Method and reagent for determining severity of respiratory infection

Abstract

To provide a method of determining severity of respiratory tract infections, and reagents that can be used for such a method.SOLUTION: A respiratory tract infection severity determination method is provided, involving measuring the level of TFPI2 in a sample collected from a patient. Antibodies that specifically recognize NT-TFPI2 and intact TFPI2 are included in reagents used in the respiratory tract infection severity determination method.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method and reagent for determining the severity of respiratory tract infections, in which tissue factor pathway inhibitor 2 (TFPI2) is measured. [Background technology]

[0002] Respiratory infections are a general term for diseases caused by pathogens, such as viruses, bacteria, fungi, and parasites, that infect the nasal cavity, pharynx, larynx, trachea, bronchi, and lungs. Respiratory infections occur when pathogens overcome the body's defense mechanisms and establish, infect, and multiply, resulting in various respiratory symptoms, such as coughing, expectoration, fever, and difficulty breathing. Severe cases may require interventions such as oxygen administration and endotracheal intubation, and in some cases may be fatal. The novel coronavirus disease (COVID-19) caused by infection with the novel coronavirus SARS-CoV-2 (SARS-CoV-2) has been spreading globally since its inception in Wuhan, China. COVID-19 is known to worsen primarily due to complications such as pneumonia, and the elderly and those with underlying conditions, such as chronic kidney disease, hypertension, and dyslipidemia, are at higher risk of developing severe symptoms.

[0003] For patients with moderate to severe symptoms who are hospitalized, blood tests such as biochemical and serological tests are performed as needed. Serological diagnostic tests such as ferritin, D-dimer, and CRP are measured for disease monitoring (Non-Patent Document 1). D-dimer is a product of plasmin-mediated degradation of stabilized fibrin in the blood coagulation / fibrinolysis system. D-dimer is a highly specific marker for diagnosing thrombosis and has been shown to be useful not only for ruling out venous thromboembolism (VTE), but also for determining the duration and termination of anticoagulant therapy and assessing the risk of recurrence. Meanwhile, elevated D-dimer levels have been reported in cancer patients, pregnant women, and postoperative patients with hyperactive coagulation systems (Non-Patent Document 2). CRP is a useful marker of systemic inflammation and is elevated in infections, cancer, collagen diseases, and other conditions. IFN-λ3 and TARC (CCL17) are also covered by health insurance in Japan as markers for predicting progression (Non-Patent Document 3).

[0004] Various treatments are being considered to prevent the COVID-19 infection from becoming severe. Among them, tocilizumab, an anti-IL-6 receptor inhibitor antibody drug for the treatment of autoimmune diseases, has been approved by the FDA. It was granted emergency use authorization in the United States and clinical trials are underway in Japan. However, it is known that in patients receiving tocilizumab, IL-6 inhibition inhibits the production of CRP and ferritin from the liver, resulting in a decrease in blood CRP and ferritin levels regardless of the progression of the disease (Non-Patent Document 4).

[0005] Tissue factor pathway inhibitor 2 (TFPI2) is a placenta-derived serine protease inhibitor containing three Kunitz-type protease inhibitor domains, identical to placental protein 5 (PP5). TFPI2 is specifically produced by clear cell carcinoma cell lines in ovarian cancer cell lines, and its gene expression in ovarian cancer patient tissues is specifically elevated only in clear cell carcinoma patients (Patent Document 1). We also disclosed a method for detecting ovarian clear cell carcinoma by measuring circulating TFPI2 (Patent Documents 2, 3, and 4, Non-Patent Documents 5, 6, and 7). However, to date, it has been unclear whether TFPI2 protein in body fluids can be applied to discriminate the severity of respiratory infections. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent No. 5224309 [Patent Document 2] Patent No. 6074676 [Patent Document 3] Patent No. 6737504 [Patent Document 4] Patent No. 6760562 [Non-patent literature]

[0007] [Non-Patent Document 1] COVID-19 Medical Care Guide, Version 5.3 [Non-patent document 2] Tripodi A.,et al.,Clin.Chem.,57, 1256-1262(2011) [Non-patent document 3] Sugiyama, M., et al. Gene766 (2021): 145145. [Non-patent document 4] Salvarani, C., et al. JAMA internal medicine 181.1 (2021): 24‐31. [Non-Patent Document 5] Arakawa, N., et al., J. Proteome Res., 2013, 12 (10), pp 4340-4350 [Non-patent document 6] Arakawa, N., et al., PloS one 11.10 (2016): e0165609. [Non-Patent Document 7] Miyagi, E., et al., Int. J. of Clinical Oncology 26.7 (2021): 1336-1344. Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a method for determining the severity of a respiratory tract infection and a reagent that can be used in said method. [Means for solving the problem]

