Treatment or preventive agent for infectious diseases

Abstract

The present invention addresses the problem of providing a useful tool for treating or preventing an infectious disease. The present invention provides a therapeutic or prophylactic agent for an infectious disease, said agent including a fusion protein or a pharmaceutically acceptable salt thereof as an active ingredient, said fusion protein being a fusion of (a) at least one polypeptide that can bind to histones to form a polypeptide aggregate and includes an amino acid sequence which is identical or substantially identical to the amino acid sequence of the N-terminal domain of pentraxin 3, and (b) the Fc section of an immunoglobulin.

Description

Technical Field

[0001] The present invention relates to a therapeutic or prophylactic agent for infectious diseases.

Background Art

[0002] Pentraxin 3 (PTX3) is a pattern recognition molecule belonging to the pentraxin family. The pentraxin family is a general term for proteins having a common pentraxin domain on the C-terminal side, and is classified into two types, short pentraxin and long pentraxin, based on the characteristics of the primary structure. C-reactive protein (CRP), serum amyloid P component (SAP), etc. belong to short pentraxins, and PTX3 belongs to long pentraxins. The primary structure of PTX3 is composed of a long N-terminal domain (18 to 178 amino acids) and a C-terminal pentraxin domain (179 to 381 amino acids) compared to short pentraxins, and its higher-order structure forms an octamer via disulfide bonds.

[0003] PTX3 is expressed in a variety of cell types by inflammatory signals, and is characterized by showing a local expression pattern, unlike CRP and SAP produced in the liver. As a characteristic production mechanism of PTX3, PTX3 stored in neutrophil granules is released extracellularly by stimulation with pathogens or Toll-like receptor (TLR) agonists. The released PTX3 functions as a constituent protein of a pathogen capture / killing structure composed of DNA and an antibacterial protein group called Neutrophil extracellular traps (NETs) (Non-Patent Document 1). PTX3 has a wide variety of functions in vivo, and for example, regulation of inflammation, innate immune response, maintenance of pregnancy, etc. have been reported (Non-Patent Document 2). PTX3 also has a function of binding to a number of proteins, and exhibits specific functions in cooperation with binding proteins.

[0004] It has been reported that PTX3 blood concentrations increase in various infections (Non-Patent Literature 4). In particular, in sepsis, PTX3 concentrations, which are normally below 2 ng / mL, rise to around 200-800 ng / mL and are known to correlate with survival rates (Non-Patent Literature 3). There are also reports that PTX3 transgenic mice are resistant to sepsis-induced lethality (Non-Patent Literature 4).

[0005] The inventors have found that the activity of binding to (aggregating with) histones and suppressing histone cytotoxicity is sufficient with the N-terminal domain of PTX3, rather than the C-terminal domain of PTX3, and have further confirmed that the 50-amino acid peptide of the N-terminal domain of PTX3 has a cytotoxicity-suppressing effect (Patent Document 1).

[0006] PTX3 is thought to directly bind to various pathogens and trigger a defense response. It is also known to directly bind to viruses such as influenza viruses and coronaviruses (SARS-CoV, MHV mouse hepatitis virus). Coronaviruses have a spike protein on their surface, which infects host cells by recognizing and binding to angiotensin-converting enzyme 2 (ACE2) on the cell membrane. SARS-CoV-2, which caused the 2020 pandemic, has a spike protein with a highly synchronic amino acid sequence compared to previous SARS and MERS viruses, and possesses a strong binding affinity to ACE2. Antibodies against this spike protein include neutralizing antibodies that inhibit the binding of the virus to ACE2 and thus inhibit infection. Furthermore, it has been pointed out that COVID-19 causes pneumonia, and in severe cases, damage to vascular endothelial cells, leading to vasculitis accompanied by cytokine storms, thrombosis, and further multi-organ failure, ultimately resulting in death (Non-Patent Literature 5 and 6). Although the detailed mechanisms are still unclear, mutations in the nucleocapsid protein of the SARS-CoV-2 virus are associated with severe disease, and thrombosis and endothelial cell damage caused by NETs (Neutrophil extracellular traps) released from neutrophils are also considered to be mechanisms of severe disease, and are targets for new drugs (Non-Patent Literature 7). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Jaillon, S., et al. (2007) J Exp Med 204, 793-804 [Non-Patent Document 2] Mantovani, A., et al. (2008) J Clin Immunol 28, 1-13 [Non-Patent Document 3] Mauri, T., et al. (2010) Intensive Care Med 36, 621-629 [Non-Patent Document 4] Dias, AA, et al. (2001) J Leukoc Biol 69, 928-936 [Non-Patent Document 5] Barnes BJ et al., JEM 217-6, 2020 [Non-Patent Document 6] Libby, P and Luscher T. European Heart Journal 41, 3038-3044, 2020 [Non-Patent Document 7] Narasaraju T et al., Frontiers in Pharmacology, 11 :870, 2020 [Patent Documents]

[0008] [Patent Document 1] WO / 2013 / 191280 publication [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] PTX3 is primarily present in neutrophils and is released as part of neutrophil extracellular traps (NETs) during inflammation, possessing antibacterial, complement-activating, and opsonizing effects. The inventors previously detected the binding of PTX3 to histones and found that it suppresses vascular endothelial cell damage caused by extracellular histones released from neutrophils in sepsis (Patent Document 1). PTX3 is a protein with a molecular weight of approximately 40,000 and forms a large complex by binding to multiple blood proteins. It was known that large molecular weight proteins are difficult to formulate, and that PTX3 is a protein that easily aggregates and is difficult to prepare. Therefore, in Patent Document 1, an attempt was made to identify the active partial peptide, and as a result, it was found that the 50-amino acid peptide at the N-terminus has an inhibitory effect on histone damage in vascular endothelial cells (Patent Document 1).

[0010] The inventors subsequently administered a 50-amino acid peptide (PTX3N50) to a mouse model, but were unable to improve the survival rate of the sepsis model mice. This suggests that there may be issues with the peptide's stability in the blood. The present invention aims to provide a useful tool for treating or preventing infectious diseases. The present invention further aims to demonstrate that PTX3 directly binds to proteins involved in infection by SARS-CoV-2, the causative virus of COVID-19, and to identify the sequence of PTX3 involved in this binding, thereby providing infection-preventive drugs, therapeutic agents, etc. [Means for solving the problem]

[0011] The inventors, through diligent research to solve the above problems, have found that by integrating the peptide of the N-terminal domain of PTX3 into an Fc fusion form, a remarkable survival effect can be obtained in a mouse sepsis model. Furthermore, the inventors have shown that PTX3, which is involved in the innate immune response, binds to the spike protein involved in coronavirus infection, and have identified an important partial peptide of PTX3 involved in this binding, demonstrating that it is possible to design a molecule that controls infection. They have also shown that the nucleocapsid protein of coronavirus damages vascular endothelial cells, similar to histones contained in NETs, ​​and that a partial peptide of PTX3 has an inhibitory effect on this. Therefore, they have shown that it is possible to suppress coronavirus infection and inhibit its severity using peptides derived from PTX3. This invention was completed based on the above findings.

