Method for assaying peptidyl glycine alpha-amidated monooxygenase (PAM) and diagnostic use thereof

Abstract

The present invention relates to a method for determining the level of PAM and / or its homoisomers and / or fragments thereof in a bodily fluid or tissue sample using an assay comprising at least one binding agent for conformational epitopes of PAM, and to the use of said method for diagnostic purposes.

Description

Technical Field

[0001] The present invention relates to a method for determining the level of PAM and / or its isoforms and / or fragments thereof in a body fluid or tissue sample using an assay comprising at least one binding agent directed against a conformational epitope of PAM, and to the use of said method for diagnostic purposes. Background Art

[0002] Bioactive peptide hormones function as signaling molecules. Most bioactive peptide hormones are synthesized from larger, inactive precursor peptides. During biosynthesis, these peptides undergo several co- and post-translational modifications, including cleavage of the signal peptide, endoproteolytic cleavage of the precursor propeptide by specific endopeptidases, primarily at basic residue pairs, removal of basic residues by carboxypeptidases, disulfide bond formation, and N- and O-glycosylation ( Eipper et al., 1993. Protein Science 2 (4): 489–97 To obtain full biological activity, more than half of the known neuroendocrine peptides require additional modification steps, including the formation of a C-terminal α-amide group ( Guembe, et al., 1999. J Histochem Cytochem 47(5): 623–36 The final step in peptide hormone biosynthesis involves the action of a bifunctional enzyme, peptidylglycine α-amidating monooxygenase (PAM). PAM specifically recognizes the C-terminal glycine residue in its substrate and cleaves glyoxylate from the C-terminal glycine residue of the peptide in a two-step enzymatic reaction, resulting in the formation of a C-terminal α-amidated peptide hormone, where the resulting α-amide group is derived from the cleaved C-terminal glycine ( Prigge et al., 2004. Science 304(5672): 864–67 This amidation reaction occurs in the lumen of the secretory granules, before the exocytosis of the amidated product ( Martinez and Treston 1996. Molecular and Cellular Endocrinol 123: 113–17 ). α-amidated peptides are, for example, adrenomedullin, substance P, vasopressin, neuropeptide Y, amylin, calcitonin, neurokinin A, etc. However, it has been previously demonstrated that PAM can also catalyze the formation of α-amides from non-peptide characteristic glycinated substrates, such as N-fatty acyl-glycine, which is converted to primary fatty acid amides (PFAMs) such as oleamide via PAM. The peptidyl glycine amidation activity that has been identified and purified has been shown to be dependent on copper and ascorbic acid ( Emeson et al., 1984. Journal of Neuroscience: 2604–13; Kumar et al., 2016. J Mol Endocrinol 56(4): T63-76; Wand et al., 1985. Neuroendocrinology 41: 482–89 ).

[0003] In humans, the PAM gene is located on chromosome 5q21.1, is 160 kb in length, and contains 25 known exons ( Gaier et al., 2014. BMC Endocrine Disorders 14At least six isoforms are known to be produced by alternative splicing (SEQ ID 1-6). PAM enzymes are found to be expressed to varying degrees in almost all mammalian cell types, with significant expression in airway epithelial cells, endothelial cells, ependymal cells, adult atria, brain, kidney, pituitary gland, gastrointestinal tract, and reproductive tissues ( Chen et al., 2018. Diabetes Obes Metab 20 Suppl 2:64-76; Oldham et al., 1992. Biochem Biophys Res Commun 184(1): 323–29; Schafer et al., 1992. J Neurosci 12(1): 222–34 ).

[0004] However, the highest areas of human PAM activity are found in the pituitary, pituitary stalk, and hypothalamus. The plasma amidation activity of healthy children under 15 years old is significantly higher than that of healthy adults ( Wand et al., 1985 Metabolism 34(11): 1044–52 ).

[0005] The precursor protein (amino acids 1-973) of the largest known PAM isoform 1 (SEQ ID No. 1) encoded by the PAM cDNA is as follows Figure 1 As shown. The N-terminal signal sequence (amino acids 1-20) ensures that the nascent PAM polypeptide is directed into the secretory cavity of the endoplasmic reticulum and is subsequently cleaved during co-translation. The PAM propeptide is then processed by the same mechanism used for the biosynthesis of integral membrane proteins and secretory proteins, which includes cleavage of the pro-region (amino acids 21-30) to ensure correct folding, disulfide bond formation, phosphorylation, and glycosylation ( Bousquet-Moore et al., 2010. J Neurosci Res 88 (12):2535-45 ).

[0006] like Figure 1 As shown in , the PAM cDNA further encodes two distinct enzymatic activities. The first, termed peptidyl-glycine α-hydroxylating monooxygenase (PHM; EC 1.14.17.3), catalyzes the conversion of a C-terminal glycine residue to α-hydroxyglycine. The second, termed peptidyl-α-hydroxyglycine α-amidating lyase (PAL; EC 4.3.2.5), catalyzes the conversion of α-hydroxyglycine to an α-amide with subsequent release of glyoxylate. The sequential action of these two separate enzymatic activities contributes to the overall peptidyl-glycine α-amidation activity. The first enzymatic activity (PHM) is located directly upstream of the proregion (amino acids 31-494 of isoform 1 (SEQ ID No. 7)). The second catalytic activity (PAL) is located within amino acids 495-817 of isoform 1 after exon 16 (SEQ ID No. 8).

[0007] like Figure 1As shown in , these two enzymatic activities can be co-encoded in one polypeptide as membrane-bound proteins (isoforms 1, 2, 5, 6; corresponding to SEQ ID Nos. 1, 2, 5, and 6) or as soluble proteins lacking a transmembrane domain (isoforms 3 and 4; corresponding to SEQ ID Nos. 3 and 4). While isoforms 1, 2, 5, and 6 remain in the outer plasma membrane after fusion of secretory vesicles with the plasma membrane (followed by endocytosis and recycling or degradation), soluble PAM isoforms lacking the TMD (isoforms 3 and 4) (amino acids 864-887) are co-secreted with the peptide hormone ( Wand et al., 1985 Metabolism 34(11): 1044–52 Furthermore, in the secretory pathway, prohormone convertase can convert membrane-bound PAM proteins into soluble PAM proteins by cleaving within the flexible region (exons 25 / 26) connecting PAL and TMD ( Bousquet-Moore et al., 2010. J Neurosci Res 88(12):2535-45 In the secretory pathway, the PHM subunit can be cleaved from the soluble or membrane-bound PAM by prohormone convertases acting on a double basic cleavage site in the exon 16 region. In addition, during endocytosis, the full-length PAM protein may also be converted to a soluble form by the action of α- and γ-secretases ( Bousquet-Moore et al., 2010. J Neurosci Res 88(12):2535-45 Membrane-bound PAMs from late endosomes can be further secreted in the form of exosomal vesicles.

[0008] PHM and PAL activities, as well as those of full-length PAM, have been measured in several human tissues and fluids. However, when allowed to react separately in the same compartment, fluid, or in vitro experimental setup, the separate activities of PHM and PAL in soluble form also result in the formation of C-terminally α-amidated products from C-terminally glycinated substrates. To date, it is not fully understood how the transfer of PHM hydroxylation products to PAL occurs. Evidence suggests that the hydroxylation products are released into solution rather than being transferred directly from PHM to PAL. Yin et al., 2011. PLoS One 6(12): e28679 ). To date, the source of circulating PAM is still unclear.

[0009] Figure 2 A partial reaction of PHM is depicted in Figure 2. PHM is a copper-dependent monooxygenase responsible for the stereospecific hydroxylation of C-terminal glycine at the α-carbon atom. In this hydroxylation reaction, ascorbic acid is believed to be the naturally occurring reducing agent, while the oxygen of the newly formed hydroxyl group is shown to be derived from molecular oxygen. Figure 2 Part of the PAL reaction is depicted in Figure 1. The catalytic action of PAL involves proton abstraction from the hydroxyglycine formed by PHM by a protein backbone-derived base and nucleophilic attack of the hydroxyl oxygen on the divalent metal, resulting in cleavage of glyoxylate and formation of the C-terminal amide.

[0010] Thus, the terms "amidation activity," "α-amidation activity," "peptidylglycine α-amidation activity," or "PAM activity," or "active PAM" refer to the continuous enzymatic activity of PHM and PAL, independent of the presence of splice variants or mixtures of splice variants or post-translationally modified PAM enzymes, or soluble, PHM or PAL activity alone, or soluble PHM and membrane-bound PAL, or a combination of all of the foregoing, as long as these forms result in the formation of an α-amidated product of peptidic or non-peptidic character from a glycinated substrate of peptidic or non-peptidic character. In other words, the terms "amidation activity," "α-amidation activity," "peptidylglycine α-amidation activity," or "PAM activity," or "active PAM" can be described as the continuous action of the enzymatic activity located within amino acids 31 to 817 of the propeptide encoded by the human PAM cDNA, independent of the presence of splice variants or mixtures thereof.

[0011] PAM activity was analyzed in several human tissues and fluids, either healthy or from samples of several diseases.

[0012] Detection of PAM activity in human body fluids primarily involves the use of radiolabeled synthetic tripeptides such as 125 ID-TyrValGly, 125 IN-acetyl-TyrValGly or a comparably modified tripeptide, and the amidated product was quantified by γ-scintillation counting ( Kapuscinski et al., 1993. Clinical Endocrinology 39(1): 51–58; Wand et al., 1985 Metabolism 34(11): 1044–52; Tsukamoto et al., 1995. Internal Medicine 34(4): 229–32; Wand et al., 1987 Neurology 37: 1057–61; Wand et al., 1985 Neuroendocrinol 41: 482– 89 In addition, substance P-Gly or truncated neuropeptide Y-Gly was used as a substrate for PAM activity assay ( Gether et al., 1991 Mol Cell Endocrinol 79(1-3): 53–63; Hyyppä et al., 1990 Pain 43: 163–68; Jeng et al., 1990 Analytical Biochemistry 185(2): 213–19 ).

[0013] The presence of α-amidating activity in human circulation was initially demonstrated by Wand et al. ( Wand et al., 1985 Metabolism 34(11): 1044–52 They reported no sex differences, but some variability in PAM activity in certain disease states: plasma PAM activity was elevated in adults with hypothyroidism and in patients with medullary thyroid carcinoma. PAM activity has been shown to be elevated in tissues of medullary thyroid carcinoma, pheochromocytoma, and pancreatic islet cell tumors, suggesting increased formation of amidated peptides in endocrine tumor tissues ( Gether et al., 1991 Mol Cell Endocrinol 79(1-3): 53–63; Wand et al., 1985 Neuroendocrinol 41: 482–89 ).

[0014] Patients with multiple endocrine neoplasia type 1 (MEN-1) and pernicious anemia show decreased plasma PAM activity compared with healthy control subjects ( Kapuscinski et al., 1993. Clin Endocrinol 39(1): 51–58 ).

[0015] Wand and colleagues demonstrated the presence of amidating activity in human cerebrospinal fluid (CSF) ( Wand et al., 1985 Neuroendocrinol 41: 482–89 ). Plasma PAM activity showed no changes in patients with Alzheimer's disease (AD) compared with healthy controls, whereas PAM activity in CSF was significantly decreased compared with that in normal samples ( Wand et al., 1987 Neurology 37: 1057–61 ). In addition, WO2015 / 103594 states that the presence of PAM proteins detected by mass spectrometry in the CSF of AD patients is reduced compared to healthy controls. In addition, ADM-NH2, one of the amidation products of PAM, was shown to be reduced in patients with prevalent and sporadic Alzheimer's disease ( WO2019 / 154900 However, to date, no direct association between circulating PAM activity and the prediction, diagnosis, or progression of AD has been reported.

[0016] The amidation activity in CSF of patients with low back pain was analyzed using 1-12 substance P-Gly (SP-Gly) as a substrate. Hyyppä et al., 1990 Pain 43: 163–68 It has been shown that PAM activity is elevated in CSF and significantly decreased in serum in patients with multiple sclerosis (MS). Tsukamoto et al., 1995. Internal Medicine 34(4): 229–32; WO2010 / 005387 ).exist( WO2014 / 118634 ) described an association between plasma activity of PAM and type 2 diabetes.

[0017] WO2021 / 170752 describes methods for determining the level (including concentration or activity) of a PAM in a body fluid sample and its use for diagnostic purposes. Specifically, the patent application shows the use of a binding agent (such as an antibody) directed against a linear peptide epitope to determine the level of a PAM.

[0018] The surprising discovery of the present invention is that the total amount (concentration) of PAM in the body fluid or tissue of a subject can be measured as the level of PAM activity using a binding agent (such as an antibody) directed against a conformational epitope of PAM for diagnosis, prognosis, prediction or monitoring of adverse events of a disease. Summary of the Invention

[0019] The subject of this application is a method for determining the level of peptidylglycine alpha-amidating monooxygenase (PAM) and / or its isomers and / or fragments in a body fluid or tissue sample using an assay comprising at least one binding agent directed against a conformational epitope of PAM.

[0020] One embodiment of the present application relates to a method for determining the level of PAM and / or its isoforms and / or fragments in a body fluid or tissue sample using an assay, wherein the assay comprises at least one binder to a conformational epitope of PAM, wherein the conformational epitope is at least 4 amino acids, preferably at least 5 amino acids.

[0021] In one embodiment of the present application, the at least one binding agent binds to a conformational epitope contained within the PHM subunit of PAM (SEQ ID No. 7) or a conformational epitope contained within the PAL subunit of PAM (SEQ ID No. 8).

[0022] In one embodiment of the present application, the at least one binding agent binds to a conformational epitope contained within the following sequence of PAM: a PHM fragment comprising amino acids 31-377 of PAM (SEQ ID No. 25), or a PAL fragment comprising amino acids 495-817 of PAM (SEQ ID No. 8).

[0023] One embodiment of the present application relates to a method for determining the level of PAM and / or its isoforms and / or fragments in a body fluid or tissue sample using an assay comprising two binding agents that bind to two different regions of PAM, wherein at least one of the two binding agents is directed against a conformational epitope of PAM.

[0024] One embodiment of the present application relates to a method for determining the level of PAM and / or its isoforms and / or fragments in a body fluid or tissue sample using an assay comprising two binding agents that bind to two different regions of PAM, wherein each of the two binding agents is directed against a conformational epitope of PAM.

[0025] In one embodiment of the present application, the first of the two binding agents binds to a conformational epitope contained within the PHM subunit of PAM (SEQ ID No. 7), and the second of the two binding agents binds to a conformational epitope contained within the PAL subunit of PAM (SEQ ID No. 8).

[0026] In one embodiment of the present application, each of the two binding agents is directed against an epitope contained within the following sequences of PAM: a PHM fragment comprising amino acids 31-377 of PAM (SEQ ID No. 25) and a PAL fragment comprising amino acids 495-817 of PAM (SEQ ID No. 8).

[0027] One embodiment of the present application relates to diagnosing or prognosing a disease in a subject and / or predicting the risk of a subject developing a disease or experiencing an adverse event and / or monitoring a disease or adverse event in a subject, which is performed by determining the level of PAM and / or its isomers and / or fragments thereof in a body fluid or tissue sample of the subject using an assay, wherein the assay comprises at least one binding agent to a conformational epitope of PAM, wherein the disease in the subject is selected from the group comprising dementia, cardiovascular disorder, kidney disease, cancer, inflammatory or infectious disease and / or metabolic disease, wherein the adverse event is selected from the group comprising cardiac event, cardiovascular event, cerebrovascular event, cancer, diabetes, infection, severe infection, sepsis-like systemic infection, sepsis and all-cause mortality.

[0028] One embodiment of the present application relates to diagnosing or prognosing a disease in a subject and / or predicting the risk of a subject developing a disease or experiencing an adverse event and / or monitoring a disease or adverse event in a subject, by determining the level of PAM and / or its isomers and / or fragments thereof in a body fluid or tissue sample of the subject using an assay, wherein the assay comprises at least one binding agent to a conformational epitope of PAM, wherein the disease in the subject is selected from the group comprising dementia, cardiovascular disorders, kidney diseases, inflammatory or infectious diseases and / or metabolic diseases, wherein the adverse event is selected from the group comprising cardiac events, cardiovascular events, cerebrovascular events, diabetes, infection, severe infection, sepsis-like systemic infection, sepsis and all-cause mortality.

[0029] One embodiment of the present application relates to a method for diagnosing or prognosing a disease in a subject and / or predicting the risk of a subject suffering from a disease or experiencing an adverse event and / or monitoring a disease or adverse event in a subject, which is performed by measuring the level of PAM and / or its isomers and / or fragments thereof in a body fluid or tissue sample of the subject, and the method comprises the following steps:

[0030] determining the level of PAM and / or its isomers and / or fragments thereof in a body fluid or tissue sample of the subject using an assay, wherein the assay comprises at least one binding agent directed against a conformational epitope of PAM,

[0031] - Comparing the measured amount to a predetermined threshold value.

[0032] wherein the subject is diagnosed as having a disease if the measured amount is below or above the predetermined threshold, or

[0033] wherein if said measured amount is below or above said predetermined threshold, the outcome of said disease is prognosed, or

[0034] wherein if the measured amount is below or above the predetermined threshold, the risk of the patient developing a disease or experiencing an adverse event is predicted, or

[0035] wherein the subject is monitored for disease or adverse events.

[0036] A preferred embodiment of the method for diagnosing or prognosing a disease in a subject and / or predicting the risk of a subject developing a disease or experiencing an adverse event and / or monitoring a disease or adverse event in a subject comprises determining the level of PAM and / or its isomers and / or fragments thereof in a body fluid or tissue sample of the subject, wherein the level of PAM and / or its isomers and / or fragments thereof is the total concentration of PAM and / or its isomers and / or fragments thereof having at least 12 amino acids in the body fluid or tissue sample of the subject, and wherein an assay comprising at least one binding agent to a conformational epitope of PAM is used.