[0009] After extensive research, the inventors discovered that blood TFPI2 levels increase with acute exacerbation in severe cases of patients with COVID-19, a respiratory infection, who have undergone tracheal intubation. They then came to the realization that TFPI2 can accurately determine the severity of respiratory infections, and completed the present invention. That is, the present invention includes the following aspects. [1] A method for determining the severity of a respiratory infection, comprising measuring the amount of TFPI2 in a sample. [2] The method according to [1], wherein the respiratory infection is determined to be serious when the measured value of the amount of TFPI2 exceeds a predetermined reference value. [3] The method according to [1] or [2], wherein the amount of TFPI2 is the sum of the amount of TFPI2-processing polypeptide and the amount of intact TFPI2. [4] The method according to any one of [1] to [3], wherein the amount of TFPI2 is measured by an antigen-antibody reaction using an antibody that binds to an antigenic determinant within the region from the 23rd aspartic acid residue to the 131st histidine residue or the 130th cysteine ​​residue of the amino acid sequence of SEQ ID NO: 1. [5] The method according to [4], wherein the antibody recognizes Kunitz domain 1 of TFPI2. [6] The method according to any one of [1] to [5], wherein the measurement is carried out using mass spectrometry. [7] A reagent for determining the severity of a respiratory infection, comprising an antibody that binds to an antigenic determinant within the region from aspartic acid at residue 23 to histidine at residue 131 or cysteine ​​at residue 130 of the amino acid sequence shown in Sequence No. 1. [Effects of the Invention]

[0010] The present invention provides a method for easily and accurately determining the severity of a respiratory infection, and Reagents are provided that can be used in the method. [Brief explanation of the drawings]

[0011] [Figure 1] Box plots of TFPI2 measurements in healthy individuals and patients with COVID-19 (from asymptomatic to severe cases). The vertical axis represents the amount of TFPI2 in the blood. [Figure 2] Box plot of CRP measurements in COVID-19 patients (from asymptomatic to severe cases). The vertical axis represents the amount of CRP in the blood. [Figure 3] Box plot of D-dimer measurements in COVID-19 patients (asymptomatic to severe cases). The vertical axis represents the amount of D-dimer in the blood. [Figure 4] A blot showing TFPI2 measurements in a group of severely ill COVID-19 patients divided by gender. The vertical axis represents the amount of TFPI2 in the blood. [Figure 5] A blot showing CRP measurements in severe COVID-19 patients divided by gender. The vertical axis represents the amount of CRP in the blood. [Figure 6] A blot showing D-dimer measurement values ​​in a group of severely ill COVID-19 patients divided by gender. The vertical axis represents the amount of D-dimer in the blood. [Figure 7] A graph showing the correlation between the maximum values ​​of TFPI2 and CRP during the blood collection period in patients with COVID-19 (from asymptomatic to severe cases). [Figure 8]A graph showing the correlation between the maximum values ​​of TFPI2 and D-dimer during the blood collection period in patients with COVID-19 (from asymptomatic to severe cases). [Figure 9] A graph showing the correlation between the maximum values ​​of CRP and D-dimer during the blood collection period in patients with COVID-19 (from asymptomatic to severe cases). [Figure 10] A graph showing the correlation between TFPI2 and CRP marker values ​​at the maximum TFPI2 value during the blood collection period in patients with COVID-19 (from asymptomatic cases to severe cases). [Figure 11] A graph showing the correlation between TFPI2 and D-dimer marker values ​​at the maximum TFPI2 value during the blood collection period in patients with COVID-19 (from asymptomatic to severe cases). [Figure 12] A graph showing the correlation between CRP and D-dimer marker values ​​at the maximum TFPI2 value during the blood collection period in patients with COVID-19 (from asymptomatic to severe cases). [Figure 13] Receiver operating characteristic (ROC) curves for TFPI2, CRP, and D-dimer in severe and non-severe COVID-19 patients. The vertical axis represents sensitivity, and the horizontal axis represents 1-specificity. [Figure 14] A graph showing changes over time during hospitalization in patients with severe COVID-19 infection. The vertical axis represents blood TFPI2 levels, and the horizontal axis represents the number of days since the onset of symptoms. [Figure 15] A graph showing changes over time during hospitalization in severe COVID-19 patients. The vertical axis represents blood TFPI2 levels, and the horizontal axis represents the number of days since the onset of symptoms. [Figure 16] A graph showing changes over time during hospitalization in patients with moderate to asymptomatic COVID-19 infection. The vertical axis represents blood TFPI2 levels, and the horizontal axis represents the number of days since the onset of symptoms. [Figure 17] A graph showing changes over time during hospitalization in a patient receiving tocilizumab (Patient Pos51). The right vertical axis represents blood TFPI2 levels, the left vertical axis represents the P / F ratio, blood CRP levels, and blood D-dimer levels, and the horizontal axis represents the number of days since the onset of symptoms. [Figure 18]A graph showing changes over time during hospitalization in a patient receiving tocilizumab (Patient Pos56). The right vertical axis represents blood TFPI2 levels, the left vertical axis represents the P / F ratio, blood CRP levels, and blood D-dimer levels, and the horizontal axis represents the number of days since the onset of symptoms. [Figure 19] A graph showing changes over time during hospitalization in a patient receiving tocilizumab (Patient Pos60). The right vertical axis represents blood TFPI2 levels, the left vertical axis represents the P / F ratio, blood CRP levels, and blood D-dimer levels, and the horizontal axis represents the number of days since the onset of symptoms. [Figure 20] A graph showing changes over time during hospitalization in a patient receiving tocilizumab (Patient Pos74). The right vertical axis represents blood TFPI2 levels, the left vertical axis represents the P / F ratio, blood CRP levels, and blood D-dimer levels, and the horizontal axis represents the number of days since the onset of symptoms. [Figure 21] A graph showing changes over time during hospitalization in a patient receiving tocilizumab (Patient Pos75). The right vertical axis represents blood TFPI2 levels, the left vertical axis represents the P / F ratio, blood CRP levels, and blood D-dimer levels, and the horizontal axis represents the number of days since the onset of symptoms. [Figure 22] A graph showing changes over time during hospitalization in a patient receiving tocilizumab (Patient Pos76). The right vertical axis represents blood TFPI2 levels, the left vertical axis represents the P / F ratio, blood CRP levels, and blood D-dimer levels, and the horizontal axis represents the number of days since the onset of symptoms. DETAILED DESCRIPTION OF THE INVENTION

