.061.06  To
对照IgG+LPSControl IgG+LPS 100100 4.354.35  To
TLR1-Fc+LPSTLR1-Fc+LPS 24.8024.80 2.422.42 p＜0.05p＜0.05
TLR2-Fc+LPSTLR2-Fc+LPS 15.9615.96 2.302.30 p＜0.05p＜0.05
TLR4-Fc+LPSTLR4-Fc+LPS 22.0922.09 2.902.90 p＜0.05p＜0.05
TLR6-Fc+LPSTLR6-Fc+LPS 39.7739.77 5.855.85 p＜0.05p＜0.05
TLR4-Fc-LALAPG+LPSTLR4-Fc-LALAPG+LPS 21.9021.90 1.941.94 p＜0.05p＜0.05
TLR1/TLR2-Fc+LPSTLR1/TLR2-Fc+LPS 29.9829.98 2.772.77 p＜0.05p＜0.05
TLR2/TLR4-Fc+LPSTLR2/TLR4-Fc+LPS 22.7222.72 1.831.83 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 17.2917.29 1.611.61 p＜0.05p＜0.05
TLR4/MD-2-Fc+LPSTLR4/MD-2-Fc+LPS 20.3820.38 2.032.03 p＜0.05p＜0.05
TLR4/CD36-Fc+LPSTLR4/CD36-Fc+LPS 27.1727.17 2.062.06 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 20.5720.57 2.562.56 p＜0.05p＜0.05
表30各处理组细胞TNFα表达水平Table 30 Cell TNFα expression levels in each treatment group
组别Group TNFα相对表达水平％Relative expression level of TNFα% SDSD p值(vs.对照IgG+LPS)p value (vs. control IgG+LPS)
空白对照(仅培养基)Blank control (medium only) 17.1317.13 1.581.58  To
对照IgGControl IgG 15.7115.71 2.152.15  To
对照IgG+LPSControl IgG+LPS 100100 5.575.57  To
TLR1-Fc+LPSTLR1-Fc+LPS 40.8540.85 6.106.10 p＜0.05p＜0.05
TLR2-Fc+LPSTLR2-Fc+LPS 32.0432.04 2.552.55 p＜0.05p＜0.05
TLR4-Fc+LPSTLR4-Fc+LPS 27.8427.84 3.893.89 p＜0.05p＜0.05
TLR6-Fc+LPSTLR6-Fc+LPS 27.5727.57 2.472.47 p＜0.05p＜0.05
TLR4-Fc-LALAPG+LPSTLR4-Fc-LALAPG+LPS 21.3521.35 1.331.33 p＜0.05p＜0.05
TLR1/TLR2-Fc+LPSTLR1/TLR2-Fc+LPS 27.3027.30 3.663.66 p＜0.05p＜0.05
TLR2/TLR4-Fc+LPSTLR2/TLR4-Fc+LPS 26.8826.88 1.811.81 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 23.4623.46 1.491.49 p＜0.05p＜0.05
TLR4/MD-2-Fc+LPSTLR4/MD-2-Fc+LPS 24.6324.63 2.642.64 p＜0.05p＜0.05
TLR4/CD36-Fc+LPSTLR4/CD36-Fc+LPS 40.7640.76 4.674.67 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 47.9247.92 3.343.34 p＜0.05p＜0.05
通过结果可以看出，二聚体免疫融合蛋白有效的抑制由于外来刺激物介导的以外周单个核细胞为代表的免疫细胞炎性渗出。It can be seen from the results that the dimeric immune fusion protein effectively inhibits the inflammatory exudation of immune cells represented by peripheral mononuclear cells mediated by foreign stimuli.
实施例12.二聚体免疫融合蛋白治疗急性肺损伤Example 12. Treatment of acute lung injury with dimeric immune fusion protein
BALB/c小鼠进行分组(n＝12)，空白组和假手术组均给与PBS，对照组给与对照IgG，各处理组给与本发明所述的二聚体免疫融合蛋白代表物，以按照10mg/kg的给药剂量尾静脉注射小鼠，每天一次连续给药3天，末次给药1h后开始造模，2％异戊巴比妥钠腹腔注射麻醉小鼠，仰卧固定于37℃恒温手术台。参照文献方法造模，造模主要步骤如下：小心剃去颈部正中毛发，酒精消毒，正中切开颈部皮肤约2cm，暴露及分离气管，利用胰岛素注射器于气管内慢慢滴注LPS5mg/kg(0.5mL/kg)，假手术组气管内滴注等量生理盐水，碘伏消毒伤口并缝合皮肤，建立小鼠急性肺损伤模型。BALB/c mice were divided into groups (n=12). Both the blank group and the sham operation group were given PBS, the control group was given control IgG, and each treatment group was given the dimer immune fusion protein representative of the present invention. The mice were injected into the tail vein at a dosage of 10 mg/kg, once a day for 3 consecutive days, and the model was started 1 hour after the last administration. The mice were anesthetized by intraperitoneal injection of 2% isopentobarbital sodium, and the mice were fixed on their back at 37. ℃ Constant temperature operating table. Refer to the literature method to make the model. The main steps of the model are as follows: carefully shave the middle hair of the neck, disinfect with alcohol, cut the neck skin about 2cm in the middle, expose and separate the trachea, use an insulin syringe to slowly instill LPS5mg/kg into the trachea (0.5mL/kg). In the sham operation group, the same amount of normal saline was instilled into the trachea, iodophor disinfected the wound and sutured the skin to establish a mouse model of acute lung injury.
造模24h后摘眼球取血，4℃冰箱静置3h后，3500r/min离心15min,分离血清，液 氮保存，待测。各组小鼠小心剪开颈部皮肤并分离气管，行气管插管。剖开胸部，结扎右支气管，用磷酸缓冲液灌洗左肺，共3次，2mL/次，收集支气管肺泡灌洗液并离心(4℃，1300r/min,5min)。After 24 hours of modeling, the eyeballs were taken and blood was collected. After standing in a refrigerator at 4°C for 3 hours, centrifuged at 3500 r/min for 15 minutes, the serum was separated, and stored in liquid nitrogen for testing. Each group of mice carefully cut the neck skin and separated the trachea, and intubated the trachea. The chest was opened, the right bronchus was ligated, and the left lung was lavaged with phosphate buffer solution for a total of 3 times, 2 mL/time. The bronchoalveolar lavage fluid was collected and centrifuged (4℃, 1300r/min, 5min).
检测如下指标：1)BCA蛋白定量试剂盒(碧云天)检测支气管肺泡灌洗液中蛋白含量,实验操作均按照试剂盒说明书进行。2)支气管肺泡灌洗液白细胞水平，用800μL0.01mol/L(pH 7.4)的PBS缓冲液重悬支气管肺泡灌洗液沉淀物,吹打均匀后,取400μL于血液分析仪中检测白细胞数目。3)肺湿干质量比(W/D)：称取左肺上叶为湿质量,将左肺上叶放入恒温干燥箱(105℃)烤72h，干燥至恒质量，并称取记录为干质量，按下列公式计算:肺湿干质量比＝湿质量/干质量。4)肺组织病理形态评分：取右肺下叶,
Figure PCTCN2020131581-appb-000006
中性甲醛浸泡固定24h,流水冲洗12h,常规石蜡包埋,切片,苏木精伊红染色,封片,显微镜下进行病理观察。选择不同视野按照标准肺炎症评分进行病理评分，评分方法参考文献[朱珊,潘灵辉,林飞,等.临床麻醉学杂志,2013,29(6).]。5)血生化指标检测血清中SOD活性和MDA的含量测定严格按照相应检测试剂盒说明书步骤进行检测。6)肺组织TGFβ1和Smad2的表达水平检测：BCA试剂盒检测肺组织匀浆总蛋白浓度,加入等量样品于电泳槽进行SDS聚丙烯酰胺凝胶电泳。经过转膜、封闭、孵育TGFβ1(1:3000)、Smad2(1:2000)一抗过夜；洗膜、孵育二抗(1:7000)(抗体均购自CST公司)、洗膜、显影,使用ImageJ软件半定量分析各条带灰度值。结果如表31～38所示： The following indicators were detected: 1) BCA protein quantification kit (Biyuntian) was used to detect the protein content in bronchoalveolar lavage fluid, and the experimental operations were carried out in accordance with the kit instructions. 2) The level of white blood cell in bronchoalveolar lavage fluid, 800μL 0.01mol/L (pH 7.4) PBS buffer is used to resuspend the bronchoalveolar lavage fluid sediment, after pipetting evenly, take 400μL in the blood analyzer to detect the number of white blood cells. 3) Lung wet-to-dry mass ratio (W/D): Weigh the upper lobe of the left lung as the wet mass. Place the upper lobe of the left lung in a constant temperature drying oven (105°C) for 72 hours, dry to constant mass, and weigh and record as The dry mass is calculated according to the following formula: lung wet-to-dry mass ratio = wet mass/dry mass. 4) Lung tissue pathological morphology score: take the lower lobe of the right lung,
Figure PCTCN2020131581-appb-000006