[0037] Another embodiment of the present application relates to a method for diagnosing or prognosing a disease in a subject and / or predicting the risk of a subject developing a disease or experiencing an adverse event and / or monitoring a subject for a disease or adverse event, which is performed by determining the level of PAM and / or its isomers and / or fragments thereof in a body fluid or tissue sample of the subject using an assay, wherein the assay comprises at least one binding agent to a conformational epitope of PAM, and wherein PAM and / or its isomers and / or fragments thereof are selected from the group comprising sequences SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8 and SEQ ID No. 10.

[0038] Those skilled in the art will appreciate that the PAM isomeric sequences shown in the sequence listing (SEQ ID Nos. 1 to 6) contain an N-terminal signal sequence (amino acids 1-20), which is cleaved prior to secretion of the protein. Therefore, in preferred embodiments, the PAM isomeric sequences (SEQ ID Nos. 1 to 6) and / or fragments thereof do not contain an N-terminal signal sequence.

[0039] Another embodiment of the present application relates to a method for diagnosing or prognosing a disease in a subject and / or predicting the risk of a subject developing a disease or experiencing an adverse event and / or monitoring a disease or adverse event in a subject, which is performed by determining the level of PAM and / or its isomers and / or fragments thereof in a body fluid or tissue sample of the subject, wherein the total concentration of PAM and / or its isomers and / or fragments thereof having at least 12 amino acids is detected using an immunoassay, wherein the immunoassay comprises at least one binder to a conformational epitope of PAM.

[0040] One embodiment of the present application relates to a method for diagnosing or prognosing a disease in a subject and / or predicting the risk of a subject developing a disease or experiencing an adverse event and / or monitoring a disease or adverse event in a subject, which is performed by determining the level of PAM and / or its isomers and / or fragments thereof in a body fluid or tissue sample of the subject using an assay, wherein the assay comprises at least one binding agent to a conformational epitope of PAM, wherein the PAM and / or its isomers and / or fragments thereof are selected from the group consisting of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8 and SEQ ID No. 10.

[0041] Another embodiment of the present application relates to a method for diagnosing or prognosing a disease in a subject and / or predicting the risk of a subject developing a disease or experiencing an adverse event and / or monitoring a subject for a disease or an adverse event, which is performed by determining the level of PAM and / or its isomers and / or fragments thereof in a body fluid or tissue sample of the subject using an assay, wherein the assay comprises at least one binding agent to a conformational epitope of PAM, wherein the risk of the subject developing a disease is determined, and wherein the subject is a healthy subject.

[0042] Another embodiment of the present application relates to a method for diagnosing or prognosing a disease in a subject and / or predicting the risk of a subject developing a disease or experiencing an adverse event and / or monitoring a disease or adverse event in a subject, which is performed by determining the level of PAM and / or its isomers and / or fragments thereof in a body fluid or tissue sample of the subject using an assay, wherein the assay comprises at least one binding agent to a conformational epitope of PAM, wherein the disease is selected from:

[0043] Dementia, wherein the dementia is selected from the group consisting of mild cognitive impairment (MCI), Alzheimer's disease, vascular dementia, mixed Alzheimer's disease and vascular dementia, dementia with Lewy bodies, frontotemporal dementia, focal dementia (including progressive aphasia), subcortical dementia (including Parkinson's disease) and secondary causes of dementia syndromes (including intracranial lesions),

[0044] Cardiovascular disorder, wherein the cardiovascular disorder may be selected from the group consisting of atherosclerosis, hypertension, heart failure (including acute heart failure and acute decompensated heart failure), atrial fibrillation, cardiovascular ischemia, cerebral ischemic injury, cardiogenic shock, stroke (including ischemic and hemorrhagic stroke and transient ischemic attack) and myocardial infarction,

[0045] Kidney disease, wherein the kidney disease may be selected from the group comprising nephrotoxicity (drug-induced nephropathy), acute kidney injury (AKI), chronic kidney disease (CKD), diabetic nephropathy, end-stage renal disease (ESRD),

[0046] Cancer, wherein the cancer may be selected from the group consisting of prostate cancer, breast cancer, lung cancer, colorectal cancer, bladder cancer, ovarian cancer, cervical cancer, skin cancer (including melanoma), stomach cancer, liver cancer, pancreatic cancer, leukemia, non-Hodgkin's lymphoma, kidney cancer, esophageal cancer, pharyngeal cancer,

[0047] an infectious disease caused by an infectious organism such as bacteria, virus, fungus or parasite, selected from the group comprising SIRS, sepsis and septic shock,

[0048] • Metabolic diseases selected from the group comprising type 1 diabetes, type 2 diabetes, metabolic syndrome.

[0049] Another specific embodiment of the present application relates to a method for determining the level of PAM and / or its isoforms and / or fragments in a body fluid or tissue sample using an assay, wherein the assay comprises two binding agents that bind to two different regions of PAM, wherein the two binding agents are directed against a conformational epitope of at least 5 amino acids in length, preferably at least 4 amino acids in length, wherein the two binding agents are directed against a conformational epitope contained within the following sequences of PAM: PHM fragment (SEQ ID No. 25) and / or PAL fragment (SEQ ID No. 8).

[0050] Another embodiment of the present application relates to the use of an antibody for determining the level of PAM and / or its isoforms and / or fragments thereof, wherein the antibody specifically binds to a conformational epitope of a sequence selected from the group consisting of a PHM fragment (SEQ ID No. 25) and / or a PAL fragment (SEQ ID No. 8).

[0051] Another preferred embodiment of the present application relates to a kit for determining PAM levels, comprising one or more antibodies that bind to a conformational epitope of a PAM sequence selected from the group comprising PHM fragment (SEQ ID No. 25) and / or PAL fragment (SEQ ID No. 8).

[0052] The object of the present invention is to provide a method for determining the level of PAM and / or its isomers and / or fragments thereof in body fluids or tissue samples.An object of the present invention is to provide corresponding assays and kits.

[0053] Another object of the present invention is to provide a method for diagnosing or prognosing a disease in a subject and / or predicting the risk of a subject developing a disease or experiencing an adverse event and / or monitoring a disease or adverse event in a subject by determining the level of PAM and / or its isomers and / or fragments thereof in a body fluid or tissue sample of the subject using an assay, wherein the assay comprises at least one binding agent to a conformational epitope of PAM.

[0054] Another important embodiment of the present invention is a method for diagnosing or prognosing a disease in a subject and / or predicting the risk of a subject developing a disease or experiencing an adverse event and / or monitoring a disease or adverse event in a subject, the method comprising:

[0055] determining the level of PAM and / or its isomers and / or fragments thereof in a body fluid or tissue sample of the subject using an assay, wherein the assay comprises at least one binding agent directed against a conformational epitope of PAM,

[0056] - Comparing the measured amount to a predetermined threshold value.

[0057] wherein the subject is diagnosed as having a disease if the measured amount is below or above the predetermined threshold, or

[0058] wherein if the measured amount is below or above the predetermined threshold, the outcome of the disease is predicted, or

[0059] wherein if the measured amount is below or above the predetermined threshold, the risk of the patient developing a disease or experiencing an adverse event is predicted, or

[0060] wherein the subject is monitored for disease or adverse events.

[0061] The threshold value is predetermined by measuring the level of PAM and / or its isomers and / or its fragments in healthy controls and calculating, for example, the corresponding 75th percentile, more preferably the 90th percentile, even more preferably the 95th percentile. If the level of the sick subject or the subject at risk of developing a disease or an adverse event is above the threshold value, the upper boundary of the 75th percentile, more preferably the 90th percentile, even more preferably the 95th percentile defines the threshold value between a healthy person and a sick patient, or a healthy person and a subject at risk of developing a disease, or a subject without a risk of an adverse event and a subject at risk of an adverse event. The threshold value is predetermined by measuring the level of PAM and / or its isomers and / or its fragments in healthy controls and calculating, for example, the corresponding 25th percentile, more preferably the 10th percentile, even more preferably the 5th percentile. If the level of the diseased subject or the subject at risk of developing the disease or experiencing an adverse event is below the threshold, the lower boundary of the 25th percentile, more preferably the 10th percentile, and even more preferably the 5th percentile defines the threshold between healthy and diseased patients, or healthy and subjects at risk of developing the disease, or subjects without adverse event risk and subjects at risk of adverse event. The level of the PAM and / or its isomers and / or fragments thereof can be measured as the total PAM concentration. The predetermined value can vary in the specific population selected, depending on certain factors such as sex, age, genetics, habits, race, etc.

[0062] Those skilled in the art will appreciate how to determine threshold values ​​from prior research. Those skilled in the art will appreciate that the specific threshold value may depend on the cohort used to calculate the predetermined threshold value, which may be used for subsequent routine testing. Those skilled in the art will appreciate that the specific threshold value may depend on the calibration used in the assay. Those skilled in the art will appreciate that the specific threshold value may depend on the sensitivity and / or specificity acceptable to the practitioner.

[0063] The sensitivity and specificity of a diagnostic test depend not only on the analytical "quality" of the test but also on the definition of an abnormal result. In practice, a receiver operating characteristic (ROC) curve is typically calculated by plotting the values ​​of a variable against their relative frequencies in "normal" (i.e., apparently healthy) and "disease" populations (i.e., patients with an infection). Depending on the specific diagnostic question being addressed, the reference group need not necessarily be "normal"; it could be a group of patients with another disease from which the target disease group should be distinguishable. For any particular marker, the distribution of marker levels in diseased and non-disease subjects may overlap. In this case, the test cannot perfectly distinguish between normal and disease with 100% accuracy, and the area of ​​overlap indicates the interval in which the test cannot distinguish between normal and disease. A threshold is chosen above which (or below which, depending on how the marker varies with disease) a test is considered abnormal, and below which a test is considered normal. The area under the ROC curve is a measure of the probability that the perceived measurement correctly identifies the disease. ROC curves can be used even when test results do not necessarily yield precise numbers. ROC curves can be generated whenever the results can be ranked. For example, the test results for a "disease" sample can be graded according to degree (e.g., 1 = low, 2 = normal, 3 = high). This grade can be correlated with the results in a "normal" population and a ROC curve generated. These methods are well known in the art (see, for example, Hartley et al., 1982). Preferably, a threshold is selected to provide an area under the ROC curve greater than about 0.5, more preferably greater than about 0.7. In this context, the term "about" refers to + / - 5% of a given measurement.

[0064] Once the threshold value is determined by using a previous study cohort and taking into account all of the above points, the practicing physician will use the predetermined threshold value for the diagnosis or prediction of the disease of the present invention and / or the method for predicting the risk of a subject suffering from a disease or experiencing an adverse event and / or monitoring a disease or adverse event, and will determine whether the subject's value is above or below the predetermined threshold value in order to perform appropriate diagnosis, prognosis, prediction or monitoring.

[0065] The thresholds described above may differ in other assays if they are systematically calibrated differently from the assay used in the present invention. Therefore, the thresholds described above should be applied to such differently calibrated assays, taking into account these calibration differences. One possibility for quantifying calibration differences is to perform a methodological comparative analysis (correlation study) between the assay in question (e.g., a PAM assay) and the corresponding biomarker assay used in the present invention by measuring the corresponding biomarker or its activity (e.g., PAM) in a sample using both methods. Another possibility is to determine the median biomarker level for a representative normal population using the test, given sufficient analytical sensitivity for the assay in question, compare the results with the median biomarker level obtained using the other assay, and recalculate the calibration based on the differences observed in this comparison. Using the calibration used in the present invention, the median plasma PAM concentration in samples from normal (healthy) subjects was measured to be 78.6 ng / mL (interquartile range [IQR] 66.4-92.5 ng / mL).

[0066] The term "diagnosis" as used herein means detecting a disease or determining the stage or extent of a disease. Typically, the diagnosis of a disease is based on the evaluation of one or more factors and / or symptoms that indicate the disease. That is, a diagnosis can be made based on the presence, absence, or amount of factors that indicate the presence or absence of the disease or disorder. Each factor or symptom that is considered to indicate a diagnosis of a particular disease does not necessarily have to be specifically associated with the particular disease, for example, there may be a differential diagnosis that can be inferred from the diagnostic factors or symptoms. Similarly, there may be a situation where factors or symptoms that indicate a particular disease are present in an individual who does not have the particular disease.

[0067] As used herein, the term "prognosis" refers to a prediction of the likely course and outcome of a clinical condition or disease (e.g., sepsis). A prognosis is typically made by evaluating disease factors or symptoms that indicate a favorable or unfavorable course or outcome of the disease. As used herein, the phrase "determining a prognosis" refers to a process by which a skilled artisan can predict the course or outcome of a clinical condition or disease in a patient. The term "prognosis" does not refer to the ability to predict the course or outcome of a clinical condition or disease with 100% accuracy. Rather, the skilled artisan will understand that the term "prognosis" refers to an increased probability of a certain course or outcome occurring; that is, a certain course or outcome is more likely to occur in patients who exhibit a given clinical condition or disease compared to individuals who do not exhibit that clinical condition or disease.

[0068] In a specific embodiment of the method of using an assay to diagnose or prognose a disease in a subject and / or predict a subject's risk of developing a disease or experiencing an adverse event and / or monitoring a subject's disease or adverse event, wherein the assay comprises at least one binding agent to a conformational epitope of a PAM, the disease is selected from the group consisting of:

[0069] Dementia, wherein the dementia is selected from the group consisting of mild cognitive impairment (MCI), Alzheimer's disease, vascular dementia, mixed Alzheimer's disease and vascular dementia, dementia with Lewy bodies, frontotemporal dementia, focal dementia (including progressive aphasia), subcortical dementia (including Parkinson's disease) and secondary causes of dementia syndromes (including intracranial lesions), and / or

[0070] Cardiovascular disorder, wherein the cardiovascular disorder may be selected from the group consisting of atherosclerosis, hypertension, heart failure (including acute heart failure and acute decompensated heart failure), atrial fibrillation, cardiovascular ischemia, cerebral ischemic injury, cardiogenic shock, stroke (including ischemic and hemorrhagic stroke and transient ischemic attack) and myocardial infarction, and / or

[0071] Kidney disease, wherein the kidney disease may be selected from the group comprising nephrotoxicity (drug-induced nephropathy), acute kidney injury (AKI), chronic kidney disease (CKD), diabetic nephropathy, end-stage renal disease (ESRD), and / or

[0072] Cancer, wherein the cancer may be selected from the group consisting of prostate cancer, breast cancer, lung cancer, colorectal cancer, bladder cancer, ovarian cancer, cervical cancer, skin cancer (including melanoma), stomach cancer, liver cancer, pancreatic cancer, leukemia, non-Hodgkin's lymphoma, kidney cancer, esophageal cancer, pharyngeal cancer, and / or

[0073] an infectious disease caused by an infectious organism such as a bacterium, virus, fungus or parasite, selected from the group consisting of SIRS, sepsis and septic shock, and / or

[0074] • Metabolic diseases selected from the group comprising type 1 diabetes, type 2 diabetes, metabolic syndrome.

[0075] In one embodiment of the present application, the disease is dementia, and the dementia is selected from the group consisting of mild cognitive impairment (MCI), Alzheimer's disease, vascular dementia, mixed Alzheimer's disease and vascular dementia, Lewy body dementia, frontotemporal dementia, focal dementia (including progressive aphasia), subcortical dementia (including Parkinson's disease) and secondary causes of dementia syndrome (including intracranial lesions).

[0076] In certain embodiments, the dementia is Alzheimer's disease.

[0077] In one embodiment of the present application, the disease is cancer, and the cancer is selected from the group consisting of prostate cancer, breast cancer, lung cancer, colorectal cancer, bladder cancer, ovarian cancer, cervical cancer, skin cancer (including melanoma), gastric cancer, liver cancer, pancreatic cancer, leukemia, non-Hodgkin's lymphoma, kidney cancer, esophageal cancer and laryngeal cancer.

[0078] In certain embodiments, the cancer is colorectal cancer and pancreatic cancer.

[0079] In one embodiment of the present application, the disease is a cardiovascular disorder, wherein the cardiovascular disorder is selected from the group consisting of atherosclerosis, hypertension, heart failure (including acute and acute decompensated heart failure), atrial fibrillation, cardiovascular ischemia, cerebral ischemic injury, cardiogenic shock, stroke (including ischemic and hemorrhagic stroke and transient ischemic attack) and myocardial infarction.

[0080] In certain embodiments, the cardiovascular disorder is heart failure (including acute heart failure and acute decompensated heart failure).

[0081] In another specific embodiment, the cardiovascular disorder is stroke (including ischemic and hemorrhagic stroke and transient ischemic attack) and myocardial infarction.

[0082] In another specific embodiment, the cardiovascular disorder is atrial fibrillation (AF).

[0083] In another specific embodiment of the present application, the disease is SIRS, sepsis or septic shock.

[0084] In another specific embodiment of the present application, the disease is type 1 diabetes, type 2 diabetes, or metabolic syndrome.

[0085] In the context of the method of the present invention, body fluids and soluble tissue extracts may be selected from blood, serum, plasma, cerebrospinal fluid (CSF), urine, saliva, sputum and pleural effusion. In a particular embodiment of the method, the sample is selected from the group comprising whole blood, serum and plasma.

[0086] The term "tissue" refers to a soluble component obtained by disrupting an organ structure using mechanical and ultrasonic forces to release intracellular components into a liquid medium. In certain embodiments, the tissue is selected from the group consisting of liver, pituitary gland, whole brain, muscle, skin (including epidermis, dermis, and subcutaneous tissue), and the like.

[0087] The term "monitoring" refers to controlling the disease or pathophysiological state of a patient, such as the risk of developing a disease or experiencing an adverse event, the severity of the disease, or the development of (detecting any changes in) the response to treatment.

