Methods for predicting sepsis and septic shock

Abstract

The subject of the present invention is a method for predicting sepsis, severe sepsis or septic shock in a patient, comprising determining the level of protachykinin A or a fragment thereof of at least 5 amino acids in a body fluid obtained from the subject and correlating the determined level of protachykinin A or a fragment thereof of at least 5 amino acids with sepsis or septic shock, wherein an elevated level above a certain threshold is predictive of sepsis, severe sepsis or septic shock.

Description

[Technical field]

[0001] The subject of the present invention is a method for predicting sepsis, severe sepsis or septic shock in a patient, comprising determining the level of protachykinin A or a fragment thereof of at least 5 amino acids in a body fluid obtained from the subject and correlating the determined level of protachykinin A or a fragment thereof of at least 5 amino acids with sepsis or septic shock, wherein an elevated level above a certain threshold is predictive of sepsis, severe sepsis or septic shock. [Background technology]

[0002] Substance P (SP) is a neuropeptide:undecapeptide that functions as a neurotransmitter and neuromodulator. It belongs to the tachykinin neuropeptide family. SP is one of five members of the tachykinin family, which in addition to SP also includes neurokinin A, neuropeptide K, neuropeptide γ, and neurokinin B. They are produced from protein precursors after differential splicing of the prepro-tachykinin A gene ( Helke et al.1990.FASEB Journal 4(6):1606-15 ). SP plays a role in nociception, inflammation, plasma extravasation, platelet and leukocyte aggregation in postcapillary venules, and leukocyte chemotactic migration through the blood vessel wall ( Otsuka M,Yoshioka K.Neurotransmitter functions of mammalian tachykinins.Physiol Rev.1993 Apr;73(2):229-308 Substance P is produced by neuronal and non-neuronal cells, including immune cells ( Brit J Pharmacol 170:1279–1292 ).

[0003] The role of SP in sepsis remains unclear. However, some studies have found that SP may play a role in the inflammatory response to sepsis through the release of proinflammatory cytokines such as interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-α ( Lotz et al.1988.Science 241:1218-1221;Laurenzi et al.1990.Scand.J.Immunol.31:529-533;Ansel et al.1993 J.Immunol.150:4478-4485;Yamaguchi et al al.2004.Inflammation.Res.53:199–204 ). Meanwhile, other studies have found that SP may have anti-inflammatory effects by decreasing TNF-α, IL-6, and inducible nitric oxide synthase (iNOS) and increasing IL-10 ( Jiang et al.2012.Neuroreport 23:786–792;Jiang et al.2013.Neuroreport 24:846–851). Furthermore, further research findings suggest that SP may play a role in microbial clearance by regulating phagocytic capacity ( Verdrengh and Tarkowski 2008.Scand.J.Immunol.67:253-259;Yang et al.2014.Crit.Care Med.42:2092-2100;Kincy-Cain and Bost 1996.J.Immunol.157:255-264;Lighvani et al al.2005.Eur.J.Immunol.35:1567-1575 ).

[0004] Circulating SP concentrations in septic patients have not been thoroughly studied. In one study of 61 septic patients, the authors found higher serum SP concentrations during the final stage of sepsis in septic patients compared to healthy controls and in non-survivors compared to survivors ( Beer et al.2002.Crit Care Med.30:1794-1798 ). Another study of 42 septic patients found that plasma SP concentrations were lower in septic patients compared to healthy controls ( Arnalich et al.1995.Life Sci 56:75-81 ). Furthermore, serum SP levels have been shown to be associated with mortality in sepsis ( Lorente et al.2015.J.Crit.Care 2015,30,924-928;Lorente et al.2017.Int J Mol Sci 18(7):1531 However, non-survivors showed lower serum SP levels compared to survivors ( Lorente et al.2015.J.Crit.Care 2015,30,924–928 ).

[0005] Furthermore, in the mouse CLP model, there was a time-dependent increase in SP, with the highest SP level observed in plasma at 1 h. SP levels in plasma decreased from 5 h and peaked again at 20 h, indicating a biphasic response of substance P ( Puneet et al.2006.J Immunol 176:3813–3820 In addition, administration of LPS to wild-type mice caused a significant increase in circulating levels of SP ( Ng et al.2008.Journal of Leukocyte Biology 83:288–295 ).

[0006] The potential role of SP in sepsis is extensive ( For reviews, see Bodkin and Fernandes 2012 Brit J Pharmacol 170:1279-1292 ). It induces many inflammatory effects related to the progression of sepsis, most of which are due to the activation of the NK1 receptor. SP is well known as an inducer of neurogenic inflammation, characterized by vasodilation, edema and leukocyte infiltration. All of these effects can be induced by SP acting on the NK1 receptor ( O’Connor et al.,2004 J Cell Physiol 201:167-180 ), which is detrimental in sepsis. NK1 activation on endothelial cells induces cell shrinkage, resulting in edema, and also leads to the production of vasodilators such as NO and prostacyclin ( Katz et al.,2003.J Vet Pharmacol Ther 26:361-368), contributing to hypotension. NK1 activation can also induce the transcription of inflammatory mediators, including chemokines, cytokines, and adhesion molecules, in several cell types ( Maggi,1997.Regul Pept 70:75-90 ). SP is also known to stimulate neutrophils for a chemotactic response to chemokines, induces expression of chemokine receptors, and the response can be inhibited with NK1 antagonists ( Sun et al.,2007.Am J Physiol Cell Physiol 293:C696-C704 These effects of SP are detrimental during sepsis because they exacerbate inflammation and lead to fatal organ damage. Edema and vasodilation may contribute to the dangerous hypotension and reduced lung function associated with poor outcomes from sepsis.

[0007] To date, human studies have been hampered by the very short half-life of SP (12 min). Conlon and Sheehan.Regul.Pept.1983;7:335-345 ). The recent development of an assay for a stable protachykinin A (PTA) fragment (N-terminal protachykinin A or NT-PTA), a surrogate for the unstable SP ( Ernst et al.Peptides 2008;29:1201-1206 ) have enabled studies into the role of this tachykinin system in human disease.

[0008] Delays in treatment of patients presenting to the emergency department (ED) with suspected infection can lead to longer hospital stays, increased morbidity, and higher infection-related mortality. Accurate assessment of the severity of the host response and the likelihood of further disease progression to sepsis, severe sepsis, septic shock, and organ dysfunction is therefore critical to direct rapid and targeted therapeutic success. Summary of the Invention [Problem to be solved by the invention]

[0009] Therefore, one object of the present invention is the use of protachykinin A or a fragment thereof to distinguish patients who are likely or at high risk of developing sepsis, severe sepsis or septic shock requiring intensive care from patients who are at low risk of needing such treatment. [Means for solving the problem]

[0010] (1) A method for predicting sepsis, severe sepsis, and / or septic shock in a patient, comprising: determining a level of pro-tachykinin A or a fragment thereof of at least 5 amino acids in a sample of bodily fluid obtained from said subject; Correlating the determined level of pro-tachykinin A or a fragment thereof of at least 5 amino acids with sepsis or septic shock; wherein elevated levels above a certain threshold are predictive of sepsis, severe sepsis and / or septic shock.

[0011] (2) The method according to (1), wherein the sample of the patient's body fluid is taken at a time when the patient shows no clinical symptoms of sepsis, severe sepsis and / or septic shock or shows mild symptoms of an infectious disease.

[0012] (3) The method according to (1) or (2), wherein the pro-tachykinin A is selected from the group comprising SEQ ID NOs: 1 to 4, and the fragment thereof is selected from the group comprising SEQ ID NOs: 5 to 12.

[0013] (4) The method according to any one of (1) to (3), wherein the level of protachykinin A or a fragment thereof of at least 5 amino acids is determined by using a binding agent for protachykinin A or a fragment thereof of at least 5 amino acids.

[0014] (5) The method according to any one of (1) to (4), wherein the binding agent is selected from the group comprising an antibody, an antibody fragment or a non-Ig scaffold that binds to pro-tachykinin A or a fragment thereof of at least 5 amino acids.

[0015] (6) The method according to any one of (1) to (5), wherein the binding agent binds to a region within an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:11, and SEQ ID NO:12.

[0016] (7) The method according to any one of (1) to (6), wherein the threshold range is 75 to 200 pmol / L, more preferably 90 to 175 pmol / L, even more preferably 100 to 150 pmol / L, and most preferably the threshold level is 120 pmol / L.

[0017] (8) The method according to any one of (1) to (7), wherein the level of protachykinin A is measured by immunoassay and the binding agent is an antibody or antibody fragment that binds to protachykinin A or a fragment thereof of at least 5 amino acids.

[0018] (9) The method according to any one of (1) to (8), wherein an assay is used that includes two binding agents that bind to two different regions within the region of pro-tachykinin A that is amino acids 3 to 22 (SEQ ID NO:11) and amino acids 21 to 36 (SEQ ID NO:12), each of said regions including at least 4 or 5 amino acids.

[0019] (10) The method according to any one of (1) to (9), wherein an assay is used to measure the level of pro-tachykinin A or a fragment thereof of at least 5 amino acids, and the assay sensitivity of the assay is <10 pmol / L, capable of quantifying pro-tachykinin A or a pro-tachykinin A fragment in a healthy subject.

[0020] (11) The method according to any one of (1) to (10), wherein the body fluid can be selected from the group including blood, serum, plasma, urine, cerebrospinal fluid (CSF), and saliva.

[0021] (12) At least one further biomarker and / or clinical parameter and / or clinical score is D-dimer, procalcitonin (PCT), C-reactive protein (CRP), lactate, penKid, ADM-NH 2, MR-proADM, NT-proBNP, BNP, presepsin, pentraxin-3 (PTX-3), CD-64, calprotectin, white blood cell count, lymphocyte count, neutrophil count, hemoglobin, platelet count, albumin, alanine transaminase, creatinine, blood urea, lactate dehydrogenase, creatinine kinase, cardiac troponin I, prothrombin time, serum ferritin, interleukin-6 (IL-6), IL-10, IL-2, IL-7, interferon gamma (IF-γ), tumor necrosis factor-α (TNF-α), granulocyte-colony stimulating factor (GCSF), IP-10, monocyte chemotactic protein 1 (MCP-1), MIP-1α, SOFA, qSOFA, APACHE II. The method according to any one of (1) to (11), wherein the determined protein is selected from the group including:

[0022] (13) The method according to any one of (1) to (12), wherein the determination is performed two or more times in one patient.

[0023] (14) The method according to any one of (1) to (13), for stratifying the subject into a risk group.