Neutral formaldehyde immersion and fixation for 24h, running water for 12h, conventional paraffin embedding, sectioning, hematoxylin and eosin staining, mounting, and pathological observation under a microscope. Different visual fields were selected for pathological scoring according to the standard pneumonia score. The scoring method refers to the literature [Zhu Shan, Pan Linghui, Lin Fei, et al. Journal of Clinical Anesthesiology, 2013, 29(6).]. 5) Detection of blood biochemical indexes The determination of SOD activity and MDA content in serum is carried out in strict accordance with the instructions of the corresponding detection kit. 6) Detection of the expression levels of TGFβ1 and Smad2 in lung tissue: BCA kit to detect the total protein concentration of lung tissue homogenate, add the same amount of sample to the electrophoresis tank for SDS polyacrylamide gel electrophoresis. After transferring the membrane, blocking, incubating TGFβ1 (1:3000), Smad2 (1:2000) primary antibody overnight; washing the membrane, incubating the secondary antibody (1:7000) (antibodies are purchased from CST company), washing the membrane, developing, use ImageJ software semi-quantitatively analyzes the gray value of each band. The results are shown in Tables 31-38:
表31各组支气管肺泡灌洗液蛋白含量Table 31 The protein content of bronchoalveolar lavage fluid in each group
组别Group 蛋白含量(g/L)Protein content (g/L) SDSD p值(vs.对照IgG+LPS)p value (vs. control IgG+LPS)
假手术组mock surgical group 0.440.44 0.050.05  To
模型组(空白对照)Model group (blank control) 3.163.16 0.460.46  To
对照IgGControl IgG 3.413.41 0.560.56  To
TLR1-Fc+LPSTLR1-Fc+LPS 0.770.77 0.040.04 p＜0.05p＜0.05
TLR2-Fc+LPSTLR2-Fc+LPS 1.311.31 0.160.16 p＜0.05p＜0.05
TLR4-Fc+LPSTLR4-Fc+LPS 0.740.74 0.070.07 p＜0.05p＜0.05
TLR6-Fc+LPSTLR6-Fc+LPS 0.470.47 0.070.07 p＜0.05p＜0.05
TLR4-Fc-LALAPG+LPSTLR4-Fc-LALAPG+LPS 0.350.35 0.020.02 p＜0.05p＜0.05
TLR1/TLR2-Fc+LPSTLR1/TLR2-Fc+LPS 0.670.67 0.010.01 p＜0.05p＜0.05
TLR2/TLR4-Fc+LPSTLR2/TLR4-Fc+LPS 1.101.10 0.110.11 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 1.401.40 0.150.15 p＜0.05p＜0.05
TLR4/MD-2-Fc+LPSTLR4/MD-2-Fc+LPS 0.210.21 0.020.02 p＜0.05p＜0.05
TLR4/CD36-Fc+LPSTLR4/CD36-Fc+LPS 0.750.75 0.060.06 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 0.300.30 0.010.01 p＜0.05p＜0.05
表32各组支气管肺泡灌洗液中性粒细胞数量Table 32 Number of neutrophils in bronchoalveolar lavage fluid in each group
组别Group 细胞数(10 8/L) Number of cells (10 8 /L) SDSD p值(vs.对照IgG+LPS)p value (vs. control IgG+LPS)
假手术组mock surgical group 1.181.18 0.110.11  To
模型组(空白对照)Model group (blank control) 9.069.06 0.930.93  To
对照IgGControl IgG 9.309.30 0.760.76  To
TLR1-Fc+LPSTLR1-Fc+LPS 2.342.34 0.170.17 p＜0.05p＜0.05
TLR2-Fc+LPSTLR2-Fc+LPS 3.253.25 0.430.43 p＜0.05p＜0.05
TLR4-Fc+LPSTLR4-Fc+LPS 3.373.37 0.270.27 p＜0.05p＜0.05
TLR6-Fc+LPSTLR6-Fc+LPS 2.052.05 0.250.25 p＜0.05p＜0.05
TLR4-Fc-LALAPG+LPSTLR4-Fc-LALAPG+LPS 2.662.66 0.370.37 p＜0.05p＜0.05
TLR1/TLR2-Fc+LPSTLR1/TLR2-Fc+LPS 4.724.72 0.430.43 p＜0.05p＜0.05
TLR2/TLR4-Fc+LPSTLR2/TLR4-Fc+LPS 3.633.63 0.310.31 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 1.031.03 0.140.14 p＜0.05p＜0.05
TLR4/MD-2-Fc+LPSTLR4/MD-2-Fc+LPS 1.391.39 0.130.13 p＜0.05p＜0.05
TLR4/CD36-Fc+LPSTLR4/CD36-Fc+LPS 1.581.58 0.170.17 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 2.072.07 0.180.18 p＜0.05p＜0.05
表33各组肺组织湿干质量比Table 33 The wet-to-dry mass ratio of lung tissues in each group
Figure PCTCN2020131581-appb-000007
Figure PCTCN2020131581-appb-000007
表34各组肺损伤评分Table 34 Lung injury score of each group
组别Group 肺组织评分Lung tissue score SDSD p值(vs.对照IgG+LPS)p value (vs. control IgG+LPS)
假手术组mock surgical group 0.250.25 0.260.26  To
模型组(空白对照)Model group (blank control) 3.083.08 0.600.60  To
对照IgGControl IgG 3.253.25 0.580.58  To
TLR1-Fc+LPSTLR1-Fc+LPS 1.251.25 0.450.45 p＜0.05p＜0.05
TLR2-Fc+LPSTLR2-Fc+LPS 1.041.04 0.450.45 p＜0.05p＜0.05
TLR4-Fc+LPSTLR4-Fc+LPS 0.750.75 0.260.26 p＜0.05p＜0.05
TLR6-Fc+LPSTLR6-Fc+LPS 0.880.88 0.430.43 p＜0.05p＜0.05
TLR4-Fc-LALAPG+LPSTLR4-Fc-LALAPG+LPS 0.990.99 0.490.49 p＜0.05p＜0.05
TLR1/TLR2-Fc+LPSTLR1/TLR2-Fc+LPS 0.960.96 0.450.45 p＜0.05p＜0.05
TLR2/TLR4-Fc+LPSTLR2/TLR4-Fc+LPS 0.880.88 0.380.38 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 0.750.75 0.340.34 p＜0.05p＜0.05
TLR4/MD-2-Fc+LPSTLR4/MD-2-Fc+LPS 0.920.92 0.360.36 p＜0.05p＜0.05
TLR4/CD36-Fc+LPSTLR4/CD36-Fc+LPS 0.880.88 0.380.38 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 0.750.75 0.400.40 p＜0.05p＜0.05
表35各组血清SOD活性Table 35 Serum SOD activity of each group
Figure PCTCN2020131581-appb-000008
Figure PCTCN2020131581-appb-000008
表36各组血清MDA含量(μmol/L)活性Table 36 Serum MDA content (μmol/L) activity of each group
组别Group SOD活性(U/ml)SOD activity (U/ml) SDSD p值(vs.对照IgG+LPS)p value (vs. control IgG+LPS)
假手术组mock surgical group 6.646.64 0.770.77  To
模型组(空白对照)Model group (blank control) 43.9043.90 3.683.68  To
对照IgGControl IgG 51.3051.30 3.453.45  To
TLR1-Fc+LPSTLR1-Fc+LPS 5.745.74 0.610.61 p＜0.05p＜0.05
TLR2-Fc+LPSTLR2-Fc+LPS 18.8518.85 2.252.25 p＜0.05p＜0.05
TLR4-Fc+LPSTLR4-Fc+LPS 8.568.56 0.750.75 p＜0.05p＜0.05
TLR6-Fc+LPSTLR6-Fc+LPS 16.0416.04 1.671.67 p＜0.05p＜0.05
TLR4-Fc-LALAPG+LPSTLR4-Fc-LALAPG+LPS 11.9411.94 1.571.57 p＜0.05p＜0.05
TLR1/TLR2-Fc+LPSTLR1/TLR2-Fc+LPS 7.197.19 0.800.80 p＜0.05p＜0.05
TLR2/TLR4-Fc+LPSTLR2/TLR4-Fc+LPS 15.5615.56 1.001.00 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 6.606.60 0.970.97 p＜0.05p＜0.05
TLR4/MD-2-Fc+LPSTLR4/MD-2-Fc+LPS 8.348.34 0.680.68 p＜0.05p＜0.05
TLR4/CD36-Fc+LPSTLR4/CD36-Fc+LPS 12.5612.56 1.551.55 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 7.917.91 0.720.72 p＜0.05p＜0.05
表37各组组织TGFβ1相对表达水平Table 37 Relative expression levels of TGFβ1 in each group
组别Group TGFβ1相对表达％Relative expression of TGFβ1% SDSD p值(vs.对照IgG+LPS)p value (vs. control IgG+LPS)
假手术组mock surgical group 5.055.05 0.260.26  To
模型组(空白对照)Model group (blank control) 101.53101.53 12.4212.42  To
对照IgGControl IgG 100100 8.818.81  To
TLR1-Fc+LPSTLR1-Fc+LPS 38.5138.51 2.262.26 p＜0.05p＜0.05
TLR2-Fc+LPSTLR2-Fc+LPS 39.7839.78 3.353.35 p＜0.05p＜0.05
TLR4-Fc+LPSTLR4-Fc+LPS 17.1317.13 2.562.56 p＜0.05p＜0.05
TLR6-Fc+LPSTLR6-Fc+LPS 20.6920.69 2.502.50 p＜0.05p＜0.05
TLR4-Fc-LALAPG+LPSTLR4-Fc-LALAPG+LPS 28.5128.51 2.282.28 p＜0.05p＜0.05
TLR1/TLR2-Fc+LPSTLR1/TLR2-Fc+LPS 12.1712.17 1.671.67 p＜0.05p＜0.05
TLR2/TLR4-Fc+LPSTLR2/TLR4-Fc+LPS 12.6412.64 1.701.70 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 28.3628.36 3.453.45 p＜0.05p＜0.05