[0088] The subject of the present invention is a method wherein said monitoring is performed in order to assess changes in the risk of developing a disease or experiencing an adverse event, changes in the severity of a disease or the response of the patient or subject to a treatment.

[0089] A particular subject of the present invention is a method, wherein said monitoring is performed in order to assess the response of said subject to the preventive and / or therapeutic measures taken.

[0090] Subject matter of the present invention is a method according to the invention, wherein said method is used for stratifying said subjects into risk groups.

[0091] As used herein, the term "risk" relates to the probability of experiencing an adverse event or effect, such as a disease or adverse event.

[0092] The term "elevated levels" refers to levels above a certain threshold level.

[0093] The term "reduced levels" refers to levels below a certain threshold level.

[0094] An "adverse event" is defined as an event that impairs an individual's health. The adverse event is not limited to, but may be selected from, the group consisting of cardiac events, cardiovascular events, cerebrovascular events, cancer, diabetes, and all-cause mortality. Adverse events include infection, severe infection, sepsis-like systemic infection, and sepsis. Adverse events are not acute externally induced adverse events and / or events resulting from externally induced trauma. Externally induced trauma includes those that may be caused by accidents, such as car accidents, and are therefore excluded from adverse events.

[0095] In a specific embodiment of the invention, the adverse event is a cardiovascular event selected from the group consisting of myocardial infarction, acute decompensated heart failure, stroke and death related to myocardial infarction, stroke or acute heart failure.

[0096] The risk of developing a disease or experiencing an adverse event refers to the risk of developing the disease or event within a certain time period. In certain embodiments, the time period is within 10 years, or within 8 years, or within 5 years, or within 2.5 years, or within 1 year, or within 6 months, or within 3 months, or within 30 days, or within 28 days.

[0097] In a specific embodiment of the present invention, the "level of PAM and / or its isomers and / or fragments thereof" is the total concentration (preferably expressed as weight / volume; w / v) of PAM comprising the sequences SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8 and SEQ ID No. 10 and / or its isomers and / or fragments thereof having at least 12 amino acids in a sample obtained from a subject.

[0098] In the present disclosure, the term "PAM" refers to the amino acid sequences of PAM isomers 1 to 6 as shown in SEQ ID Nos. 1 to 6. In some aspects, the PAMs disclosed herein have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequences of SEQ ID Nos. 1 to 6.

[0099] In some aspects, the PAM is a functional fragment (i.e., PHM (SEQ ID No. 7) or PAL (SEQ ID No. 8), a PAM that retains at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the PAM activity of the corresponding full-length PAM). In some aspects, the PAM is a variant or derivative of a PAM disclosed herein.

[0100] In a specific embodiment of the invention, the peptidylglycine α-amidating monooxygenase is an active PAM.

[0101] The percent identity of an amino acid or nucleic acid sequence, or the term "% sequence identity," is defined herein as the percentage of residues in a candidate amino acid or nucleic acid sequence that are identical to the residues in the reference sequence, after aligning the candidate sequence with the reference sequence and introducing gaps (if necessary) to achieve the maximum percent identity. In a preferred embodiment, the calculation of at least the percent sequence identity is performed without introducing gaps. Methods and computer programs for alignment are well known in the art, such as "Align 2" or the BLAST service of the National Center for Biotechnology Information (NCBI).

[0102] In a specific embodiment of the invention, the levels of PAM and / or its isomers and / or fragments thereof are determined using an assay, wherein the assay is a sandwich assay, preferably a fully automated assay, wherein the assay comprises at least one binding agent directed against a conformational epitope of PAM.

[0103] In one embodiment of the present invention, it can be a so-called POC test (point-of-care test), which is a testing technology that allows testing to be performed near the patient in less than an hour without the need for a fully automated assay system. An example of such a technology is the immunochromatographic testing technology.

[0104] In one embodiment of the invention, the assay is a sandwich immunoassay using any type of detection technology, including but not limited to enzyme labels, chemiluminescent labels, and electrochemiluminescent labels, preferably a fully automated assay. In one embodiment of the invention, the assay is an enzyme-labeled sandwich assay. Examples of automated or fully automated assays include those available for use with one of the following systems: Roche Elecsys®, Abbott Architect®, Siemens Centauer®, Brahms Kryptor®, Biomerieux Vidas®, Alere Triage®, and OrthoClinical Diagnostics Vitros®.

[0105] In a specific embodiment of the invention, at least one of the two binding agents is labeled to facilitate detection.

[0106] Preferred detection methods include various formats of immunoassays, such as radioimmunoassays (RIA), homogeneous enzyme multiplication immunoassays (EMIT), chemiluminescent and fluorescent immunoassays, enzyme-linked immunoassays (ELISA), Luminex-based bead arrays, protein microarray assays, and rapid test formats, such as immunochromatographic strip tests.

[0107] In a preferred embodiment, the label is selected from the group consisting of chemiluminescent labels, enzyme labels, fluorescent labels, and radioiodine labels.

[0108] The assay can be a homogeneous or heterogeneous assay, a competitive or non-competitive assay. In one embodiment, the assay takes 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 to a second antibody. The first antibody can be bound to a solid phase, such as a bead, the surface of a well or other container, a chip or a strip, and the second antibody is an antibody labeled, for example, 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 basic principles and procedures associated with "sandwich assays" are well-established and known to those skilled in the art ( Immunity Test The Immunoassay Handbook, ed. David Wild, Elsevier LTD, Oxford; 3rd edition (May 2005); Hultschig et al., 2006. Curr Opin Chem Biol. 10 (1): 4-10 ).

[0109] In another embodiment, the assay comprises two capture molecules, preferably antibodies, both present as dispersions in a liquid reaction mixture, wherein a first labeling component is attached to the first capture molecule, wherein the first labeling component is part of a labeling system based on fluorescence or chemiluminescence quenching or amplification, and a second labeling component of the labeling system is attached to the second capture molecule, such that a measurable signal is generated upon binding of the two capture molecules to the analyte, thereby enabling detection of the formed sandwich complex in a solution containing the sample.

[0110] In another embodiment, the labeling system comprises a rare earth cryptate or rare earth chelate in combination with a fluorescent dye or a chemiluminescent dye, in particular a dye of the cyanine type.

[0111] In the context of the present invention, fluorescence-based assays include the use of dyes which may, for example, be selected from the group comprising: 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 green, rhodamine red, rhodamine 110, BODIPY dyes such as BODIPY TMR, Oregon Green, coumarins such as umbelliferone, benzylimines such as Hoechst 33258, phenanthridines such as Texas Red, Yakima Yellow, Alexa Fluor, PET, ethidium bromide, acridine dyes, carbazole dyes, phenoxazine dyes, porphyrin dyes, polymethine dyes, etc.

[0112] In the context of the present invention, chemiluminescence-based assays involve the use of dyes based on the physical principles described for chemiluminescent materials in: Kirk-Othmer, Encyclopedia of Chemical Technology Chemical Technology), 4th edition, 1993. John Wiley & Sons, Vol.15: 518-562, cited by Incorporated herein, including citations on pages 551-562 Preferred chemiluminescent dyes are acridinium esters.

[0113] As used herein, an "assay" or "diagnostic assay" may be of any type used in the field of diagnostics. Such an assay may be based on the binding of an analyte to be detected to one or more capture probes having a certain affinity. A binding agent useful for determining the level of a PAM and / or its isomers and / or fragments thereof exhibits at least 10 affinity for a PAM and / or its isomers and / or fragments thereof. 7 M -1 , preferably 10 8 M -1 The affinity constant is preferably greater than 10 9 M -1 , most preferably greater than 10 10 M -1 It will be appreciated by those skilled in the art that it may be considered to compensate for the lower affinity by administering a higher dose of the compound, and such measures would not be beyond the scope of the present invention.

[0114] In the context of the present invention, a "binding agent molecule" is a molecule that can be used to bind to a target molecule or target molecule, i.e., an analyte (i.e., in the context of the present invention, a PAM and its isomers and fragments). Therefore, the binding agent molecule must be sufficiently adapted in terms of steric and surface characteristics (e.g., surface charge, hydrophobicity, hydrophilicity, the presence or absence of Lewis donors and / or acceptors) to specifically bind to the target molecule or target molecule. Thus, the binding can be mediated, for example, by ionic bonds, van der Waals forces, π-π interactions, σ-π interactions, hydrophobic interactions, hydrogen bonds, or a combination of two or more of the aforementioned interactions between the capture molecule and the target molecule or target molecule.

[0115] In the context of the present invention, the binding agent molecule can be, for example, selected from the group comprising nucleic acid molecules, carbohydrate molecules, PNA molecules, proteins, antibodies, peptides or glycoproteins. Preferably, the binding agent molecule is an antibody, including fragments thereof having sufficient affinity for the target or target molecule, and including recombinant antibodies or recombinant antibody fragments, as well as chemically and / or biochemically modified derivatives of the antibody or fragments derived from the variant chain.

[0116] In certain embodiments, the binding agent may be selected from an antibody, an antibody fragment, or a non-IgG scaffold.

[0117] The basic structural unit of an antibody is typically a tetramer composed of two identical pairs of immunoglobulin chains, each pair comprising one light chain and one heavy chain. Within each chain pair, the light and heavy chain variable regions bind to the antigen, while the constant regions mediate effector functions. Immunoglobulins also exist in various other forms, including, for example, Fv, Fab, and (Fab')2, as well as bifunctional hybrid antibodies and single chains (e.g., Lanzavecchia et al., 1987; Huston et al., 1988; Bird et al., 1988; Hood et al., 1984; Hunkapiller and Hood, 1986). The variable region of an immunoglobulin light or heavy chain consists of a framework region separated by three hypervariable regions, also known as complementarity-determining regions (CDRs) (see Kabat et al., 1983). As described above, the CDRs are primarily responsible for binding to the antigen epitope. An immune complex is an antibody (e.g., a monoclonal antibody, chimeric antibody, humanized antibody, or human antibody) or functional antibody fragment that specifically binds to an antigen.

[0118] Chimeric antibodies are antibodies whose light and heavy chain genes are constructed, typically through genetic engineering, from immunoglobulin variable and constant region genes belonging to different species. For example, the variable segments from genes for a mouse monoclonal antibody can be linked to human constant segments, such as kappa and gamma 1 or gamma 3. Thus, in one example, a therapeutic chimeric antibody is a hybrid protein composed of a variable region or antigen-binding domain from a mouse antibody and a constant region or effector domain from a human antibody, although other mammalian species can also be used, or the variable regions can be prepared using molecular techniques. Methods for preparing chimeric antibodies are well known in the art, see, for example, U.S. Patent No. 5,807,715. A "humanized" immunoglobulin is an immunoglobulin that includes human framework regions and one or more CDRs from a non-human (e.g., mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is called the "donor," and the human immunoglobulin providing the framework regions is called the "acceptor." In one embodiment, all CDRs are derived from the donor immunoglobulin in the humanized immunoglobulin. Constant regions are not necessarily present, but if present, they must be substantially identical to human immunoglobulin constant regions, i.e., have an identity of at least about 85-90%, such as about 95% or higher. Thus, all parts of a humanized immunoglobulin, except the CDRs, are substantially identical to corresponding parts of a natural human immunoglobulin sequence. A "humanized antibody" is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The acceptor framework region of a humanized immunoglobulin or antibody may have a limited number of amino acid substitutions taken from the donor framework region. Humanized or other monoclonal antibodies may have additional conservative amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin functions. Exemplary conservative substitutions are, for example, the following substitutions: gly, ala; val, ile, leu; asp, glu; asn, gln; ser, thr; lys, arg; and phe, tyr. Humanized immunoglobulins can be constructed using genetic engineering (see, for example, U.S. Patent No. 5,585,089). Human antibodies are antibodies in which the light chain and heavy chain genes are derived from humans. Human antibodies can be produced using methods known in the art. Human antibodies can be produced by immortalizing human B cells that secrete the antibody of interest. Immortalization can be achieved, for example, by EBV infection or by fusing human B cells with myeloma or hybridoma cells to produce tri-hybridoma cells. Human antibodies can also be produced by phage display methods (see, for example, PCT Publication No. WO91 / 17271; PCT Publication No. WO92 / 001047; PCT Publication No. WO92 / 20791, which are incorporated herein by reference), or selected from a human combinatorial monoclonal antibody library (see the Morphosys website).Human antibodies can also be prepared by using transgenic animals carrying human immunoglobulin genes (see, eg, PCT Publication No. WO 93 / 12227; PCT Publication No. WO 91 / 10741, which are incorporated herein by reference).

[0119] Thus, the PAM antibody may have a form known in the art, examples being human antibodies, monoclonal antibodies, humanized antibodies, chimeric antibodies, and CDR-grafted antibodies. In a preferred embodiment, the antibody according to the present invention is a recombinantly produced antibody, such as a typical full-length immunoglobulin such as IgG, or an antibody fragment containing at least the F variable domain of a heavy chain and / or a light chain, such as a chemically coupled antibody (antigen-binding fragment), including but not limited to a Fab fragment, including a Fab minibody, a single-chain Fab antibody, a monovalent Fab antibody with an epitope tag, such as Fab-V5Sx2; a bivalent Fab (minibody) dimerized using the CH3 domain; a bivalent Fab or a multivalent Fab, for example formed by multimerization with the help of a heterologous domain, such as by dimerization of a dHLX domain, such as Fab-dHLX-FSx2; a F(ab')2 fragment, a scFv fragment, a multimeric multivalent or / and multispecific scFv fragment, a bivalent and / or bispecific diabody, BITE® (bispecific T-cell engager), a triabody, a multivalent antibody, for example from a class different from G; a single domain antibody, such as a nanobody derived from camel or fish immunoglobulins, and many other antibodies.

[0120] In addition to anti-PAM antibodies, other biopolymer scaffolds are known in the art that can complex target molecules and have been used to generate highly target-specific biopolymers. Examples are aptamers, spiegelmers, anticalins, and conotoxins.

[0121] Non-Ig scaffolds can be protein scaffolds and are useful as antibody mimics in that they are able to bind to ligands or antigens. The non-Ig scaffold may be selected from the group comprising a tetranectin-based non-Ig scaffold (e.g. described in US 2010 / 0028995), a fibronectin scaffold (e.g. described in EP 1266 025), a lipocalin-based scaffold (e.g. described in WO 2011 / 154420), a ubiquitin scaffold (e.g. described in WO 2011 / 073214), a transferrin scaffold (e.g. described in US 2004 / 0023334), a protein A scaffold (e.g. described in EP 2231860), an ankyrin repeat-based scaffold (e.g. described in WO 2010 / 060748), a microprotein (preferably a cystine knot-forming microprotein) scaffold (e.g. described in EP 2314308), a Fyn SH3 domain-based scaffold (e.g. described in WO 2011 / 023685), an EGFR-A domain-based scaffold (e.g. described in WO 2011 / 073214). 2005 / 040229) and Kunitz domain-based scaffolds (e.g., described in EP 1941867). Non-Ig scaffolds can be peptide aptamers or oligonucleotide aptamers. Aptamers are typically obtained by screening them from large random sequence libraries and can be short oligonucleotides (DNA, RNA, or XNA; Xu et al., 2010; Deng et al., 2014) or short variable peptide domains attached to a protein scaffold (Li et al., 2011).

[0122] Chemiluminescent labels may be acridinium ester labels, steroid labels including isoluminol labels, and the like.

[0123] Enzyme markers can be lactate dehydrogenase (LDH), creatine kinase (CPK), alkaline phosphatase, aspartate aminotransferase (AST), alanine aminotransferase (ALT), acid phosphatase, glucose-6-phosphate dehydrogenase, etc.

[0124] In one embodiment of the invention, at least one of the two binding agents is bound to a solid phase, such as magnetic particles and polystyrene surfaces.

[0125] The subject of the present invention is a method for determining the level of PAM and / or its isomers and / or fragments in a body fluid sample using an assay, wherein the assay comprises two binding agents that bind to two different epitopes of PAM, wherein the two binding agents are directed against epitopes of at least 5 amino acids in length, preferably at least 4 amino acids in length.

[0126] An epitope, also known as an antigenic determinant, is the portion of an antigen (such as a peptide or protein) that is recognized by the immune system, particularly antibodies. For example, an epitope is a specific segment of an antigen that binds to an antibody. The portion of the antibody that binds to the epitope is called a paratope. Protein antigen epitopes are divided into two categories based on their structure and interaction with the paratope: conformational epitopes and linear epitopes.

[0127] A linear or continuous epitope is an epitope that is recognized by an antibody because of its linear amino acid sequence or primary structure and is formed by the 3-D conformation adopted by the interaction of adjacent amino acid residues. Conformational and linear epitopes interact with the complement on the basis of the 3-D conformation adopted by the epitope, which is determined by the surface features of the epitope residues involved and the shape or tertiary structure of other segments of the antigen. In contrast, conformational epitopes are formed by the 3-D conformation adopted by the interaction of amino acid residues that are not continuous in sequence but are in close proximity in three-dimensional space.

[0128] For conformational epitopes, discontinuous amino acid segments are brought together to form antibody binding sites during protein folding. The binding of antibodies to these epitopes depends on the correct formation of the three-dimensional shape or tertiary structure of the protein antigen ( Barlow et al., 1986. Continuous and discontinuous protein antigenic determinants protein antigenic determinants), Nature 322: 747–748 When a protein (such as an enzyme) is denatured, the secondary, tertiary, and (in the case of subunits) quaternary structures are altered, leaving only the peptide bonds of the primary structure between amino acids intact. Because all levels of a protein's structure contribute to its function, once denatured, the protein or enzyme can no longer perform its function. If a protein is denatured, the 3D conformation is lost, and conformational epitopes are no longer exposed, making binding to specific binding agents impossible.