[0024] (15) A point-of-care device for carrying out the method according to any one of (1) to (14), comprising at least two antibodies or antibody fragments against amino acids 3 to 22 (SEQ ID NO:11) and amino acids 21 to 36 (SEQ ID NO:12).

[0025] (16) A kit for carrying out the method according to any one of (1) to (14), comprising at least two antibodies or antibody fragments against amino acids 3 to 22 (SEQ ID NO:11) and amino acids 21 to 36 (SEQ ID NO:12). [Brief description of the drawings]

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[0037] It was a surprising discovery of the present invention that pro-tachykinin A or a fragment thereof is an early biomarker for predicting that a patient will subsequently develop sepsis, severe sepsis and septic shock. The term "early biomarker" means that the level of the biomarker pro-tachykinin A or a fragment thereof is elevated in a patient before the patient develops sepsis, severe sepsis or septic shock.

[0038] The term "subject" as used herein refers to a living human or non-human organism. Preferably, as used herein, a subject is a human subject. A subject may be healthy or diseased, unless otherwise specified.

[0039] The term "elevated level" means a level above a certain threshold level.

[0040] The "body fluid" may be selected from the group comprising blood, serum, plasma, urine, cerebrospinal fluid (CSF) and saliva. In one particular embodiment, the body fluid according to the invention is a blood sample. The blood sample may be selected from the group comprising whole blood, serum and plasma. In a particular embodiment of the diagnostic method, the sample is selected from the group comprising human citrated plasma, heparinized plasma and EDTA plasma.

[0041] The term "prediction" refers to the prognosis of an outcome or a particular risk for a subject. It can also include an estimation of the likelihood of recovery or adverse outcome for the subject.

[0042] The method of the invention may also be used for monitoring, therapy monitoring, therapy guidance and / or therapy control. In the context of the present application, "monitoring" relates to following a patient and potentially arising complications, for example to analyze the progress of a healing process or the impact of a particular treatment or therapy on the patient's health status.

[0043] The term "therapeutic monitoring" or "therapeutic control" in the context of the present invention refers to the monitoring and / or adjustment of the therapeutic treatment of the patient, for example by obtaining feedback on the effectiveness of the treatment. As used herein, the term "therapeutic guidance" refers to the application of a particular treatment, therapeutic action or medical intervention based on the values / levels of one or more biomarkers and / or clinical parameters and / or clinical scores. This includes adjusting the treatment or discontinuing the treatment.

[0044] In the present invention, the terms "risk assessment" and "risk stratification" relate to grouping subjects into different risk groups according to their further prognosis. Risk assessment also relates to stratification for applying preventive and / or therapeutic measures. The term "therapeutic stratification" particularly relates to grouping or classifying patients into different groups, such as risk groups or treatment groups, which receive specific differential therapeutic measures depending on the classification. The term "therapeutic stratification" also relates to grouping or classifying patients with infectious diseases or symptoms of infectious diseases into groups that do not need to receive specific therapeutic measures.

[0045] Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection ( Singer et al.2016.JAMA 315(8):801-810(see ). Organ dysfunction can be identified as an acute change in total SOFA score ≥ 2 points as a result of infection. The baseline SOFA score can be assumed to be 0 in patients not known to have pre-existing organ dysfunction. A SOFA score ≥ 2 reflects an overall mortality risk of approximately 10% in the overall hospital population with suspected infection. Even patients with moderate dysfunction may deteriorate further, highlighting the severity of this condition and the need for prompt and appropriate intervention if not already initiated. Sepsis is a life-threatening condition that occurs when the body's response to infection damages its own tissues and organs. Patients with suspected infection who are likely to have a prolonged ICU stay or die in hospital can be rapidly identified at the bedside by qSOFA, i.e., altered mental status, systolic blood pressure ≤ 100 mm Hg, or respiratory rate ≥ 22 / min.

[0046] Septic shock is a subset of sepsis in which the underlying circulatory and cellular / metabolic abnormalities are severe enough to substantially increase mortality. Patients with septic shock can be identified by the clinical constructs of sepsis with persistent hypotension requiring vasopressors to maintain a mean arterial pressure (MAP) ≥ 65 mm Hg and with serum lactate levels > 2 mmol / L (18 mg / dL) despite adequate volume resuscitation. With these criteria, hospital mortality is greater than 40%.

[0047] The term "sepsis" as used in the context of the present application relates to all possible stages in the development of sepsis.

[0048] The term "sepsis" also includes severe sepsis or septic shock based on the definition of SEPSIS-2 ( Bone et al.1992. Crit Care Med 20(6):864-874. The term "sepsis" also includes subjects included in the definition of SEPSIS-3 ( Singer et al.2016 JAMA 315(8):801-810). As used herein, organ dysfunction refers to a state or health condition in which an organ does not perform its expected function. "Organ failure" refers to organ dysfunction to the extent that normal homeostasis cannot be maintained without external clinical intervention. The organ failure may relate to an organ selected from the group including kidney, liver, heart, lungs, nervous system. In contrast, organ function refers to the expected function of the respective organ within the physiological range. A person skilled in the art knows the respective function of the organ under medical examination.

[0049] Organ dysfunction can be defined by the Sequential Organ Failure Assessment score (SOFA score) or its components. The SOFA score, formerly known as the Sepsis-Associated Organ Failure Assessment score (SOFA score), is Singer et al.2016.JAMA 315(8):801-10 ) is used to track the condition of people admitted to the intensive care unit (ICU) to determine the degree of organ function or failure rate of a person. The score is based on six different scores, one each for the respiratory, cardiovascular, hepatic, coagulation, renal and nervous systems, each scored from 0 to 4, with increasing scores reflecting worsening organ dysfunction. The assessment criteria for the SOFA score are described, for example, in Lamden et al. (for review, see Lambden et al.2019.Critical Care 23:374 (see, for example, US 2005 / 0139916). The SOFA score can traditionally be calculated upon admission to the ICU and every 24 hours thereafter. In particular, the organ dysfunction is selected from the group including renal failure, cardiac dysfunction, hepatic dysfunction or airway dysfunction.

[0050] The quickSOFA score (qSOFA) was introduced by the Sepsis-3 group in February 2016 as a simplified version of the SOFA score as an early method to identify patients at high risk for poor outcomes due to infection ( Angus et al.2016.Critical Care Medicine.44(3 ) :e113-e121). qSOFA greatly simplifies the SOFA score by including only its three clinical criteria and by including "any altered mental effects" instead of requiring a GCS <15. qSOFA can be easily and quickly repeated serially on patients. Scores range from 0 to 3 points. 1 point is awarded for: low blood pressure (SBP ≤ 100 mm Hg), high respiratory rate (≥ 22 breaths / min), and altered mental effects (GCS ≤ 15). The presence of 2 or more qSOFA points near the onset of infection was associated with a greater risk of death or prolonged intensive care unit admission. These are outcomes that are more common in infected patients who may be septic than in patients with uncomplicated infection. Based on these findings, the Third International Consensus Definitions for Sepsis recommends qSOFA as a brief prompt to identify infected patients outside the ICU who are likely to be septic ( Seymour et al.2016.JAMA 315(8 ) :762-774 ).

[0051] In a preferred embodiment of the invention, the method is defined by the prediction of sepsis, severe sepsis and / or septic shock in a patient population for which it was previously difficult, if not impossible, to determine by routine clinical and / or molecular diagnostic means whether there is a serious risk of a serious deterioration of a medical condition, thereby requiring hospitalization. This patient population may be considered as patients with mild symptoms of an infectious disease, or patients without symptoms of a severe infectious disease (e.g. without symptoms of sepsis).

[0052] In one embodiment, at the time of sample provision, the patient exhibits mild symptoms of an infectious disease, corresponding to a qSOFA score of 0 or 1.

[0053] In one embodiment, at the time of providing the sample, the patient does not exhibit clinical symptoms of sepsis, severe sepsis and / or septic shock.

[0054] In one embodiment, at the time of sample provision, the patient has been diagnosed with an infectious disease of bacterial, viral, fungal, or parasitic origin.

[0055] In one embodiment, at the time of sample provision, the patient has been diagnosed with community-acquired pneumonia (CAP) or urinary tract infection (UTI).

[0056] It was quite surprising that patients who only show mild symptoms of infectious disease, e.g. those who correspond to a qSOFA score of 0 or 1, and patients who show symptoms of infectious disease but do not show the presence of sepsis, can be classified as needing hospitalization based on determining a high-risk level of pro-tachykinin A or a fragment thereof according to the present invention. This represents a great advantage of the method of the present invention, since a specialist physician would not expect that a patient with mild symptoms of infectious disease and / or symptoms not suggestive of sepsis would need hospitalization, e.g. to monitor the progression of the symptoms of infectious disease and to carry out treatment. Such patients are usually examined by medical staff and can then be released from continuous medical observation, e.g. with a treatment prescription that can be carried out without specialist medical supervision. However, the method of the present invention allows to identify by objective means patients who do not show severe symptoms of sepsis but still need hospitalization.

[0057] In one embodiment of the invention, the method is used to stratify the patient into risk groups.

[0058] In one embodiment, a high risk level of PTA or a fragment thereof indicates that the patient is at risk of developing sepsis, severe sepsis and / or septic shock within 48 hours, 24 hours, 12 hours, 6 hours, 4 hours, preferably 2 hours, and a low risk level of PTA or a fragment thereof indicates that the patient is not at risk of developing sepsis, severe sepsis or septic shock within 48 hours, 24 hours, 12 hours, 6 hours, 4 hours, preferably 2 hours.

[0059] In one embodiment, the level of PTA or a fragment thereof in the body fluid sample is indicative of the risk of developing sepsis, severe sepsis and / or septic shock in a patient requiring hospitalization.

[0060] In one embodiment, a high risk level of PTA or a fragment thereof indicates that the patient is at risk of developing sepsis requiring hospitalization, severe sepsis and / or septic shock within 48 hours, 24 hours, 12 hours, 6 hours, 4 hours, preferably 2 hours, and a low risk level of PTA or a fragment thereof indicates that the patient is not at risk of developing sepsis requiring hospitalization, severe sepsis and / or septic shock within 48 hours, 24 hours, 12 hours, 6 hours, 4 hours, preferably 2 hours.

[0061] In another embodiment of the invention, the method is used to stratify the patient into groups for early treatment (e.g., need for antibiotic administration), where the term "early" is defined as the time of treatment before the patient shows clinical signs and symptoms of sepsis, severe sepsis and / or septic shock or before the patient is diagnosed with sepsis, severe sepsis and / or septic shock.