[0012] In other words, the present invention relates to the following: <1> A therapeutic or prophylactic agent for infectious diseases comprising, as an active ingredient, a fusion protein or a pharmacoagulably accepted salt thereof of (a) at least one polypeptide having an amino acid sequence identical or substantially identical to the amino acid sequence of the N-terminal domain of pentraxin 3, which can bind to histones to form polypeptide aggregates, and (b) the Fc portion of an immunoglobulin. <2> The amino acid sequence of the N-terminal domain of pentraxin 3 is a partial sequence of 15 amino acids or more in length from the amino acid sequence represented by Sequence ID No. 2. <1> The therapeutic or prophylactic agents described above. <3> The amino acid sequence of the N-terminal domain of pentraxin 3 is a partial sequence of 15 amino acids or more in length from amino acid positions 1 to 120 of the amino acid sequence represented by SEQ ID NO: 2. <1> or <2> Therapeutic or prophylactic agents listed below: <4> The amino acid sequence of the N-terminal domain of pentraxin 3 contains any of the following regions: <1> from <3> Any of the following therapeutic or prophylactic agents: (1) The region consisting of amino acids 18-67 of the amino acid sequence represented by Sequence ID No. 2, (2) The region consisting of amino acids at positions 18 to 37 of the amino acid sequence represented by SEQ ID NO: 2, (3) The region consisting of amino acids at positions 38 to 57 of the amino acid sequence represented by SEQ ID NO: 2, (4) The region consisting of amino acids at positions 58 to 77 of the amino acid sequence represented by SEQ ID NO: 2, (5) The region consisting of amino acids at positions 78 to 97 of the amino acid sequence represented by SEQ ID NO: 2, (6) The region consisting of amino acids at positions 98 to 117 of the amino acid sequence represented by SEQ ID NO: 2, (7) The region consisting of amino acids at positions 85 to 144 of the amino acid sequence represented by SEQ ID NO: 2, and (8) The region consisting of amino acids at positions 119 to 176 of the amino acid sequence represented by SEQ ID NO: 2. <5> The therapeutic or prophylactic agent according to any one of <2> to <4>, wherein at least one cysteine residue in the amino acid sequence represented by SEQ ID NO: 2 is substituted with another amino acid residue. <6> The therapeutic or prophylactic agent according to any one of <2> to <5>, wherein the cysteine residues at positions 47 and 49 in the amino acid sequence represented by SEQ ID NO: 2 are substituted with other amino acid residues. <7> The therapeutic or prophylactic agent according to <5> or <6>, wherein the other amino acid residue is a serine residue. <8> (a) At the C-terminus of a polypeptide containing an amino acid sequence identical or substantially identical to the amino acid sequence of the N-terminal domain of pentraxin 3 that can bind to histones to form polypeptide aggregates, (b) the N-terminus of the immunoglobulin Fc portion is fused. The therapeutic or prophylactic agent according to any one of <1> to <7>. <9> The therapeutic or prophylactic agent according to any one of <1> to <8>, wherein the infectious disease is sepsis. <10> The therapeutic or prophylactic agent according to any one of <1> to <8>, wherein the infectious disease is COVID-19 infection.

Advantages of the Invention

[0013] According to the present invention, by administering a fusion protein of the N-terminal domain of pentraxin 3 and Fc to a patient, an infectious disease can be treated or prevented.

Brief Description of Drawings

[0014] [Figure 1] Figure 1 shows the design of the PTX3-Fc fusion protein. [Figure 2] Figure 2 shows the inhibitory effect of the PTX3-Fc fusion protein on histone-induced vascular endothelial cell injury. [Figure 3] Figure 3 shows the improvement of the survival rate of septic model mice by the PTX3-Fc fusion protein. [Figure 4] Figure 4 shows the inhibitory effect of 20-amino acid peptides (100 μg / ml) of five types of PTX3 on histone H3 (100 μg / ml) injury. [Figure 5] Figure 5 shows the inhibitory effect of 20-amino acid peptides (100 μg / ml) of five types of PTX3 on histone H4 (100 μg / ml) injury. [Figure 6] Figure 6 shows the design of the PTX3-Fc fusion protein containing 60 amino acids. [Figure 7] Figure 7 shows the results of the binding experiment between various 60-amino acid PTX3-Fc fusion proteins and the spike protein (SPN-C52H8). [Figure 8] Figure 8 shows the vascular endothelial cell (HUVEC) injury activity of the coronavirus nucleocapsid protein. H2O: water, Opti-MEM (HUVEC culture medium), HCoV-NL63: viral nucleocapsid (N) protein, SARS-CoV: viral N protein, SARS-CoV-2: viral N protein [Figure 9] Figure 9 shows the results of the binding experiment between various 60-amino acid PTX3-Fc fusion proteins and the N protein (NP) of SARS-CoV-2. [Figure 10]Figure 10 shows the inhibitory effect of the 60PTX3-Fc protein on the HUVEC-damaging activity of the SARS-CoV-2 N protein (NP). [Modes for carrying out the invention]

[0015] Embodiments of the present invention will be described below. In the embodiment of the present invention, a PTX3-Fc fusion protein (PTX3(N50)-Fc), obtained by fusing the N-terminal 50 amino acid residues of PTX3 with the immunoglobulin Fc moiety, and its amino acid variants were purified from the culture medium of CHO recombinant cells using an affinity column (Figure 1). PTX3(N50)-Fc and its amino acid variants bound to histones and suppressed histone-induced vascular endothelial cell damage (Figure 2). Furthermore, in experiments using a sepsis model mouse administered LPS, both PTX3(N50)-Fc and its amino acid variants showed a significant life-prolonging effect, but the amino acid variants were found to have a higher life-prolonging effect. As described above, the PTX3-Fc fusion protein (PTX3(N50)-Fc) and its amino acid variants have been shown to be useful as therapeutic or prophylactic agents for infections such as sepsis.