[0129] In one embodiment of the present invention, the binding agent to a conformational epitope of PAM does not bind to denatured PAM or denatured PAM subunits (eg, PAL or PHM).

[0130] In certain embodiments, the binder to a conformational epitope of PAM does not bind to denatured PAM or denatured PAM subunits (eg, PAL or PHM) using Western blotting techniques as described in Example 2.

[0131] In another embodiment of the present invention, the binder to the conformational epitope of PAM binds to an enzymatically active PAM or an enzymatically active PAM subunit (such as PAL or PHM), but does not bind to an enzymatically inactive PAM or an enzymatically inactive PAM subunit (such as PAL or PHM).

[0132] Binding agents (e.g., antibodies) can be generated using various immunization strategies. Classical protein immunization strategies typically rely on synthetic peptides, large fragments of bacterial or mammalian cell origin, full-length recombinant proteins, or purified native proteins as the source of immunogens. Commonly used peptides, ranging in size from 12-20 amino acid residues, rarely encompass more than one epitope and may lack secondary and tertiary conformational structure. Therefore, due to the unstructured nature of peptides, anti-peptide antibodies often lack the ability to bind native proteins. Full-length protein antigens overcome many of the limitations attributed to peptides. Inherently, they contain surface areas, multiple immunogenic epitopes, and, even when synthesized in prokaryotic systems, may fold (at least partially) to form a native structure. More innovative approaches, such as DNA (or "genetic") immunization, have emerged as alternative and / or complementary tools to classical antibody generation strategies. DNA immunization employs an expression plasmid encoding a selected antigen to immunize an animal. Transfected tissues of the immunized animal express the antigen, subsequently eliciting an antibody response. DNA immunization using sequences encoding polypeptide protein regions combines the advantages of full-length proteins and peptides and immunization methods, providing immunogens that encompass relatively large regions of the target protein, have the potential for multiple epitopes, and are more accessible than full-length proteins. Brown et al., 2011. PLoS One. 6(12): e28718 ).

[0133] In one embodiment of the present invention, the binding agent is produced using large fragment proteins, full-length proteins or DNA immunization technology.

[0134] Large fragment proteins are defined as peptide sequences of at least 100 amino acids, more preferably at least 150 amino acids, even more preferably at least 200 amino acids, even more preferably at least 250 amino acids, most preferably at least 300 amino acids.

[0135] In a specific embodiment, the large fragment protein is SEQ ID No. 7 and / or SEQ ID No. 8 and / or SEQ ID No. 25.

[0136] Another embodiment of the present invention relates to a method for preparing antibodies against conformational epitopes of PAM.

[0137] A method for producing an antibody targeting a conformational epitope comprises the following steps:

[0138] Synthesize DNA encoding full-length PAM, PHM subunits, PAL subunits, or enzymatically active PAM protein fragments, and / or

[0139] Cloning DNA encoding full-length PAM, PHM subunits, PAL subunits, or enzymatically active PAM protein fragments into an expression vector, and / or

[0140] Transfect the expression vector into a suitable cell line, and / or

[0141] Purification of the expressed PAM construct (e.g., using nickel affinity chromatography for polyhistidine C-terminally truncated constructs, or using anion exchange chromatography), and / or

[0142] Testing the activity of the expressed PAM construct, and / or

[0143] Immunization of animals with an enzymatically active full-length PAM, PHM subunit, PAL subunit, or an enzymatically active PAM protein fragment (e.g., using fusion techniques between immunized Balb / c mouse spleen cells and SP2 / 0 myeloma cells), and / or

[0144] Screening for hybridoma cell lines that secrete specific monoclonal antibodies against enzymatically active full-length PAM, enzymatically active PAM protein fragments, PHM subunits, or PAL subunits (e.g., using ELISA assays or flow cytometry), and / or

[0145] Purify the antibody from a positively characterized cell line (e.g. using protein A chromatography).

[0146] Another method for generating antibodies targeting conformational epitopes comprises the following steps:

[0147] Synthesize DNA encoding an enzymatically active full-length PAM, an enzymatically active PHM subunit, an enzymatically active PAL subunit, or an enzymatically active PAM protein fragment, and / or

[0148] Incorporating the synthesized DNA into a plasmid vector for DNA immunization, and / or

[0149] Immunizing a host animal with the plasmid DNA using a delivery method such as a gene gun, electroporation, or intramuscular injection, and / or

[0150] fusing spleen cells from said animal with myeloma cells to produce hybridoma cell lines, and / or

[0151] Screening for hybridoma cell lines that secrete specific monoclonal antibodies against enzymatically active full-length PAM, enzymatically active PHM subunits, enzymatically active PAL subunits, or enzymatically active PAM protein fragments (e.g., using ELISA assays or flow cytometry), and / or

[0152] Purify the antibody from a positively characterized cell line (e.g. using protein A chromatography).

[0153] Another embodiment of the present invention relates to a method for screening for conformational antibodies.

[0154] Methods for screening antibodies that bind to conformational epitopes of an enzymatically active full-length PAM, an enzymatically active PHM subunit, an enzymatically active PAL subunit, or an enzymatically active PAM protein fragment may include, but are not limited to, the following methods:

[0155] Western blot analysis, and / or

[0156] Native PAGE analysis, and / or

[0157] Surface plasmon resonance (SPR) and related techniques, and / or

[0158] Co-crystallization analysis, and / or

[0159] Enzyme-linked immunosorbent assay (ELISA) and / or

[0160] Co-elution analysis.

[0161] In one embodiment, the method for screening for antibodies that bind to a conformational epitope of an enzymatically active full-length PAM, an enzymatically active PHM subunit, an enzymatically active PAL subunit, or an enzymatically active PAM protein fragment is a western blot analysis comprising the steps of:

[0162] Prepare protein samples containing enzymatically active full-length PAM, enzymatically active PHM subunits, enzymatically active PAL subunits, or enzymatically active PAM protein fragments in a denaturing sample buffer (e.g., containing sodium dodecyl sulfate (SDS)) and then heat them to above 70°C to ensure protein denaturation, and / or

[0163] Perform sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to separate proteins by molecular weight and transfer the proteins from the gel to a nitrocellulose membrane, and / or

[0164] Blocking the nitrocellulose membrane with albumin to prevent nonspecific binding, and / or

[0165] Incubate the nitrocellulose membrane with potential conformational antibody candidates, and / or

[0166] Wash the nitrocellulose membrane to remove unbound material, and / or

[0167] Addition of secondary antibodies conjugated to enzymes or fluorescent tags, and / or

[0168] Detection signal.

[0169] Therefore, the absence of signal in the Western blot indicates that the developed antibodies recognize a conformational epitope of the enzymatically active full-length PAM, the enzymatically active PHM subunit, the enzymatically active PAL subunit, or the enzymatically active PAM protein fragment.

[0170] In one embodiment, a method for screening for antibodies that bind to a conformational epitope of an enzymatically active full-length PAM, an enzymatically active PHM subunit, an enzymatically active PAL subunit, or an enzymatically active PAM protein fragment is native PAGE analysis, comprising the steps of:

[0171] Prepare samples without denaturants and without heating, and / or

[0172] Perform non-denaturing (native) polyacrylamide gel electrophoresis (PAGE) to separate proteins by molecular weight and transfer the proteins from the gel to a nitrocellulose membrane, and / or

[0173] Blocking the nitrocellulose membrane with albumin to prevent nonspecific binding, and / or

[0174] Incubate the nitrocellulose membrane with potential conformational antibody candidates, and / or

[0175] Wash the nitrocellulose membrane to remove unbound material, and / or

[0176] Addition of secondary antibodies conjugated to enzymes or fluorescent tags, and / or

[0177] Detection signal.

[0178] Detection of the signal indicates that the developed antibody recognizes a conformational epitope of an enzymatically active full-length PAM, an enzymatically active PHM subunit, an enzymatically active PAL subunit, or an enzymatically active PAM protein fragment.

[0179] In one embodiment, the method for screening antibodies that bind to a conformational epitope of an enzymatically active full-length PAM, an enzymatically active PHM subunit, an enzymatically active PAL subunit, or an enzymatically active PAM protein fragment is surface plasmon resonance (SPR), comprising the steps of:

[0180] Immobilize native and denatured forms of PAM protein (enzymatically active full-length PAM, enzymatically active PHM subunits, enzymatically active PAL subunits, or enzymatically active PAM protein fragments) on an SPR chip, and / or

[0181] Applying antibodies to the chip, and / or

[0182] Real-time measurement integration, and / or

[0183] Comparative binding kinetics between native and denatured PAM proteins.

[0184] Binding to the native form of the PAM protein but not to the denatured form of the PAM protein indicates specificity of the corresponding antibody for the conformational epitope.

[0185] In one embodiment, a method for screening for antibodies that bind to a conformational epitope of an enzymatically active full-length PAM, an enzymatically active PHM subunit, an enzymatically active PAL subunit, or an enzymatically active PAM protein fragment is a co-crystallization analysis comprising the steps of:

[0186] Mixing the antibody to be tested with the antigen (enzymatically active full-length PAM, enzymatically active PHM subunit, enzymatically active PAL subunit, or enzymatically active PAM protein fragment), and / or

[0187] Screening of crystallization conditions, and / or

[0188] After obtaining the crystals, X-ray diffraction was performed to determine the structure.

[0189] Structural analysis confirmed the interaction of the antibody with a conformational epitope on the antigen.

[0190] In one embodiment, the method for screening antibodies that bind to a conformational epitope of an enzymatically active full-length PAM, an enzymatically active PHM subunit, an enzymatically active PAL subunit, or an enzymatically active PAM protein fragment is an enzyme-linked immunosorbent assay (ELISA) comprising the following steps:

[0191] Coating a solid phase (e.g., a well in a microtiter plate) with both native antigen (enzymatically active full-length PAM, enzymatically active PHM subunit, enzymatically active PAL subunit, or enzymatically active PAM protein fragment) and denatured antigen, and / or

[0192] A blocking step to prevent nonspecific binding, and / or

[0193] Add the antibody to be tested, and / or

[0194] Washing steps to remove unbound antibody, and / or

[0195] Add enzyme-linked secondary antibody and substrate, and / or

[0196] ·Measure enzyme reactions.

[0197] The enzymatic reaction was measured, and differential binding to native and denatured antigens indicated a preference for conformational epitopes.

[0198] In one embodiment, a method for screening for antibodies that bind to a conformational epitope of an enzymatically active full-length PAM, an enzymatically active PHM subunit, an enzymatically active PAL subunit, or an enzymatically active PAM protein fragment is a co-elution assay comprising the steps of:

[0199] mixing the antibody with the antigen (enzymatically active full-length PAM, enzymatically active PHM subunit, enzymatically active PAL subunit, or enzymatically active PAM protein fragment) in its native or denatured state (e.g., by heating to >70°C), and / or

[0200] Incubate the mixture for a period of time, and / or

[0201] The mixture was applied to HPLC based size exclusion chromatography and the elution profile was analyzed.

[0202] Due to the larger molecular weight of the antibody-antigen complex, the antibody-antigen complex will elute faster from the SEC column, resulting in additional peaks in the chromatogram, while the peaks of unreacted antigen and antibody will have lower intensities. If the antibody reacts with a linear epitope, the elution peak of the antibody-antigen complex will only be formed when a denatured antigen is used. If the antibody recognizes a conformational epitope, the elution peak of the antibody-antigen complex will only be formed when a non-denatured antigen is used.

[0203] In one embodiment of the present invention, the conformational epitope is related to the following sequences of PAM: SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 10 and SEQ ID No. 25.

[0204] The conformational epitope may comprise at least 6 amino acids, preferably at least 5 amino acids, most preferably at least 4 amino acids.

[0205] In one embodiment of the invention, the first and second binding agents bind to conformational epitopes contained within the following PAM sequences: SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6 and SEQ ID No. 10.

[0206] In one embodiment of the invention, the first and second binding agents bind to a conformational epitope contained within the PAL subunit of PAM (SEQ ID No. 8).

[0207] In one embodiment of the invention, the first and second binding agents bind to a conformational epitope contained within the PHM subunit of PAM (SEQ ID No. 7).

[0208] In a specific embodiment of the invention, the first binding agent binds to a conformational epitope contained within the PAL subunit of PAM (SEQ ID No. 8), and the second binding agent binds to a conformational epitope contained within the PHM subunit of PAM (SEQ ID No. 25).

[0209] The level of PAM and / or its isomers and / or fragments thereof is determined using at least two binding agents, wherein at least one of the binding agents is directed against a conformational epitope contained within the following sequences of PAM: SEQ ID No. 8 and / or SEQ ID No. 25.

[0210] One embodiment of the present application relates to a kit for performing a method of diagnosing or prognosing a disease in a subject and / or predicting the risk of developing a disease or an adverse event in a subject and / or monitoring a disease or an adverse event in a subject, wherein the kit comprises at least two binding agents to conformational epitopes within the following PAM sequences: SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 10 and SEQ ID No. 25.

[0211] A specific embodiment of the present application relates to a kit for detecting PAM levels, comprising one or more binding agents that bind to a conformational epitope within a PAM sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 10, and SEQ ID No. 25.

[0212] Another embodiment of the present invention relates to a method for obtaining a monoclonal antibody.

[0213] In all of the following embodiments, the term monoclonal antibody is intended to include monoclonal antibodies as well as fragments of monoclonal antibodies, such as the fragments described in detail herein, and more particularly monoclonal antibodies.

[0214] hybridoma

[0215] In another aspect, the antibody according to the present invention is a monoclonal antibody, which can be obtained by a method comprising the steps of:

[0216] i) fusing antibody-secreting cells from an animal previously immunized with the antigen with myeloma cells to obtain a large number of hybridomas, and / or

[0217] ii) isolating hybridomas producing the desired monoclonal antibody from the plurality of hybridomas.

[0218] In certain embodiments, the antibodies according to the present invention are monoclonal antibodies, which can be obtained by isolating a hybridoma that produces the desired monoclonal antibody from a plurality of hybridomas produced by fusing antibody-secreting cells from an animal previously immunized with an antigen with myeloma cells to obtain a plurality of hybridomas.

[0219] Specifically, the desired monoclonal antibody is particularly one that is 7 M -1 , preferably 10 8 M -1 A monoclonal antibody that binds to the antigen with an affinity greater than 10 9 M -1 , most preferably above 10 10 M -1 .

[0220] To determine the affinity of an antibody for its target (e.g., PAM), the binding kinetics of the target to the immobilized antibody can be determined using label-free surface plasmon resonance using a Biacore 2000 system (GE Healthcare Europe GmbH, Freiburg, Germany).

[0221] In certain embodiments of the method for obtaining an antibody, in step i), the animal is a mammal, particularly a rabbit, mouse or rat, more particularly a mouse, more particularly a Balb / c mouse.

[0222] In certain embodiments of the method for obtaining antibodies, in step i), the antibody-secreting cells are splenocytes, more particularly activated B cells.

[0223] In certain embodiments of the method for obtaining an antibody, in step i), the fusion involves the use of polyethylene glycol.

[0224] In certain embodiments of the method for obtaining an antibody, in step i), the myeloma is derived from a mammal, and in certain embodiments, from the same mammalian species from which the large number of antibody-secreting cells is obtained. In certain specific embodiments of the method for obtaining an antibody, in step i), the myeloma cell is an SP2 / 0 cell line.

[0225] In certain embodiments of the method for obtaining an antibody, the fusion in step i) comprises PEG-assisted fusion, Sendai virus-assisted fusion, or current-assisted fusion.

[0226] In certain embodiments of the method for obtaining antibodies, the isolating in step ii) comprises performing an antibody capture assay, an antigen capture assay and / or a functional screen.

[0227] In certain embodiments of the method for obtaining an antibody, in step ii), isolating the hybridoma producing the desired monoclonal antibody may involve cloning and recloning the hybridoma using a limiting dilution technique.

[0228] In one embodiment, the antigen capture assay comprises:

[0229] a) binding the produced antibody to a substrate, in particular a solid substrate, and / or

[0230] b) allowing the antigen to bind to the antibody, and / or

[0231] c) removal of unbound antigen by washing, and / or

[0232] d) detecting bound antigen;

[0233] Alternatively, the antigen capture assay comprises:

[0234] a) allowing an antigen to bind to said produced antibody to form an antibody-antigen complex, and / or

[0235] b) binding the antibody-antigen complex to a substrate, in particular a solid substrate, and / or

[0236] c) removal of unbound antigen by washing, and / or

[0237] d) Detection of bound antigen.

[0238] In one embodiment, the separation in step ii) comprises performing enzyme-linked immunosorbent assay, fluorescence-activated cell sorting, cell staining, immunoprecipitation and / or western blotting.

[0239] In one embodiment, detection of the antibody or antigen is accomplished using an immunoassay.

[0240] In one embodiment, the animal is a transgenic animal, in particular a transgenic mouse (in which, in particular, the mouse immunoglobulin (Ig) loci have been replaced by human loci within the genome of the transgenic animal), such as a HuMabMouse or a XenoMouse.

[0241] In one embodiment, the antigen comprises a peptide as described in Table 1 herein, which in certain embodiments (particularly for immunization) may be coupled to a protein, particularly a serum protein, more particularly serum albumin, more particularly BSA.