[0062] In one embodiment, the level of PTA or a fragment thereof in the bodily fluid sample indicates that the patient requires frequent monitoring and / or critical care, hi one embodiment, patients with high risk levels require medical treatment provided in a hospital setting.

[0063] Examples of these treatments include, but are not limited to, fluid therapy, vasopressors, intravenous antibodies, and in some embodiments essentially any treatment beyond oral antibodies that can be self-administered at home.

[0064] Depending on the outcome of the method of the present invention, embodiments of the method may include subsequent therapeutic decisions and / or treatment actions. Such therapeutic decisions may include the initiation, alteration or modification of medical treatment. Preferably, if the method of the present invention indicates progression to a condition requiring in-hospital treatment, appropriate therapeutic measures, such as the initiation or alteration of a particular medication or fluid therapy, may be initiated.

[0065] Any of the therapies, medical procedures or treatments disclosed herein can be used in the context of the methods of the invention as a subsequent treatment decision or treatment, especially if the treatment measures are specifically administered in the hospital, including but not limited to intravenous fluid therapy, dialysis, management of electrolyte abnormalities (especially potassium, calcium and phosphorus). Furthermore, sustained intensive observation and care of the patient may be indicated, potentially for extended periods such as days, weeks or months. This may involve keeping or transferring the patient to the ICU and / or prolonging the patient's stay in the ICU.

[0066] On the other hand, if the results of the methods of the present invention indicate that there is no risk of developing sepsis, severe sepsis, or septic shock requiring hospitalization, then specific treatment measures for such complications may not be required or less severe, self-administered treatments may be prescribed.

[0067] Pro-tachykinin A or a fragment thereof is particularly for the aforementioned medical utility in all patient populations attending emergency departments.

[0068] Throughout this specification, the terms pro-tachykinin and pro-tachykinin A (PTA) are used interchangeably. This term includes all splice variants of pro-tachykinin A, namely αPTA, βPTA, γPTA and δPTA. Throughout this specification, the term fragments of pro-tachykinin A should be understood to also include substance P and neurokinin A, neuropeptide K, neuropeptide γ, and neurokinin B, unless otherwise specified.

[0069] The term "determining the level of pro-tachykinin, its splice variants including substance P and neurokinin or its fragments of at least 5 amino acids" generally means that the immunoreactivity to a region within said molecule is determined. This means that a particular fragment does not have to be measured selectively. It is understood that the binding agent used to determine the level of pro-tachykinin or its fragments of at least 5 amino acids including substance P and neurokinin will bind to any fragment that includes the binding region of the binding agent. The binding agent may be an antibody or antibody fragment or a non-IgG scaffold.

[0070] A subject of the present invention is a method, in which the level of pro-tachykinin A or of a fragment thereof of at least 5 amino acids is determined by using a binding agent for pro-tachykinin A or of a fragment thereof of at least 5 amino acids.

[0071] In one embodiment of the invention, the binding agent is selected from the group comprising an antibody, an antibody fragment or a non-Ig scaffold that binds to pro-tachykinin A or a fragment thereof of at least 5 amino acids.

[0072] Alternative splicing of the PTA gene transcript generates four distinct PTA-mRNA molecules, designated αPTA, βPTA, γPTA, and δPTA, respectively. Harmar et al.1990.FEBS Lett 275:22-4;Kawaguchi et al.1986.Biochem Biophys Res Comm 139:1040-6;Nawa et al.1984.Nature 312:729-34 ), which differ in their exon combinations. All seven exons are contained only in the β-PTA mRNA. However, the first three exons encoding the SP and a common N-terminal region of 37 amino acids (SEQ ID NO:5) are present in all PTA precursor molecules.

[0073] Alternative splicing gives rise to the following pro-tachykinin A sequence: SEQ ID NO:1 (isoform αPTA) EEIGANDDLNYWSDWYDSDQIKEELPEPFEHLLQRIARRPKPQQFFGLMGKRDADSSIEKQVALLKALYGHGQISHKMAYERSAMQNYERRR SEQ ID NO:2 (isoform βPTA) EEIGANDDLNYWSDWYDSDQIKEELPEPFEHLLQRIARRPKPQQFFGLMGKRDADSSIEKQVALLKALYGHGQISHKRHKTDSFVGLMGKRALNSVAYERSAMQNYERRR SEQ ID NO:3 (isoform gamma PTA) EEIGANDDLNYWSDWYDSDQIKEELPEPFEHLLQRIARRPKPQQFFGLMGKRDAGHGQISHKRHKTDSFVGLMGKRALNSVAYERSAMQNYERRRSEQ SEQ ID NO: 4 (isoform δPTA) EEIGANDDLNYWSDWYDSDQIKEELPEPFEHLLQRIARRPKPQQFFGLMGKRDAGHGQISHKMAYERSAMQNYERRR Fragments of pro-tachykinin A which can be measured in body fluids can, for example, be selected from the group of the following fragments: SEQ ID NO:5 (Pro-tachykinin A 1-37, P37, NT-PTA) EEIGANDDLNYWSDWYDSDQIKEELPEPFEHLLQRIA SEQ ID NO:6 (Substance P) RPKPQQFFGLM(-NH 2 ) SEQ ID NO: 7 (Neuropeptide K) DADSSIEKQVALLKALYGHGQISHKRHKTDSFVGLM(-NH 2 ) SEQ ID NO:8 (Neuropeptide gamma) GHGQISHKRHKTDSFVGLM(-NH 2 ) SEQ ID NO:9 (Neurokinin B) HKTDSFVGLM(-NH 2 ) SEQ ID NO:10 (C-terminal flanking peptide, PTA 92-107). ALNSVAYERSAMQNYE SEQ ID NO:11 (PTA 3-22) GANDDLNYWSDWYDSDQIK SEQ ID NO:12 (PTA 21-36) IKEELPEPFEHLLQRI

[0074] Determining the level of pro-tachykinin A or a fragment thereof may mean determining the immunoreactivity to PTA or a fragment thereof, including substance P and neurokinins. Depending on the binding domain, the binding agent used for the determination of PTA or a fragment thereof may bind to more than one of the molecules shown above. This will be clear to the skilled person.

[0075] In a more specific embodiment of the invention, the fragment of PTA may be selected from SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12.

[0076] In a more specific embodiment of the method according to the invention, the level of peptide 37 (P37), also called PTA 1-37 or NT-PTA, SEQ ID NO: 5, is determined. In an even more specific embodiment according to the invention, at least one or two binding agents are used which bind to PTA 1-37 (NT-PTA), SEQ ID NO: 5, in case of two or more binding agents, they preferably bind to two different regions within PTA 1-37 (NT-PTA), SEQ ID NO: 5. The binding agent may preferably be an antibody or a binding fragment thereof.

[0077] In even more specific embodiments, the binding agents are used to determine which variants and fragments thereof bind to one or both of the following regions within PTA, PTA 1-37 (NT-PTA): PTA 3-22 (GANDDLNYWSDWYDSDQIK, which is SEQ ID NO: 11) and PTA 21-36 (IKEELPEPFEHLLQRI, which is SEQ ID NO: 12), respectively.

[0078] Thus, in accordance with the present invention, the level of an immunoreactive analyte by using at least one binding agent that binds to a region within the amino acid sequence of any of the above peptides and peptide fragments (i.e., pro-tachykinin A (PTA) and fragments according to any of sequences 1-12) is determined in a body fluid obtained from the subject and correlates with certain embodiments of clinical relevance.

[0079] In a more specific embodiment of the method according to the invention, the level of PTA 1-37 is determined (SEQ ID NO: 5: NT-PTA).

[0080] In more specific embodiments, the level of the immunoreactive analyte is determined by using at least one binding agent that binds to pro-tachykinin A or a fragment thereof of at least 5 amino acids, and correlates with the above-mentioned embodiments according to the invention to the following specific embodiments of clinical relevance: - Correlating the level of the immunoreactive analyte with sepsis or septic shock, where elevated levels above a certain threshold are predictive of sepsis, severe sepsis or septic shock.

[0081] Alternatively, the levels of any of the above analytes may be determined by other analytical methods, such as mass spectrometry.

[0082] The subject of the present application is a method for predicting sepsis, severe sepsis and / or septic shock in a patient, comprising: determining a level of pro-tachykinin A or a fragment thereof of at least 5 amino acids in a body fluid obtained from the subject; Correlating the determined level of pro-tachykinin A or a fragment thereof of at least 5 amino acids with sepsis or septic shock; wherein elevated levels above a certain threshold are predictive of sepsis, severe sepsis and / or septic shock.

[0083] One embodiment of the present application relates to a method for predicting sepsis, severe sepsis or septic shock in a patient, wherein a sample of a body fluid of the patient is taken at a time when the patient does not show clinical symptoms of sepsis, severe sepsis and / or septic shock.

[0084] Another embodiment of the present application relates to a method for predicting sepsis, severe sepsis and / or septic shock in a patient, wherein the pro-tachykinin A is selected from the group comprising SEQ ID NOs: 1-4 and the fragment thereof is selected from the group comprising SEQ ID NOs: 5-12.

[0085] Another embodiment of the present application relates to a method for predicting sepsis, severe sepsis and / or septic shock in a patient, wherein the level of pro-tachykinin A or a fragment thereof of at least 5 amino acids is determined by using a binding agent for pro-tachykinin A or a fragment thereof of at least 5 amino acids.

[0086] Another particular embodiment of the present application relates to a method for predicting sepsis, severe sepsis and / or septic shock in a patient, wherein the binding agent is selected from the group comprising an antibody, an antibody fragment or a non-Ig scaffold that binds to pro-tachykinin A or a fragment thereof of at least 5 amino acids.

[0087] Another preferred embodiment of the present application relates to a method for predicting sepsis, severe sepsis and / or septic shock in a patient, wherein the binding agent binds to a region within an amino acid sequence selected from the group comprising SEQ ID NO:5, SEQ ID NO:11 and SEQ ID NO:12.

[0088] Another embodiment of the present application relates to a method for predicting sepsis, severe sepsis and / or septic shock in a patient, wherein the threshold range is 75-200 pmol / L, more preferably 90-175 pmol / L, even more preferably 100-150 pmol / L, and most preferably the threshold level is 120 pmol / L.

[0089] A further embodiment of the present application relates to a method for predicting sepsis, severe sepsis and / or septic shock in a patient, wherein the level of protachykinin A is measured by immunoassay and the binding agent is an antibody or antibody fragment that binds to protachykinin A or a fragment thereof of at least 5 amino acids.