TLR4/MD-2-Fc+LPSTLR4/MD-2-Fc+LPS 15.7315.73 0.910.91 p＜0.05p＜0.05
TLR4/CD36-Fc+LPSTLR4/CD36-Fc+LPS 39.0639.06 5.505.50 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 5.045.04 0.310.31 p＜0.05p＜0.05
表38各组组织Smad2相对表达水平Table 38 Relative expression levels of Smad2 in each group
组别Group Smad2相对表达％Smad2 relative expression% SDSD p值(vs.对照IgG+LPS)p value (vs. control IgG+LPS)
假手术组mock surgical group 11.0711.07 0.810.81  To
模型组(空白对照)Model group (blank control) 3.383.38 0.230.23  To
对照IgGControl IgG 100100 7.667.66  To
TLR1-Fc+LPSTLR1-Fc+LPS 16.8616.86 1.431.43 p＜0.05p＜0.05
TLR2-Fc+LPSTLR2-Fc+LPS 35.9135.91 4.924.92 p＜0.05p＜0.05
TLR4-Fc+LPSTLR4-Fc+LPS 15.6415.64 1.481.48 p＜0.05p＜0.05
TLR6-Fc+LPSTLR6-Fc+LPS 29.2629.26 3.733.73 p＜0.05p＜0.05
TLR4-Fc-LALAPG+LPSTLR4-Fc-LALAPG+LPS 14.4814.48 1.301.30 p＜0.05p＜0.05
TLR1/TLR2-Fc+LPSTLR1/TLR2-Fc+LPS 13.2413.24 1.761.76 p＜0.05p＜0.05
TLR2/TLR4-Fc+LPSTLR2/TLR4-Fc+LPS 29.8029.80 2.922.92 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 10.2810.28 1.261.26 p＜0.05p＜0.05
TLR4/MD-2-Fc+LPSTLR4/MD-2-Fc+LPS 25.0025.00 2.772.77 p＜0.05p＜0.05
TLR4/CD36-Fc+LPSTLR4/CD36-Fc+LPS 32.6532.65 2.972.97 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 17.9117.91 0.980.98 p＜0.05p＜0.05
这些实验证实，本发明所描述的二聚体免疫融合蛋白具有降低急性炎症渗出、抑制白细胞渗出、减少组织水肿、减轻组织损伤、增强SOD活性、降低血清MDA含量、抑制Smad2和TGFβ1表达，具有较强的抗炎、抗LPS作用。可以治疗急性脏器炎症损伤。These experiments confirmed that the dimeric immune fusion protein described in the present invention can reduce acute inflammation exudation, inhibit leukocyte exudation, reduce tissue edema, reduce tissue damage, enhance SOD activity, reduce serum MDA content, and inhibit the expression of Smad2 and TGFβ1, It has strong anti-inflammatory and anti-LPS effects. It can treat acute organ inflammation damage.
实施例13在盲肠结扎穿孔服毒症的小鼠模型中施用二聚体免疫融合蛋白的方案概述Example 13 Overview of the protocol for administering the dimer immune fusion protein in a mouse model of cecal ligation and perforation poisoning
C57雄性小鼠，手术如下：使用短效的异氟院进行麻醉，以使麻醉对心血管功能的有害作用最小化。手术过程包括开始在胸板下方2cm处的5cm的中线开腹。将盲肠隔离在腹腔外的无菌纱布上，以避免血管损伤。之后利用2-0薇乔线(vicry1)在紧接回盲瓣的下方结扎，并保持肠的连续性。然后将盲肠内容物挤到盲肠的一端。利用20号针将盲肠刺破3次，然后于动从每个刺破位置单滴挤出排泄物质。然后将腹腔封闭两层，之后是流体复苏，并将动物放回合适的笼中。假手术组小鼠除不进行盲肠结扎穿孔外，其余手术步骤完全相同。在手术后2小时、6小时和12小时检查动物。允许动物随意饮食。手术后12小时，将动物分为空白组(注射PBS)或静脉注射包含活性成分为本发明所述的免疫融合代表药物组合物，n＝10，剂量为10mg/kg，每天连续给给药。每12小时观察并记录小鼠生存情况，直至手术后7天。手术的48小时时采集一次小鼠血样，用ELISA试剂盒(CST)检测各组血浆中TNFα的水平。结果如表39、40所示：For C57 male mice, the operation is as follows: Use a short-acting isoflurane for anesthesia to minimize the harmful effects of anesthesia on cardiovascular function. The surgical procedure included a 5cm midline laparotomy starting 2cm below the chest plate. Isolate the cecum on a sterile gauze outside the abdominal cavity to avoid vascular damage. Afterwards, a 2-0 Vicryl line (vicry1) was used to ligate immediately below the ileocecal valve and maintain the continuity of the intestine. Then squeeze the contents of the cecum to one end of the cecum. Use a 20-gauge needle to puncture the cecum 3 times, and then squeeze a single drop of excretion from each puncture site. The abdominal cavity is then sealed in two layers, followed by fluid resuscitation, and the animal is returned to the appropriate cage. The mice in the sham-operated group did not undergo cecal ligation and perforation, and the rest of the surgical procedures were exactly the same. The animals were checked at 2 hours, 6 hours and 12 hours after the operation. Allow animals to eat and drink at will. Twelve hours after the operation, the animals were divided into a blank group (injected with PBS) or intravenously injected with the active ingredient as the representative pharmaceutical composition of the immune fusion described in the present invention, n=10, a dose of 10 mg/kg, and continuous administration every day. The survival of the mice was observed and recorded every 12 hours until 7 days after the operation. A mouse blood sample was collected 48 hours after the operation, and the level of TNFα in the plasma of each group was detected with an ELISA kit (CST). The results are shown in Tables 39 and 40:
表39小鼠存活率(％)Table 39 Survival rate of mice (%)
组别Group 2h2h 24h24h 2天2 days 3天3 days 4天4 days 5天5 days 6天6 days 7天7 days
假手术组mock surgical group 100100 100100 100100 100100 100100 100100 100100 100100
模型组(空白对照)Model group (blank control) 100100 9090 3030 2020 2020 2020 2020 2020
对照IgGControl IgG 100100 100100 2020 2020 2020 2020 2020 2020
TLR1-FcTLR1-Fc 100100 100100 100100 9090 8080 8080 7070 6060
TLR2-FcTLR2-Fc 100100 9090 8080 8080 7070 7070 7070 7070
TLR4-FcTLR4-Fc 100100 9090 8080 8080 6060 6060 6060 6060
TLR2/TLR4-FcTLR2/TLR4-Fc 100100 100100 7070 7070 6060 6060 6060 5050
TLR4/TLR6-FcTLR4/TLR6-Fc 100100 100100 8080 6060 6060 6060 6060 5050
TLR4/MD-2-FcTLR4/MD-2-Fc 100100 100100 8080 7070 6060 6060 6060 5050
TLR4/CD36-FcTLR4/CD36-Fc 100100 100100 6060 6060 6060 6060 6060 5050
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 100100 100100 6060 6060 6060 6060 6060 5050
表40 TNFα表达水平Table 40 TNFα expression level
组别Group TNFα(pg/ml)TNFα(pg/ml) SDSD p值(vs.对照IgG+LPS)p value (vs. control IgG+LPS)
假手术组mock surgical group 27.4927.49 3.153.15 p＜0.05p＜0.05
模型组(空白对照)Model group (blank control) 793.47793.47 62.4462.44 p＜0.05p＜0.05
对照IgGControl IgG 779.51779.51 64.6664.66 p＜0.05p＜0.05
TLR1-FcTLR1-Fc 110.02110.02 15.1815.18 p＜0.05p＜0.05
TLR2-FcTLR2-Fc 149.20149.20 15.2415.24 p＜0.05p＜0.05
TLR4-FcTLR4-Fc 159.25159.25 22.4022.40 p＜0.05p＜0.05
TLR2/TLR4-FcTLR2/TLR4-Fc 198.55198.55 27.0127.01 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 296.06296.06 29.3929.39 p＜0.05p＜0.05
TLR4/MD-2-FcTLR4/MD-2-Fc 305.86305.86 26.4926.49 p＜0.05p＜0.05
TLR4/CD36-FcTLR4/CD36-Fc 337.32337.32 44.6744.67 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 123.03123.03 7.657.65 p＜0.05p＜0.05
这些实验证实，本发明所描述的二聚体免疫融合蛋白具有减轻脓毒血症细胞因子、提高患者对抗脓毒血症的存活率。可以治疗急性脏器炎症损伤。These experiments confirmed that the dimeric immune fusion protein described in the present invention can reduce septic cytokines and improve the survival rate of patients against sepsis. It can treat acute organ inflammation damage.
