WO2021109914A1 - Protéine de fusion immunitaire dimère, composition pharmaceutique et utilisation - Google Patents

Protéine de fusion immunitaire dimère, composition pharmaceutique et utilisation 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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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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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.

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Abstract

L'invention concerne une protéine de fusion immunitaire dimère soluble, comprenant une première chaîne polypeptidique dimérisée et une seconde chaîne polypeptidique dimérisée. La formule générale développée de la première chaîne polypeptidique est Z1-Z2, et la formule générale développée de la seconde chaîne polypeptidique est Y1-Y2, Z1 étant (i) un domaine extracellulaire d'un premier récepteur de reconnaissance de motif ou un variant fonctionnel ou un fragment de celui-ci, ou (ii) un premier co-récepteur ou un variant fonctionnel ou un fragment de celui-ci ; Z2 étant un domaine dimérisé ou un variant fonctionnel ou un fragment de celui-ci ; Y1 étant (i) un domaine extracellulaire d'un second récepteur de reconnaissance de motif ou un variant fonctionnel ou un fragment de celui-ci, ou (ii) un second co-récepteur ou un variant fonctionnel ou un fragment de celui-ci ; et Y2 étant un domaine dimérisé ou un variant fonctionnel ou un fragment de celui-ci. La protéine dimère peut bloquer l'invasion de pathogènes et limiter la génération et l'expansion de médiateurs inflammatoires, et peut être utilisée pour préparer des produits destinés à la prévention ou au traitement de maladies associées aux médiateurs inflammatoires.
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