[0090] Another embodiment of the present application relates to a method for predicting sepsis, severe sepsis and / or septic shock in a patient, in which an assay is used comprising two binding agents that bind to two different regions within the region of pro-tachykinin A which are amino acids 3-22 (SEQ ID NO:11) and amino acids 21-36 (SEQ ID NO:12), each of which comprises at least 4 or 5 amino acids.

[0091] Another particular embodiment of the present application relates to a method for predicting sepsis, severe sepsis and / or septic shock in a patient, wherein an assay is used to determine the level of pro-tachykinin A or a fragment thereof of at least 5 amino acids, the assay sensitivity of which is capable of quantifying pro-tachykinin A or a pro-tachykinin A fragment in healthy subjects is <10 pmol / L.

[0092] Another embodiment of the present application relates to a method for predicting sepsis, severe sepsis and / or septic shock in a patient, wherein the body fluid may be selected from the group comprising blood, serum, plasma, urine, cerebrospinal fluid (CSF) and saliva.

[0093] Another embodiment of the present application relates to a method for predicting sepsis, severe sepsis and / or septic shock in a patient, further comprising: determining whether at least one biomarker and / or clinical parameter and / or clinical score is D-dimer, procalcitonin (PCT), C-reactive protein (CRP), lactate, penKid, ADM-NH 2, MR-proADM, NT-proBNP, BNP, presepsin, pentraxin-3 (PTX-3), CD-64, calprotectin, white blood cell count, lymphocyte count, neutrophil count, hemoglobin, platelet count, albumin, alanine transaminase, creatinine, blood urea, lactate dehydrogenase, creatinine kinase, cardiac troponin I, prothrombin time, serum ferritin, interleukin-6 (IL-6), IL-10, IL-2, IL-7, interferon gamma (IF-γ), tumor necrosis factor alpha (TNF-α), granulocyte-colony stimulating factor (GCSF), IP-10, monocyte chemoattractant protein 1 (MCP-1), MIP-1α, SOFA, qSOFA, and APACHE II.

[0094] Another preferred embodiment of the present application relates to a method for predicting sepsis, severe sepsis and / or septic shock in a patient, wherein said determination is performed two or more times in one patient.

[0095] One embodiment of the present application relates to a method for predicting sepsis, severe sepsis and / or septic shock in a patient in order to stratify said subject into risk groups.

[0096] The subject of the present application is also a point-of-care (POC) device for carrying out a method for predicting sepsis, severe sepsis and / or septic shock in a patient, the point-of-care device comprising at least two antibodies or antibody fragments against amino acids 3 to 22 (SEQ ID NO:11) and amino acids 21 to 36 (SEQ ID NO:12).

[0097] The subject of the present application is also a kit for carrying out a method for predicting sepsis, severe sepsis and / or septic shock in a patient, said kit comprising at least two antibodies or antibody fragments against amino acids 3 to 22 (SEQ ID NO:11) and amino acids 21 to 36 (SEQ ID NO:12).

[0098] The subject matter of the present application also relates to a method for assessing a patient's risk of suffering from sepsis, severe sepsis and / or septic shock, comprising: determining a level of pro-tachykinin A or a fragment thereof of at least 5 amino acids in a body fluid obtained from the subject; Correlating the determined level of pro-tachykinin A or a fragment thereof of at least 5 amino acids with sepsis or septic shock; wherein elevated levels above a certain threshold indicate a risk of developing sepsis, severe sepsis and / or septic shock.

[0099] One embodiment of the present application relates to a method for assessing a patient's risk of suffering from sepsis, severe sepsis and / or septic shock, wherein a sample of a body fluid from the patient is taken at a time when the patient is not showing clinical symptoms of sepsis, severe sepsis and / or septic shock.

[0100] Another embodiment of the present application relates to a method for assessing a patient's risk of suffering from sepsis, severe sepsis and / or septic shock, wherein the pro-tachykinin A is selected from the group comprising SEQ ID NOs: 1-4 and the fragment thereof is selected from the group comprising SEQ ID NOs: 5-12.

[0101] Another embodiment of the present application relates to a method for assessing a patient's risk of suffering from sepsis, severe sepsis and / or septic shock, wherein the level of pro-tachykinin A or a fragment thereof of at least 5 amino acids is determined by using a binding agent for pro-tachykinin A or a fragment thereof of at least 5 amino acids.

[0102] Another particular embodiment of the present application relates to a method for assessing the risk of a patient to suffer from sepsis, severe sepsis and / or septic shock, wherein the binding agent is selected from the group comprising an antibody, an antibody fragment or a non-Ig scaffold that binds to pro-tachykinin A or a fragment thereof of at least 5 amino acids.

[0103] Another preferred embodiment of the present application relates to a method for assessing a patient's risk of suffering from sepsis, severe sepsis and / or septic shock, wherein the binding agent binds to a region within an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:11 and SEQ ID NO:12.

[0104] Another embodiment of the present application relates to a method for assessing a patient's risk of suffering from sepsis, severe sepsis and / or septic shock, wherein the threshold range is 75-200 pmol / L, more preferably 90-175 pmol / L, even more preferably 100-150 pmol / L, and most preferably the threshold level is 120 pmol / L.

[0105] A further embodiment of the present application relates to a method for assessing a patient's risk of suffering from sepsis, severe sepsis and / or septic shock, wherein the level of protachykinin A is measured by immunoassay and the binding agent is an antibody or antibody fragment that binds to protachykinin A or a fragment thereof of at least 5 amino acids.

[0106] Another embodiment of the present application relates to a method for assessing a patient's risk of suffering from sepsis, severe sepsis and / or septic shock, in which an assay is used comprising two binding agents that bind to two different regions within the region of pro-tachykinin A which are amino acids 3-22 (SEQ ID NO:11) and amino acids 21-36 (SEQ ID NO:12), each of which comprises at least 4 or 5 amino acids.

[0107] Another particular embodiment of the present application relates to a method for assessing a patient's risk of suffering from sepsis, severe sepsis and / or septic shock, wherein an assay is used to determine the level of pro-tachykinin A or a fragment thereof of at least 5 amino acids, the assay sensitivity of which is capable of quantifying pro-tachykinin A or a pro-tachykinin A fragment in healthy subjects is <10 pmol / L.

[0108] Another embodiment of the present application relates to a method for assessing a patient's risk of suffering from sepsis, severe sepsis and / or septic shock, wherein the body fluid may be selected from the group comprising blood, serum, plasma, urine, cerebrospinal fluid (CSF) and saliva.

[0109] Another embodiment of the present application relates to a method for assessing a patient's risk of suffering from sepsis, severe sepsis and / or septic shock, further comprising measuring at least one biomarker and / or clinical parameter and / or clinical score, such as D-dimer, procalcitonin (PCT), C-reactive protein (CRP), lactate, penKid, ADM-NH 2 , MR-proADM, NT-proBNP, BNP, presepsin, pentraxin-3 (PTX-3), CD-64, calprotectin, white blood cell count, lymphocyte count, neutrophil count, hemoglobin, platelet count, albumin, alanine transaminase, creatinine, blood urea, lactate dehydrogenase, creatinine kinase, cardiac troponin I, prothrombin time, serum ferritin, interleukin-6 (IL-6), IL-10, IL-2, IL-7, interferon gamma (IF-γ), tumor necrosis factor alpha (TNF-α), granulocyte-colony stimulating factor (GCSF), IP-10, monocyte chemoattractant protein 1 (MCP-1), MIP-1α, SOFA, qSOFA, and APACHE II.

[0110] Another preferred embodiment of the present application relates to a method for assessing a patient's risk of suffering from sepsis, severe sepsis and / or septic shock, wherein said determination is performed on two or more occasions in one patient.

[0111] One embodiment of the present application relates to a method for assessing a patient's risk of suffering from sepsis, severe sepsis and / or septic shock in order to stratify said subject into risk groups.

[0112] The subject of the present application is also a point-of-care device for carrying out a method for assessing a patient's risk of suffering from sepsis, severe sepsis and / or septic shock, the point-of-care device comprising at least two antibodies or antibody fragments against amino acids 3 to 22 (SEQ ID NO:11) and amino acids 21 to 36 (SEQ ID NO:12).

[0113] The subject of the present application is also a kit for carrying out a method for assessing a patient's risk of suffering from sepsis, severe sepsis and / or septic shock, the kit comprising at least two antibodies or antibody fragments against amino acids 3 to 22 (SEQ ID NO:11) and amino acids 21 to 36 (SEQ ID NO:12).

[0114] The subject of the present invention is therefore a method for assessing the risk of suffering sepsis, severe sepsis and / or septic shock in a patient, comprising: determining the level of an immunoreactive analyte by using at least one binding agent that binds to a region within the amino acid sequence of a peptide selected from the group consisting of peptides and fragments of SEQ ID NOs: 1-12 in a body fluid obtained from the subject; correlating the level of pro-tachykinin or a fragment thereof with sepsis, severe sepsis and / or septic shock; wherein elevated levels above a certain threshold indicate a risk of developing sepsis, severe sepsis and / or septic shock. In a more specific embodiment of the present application, the level of an immunoreactive analyte in a body fluid obtained from the subject by using at least one binding agent that binds to a region within the amino acid sequence of Pro-tachykinin 1-37, N-terminal Pro-tachykinin A fragment, NT-PTA (SEQ ID NO: 5).

[0115] In a particular embodiment of the present application, the level of pro-tachykinin A or a fragment thereof is measured in an immunoassay using an antibody or a fragment of an antibody that binds to pro-tachykinin A or a fragment thereof. An immunoassay that may be useful for determining the level of pro-tachykinin A or a fragment thereof of at least 5 amino acids may include the steps outlined in Example 1. All thresholds and values ​​need to be seen in correlation with the test and calibration used according to Example 1. A person skilled in the art may know that the absolute value of the thresholds may be influenced by the calibration used. This means that all values ​​and thresholds shown herein should be understood in the context of the calibration used herein (Example 1).

[0116] According to the invention, the diagnostic binding agent for pro-tachykinin A is selected from the group consisting of antibodies, e.g. IgG, typical full length immunoglobulins or antibody fragments containing at least the F variable domain of the heavy and / or light chain, e.g. as chemically conjugated antibodies (fragment antigen binding), including but not limited to Fab minibodies, single chain Fab antibodies, monovalent Fab antibodies with an epitope tag, e.g. Fab fragments comprising Fab-V5Sx2; bivalent Fab (mini antibodies) dimerized with a CH3 domain; bivalent Fab or multivalent Fab formed, e.g. via multimerization with heterologous domains, e.g. via dimerization of dHLX domains, e.g. Fab-dHLX-FSx2; F(ab')2 fragments, scFv fragments, multimerized multivalent or / and multispecific scFv fragments, bivalent and / or bispecific diabodies, BITE® (bispecific T cell engagers), trifunctional antibodies, e.g. multivalent antibodies of a class different from G; single domain antibodies, e.g. nanobodies derived from camelid or fish immunoglobulins.