实施例14二聚体免疫融合蛋白对血吸虫感染和感染导致肝脏纤维化的作用Example 14 The effect of dimeric immune fusion protein on schistosoma infection and liver fibrosis caused by infection
选取Balb/c小鼠，体重20～25g。日本血吸虫感染性钉螺由江苏血吸虫病防治研究所提供。实验动物随机分组，每组10只小鼠，分别为健康对照组(空白组)、感染对照组(模型组)、对照IgG组合二聚体免疫融合蛋白处理各组。除空白组外，其他各组采用腹部贴片法每只小鼠感染(30±2)条尾蚴。自感染后42d开始，连续给药30d。二聚体免疫融合蛋白和对照IgG给药量为10mg/kg,3d一次，最后一次给药24h后，在吸入式异氟烷麻醉机麻醉下处死动物，采集血样，分离血清用于检测。取出小鼠肝脏后，在预冷的生理盐水中清洗2次以去除残余血渍。每个肝脏称重后立即切割为几部分，一部分置于4％中性多聚甲醛溶液中固定，用于后续组织病理学检测，另一部分经液氮速冻后置于-80℃冰箱中保存用于后续分子生物学检测。Choose Balb/c mice, weighing 20-25g. Oncomelania snails infected by Schistosoma japonicum were provided by Jiangsu Institute of Schistosomiasis Control. The experimental animals were randomly divided into groups with 10 mice in each group, namely the healthy control group (blank group), the infection control group (model group), and the control IgG combined dimer immune fusion protein treatment groups. Except for the blank group, each mouse in the other groups was infected with (30±2) cercariae by abdominal patch method. Starting from 42 days after infection, the drug was continuously administered for 30 days. The dose of dimer immune fusion protein and control IgG was 10 mg/kg, once every 3 days, 24 hours after the last administration, the animals were sacrificed under anesthesia with an inhaled isoflurane anesthesia machine, blood samples were collected, and the serum was separated for testing. After the mouse liver was taken out, it was washed twice in pre-cooled normal saline to remove residual blood stains. Each liver is cut into several parts immediately after being weighed, one part is fixed in 4% neutral paraformaldehyde solution for subsequent histopathological examination, and the other part is quick-frozen in liquid nitrogen and stored in a -80℃ refrigerator For subsequent molecular biology testing.
血清肝功能指标ALT、AST，血清肝纤维化指标透明质酸(HA)、层黏连蛋白(LN) 含量，肝组织氧化损伤指标SOD、GSH PX、GSH R、MDA、GSH及羟脯氨酸含量检测均按检测试剂盒说明书执行，结果如表41～49所示。Serum liver function indexes ALT, AST, serum liver fibrosis indexes hyaluronic acid (HA), laminin (LN) content, liver tissue oxidative damage indexes SOD, GSH PX, GSH R, MDA, GSH and hydroxyproline The content detection is performed according to the instructions of the detection kit, and the results are shown in Tables 41 to 49.
表41各组小鼠肝功能情况Table 41 Liver function of mice in each group
组别Group ALT水平(U/L)ALT level (U/L) SDSD p值p value
空白对照Blank control 26.0526.05 3.163.16  To
模型组Model group 124.47124.47 13.3513.35  To
对照IgGControl IgG 126.48126.48 7.097.09  To
TLR1-FcTLR1-Fc 45.9945.99 2.562.56 p＜0.05p＜0.05
TLR2-FcTLR2-Fc 41.3341.33 5.825.82 p＜0.05p＜0.05
TLR4-FcTLR4-Fc 22.3122.31 2.202.20 p＜0.05p＜0.05
TLR2/TLR4-FcTLR2/TLR4-Fc 33.6733.67 2.272.27 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 45.3745.37 2.432.43 p＜0.05p＜0.05
TLR4/MD-2-FcTLR4/MD-2-Fc 50.4950.49 5.875.87 p＜0.05p＜0.05
TLR4/CD36-FcTLR4/CD36-Fc 25.9425.94 2.722.72 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 55.2455.24 7.197.19 p＜0.05p＜0.05
表42各组小鼠肝功能情况Table 42 Liver function of mice in each group
组别Group AST水平(U/L)AST level (U/L) SDSD p值p value
空白对照Blank control 25.5325.53 2.172.17  To
模型组Model group 132.65132.65 11.4111.41  To
对照IgGControl IgG 121.00121.00 6.436.43  To
TLR1-FcTLR1-Fc 72.0972.09 9.439.43 p＜0.05p＜0.05
TLR2-FcTLR2-Fc 46.3546.35 3.453.45 p＜0.05p＜0.05
TLR4-FcTLR4-Fc 47.5247.52 5.055.05 p＜0.05p＜0.05
TLR2/TLR4-FcTLR2/TLR4-Fc 69.3069.30 7.457.45 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 77.1377.13 4.374.37 p＜0.05p＜0.05
TLR4/MD-2-FcTLR4/MD-2-Fc 34.2734.27 6.006.00 p＜0.05p＜0.05
TLR4/CD36-FcTLR4/CD36-Fc 55.7355.73 4.184.18 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 58.5958.59 7.557.55 p＜0.05p＜0.05
表43各组小鼠血清透明质酸水平情况Table 43 Serum hyaluronic acid levels of mice in each group
组别Group 透明质酸水平(ng/ml)Hyaluronic acid level (ng/ml) SDSD p值p value
空白对照Blank control 77.0777.07 5.555.55  To
模型组Model group 590.84590.84 33.3433.34  To
对照IgGControl IgG 534.82534.82 33.0033.00  To
TLR1-FcTLR1-Fc 105.72105.72 12.5812.58 p＜0.05p＜0.05
TLR2-FcTLR2-Fc 199.62199.62 19.9219.92 p＜0.05p＜0.05
TLR4-FcTLR4-Fc 140.74140.74 36.1336.13 p＜0.05p＜0.05
TLR2/TLR4-FcTLR2/TLR4-Fc 190.99190.99 27.7627.76 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 124.67124.67 6.546.54 p＜0.05p＜0.05
TLR4/MD-2-FcTLR4/MD-2-Fc 198.84198.84 29.2329.23 p＜0.05p＜0.05
TLR4/CD36-FcTLR4/CD36-Fc 112.62112.62 11.1311.13 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 196.84196.84 24.0924.09 p＜0.05p＜0.05
表44各组小鼠血清层黏连蛋白水平情况Table 44 Serum laminin levels of mice in each group
组别Group LN水平(ng/ml)LN level (ng/ml) SDSD p值p value
空白对照Blank control 30.8330.83 3.823.82  To
模型组Model group 150.79150.79 21.0621.06  To
对照IgGControl IgG 145.08145.08 8.688.68  To
TLR1-FcTLR1-Fc 51.7151.71 6.026.02 p＜0.05p＜0.05
TLR2-FcTLR2-Fc 21.3021.30 1.911.91 p＜0.05p＜0.05
TLR4-FcTLR4-Fc 23.9923.99 1.331.33 p＜0.05p＜0.05
TLR2/TLR4-FcTLR2/TLR4-Fc 33.9433.94 4.304.30 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 39.1039.10 2.242.24 p＜0.05p＜0.05
TLR4/MD-2-FcTLR4/MD-2-Fc 56.2356.23 8.178.17 p＜0.05p＜0.05
TLR4/CD36-FcTLR4/CD36-Fc 34.2434.24 2.742.74 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 31.4731.47 2.022.02 p＜0.05p＜0.05
表45各组小鼠肝脏中羟脯氨酸水平情况Table 45 The level of hydroxyproline in the liver of each group of mice
组别Group 羟脯氨酸(μg/mg)Hydroxyproline (μg/mg) SDSD p值p value
空白对照Blank control 0.010.01 0.000.00  To
模型组Model group 0.140.14 0.000.00  To
对照IgGControl IgG 0.130.13 0.000.00  To
TLR1-FcTLR1-Fc 0.050.05 0.000.00 p＜0.05p＜0.05
TLR2-FcTLR2-Fc 0.060.06 0.000.00 p＜0.05p＜0.05
TLR4-FcTLR4-Fc 0.020.02 0.000.00 p＜0.05p＜0.05
TLR2/TLR4-FcTLR2/TLR4-Fc 0.030.03 0.000.00 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 0.020.02 0.000.00 p＜0.05p＜0.05
TLR4/MD-2-FcTLR4/MD-2-Fc 0.010.01 0.000.00 p＜0.05p＜0.05
TLR4/CD36-FcTLR4/CD36-Fc 0.050.05 0.000.00 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 0.020.02 0.000.00 p＜0.05p＜0.05
表46各组小鼠肝脏氧化损伤指标Table 46 Liver oxidative damage indexes of mice in each group
Figure PCTCN2020131581-appb-000009
Figure PCTCN2020131581-appb-000009
表47各组小鼠肝脏中虫卵肉芽肿面积占比Table 47 Percentage of egg granuloma area in the liver of each group of mice
组别Group 肉芽肿面积比％Granuloma area ratio% SDSD p值p value
空白对照Blank control 00 00  To
模型组Model group 65.1865.18 7.357.35  To
对照IgGControl IgG 59.7759.77 4.864.86  To
TLR1-FcTLR1-Fc 11.5111.51 0.690.69 p＜0.05p＜0.05
TLR2-FcTLR2-Fc 13.2513.25 1.281.28 p＜0.05p＜0.05
TLR4-FcTLR4-Fc 13.9113.91 1.621.62 p＜0.05p＜0.05