[0012] <1> The method of determining the severity of a respiratory infection of the present invention Respiratory infections are diseases caused by pathogens infecting organs such as the nasal cavity, pharynx, trachea, bronchi, and alveoli. Typical pathogens include viruses, bacteria, fungi, and parasites. Viruses include coronaviruses, influenza viruses, respiratory syncytial viruses, adenoviruses, and cytomegaloviruses. Coronaviruses that infect humans include alphacoronaviruses (229E, NL63) and betacoronaviruses (OC43, HKU1, SARS-CoV, MERS-CoV, and SARS-CoV-2). SARS-CoV is the causative agent of severe acute respiratory syndrome (SARS) that emerged in 2002, and SARS-CoV-2 is the causative agent of novel coronavirus disease (COVID-19) that emerged in 2019. Bacteria known to cause respiratory infections include Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, Staphylococcus aureus, Legionella spp., Mycoplasma gondii, and Pseudomonas aeruginosa.

[0013] A first aspect of the present invention is a method for determining the severity of a respiratory infection, which involves measuring the amount of TFPI2 in a sample. This method is based on the fact that the amount of TFPI2 increases with the severity of the infection in biological samples, such as blood, from patients with respiratory infections compared to healthy individuals. Measurement of the amount of TFPI2 in a sample is usually performed in vitro. As shown in the Examples below, this method allows for highly accurate determination of the severity of a respiratory infection.

[0014] The present invention is applicable to patients suspected of having a serious respiratory infection or who have already been definitively diagnosed with a respiratory infection, including those who show signs of decreased pulmonary gas exchange function due to bronchitis or pneumonia (such as dyspnea, rapid breathing, cyanosis, decreased percutaneous arterial oxygen saturation (SpO2), decreased arterial oxygen partial pressure (PaO2)), those who show signs of pneumonia on chest X-rays or CT scans, and those who have progressed to acute respiratory distress syndrome or sepsis.

[0015] The method of the present invention includes the step of estimating the severity of a respiratory infection, but does not include the final determination of the severity of the respiratory infection. A physician will diagnose the severity of the respiratory infection and formulate a treatment plan by comprehensively taking into account the results of the determination by the method of the present invention.

[0016] The TFPI2 measured in the present invention is not particularly limited, and may be, for example, intact TFPI2 (hereinafter also referred to as "I-TFPI2"), TFPI2 processing polypeptide (hereinafter also referred to as "NT-TFPI2"), or both. The amino acid sequence based on the cDNA of human TFPI2 is shown in SEQ ID NO: 1. In SEQ ID NO: 1, the portion from the initiation methionine to the 22nd residue, glycine, is a signal peptide. "Intact TFPI2" refers to the peptide represented by residues 23 to 235 of the amino acid sequence of SEQ ID NO:1.

[0017] Furthermore, as described in Patent Document 3, "NT-TFPI2" is an intact TFPI. NT-TFPI2 refers to a peptide fragment containing Kunitz domain 1 located at the N-terminus of TFPI2. More specifically, NT-TFPI2 is a peptide containing at least the sequence from aspartic acid at residue 23 to histidine at residue 131 or cysteine ​​at residue 130 of the amino acid sequence of SEQ ID NO: 1, or a peptide containing an amino acid sequence having 80% or more identity to the above sequence. The identity is preferably 90% or more, more preferably 95% or more. This polypeptide may also be a polypeptide consisting of the above sequence in which one or several amino acids have been deleted, substituted, inserted, and / or added. Here, "several" preferably refers to 2 to 20, more preferably 2 to 10, and even more preferably 2 to 5. The sequence may have other peptide fragments on both sides, but preferably does not have an antigenic determinant for an antibody that recognizes Kunitz domain 3 of TFPI2.