[0016] The present invention provides a therapeutic or prophylactic agent for infectious diseases. (a) at least one polypeptide having an amino acid sequence identical or substantially identical to the amino acid sequence of the N-terminal domain of pentraxin 3, which can bind to histones to form polypeptide aggregates, (b) With the Fc portion of immunoglobulin, It contains a fusion protein, or a pharmacologically acceptable salt thereof, as an active ingredient.

[0017] A polypeptide containing an amino acid sequence identical or substantially identical to the amino acid sequence of the N-terminal domain of pentraxin 3, which can bind to histones to form polypeptide aggregates, may be referred to below as "polypeptide used in the present invention."

[0018] PTX3 is a known protein belonging to a protein family generally called the pentraxin family, and specifically to the long pentraxin family. In this invention, PTX3 is usually derived from vertebrates.

[0019] Examples of vertebrates include mammals, birds, fish, amphibians, and reptiles. Mammals are not particularly limited, but examples include rodents such as mice, rats, hamsters, and guinea pigs, and laboratory animals such as rabbits; livestock such as pigs, cows, goats, horses, sheep, and minks; pets such as dogs and cats; and primates such as humans, monkeys, rhesus macaques, marmosets, orangutans, and chimpanzees. Examples of birds include chickens, quail, ducks, geese, turkeys, ostriches, emus, ostriches, guinea fowl, and pigeons. The vertebrates are preferably mammals, and more preferably humans.

[0020] In this specification, "derived from organism X" for polypeptides and polynucleotides means that the amino acid sequence of the polypeptide or the nucleic acid sequence of the polynucleotide is identical to the amino acid sequence of the polypeptide or the nucleic acid sequence of the polynucleotide that is naturally expressed in organism X.

[0021] Human-derived PTX3 typically consists of a single-chain polypeptide with a total length of 381 amino acids. A representative amino acid sequence of human-derived PTX3 polypeptide is registered as Genebank Accession No. AAH39733 (SEQ ID NO: 2). In addition, a representative nucleotide sequence encoding human-derived PTX3 polypeptide is registered as Genebank Accession No. BC039733 (SEQ ID NO: 1).

[0022] Typically, PTX3 polypeptides expressed in cells undergo cleavage of their N-terminal signal peptide during the process of secretion into the extracellular space, becoming mature PTX3 polypeptides. In this specification, PTX3 polypeptides are preferably mature PTX3 polypeptides. For example, in the amino acid sequence of human-derived PTX3, the amino acid segment from the N-terminus (1st to 17th amino acids) is a signal peptide, which is cleaved during the process of secretion into a mature polypeptide. Therefore, human-derived mature PTX3 polypeptides typically include the amino acid sequence from position 18 to 381 of the amino acid sequence represented by Sequence ID No. 2.

[0023] In the present invention, the N-terminal domain of PTX3 is the region or a part of the region that is N-terminal to the pentraxin domain of the PTX3 polypeptide (preferably a mature PTX3 polypeptide) described above. The pentraxin domain is a domain common to members of the pentraxin superfamily such as CRP (C-reactive protein) and SAP (Serum amyloid P component), and is registered in the NCBI Conserved Domains as accession number cd00152. Therefore, a person skilled in the art can identify the pentraxin domain of any PTX3 based on its sequence information and then identify the N-terminal domain of that PTX3. Typically, the pentraxin domain of human-derived PTX3 corresponds to the region consisting of amino acids 179 to 380 of the amino acid sequence represented by SEQ ID NO: 2. Therefore, the N-terminal domain of human-derived PTX3 is usually the region or a part of the region consisting of amino acids 1 to 178 of the amino acid sequence represented by SEQ ID NO: 2, and preferably the region or a part of the region consisting of amino acids 18 to 178 of the amino acid sequence represented by SEQ ID NO: 2. If PTX3 is of human origin, its N-terminal domain is preferably the amino acid region from the 18th to the 178th amino acid from the N-terminus. The amino acid sequence for the region consisting of the 18th to the 178th amino acids of the amino acid sequence represented by SEQ ID NO: 2 is shown in SEQ ID NO: 3.

[0024] When the N-terminal domain of PTX3 is part of the region of the PTX3 polypeptide that is N-terminal to the pentraxin domain, its length is not particularly limited as long as it has the activity to bind to histones and form polypeptide aggregates, but it is at least 8 amino acids, for example 10 amino acids or more, preferably 15 amino acids or more, and more preferably 20 amino acids or more. The length of the amino acids may be 30 amino acids or more, or even 50 amino acids or more.

[0025] The amino acid sequence of the N-terminal domain of PTX3 is preferably a partial sequence of at least eight consecutive amino acids (for example, 10 or more amino acids, preferably 15 or more amino acids, more preferably 20 or more amino acids) from the amino acid sequence from position 1 to 120 of the amino acid sequence represented by Sequence ID No. 2. The amino acid sequence of the N-terminal domain of PTX3 is preferably an amino acid sequence consisting of the amino acids from the Xth to the Yth position of the amino acid sequence represented by Sequence ID No. 2. Here, X is preferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 245, 46, or 47, and Y is preferably 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, or 58.

[0026] In one example of the present invention, the amino acid sequence of the N-terminal domain of PTX3 includes any of the following regions. (1) The region consisting of amino acids 18-67 of the amino acid sequence represented by Sequence ID No. 2, (2) The region consisting of amino acids 18 to 37 of the amino acid sequence represented by Sequence ID No. 2, (3) The region consisting of amino acids 38 to 57 of the amino acid sequence represented by Sequence ID No. 2, (4) The region consisting of amino acids 58 to 77 of the amino acid sequence represented by Sequence ID No. 2, (5) The region consisting of amino acids 78 to 97 of the amino acid sequence represented by Sequence ID No. 2, (6) The region consisting of amino acids 98 to 117 of the amino acid sequence represented by Sequence ID No. 2, (7) The region consisting of amino acids 85 to 144 of the amino acid sequence represented by Sequence ID No. 2, and (8) The region consisting of amino acids 119 to 176 of the amino acid sequence represented by Sequence ID No. 2.

[0027] The polypeptide used in the present invention contains an amino acid sequence that is identical or substantially identical to the N-terminal domain of PTX3. An amino acid sequence substantially identical to the N-terminal domain of PTX3 includes amino acid sequences having 50% or more, preferably 60% or more, more preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, particularly preferably 95% or more, and most preferably 99% or more identity with the N-terminal domain of PTX3. Here, "identity" means the percentage (%) of identical amino acids relative to all overlapping amino acid residues in the optimal alignment when the two amino acid sequences are aligned using a mathematical algorithm known in the art.