[0117] In certain embodiments of the present application, the level of pro-tachykinin A or a fragment thereof is measured in an assay using a binding agent selected from the group comprising antibodies, antibody fragments, aptamers, non-Ig scaffolds that bind to pro-tachykinin A or a fragment thereof, as described in more detail below.

[0118] A binding agent that can be used to determine the level of protachykinin A or a fragment thereof has a binding affinity of at least 10 to protachykinin A or a fragment thereof. 7 M -1 , preferably 10 8 M -1 and the preferred affinity constant is 10 9 M -1 Greater than 10, most preferably 10 M -1 The binding affinity is greater than the affinity of the compound. Those skilled in the art know that it is possible to consider to compensate for lower affinity by applying a higher dose of the compound, and this measure does not fall outside the scope of the present invention. Binding affinity can be determined, for example, using the Biacore method provided as a service analysis at Biaffin, Kassel, Germany (http: / / www.biaffin.com / de / ).

[0119] To determine the affinity of the antibodies, the binding kinetics of the PTA splice variants or fragments thereof to the immobilized antibodies was determined by label-free surface plasmon resonance using a Biacore 2000 system (GE Healthcare Europe GmbH, Freiburg, Germany). Reversible immobilization of the antibodies was performed using anti-mouse Fc antibodies covalently coupled at high density to the CM5 sensor surface according to the manufacturer's instructions (Mouse Antibody Capture Kit; GE Healthcare). Lorenz et al.,” Functional Antibodies Targeting IsaA of Staphylococcus aureus Augment Host Immune Response and Open New Perspectives for Antibacterial Therapy”;Antimicrob Agents Chemother.2011 January;55(1):165-173 ).

[0120] The assay can be calibrated with synthetic (for our experiments we used synthetic P37, SEQ ID NO:5) or recombinant PTA splice variants or fragments thereof.

[0121] In addition to antibodies, other biopolymer scaffolds are known in the art to complex target molecules and have been used to generate highly target-specific biopolymers. Examples are aptamers, spiegelmers, anticalins and conotoxins. Non-Ig scaffolds may be protein scaffolds and can bind ligands or antigens, and thus can be used as antibody mimics. Non-Ig scaffolds include tetranectin-based non-Ig scaffolds (e.g., U.S. Patent Application Publication No. 2010 / 0028995 ), fibronectin scaffolds (e.g., European Patent No. 1266025 ); lipocalin-based scaffolds (e.g., International Publication No. 2011 / 154420 ubiquitin scaffolds (e.g., International Publication No. 2011 / 073214 ), metastatic scaffolds (e.g., US Patent Application Publication No. 2004 / 0023334 ), protein A scaffolds (e.g., European Patent No. 2231860 ), ankyrin repeat-based scaffolds (e.g., International Publication No. 2010 / 060748 ), a microprotein, preferably a cystine-knot forming microprotein) scaffold (e.g., European Patent No. 2314308 ), Fyn SH3 domain-based scaffolds (e.g., International Publication No. 2011 / 023685 (described in, for example, International Publication No. 2005 / 040229 ) and Kunitz domain-based scaffolds (e.g., European Patent No. 1941867 The compound may be selected from the group including those described in

[0122] Another preferred embodiment of the present application relates to a method for predicting sepsis, severe sepsis and / or septic shock in a patient, wherein the threshold level of PTA or a fragment thereof is 75-200 pmol / L, more preferably 90-175 pmol / L, even more preferably 100-150 pmol / L, and most preferably said threshold level is 120 pmol / L.

[0123] Another preferred embodiment of the present application relates to a method for assessing a patient's risk of suffering from sepsis, severe sepsis and / or septic shock, wherein the threshold level of PTA or a fragment thereof is 75-200 pmol / L, more preferably 90-175 pmol / L, even more preferably 100-150 pmol / L, and most preferably the threshold level is 120 pmol / L.

[0124] In another preferred embodiment, the threshold value for PTA or a fragment thereof may be the upper normal range (99th percentile, 107 pmol / L).

[0125] In one particular embodiment of the present application, the level of pro-tachykinin A or a fragment thereof is measured by immunoassay and the binding agent is an antibody or antibody fragment that binds to pro-tachykinin A or a fragment thereof of at least 5 amino acids.

[0126] In one particular embodiment of the present application, the assay used comprises two binding agents that bind to two different regions within the region of pro-tachykinin A that is amino acids 3-22 (sequence, SEQ ID NO:11) and amino acids 21-36 (sequence, SEQ ID NO:12), each of the regions comprising at least 4 or 5 amino acids.

[0127] In one embodiment of the present application of an assay for measuring pro-tachykinin A or pro-tachykinin A fragments in a body fluid sample according to the present invention, the assay sensitivity of the assay capable of quantifying pro-tachykinin A or pro-tachykinin A fragments in healthy subjects is <20 pmol / L, preferably <10 pmol / L, more preferably <5 pmol / L.

[0128] The subject of the present invention is the use of at least one binding agent that binds to a region within the amino acid sequence of a peptide selected from the group comprising the peptides and fragments of SEQ ID NOs: 1 to 12 in a body fluid obtained from a subject, in a method for predicting sepsis, severe sepsis and / or septic shock in a patient.

[0129] The subject of the present invention is the use of at least one binding agent that binds to a region within the amino acid sequence of a peptide selected from the group comprising the peptides and fragments of SEQ ID NOs: 1 to 12 in a body fluid obtained from a subject, in a method for assessing the risk of developing sepsis, severe sepsis and / or septic shock in a patient.

[0130] In one embodiment of the invention, the binding agent is selected from the group comprising antibodies, antibody fragments or non-Ig scaffolds that bind to pro-tachykinin A or a fragment thereof of at least 5 amino acids. In a particular embodiment, the at least one binding agent binds to a region having a sequence selected from the group comprising SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12. In a particular embodiment, the binding agent does not bind to SEQ ID NOs: 6, 7, 8 and 9. In a particular embodiment, the at least one binding agent binds to a region having a sequence selected from the group comprising SEQ ID NOs: 1, 2, 3, 4, 5, 11 and 12. In another particular embodiment, the at least one binding agent binds to a region having a sequence selected from the group comprising SEQ ID NOs: 5, 11 and 12. In another very particular embodiment, the binding agent binds to pro-tachykinin A 1-37, N-terminal pro-tachykinin A fragment, NT-PTA (SEQ ID NO: 5).

[0131] In a more specific embodiment of the present application, the at least one binding agent binds to regions within the amino acid sequence of Pro-tachykinin A 1-37, N-terminal Pro-tachykinin A fragment, NT-PTA (SEQ ID NO:5) in a body fluid obtained from the subject, more specifically amino acids 3-22 (GANDDLNYWSDWYDSDQIK, SEQ ID NO:11) and / or amino acids 21-36 (IKEELPEPFEHLLQRI, SEQ ID NO:12), each of which regions comprises at least 4 or 5 amino acids.

[0132] Thus, according to the method, the level of immunoreactivity of the binding agent is determined in a body fluid obtained from the subject. The level of immunoreactivity means the concentration of an analyte, which is determined quantitatively, semi-quantitatively or qualitatively by the binding reaction of the binding agent to such analyte, and preferably the binding agent is at least 10 8 M -1 has an affinity constant for binding to the analyte of 0.05, the binding agent may be an antibody or antibody fragment or a non-IgG backbone, and the binding reaction is an immunoassay.

[0133] A subject of the present invention is also a method for predicting sepsis, severe sepsis and / or septic shock in a patient according to any of the preceding embodiments, in which the level of pro-tachykinin A or a fragment thereof of at least 5 amino acids in a body fluid obtained from said subject is used either alone or in combination with other useful biomarkers and / or clinical parameters and / or clinical scores, which may be selected from the following options: - comparison with the median level of protachykinin A or a fragment thereof of at least 5 amino acids in body fluids obtained from said subjects in a given ensemble of samples in a population of "healthy" or "apparently healthy" subjects; - comparison with quartiles of levels of protachykinin A or a fragment thereof of at least 5 amino acids in body fluids obtained from subjects in a given ensemble of samples in a population of "healthy" or "apparently healthy" subjects; • Calculation based on Cox Proportional Hazards analysis or by using risk index calculations such as NRI (Net Reclassification Index), IDI (Integrated Discrimination Index), etc.

[0134] A subject of the present invention is also a method for assessing the risk of suffering sepsis, severe sepsis and / or septic shock in a patient according to any of the preceding embodiments, in which the level of pro-tachykinin A or a fragment thereof of at least 5 amino acids in a body fluid obtained from said subject is used either alone or in combination with other useful biomarkers and / or clinical parameters and / or clinical scores, which may be selected from the following options: - comparison with the median level of protachykinin A or a fragment thereof of at least 5 amino acids in body fluids obtained from said subjects in a given ensemble of samples in a population of "healthy" or "apparently healthy" subjects; - comparison with quartiles of levels of protachykinin A or a fragment thereof of at least 5 amino acids in body fluids obtained from subjects in a given ensemble of samples in a population of "healthy" or "apparently healthy" subjects; • Calculation based on Cox Proportional Hazards analysis or by using risk index calculations such as NRI (Net Reclassification Index), IDI (Integrated Discrimination Index), etc.

[0135] The at least one further biomarker and / or clinical parameter and / or clinical score is D-dimer, procalcitonin (PCT), C-reactive protein (CRP), lactate, penKid, ADM-NH 2, MR-proADM, NT-proBNP, BNP, presepsin, pentraxin-3 (PTX-3), CD-64, calprotectin, white blood cell count, lymphocyte count, neutrophil count, hemoglobin, platelet count, albumin, alanine transaminase, creatinine, blood urea, lactate dehydrogenase, creatinine kinase, cardiac troponin I, prothrombin time, serum ferritin, interleukin-6 (IL-6), IL-10, IL-2, IL-7, interferon gamma (IF-γ), tumor necrosis factor alpha (TNF-α), granulocyte-colony stimulating factor (GCSF), IP-10, monocyte chemoattractant protein 1 (MCP-1), MIP-1α, SOFA, qSOFA, APACHE II.