[0018] In the present invention, patient-derived specimens (test samples) include blood components such as whole blood, blood cells, serum, and plasma; cell or tissue extracts; urine; cerebrospinal fluid; tracheal aspirate; and bronchoalveolar lavage fluid. Lung tissue biopsy samples may also be used, in which case the extract or culture supernatant of the biopsy sample is measured. Body fluids such as blood components, urine, cerebrospinal fluid, tracheal aspirate, and bronchoalveolar lavage fluid are preferred as specimens because they are simple and non-invasive. Considering the ease of specimen collection, risk of secondary infection, and versatility for other test items, blood components are particularly preferred. The dilution ratio of the specimen may be selected from undiluted to 100-fold dilution, depending on the type and condition of the specimen. In the method of the present invention for determining the severity of respiratory tract infections, it is preferable to use the method of measuring TFPI2 according to the present invention in combination with other markers that are commonly used in the diagnosis of respiratory tract infections. There are no particular limitations on the way in which they are combined.

[0019] In the method for determining the severity of respiratory tract infection of the present invention, the markers other than TFPI2 used in combination may be appropriately selected from markers measured by conventional biochemical or immunological methods, and examples include CRP, D-dimer, ferritin, LDH, procalcitonin, IFN-λ3, TARC, IL-6, etc. Among these, CRP, D-dimer, and ferritin, which are commonly used as monitoring markers for COVID-19 infection, are preferred because their clinical usefulness has been widely reported. Furthermore, the markers used in combination with TFPI2 in the method for determining the severity of respiratory tract infection of the present invention may be only one type, or two or more types.

[0020] Furthermore, the timing of specimen collection in the present invention is not particularly limited. For example, specimens may be collected at any time between the time of hospitalization and follow-up observation after a definitive diagnosis of respiratory tract infection, and specimens collected at any stage, such as before or after a definitive diagnosis or before or after the start of treatment, can be subjected to the method of the present invention.

[0021] In the discrimination method of the present invention, it is preferable to determine that a respiratory infection is serious when the amount of TFPI2 obtained by measurement exceeds a predetermined reference value (cutoff value). Here, the amount of TFPI2 may be the amount of intact TFPI2, the amount of NT-TFPI2, or the sum of the amounts of intact TFPI2 and NT-TFPI2, but the sum of the amounts of intact TFPI2 and NT-TFPI2 is more preferred from the viewpoints of achieving both ease of measurement and sufficient sensitivity and specificity. The reference value used for the discrimination may be either a measured value or a converted concentration value. The converted concentration value is a value converted from the measured value based on a calibration curve prepared using TFPI2 as a standard sample. The cutoff value for discriminating the severity of respiratory infections can be determined by measuring the severity of respiratory infections in both severe and non-severe cases and by receiver operating characteristic (ROC) curve analysis to determine the optimum sensitivity and specificity. For example, the cutoff value for discriminating the severity of TFPI2 is 548.3 pg / mL, which is the value at which the sensitivity and specificity are maximized, as shown in the Examples below. It may be set to mL, but this is not the case.

[0022] Hereinafter, the method for measuring TFPI2 will be explained. In the present invention, the amount of NT-TFPI2 or the amount of intact TFPI2 in a sample may be measured separately, or the values ​​may be summed to determine the total amount. Alternatively, the total amount of NT-TFPI2 and intact TFPI2 in a sample may be measured using a measurement system that can simultaneously measure the total amount. Alternatively, as described below, the amount of NT-TFPI2 may be measured indirectly from the total amount measured by both measurements and the amount of intact TFPI2 alone.

[0023] In the methods of the present invention, the method for measuring the amount of NT-TFPI2 and / or intact TFPI2 is not particularly limited, and examples thereof include a method utilizing an antigen-antibody reaction using an antibody that recognizes NT-TFPI2 and / or intact TFPI2, and a method utilizing mass spectrometry. (a) A competitive method using a labeled analyte and an antibody that recognizes the analyte, utilizing the competitive binding of the labeled analyte and the analyte contained in the sample to the antibody. (b) A method using surface plasmon resonance in which a sample is brought into contact with a chip onto which an antibody that recognizes the target of measurement is immobilized, and a signal dependent on the binding between the antibody and the target of measurement is detected. (c) Fluorescence polarization immunoassay, which uses an antibody that recognizes a fluorescently labeled analyte and utilizes the fact that the degree of fluorescence polarization increases when the antibody binds to the analyte. (d) The sandwich method uses two types of antibodies (one of which is labeled) that recognize the target substance and have different antigenic determinants, and forms a three-component complex between the two antibodies and the target substance. (e) A method in which the target substance in the sample is concentrated using an antibody that recognizes the target substance as a pretreatment, and then the polypeptide of the binding protein is detected using a mass spectrometer or the like. Methods (d) and (e) are simple and versatile, but method (d) is more preferable for processing multiple samples because the techniques for reagents and equipment are well established.