[0028] Examples of amino acid sequences substantially identical to the amino acid sequence of the N-terminal domain of PTX3 include: (1) an amino acid sequence in which one or more amino acids (preferably about 1 to 30, preferably about 1 to 10, and more preferably one or two) are deleted from the amino acid sequence of the N-terminal domain of PTX3; (2) an amino acid sequence in which one or more amino acids (preferably about 1 to 30, preferably about 1 to 10, and more preferably one or two) are added to the amino acid sequence of the N-terminal domain of PTX3; (3) an amino acid sequence in which one or more amino acids (preferably about 1 to 30, preferably about 1 to 10, and more preferably one or two) are inserted into the amino acid sequence of the N-terminal domain of PTX3; (4) an amino acid sequence in which one or more amino acids (preferably about 1 to 30, preferably about 1 to 10, and more preferably one or two) are replaced with other amino acids; or (5) an amino acid sequence that combines these.

[0029] As described above, when an amino acid sequence is inserted, deleted, added, or substituted, the location of such insertion, deletion, addition, or substitution is not particularly limited, as long as the polypeptide having such an amino acid sequence has the activity to bind to histones and form polypeptide aggregates. Examples of amino acid sequences substantially identical to the amino acid sequence of the N-terminal domain of PTX3 usable in the present invention include the amino acid sequences of its homologs in other vertebrates other than humans mentioned above.

[0030] In the present invention, it is preferable that at least one cysteine ​​residue in the amino acid sequence represented by SEQ ID NO: 2 is substituted with another amino acid residue. More preferably, the cysteine ​​residues at positions 47 and 49 in the amino acid sequence represented by SEQ ID NO: 2 are substituted with another amino acid residue. The other amino acid residue is preferably a serine residue.

[0031] As a polypeptide containing an amino acid sequence substantially identical to the amino acid sequence of the N-terminal domain of PTX3, a polypeptide containing an amino acid sequence substantially identical to the amino acid sequence of the N-terminal domain of PTX3 and having substantially the same activity as the polypeptide containing the amino acid sequence of the N-terminal domain of PTX3 is preferred.

[0032] Examples of substantially identical activity include the activity of binding to histones and forming polypeptide aggregates. Here, "substantially identical" means that their properties are qualitatively (for example, physiologically or pharmacologically) identical. Therefore, it is preferable that the activity of polypeptides consisting of substantially identical amino acid sequences is equivalent, but the degree of activity (for example, about 0.01 to about 100 times, preferably about 0.1 to about 10 times, more preferably 0.5 to 2 times) and quantitative elements such as the molecular weight of the polypeptides may differ.

[0033] The length of the polypeptide used in the present invention is not particularly limited as long as it has the activity to bind to histones and form polypeptide aggregates, but from the viewpoint of ease of preparation and polypeptide stability, it is, for example, 200 amino acids or less, preferably 100 amino acids or less, more preferably 50 amino acids or less, and even more preferably 30 amino acids or less.

[0034] Examples of polypeptides used in the present invention include an amino acid sequence consisting of amino acids 18 to 67 of the amino acid sequence represented by Sequence ID No. 2, and an amino acid sequence in which the cysteine ​​residues at positions 47 and 49 in the amino residual sequence are substituted with other amino acid residues (for example, serine residues).

[0035] The polypeptide used in the present invention has the activity to bind to histones and form polypeptide aggregates. Herein, "forming aggregates" means that the N-terminal domain of PTX3 and histones bind through specific interactions to form a dense, water-insoluble aggregate state. Furthermore, "polypeptide aggregate" in this specification means a water-insoluble, bulky aggregate comprising the N-terminal domain of PTX3 and histones.

[0036] Histones are a type of protein that makes up eukaryotic chromatin (chromosomes) and have the activity to bind to DNA. In this specification, histones are usually derived from vertebrates, preferably mammals, and most preferably humans. Histones include H1, H2A, H2B, H3, and H4. The N-terminal domain of PTX3 and the polypeptide of the present invention usually have the activity to bind to at least one histone selected from the group consisting of H1, H2A, H2B, H3, and H4, preferably at least one histone selected from the group consisting of H1, H3, and H4, and more preferably H1, H3, and H4, respectively, to form polypeptide aggregates.

[0037] The presence or absence of activity in binding to histones to form polypeptide aggregates can be confirmed, for example, by visual observation of polypeptide aggregate formation. For example, if equal volumes of a 1.0 mg / ml histone solution (in buffer (150 mM NaCl, 20 mM HEPES, 4 mM CaCl2, 0.005% surfactant P20 (pH 7.4))) and a 1.0 mg / ml polypeptide solution to be evaluated (in the buffer) are mixed, and the presence of particulate matter can be confirmed visually, it can be determined that the polypeptide to be evaluated has the activity to bind to histones to form polypeptide aggregates. Visual observation can be further confirmed by using an electron microscope to more clearly confirm the formation of polypeptide aggregates.

[0038] Furthermore, the presence or absence of activity in binding to histones to form polypeptide aggregates can also be confirmed by measuring the UV-Vis absorption spectrum. For example, when equal amounts of a 0.1 mg / ml histone solution (in buffer (150 mM NaCl, 20 mM HEPES, 4 mM CaCl2, 0.005% surfactant P20 (pH 7.4))) and various concentrations of the polypeptide under evaluation (in the buffer) are mixed and the spectrum is measured, if a dose-dependent increase in the UV-Vis light (e.g., 310 nm) absorption spectrum due to scattering of aggregates is observed, or if an increase in the absorption spectrum is observed compared to histone only, the N-terminal domain of PTX3 only, or the polypeptide of the present invention only at the same concentration, it can be determined that the polypeptide under evaluation has the activity to bind to histones to form polypeptide aggregates.

[0039] Furthermore, the presence or absence of activity to bind to histones and form polypeptide aggregates can also be confirmed using immunochromatography, Octarony's method, and immunoturbidimetric analysis.

[0040] When polypeptide aggregate formation is confirmed using at least one of the above methods, the polypeptide being evaluated is judged to have the activity to bind to histones and form polypeptide aggregates.

[0041] In the present invention, (a) at least one polypeptide having an amino acid sequence identical or substantially identical to the amino acid sequence of the N-terminal domain of pentraxin 3, which can bind to histones to form polypeptide aggregates, (b) With the Fc portion of immunoglobulin, Use a fusion protein.