TLR2/TLR4-FcTLR2/TLR4-Fc 9.389.38 0.530.53 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 17.6617.66 1.551.55 p＜0.05p＜0.05
TLR4/MD-2-FcTLR4/MD-2-Fc 15.0415.04 1.951.95 p＜0.05p＜0.05
TLR4/CD36-FcTLR4/CD36-Fc 13.8513.85 1.221.22 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 6.216.21 0.520.52 p＜0.05p＜0.05
表48各组小鼠肝脏中TGFβα表达水平Table 48 The expression level of TGFβα in the liver of each group of mice
组别Group TGFβ相对表达％Relative expression of TGFβ% SDSD p值p value
空白对照Blank control 9.229.22 1.221.22  To
模型组Model group 93.9093.90 5.795.79  To
对照IgGControl IgG 100100 7.887.88  To
TLR1-FcTLR1-Fc 12.5812.58 1.391.39 p＜0.05p＜0.05
TLR2-FcTLR2-Fc 13.1413.14 0.730.73 p＜0.05p＜0.05
TLR4-FcTLR4-Fc 11.8911.89 1.031.03 p＜0.05p＜0.05
TLR2/TLR4-FcTLR2/TLR4-Fc 19.7219.72 2.422.42 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 19.9219.92 1.201.20 p＜0.05p＜0.05
TLR4/MD-2-FcTLR4/MD-2-Fc 19.5319.53 0.720.72 p＜0.05p＜0.05
TLR4/CD36-FcTLR4/CD36-Fc 29.1229.12 2.602.60 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 13.5213.52 0.510.51 p＜0.05p＜0.05
表49各组小鼠肝脏中αSMA表达水平Table 49 The expression level of αSMA in the liver of each group of mice
组别Group TGFβ相对表达％Relative expression of TGFβ% SDSD p值p value
空白对照Blank control 1.651.65 0.190.19  To
模型组Model group 99.4799.47 9.409.40  To
对照IgGControl IgG 100100 10.5510.55  To
TLR1-FcTLR1-Fc 13.5413.54 1.341.34 p＜0.05p＜0.05
TLR2-FcTLR2-Fc 5.425.42 0.360.36 p＜0.05p＜0.05
TLR4-FcTLR4-Fc 10.3210.32 2.022.02 p＜0.05p＜0.05
TLR2/TLR4-FcTLR2/TLR4-Fc 8.728.72 1.111.11 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 2.942.94 0.320.32 p＜0.05p＜0.05
TLR4/MD-2-FcTLR4/MD-2-Fc 16.2216.22 2.222.22 p＜0.05p＜0.05
TLR4/CD36-FcTLR4/CD36-Fc 19.9919.99 1.061.06 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 19.0519.05 1.161.16 p＜0.05p＜0.05
这些实验证实，本发明所描述的二聚体免疫融合蛋白具有抑制血吸虫肝脏定植、减少肝脏纤维化、减少脏器炎症性损伤、可以作为对抗脏器纤维化的产品。These experiments confirmed that the dimeric immune fusion protein described in the present invention can inhibit schistosomiasis liver colonization, reduce liver fibrosis, reduce organ inflammatory damage, and can be used as a product against organ fibrosis.
实施例15二聚体免疫融合蛋白治疗小鼠子宫内膜损伤模型Example 15 Treatment of mouse endometrial injury model with dimer immune fusion protein
(1)动物子宫内膜损伤模型的构建(C57小鼠)(1) Construction of animal endometrial injury model (C57 mice)
取8周龄大的雌性小鼠分为组，每组10只小鼠，采用双重(感染+机械)损伤法构建子宫内膜损伤模型，即小鼠麻醉后，取下腹部正中长约2cm纵切口进腹，于子宫中下1/3处作0.5cm纵行切口，采用子宫内膜刮勺搔刮中上段子宫腔，当刮勺进出子宫凹凸感消失，四壁感觉粗糙时，停止刮宫，刮宫后宫腔留置脂多糖棉线，缝合腹部切口，48h后取出脂多糖棉线。Eight-week-old female mice were divided into groups, each with 10 mice, and the double (infection + mechanical) injury method was used to construct an endometrial injury model, that is, after the mice were anesthetized, the median length of the abdomen was removed and the longitudinal length was about 2 cm. The incision is made into the abdomen, and a 0.5cm longitudinal incision is made at the middle and lower 1/3 of the uterus, and the upper and middle uterine cavity is scraped with an endometrial spatula. When the spatula enters and exits the uterus, the unevenness disappears and the four walls feel rough, stop curettage. Lipopolysaccharide cotton thread was left in the uterine cavity after curettage, the abdominal incision was sutured, and the lipopolysaccharide cotton thread was taken out 48 hours later.
建模完成后，设置空白对照组(假手术组)、仅注射生理盐水(模型)组以及处理组。处 理组分别经脉施用10mg/kg本发明所述二聚体免疫融合蛋白的代表，3天一次，共施用3次。在小鼠动情3个周期后将小鼠与雄性小鼠进行交配。1个月后取材行HE染色和Masson染色进行子宫内膜组织功能学评估，3个月后行小鼠妊娠结果评估。结果：术后1个月组织功能学评估显示，与各对照组相比，免疫二聚体处理各组的纤维化程度明显减少(表50)；与各对照组相比，外泌体腺体数量均高于各对照组。妊娠结果评估显示，免疫二聚体处理各组的妊娠率高于各对照组，结果如表51所示。After the modeling is completed, a blank control group (sham operation group), a saline injection only (model) group, and a treatment group are set up. The treatment group was administered 10 mg/kg representative of the dimer immune fusion protein of the present invention via the vein, once every 3 days, for a total of 3 administrations. The mice were mated with male mice after 3 cycles of estrus. One month later, the specimens were collected for HE staining and Masson staining to evaluate the function of the endometrium. Three months later, the mouse pregnancy results were evaluated. Results: 1 month after operation, the histological function evaluation showed that compared with each control group, the degree of fibrosis in each group treated with immunodimer was significantly reduced (Table 50); compared with each control group, the exosomal glands The numbers are higher than those of each control group. The evaluation of pregnancy results showed that the pregnancy rate of each group treated with immunodimer was higher than that of each control group. The results are shown in Table 51.
表50各组小鼠子宫内膜纤维化程度Table 50 The degree of endometrial fibrosis in each group of mice
组别Group 相对纤维化面积％Relative fibrosis area% SDSD p值(比对照IgG)p value (compared to control IgG)
空白对照组Blank control group 2.052.05 0.230.23  To
模型组Model group 97.4697.46 9.419.41  To
对照IgGControl IgG 100100 10.5810.58  To
TLR1-FcTLR1-Fc 17.8217.82 2.402.40 p＜0.05p＜0.05
TLR2-FcTLR2-Fc 24.2924.29 1.561.56 p＜0.05p＜0.05
TLR4-FcTLR4-Fc 40.7740.77 2.892.89 p＜0.05p＜0.05
TLR2/TLR4-FcTLR2/TLR4-Fc 22.3922.39 2.312.31 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 16.9716.97 1.671.67 p＜0.05p＜0.05
TLR4/MD-2-FcTLR4/MD-2-Fc 25.0325.03 1.701.70 p＜0.05p＜0.05
TLR4/CD36-FcTLR4/CD36-Fc 31.3031.30 2.932.93 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 32.9332.93 2.402.40 p＜0.05p＜0.05
表51各组小鼠妊娠结果分析Table 51 Analysis of the results of pregnancy in each group of mice
组别Group 怀孕率％Pregnancy rate% p值(比对照IgG)p value (compared to control IgG)
空白对照组Blank control group 100100  To
模型组Model group 1010  To
对照IgGControl IgG 2020  To
对照IgGControl IgG 7070 p＜0.05p＜0.05
TLR1-FcTLR1-Fc 8080 p＜0.05p＜0.05
TLR2-FcTLR2-Fc 7070 p＜0.05p＜0.05
TLR4-FcTLR4-Fc 8080 p＜0.05p＜0.05
TLR2/TLR4-FcTLR2/TLR4-Fc 7070 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 8080 p＜0.05p＜0.05
TLR4/MD-2-FcTLR4/MD-2-Fc 8080 p＜0.05p＜0.05
TLR4/CD36-FcTLR4/CD36-Fc 7070 p＜0.05p＜0.05
这些实验证实，本发明所描述的二聚体免疫融合蛋白具有抑制减少脏器炎症性损伤、 抑制脏器慢性炎症，尤其是子宫内膜炎症和纤维化，可以作为对抗脏器纤维化的产品、提高不孕不育疾病的治疗。These experiments confirmed that the dimeric immune fusion protein described in the present invention can inhibit organ inflammatory damage, inhibit chronic inflammation of organs, especially endometrial inflammation and fibrosis, and can be used as a product to combat organ fibrosis. Improve the treatment of infertility diseases.