[0024] Specific examples of methods for measuring the amount of NT-TFPI2 and / or intact TFPI2 using an antigen-antibody reaction include the following. (A) A method of measuring the total amount of NT-TFPI2 and intact TFPI2 using an antibody that recognizes both NT-TFPI2 and intact TFPI2 (NT+I-TFPI2 measurement system). The antibody that recognizes both NT-TFPI2 and intact TFPI2 is preferably an antibody that binds to an antigenic determinant within the region from aspartic acid at residue 23 to histidine at residue 131 or cysteine ​​at residue 130 in the TFPI2 amino acid sequence represented by SEQ ID NO: 1, and more preferably an antibody that has an antigen recognition site that binds to an antigenic determinant within Kunitz domain 1 of TFPI2. Furthermore, when the sandwich method described above is used in this method, usually two types of antibodies with different antigenic determinants are used.

[0025] (B) A method of measuring the amount of intact TFPI2 alone using an antibody that recognizes intact TFPI2 but not NT-TFPI2 (I-TFPI2 measurement system). The antibody that recognizes intact TFPI2 but not NT-TFPI2 is preferably an antibody that has an antigen-recognition site in Kunitz domain 3 of TFPI2. When the sandwich method described above is used in this method, two types of antibodies with different antigenic determinants are usually used, at least one of which is an antibody that recognizes intact TFPI2 but not NT-TFPI2, and the other may be an antibody that recognizes intact TFPI2 but not NT-TFPI2, or an antibody that recognizes both NT-TFPI2 and intact TFPI2.

[0026] (C) A method for calculating the amount of NT-TFPI2 alone by subtracting the amount of intact TFPI2 alone measured with the I-TFPI2 measurement system in (B) from the total amount of NT-TFPI2 and intact TFPI2 measured with the NT+I-TFPI2 measurement system in (A).

[0027] (D) A method for measuring the amount of NT-TFPI2 alone using an antibody that recognizes NT-TFPI2 but not intact TFPI2. The antibody that recognizes NT-TFPI2 but not intact TFPI2 includes, for example, an antibody that specifically recognizes the peptide sequence at the C-terminus of NT-TFPI2. When using the sandwich method described above, for example, the antibody is used as a solid-phase antibody, and an antibody having a recognition site in Kunitz domain 1 is used as a detection antibody.

[0028] In the method of the present invention for determining the severity of a respiratory infection, the amount of NT-TFPI2 alone measured by the above-mentioned methods (C) and (D) may be used as the criterion for determination. However, the latter method is more preferable because it provides sufficient sensitivity and specificity when the total amount of NT-TFPI2 and intact TFPI2 measured by method (A) is used as the criterion for determination, and the antibody is easy to obtain and the measurement is simple in one step.

[0029] Antibodies that recognize NT-TFPI2 and / or intact TFPI2 can be obtained by immunizing animals with immunogens such as NT-TFPI2 polypeptides or proteins, oligopeptides consisting of partial regions of intact TFPI2 polypeptides or TFPI2 proteins, or polynucleotides encoding intact or partial regions of NT-TFPI2 polypeptides or TFPI2 proteins. The proteins or oligopeptides or polypeptides may not reflect the three-dimensional structure of TFPI2 in vivo, or their structure may change during their preparation. Therefore, the obtained antibodies may not have high specificity or binding strength for the desired in vivo TFPI2, and even if an assay system is constructed using the antibodies, the TFPI2 concentration in a sample may not be accurately quantified.

[0030] On the other hand, using an expression vector containing a polynucleotide encoding an intact or partial region of a TFPI2 polypeptide or intact TFPI2 protein as an immunogen is more preferable because the intact or partial region of the TFPI2 polypeptide or intact TFPI2 protein is expressed in the body of the immunized animal, eliciting an immune response, thereby obtaining antibodies with high specificity and binding strength (i.e., high affinity) for TFPI2 in the sample. The animal used for immunization is not particularly limited as long as it has the ability to produce antibodies, and may be a mammal that is normally used for immunization, such as a mouse, rat, or rabbit, or may be a bird such as a chicken.

[0031] Furthermore, TFPI1, which is known to be a homologue of TFPI2, is also present in the blood. Therefore, it is desirable to use an antibody that specifically recognizes only TFPI2 without cross-reacting with TFPI1.

[0032] The antibody that recognizes TFPI2 may be a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody.

[0033] Hybridoma cells producing antibodies that recognize TFPI2 can be established by any method appropriately selected from established techniques. For example, B cells are collected from an animal immunized by the method described above, and the B cells are fused with myeloma cells electrically or in the presence of polyethylene glycol, followed by selection of hybridoma cells that produce the desired antibodies in HAT medium, and then monocloning the selected hybridoma cells by limiting dilution to establish hybridoma cells that produce monoclonal antibodies that recognize TFPI2. The selection of the monoclonal antibody that recognizes TFPI2 used in the present invention is based on the host expression system. This may be done based on the affinity for GPI (glycosylphosphatidylinositol)-anchored TFPI2 or secreted TFPI2.