[0042] Immunoglobulin molecules are formed by disulfide bonds (SS bonds) between two heavy chains and two light chains. The heavy chain of an immunoglobulin consists of a variable region and a constant region. In mammals, there are five types of constant regions: α, δ, ε, γ, and μ, and based on the differences in these constant regions, there are five classes of antibodies (IgA for α, IgD for δ, IgE for ε, IgG for γ, and IgM for μ). The constant regions of α, δ, and γ consist of three domains of approximately 340 amino acids, while the constant regions of μ and ε consist of four domains of approximately 440 amino acids. The light chain of an immunoglobulin also consists of a variable region and a constant region. In mammals, there are two types of light chains, λ (Lambda) and κ (Kappa), based on the differences in their constant regions. The Fc portion of an immunoglobulin is the part composed of the constant regions of the heavy chain and the light chain.

[0043] The immunoglobulin can be any of IgA, IgD, IgE, IgG, or IgM, but IgG is preferred. The isotype (subclass) of the immunoglobulin is also not particularly limited. For example, in the case of IgG, any of IgG1, IgG2a, IgG2b, IgG3, or IgG4 may be used.

[0044] The amino acid sequences of the Fc region of the various immunoglobulins described above are publicly known. That is, genes encoding the Fc region of immunoglobulins have already been isolated and identified in large numbers in mammals, including humans. Numerous nucleotide sequences have also been reported. For example, sequence information of the nucleotide sequences containing the Fc regions of human IgG1, IgG2, IgG3, and IgG4 is available in public DNA databases such as NCBI, and is registered with access numbers AJ294730, AJ294731, AJ294732, and AJ294733, respectively. Therefore, those skilled in the art can design primers or probes specific to the Fc region and obtain and use cDNA encoding the Fc region using general molecular biological techniques. In this case, the animal species and subtype of the gene encoding the Fc region are not particularly limited, but genes encoding the Fc region such as human IgG1 or IgG2, or mouse IgG2a or IgG2b, which have strong binding affinity to protein A / G, are preferred.

[0045] The binding order between the peptide and the immunoglobulin Fc moiety used in this invention is not particularly limited. For example, the N-terminus of the immunoglobulin Fc moiety can be fused to the C-terminus of the peptide used in this invention.

[0046] The peptide and the immunoglobulin Fc portion used in the present invention may be fused via a linker sequence. As the linker sequence, for example, an amino acid sequence of 3 to 20 amino acid residues, preferably 3 to 10 amino acid residues (one example being GGGGS) can be used.

[0047] A single polypeptide ("a polypeptide used in the present invention") containing an amino acid sequence identical or substantially identical to the amino acid sequence of the N-terminal domain of pentraxin 3, which can bind to histones to form polypeptide aggregates) may be used, or two or more may be used.

[0048] In other words, when using one peptide in the present invention, one molecule of the peptide is fused with the Fc portion of one molecule of immunoglobulin. When using two or more peptides in the present invention, two or more molecules of the peptide are fused with the Fc portion of one molecule of immunoglobulin.

[0049] The fusion protein in the present invention may be a free form or a pharmacologically acceptable salt. Such salts may be salts with pharmacologically acceptable acids or bases. Examples of such salts include salts with inorganic acids (e.g., hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), salts with organic acids (e.g., acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid), alkali metal salts (e.g., sodium salts, potassium salts), alkaline earth metal salts (e.g., calcium salts, barium salts), magnesium salts, aluminum salts, and the like.

[0050] The fusion protein in this invention is preferably isolated. "Isolation" means that an operation has been performed to remove factors other than the target component. The purity of the isolated polypeptide X (percentage of polypeptide X to the total polypeptide weight) is usually 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably substantially 100%.

[0051] The fusion protein in the present invention can be produced by culturing a transformant into which an expression vector containing a polynucleotide comprising the nucleotide sequence encoding the fusion protein or its complementary sequence has been introduced using a method known to the present day, and then separating the polypeptide from the resulting culture.

[0052] The polynucleotide containing the nucleotide sequence or complementary sequence encoding the fusion protein may be DNA, RNA, or a DNA / RNA chimera, but is preferably DNA. The nucleic acid may also be double-stranded or single-stranded. If double-stranded, it may be double-stranded DNA, double-stranded RNA, or a DNA:RNA hybrid.

[0053] DNA containing a nucleotide sequence or complementary sequence encoding a fusion protein can include chromosomal DNA, cDNA, synthetic DNA, or combinations thereof. The chromosomal DNA and cDNA encoding the polypeptide can be directly amplified by Polymerase Chain Reaction (PCR) or Reverse Transcriptase-PCR (RT-PCR) using chromosomal DNA fractions and total RNA or mRNA fractions prepared from the aforementioned cells or tissues as templates. Alternatively, the chromosomal DNA and cDNA encoding the polypeptide used in this invention can be cloned from known chromosomal DNA libraries and cDNA libraries prepared by inserting chromosomal DNA, cDNA, and total RNA or mRNA fragments prepared from the aforementioned cells or tissues into a suitable vector, using colony or plaque hybridization or PCR, respectively.

[0054] The fusion protein in this invention can also be produced according to known peptide synthesis methods. These peptide synthesis methods may be either solid-phase or liquid-phase. The desired fusion protein can be produced by condensing a partial peptide or amino acid that can constitute the fusion protein with the remaining portion, and removing the protecting group if the product has one. Condensation and removal of the protecting group can be carried out according to known methods.

[0055] The fusion protein obtained as described above can be purified by known methods. Examples of purification methods include solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, and combinations thereof. Furthermore, if the polypeptide obtained by the above method is in the form of a free form, the free form can be converted to a suitable salt (preferably a pharmacologically acceptable salt) by known methods or similar methods. Conversely, if the polypeptide is obtained as a salt, the salt can be converted to a free form or another salt (preferably a pharmacologically acceptable salt) by known methods or similar methods.

[0056] The fusion protein used in the present invention or a pharmacoagulably acceptable salt thereof can be mixed with a pharmacoagulably acceptable carrier as needed to form a pharmaceutical composition which can be used as a therapeutic or prophylactic agent for infectious diseases. By administering a prophylactic or therapeutically effective amount of the fusion protein of the present invention or a pharmacoagulably acceptable salt thereof to a mammal, infectious diseases in the mammal (preferably a human) can be prevented or treated. The target mammal is preferably a human, but other mammals may also be used. Examples of such mammals include mice, rats, rabbits, dogs, cats, horses, sheep, cattle, goats, pigs, miniature pigs, hairless pigs, monkeys, and the like.

[0057] Infectious diseases are a general term for illnesses caused by infection with pathogens such as bacteria, fungi, viruses, parasites, and abnormal prions. The term "infectious disease" encompasses a series of events, including infections that do not cause symptoms (asymptomatic infections) and those that develop symptoms later. Infectious diseases can occur in various organs of the body, such as encephalitis, rhinitis, pharyngitis, pneumonia, infectious endocarditis, hepatitis, and enteritis, primarily causing inflammation in the infected organ. Novel coronavirus infection (COVID-19) is also one type of infectious disease.