[0242] In a preferred embodiment, the antibody according to the present invention is a monoclonal antibody obtainable by a method comprising the following steps:

[0243] i) fusing splenocytes from Balb / c mice previously immunized with the peptides described in Table 1 herein with SP2 / 0 myeloma cells using polyethylene glycol to obtain a large number of hybridomas, and / or

[0244] ii) isolating hybridomas producing the desired monoclonal antibody from the plurality of hybridomas;

[0245] More preferably, the method comprises:

[0246] 1) Grow the hybridomas in HAT medium [RPMI 1640 medium supplemented with 20% fetal bovine serum and HAT supplement] for a first period of time (typically 2 weeks), and / or

[0247] 2) Subsequent replacement of HAT medium with HT medium for multiple passages (especially 3), and / or

[0248] 3) then returning to normal cell culture medium for a second period of time, in particular until the end of three weeks post-confluency, and / or

[0249] 4) Preliminary screening of cell culture supernatants for antigen-specific IgG antibodies, and / or

[0250] 5) expanding microcultures of cells that tested positive in 4), and / or

[0251] 6) Retest the cell culture supernatant of the microculture for antigen-specific IgG antibodies, and / or

[0252] 7) Cloning and recloning of cultures that tested positive in 6) using limiting dilution techniques, and / or

[0253] 8) optionally determining the isotype of the clones obtained from 7), and / or

[0254] 9) Optionally purify the antibody by protein A.

[0255] Phage display

[0256] In another aspect, the antibody according to the present invention is a monoclonal antibody obtainable by a method comprising the steps of:

[0257] i) isolating at least one antibody having affinity for the antigen from the antibody gene library, and / or

[0258] ii) producing at least one cell line expressing said at least one antibody, and / or

[0259] iii) isolating the at least one antibody from the culture of the at least one cell line obtained in step ii).

[0260] Specifically, an antibody having affinity for an antigen is one that has a binding affinity of at least 10 7 M -1 , preferably 10 8 M -1 More preferably, the affinity is greater than 10 9 M -1 , most preferably greater than 10 10 M -1 .

[0261] In one embodiment, the antibody according to the present invention is a monoclonal antibody, which can be obtained by isolating at least one antibody from a culture derived from at least one cell line expressing at least one antibody having affinity for an antigen from an antibody gene library.

[0262] In one embodiment, the antigen comprises a peptide as described in Table 1 herein, which in certain embodiments may be bound to a solid phase.

[0263] In certain embodiments of the method for obtaining antibodies, in step i), the antibody gene library is a natural antibody gene library, in particular a human natural antibody gene library, more specifically, in the library, antibodies are displayed by phage display, i.e., displayed on phages comprising nucleotide sequences encoding such corresponding antibodies; more specifically, it is library HAL7, HAL 8 or HAL 9, more specifically, it is a library comprising the human natural antibody gene library HAL7 / 8.

[0264] In certain embodiments of the methods for obtaining antibodies, in step i), the screening involves the use of an antigen, particularly an antigen containing a tag, more particularly a biotin tag, attached via two different spacers. In certain embodiments, such a screening strategy involves a mixture of screening rounds using nonspecifically bound antigen and antigen specifically bound via the tag (in the case of a biotin tag, the antigen is bound to streptavidin). In this way, background from nonspecific binders can be minimized.

[0265] In certain embodiments of the method for obtaining antibodies, in step i), in embodiments wherein the library is a phage display library, the antibodies are isolated by isolating phage that present the antibodies (and comprise a nucleotide sequence encoding the antibodies).

[0266] In certain embodiments of the method for obtaining an antibody, in step ii), the cell strain is generated by introducing a nucleotide sequence encoding the antibody, and in embodiments where the library in step i) is a phage display library, the isolated phage from step i) can be used to generate a bacterial strain, such as an Escherichia coli (E. coli) strain, that expresses the antibody.

[0267] In certain embodiments of the method for obtaining antibodies, in step iv), in embodiments wherein the library in step i) is a phage display library and wherein a bacterial strain is produced in step ii), the antibodies may be isolated from the supernatant of the culture.

[0268] It should be understood that when used to describe a method for obtaining an antibody, the term "an antibody" in the expression "at least one antibody" may specifically include more than one antibody molecule having the same amino acid sequence. This understanding applies mutatis mutandis to the term "a cell line."

[0269] In certain embodiments of the method for obtaining antibodies, more than one antibody (referring to multiple antibodies each having a different amino acid sequence) is isolated in step i), and thus more than one cell line is generated in step ii). This method may involve screening clones that test positive for binding to the antigen, for example, by a binding assay, such as an ELISA assay involving the antigen, and cells that test positive for binding to the antigen can be isolated to generate a monoclonal cell line.

[0270] In a preferred embodiment, the antibody according to the present invention is a monoclonal antibody obtainable by a method comprising the following steps:

[0271] i) isolating at least one antibody having affinity for an antigen from an antibody gene library comprising a human natural antibody gene library HAL7 / 8 by eluting phage carrying said antibody from said library, and / or

[0272] ii) producing at least one E. coli cell line expressing said at least one antibody, and / or

[0273] iii) isolating the at least one antibody from the culture supernatant of the at least one E. coli cell line obtained in step ii).

[0274] In yet another aspect, antibody fragments according to the present invention are produced by methods involving enzymatic digestion of antibodies. In certain embodiments, this method produces, for example, Fab or F(ab)2 antibody fragments. In certain embodiments, this method involves digestion with pepsin or papain, wherein the enzyme is optionally immobilized on the surface.

[0275] In certain embodiments, antibodies can be humanized by CDR grafting, particularly by a process involving the following steps:

[0276] - extracting RNA from a hybridoma expressing the antibody of interest (e.g., obtained by the methods described herein), and / or

[0277] - amplifying the extracted RNA by RT-PCR, in particular using primer sets specific for the heavy and light chains of the antibody of interest, to obtain DNA products, and / or

[0278] - further amplifying the DNA product by PCR, in particular using a semi-nested primer set specific for the antibody variable region, and / or

[0279] - determining the sequence of said DNA product, and / or

[0280] - Aligning the sequences with homologous human framework sequences to determine the humanized variable heavy and variable light chain sequences (of the desired antibody).

[0281] In certain embodiments, the antibody can be humanized as follows: RNA extracted from a hybridoma expressing the target antibody is amplified by RT-PCR (particularly using a specific primer set for the heavy chain and light chain of the target antibody), and the DNA obtained therefrom is further amplified by PCR (particularly using a specific semi-nested primer set for the variable region of the antibody), and the sequence of the DNA product thus obtained is aligned with the homologous human framework sequence to determine the sequence of the humanized variable heavy chain and variable light chain (of the desired antibody).

[0282] In certain embodiments, antibodies can be humanized by:

[0283] - Determination of the complementarity determining regions (CDRs), which can be achieved by analyzing the structural interactions of the framework regions (FRs) with the CDRs and the antigen, and / or

[0284] - Grafting the CDR sequences into human framework regions.

[0285] In certain embodiments, antibodies can be humanized by grafting framework (FR) sequences, which may preferably be determined by analyzing the structural interactions of the CDRs with the antigen, into human framework regions.

[0286] In certain embodiments, changes in the amino acid sequence of the CDRs or FRs can be introduced to maintain structural interactions with the antigen that might otherwise be disrupted by the introduction of human FR sequences, for example, by random approaches using phage display libraries or by directed approaches guided by molecular modeling.

[0287] DNA sequences encoding the antibodies identified as detailed herein can be transferred into cells by known genetic engineering techniques and used to produce said antibodies.

[0288] Producing antibodies

[0289] In another aspect, the antibody according to the invention is a monoclonal antibody obtainable by the method described herein, which is produced by a method comprising the steps of:

[0290] - cultivating a cell line comprising a nucleotide sequence encoding the antibody;

[0291] - isolating said antibody from said culture.

[0292] In another specific aspect, the antibody according to the invention is a monoclonal antibody obtainable by the method described herein, which is produced by isolating said antibody from a culture of a cell line comprising a nucleotide sequence encoding said antibody.

[0293] In certain embodiments of the method, the cell strain is produced as described above and can include bacterial cells, such as Gram-negative bacteria such as Escherichia coli, Proteus mirabilis or Pseudomonas putidas, Gram-positive bacteria such as Bacillus brevis, Bacillus subtilis, Bacillus megaterium, Lactobacillus such as Lactobacillus zeae / Lactobacillus casei or Lactobacillus paracasei, or Streptomyces such as Streptomyces lividans; eukaryotic cells, such as yeast such as Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha. polymorpha), Schizosaccharomyces pombe, Schwanniomyces occidentalis, Kluyveromyces lactis, or Yarrowia lipolytica; fungi, for example, filamentous fungi such as those of the genus Trichoderma or Aspergillus, such as A. niger (e.g., subgenus A. awamori) and Aspergillus oryzae, Trichoderma reesei, or fungi such as C. lucknowense; protozoa, for example, Leishmania such as L. tarentolae; insect cells, for example, insect cells transfected with a baculovirus (e.g., AcNPV), such as insect cell lines from Spodoptera frugiperda, such as Sf-9 or Sf-21, or cells from Drosophila melanogaster. melanogaster ) cell lines such as DS2, or cell lines from Trichopulsia ni such as High Five cells (BTI-TN-5B1-4); mammalian cells, for example, hamster, such as Chinese hamster ovary cells such as K1-, DukX B11-, DG44, Lec13 or BHK, mouse, such as mouse myeloma cells such as NS0, Homo sapiens, such as Per.C6, AGE1.HN, HEK293.

[0294] In certain embodiments of the methods, the cell can be a hybridoma cell, eg, a hybridoma cell as described herein.

[0295] In certain embodiments of the method, the culture can be carried out in static suspension culture, stirred suspension culture, membrane-based culture, matrix-based culture or high cell density bioreactor; the container for such culture can be selected from the group consisting of: T-flask, roller culture, spinner culture, stirred tank bioreactor, airlift bioreactor, static membrane-based or matrix-based culture system, suspension bioreactor, fluidized bed bioreactor, ceramic bioreactor, perfusion system and hollow fiber bioreactor.

[0296] In certain embodiments of the methods, the cells may be immobilized on a matrix.

[0297] High cell density bioreactors are specifically capable of producing more than 10 8 The cell density of the culture system is 10 cells / ml.

[0298] In another aspect, the antibody according to the invention is a monoclonal antibody obtainable by the method described herein, which is produced by a method comprising the steps of:

[0299] - producing transgenic plants or animals comprising a nucleotide sequence encoding said antibody, and / or

[0300] - isolating said antibody from said plant or animal or a secretion or product of said plant or animal.

[0301] In a certain further aspect, the antibody according to the invention is a monoclonal antibody obtainable by the method described herein, which is produced by isolating the antibody from a transgenic plant or transgenic animal having a nucleotide sequence encoding the antibody, or from a secretion or product of the transgenic plant or transgenic animal.

[0302] The mammals may be selected from chickens, mice, rats, rabbits, cows, goats, sheep, and pigs; the secretions or products may be milk or eggs; and the plants may be selected from tobacco (N. tabacum or N. benthamiana), duckweed (Lemna minor), Chlamydomonas reinhardtii, rice, Arabidopsis thaliana, alfalfa (Medicago sativa), lettuce, and corn.

[0303] In certain embodiments, the antibodies can be isolated by physicochemical fractionation such as size exclusion chromatography, precipitation such as using ammonium sulfate, ion exchange chromatography, immobilized metal chelate chromatography gel filtration, zone electrophoresis; based on their classification, for example, by binding to bacterial proteins A, G or L, japonin; antigen-specific affinity purification by immobilized ligand / antigen; if necessary, low molecular weight components can be removed by methods such as dialysis, desalting and diafiltration.

[0304] In some embodiments, the antibody is encoded by a nucleotide sequence that is a reverse transcript of an amino acid sequence from an antibody produced by one of the processes described herein.

[0305] Against the above background, the following consecutively numbered embodiments provide further specific aspects of the present invention:

[0306] 1. A method for determining the level of peptidylglycine alpha-amidating monooxygenase (PAM) and / or its isoforms and / or fragments in a body fluid or tissue sample using an assay, wherein the assay comprises at least one binding agent directed against a conformational epitope of PAM.

[0307] 2. A method for determining the level of peptidylglycine α-amidating monooxygenase (PAM) and / or its isomers and / or fragments in a body fluid or tissue sample according to embodiment 1, wherein the assay comprises two binding agents that bind to two different regions of PAM, wherein each of the two binding agents is directed against a conformational epitope of PAM.

[0308] 3. A method for determining the level of peptidylglycine α-amidating monooxygenase (PAM) and / or its isomers and / or fragments in a body fluid or tissue sample according to embodiment 2, wherein the first of the two binding agents binds to a conformational epitope contained within the PHM subunit of PAM (SEQ ID No. 7), and the second of the two binding agents binds to a conformational epitope contained within the PAL subunit of PAM (SEQ ID No. 8).

[0309] 4. A method for determining the level of peptidylglycine α-amidating monooxygenase (PAM) and / or its isomers and / or fragments in a body fluid or tissue sample according to embodiment 2 or 3, wherein each of the two binding agents is directed against an epitope contained within the following sequences of PAM: SEQ ID No. 25 (PHM, amino acids 31-377 of SEQ ID No. 1) and SEQ ID No. 8 (PAL, amino acids 495-817 of SEQ ID No. 1) of human PAM.

[0310] 5. The method for determining the level of peptidylglycine α-amidating monooxygenase (PAM) and / or its isomers and / or fragments in a body fluid or tissue sample according to any one of embodiments 1 to 4, wherein the conformational epitope has at least 4 amino acids, preferably at least 5 amino acids.

[0311] 6. A method for determining the level of peptidylglycine alpha-amidating monooxygenase (PAM) and / or its isomers and / or fragments in a body fluid or tissue sample according to any one of embodiments 1 to 5, wherein the binding agent does not bind to denatured PAM or denatured PAM subunits (e.g., PAL or PHM).

[0312] 7. A method for determining the level of peptidylglycine α-amidating monooxygenase (PAM) and / or its isomers and / or fragments in a body fluid or tissue sample according to embodiments 1 to 6, wherein the binding agent does not bind to denatured PAM or denatured PAM subunits (e.g., PAL or PHM) when using Western blotting techniques.

[0313] 8. A method for determining the level of peptidylglycine α-amidating monooxygenase (PAM) and / or its isomers and / or fragments in a body fluid or tissue sample according to any one of embodiments 1 to 7, wherein the binder to a conformational epitope of PAM binds to an enzymatically active PAM or an enzymatically active PAM subunit (e.g., PAL or PHM), but does not bind to an enzymatically inactive PAM or an enzymatically inactive PAM subunit (e.g., PAL or PHM).

[0314] 9. The method for determining the level of peptidylglycine alpha-amidating monooxygenase (PAM) and / or its isomers and / or fragments in a body fluid or tissue sample according to any one of embodiments 1 to 8, wherein the at least one binding agent is selected from an antibody, an antibody fragment or a non-IgG scaffold.

[0315] 10. The method for determining the level of peptidylglycine alpha-amidating monooxygenase (PAM) and / or its isoforms and / or fragments in a body fluid or tissue sample according to any one of embodiments 1 to 9, wherein the at least one binding agent is produced using a large fragment protein, a full-length protein or DNA immunoassay technology.

[0316] 11. A method for diagnosing or prognosing a disease in a patient and / or predicting the risk of a patient developing a disease or experiencing an adverse event and / or monitoring a patient's disease or adverse event, said method being performed by determining the level of peptidylglycine α-amidating monooxygenase (PAM) and / or its isomers and / or fragments thereof in a body fluid or tissue sample of said patient according to any one of embodiments 1 to 10.

[0317] wherein the disease in the patient is selected from the group comprising dementia, cardiovascular disorders, kidney diseases, cancer, inflammatory or infectious diseases and / or metabolic diseases,

[0318] The adverse events are selected from the group consisting of cardiac events, cardiovascular events, cerebrovascular events, cancer, diabetes, infection, serious infection, sepsis-like systemic infection, sepsis, and all-cause mortality.

[0319] 12. A method for diagnosing or prognosing a disease in a patient and / or predicting the risk of a patient developing a disease or experiencing an adverse event and / or monitoring a patient's disease or adverse event, said method being performed by determining the level of peptidylglycine α-amidating monooxygenase (PAM) and / or its isomers and / or fragments thereof in a body fluid or tissue sample of said patient according to any one of embodiments 1 to 11, said method comprising the following steps:

[0320] Determining the level of PAM and / or its isomers and / or fragments thereof in a body fluid or tissue sample of said patient,

[0321] comparing the measured amount with a predetermined threshold value,

[0322] wherein if the measured amount is below or above the predetermined threshold, the patient is diagnosed as having a disease, or

[0323] wherein if said measured amount is below or above said predetermined threshold, the outcome of said disease is prognosed, or

[0324] wherein if the measured amount is below or above the predetermined threshold, the risk of the patient developing a disease or experiencing an adverse event is predicted, or

[0325] wherein the patient is monitored for disease or adverse events.

[0326] 13. The method according to embodiments 1 and 12, wherein the level of PAM and / or its isomers and / or its fragments is the total concentration of PAM and / or its isomers and / or fragments having at least 12 amino acids in the body fluid or tissue sample of the patient.

[0327] 14. The method according to any one of embodiments 1 to 13, wherein the total concentration of the PAM and / or its isomers and / or fragments thereof having at least 12 amino acids is detected using an immunoassay.

[0328] 15. A method for diagnosing or prognosing a disease in a patient and / or predicting the risk of a patient developing a disease or experiencing an adverse event and / or monitoring a patient for a disease or an adverse event, the method being performed by determining the level of a PAM and / or its isomers and / or fragments thereof in a body fluid or tissue sample of the patient according to any one of embodiments 1 to 14, wherein the PAM and / or its isomers and / or fragments thereof are selected from the group consisting of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8 and SEQ ID No. 10.

[0329] 16. A method for diagnosing or prognosing a disease in a patient and / or predicting the risk of a patient developing a disease or experiencing an adverse event and / or monitoring a patient for a disease or an adverse event, said method being performed by determining the level of a PAM and / or its isomers and / or fragments thereof in a body fluid or tissue sample of said patient according to any one of embodiments 1 to 15, wherein the risk of the patient developing a disease is determined, and wherein said patient is a healthy patient.

[0330] 17. The method of embodiment 16, wherein the disease is selected from Alzheimer's disease, colorectal cancer, and pancreatic cancer.