[0034] The host is not particularly limited and may be appropriately selected from microbial cells such as Escherichia coli and yeast, insect cells, animal cells, or cell-free expression systems developed based on wheat germ or mammalian cells, which are commonly used by those skilled in the art for protein expression. However, mammalian cells are preferred, as they are capable of expressing proteins with a structure similar to that of native TFPI2 through post-translational modifications such as disulfide bonds or glycosylation. Examples of mammalian cells include the conventionally used human embryonic kidney (HEK) 293T cell line, monkey kidney COS7 cell line, Chinese hamster ovary (CHO) cells, and cancer cells isolated from humans.

[0035] The antibodies used in the present invention can be purified by any method appropriately selected from established techniques. For example, antibody-producing hybridoma cells established by the above-described method are cultured, the culture supernatant is collected, and the antibodies are concentrated by ammonium sulfate precipitation, if necessary, and then purified by affinity chromatography and / or ion exchange chromatography using a carrier on which protein A, protein G, protein L, or the like is immobilized.

[0036] The labeled antibody used in the antigen-antibody reaction by the sandwich method described above can be obtained by labeling the antibody purified by the method described above with an enzyme such as peroxidase or alkaline phosphatase, and the labeling can be performed using a method for which the technology is well established.

[0037] In the method of the present invention, the method for measuring the amount of TFPI2 using mass spectrometry will be specifically described below. When the sample is blood, it is preferable to pretreat the blood by removing major proteins such as albumin, immunoglobulin, and transferrin, which are abundant in the blood, using Agilent Human 14 or the like, followed by further fractionation by ion exchange, gel filtration, reverse-phase HPLC, etc. Alternatively, TFPI2 can be specifically recovered by an immunological technique using an anti-TFPI2 antibody.

[0038] Measurements can be performed using tandem mass spectrometry (MS / MS), liquid chromatography-tandem mass spectrometry (LC / MS / MS), matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF / MS), surface-enhanced laser desorption ionization mass spectrometry (SELDI-MS), etc.

[0039] The method for determining the severity of a respiratory tract infection of the present invention can be applied to a method for treating a respiratory tract infection. That is, the present invention provides a method for treating a respiratory tract infection in a subject, comprising: (i) identifying the subject as a patient with a severe respiratory infection based on the measured value of the amount of TFPI2 exceeding a predetermined reference value; and (ii) administering a treatment to the identified subject. In a preferred embodiment of the method for treating a respiratory infection, the amount of TFPI2 is the sum of the amount of TFPI2 processing polypeptide and the amount of intact TFPI2.

[0040] In the step (i), the amount of TFPI2 is preferably measured by measuring the amount of TFPI2 between the 23rd residue aspartic acid and the 131st residue histidine in the amino acid sequence of SEQ ID NO: 1. This is carried out by an antigen-antibody reaction using an antibody that binds to an antigenic determinant within the region up to cysteine ​​residue 0. More preferably, the antibody is an antibody that recognizes Kunitz domain 1 of TFPI2. In the identification step (i), the amount of TFPI2 may be measured by mass spectrometry.

[0041] The treatment in the step (ii) includes, but is not limited to, drug therapy, surgical therapy, respiratory management using an artificial respirator or extracorporeal membrane oxygenation (ECMO), and the like.

[0042] <2> Reagent for determining the severity of respiratory infections of the present invention When the reagent of the present invention is used in the sandwich method described above, it is preferable that the antibodies contain two types of antibodies with different antigenic determinants. The antibody contained in the reagent of the present invention may be an antibody itself, may be labeled, or may be immobilized on a solid phase.

[0043] The reagent of the present invention will be specifically described below when used in the two-step sandwich method, which is one embodiment of the sandwich method described above, although the present invention is not limited thereto. First, the reagent of the present invention can be prepared by the following methods (I) to (III). (I) First, of two types of antibodies (hereinafter referred to as "antibody 1" and "antibody 2") that recognize TFPI2 and have different antigenic determinants, antibody 1 is bound to a carrier capable of B / F (Bound / Free) separation, such as an immunoplate or magnetic particles. The binding method may be physical binding using a hydrophobic bond, or chemical binding using a linker reagent that can crosslink two substances.

[0044] (II) After binding the antibody 1 to the carrier, in order to prevent non-specific binding, the surface of the carrier is blocked with bovine serum albumin, skim milk, a commercially available blocking agent for immunoassays, or a chemically synthesized polymer for inhibiting protein adsorption, to prepare the primary reagent.

[0045] (III) The other antibody 2 is labeled, and a solution containing the resulting labeled antibody is prepared as a secondary reagent. Substances that can be used to label antibody 2 include enzymes such as peroxidase and alkaline phosphatase, fluorescent substances, chemiluminescent substances, radioisotopes, and other substances that can be detected by a detection device, as well as substances that specifically bind to biotin, such as avidin. Furthermore, the solution of the secondary reagent is preferably a buffer solution that allows for good antigen-antibody reactions, such as phosphate buffer or Tris-HCl buffer. The reagent of the present invention prepared in this manner may be lyophilized if necessary.