[0058] Sepsis is a condition in which an infectious microorganism and its toxins act on the body beyond the site of infection, triggering a severe systemic inflammatory response. Sepsis is a systemic inflammatory response syndrome, and without treatment, it can lead to shock, multiple organ failure, disseminated intravascular coagulation (DIC), and eventually death. Since sepsis often develops as a complication of a weakened immune system, the treatment outcomes are not always favorable. The infectious disease treatment or preventive agent of the present invention can preferably be used as a treatment or preventive agent for sepsis.

[0059] In infectious diseases, acute organ injury may occur. Examples of acute organ injury include acute lung injury, acute kidney injury, acute liver injury, intestinal dysfunction, DIC (disseminated intravascular coagulation), ARDS (acute respiratory distress syndrome), circulatory collapse (septic shock), and septic encephalopathy. The infectious disease treatment or preventive agent of the present invention can preferably be used as a treatment or preventive agent for acute organ injury.

[0060] In the present invention, pharmacologically acceptable carriers used in pharmaceutical compositions include various organic or inorganic carrier substances commonly used as formulation materials, such as excipients, lubricants, binders, and disintegrants in solid formulations; and solvents, solubilizers, suspending agents, isotonic agents, buffers, and analgesics in liquid formulations. Pharmaceutical additives such as preservatives, antioxidants, colorants, and sweeteners may also be used as needed. These carriers can be compounds known to be usable in pharmaceutical compositions, and commercially available products are preferably used. Furthermore, the amounts of each carrier can be appropriately determined by those skilled in the art.

[0061] Examples of dosage forms for the above-mentioned pharmaceutical compositions include oral preparations such as tablets, capsules (including soft capsules and microcapsules), granules, powders, syrups, emulsions, and suspensions; and parenteral preparations such as injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections, etc.), topical preparations (e.g., nasal administration preparations, transdermal preparations, ointments, etc.), suppositories (e.g., rectal suppositories, vaginal suppositories, etc.), pellets, drip preparations, and sustained-release preparations (e.g., sustained-release microcapsules, etc.). Such pharmaceutical compositions can be manufactured by methods commonly used in the pharmaceutical technology field, such as those described in the Japanese Pharmacopoeia.

[0062] The dosage of the therapeutic or prophylactic agent for infectious diseases in the present invention is preferably an amount sufficient for the fusion protein of the present invention or a pharmacoacceptable salt thereof to bind to histones and form aggregates in the body of the target mammal (e.g., in the blood) to neutralize the histones. When the therapeutic or prophylactic agent of the present invention is administered parenterally, the dosage will vary depending on the target patient, target organ, symptoms, and method of administration. For example, in a patient weighing 60 kg, the amount of the polypeptide of the present invention is approximately 10 to 1000 mg per day, preferably approximately 100 to 500 mg, and more preferably approximately 200 to 400 mg. Even when the target patient is not human, the amount can be administered converted to a per 60 kg body weight.

[0063] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples. [Examples]

[0064] <Example 1> Construction, expression, and purification of PTX3-Fc fusion protein An expression construct for a PTX3-Fc fusion protein, which ligates the N-terminal (1-50 amino acid residues) peptide of pentraxin 3 (PTX3) with the immunoglobulin Fc portion, was prepared as follows: The sequence encoding the PTX partial-length peptide, in which GGGGS is ligated to the C-terminus of 50 amino acids (18-67 amino acid residues, with cysteine ​​residues at positions 47 and 49 replaced with serine residues), was totally synthesized using a sequence optimized for mammalian cell expression. This sequence was then recloned into the XhoI / SpeI site of the pCAG-Hyg mIgG1-Fc vector (Wako) to produce the expression vector PTX3(N50)-Fc (Figure 1a).

[0065] Similarly, a sequence encoding a PTX partial-length peptide, consisting of 50 amino acids of PTX3 (a mutant in which cysteine ​​residues 18-67 and 47th and 49th are replaced with serine residues) with GGGGS ligated to the C-terminus, was totally synthesized using a sequence optimized for mammalian cell expression. This sequence was then recloned into the XhoI / SpeI site of the pCAG-Hyg mIgG1-Fc vector (Wako) to produce the expression vector PTX3(N50)CS-Fc (Figure 1b).

[0066] Each expression vector was transfected into CHO cells using the FreeStyleMAX CHO system (Invitrogen), and stable cells were established using Hygromycin B. The respective fusion proteins (PTX3(N50)-Fc and PTX3(N50)CS-Fc) were purified from the culture supernatant of each stable cell using 1 mL of HisTrap Protein A (GE Healthcare Life Sciences).

[0067] <Example 2> Inhibition of vascular endothelial cell damage by histones using PTX3-Fc fusion protein Using the two purified PTX3-Fc fusion proteins (PTX3(N50)-Fc and PTX3(N50)CS-Fc) described above, the inhibitory effect of histones on vascular endothelial cell (human umbilical vein endothelial cell: HUVEC) damage was analyzed by measuring the uptake of propidium iodide (PI) by HUVECs.

[0068] HUVEC cells were incubated in Opti-MEM (Thermo Fisher Scientific) medium with PTX3-Fc fusion protein (PTX3(N50)-Fc or PTX3(N50)CS-Fc) and calf thymus histones (CTH; 50 μg / ml) (Sigma) at 37°C for 1 hour. Then, PI (10 μg / ml) was added and incubated at 37°C for 5 minutes. After washing with phosphate buffer (PBS), the cells were detached using 1 mM EDTA supplemented with 0.2% PLURONIC® F-68 (Gibco) prepared in PBS, and then detached using Guava easyCyte. TM Analysis was performed using a flow cytometer (Luminex). The degree of cytotoxicity (level of PI uptake by cells) was evaluated by mean fluorescence intensity (MFI).

[0069] As a result, both PTX3-Fc fusion protein, PTX3(N50)-Fc, and PTX3(N50)CS-Fc showed an inhibitory effect on histone damage in vascular endothelial cells (Figure 2).

[0070] <Example 3> Improvement of sepsis model mouse survival rate by PTX3-Fc fusion protein Male C57BL / 6 mice were intraperitoneally administered 2.7 mg / kg of PTX3-Fc fusion protein (PTX3(N50)-Fc or PTX3(N50)CS-Fc), followed by intraperitoneal administration of 11 mg / kg of LPS 2 hours later, and their survival was observed for 7 days.

[0071] As a result, a significant improvement in survival rate was observed for the PTX3-Fc fusion protein, PTX3(N50)CS-Fc (Figure 3).