[0331] 18. The method of any one of embodiments 1 to 17 for determining the level of peptidylglycine alpha-amidating monooxygenase (PAM) and / or its isomers and / or fragments in a body fluid or tissue sample, wherein the level is the level of active PAM.

[0332] 19. Use of an antibody for determining the level of PAM and / or its isomers and / or fragments thereof, wherein the antibody is directed against a conformational epitope contained within the following sequences of PAM: PHM fragment (amino acids 31-377 of PAM) (SEQ ID No. 25) and / or PAL fragment (amino acids 495-817 of PAM) (SEQ ID No. 8).

[0333] 20. Use of the antibody according to embodiment 19 for determining the level of PAM and / or its isoforms and / or fragments thereof, wherein the antibody does not bind to denatured PAM or denatured PAM subunits (e.g., PAL or PHM).

[0334] 21. Use of the antibodies according to embodiments 19 and 20 for determining the level of PAM and / or its isoforms and / or fragments thereof, wherein the antibody against a conformational epitope of PAM binds to an enzymatically active PAM or an enzymatically active PAM subunit (e.g., PAL or PHM), but does not bind to an enzymatically inactive PAM or an enzymatically inactive PAM subunit (e.g., PAL or PHM).

[0335] 22. A kit for determining the level of PAM and / or its isoforms and / or fragments thereof, comprising one or more antibodies that bind to PAM, said antibodies being directed against a conformational epitope of PAM contained within the following sequences: PHM fragment (amino acids 31-377 of PAM) (SEQ ID No. 25) and / or PAL fragment (amino acids 495-817 of PAM) (SEQ ID No. 8).

[0336] 23. A kit for determining the level of PAM and / or its isoforms and / or fragments thereof according to embodiment 22, comprising one or more antibodies that bind to PAM, wherein the antibodies do not bind to denatured PAM or denatured PAM subunits (e.g., PAL or PHM).

[0337] 24. A kit for determining the level of PAM and / or its isoforms and / or fragments thereof according to embodiments 22 and 23, comprising one or more antibodies that bind to PAM, wherein the antibodies against a conformational epitope of PAM bind to an enzymatically active PAM or an enzymatically active PAM subunit (e.g., PAL or PHM), but do not bind to an enzymatically inactive PAM or an enzymatically inactive PAM subunit (e.g., PAL or PHM). BRIEF DESCRIPTION OF THE DRAWINGS

[0338] Figure 1 : Schematic representation of PAM isoform 1. The bold black arrow indicates the cleavage site at the dibasic amino acid.

[0339] Figure 2 : Enzyme reaction catalyzed by PAM.

[0340] Figure 3 Figure 3: Alpha Fold-predicted structural basis for antibody production in mice against the human PAM protein (Uniprot ID: P19021). A: The full-length structure of the protein, with the PHM and PAL domains highlighted in dark gray and the unstructured regions and transmembrane domain in light gray. Conformational antibodies are generated by immunization with a stable, well-structured protein construct (B), while antibodies against linear epitopes are generated by immunization with an unstructured synthetic peptide (black marker, C).

[0341] Figure 4 Characterization of Antibodies in the PAM-LIA Assay: Samples were prepared using 15 ng / mL of recombinant full-length PAM in EDTA-spiked human plasma and 1x PBS, with a total load of 200 ng. Controls included antibodies against linearized PHM (Pep 14, SEQ ID No. 24) and PAL (Pep 4, SEQ ID No. 14) peptides. The expected molecular weight of full-length PAM is approximately 90 kDa.

[0342] Figure 5 : Reactivity of antibody cell lines tested in ELISA assays to recombinant PAL (A) and PHM (C) subunits and full-length PAM (B, D).

[0343] Figure 6 AM: Typical calibration curve of a PAM sandwich immunoassay. AJ used recombinant PAM as a calibrant in a setup with an antibody against a linearized peptide. (A) Solid phase: antibody against peptide 10 (SEQ ID No. 20), tracer: antibody against peptide 9 (SEQ ID No. 19); (B) Solid phase: antibody against peptide 10 (SEQ ID No. 20), tracer: antibody against peptide 10 (SEQ ID No. 20); (C) Solid phase: antibody against peptide 9 (SEQ ID No. 19), tracer: antibody against peptide 10 (SEQ ID No. 20); (D) Solid phase: antibody against recombinant PAM (SEQ ID No. 10), tracer: antibody against recombinant PAM (SEQ ID No. 10); (E) Solid phase: antibody against peptide 10 (SEQ ID No. 20), tracer: antibody against recombinant PAM (SEQ ID No. 10); (F) Solid phase: antibody against peptide 13 (SEQ ID No. 23), tracer: antibody against peptide 10 (SEQ ID No. (G) solid phase: antibody against peptide 14 (SEQ ID No. 24), tracer: antibody against peptide 13 (SEQ ID No. 23); (H) solid phase: antibody against recombinant PAM (SEQ ID No. 10), tracer: antibody against peptide 13 (SEQ ID No. 23); (I) solid phase: antibody against peptide 13 (SEQ ID No. 23), tracer: antibody against peptide 9 (SEQ ID No. 19); (J) solid phase: antibody against peptide 10 (SEQ ID No. 20), tracer: antibody against peptide 13 (SEQ ID No. 23). K and L used native PAM (EDTA plasma) as a calibrant: (K) Solid phase: antibody against peptide 14 (SEQ ID No. 24), tracer: antibody against peptide 13 (SEQ ID No. 23); (L) Solid phase: antibody against peptide 10 (SEQ ID No. 20), tracer: antibody against peptide 13 (SEQ ID No. 23). M used recombinant PAM as a calibrant in a setup utilizing antibodies against conformational peptides. Solid phase: antibody against the PAL subunit, tracer: antibody against the PHM subunit.

[0344] Figure 7 : Frequency distribution (histogram) of PAM concentrations (conformational PAM-LIA) in healthy individuals (n=4106).

[0345] Figure 8: PAM concentration (RLU) and α-amidation activity (AMA) (ng / L) in matched EDTA and Li-heparin plasma using a conformational antibody (A) and an antibody against a linearized peptide (B). h) Correlation, n is the number of participants, and r is the Spearman correlation coefficient.

[0346] Figure 9 : PAM concentration (ng / mL) and α-amidation activity (AMA) (µg / L) in matched EDTA and Li-heparin plasma h) Correlation, n is the number of participants, and r is the Spearman correlation coefficient.

[0347] Figure 10 : Representative calibration curve of recombinant PAM (AMA).

[0348] Figure 11 : Frequency distribution of AMA in self-reported healthy individuals (n=120).

[0349] Figure 12 : Alzheimer's disease incidence in a large, prospective population-based cohort measured in PAM-LIA and PAM-AMA, with cutoffs of 93.2 ng / mL and 14.4 µg / L, respectively h. Significance was calculated using the Gehan-Breslow-Wilcoxon test.

[0350] Figure 13 : PAM concentrations (ng / mL) in sepsis and healthy cohorts measured in PAM-LIA. Significance was calculated using a two-tailed Mann-Whitney test.

[0351] Figure 14: HPLC elution profiles showing (A) recombinantly produced active PAL subunit, (B) anti-PAL bound conformation antibody, (C) complex of anti-PAL bound conformation antibody and PAL subunit, (D) recombinantly produced active PHM subunit, (E) anti-PHM bound conformation antibody, and (F) complex of anti-PHM bound conformation antibody and PHM subunit.

[0352] Figure 15 : SDS-PAGE analysis of HPLC elution fractions. Lane 1: Recombinantly produced active PAL subunit. Lane 2: Recombinantly produced active PHM subunit. Lane 3: Conformational antibody (Note: anti-PHM and anti-PAL antibodies produce the same pattern; anti-PAL antibody is not shown). Lane 4: Prestained protein marker used as a molecular weight standard. Lane 5: Complex of anti-PAL-bound conformational antibody with PAL subunit. Lane 6: Complex of anti-PHM-bound conformational antibody with PHM subunit. DETAILED DESCRIPTION

[0353] Example

[0354] Example 1 – Production of recombinant PAM

[0355] 1.1. Preparation of full-length PAM for immunization

[0356] Variant A: PAM cDNA was synthesized according to Uniprot accession number P19021 and encodes amino acids 21-834 of the PAM protein, including codon optimization for expression in mammalian cells. The PAM signal sequence was replaced with the human serum albumin signal sequence (MKWVTFISLLFLFSSAYSFR [SEQ ID No. 9]). A hexahistidine tag was added to the C-terminus of PAM via a GS linker. The sequence of the recombinant PAM (amino acids 21-834 of PAM without the signal sequence and hexahistidine tag) is shown in SEQ ID No. 10. The cDNA was cloned into an expression vector (plasmid DNA) using 5'-NotI and 3' HindIII restriction sites. The expression vector carrying the cDNA for PAM expression was replicated in E. coli and prepared from E. coli with low endotoxin levels.

[0357] HEK-INV cells were transfected with the expression vectors using INVect transfection reagent in serum-free suspension culture. Transfection efficiency was controlled by co-transfection with a GFP (green fluorescent protein) expression vector. Cells were cultured at 37°C and 5% CO2 in the presence of valproic acid and penicillin-streptomycin. When viability reached <60%, cells were harvested by centrifugation (>2000 g, 30-45 minutes, 2-8°C). Cell culture supernatant (CCS) was washed five times with 100 mM Tris / HCl (pH 8.0) by tangential flow filtration (TFF, 30 kDa cutoff).

[0358] Purification of recombinant PAM involved applying buffer-exchanged CCS to Q-sepharose fast-flow resin (GE Healthcare) and eluting with a NaCl gradient (up to 2 M). Fractions containing amidation activity were pooled and applied to a Superdex 200pg (GE Healthcare) size exclusion chromatography column using an elution buffer of 100 mM Tris / HCl, 200 mM NaCl, pH 8.0. Fractions containing amidation activity were pooled, dialyzed against 100 mM Tris-HCl, 200 mM NaCl, pH 8.0, and sterile filtered (0.2 µm). Endotoxin load was determined using a Charles River PTS Endosafe system and was less than 5 EU / mL.

[0359] Variant B: The second construct of the full-length PAM used for immunization was commercially obtained from SinoBiological and comprises residues 31-973 of the human PAM (UniProtKB: P19021-1, SEQ ID No. 1), lacking the amino acid sequence from positions 388 to 494, and carrying a deca-histidine tag at the C-terminus.

[0360] 1.2. Production of Soluble Catalytic Cores of Single PAM Subunits for Immunization

[0361] Soluble PHM and PAL subunits containing residues 31-377 (SEQ ID No. 25) and residues 495-817 (SEQ ID No. 8), respectively, of the human PAM (UniProtKB: P19021-1) were synthesized and expressed in transiently transfected human kidney 293 (HEK293) cells. Both constructs were N-terminally truncated and linked to a decaphidine tag via a GS linker, followed by a TEV cleavage site. The signal sequences of the PHM and PAL constructs were replaced with the human serum albumin signal sequence (MKWVTFISLLFLFSSAYSFR [SEQ ID No. 9]). The cDNAs were cloned into expression vectors (plasmid DNA) using 5'-NotI and 3' HindIII restriction sites. The expression vectors carrying the cDNAs for PHM and PAL were replicated in Escherichia coli and prepared from E. coli to low endotoxin levels.

[0362] Transfection and culture of transfected HEK-INV cells with PAL or PHM constructs were identical to the full-length PAM method described in variant A (see above).

[0363] The overexpressed recombinant constructs PHM and PAL were purified by cobalt affinity chromatography, with sample purity exceeding 95% as determined by capillary gel electrophoresis. Elution fractions obtained from the protein purification process were analyzed by Western blotting. Specifically, the elution fractions were loaded onto a sodium dodecyl sulfate (SDS) gel and transferred to a nitrocellulose membrane. The membrane was probed with an anti-His antibody to detect the target protein. Fractions containing PHM or PAL protein were pooled, dialyzed against 50 mM Tris-HCl, 150 mL NaCl (pH 8.0), and sterile filtered (0.2 µm). Endotoxin load was determined using a Charles River PTS Endosafe system and was less than 5 EU / mL.

[0364] Example 2 – Preparation of antibodies targeting linear and conformational epitopes

[0365] Can be based on Figure 3 The procedure described in , two types of antibodies are robustly generated in mice - antibodies against linear epitopes and antibodies against conformational epitopes. First, the structure of the protein should be known or predicted. In this specific case, the structure of the protein is predicted using the program AlphaFold, which does not require any prior knowledge of a structure that has been solved using X-ray crystallography, but only the protein sequence. This allows for unbiased structure prediction. Once the structure is predicted, solid fragments with minimal unstructured regions are required to produce conformational antibodies. For antibodies against linear epitopes, unstructured and disordered regions are required because they are also linear in vivo.

[0366] The unstructured region is identified and produced in vivo, such as by synthesis or recombinant expression. Finally, the solid fragment and synthetic peptide are used for immunization. Through this process, mice can produce antibodies against both linear and conformational epitopes, which can be used in various applications.

[0367] Anti-PAM antibodies targeting linear epitopes were synthesized as follows:

[0368] PAM peptides for immunization, see Table 1 (Peptides & Elephants, Hennigsdorf, Germany), were synthesized with an additional C-terminal cysteine ​​residue (if cysteine ​​was not present in the selected PAM sequence) for coupling to bovine serum albumin (BSA). The peptides were covalently linked to BSA using Sulfolink coupling gel (Perbio-science, Bonn, Germany). The coupling procedure was performed according to the Perbio manual.

[0369] Table 1: PAM Immunity Peptides

[0370]

[0371] According to SEQ ID No. 1; amino acid (aa)

[0372] Balb / c mice were injected intraperitoneally (ip) with 100 µg of PAM peptide-BSA conjugate (emulsified in TiterMaxGold adjuvant) on day 0, 100 µg and 100 µg (emulsified in complete Freund's adjuvant) on day 14, and 50 µg and 50 µg (emulsified in incomplete Freund's adjuvant) on days 21 and 28, respectively. On day 45, animals received an intravenous (iv) injection of 50 µg of PAM peptide-BSA conjugate dissolved in saline. Three days later, mice were sacrificed and immune cell fusion was performed.

[0373] Anti-PAM antibodies directed against conformational epitopes according to the present invention were synthesized as follows: Soluble PHM and PAL proteins (SEQ ID No. 25 and 8, respectively) and two constructs of recombinant full-length PAM (variants A (SEQ ID No. 10) and B) were prepared for immunization as described in Example 1.

[0374] Balb / c mice were injected intraperitoneally with 100 µg of PAL, PHM, or full-length PAM protein on day 0, followed by 100 µg on day 14, and 50 µg on days 21 and 28. A single intravenous injection of 50 µg of recombinant protein was administered on day 45. Three days later, mice were sacrificed, and immune cell fusion was performed.

[0375] Spleen cells 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 seconds. After washing, the cells were seeded in 96-well cell culture plates. Hybridoma clones were selected by growth in HAT medium (RPMI 1640 medium supplemented with 20% fetal bovine serum and HAT supplement). After one week, the HAT medium was replaced with HT medium for three passages before returning to normal cell culture medium.

[0376] Two weeks after fusion, cell culture supernatants were initially screened for IgG antibodies that bind to recombinant PHM, PAL, and full-length PAM. Recombinant PAM (SEQ ID No. 10), PHM (SEQ ID No. 25), and PAL (SEQ ID No. 8) were immobilized in 96-well plates (100 ng / well) and incubated with 50 µl of cell culture supernatant per well for 2 hours at room temperature. After washing, 50 µl / well of POD-rabbit anti-mouse IgG antibody was added and incubated at room temperature for 1 hour.

[0377] After the following wash step, 50 µl of chromogen solution (3.7 mM o-phenylenediamine in citrate / hydrogen phosphate buffer, 0.012% H₂O₂) was added to each well, incubated at room temperature for 15 minutes, and the color development reaction was stopped by adding 50 µl of 4N sulfuric acid. The absorbance was measured at 490 nm.

[0378] Microcultures that test positive are transferred to 24-well plates for expansion. After retesting, selected cultures are cloned and recloned using limiting dilution techniques and their isotypes determined.

[0379] Antibodies raised against recombinant human PAM, PHM, or PAL or PAM peptides were generated by standard antibody production methods ( Marx et al. 1997 ) and purified by protein A. The antibody purity was ≥ 90% based on SDS gel electrophoresis analysis.

[0380] Results: Use Figure 3 Antibodies generated by the described technique were tested by western blotting to distinguish between conformational antibodies and antibodies against linearized peptides. Recombinant full-length PAM was spiked into EDTA plasma or dissolved in 1xPBS and linearized by diluting in SDS loading dye supplemented with β-mercaptoethanol and heating at 95°C for at least 10 minutes. The samples were then loaded onto SDS-PAGE (200 ng of recombinant PAM per load) and transferred to the membrane for Western blotting. As primary antibodies, 2 µg / mL of conformational antibodies or antibodies against linear epitopes were used, while the secondary antibody was a rabbit anti-mouse antibody conjugated to horseradish peroxidase. When the linearized antibody was used for detection, strong signals between 70 and 100 kDa were observed in both the PAM samples spiked with EDTA and the PAM samples in PBS, which was expected since the full-length protein has a molecular weight of 90 kDa ( Figure 4 However, when antibodies against the conformational epitope were used for detection, no signal was observed in either sample ( Figure 4This demonstrates the conformational nature of these antibodies, as the PAM should not exhibit any tertiary structure when subjected to SDS analysis, and therefore the antibodies cannot bind.

[0381] To evaluate the ability of hybridoma cell lines to bind to target antigens, dilutions of cell culture supernatants were tested. Conformational antibodies raised against PAL or PHM subunits exhibited significantly high affinity binding to their corresponding protein targets as well as to the full-length PAM. Figure 5 When a universal protein with a His tag was used as a target, no signal was detected.