[0046] In the case of the one-step sandwich method, antibody 1 is bound to a carrier and subjected to a blocking treatment in the same manner as in (I) to (II) above, and a buffer solution containing labeled antibody 2 is further added to the antibody-immobilized carrier to prepare a reagent.

[0047] Next, to detect and measure TFPI2 by the two-step sandwich method using the reagent obtained by the above-mentioned method, the following methods (IV) to (VI) may be used. (IV) The primary reagent prepared in (II) is brought into contact with the sample for a certain period of time at a certain temperature. The reaction conditions are a temperature range of 4°C to 40°C and a time range of 5 to 180 minutes.

[0048] (V) Unreacted materials are removed by B / F separation, and then the mixture is contacted with the secondary reagent prepared in (III) for a certain time and at a certain temperature to form a sandwich complex. The reaction conditions are a temperature range of 4°C to 40°C and a reaction time of 5 to 180 minutes.

[0049] (VI) Unreacted substances are removed by B / F separation, the labeled substance of the labeled antibody is quantified, and human TFPI2 in the sample is quantified using a calibration curve prepared using a TFPI2 solution of known concentration as a standard.

[0050] The amounts of reagent components such as antibodies contained in the reagent of the present invention may be appropriately determined depending on various conditions such as the amount of sample, the type of sample, the type of reagent, the measurement method, etc. Specifically, for example, when TFPI2 level is measured by the sandwich method using 20 μL of serum or plasma as a sample as described below, the amount of antibody bound to the carrier may be 100 ng to 1000 μg and the amount of labeled antibody may be 2 ng to 20 μg per reaction system in which 20 μL of the sample is reacted with the antibody.

[0051] The reagent of the present invention can be used for manual measurement and for measurement using an automated immunodiagnostic device, and measurement using an automated immunodiagnostic device is particularly preferred because it allows measurement without being affected by endogenous measurement-interfering factors or competing enzymes contained in the sample and enables TFPI2 in the sample to be quantified in a short period of time.

[0052] Another aspect of the present invention is the use of a reagent for measuring the amount of TFPI2 in the manufacture of a reagent for determining the severity of a respiratory tract infection. Another aspect of the present invention is the in vitro use of a reagent for measuring the amount of TFPI2 in determining the severity of a respiratory tract infection. Here, the reagent for measuring the amount of TFPI2 is preferably a reagent for measuring the sum of the amount of TFPI2-processing polypeptide and the amount of intact TFPI2. The reagent for measuring the amount of TFPI2 is preferably an antibody that binds to an antigenic determinant in the region from aspartic acid at residue 23 to histidine at residue 131 or cysteine ​​at residue 130 in the amino acid sequence of SEQ ID NO: 1, more preferably an antibody that recognizes Kunitz domain 1 of TFPI2. [Example]

[0053] EXAMPLES In the following, examples are shown to specifically explain the present invention, but these examples are merely examples of the present invention and the present invention is not limited to these examples.

[0054] Example 1: Evaluation of clinical specimens The breakdown of the clinical samples used in this example is shown in Table 1. Sera from 241 healthy individuals (102 males and 139 females) and sera from asymptomatic to severe cases of COVID-19 (42 males) were collected. Of the 15 cases (15 women, 15 dialysis patients excluded), all cases except for healthy male subjects were collected at Yokohama City University. The samples were collected from subjects who provided informed consent and were approved by the Yokohama City University Ethics Committee. Healthy male volunteer samples were collected from subjects who provided informed consent and were approved by the Tosoh Corporation In-house Ethics Committee.

[0055] [Table 1]

[0056] The TFPI2 measurement reagent used was E-test "TOSOH" 2 (TFPI2) (manufactured by Tosoh Corporation: manufacturing and sales notification number 30200EZX00040000), and the evaluation device used was the fully automated enzyme immunoassay device AIA-2000 (manufactured by Tosoh Corporation: manufacturing and sales notification number 13B3X90002000009). TFPI2 measurement using the fully automated enzyme immunoassay device AIA-2000 was performed as follows.

[0057] (1) 20 μL of sample and 100 μL of diluent containing surfactant are automatically dispensed into a container containing a TFPI2 measurement reagent. (2) Carry out an antigen-antibody reaction at a constant temperature of 37°C for 10 minutes. (3) After B / F separation, wash eight times with a buffer solution containing a surfactant. (4) 4-Methylumbelliferyl phosphate was added, and the concentration of 4-methylumbelliferone produced by alkaline phosphatase per unit time was taken as the measured value (TFPI2 intensity, nmol / (L·s)). (5) A calibration curve was created using the standard provided with the product, and the TFPI2 concentration in the sample was calculated. Blood CRP and D-dimer values ​​were quoted from the measurements recorded in the electronic medical records obtained through insured medical treatment.