[0072] <Example 4> Effect of PTX3 20 amino acid peptide on histone-induced vascular endothelial cell damage The inhibitory effect of 20 amino acid residue (aa) peptides of PTX3 (PTX3(18-37aa), PTX3(38-57aa), PTX3(58-77aa), PTX3(78-97aa), PTX3(98-117aa)) on histone damage to HUVECs was analyzed.

[0073] HUVEC cells were incubated in Opti-MEM (Thermo Fisher Scientific) medium with PTX3 20aa (100 μg / ml) and recombinant histone H3 (100 μg / ml) (New England BioLab) or recombinant histone H4 (100 μg / ml) (New England BioLab) at 37°C for 1 hour. Then, PI (10 μg / ml) was added and incubated at 37°C for 5 minutes. After washing the cells with PBS, they were detached using 1 mM EDTA with 0.2% PLURONIC® F-68 added in PBS and then detached using Guava easyCyte. TM The cells were analyzed using a flow cytometer. The degree of cell damage was assessed by MFI (Metabolic Factor Injection). The results are shown in Figures 4 and 5.

[0074] <Example 5> Binding of SARS-CoV-2 virus spike protein to PTX3 The binding activity of pentraxin 3 (PTX3) to the SARS-CoV-2 virus spike protein (ACROBiosystems: SPN-C52H8) was measured by ELISA (Figure 6).

[0075] (Experimental method) (1) Expression and purification of recombinant PTX3 To investigate the sites involved in N-terminal protein binding, five types of Fc fusion proteins consisting of partial peptides (60 amino acids) were created. The cDNA constructs for PTX3-Fc fusion proteins (60PTX_1Fc, 60PTX_1CSFc, 60PTX_2Fc, 60PTX_3Fc, 60PTX_4Fc) were totally synthesized using codons optimized for mammalian cell expression, with GGGGS attached to the C-terminus of 60 amino acids of PTX3 (18-77, and with the cysteine ​​residues at positions 47 and 49 of 18-77 mutated to serine residues, and amino acid residues 51-110, 85-144, and 119-178, respectively). This was then recloned into the XhoI / SpeI site of the pCAG-Hyg mIgG1-Fc expression vector (Wako). TM ExpiCHO TM Expression vectors were transfected into CHO cells using the Expression System (ThermoFisher), and each Fc fusion protein was purified from the culture supernatant using Protein A agarose (GE Healthcare Life Sciences).

[0076] Figure 6 shows the amino acid residue locations of each PTX3Fc fusion protein. 60PTX_1CSFc is a mutant in which the cysteine ​​residues at positions 47 and 49 have been mutated to serine residues.

[0077] These 60PTX_Fc fusion proteins were purified from the culture supernatant prepared in a CHO cell expression system using a Protein A column and used in binding experiments with spike proteins.

[0078] (2) Binding assay (ELISA) of spike protein and recombinant 60PTX_Fc fusion protein SARS-CoV-2 virus spike protein (ACROBiosystems: SPN-C52H8) was adjusted to a concentration of 2 μg / mL in Tris buffer (TBS) / 4 mM CaCl2 and added to a 96-well ELISA plate, where it was immobilized overnight at 4°C. The solution was discarded, blocking buffer (TBS / 4 mM CaCl2, 0.1% Triton-X100, 1% BSA) was added, and the plate was incubated at room temperature for 2 hours. After washing four times with washing buffer (TBS, 0.1% Triton-X100), 60PTX_Fc fusion protein diluted to various concentrations with blocking buffer was added to each well, and the plate was reacted at room temperature for 1 hour. After washing four times with washing buffer, detection antibody diluted with blocking buffer was added, and the plate was reacted at room temperature for 1 hour. Anti-mouse IgG antibody was used as the detection antibody for 60PTX3_Fc fusion. After washing six times with washing buffer, TMB solution was added and a color reaction was carried out for 30 minutes, and the absorbance at 450 nm was measured.

[0079] (result) The results are shown in Figure 7. As shown in Figure 7, 60PTX_3Fc and 60PTX_4Fc showed binding activity to the spike protein. Based on these results, it is thought that PTX3 has a site at its N-terminus (85-178 aa) that binds to the spike protein of the novel coronavirus. Therefore, it is thought that PTX3 directly binds to the coronavirus, inhibits infection, and is involved in the defense response.

[0080] <Example 6> Regarding the vascular endothelial cell damage effect of coronavirus nucleocapsid protein In addition to the spike (S) protein, the nucleocapsid (N) protein is known to be a major component of the coronavirus. The N protein binds to the viral genomic RNA to form a ribonucleocapsid and is thought to be a multifunctional protein involved in viral assembly, germination, envelope formation, replication, and the regulation of the host cell cycle, translation regulation, and inhibition of interferon production. It is also known that serum N protein is detected in SARS-CoV-2 infected individuals from the early stages of the disease, and that anti-N protein antibodies are detected at high concentrations in antiserum along with anti-S protein antibodies. These findings suggest that the N protein is deeply involved in the pathogenesis of COVID-19.

[0081] Coronaviruses are generally known as common cold viruses, with (HCov)-HKU1, HCoV-NL63, HCoV-OC43, and HCoV-229E accounting for about 20% of colds and causing mild symptoms. SARS and MERS, caused by SARS-CoV and MERS-CoV, have high mortality rates of 9% and 36%, respectively. SARS-CoV-2 is considered to be highly infectious among coronaviruses, but the mortality rate for COVID-19 varies widely depending on region, age, and underlying health conditions, ranging from less than 1% to over 10%.

[0082] In this example, we observed vascular endothelial cell damage caused primarily by extracellular histones derived from neutrophils and the protective effect of PTX3 against this damage. Since histones are representative nucleoproteins that interact with DNA, and it is presumed that the N protein of coronaviruses has a similar effect, we measured the cytotoxic activity of N proteins derived from various coronaviruses using human vascular endothelial cells (HUVECs).

[0083] (Experimental method) The measurement is the same as in the histone HUVEC impairment assay, where the uptake of a dye (propidium iodide, PI) is measured by flow cytometry and the increase in mean fluorescence intensity (MFI) is observed. Instead of histones (whole histones or H3, H4), 100 μg / ml each of the following was used: HCoV-NL63 virus nucleocapsid (N) protein (Human coronavirus (YP_003771.1)(Met1-His377)), SARS-CoV virus N protein (Human SARS Coronavirus (SARS-CoV) (NP_828858.1)(Met1-Ala422), and SARS-CoV-2 virus N protein (SARS-CoV-2 (2019-nCoV) (YP_009724397.2)(Met1-Ala419)(335Gly / Ala)) (Sino Logical Inc.).