实施例16.二聚体免疫融合蛋白对自发性流产模型的治疗作用Example 16. Therapeutic effect of dimeric immune fusion protein on spontaneous abortion model
利用CBA/J雌性小鼠和DBA/2J雄性小鼠建立应激流产模型，该流产模型为经典的母胎免疫耐受障碍的研究模型，建立方法、实验方法和观察时间点等同文献(Blois S M,et al..Nature Medicine,2007,13(12):1450-1457.)。CBA/J雌性小鼠合笼前分为阴性对照组，应激压力组，处理组。处理组分别经脉施用10mg/kg本发明所述二聚体免疫融合蛋白，3天一次，共施用3次。第一次施用后3天后合笼。确定阴栓怀孕后立即将小鼠分笼(有效n＝10)。CBA/J female mice and DBA/2J male mice were used to establish a stress abortion model. This abortion model is a classic research model of maternal-fetal immune tolerance. The establishment methods, experimental methods and observation time points are equivalent to the literature (Blois S M ,et al.. Nature Medicine, 2007,13(12):1450-1457.). CBA/J female mice were divided into negative control group, stress pressure group, and treatment group before being caged. The treatment group was administered 10 mg/kg of the dimer immune fusion protein of the present invention via the vein, once every 3 days, for a total of 3 administrations. The cages were closed 3 days after the first application. The mice were caged immediately after the vaginal plug was pregnant (effective n=10).
实验结果显示(表52)，治疗组的流产率显著低于应激压力流产组，说明采用二聚体免疫融合蛋白具有良好的治疗效果。The experimental results show (Table 52) that the abortion rate of the treatment group was significantly lower than that of the stress-stressed abortion group, indicating that the use of dimer immune fusion protein has a good therapeutic effect.
表52各组小鼠胚胎吸收率(流产)分析Table 52 Analysis of mouse embryo absorption rate (abortion) in each group
组别Group 胚胎吸收率Embryo Absorption Rate SDSD p值(比应激压力+对照IgG)p value (specific stress + control IgG)
空白对照组Blank control group 7.867.86 6.806.80  To
应激压力组Stress group 48.0448.04 5.485.48  To
对照IgGControl IgG 50.8350.83 11.4211.42  To
TLR1-FcTLR1-Fc 19.7619.76 7.367.36 p＜0.05p＜0.05
TLR2-FcTLR2-Fc 21.1921.19 7.577.57 p＜0.05p＜0.05
TLR4-FcTLR4-Fc 17.1417.14 9.279.27 p＜0.05p＜0.05
TLR2/TLR4-FcTLR2/TLR4-Fc 18.3918.39 6.586.58 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 22.8622.86 8.418.41 p＜0.05p＜0.05
TLR4/MD-2-FcTLR4/MD-2-Fc 21.0121.01 8.768.76 p＜0.05p＜0.05
TLR4/CD36-FcTLR4/CD36-Fc 16.2516.25 8.128.12 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 16.0716.07 8.258.25 p＜0.05p＜0.05
进一步将所述对照组、应激压力组和处理组的小鼠主动脉旁淋巴结分离，检测其中的Foxp3阳性T辅助淋巴细胞水平。分离蜕膜组织中，检测组织中的TNFα的水平；分离和检测的方法同文献(Kim B J,et al..Proceedings of the National Academy of Sciences,2015,112(5):1559-1564。结果显示二聚体免疫融合蛋白处理可以有效增加Foxp3阳性T辅助淋巴细胞水平、降低组织TNFα的水平(表53)。The para-aortic lymph nodes of the mice in the control group, the stress pressure group, and the treatment group were further separated, and the level of Foxp3-positive T helper lymphocytes in them was detected. In the separation of decidua tissue, the level of TNFα in the tissue is detected; the method of separation and detection is the same as that in the literature (Kim B J, et al.. Proceedings of the National Academy of Sciences, 2015, 112(5): 1559-1564. Results Shows that dimer immune fusion protein treatment can effectively increase the level of Foxp3-positive T helper lymphocytes and reduce the level of tissue TNFα (Table 53).
表53各组小鼠Foxp3％表达分析Table 53 Foxp3% expression analysis of mice in each group
Figure PCTCN2020131581-appb-000010
Figure PCTCN2020131581-appb-000010
Figure PCTCN2020131581-appb-000011
Figure PCTCN2020131581-appb-000011
表54各组TNFα组织表达分析Table 54 Tissue expression analysis of TNFα in each group
组别Group 相对TNFα表达％Relative TNFα expression% SDSD p值(vs应激压力+对照IgG)p value (vs stress pressure + control IgG)
空白对照组Blank control group 9.739.73 0.500.50  To
应激压力组Stress group 92.6092.60 5.195.19  To
对照IgGControl IgG 100100 5.565.56  To
TLR1-FcTLR1-Fc 13.1013.10 1.441.44 p＜0.05p＜0.05
TLR2-FcTLR2-Fc 15.5015.50 1.831.83 p＜0.05p＜0.05
TLR4-FcTLR4-Fc 18.9118.91 2.452.45 p＜0.05p＜0.05
TLR2/TLR4-FcTLR2/TLR4-Fc 11.2011.20 0.770.77 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 19.2719.27 2.802.80 p＜0.05p＜0.05
TLR4/MD-2-FcTLR4/MD-2-Fc 12.8512.85 1.051.05 p＜0.05p＜0.05
TLR4/CD36-FcTLR4/CD36-Fc 37.5437.54 1.461.46 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 20.2920.29 2.922.92 p＜0.05p＜0.05
这些实验证实，本发明所描述的二聚体免疫融合蛋白具有抑制减少脏器炎症性因子的异常，对抗异常免疫紊乱、提高不孕不育疾病的治疗。These experiments confirmed that the dimeric immune fusion protein described in the present invention can inhibit and reduce the abnormality of organ inflammatory factors, fight against abnormal immune disorders, and improve the treatment of infertility diseases.
实施例17:二聚体免疫融合蛋白对狼疮小鼠模型的治疗作用Example 17: Therapeutic effect of dimeric immune fusion protein on lupus mouse model
狼疮性肾炎作为一种代表性免疫系统疾病，发病率约为50/10万，在我国约占人口的0.7‰。90％以上狼疮性肾炎见于女性，主要为青、中年女性，一般认为30岁以下者肾脏受累率高，约有70％病人有不同程度的肾损害临床表现，以程度不等的蛋白尿、镜下血尿为多见，常伴有管型尿及肾功能损害，严重影响患者的正常生活。As a representative disease of the immune system, lupus nephritis has an incidence of about 50/100,000, which accounts for about 0.7‰ of the population in my country. More than 90% of lupus nephritis is seen in women, mainly young and middle-aged women. It is generally believed that people under 30 have a high renal involvement rate. About 70% of patients have clinical manifestations of renal damage to varying degrees, with varying degrees of proteinuria, Hematuria under microscope is more common, often accompanied by tubular urine and renal function damage, which seriously affects the normal life of patients.
狼疮小鼠模型多采用NZB雌鼠与NZW雄鼠杂交产生，杂交第一代(NZB×NZW)F1，能产生包括狼疮性肾炎在内的典型狼疮症状，是目前公认的研究狼疮性肾炎动物模型之一。模型的建立参考非专利文献Brinks et al.Circ Res(2010)107:1140-1149。然后将小鼠分组为各蜕膜NK细胞处理组和对照组，每组n＝10。Lupus mouse models are mostly produced by crossing NZB female mice and NZW male mice. The first-generation (NZB×NZW) F1 hybridization can produce typical lupus symptoms including lupus nephritis. It is currently recognized as an animal model for studying lupus nephritis. one. The establishment of the model refers to the non-patent literature Brinks et al. Circ Res (2010) 107:1140-1149. The mice were then divided into decidual NK cell treatment groups and control groups, n=10 in each group.
二聚体免疫融合蛋白的施用剂量为:10mg/kg，一周两次尾静脉注射，连续注射十周。 对照组注射对照IgG，剂量同处理组。The administration dose of the dimer immune fusion protein is: 10 mg/kg, twice a week by tail vein injection for ten consecutive weeks. The control group was injected with control IgG at the same dose as the treatment group.
于第30周分别检测各处理组自身抗体水平，各处理组较对照组抗dsDNA抗体、抗组蛋白抗体显著减少，而总IgG水平不变，证实处理对自身免疫性抗体有抑制生成的作用。如表11-13所示。At the 30th week, the autoantibody level of each treatment group was detected. Compared with the control group, the anti-dsDNA antibody and anti-histone antibody of each treatment group were significantly reduced, while the total IgG level remained unchanged, confirming that the treatment can inhibit the production of autoimmune antibodies. As shown in Table 11-13.
于40周统计蛋白尿发生率，50周统计存活率，小鼠死亡立即进行组织学研究做病理评分，存活小鼠在50周同一处死，肾脏组织学研究进行病理评分，评分方法参考文献[Liu S,et al.Clinical Immunology,2019,203:72-80.]。如表55-60所示，各处理组较对照组有效减少蛋白尿水平、减轻肾脏炎症和病理破坏、提高狼疮小鼠存活力。The incidence of proteinuria was counted at 40 weeks, and the survival rate was counted at 50 weeks. Mice died immediately for histological study for pathological scoring. Surviving mice were sacrificed at the same time at 50 weeks. Kidney histological study was for pathological scoring. References for scoring method S, et al. Clinical Immunology, 2019, 203:72-80.]. As shown in Table 55-60, compared with the control group, each treatment group effectively reduced proteinuria levels, reduced kidney inflammation and pathological damage, and improved the viability of lupus mice.