[0136] The threshold level can be obtained, for example, from a Kaplan-Meier analysis in which the occurrence of the disease correlates with the quartiles of the biomarkers in the population. According to this analysis, subjects with biomarker levels above the 75th percentile are at significantly higher risk of suffering from the disease according to the invention. This result is further supported by a Cox regression analysis fully adjusted for classical risk factors. The highest quartile relative to all other subjects is highly significantly associated with an increased risk of suffering from the disease according to the invention.

[0137] Other preferred cut-off values ​​are, for example, the 90th, 95th or 99th percentile of the normal population.By using a percentile higher than the 75th percentile, the number of identified false positive subjects is reduced, but it may not be possible to identify subjects with medium risk, even though they are still at high risk.Therefore, the cut-off value can be adopted depending on whether it is considered more appropriate to identify the majority of risk subjects at the expense of also identifying "false positives", or whether it is considered more appropriate to identify mainly high risk subjects at the expense of missing some subjects with medium risk.

[0138] The above thresholds may be different for other assays if they are calibrated differently from the assay system used in the present invention. The above thresholds should therefore be applied to such differently calibrated assays, taking into account the calibration difference. One possibility to quantify the calibration difference is a method comparison analysis (correlation) of the assay in question (e.g. NT-PTA assay) with each biomarker assay used in the present invention by measuring the respective biomarker (e.g. NT-PTA) in samples using both methods. Another possibility is to use the assay in question to determine the median biomarker level of a representative normal population, assuming that the test has sufficient analytical sensitivity, compare the result with the mean biomarker level (see Example 2), and recalculate the calibration based on the difference obtained by this comparison. The calibration used in the present invention was used to measure samples from normal (healthy) subjects: the mean plasma NT-PTA was 55.2 pmol / L (SD ± 17.8 pmol / L).

[0139] A variety of immunoassays are known and can be used in the assays and methods of the invention, including radioimmunoassays ("RIA"), homogeneous enzyme amplified immunoassays ("EMIT"), enzyme-linked immunosorbent assays ("ELISA"), apoenzyme reactivation immunoassays ("ARIS"), chemiluminescent and fluorescent immunoassays, Luminex-based bead arrays, protein microarray assays, and rapid test formats such as immunochromatographic strip tests ("dipstick immunoassays") and immunochromatographic assays.

[0140] In one embodiment of the present invention, such an assay is a sandwich immunoassay using any type of detection technology, including but not limited to enzyme labeling, chemiluminescent labeling, electrochemiluminescent labeling, and is preferably a fully automated assay. In one embodiment of the present invention, such an assay is an enzyme-labeled sandwich assay. Examples of automated or fully automated assays include assays that can be used in one of the following systems: Roche Elecsys®, Abbott Architect®, Siemens Centauer®, Brahms Kryptor®, Biomerieux Vidas®, Alere Triage®.

[0141] In one embodiment of the present invention, it may be a so-called POC test (Point of Care), which is a testing technology that allows the test to be performed near the patient within less than an hour without the need for a fully automated assay system. An example of this technology is the immunochromatography testing technology.

[0142] In one embodiment of the invention, at least one of the two binding agents is labeled for detection.

[0143] In a preferred embodiment, the label is selected from the group comprising a chemiluminescent label, an enzyme label, a fluorescent label, a radioactive iodine label.

[0144] The assays may be homogeneous or heterogeneous assays, competitive and non-competitive assays. In one embodiment, the assay is in the form of a sandwich assay, which is a non-competitive immunoassay, in which the molecule to be detected and / or quantified is bound to a first antibody and a second antibody. The first antibody may be bound to a solid phase, e.g., a bead, a surface of a well or other container, a chip or a strip, and the second antibody is an antibody labeled, e.g., with a dye, a radioisotope, or a reactive or catalytically active moiety. The amount of labeled antibody bound to the analyte is then measured by an appropriate method. The general compositions and procedures involved in "sandwich assays" are well established and known to those skilled in the art (see, e.g., US Pat. No. 5,399,111). The Immunoassay Handbook,Ed.David Wild,Elsevier LTD,Oxford;3rd ed.(May 2005),ISBN-13:978-0080445267;Hultschig C et al.,Curr Opin Chem Biol.2006 Feb;10(1):4-10.PMID:16376134 ).

[0145] In another embodiment, the assay comprises two capture molecules, preferably antibodies, both present as dispersion in a liquid reaction mixture, a first label component attached to the first capture molecule, said first label component being part of a labeling system based on fluorescence or chemiluminescence quenching or amplification, and a second label component of said marking system attached to the second capture molecule, such that, upon binding of both capture molecules to the analyte, a measurable signal is generated that allows detection of the sandwich complex formed in the solution containing the sample.

[0146] In another embodiment, the labeling system comprises a rare earth cryptate or rare earth chelate in combination with a fluorescent or chemiluminescent dye, particularly a cyanine type dye.

[0147] In the context of the present invention, fluorescence-based assays include, for example, FAM (5- or 6-carboxyfluorescein), VIC, NED, fluorescein, fluorescein isothiocyanate (FITC), IRD-700 / 800, cyanine dyes such as CY3, CY5, CY3.5, CY5.5, Cy7, xanthene, 6-carboxy-2',4',7',4,7-hexachlorofluorescein (HEX), TET, 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (JOE), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 5-carboxyrhodamine-6G (R6G5), 6-carboxyrhodamine-6G (RG6), rhodamine, rhodamine glow, rhodamine red, rhodamine 110, BODIPY dyes, e.g., BODIPY TMR, Oregon Green, coumarins such as umbelliferone, benzimides such as Hoechst 33258; phenanthridines, e.g., Texas Red, Yakima Yellow, Alexa Fluor, PET, ethidium bromide, acridinium dyes, carbazole dyes, phenoxazine dyes, porphyrin dyes, polymethine dyes, and the like.

[0148] In the context of the present invention, chemiluminescence-based assays include the use of dyes based on the physical principles described for chemiluminescent materials (see, for example, US Pat. No. 6,313,992, incorporated herein by reference, including citations at pages 551-562). Kirk-Othmer, Encyclopedia of chemical technology, 4th ed., executive editor, JIKroschwitz; editor, M. Howe-Grant, John Wiley&Sons, 1993, vol. 15, p. 518-562 ). Preferred chemiluminescent dyes are acridinium esters.

[0149] As referred to herein, an "assay" or "diagnostic assay" can be of any type applied in the field of diagnostics. Such an assay can be based on the binding of the analyte to be detected to one or more capture probes with a specific affinity. For the interaction between the capture molecule and the target molecule or molecule of interest, the affinity constant is preferably greater than 10 8 M -1 Greater than.

[0150] In the context of the present invention, a "binder molecule" is a molecule that can be used to bind a target molecule or molecule of interest from a sample, i.e., an analyte (in the context of the present invention, Pro-Tachyinin A and its fragments). Thus, the binder molecule must be appropriately shaped, both in terms of space and surface features such as surface charge, hydrophobicity, hydrophilicity, presence or absence of Lewis donors and / or acceptors, in order to specifically bind to the target molecule or molecule of interest. Thereby, the binding can be mediated, for example, by ionic, van der Waals, pi-pi, sigma-pi, hydrophobic or hydrogen bond interactions, or a combination of two or more of the aforementioned interactions between the capture molecule and the target molecule or molecule of interest. In the context of the present invention, the binder molecule may be selected from the group including, for example, a nucleic acid molecule, a carbohydrate molecule, a PNA molecule, a protein, an antibody, a peptide or a glycoprotein. Preferably, the binder molecule is an antibody, including a fragment of an antibody having sufficient affinity for the target or molecule of interest, including recombinant antibodies or recombinant antibody fragments, as well as chemically and / or biochemically modified derivatives of said antibody or its fragments derived from a variant chain having a length of at least 12 amino acids.

[0151] The chemiluminescent label may be an acridinium ester label, a steroid label with an isoluminol label, and the like.

[0152] The enzyme label may be lactate dehydrogenase (LDH), creatine kinase (CPK), alkaline phosphatase, aspartate aminotransferase (AST), alanine aminotransferase (ALT), acid phosphatase, glucose-6-phosphate dehydrogenase, and the like.

[0153] In one embodiment of the invention, at least one of the two binding agents is bound to a solid phase as a magnetic particle and a polystyrene surface.

[0154] In one embodiment of the assay for determining pro-tachykinin A or fragments in a body fluid sample according to the invention, such an assay is a sandwich assay, preferably a fully automated assay. It can be a fully automated or manual ELISA. It can also be a so-called POC test (point of care). Examples of automated or fully automated assays include assays that can be used in one of the following systems: Roche Elecsys®, Abbott Architect®, Siemens Centauer®, Brahms Kryptor®, Biomerieux Vidas®, Alere Triage®. Examples of test formats are provided above.

[0155] In one embodiment of the assay for determining pro-tachykinin A or a fragment in a body fluid sample according to the invention, at least one of the two binding agents is labeled for detection. Examples of labels are provided above.

[0156] In one embodiment of the assay for determining pro-tachykinin A or a fragment in a sample of a body fluid according to the invention, at least one of the two binding agents is bound to a solid phase, examples of which are provided above.

[0157] In one embodiment of the assay for measuring pro-tachykinin A or a fragment in a body fluid sample according to the invention, the label is selected from the group comprising a chemiluminescent label, an enzymatic label, a fluorescent label, a radioactive iodine label. A further subject of the invention is a kit comprising an assay according to the invention, the components of which may be contained in one or more containers.

[0158] In one embodiment, a subject of the present invention is a point-of-care device for carrying out a method according to the invention, said point-of-care device comprising at least one antibody or antibody fragment directed against either amino acids 3 to 22 (GANDDLNYWSDWYDSDQIK, SEQ ID NO: 11) or amino acids 21 to 36 (IKEELPEPFEHLLQRI, SEQ ID NO: 12), each of said regions comprising at least 4 or 5 amino acids.

[0159] In one embodiment, a subject of the present invention is a point-of-care device for carrying out a method according to the invention, said point-of-care device comprising at least two antibodies or antibody fragments against amino acids 3 to 22 (GANDDLNYWSDWYDSDQIK, SEQ ID NO: 11) and amino acids 21 to 36 (IKEELPEPFEHLLQRI, SEQ ID NO: 12), each of said regions comprising at least 4 or 5 amino acids.

[0160] In one embodiment, the subject of the invention is a kit or a kit for implementing a method according to the invention, said kit comprising at least one antibody or antibody fragment directed against either amino acids 3 to 22 (GANDDLNYWSDWYDSDQIK, SEQ ID NO: 11) or amino acids 21 to 36 (IKEELPEPFEHLLQRI, SEQ ID NO: 12), each of said regions comprising at least 4 or 5 amino acids.