[0058] <Example 2> Comparison of TFPI2, CRP, and D-dimer in healthy individuals and patients with COVID-19 Boxplots of TFPI2, CRP, and D-dimer (maximum values ​​during the blood sampling period) in healthy individuals and COVID-19 patients are shown in Figures 1 to 3. TFPI2 levels tended to be significantly higher in some severe cases and critically ill patients compared to healthy individuals, asymptomatic COVID-19 patients, and mild cases. CRP and D-dimer levels also tended to increase with the severity of the condition.

[0059] <Example 3> Gender differences in severe COVID-19 patients Plots of TFPI2, CRP, and D-dimer (maximum values ​​during the blood sampling period) when severe COVID-19 patients were classified by gender are shown in Figures 4 to 6. It was shown that none of the markers were affected by gender differences.

[0060] <Example 4> Correlation between TFPI2, CRP, and D-dimer in patients with COVID-19 For all COVID-19 patients, correlations between TFPI2, CRP, and D-dimer using the maximum sample collection period are shown in Table 2 and Figures 7-9, while correlations between CRP and D-dimer in samples showing maximum TFPI2 values ​​are shown in Table 3 and Figures 10-12. While no correlation was observed with any of the markers, a strong correlation was observed between CRP and D-dimer, particularly at the maximum sample collection period (correlation coefficient: 0.69). These results suggest that TFPI2 may be an indicator independent of CRP or D-dimer.

[0061] [Table 2]

[0062] [Table 3]

[0063] <Example 5> Comparison of the performance of TFPI2, CRP, and D-dimer in discriminating the severity of COVID-19 (ROC analysis) The ROC analysis results and area under the curve (AUC) of TFPI2, CRP, and D-dimer in severe and non-severe cases (asymptomatic to severe) of COVID-19 are shown in Figure 13. TFPI2 showed the highest AUC of 0.8953, demonstrating its ability to discriminate the severity of COVID-19.

[0064] <Example 6> Comparison of the performance of TFPI2, CRP, and D-dimer in determining the severity of COVID-19 infection (sensitivity / specificity) Table 4 shows the sensitivity / specificity of TFPI2, CRP, and D-dimer when the maximum value of the Youden index (specificity + sensitivity - 1) was set as the cutoff value based on the results of the ROC analysis described in Example 5. TFPI2 (cutoff value: 548.3 pg / mL) had a sensitivity of 100.0% and a specificity of 82.6%, demonstrating that it has a high or equivalent ability to discriminate severity compared to CRP or D-dimer.

[0065] [Table 4]

[0066] <Example 7> Changes in TFPI2 over time in patients with COVID-19 COVID-19 patients were classified into severe, critical, and moderate / mild / asymptomatic groups, and the changes in TFPI2 over time from the day of onset are shown in Figures 14 to 16. In severe cases, TFPI2 fluctuated dramatically between 10 and 30 days after onset, with a tendency to decrease from 30 days onward. Although TFPI2 levels increased in some severe cases, TFPI2 levels generally tended to remain low in many severe patients and moderate / mild / asymptomatic patients.

[0067] <Example 8> Changes over time in TFPI2, CRP, D-dimer, and oxygenation index (P / F ratio) in patients administered tocilizumab The time course of TFPI2, CRP, D-dimer, and P / F ratio from the day of onset in six patients receiving tocilizumab is shown in Figures 17 to 22. In patients receiving tocilizumab, CRP levels uniformly decreased, but TFPI2 and D-dimer levels tended to fluctuate. [Industrial Applicability]

[0068] The present invention provides a simple blood test method for determining the severity of respiratory tract infections that places a relatively small burden on patients. TFPI2, which does not correlate with the existing markers CRP or D-dimer, is expected to contribute to the treatment of COVID-19, an emerging infectious disease, and is therefore extremely useful industrially.

Claims

1. measuring the amount of TFPI2 and the amount of CRP and / or D-dimer in a sample; the specimen is a blood component, the amount of TFPI2 is the sum of the amount of TFPI2 processing polypeptide and the amount of intact TFPI2; The method provides information for determining the severity of novel coronavirus infection (SARS-CoV-2), in which the novel coronavirus infection (SARS-CoV-2) is deemed to be severe when the measured values ​​of the TFPI2 amount and the CRP amount and / or the D-dimer amount exceed predetermined reference values.

2. The method according to claim 1, wherein the amount of TFPI2 is measured by an antigen-antibody reaction using an antibody that binds to an antigenic determinant within the region from the 23rd aspartic acid residue to the 131st histidine residue or the 130th cysteine ​​residue of the amino acid sequence of SEQ ID NO:

1.

3. The method according to claim 2, wherein the antibody is an antibody that recognizes Kunitz domain 1 of TFPI2.

4. The method according to any one of claims 1 to 3, wherein the measurement is carried out using mass spectrometry.

5. A combination of reagents for determining the severity of novel coronavirus infection (SARS-CoV-2), which is used on blood component samples, includes a reagent containing an antibody that binds to an antigenic determinant within the region from the 23rd aspartic acid residue to the 131st histidine residue or the 130th cysteine ​​residue of the amino acid sequence shown in SEQ ID NO: 1, and a reagent for measuring the amount of CRP and / or D-dimer.

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