表55 30周各组小鼠血清抗dsDNA抗体Table 55 Serum anti-dsDNA antibodies of mice in each group at 30 weeks
Figure PCTCN2020131581-appb-000012
Figure PCTCN2020131581-appb-000012
表56 30周各组小鼠血清抗组蛋白抗体Table 56 Serum anti-histone antibodies of mice in each group at 30 weeks
Figure PCTCN2020131581-appb-000013
Figure PCTCN2020131581-appb-000013
表57 30周各组小鼠血清总IgG水平Table 57 Serum total IgG levels of mice in each group at 30 weeks
Figure PCTCN2020131581-appb-000014
Figure PCTCN2020131581-appb-000014
表58 40周各组小鼠蛋白尿发生率Table 58 The incidence of proteinuria in mice in each group at 40 weeks
组别Group 蛋白尿发生率％The incidence of proteinuria% p值p value
空白组(模型组)Blank group (model group) 100100  To
对照IgGControl IgG 100100  To
TLR1-FcTLR1-Fc 4040 p＜0.05p＜0.05
TLR2-FcTLR2-Fc 3030 p＜0.05p＜0.05
TLR4-FcTLR4-Fc 3030 p＜0.05p＜0.05
TLR2/TLR4-FcTLR2/TLR4-Fc 3030 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 2020 p＜0.05p＜0.05
TLR4/MD-2-FcTLR4/MD-2-Fc 3030 p＜0.05p＜0.05
TLR4/CD36-FcTLR4/CD36-Fc 3030 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 3030 p＜0.05p＜0.05
表59 50周各组小鼠存活率Table 59 Survival rate of mice in each group at 50 weeks
组别Group 存活率％Survival rate% p值p value
空白组(模型组)Blank group (model group) 1010  To
对照IgGControl IgG 2020  To
TLR1-FcTLR1-Fc 8080 p＜0.05p＜0.05
TLR2-FcTLR2-Fc 8080 p＜0.05p＜0.05
TLR4-FcTLR4-Fc 7070 p＜0.05p＜0.05
TLR2/TLR4-FcTLR2/TLR4-Fc 8080 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 8080 p＜0.05p＜0.05
TLR4/MD-2-FcTLR4/MD-2-Fc 8080 p＜0.05p＜0.05
TLR4/CD36-FcTLR4/CD36-Fc 7070 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 8080 p＜0.05p＜0.05
表60各组肾脏病理评分小鼠存活率Table 60 Survival rate of mice with renal pathology score in each group
组别Group 病理评分Pathology score SDSD p值p value
空白组(模型组)Blank group (model group) 3.203.20 0.260.26  To
对照IgGControl IgG 3.303.30 0.260.26  To
TLR1-FcTLR1-Fc 0.850.85 0.470.47 p＜0.05p＜0.05
TLR2-FcTLR2-Fc 0.800.80 0.350.35 p＜0.05p＜0.05
TLR4-FcTLR4-Fc 0.750.75 0.260.26 p＜0.05p＜0.05
TLR2/TLR4-FcTLR2/TLR4-Fc 1.051.05 0.440.44 p＜0.05p＜0.05
TLR4/TLR6-FcTLR4/TLR6-Fc 1.101.10 0.700.70 p＜0.05p＜0.05
TLR4/MD-2-FcTLR4/MD-2-Fc 1.101.10 0.660.66 p＜0.05p＜0.05
TLR4/CD36-FcTLR4/CD36-Fc 0.800.80 0.350.35 p＜0.05p＜0.05
TLR2/Dectin-1-FcTLR2/Dectin-1-Fc 0.950.95 0.440.44 p＜0.05p＜0.05
综上，在狼疮小鼠模型中，本发明所述的免疫二聚体融合蛋白可以减少自身免疫抗体、减少脏器病理性炎症渗出、对于免疫系统疾病均具有良好的治疗效果，有利于后续的临床试验的开展。In summary, in the lupus mouse model, the immune dimer fusion protein of the present invention can reduce autoimmune antibodies, reduce pathological inflammatory exudation of organs, and has a good therapeutic effect on immune system diseases, which is beneficial to follow-up The development of clinical trials.
本发明中涉及的未说明部分与现有技术相同或采用现有技术加以实现。申请人声明，本发明通过上述实施例来说明本发明的详细方法，但本发明并不局限于上述详细方法，即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。The unspecified parts involved in the present invention are the same as the prior art or implemented by the prior art. The applicant declares that the present invention uses the above-mentioned embodiments to illustrate the detailed methods of the present invention, but the present invention is not limited to the above-mentioned detailed methods, which does not mean that the present invention must rely on the above-mentioned detailed methods to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

Claims (10)

  1. 一种二聚体免疫融合蛋白，其特征在于，包含二聚化的第一条多肽链和第二条多肽链，所述第一条多肽链的结构通式为Z1-Z2，所述第二条多肽链的结构通式为Y1-Y2，A dimeric immune fusion protein, which is characterized in that it comprises a dimerized first polypeptide chain and a second polypeptide chain, the structural formula of the first polypeptide chain is Z1-Z2, and the second polypeptide chain The general structural formula of a polypeptide chain is Y1-Y2,
    其中，Z1是(i)第一种模式识别受体的细胞外结构域或其功能变体或片段，或(ii)第一种共受体或其功能变体或片段；Z2是二聚化结构域或其功能变体或片段，Y1是(i)第二种模式识别受体的细胞外结构域或其功能变体或片段，或(ii)第二种共受体或其功能变体或片段；Y2是二聚化结构域或其功能变体或片段。Among them, Z1 is (i) the extracellular domain of the first pattern recognition receptor or its functional variant or fragment, or (ii) the first co-receptor or its functional variant or fragment; Z2 is dimerization Domain or its functional variant or fragment, Y1 is (i) the extracellular domain of the second pattern recognition receptor or its functional variant or fragment, or (ii) the second co-receptor or its functional variant Or fragment; Y2 is a dimerization domain or a functional variant or fragment thereof.
  2. 根据权利要求1所述的二聚体免疫融合蛋白，其特征在于：The dimeric immune fusion protein according to claim 1, wherein:
    其中，当Z1和Y1均为模式识别受体的细胞外结构域或其功能变体或片段时，所述第一模式识别受体和所述第二模式识别受体分别选自：TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、Dectin-1、Dectin-2、Mincle、CLEC2、CLEC5A、CLEC12A、DCIR以及CLECSF8中的任一个；Wherein, when both Z1 and Y1 are the extracellular domains of pattern recognition receptors or functional variants or fragments thereof, the first pattern recognition receptor and the second pattern recognition receptor are respectively selected from: TLR1, TLR2 , TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, Dectin-1, Dectin-2, Mincle, CLEC2, CLEC5A, CLEC12A, DCIR and CLECSF8;
    当Z1和Y1均为共受体或其功能变体或片段时，第一共受体和第二共受体分别选自CD14、MD-2、LBP以及CD36中的任一个。When both Z1 and Y1 are co-receptors or functional variants or fragments thereof, the first co-receptor and the second co-receptor are selected from any one of CD14, MD-2, LBP, and CD36, respectively.
  3. 根据权利要求1所述的二聚体免疫融合蛋白，其特征在于：The dimeric immune fusion protein according to claim 1, wherein:
    其中，Z2和Y2为IgG的Fc片段或改变其生物活性的Fc突变体，或利用Knob-in-hole技术、改变电荷极性的ART-Ig技术或BiMab技术构建的异源二聚IgG-Fc片段。Among them, Z2 and Y2 are Fc fragments of IgG or Fc mutants that change its biological activity, or a heterodimeric IgG-Fc constructed using Knob-in-hole technology, ART-Ig technology that changes charge polarity, or BiMab technology Fragment.
  4. 根据权利要求1所述的二聚体免疫融合蛋白，其特征在于：The dimeric immune fusion protein according to claim 1, wherein:
    其中，二聚化结构域Z2和Y2还包含有肽接头，所述肽接头由15-32个氨基酸残基组成，该氨基酸残基包含多个甘氨酸残基、一至八个半胱氨酸残基以及至少一个丝氨酸残基。Wherein, the dimerization domains Z2 and Y2 also contain a peptide linker, the peptide linker is composed of 15-32 amino acid residues, the amino acid residues include multiple glycine residues, one to eight cysteine residues And at least one serine residue.