[0161] In one embodiment, a subject of the present invention is a kit for carrying out a method according to the invention, said kit comprising at least two antibodies or antibody fragments directed against amino acids 3 to 22 (GANDDLNYWSDWYDSDQIK, SEQ ID NO: 11) and amino acids 21 to 36 (IKEELPEPFEHLLQRI, SEQ ID NO: 12), each of said regions comprising at least 4 or 5 amino acids.

[0162] The method of the present invention may be partially computer-implemented. For example, the step of comparing the detected level of a marker, such as NT-PTA, with a reference and / or threshold level may be implemented in a computer system. For example, the determined value may be input into the computer system (either manually by a medical professional or automatically from a device where the respective marker level is determined). The computer system may be directly at the point of care (e.g., intensive care unit or ED) or at a remote location connected via a computer network (e.g., via the Internet or a dedicated medical cloud system that can be optionally combined with other IT systems or platforms such as a hospital information system (HIS)). Alternatively or additionally, the associated treatment guidance and / or treatment stratification is displayed and / or printed for the user (typically a medical professional such as a doctor).

[0163] The following embodiments are also subject of the present invention. 1. A method for predicting sepsis, severe sepsis and / or septic shock in a patient, comprising: determining a level of pro-tachykinin A or a fragment thereof of at least 5 amino acids in a sample of bodily fluid obtained from the subject; Correlating the determined level of pro-tachykinin A or a fragment thereof of at least 5 amino acids with sepsis or septic shock; wherein elevated levels above a certain threshold are predictive of sepsis, severe sepsis and / or septic shock. 2. The method of embodiment 1, wherein the sample of the patient's body fluid is taken at a time when the patient shows no clinical symptoms of sepsis, severe sepsis and / or septic shock, or shows mild symptoms of an infectious disease. 3. The method according to embodiment 1 or 2, wherein said pro-tachykinin A is selected from the group comprising SEQ ID NOs: 1-4 and said fragments thereof are selected from the group comprising SEQ ID NOs: 5-12. 4. The method according to any one of the preceding embodiments, wherein the level of pro-tachykinin A or a fragment thereof of at least 5 amino acids is determined by using a binding agent for pro-tachykinin A or a fragment thereof of at least 5 amino acids. 5. The method according to any one of the preceding embodiments, wherein the binding agent is selected from the group comprising an antibody, an antibody fragment or a non-Ig scaffold that binds to pro-tachykinin A or a fragment thereof of at least 5 amino acids. 6. The method of any one of embodiments 1 to 5, wherein the binding agent binds to a region within an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:11 and SEQ ID NO:12. 7. The method according to any one of embodiments 1 to 6, wherein the threshold range is 75 to 200 pmol / L, more preferably 90 to 175 pmol / L, even more preferably 100 to 150 pmol / L, and most preferably the threshold level is 120 pmol / L. 8. The method of any one of the preceding embodiments, wherein the level of pro-tachykinin A is measured by immunoassay and the binding agent is an antibody or antibody fragment that binds to pro-tachykinin A or a fragment thereof of at least 5 amino acids. 9. The method of any one of embodiments 1-8, wherein an assay is used that comprises two binders that bind to two different regions within the region of pro-tachykinin A that is amino acids 3 to 22 (SEQ ID NO:11) and amino acids 21 to 36 (SEQ ID NO:12), each of said regions comprising at least 4 or 5 amino acids. 10. The method according to any one of the preceding embodiments, wherein an assay is used to determine the level of pro-tachykinin A or a fragment thereof of at least 5 amino acids, and the assay sensitivity of the assay is capable of quantifying pro-tachykinin A or a fragment of pro-tachykinin A in a healthy subject and is <10 pmol / L. 11. The method according to any one of the preceding embodiments, wherein the body fluid may be selected from the group comprising blood, serum, plasma, urine, cerebrospinal fluid (CSF), and saliva. 12. At least one further biomarker and / or clinical parameter and / or clinical score is D-dimer, procalcitonin (PCT), C-reactive protein (CRP), lactate, penKid, ADM-NH 2 , MR-proADM, NT-proBNP, BNP, presepsin, pentraxin-3 (PTX-3), CD-64, calprotectin, white blood cell count, lymphocyte count, neutrophil count, hemoglobin, platelet count, albumin, alanine transaminase, creatinine, blood urea, lactate dehydrogenase, creatinine kinase, cardiac troponin I, prothrombin time, serum ferritin, interleukin-6 (IL-6), IL-10, IL-2, IL-7, interferon gamma (IF-γ), tumor necrosis factor alpha (TNF-α), granulocyte colony stimulating factor (GCSF), IP-10, monocyte chemotactic protein 1 (MCP-1), MIP-1α, SOFA, qSOFA, APACHE II. 13. The method according to any one of embodiments 1 to 12, wherein said determining is carried out more than once in one patient. 14. The method of any one of embodiments 1 to 13, for stratifying the subject into risk groups. 15. A point-of-care device for carrying out the method according to any one of embodiments 1 to 14, comprising at least two antibodies or antibody fragments against amino acids 3 to 22 (SEQ ID NO: 11) and amino acids 21 to 36 (SEQ ID NO: 12). 16. A kit for carrying out the method according to any one of embodiments 1 to 14, comprising at least two antibodies or antibody fragments against amino acids 3 to 22 (SEQ ID NO: 11) and amino acids 21 to 36 (SEQ ID NO: 12). EXAMPLES

[0164] Example 1 - Antibody Development Peptides / Conjugates for Immunization For conjugation of the peptide to bovine serum albumin (BSA), the immunizing peptide was synthesized with an additional N-terminal cysteine ​​residue (JPT Technologies, Berlin, Germany). The peptide was covalently bound to BSA by using Sulfo-SMCC (Perbio-science, Bonn, Germany). The coupling procedure was performed according to the Perbio manual.

[0165] [Table 1]

[0166] Production of monoclonal antibodies BALB / c mice were immunized with 100 μg of peptide-BSA-conjugate (emulsified in 100 μl of complete Freund's adjuvant) on days 0 and 14, and with 50 μg (in 100 μl of incomplete Freund's adjuvant) on days 21 and 28. Three days before the fusion experiments were performed, animals received one intraperitoneal injection and one intravenous injection of 50 μg of the conjugate dissolved in 100 μl of saline. Splenocytes from immunized mice and cells of the myeloma cell line SP2 / 0 were fused with 1 mL of 50% polyethylene glycol at 37° C. for 30 s. After washing, the cells were seeded in 96-well cell culture plates. Hybrid clones were selected by growth in HAT medium (RPMI 1640 culture medium supplemented with 20% fetal bovine serum and HAT supplement). After 2 weeks, the HAT medium was replaced with HT medium for 3 passages, and then returned to normal cell culture medium. Cell culture supernatants were primarily screened for antigen-specific IgG antibodies 3 weeks after fusion. Positive microcultures were transferred to 24-well plates for expansion. After retesting, selected cultures were cloned and recloned using limiting dilution method, and isotypes were determined ( Lane,RD1985:J.Immunol.Meth.81:223-228;Ziegler,B.et al.1996:Horm.Metab.Res.28:11-15 ). Antibodies were generated by standard antibody generation methods ( Marx et al.1997, Monoclonal Antibody Production ATLA 25,121 ) and purified by protein A chromatography. Antibody purity was >95% based on SDS gel electrophoresis analysis.

[0167] Antibody labeling and coating Labeled compound (tracer, anti-PTA 3-22): 100 μg (100 μl) antibody (1 mg / ml in PBS, pH 7.4) was mixed with 10 μl acridinium NHS-ester (1 mg / ml in acetonitrile, InVent GmbH, Germany) and incubated for 20 min at room temperature. The labeled antibody was purified by gel filtration HPLC on a Bio-Sil SEC 400-5 (Bio-Rad Laboratories, Inc., USA). The purified labeled antibody was diluted in (300 mmol / l potassium phosphate, 100 mmol / l NaCl, 10 mmol / l Na-EDTA, 5 g / l bovine serum albumin, pH 7.0). The final concentration was approximately 800.000 relative light units (RLU) of labeled compound (approximately 20 ng of labeled antibody) per 200 μl. Acridinium ester chemiluminescence was measured by using an AutoLumat LB 953 (Berthold Technologies GmbH & Co. KG).

[0168] Coating (solid phase, anti-PTA 22-36): Polystyrene tubes (Greiner Bio-One International AG, Austria) were coated with the antibody (1.5 μg antibody / 0.3 mL 100 mmol / l NaCl, 50 mmol / l Tris / HCl, pH 7.8) for 18 h at room temperature. After blocking with 5% bovine serum albumin, the tubes were washed with PBS (pH 7.4) and dried under vacuum.

[0169] Pro-tachykinin A immunoassay and calibration 50 μl of sample (or calibrator) was pipetted into the coated tube, and after adding the labeled antibody (200 μl), the tube was incubated for 2 h at 18-25 °C. Unbound tracer was removed by washing five times (1 mL each) with a washing solution (20 mmol / l PBS, pH 7.4, 0.1% Triton X-100). The labeled antibody bound to the tube was measured by using a Luminometer LB 953, Berthold, Germany. A K of 20 mM was used. 2 PO 4 The assay was calibrated using dilutions of synthetic P37 diluted in 6 mM EDTA, 0.5% BSA, 50 μM amastatin, 100 μM leupeptin, pH 8.0. PTA control plasma is available from ICI-diagnostics, Berlin, Germany. Figure 1 shows a typical PTA dose / signal curve.

[0170] The analytical assay sensitivity was 4.4 pmol / L (median signal generated by 20 measurements of 0 calibrators (no PTA added) + 2 standard deviations (SD); corresponding PTA concentrations were calculated from the standard curve).

[0171] Example 2 - NT-PTA in healthy subjects EDTA plasma samples from fasting healthy subjects (n=4435, mean age 56 years) were measured using the NT-PTA assay. The mean NT-PTA value in the population was 55.2 pmol / L, with a standard deviation of ±17.8 pmol / L, and the lowest value was 9.07 pmol / L, with a 99. th The percentile was 107.6 pmol / L. The assay sensitivity was 4.4 pmol / L, so all values ​​were detectable by the assay. The distribution of PTA values ​​in healthy subjects is shown in Figure 2.