[0382] Example 3 – PAM immunoassay

[0383] As described in Example 2, antibodies were generated against recombinant PAM (variant A (SEQ ID No. 10) and variant B, Example 1), as well as against its recombinant subunits (SEQ ID No. 25 and No. 8, Example 1) and linear PAM peptides (SEQ ID Nos. 11 to 24).

[0384] The technology used is a sandwich luminescent immunoassay based on acridinium ester labeling.

[0385] 3.1. Labeled compounds (tracers)

[0386] Purified antibodies (0.2 g / L) were labeled by incubation with a 1:5 mol / L ratio of MACN-acridinium-NHS ester (1 g / L, InVent GmbH) in 10% labeling buffer (500 mmol / L sodium phosphate, pH 8.0) at 22°C for 20 minutes. The corresponding antibodies were separated from free label by adding 5% 1 mol / L Tris-HCl (pH 8.0) for 10 minutes. The purified labeled antibodies were diluted in 300 mmol / L potassium phosphate, 100 mmol / L NaCl, 10 mmol / L Na-EDTA, and 5 g / L bovine serum albumin (pH 7.0). The final concentration was approximately 20 ng of labeled antibody per 150 μL.

[0387] Solid phase

[0388] White polystyrene microtiter plates (Greiner Bio-One International AG) were coated with the corresponding antibodies (2 μg / well in 0.2 mL, 50 mmol / L Tris-HCl, 100 mmol / L NaCl, pH 7.8) for 18 h at 20°C. After blocking with 30 g / L Karion, 5 g / L BSA (protease-free), 6.5 mmol / L potassium dihydrogen phosphate, and 3.5 mmol / L sodium dihydrogen phosphate (pH 6.5), the plates were vacuum dried.

[0389] Calibration

[0390] The assay was calibrated using dilutions of commercially available recombinant PAM (Example 1, variant B). Typical concentrations ranged from 1-1000 ng / mL.

[0391] 3.4. PAM immunoassay:

[0392] 3.4.1. PAM-LIA

[0393] One-step version: Pipette at least 10 μL of sample / calibrator into a precoated microtiter plate. Add 200 μL of labeled antibody-containing buffer (300 mmol / L potassium phosphate, 100 mmol / L NaCl, 10 mmol / L Na-EDTA, 50 μmol / L aprotinin, 100 μmol / L leupeptin, 0.1% bovine IgG, 0.02% mouse IgG, 0.5% BSA, pH 7.0). Incubate the microtiter plate at room temperature (20°C) with agitation at 600 rpm for at least 3 h. Remove unbound tracer by washing five times (350 μL per well) with wash solution (20 mmol / L PBS, 1 g / L Triton X-100, pH 7.4).

[0394] The bound chemiluminescent signal in the wells was measured using a Centro LB 960 microtiter plate luminescence reader (Berthold Technologies) with a duration of 1 second per well.

[0395] Two-step version: Pipette at least 10 μL of sample / calibrator into a pre-coated microtiter plate. After adding 200 μL of buffer (as described for the one-step version), incubate the microtiter plate at 2-8°C with agitation at 600 rpm for 15-20 hours. Remove unbound sample by washing four times with wash solution (350 μL per well each time), then add 200 μL of tracer material and incubate the microtiter plate at room temperature (20°C) for 2 hours. Remove unbound tracer by washing four times with wash solution (350 μL per well each time). Measure the bound chemiluminescent signal in each well using a Centro LB 960 Microtiter Plate Luminescence Reader (Berthold Technologies) for 1 second per well.

[0396] Results: The exemplary antibody production process described in Example 2 ( Figure 3 ) provides a robust method for generating antibodies suitable for immunoassay applications. These antibodies can be used in various combinations, either as solid-phase antibodies or as tracer antibodies, allowing for versatile assay design. Table 2 shows the signal-to-noise analysis results measured in various body fluids and tissue extracts.

[0397] Table 2: Signal-to-noise ratios of PAMs measured in serum, EDTA plasma, Li-heparin plasma, pituitary extracts, and recombinant PAMs. Clones 1 to 4 raise antibodies against a conformational epitope of PAL, while clones 5 to 7 raise antibodies against a conformational epitope of the PHM subunit. RLU signals are presented to the third or fourth decimal place.

[0398]

[0399] The conformational antibody-based ELISA demonstrated high signal linearity between 1 and 1000 ng / mL and was also found to be suitable for measuring protein targets in serum, plasma, and tissue extract samples (e.g., pituitary). The samples were unmatched. The background signal of the immunoassay was found to be approximately 192 RLU when using the conformational antibody.

[0400] The mean intra-assay CV was 2.2% [1.3% - 3.8%], and the mean inter-assay CV was 6.7% [2.8% - 12.9%]. The limit of interest (LOD) and limit of quantity (LOQ) were 189 pg / mL and 250 pg / mL, respectively. The accuracy of the PAM-LIA assay was determined by spiking analyte-depleted EDTA plasma with known concentrations of recombinant PAM and ranged from 90.3% to 99.2%. Assay linearity was assessed by dilution and mixing. In the first case, the mean deviations between the measured and target concentrations for samples with starting PAM concentrations of 91.2 ng / mL, 323.5 ng / mL, and 684.7 ng / mL were 13.2% [8.9% - 17.9%], 1.2% [3.7% - 8.2%], and 5.2% [0.4% - 8.8%], respectively. In the second case, the measured concentrations of PAM deviated from the expected concentrations by an average of 4.9% [0.7% - 10.2%].

[0401] Typical calibration curves for LIA using linear and conformational antibodies are shown in Figure 6A -L and Figure 6M middle. Figure 7 Figure 2 shows the distribution of PAM concentrations (PAM-LIA) in serum samples from n = 4106 individuals (a randomly selected subcohort of individuals without a history of cardiovascular disease from the Malmö Prevention Project (MPP), a prospective, population-based study in Sweden). The mean PAM-LIA was 77.8 ng / mL [SD = 19.0]. The median plasma PAM concentration was 78.6 ng / mL (interquartile range [IQR] 66.4 - 92.5 ng / mL). The 10th and 90th percentiles were 56.3 and 106.6 ng / mL, respectively. The 2.5th, 97.5th, and 99th percentiles were 45, 123.5, and 135.5 ng / mL, respectively.

[0402] PAM amidation activity (AMA, in ng / mL) was studied using both conformational and linearized peptide antibodies in different immunoassay settings. h) and PAM concentration (LIA, in ng / mL). The results showed that when two types of antibodies were used, the correlation was significant ( Figure 8). However, the correlation between AMA and PAM concentration (LIA) was significantly higher when PAM levels were measured in EDTA plasma samples using conformational antibodies (r=0.809, p<0.0001) compared to linear antibodies in the immunoassay setting (r=0.431, p=0.014). Statistical analysis of the data showed that the higher correlation coefficients and lower p-values ​​obtained for the ELISA setting using conformational antibodies compared to antibodies against linear epitopes for a cohort of comparable size indicate a more stable and significant relationship between the variables and enhance the confidence and validity of the results. For the immunoassay using antibodies against conformational epitopes, the correlation remained highly significant when measured in plasma in a large sub-cohort of n=4850 individuals of MPP, with r=0.71 (p<0.0001) ( Figure 9 ).

[0403] Example 4 – PAM activity assay

[0404] Human serum or Li-heparinized plasma from self-reported healthy volunteers was used as a source of human native PAM. Each sample (20 µl) was diluted two-fold in 100 mM Tris-HCl in duplicate. The amidation reaction was initiated by adding 160 µl of PAM reaction buffer (100 mM Tris-HCl, pH 7.5, 6.25 µM CuSO4, 2.5 mM L-ascorbic acid, 125 µg / mL catalase, 62.5 µM aprotinin, 250 µM leupeptin, 36 ng / mL synthetic ADM-Gly, and 375 µg / mL NT-ADM antibody). The 100 µl sample from each duplicate reaction was then combined and transferred to 20 µl of 200 mM EDTA to terminate the amidation reaction and generate a reaction time point of t = 0 minutes, followed by incubation at 37°C for 40 minutes. The unfinished reaction was then stopped with 10 µl of 200 mM EDTA. To determine PAM activity, the sphingotest® bio-ADM immunoassay ( Weber et al., 2017 ) quantifies bio-ADM, a reaction product, in each sample. The amidation assay is calibrated using a six-point calibration curve generated with a recombinant human PAM of known activity. Samples and calibrators are processed identically. The relative light units (RLU) measured for each sample using the sphingotest® bio-ADM immunoassay (t40 min - t0 min) are fitted to the RLU of the calibrator (t40 min - t0 min) to determine the PAM activity in the sample. PAM activity is described as "adrenomedullin maturation activity" (AMA) and is expressed as µg of bio-ADM formed per liter of sample per hour.

[0405] Figure 10 A typical PAM calibration curve is shown in . Figure 11 The distribution of AMA in Li-heparin samples from 120 self-reported healthy volunteers is shown. The median [IQR] of Li-heparin AMA was 18.4 µg / (L h) [13.5-21.9]. The 10th and 90th percentiles were 10.5 and 24.2 µg / (L, respectively). h). The 2.5th, 97.5th, and 99th percentiles were 8.1, 31.6, and 40.8 µg / (L, respectively). h).

[0406] Example 5 – Disease prediction in healthy subjects

[0407] According to Example 3, the concentration of PAM showed a strong correlation with its activity. Therefore, it can be used as an additional, simpler method to measure PAM levels in a high-throughput manner for clinical applications.

[0408] Study Cohort

[0409] The Malmö Prevention Project (MPP) was funded in the mid-1970s to explore CV risk factors in the general population and recruited 33,346 individuals living in Malmö ( Fedorowski et al., 2010. Eur Heart J 31: 85– 91 Between 2002 and 2006, a total of 18,240 original participants responded to the invitation (participation rate 70.5%) and underwent screening, which included a comprehensive physical examination and blood sample collection ( Fava et al. 2013. Hypertension 2013; 61: 319–26 In this study, the repeat examination in the MPP was considered baseline. Subjects with preexisting CVD at baseline were excluded. Informed consent was obtained from all participants, and the Ethics Committee of Lund University (Lund, Sweden) approved the study protocol (see Tables 3 and 4 for detailed patient characteristics).

[0410] Table 3: Baseline clinical characteristics of the analyzed subjects grouped according to the quartile (Q) of AMA at baseline.

[0411]

[0412] N / A: Not applicable

[0413] Table 4: Baseline clinical characteristics grouped by quartile (Q) of PAM concentrations measured by the PAM-LIA assay. na not applicable, ns not significant.

[0414]

[0415] Statistical Analysis: Values ​​are presented as mean and standard deviation, median and interquartile range (IQR), or count and percentage, as appropriate. Intergroup comparisons of continuous variables were performed with the use of the Kruskal-Wallis test. Biomarker data were log-transformed. Cox proportional hazards regression was used to analyze the effects of risk factors on survival in both univariate and multivariate analyses. The proportional hazards assumption was tested for all variables. For continuous variables, hazard ratios (HRs) were standardized to describe the HR for a biomarker change of one IQR. The 95% confidence intervals (CIs) and chi-square significance levels (Wald test) for risk factors are presented. The predictive value of each model was assessed using the model likelihood ratio chi-square statistic. The concordance index (C-index) was presented as an effect measure. It is equivalent to the area under the curve (AUC) used for binary outcomes. For multivariate models, a bootstrap-corrected version of the C-index is presented. Survival curves were constructed using the Kaplan-Meier method for illustrative purposes. To test the independence of PAM from clinical variables, we used the likelihood ratio chi-square test for nested models. All statistical tests were two-sided, and a two-sided p value of 0.05 was considered significant.

[0416] 5.2. Prediction of Alzheimer’s disease

[0417] A sample of 3716 individuals with information on dementia diagnoses (n = 169 patients with incident AD) was selected. Information on dementia diagnoses was requested from the Swedish National Patient Register (SNPR). Diagnoses in this register are collected according to International Classification of Diseases (ICD) codes, revisions 290, 293 (ICD-8), 290, 331 (ICD-9), or F00, F01, F03, G30 (ICD-10). The SNPR includes all inpatient care in Sweden since 1987 and also contains data on outpatient visits recorded after 2000, including day surgery and psychiatric care by both private and public caregivers. All-cause dementia was diagnosed according to the criteria of the Diagnostic and Statistical Manual of Mental Disorders (DSM)-III, while DSM-IV criteria were applied for the diagnosis of Alzheimer's disease and vascular dementia. Diagnoses were verified through a thorough review of medical records and available neuroimaging data. A study physician assigned a final diagnosis to each patient, and a geriatrician specializing in cognitive impairment was consulted in unresolved cases. PAM activity (AMA) and PAM concentration (LIA) were determined as described in Examples 4 and 3, respectively. When both measurements were applied, the incident AD group (n=169) in the MPP cohort had significantly lower PAM activity and PAM concentrations compared with the non-AD group (p=0.01). For analysis, the LIA cutoff value of 93.2 ng / mL (groups 1 and 2 included 2779 and 937 individuals, respectively) and the AMA cutoff value of 14.4 μg / L were used. h (Group 1 and Group 2 contain 2779 and 937 individuals, respectively). Figure 12 As shown in the results, the PAM-LIA assay had a higher predictive ability for incident AD (HR = 0.58 (0.41-0.82, p = 0.003)) compared with AMA (HR = 0.73 (0.51-1.02, p = 0.03)).

[0418] Example 6 – Prognosis and surveillance

[0419] 6.1. Study Cohort AdrenOSS-1 for AMA Measurement

[0420] AdrenOSS-1 is a European prospective observational study. 583 patients were enrolled in 24 centers in five countries (France, Belgium, the Netherlands, Italy, and Germany) between June 2015 and May 2016. The study protocol was approved by the local ethics committees and was conducted in accordance with the Declaration of Helsinki. The study enrolled patients aged 18 years and older who (1) were admitted to the ICU with sepsis or septic shock or (2) were transferred from another ICU with sepsis and septic shock within 24 hours of admission. Sepsis and organ failure were defined according to the 2001 definition ( Levy et al., 2003. 2001 SCCM / ESICM / ACCP / ATS / SIS International Sepsis Definitions Conference. Crit Care Med. 31(4):1250–6 ), the included patients were stratified by severe sepsis and septic shock. The term “sepsis” refers to the updated definition of Sepsis-3 ( Singer et al., 2016, Sepsis The Third International Consensus Definition of Septic Shock and Septic Shock Definitions for Sepsis and Septic Shock) (Sepsis-3), JAMA. 315(8):801–10 Patients were treated according to local practice, and treatments and procedures were registered. The primary endpoint was 28-day mortality. Secondary endpoints involved organ failure (as defined by the Sequential Organ Failure Assessment [SOFA] score) and organ support, use of vasopressors / inotropes, fluid balance, and use of renal replacement therapy (RRT).

[0421] After admission, demographics (age, sex), body mass index, presence of septic shock, type of ICU admission, organ dysfunction score (SOFA, Acute Physiology Assessment and Chronic Health Evaluation II [APACHE II]), origin of sepsis, prior comorbidities (ie, conditions treated within the past year), past medical history, laboratory values, and organ support were recorded, and blood was drawn for measurement of bio-ADM and other markers. After enrollment, the following data were collected daily during the first week: SOFA score, antimicrobial therapy, fluid balance, ventilatory status, Glasgow Coma Scale score, central venous pressure, need for RRT, invasive procedures for sepsis control, and vasopressor / inotropic therapy. In addition, discharge status and mortality were recorded on day 28 after ICU admission.

[0422] 6.2. Sepsis Study Cohorts Used for LIA Measurement

[0423] The sepsis cohort consisted of 12 individuals whose plasma was drawn while admitted to the ICU with overt sepsis.

[0424] 6.3. Self-reported health cohort

[0425] For both assays, the healthy cohort consisted of n = 98 (AMA) and n = 12 (LIA) individuals of varying age and sex with no history of overt disease.

[0426] 6.4. Outcome of Sepsis

[0427] AMA in the AdrenOSS-I subcohort (n=98) showed that compared with healthy individuals (AMA 17.0 μg / L h [SD=30.8]) compared with AMA (27.7 μg / L in the sepsis group h [SD = 55.8]) were significantly higher (p < 0.0001). (Similarly, PAM concentrations (LIA) were significantly lower in healthy individuals (96.6 ng / mL [SD = 11.5], n = 12) compared with the sepsis cohort (174.5 ng / mL [SD = 28.8]) (p < 0.001) ( Figure 13 Compared with the corresponding healthy cohort, the PAM concentration (LIA) was found to be 1.8-fold elevated in sepsis, whereas the AMA concentration was only 1.6-fold elevated. These results suggest that PAM-LIA is a suitable and convenient potential predictive method for sepsis.

[0428] Example 7: Protocol for Extracting Soluble PAM from Human Tissue Samples

[0429] Tissue samples were extracted as follows: 1 gram of liver or pituitary tissue was obtained. The frozen tissue sample was then ground into a fine powder using a cryogenic grinder or mortar and pestle, keeping the powder on dry ice throughout the process. The ground powder was dissolved in lysis buffer (50 mM Tris / HCl pH 7.4, 0.5% Triton, complete protease inhibitor cocktail) using a homogenizer or sonicator (0.5 cycles, 60% amplitude, 60 seconds). The mixture was centrifuged twice at 5,000 x g at 4°C for 5 minutes each to separate the soluble and insoluble fractions. After each centrifugation, the pellet was discarded, and the supernatant was collected for a final centrifugation at 20,000 x g at 4°C for 60 minutes. The supernatant, containing the extracted liver or pituitary protein, was transferred to a new container (e.g., a 1.5 mL Eppendorf tube). The supernatant was stored at -80°C until ready for use, and the container was appropriately labeled with the sample name, extraction date, and storage temperature.