[0084] (result) As shown in Figure 8, when N protein (100 μg / ml) derived from each coronavirus was added, a significant increase in MFI was observed, indicating HUVEC-mediated cytotoxicity. In this assay, no correlation was found between viral lethality and the increase in MFI (i.e., cytotoxic activity) caused by each N protein. The results above suggest that if the coronavirus N protein is produced in large quantities and released into the bloodstream, it can lead to cell damage, potentially causing severe pneumonia, vasculitis, and even organ damage. Therefore, it is thought that inhibiting the N protein, similar to extracellular histones, could provide a therapeutic agent that can prevent the progression of coronavirus infection into severe cases.

[0085] <Example 7> Suppression of coronavirus N protein-induced cytotoxicity by PTX3 partial peptide (1) SARS-CoV-2 virus N protein, SARS-CoV-2 (2019-nCoV) Nucleocapsid-His recombinant Protein (#40588-V08B), was adjusted to a concentration of 2 μg / mL in Tris buffer (TBS) / 4 mM CaCl2, and 50 μL / well was added to a 96-well ELISA plate and immobilized overnight at 4°C. The solution was discarded, and the plates were washed three times with washing buffer (TBS / 4 mM CaCl2, 0.1% Triton-X100), followed by the addition of blocking buffer (TBS / 4 mM CaCl2, 0.1% Triton-X100, 1% BSA), and incubated at room temperature for 2 hours. After four washes with washing buffer, various 60PTX3_Fc fusion proteins diluted to different concentrations in blocking buffer were added to each well, and the plates were incubated at room temperature for 1 hour. After washing four times with washing buffer, the detection antibody (HRP-labeled anti-mouse IgG antibody) diluted with blocking buffer was added and the mixture was reacted at room temperature for 1 hour. After washing six times with washing buffer, TMB solution was added and a color reaction was carried out for 30 minutes, and the absorbance at 450 nm (A450) was measured.

[0086] (result) As shown in Figure 9, weak binding activity was observed with 60PTX_1Fc and 60PTX_1CSFc, while stronger binding activity to the SARS-CoV-2 virus N protein was observed with 60PTX_3Fc and 60PTX_4Fc fusion proteins. Based on these results, it is thought that PTX3 has a binding site for the N protein of the novel coronavirus at its N-terminus (85-178a.a.). Therefore, PTX3 is thought to directly bind to the coronavirus N protein, and to bind to N proteins exposed from viral particles or leaked from cells, thereby suppressing the elimination of the virus or N protein and the cytotoxic effects of viral components such as the N protein and RNA genome, and thus playing a role in the body's defense against viral attack.

[0087] (2) We investigated the suppression of HUVEC damaging activity of N protein by the 60PTX_Fc fusion protein purified in Example 5. (Experimental method) Measurements were performed by measuring the uptake of the pigment (PI) using flow cytometry and comparing the increase in MFI values. 10,000 HUVEC cells were seeded per well in a 96-well plate and cultured for 2 days in Opti-MEM medium (with 10% FCS). After washing twice with phosphate buffer (PBS), Opti-MEM medium alone was added, supplemented with 100 μg / ml of N protein (NP), NP, and various 60PTX_Fc fusion proteins. 100 μg / ml of SARS-CoV-2 virus N protein (SARS-CoV-2(2019-nCoV)(YP_009724397.2)(Met1-Ala419)(335Gly / Ala))(Sino Logical Inc.) was used as the NP. After incubation at 37°C for 1 hour, PI (30 μg / ml) was added, and after incubation for 5 minutes, the cells were washed with PBS, harvested in 1 mM MEDTA and 0.2% PLURONIC, and measured using a flow cytometer (CyteFlex; Beckman Coulter).

[0088] In Figure 10, MFI represents Mean Fluorescence Intensity. As shown in Figure 10, administration of 60PTX_2Fc, 60PTX_3Fc, and 60PTX_4Fc showed a significant inhibitory effect (p<0.05) on N protein-induced cytotoxicity.

[0089] Based on the results of Examples 5-7 above, the region of PTX3, particularly the area containing amino acid residues 85 to 178, is thought to bind to the S and N proteins, which are the major proteins of SARS-CoV-2, and is considered useful for trapping the virus, suppressing infection, and preventing severe illness. In other words, it is useful as a therapeutic agent for COVID-19. [Industrial applicability]

[0090] The therapeutic or preventive agent of the present invention is useful in the field of pharmaceuticals for treating or preventing infectious diseases.

Claims

1. A therapeutic or prophylactic agent for infectious diseases containing as an active ingredient a fusion protein or a pharmacoagulably accepted salt thereof of (a) at least one polypeptide having the same amino acid sequence as the N-terminal domain of pentraxin 3, or a variant in which a cysteine ​​residue in the said amino acid sequence is substituted with another amino acid residue, and (b) the Fc portion of an immunoglobulin, wherein the amino acid sequence of the N-terminal domain of pentraxin 3 is The region consisting of amino acids 18-67 of the amino acid sequence represented by Sequence ID No. 2; The region consisting of amino acids 85 to 144 of the amino acid sequence represented by Sequence ID No. 2; or The region consisting of amino acids 119 to 176 of the amino acid sequence represented by Sequence ID No. 2; It is one of the following: A treatment or preventative agent for infectious diseases.

2. The therapeutic or prophylactic agent according to claim 1, wherein at least one cysteine ​​residue in the amino acid sequence represented by SEQ ID NO: 2 is substituted with another amino acid residue.

3. The therapeutic or prophylactic agent according to claim 1 or 2, wherein the cysteine ​​residues at positions 47 and 49 in the amino acid sequence represented by Sequence ID No. 2 are substituted with other amino acid residues.

4. The therapeutic or prophylactic agent according to claim 2 or 3, wherein the other amino acid residue is a serine residue.

5. The therapeutic or prophylactic agent according to any one of claims 1 to 4, wherein the N-terminus of the immunoglobulin Fc portion (b) is fused to the C-terminus of at least one polypeptide which is identical to the amino acid sequence of the N-terminal domain of pentraxin 3 that can bind to histones to form polypeptide aggregates, or a mutant in which a cysteine ​​residue in the said amino acid sequence is replaced with another amino acid residue.

6. The therapeutic or prophylactic agent according to any one of claims 1 to 5, wherein the aforementioned infection is sepsis.

7. The therapeutic or prophylactic agent according to any one of claims 1 to 5, wherein the aforementioned infectious disease is COVID-19 infection.

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