[0172] Example 3 - Endotoxin studies in humans Thirty-two healthy male applicants (mean (±SE) age 23.9 [±0.7 years]) were admitted to the clinical research unit of the hospital. All screening tests were normal. Participants were challenged at t=0 hours with LPS (Escherichia coli lipopolysaccharide, 0311.H10:k) as a bolus intravenous injection at a standardized dose of 4 ng / kg. Blood was collected from 2 hours before LPS injection and at intervals up to 24 hours thereafter. Blood was immediately centrifuged (4°C, 10 min, 3000 rpm) and plasma was stored at -20°C until assayed. Determination of different biomarkers of inflammation interleukin-6 (IL-6), tumor necrosis factor alpha (TNFα), and procalcitonin (PCT) as well as N-terminal protachykinin A (NT-PTA) in endotoxin-treated test human subjects showed a time-dependent course of the concentration of the substances in the blood (see Figure 3).

[0173] First, an increase in TNFα occurs as expected about 1 hour after injection of endotoxin. This is followed shortly by an increase in the cytotoxin IL-6 (about 1.5 hours after injection of endotoxin). After 3 hours, the concentrations of TNFα and IL-6 are decreasing, but surprisingly, an increase in A-peptide concentration now occurs, reaching starting levels only after about 7 hours. PCT shows an increase in concentration after 5 hours, which increases steadily during the further course. NT-PTA secretion is inducible by injection of endotoxin alone, and this secretion is one event in the immune cascade between TNFα / IL-6 and PCT.

[0174] PCT is an established marker for sepsis diagnosis and indicates the onset of sepsis in this study. However, NT-PTA concentrations increase approximately 3 hours before the sepsis biomarker PCT increases. Therefore, it can be concluded that NT-PTA is a marker for sepsis prediction.

[0175] Example 4 - PTA for prediction of sepsis within 48 hours in patients with CAP A cohort of 218 subjects hospitalized with community-acquired pneumonia (CAP) was studied. The primary outcomes were sepsis and severe sepsis, defined according to the International Consensus Conference criteria ( Singer et al.2016.JAMA 315(8):801-10 ). A total of 105 patients developed sepsis / severe sepsis within 48 hours. NT-PTA was measured in EDTA plasma samples at the time of admission. Patients who developed sepsis / severe sepsis within 48 hours after admission had significantly higher NT-PTA concentrations (p=0.0007) compared to patients who did not develop sepsis / severe sepsis (Figure 4), with an AUC of 0.632 (p=0.08) (Figure 5). Table 2 shows exemplary cutoff values ​​with their respective sensitivity and specificity.

[0176] [Table 2]

[0177] Example 5 - PTA in patients with suspected sepsis We investigated 712 patients admitted with suspected sepsis. Inclusion criteria were age ≥ 18 years, qSOFA at least 1 (GCS < 15), respiratory rate ≥ 22 / min, and systolic blood pressure ≤ 100 mm Hg. One hundred and ninety-eight patients developed sepsis or septic shock after admission. NT-proTA was measured in EDTA plasma samples at admission (Figure 6). Patients who developed sepsis had significantly higher NT-proTA concentrations (median 125.8 pmol / L [IQR 77.8-237.8]) than patients who did not develop sepsis (median 76.9 pmol / L [55.5-115.2]). Furthermore, patients who further developed septic shock (n = 23) had significantly elevated NT-proTA levels (209.2 pmol / L [118.7-402.3]) compared with patients who did not develop septic shock or sepsis. ROC plot analysis revealed an AUC of 0.703 (p<0.0001) for NT-proTA to distinguish between patients who developed sepsis or septic shock after hospitalization and those who did not develop sepsis or septic shock (Figure 7).

[0178] Table 3 shows exemplary cut-off values ​​with their respective sensitivity and specificity for distinguishing patient groups (never developing sepsis / septic shock vs. developing sepsis / septic shock).

[0179] [Table 3]

[0180] ROC plot analysis for discriminating between patients who would develop septic shock after hospitalization and those who would not reveal an AUC of 0.757 (p<0.0001) for NT-proTA (Figure 8).

[0181] Table 4 shows exemplary cutoff values ​​with their respective sensitivity and specificity for distinguishing patient groups (not developing septic shock vs. developing septic shock).

[0182] [Table 4]

[0183] Example 6 - PTA in patients admitted to the emergency department (ED) This was a prospective observational study enrolling 97 consecutive patients admitted to the emergency department of the Sant'Andrea Hospital in Rome for acute pathological conditions and further hospitalization. For each enrolled patient, clinical laboratory data and plasma NT-proTA levels were collected on arrival. Patient characteristics are summarized in Table 5. After discharge, a 60-day follow-up by telephone was performed.

[0184] [Table 5]

[0185] The survival rate was 81.4%, and the event (death) mainly occurred in the first week after hospitalization. NT-proTA was measured at the time of admission. The inventors correlated the initial PTA value with in-hospital mortality. NT-proTA is highly prognostic for the outcome in ED patients admitted (AUC / C index 0.795; p < 0.00001). Figure 9 shows Kaplan-Meier plots for the survival of ED patients by a) quartiles of PTA at admission and b) a cutoff of 100 pmol / L of NT-proTA at admission.

[0186] Procalcitonin was measured at the time of admission and 24 / 48 and 72 hours later. The PCT value was used as a surrogate for the diagnosis of sepsis. The sepsis categories were defined as follows. High-high (HH) - developed sepsis and has not yet resolved, low-high (LH) - has developed sepsis, high-low (HL) - sepsis has resolved, low-low (LL) - no sepsis. Changes were defined as follows: when PCT t0 < 1 ng / mL and the maximum PCT value at t24 - t72 > 1 ng / mL, low-high (LH); when PCT t0 > 1 ng / mL and the maximum PCT value at t24 - t72 < 75% of t0, high-low (HL); when PCT t0 < 1 ng / mL at all time points, low-low (LL); when PCT t0 > 1 ng / mL at all time points, high-high (HH). The NT-proTA values (within 72 hours, p = 0.049) for the sepsis categories are shown in Figure 10. The NT-proTA concentrations in each subgroup are shown in Table 6.

[0187]

Table 6

[0188] Patients with sepsis who do not resolve within the next 72 hours (HH) show higher NT-proTA values than patients who develop sepsis within 72 hours (LH), which is also higher than the NT-proTA values in patients with resolved sepsis (HL) and patients without sepsis (LL).

[0189] Table 7 shows patient subgroups and the percentage of patients above exemplary cutoff values ​​of 100, 120 and 140 pmol / L, respectively.

[0190] [Table 7]

Claims

1. A method for predicting sepsis, severe sepsis and / or septic shock in a patient, comprising: ● determining the level of pro-kininin A or a fragment thereof of at least 5 amino acids in a sample of body fluid obtained from said subject; ● correlating the determined level of pro-kininin A or a fragment thereof of at least 5 amino acids with sepsis or septic shock; wherein an elevated level above a certain threshold predicts sepsis, severe sepsis and / or septic shock.

2. The method according to claim 1, wherein said sample of body fluid of said patient is taken at a time when said patient does not exhibit clinical symptoms of sepsis, severe sepsis and / or septic shock or exhibits mild symptoms of an infectious disease.

3. The method according to claim 1 or 2, wherein said pro-kininin A is selected from the group consisting of SEQ ID NOs: 1 to 4, and said fragment thereof is selected from the group consisting of SEQ ID NOs: 5 to 12.

4. The method according to claim 1 or 2, wherein the level of pro-kininin A or a fragment thereof of at least 5 amino acids is determined by using a binding agent for pro-kininin A or a fragment thereof of at least 5 amino acids.

5. The method according to claim 1 or 2, wherein said binding agent is selected from the group consisting of an antibody, an antibody fragment or a non-Ig scaffold that binds to pro-kininin A or a fragment thereof of at least 5 amino acids.

6. The method according to claim 1 or 2, wherein said binding agent binds to a region within an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11 and 12.

7. The method according to claim 1 or 2, wherein said threshold range is 75 to 200 pmol / L, more preferably 90 to 175 pmol / L, even more preferably 100 to 150 pmol / L, and most preferably said threshold level is 120 pmol / L.

8. The method according to claim 1 or 2, wherein the level of pro-kininin A is measured by immunoassay, and said binding agent is an antibody or an antibody fragment that binds to pro-kininin A or a fragment thereof of at least 5 amino acids.

9. An assay is used that comprises two binding agents that bind to two different regions within the region of pro-kininin A that is amino acids 3 to 22 (SEQ ID NO: 11) and amino acids 21 to 36 (SEQ ID NO: 12), each of said regions containing at least 4 or 5 amino acids. The method according to claim 1 or 2.

10. An assay is used to measure the level of pro-tachykinin A or a fragment thereof of at least 5 amino acids, and the assay sensitivity of the assay can quantify pro-tachykinin A or a pro-tachykinin A fragment in a healthy subject and is <10 pmol / L. The method according to claim 1 or 2.

11. The method according to claim 1 or 2, wherein the body fluid can be selected from the group consisting of blood, serum, plasma, urine, cerebrospinal fluid (CSF), and saliva.

12. At least one additional biomarker and / or clinical parameter and / or clinical score is D-dimer, procalcitonin (PCT), C-reactive protein (CRP), lactate, penKid, ADM-NH ２ , MR-proADM, NT-proBNP, BNP, presepsin, pentraxin-3 (PTX-3), CD-64, calprotectin, white blood cell count, lymphocyte count, neutrophil count, hemoglobin, platelet count, albumin, alanine transaminase, creatinine, blood urea, lactate dehydrogenase, creatine kinase, cardiac troponin I, prothrombin time, serum ferritin, interleukin-6 (IL-6), IL-10, IL-2, IL-7, interferon gamma (IF-γ), tumor necrosis factor-α (TNF-α), granulocyte colony-stimulating factor (G-CSF), IP-10, monocyte chemoattractant protein 1 (MCP-1), MIP-1α, SOFA, qSOFA, APACHE II, and can be determined to be selected from the group consisting of, the method according to claim 1 or 2.

13. The method according to claim 1 or 2, wherein the determination is performed two or more times in one patient.

14. The method according to claim 1 or 2 for stratifying the subject into a risk group.

15. A point-of-care device for carrying out the method according to claim 1 or 2, comprising at least two antibodies or antibody fragments against amino acids 3-22 (SEQ ID NO: 11) and amino acids 21-36 (SEQ ID NO: 12).

16. A kit for carrying out the method according to claim 1 or 2, comprising at least two antibodies or antibody fragments against amino acids 3-22 (SEQ ID NO: 11) and amino acids 21-36 (SEQ ID NO: 12).

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