  5. 根据权利要求1所述的二聚体免疫融合蛋白，其特征在于，The dimeric immune fusion protein of claim 1, wherein:
    其中，当Z1和Y1均为模式识别受体的细胞外结构域或其功能变体或片段时，Z1和Y1具有SEQ ID NO.1～18所示的氨基酸序列中的任一个序列；Wherein, when Z1 and Y1 are both extracellular domains of pattern recognition receptors or functional variants or fragments thereof, Z1 and Y1 have any one of the amino acid sequences shown in SEQ ID NO. 1-18;
    当Z1和Y1均为共受体或其功能变体或片段时，Z1和Y1具有SEQ ID NO.19～22所示的氨基酸序列中的任一个序列；When both Z1 and Y1 are co-receptors or functional variants or fragments thereof, Z1 and Y1 have any one of the amino acid sequences shown in SEQ ID NOs. 19-22;
    Z2和Y2具有SEQ ID NO.23-29所示的氨基酸序列中的任一个序列。Z2 and Y2 have any one of the amino acid sequences shown in SEQ ID NO. 23-29.
  6. 根据权利要求5所述的二聚体免疫融合蛋白，其特征在于：The dimeric immune fusion protein of claim 5, wherein:
    其中，Z1和Y1来源相同，均为TLR1、TLR2、TLR4、TLR6、Dectin-1、Dectin-2的细胞外结构域或其功能变体或片段，Among them, Z1 and Y1 are of the same origin, and both are the extracellular domains of TLR1, TLR2, TLR4, TLR6, Dectin-1, Dectin-2 or functional variants or fragments thereof,
    当Z1和Y1均为TLR1细胞外结构域或其功能变体或片段时，Z1-Z2多肽链和Y1-Y2多肽链的序列与SEQ ID NO:30或SEQ ID NO:31所示的氨基酸序列具有至少95％的同一性；When both Z1 and Y1 are TLR1 extracellular domains or functional variants or fragments thereof, the sequence of the Z1-Z2 polypeptide chain and Y1-Y2 polypeptide chain is the same as the amino acid sequence shown in SEQ ID NO: 30 or SEQ ID NO: 31 Have at least 95% identity;
    当Z1和Y1均为TLR2细胞外结构域或其功能变体或片段时，Z1-Z2多肽链和Y1-Y2多肽链的序列与SEQ ID NO:32或SEQ ID NO:33所示的氨基酸序列具有至少95％的同一 性；When both Z1 and Y1 are TLR2 extracellular domains or functional variants or fragments thereof, the sequence of the Z1-Z2 polypeptide chain and the Y1-Y2 polypeptide chain is the same as the amino acid sequence shown in SEQ ID NO: 32 or SEQ ID NO: 33 Have at least 95% identity;
    当Z1和Y1均为TLR4细胞外结构域或其功能变体或片段时，Z1-Z2多肽链和Y1-Y2多肽链的序列与SEQ ID NO:34或SEQ ID NO:35所示的氨基酸序列具有至少95％的同一性；When both Z1 and Y1 are TLR4 extracellular domains or functional variants or fragments thereof, the sequence of the Z1-Z2 polypeptide chain and Y1-Y2 polypeptide chain is the same as the amino acid sequence shown in SEQ ID NO: 34 or SEQ ID NO: 35 Have at least 95% identity;
    当Z1和Y1均为TLR6细胞外结构域或其功能变体或片段时，Z1-Z2多肽链和Y1-Y2多肽链的序列与SEQ ID NO:36或SEQ ID NO:37所示的氨基酸序列具有至少95％的同一性；When both Z1 and Y1 are TLR6 extracellular domains or functional variants or fragments thereof, the sequence of the Z1-Z2 polypeptide chain and Y1-Y2 polypeptide chain is the same as the amino acid sequence shown in SEQ ID NO: 36 or SEQ ID NO: 37 Have at least 95% identity;
    当Z1和Y1均为Dectin-1细胞外结构域或其功能变体或片段时，Z1-Z2多肽链和Y1-Y2多肽链的序列与SEQ ID NO:48或SEQ ID NO:58所示的氨基酸序列具有至少95％的同一性；When both Z1 and Y1 are Dectin-1 extracellular domains or functional variants or fragments thereof, the sequences of the Z1-Z2 polypeptide chain and Y1-Y2 polypeptide chain are the same as those shown in SEQ ID NO: 48 or SEQ ID NO: 58 The amino acid sequence has at least 95% identity;
    当Z1和Y1均为Dectin-2细胞外结构域或其功能变体或片段时，Z1-Z2多肽链和Y1-Y2多肽链的序列与SEQ ID NO:49所示的氨基酸序列具有至少95％的同一性。When both Z1 and Y1 are Dectin-2 extracellular domains or functional variants or fragments thereof, the sequences of the Z1-Z2 polypeptide chain and Y1-Y2 polypeptide chain are at least 95% of the amino acid sequence shown in SEQ ID NO: 49的identity.
  7. 根据权利要求5所述的二聚体免疫融合蛋白，其特征在于：The dimeric immune fusion protein of claim 5, wherein:
    其中，Z1和Y1来源于不同的细胞外结构域：Among them, Z1 and Y1 are derived from different extracellular domains:
    Z1-Z2多肽链序列与SEQ ID NO.38、SEQ ID NO.39、SEQ ID NO.41、SEQ ID NO.46、SEQ ID NO.42、SEQ ID NO.48、SEQ ID NO.50或SEQ ID NO.52所示的氨基酸序列具有至少95％的同一性；The Z1-Z2 polypeptide chain sequence is the same as SEQ ID NO.38, SEQ ID NO.39, SEQ ID NO.41, SEQ ID NO.46, SEQ ID NO.42, SEQ ID NO.48, SEQ ID NO.50 or SEQ The amino acid sequence shown in ID NO.52 has at least 95% identity;
    Y1-Y2多肽链序列与SEQ ID NO.39、SEQ ID NO.40、SEQ ID NO.41、SEQ ID NO.42、SEQ ID NO.43、SEQ ID NO.44、SEQ ID NO.45、SEQ ID NO.47、SEQ ID NO.50、SEQ ID NO.51、SEQ ID NO.53、SEQ ID NO.54、SEQ ID NO.55、SEQ ID NO.56或SEQ ID NO.57所示的氨基酸序列具有至少95％的同一性。The Y1-Y2 polypeptide chain sequence is the same as SEQ ID NO.39, SEQ ID NO.40, SEQ ID NO.41, SEQ ID NO.42, SEQ ID NO.43, SEQ ID NO.44, SEQ ID NO.45, SEQ ID NO.47, SEQ ID NO.50, SEQ ID NO.51, SEQ ID NO.53, SEQ ID NO.54, SEQ ID NO.55, SEQ ID NO.56 or SEQ ID NO.57 The sequence has at least 95% identity.
  8. 含有权利要求1～6任一项所述的二聚体免疫融合蛋白的药物组合物，其特征在于，还包括药学上可接受的药物载体。A pharmaceutical composition containing the dimeric immune fusion protein according to any one of claims 1 to 6, characterized in that it further comprises a pharmaceutically acceptable pharmaceutical carrier.
  9. 权利要求1～6任一项所述的二聚体免疫融合蛋白在制备诊断或治疗去除炎性介质相关疾病药物、试剂、试剂盒中的用途。Use of the dimeric immune fusion protein according to any one of claims 1 to 6 in the preparation of drugs, reagents and kits for diagnosis or treatment of diseases related to the removal of inflammatory mediators.
  10. 根据权利要求8所述的二聚体免疫融合蛋白在制备治疗炎性介质相关性疾病药物中的用途，其特征在于：The use of the dimeric immune fusion protein according to claim 8 in the preparation of a medicine for the treatment of diseases related to inflammatory mediators, characterized in that:
    其中，所述治疗炎性介质相关性疾病药物为能够结合病原微生物表面分子、细胞壁或细胞表面成分的药物；直接杀伤病原微生物或限制病原微生物生长的药物；抑制或减少炎性介质或细胞因子过度表达的药物；减少慢性炎性介质损伤或减轻脏器炎症纤维化作用的药物；抑制局部免疫耐受紊乱的药物；或抑制自身免疫耐受异常介导的炎性介质的药物。Wherein, the drugs for treating inflammatory mediator-related diseases are drugs that can bind to the surface molecules, cell walls, or cell surface components of pathogenic microorganisms; drugs that directly kill pathogenic microorganisms or restrict the growth of pathogenic microorganisms; inhibit or reduce excessive inflammatory mediators or cytokines Expressed drugs; drugs that reduce chronic inflammatory mediator damage or reduce organ inflammatory fibrosis; drugs that inhibit local immune tolerance disorders; or drugs that inhibit inflammatory mediators mediated by abnormal autoimmune tolerance.
    所述炎性介质包括病原微生物、酶、细胞因子、前列腺素、类花生酸、自三烯类、激肽类、补体、凝血因子、内毒素、肠毒素、脂多糖、诱导细胞凋亡的物质、胆汁盐、脂肪酸、磷脂、氧化副产物、活性氧簇、氧自由基、表面活性剂、离子、刺激性物质、细胞碎片、干扰素、免疫调节性抗体中的任一种或至少两种的组合。The inflammatory mediators include pathogenic microorganisms, enzymes, cytokines, prostaglandins, eicosanoids, autotrienes, kinins, complement, coagulation factors, endotoxins, enterotoxins, lipopolysaccharides, and substances that induce apoptosis Any one or at least two of, bile salts, fatty acids, phospholipids, oxidation by-products, reactive oxygen species, oxygen free radicals, surfactants, ions, irritating substances, cell debris, interferons, immunomodulatory antibodies combination.
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