[0430] Example 8: Antibodies against conformational epitopes of active PAM or active PAM subunits

[0431] As described in Example 2, antibodies were generated against the full-length PAM and subunits PHM and PAL. The antibodies were tested for binding to conformational or linear epitopes, respectively. Western blot analysis (as described in Example 2), ELISA, and co-elution HPLC were used to characterize the epitopes of the antibodies (see Table 5).

[0432] To confirm that the antibody binds to the conformational epitope by ELISA assay, the following experiments were performed:

[0433] Variant 1

[0434] Solid phase antibody coating As described in detail in Example 3, polystyrene microtiter plates were coated with antibodies against conformational or linear epitopes of PAL or PHM subunits and then blocked with 30 g / L Karion, 5 g / L BSA (protease-free), 6.5 mmol / L potassium dihydrogen phosphate, 3.5 mmol / L sodium dihydrogen phosphate (pH 6.5).

[0435] Protein linearization To linearize the full-length PAM, denature it by incubating with a 5% SDS solution and heating at 80°C for 10 minutes, or by treating it with a 3 M urea solution at room temperature for 1 hour. Subsequently, the linearized protein solution was diluted 200-fold with 1x PBS and then incubated with the solid-phase antibody. To verify that the diluted urea and SDS concentrations did not interfere with the binding of the native conformation protein, a concentration of 0.025% SDS and a concentration of 15 mM urea were added to the native full-length PAM. Binding efficiency was then assessed in a PAM immunoassay under standard conditions, as outlined in Example 4.

[0436] Testing of native conformation As described in Example 4, the native PAM conformation was confirmed by performing HPLC on 200 μg of purified full-length PAM protein and evaluating its elution profile and activity.

[0437] PAM immunoassay As described in Example 3, linearized full-length PAM or PAM in native conformation was incubated with solid-phase antibody at room temperature for 3 hours under standard assay conditions (including the corresponding tracer antibody).

[0438] result : The presence of 0.025% SDS and 15 mM urea in the diluent did not affect the binding of the full-length PAM in its native conformation under standard conditions. When using an antibody against the conformational epitope, no chemiluminescent signal was detected for the linearized full-length protein, indicating that the assay specifically recognizes the protein in its native conformation and not its linearized form. When using an antibody against the linear epitope, a chemiluminescent signal was detected for the linearized protein, but no signal was detected when using the conformational antibody. As expected, a chemiluminescent signal was detected for the linearized protein when using an antibody against the linear epitope, but no signal was detected when using the conformational antibody.

[0439] Variant 2:

[0440] Protein coating and blockingPolystyrene microtiter plates were coated with 2.5 μg of full-length PAM, PAL, or PHM subunits in their native conformation per well and then blocked as described for antibodies in Example 3 (omitting vacuum drying). To confirm that the coated proteins retained their native conformation, the amidation activity of full-length PAM was measured as described in Example 4: for PHM-coated proteins, 2.5 μg of PAL subunits were added to the reaction buffer; for PAL-coated proteins, 2.5 μg of PHM subunits were added to the reaction buffer and measured.

[0441] Protein linearization : Full-length PAM, PAL, or PHM subunit protein was linearized as described in Variant 1 and coated onto polystyrene microtiter plates using the coating method described above for native conformation proteins.

[0442] PAM immunoassay As detailed in Example 3, anti-PAL or anti-PHM tracer antibodies were applied to pre-coated polystyrene microtiter plates and incubated under standard PAM immunoassay conditions.

[0443] result : As with variant 1, no chemiluminescent signal was detected for the linearized full-length protein, confirming that the antibody against the conformational epitope is specific for the correctly folded protein over its linearized form.

[0444] As expected, the chemiluminescent signal of the linearized protein was detected when the antibody against the linear epitope was used, but not when the conformational antibody was used.

[0445] To confirm the antibody-antigen binding conformational epitope by analyzing the elution HPLC profile of the antibody-antigen complex, the following experiments were performed:

[0446] A total of 78 µg of full-length PAM in its native conformation or linearized form (linearized as described above) was incubated with 122 µg of an antibody that recognizes a conformational epitope of the PAL or PHM subunit. This incubation was performed in 500 µL of 1xPBS at room temperature for one hour. Similarly, 45 µg of native or linearized PAL or PHM subunits were incubated with 155 µg of the corresponding antibody against their conformational epitope using the same buffer and conditions. Following incubation, the mixture was applied to an HPLC column (Protein KW-803, Fa Shodex) at a flow rate of 0.5 mL / min for 30 minutes. The elution was fractionated and analyzed by SDS-PAGE. As controls, the antibody alone and individual PAL and PHM subunits were separated by HPLC and subjected to SDS-PAGE under the same conditions.

[0447] result For all tested antibodies targeting conformational epitopes, stable antibody-antigen complex formation was confirmed by elution HPLC profiles and SDS-PAGE analysis of elution fractions, whereas this was not the case for the linearized forms. As shown in Figure 14, chromatographic profiles of both the PAL and PHM subunits and their specific conformational antibodies were analyzed using size exclusion chromatography to estimate the molecular weights of the subunit-antibody complexes. The PAL subunit, with a molecular weight of 42 kDa, exhibited a major peak at 16.56 minutes (Figure 14A). The antibody targeting the conformational PAL epitope, with an expected molecular weight of 150 kDa, exhibited a major elution peak at 11.91 minutes (Figure 14B). After mixing the PAL subunits with their corresponding antibodies, the elution profiles exhibited a shifted peak at 11.02 minutes, corresponding to the PAL-antibody complex, and an additional peak at 16.96 minutes, representing free antibody not involved in complex formation (Figure 14C).

[0448] Likewise, the individual 41 kDa PHM subunit was characterized as a peak at 16.80 minutes ( FIG. 14D ). The elution peak of an antibody directed against the conformational PHM epitope was at 11.93 minutes ( FIG. 14E ).

[0449] After mixing the PHM subunits with their corresponding antibodies, the elution profile showed a major shifted peak at 11.49 min and a separate peak at 16.85 min corresponding to excess unbound antibody ( FIG. 14F ).

[0450] like Figure 15 The eluted fractions were analyzed by SDS-PAGE as shown in . The presence of bands corresponding to individual PAL or PHM subunits and the heavy and light chains of the antibody in the antigen-antibody complex fractions indicated successful complex formation, as demonstrated in lanes 5 and 6.

[0451] Table 5: Summary of antibodies developed against different PAM antigens and conformational epitope binding assays.

[0452]

[0453] sequence

[0454] SEQ ID No. 1 - Prepro-PAM isomer 1 AS 1-973

[0455]

[0456] SEQ ID No. 2 - Prepro-PAM isomer 2 AS 1-868

[0457]

[0458] SEQ ID No. 3 - Prepro-PAM isoform 3 AS (amino acids 829-896 of SEQ ID No. 1 deleted)

[0459]

[0460] SEQ ID No. 4 - Prepro-PAM isoform 4 (amino acids 829-914 of SEQ ID No. 1 deleted)

[0461]

[0462] SEQ ID No. 5 - Prepro-PAM isoform 5 (isoform 1 with an extra aa in position 896)

[0463]

[0464] SEQ ID No. 6 - Prepro-PAM isoform 6 (amino acids 897-914 of SEQ ID No. 1 deleted)

[0465]

[0466] SEQ ID No. 7 – PHM subunit of PAM

[0467]

[0468] SEQ ID No. 8 - PAL subunit of PAM

[0469]

[0470] SEQ ID No. 9 - Human serum albumin signal sequence

[0471]

[0472] SEQ ID No. 10 - Sequence of recombinant human PAM

[0473]

[0474] SEQ ID No. 11 - Peptide 1 (aa 42-56 of PAM SEQ ID No. 1)

[0475]

[0476] SEQ ID No. 12 - Peptide 2 (aa 109-128 of PAM SEQ ID No. 1)

[0477]

[0478] SEQ ID No. 13 - Peptide 3 (aa 168-180 of PAM SEQ ID No. 1)

[0479]

[0480] SEQ ID No. 14 - Peptide 4 (aa 204-216 of PAM SEQ ID No. 1)

[0481]

[0482] SEQ ID No. 15 - Peptide 5 (aa 329-342 of PAM SEQ ID No. 1)

[0483]

[0484] SEQ ID No. 16 - Peptide 6 (aa 291-310 of PAM SEQ ID No. 1)

[0485]

[0486] SEQ ID No. 17 - Peptide 7 (aa 234-244 of PAM SEQ ID No. 1)

[0487]

[0488] SEQ ID No. 18 - Peptide 8 (aa 261-276 of PAM SEQ ID No. 1)

[0489]

[0490] SEQ ID No. 19 - Peptide 9 (aa 530-557 of PAM SEQ ID No. 1)

[0491]

[0492] SEQ ID No. 20 - Peptide 10 (aa 611-631 of PAM SEQ ID No. 1)

[0493]

[0494] SEQ ID No. 21 - Peptide 11 (aa 562-579 of PAM SEQ ID No. 1)

[0495]

[0496] SEQ ID No. 22 - Peptide 12 (aa 745-758 of PAM SEQ ID No. 1)

[0497]

[0498] SEQ ID No. 23 - Peptide 13 (aa 669-687 of PAM SEQ ID No. 1)

[0499]

[0500] SEQ ID No. 24 - Peptide 14 (aa 710-725 of PAM SEQ ID No. 1)

[0501]

[0502] SEQ ID No. 25 – PHM fragment (aa 31-377 of PAM SEQ ID No. 1)

[0503]

Claims

1. A method for determining the level of peptidylglycine alpha-amidating monooxygenase (PAM) and / or its isomers and / or fragments in a body fluid or tissue sample using an assay, wherein the assay comprises at least one binding agent directed against a conformational epitope of PAM.

2. A method for determining the level of peptidylglycine α-amidating monooxygenase (PAM) and / or its isomers and / or fragments in a body fluid or tissue sample according to claim 1, wherein the assay comprises two binding agents that bind to two different regions of PAM, wherein each of the two binding agents is directed against a conformational epitope of PAM.

3. The method for determining the level of peptidylglycine α-amidating monooxygenase (PAM) and / or its isomers and / or fragments in a body fluid or tissue sample according to claim 2, wherein the first of the two binding agents binds to a conformational epitope contained within the PHM subunit of PAM (SEQ ID No. 7), and the second of the two binding agents binds to a conformational epitope contained within the PAL subunit of PAM (SEQ ID No. 8).

4. A method for determining the level of peptidylglycine α-amidating monooxygenase (PAM) and / or its isomers and / or fragments in a body fluid or tissue sample according to claim 2 or 3, wherein each of the two binding agents is directed against an epitope contained within the following sequences of PAM: SEQ ID No. 25 (PHM, amino acids 31-377 of SEQ ID No. 1) and SEQ ID No. 8 (PAL, amino acids 495-817 of SEQ ID No. 1) of human PAM.

5. The method for determining the level of peptidylglycine α-amidating monooxygenase (PAM) and / or its isomers and / or fragments in a body fluid or tissue sample according to any one of claims 1 to 4, wherein the conformational epitope has at least 4 amino acids, preferably at least 5 amino acids.

6. A method for determining the level of peptidylglycine alpha-amidating monooxygenase (PAM) and / or its isomers and / or fragments in a body fluid or tissue sample according to any one of claims 1 to 5, wherein the binding agent does not bind to denatured PAM or denatured PAM subunits (e.g., PAL or PHM).

7. A method for determining the level of peptidylglycine alpha-amidating monooxygenase (PAM) and / or its isomers and / or fragments in a body fluid or tissue sample according to claims 1 to 6, wherein the binding agent does not bind to denatured PAM or denatured PAM subunits (e.g. PAL or PHM) when using Western blotting techniques.

8. A method for determining the level of peptidylglycine α-amidating monooxygenase (PAM) and / or its isomers and / or fragments in a body fluid or tissue sample according to any one of claims 1 to 7, wherein the binding agent against a conformational epitope of PAM binds to an enzymatically active PAM or an enzymatically active PAM subunit (e.g., PAL or PHM), but does not bind to an enzymatically inactive PAM or an enzymatically inactive PAM subunit (e.g., PAL or PHM).

9. The method for determining the level of peptidylglycine alpha-amidating monooxygenase (PAM) and / or its isomers and / or fragments in a body fluid or tissue sample according to any one of claims 1 to 8, wherein the at least one binding agent is selected from an antibody, an antibody fragment or a non-IgG scaffold.

10. A method for determining the level of peptidylglycine alpha-amidating monooxygenase (PAM) and / or its isomers and / or fragments in a body fluid or tissue sample according to any one of claims 1 to 9, wherein the at least one binding agent is produced using large fragment proteins, full-length proteins or DNA immunoassay techniques.

11. A method for diagnosing or prognosing a disease in a patient and / or predicting the risk of a patient developing a disease or experiencing an adverse event and / or monitoring a disease or adverse event in a patient, said method being performed by determining the level of peptidylglycine α-amidating monooxygenase (PAM) and / or its isomers and / or fragments thereof in a body fluid or tissue sample of the patient according to any one of claims 1 to 10. wherein the disease in the patient is selected from the group comprising dementia, cardiovascular disorders, kidney diseases, cancer, inflammatory or infectious diseases and / or metabolic diseases, The adverse events are selected from the group consisting of cardiac events, cardiovascular events, cerebrovascular events, cancer, diabetes, infection, serious infection, sepsis-like systemic infection, sepsis, and all-cause mortality.

12. A method for diagnosing or prognosing a disease in a patient and / or predicting the risk of a patient suffering from a disease or experiencing an adverse event and / or monitoring a patient's disease or adverse event, said method being performed by determining the level of peptidylglycine α-amidating monooxygenase (PAM) and / or its isomers and / or fragments thereof in a body fluid or tissue sample of said patient according to any one of claims 1 to 11, said method comprising the following steps: Determining the level of PAM and / or its isomers and / or fragments thereof in a body fluid or tissue sample of said patient, comparing the measured amount with a predetermined threshold value, wherein if the measured amount is below or above the predetermined threshold, the patient is diagnosed as having a disease, or wherein if said measured amount is below or above said predetermined threshold, the outcome of said disease is prognosed, or wherein if the measured amount is below or above the predetermined threshold, the risk of the patient developing a disease or experiencing an adverse event is predicted, or wherein the patient is monitored for disease or adverse events.

13. The method according to claims 1 and 12, wherein the level of PAM and / or its isomers and / or fragments thereof is the total concentration of PAM and / or its isomers and / or fragments thereof having at least 12 amino acids in the body fluid or tissue sample of the patient.

14. The method according to any one of claims 1 to 13, wherein the total concentration of the PAM and / or its isomers and / or fragments thereof having at least 12 amino acids is detected using an immunoassay.

15. A method for diagnosing or prognosing a disease in a patient and / or predicting the risk of a patient developing a disease or experiencing an adverse event and / or monitoring a patient for a disease or an adverse event, said method being performed by determining the level of a PAM and / or its isomers and / or fragments thereof in a body fluid or tissue sample of said patient according to any one of claims 1 to 14, wherein said PAM and / or its isomers and / or fragments thereof are selected from the group consisting of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8 and SEQ ID No.

10.

16. A method for diagnosing or prognosing a disease in a patient and / or predicting the risk of a patient developing a disease or experiencing an adverse event and / or monitoring a patient for a disease or adverse event, said method being performed by determining the level of a PAM and / or its isomers and / or fragments thereof in a body fluid or tissue sample of said patient according to any one of claims 1 to 15, wherein the risk of the patient developing a disease is determined, wherein said patient is a healthy patient.

17. The method of claim 16, wherein the disease is selected from the group consisting of Alzheimer's disease, colorectal cancer, and pancreatic cancer.

18. A method for determining the level of peptidylglycine alpha-amidating monooxygenase (PAM) and / or its isomers and / or fragments in a body fluid or tissue sample according to any one of claims 1 to 17, wherein the level is the level of active PAM.

19. Use of an antibody for determining the level of PAM and / or its isomers and / or fragments thereof, wherein the antibody is directed against a conformational epitope contained within the following sequences of PAM: PHM fragment (amino acids 31-377 of PAM) (SEQ ID No. 25) and / or PAL fragment (amino acids 495-817 of PAM) (SEQ ID No. 8).

20. Use of the antibody according to claim 19 for determining the level of PAM and / or its isomers and / or fragments thereof, wherein the antibody does not bind to denatured PAM or denatured PAM subunits (such as PAL or PHM).

21. Use of the antibody according to claims 19 and 20 for determining the level of PAM and / or its isomers and / or fragments thereof, wherein the antibody directed against a conformational epitope of PAM binds to enzymatically active PAM or an enzymatically active PAM subunit (e.g. PAL or PHM), but does not bind to inactive PAM or an inactive PAM subunit (e.g. PAL or PHM).

22. A kit for determining the level of PAM and / or its isoforms and / or fragments thereof, comprising one or more antibodies that bind to PAM, said antibodies being directed against a conformational epitope of PAM contained in the following sequences: PHM fragment (amino acids 31-377 of PAM) (SEQ ID No. 25) and / or PAL fragment (amino acids 495-817 of PAM) (SEQ ID No. 8).

23. A kit for determining the level of PAM and / or its isoforms and / or fragments thereof according to claim 22, comprising one or more antibodies that bind to PAM, wherein the antibodies do not bind to denatured PAM or denatured PAM subunits (e.g., PAL or PHM).

24. A kit for determining the level of PAM and / or its isomers and / or fragments thereof according to claims 22 and 23, comprising one or more antibodies that bind to PAM, wherein the antibodies directed against a conformational epitope of PAM bind to enzymatically active PAM or an enzymatically active PAM subunit (e.g., PAL or PHM), but do not bind to inenzymatically inactive PAM or an inenzymatically inactive PAM subunit (e.g., PAL or PHM).

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