A combination of a free N-terminus-binding anti-ADM antibody and vitamin C to promote the transfer from ADM-Gly to bio-ADM in patients with an ADM-Gly / bio-ADM ratio exceeding a threshold.

Abstract

This provides an anti-ADM antibody that binds to the free N-terminus to promote the transfer from ADM-Gly to bio-ADM. [Solution] An anti-adrenomedullin (ADM) antibody or fragment or anti-ADM non-Ig scaffold used to promote the conversion of ADM-Gly to ADM-NH2 in circulating ADM-Gly in the treatment of a disease or condition selected from the group including severe infection, shock, acute heart failure, myocardial infarction, stroke, organ dysfunction, or dementia, characterized in that it binds to the N-terminal and / or central region (amino acids 1-42) of ADM-Gly and / or ADM-NH2, and the ratio of pro-adrenomedullin or its fragment to ADM-NH2 in a patient body fluid sample exceeds a certain threshold.

Description

[Technical Field]

[0001] The subject of the present invention is an anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold, which is used in a patient to promote the conversion from ADM-Gly to ADM-NH2, the patient being characterized in that the ratio of pro-adrenomedullin or its fragment to ADM-NH2 (SEQ ID NO: 20) in a body fluid sample exceeds a certain threshold, the pro-adrenomedullin or its fragment is selected from the group consisting of PAMP (SEQ ID NO: 32), MR-proADM (SEQ ID NO: 33), ADM-Gly (SEQ ID NO: 21), and CT-proADM (SEQ ID NO: 34), the anti-ADM antibody or anti-ADM fragment or anti-ADM non-Ig scaffold binds to the N-terminal and / or central region (amino acids 1-42) of ADM-Gly and / or ADM-NH2: YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVA (SEQ ID NO: 23). [Background technology]

[0002] The peptide adrenomedullin (ADM) was first described in 1993 as a novel antihypertensive peptide containing 52 amino acids (Kitamura et al., 1993. Biochem Biophys Res Comm 192 (2): 553-560), and was isolated from a human pheochromocytoma cell line (SEQ ID NO: 20). In the same year, a cDNA encoding a precursor peptide containing 185 amino acids, and the entire amino acid sequence of this precursor peptide, were also described. In particular, the precursor peptide containing a 21-amino acid signal sequence at the N-terminus is called "pre-proadrenomedullin" (pre-proADM). In this specification, all identified amino acid positions are usually associated with pre-proADM containing 185 amino acids. Subsequently, pre-proADM is converted to pro-ADM (SEQ ID NO: 31) containing 164 amino acids by cleavage of the N-terminal signal peptide. pro-ADM is further processed to obtain the 20 peptides at the N-terminus of pro-ADM (PAMP; SEQ ID NO: 32), the central region pro-ADM (MR-proADM; SEQ ID NO: 33), the adrenotensin pro-ADM153-185 (CT-proADM; SEQ ID NO: 34), and immature ADM, as well as C-terminal glycine-extended ADM (ADM-Gly; SEQ ID NO: 21). This is then converted to the mature, bioactive form of ADM (Bio-ADM; ADM-NH2; SEQ ID NO: 20) by enzymatic amidation of its C-terminus. More than half of known neuropeptides and endocrine peptides require the formation of a C-terminal α-amide group to achieve full bioactivity (Guembe et al. 1999. J Histochem Cytochem 47(5): 623-36; Vishwanatha et al. 2013. Handbook of Biologically Active Peptides Peptidylglycine Amidating Monoxygenase (PAM). Second Edi. Elsevier Inc.). This final step in peptide hormone biosynthesis involves the action of peptidylglycine α-amide-forming monooxygenase (PAM), a bifunctional enzyme that specifically recognizes the C-terminal glycine (CT-Gly) residue in its substrate.PAM cleaves glyoxylic acid from the peptide CT-Gly residue in a two-step enzymatic reaction, leading to the production of an α-amidated peptide hormone at the C-terminus, with the resulting α-amide group derived from the cleaved CT-Gly (Prigge et al. 2000. Cellular and Molecular Life Sciences 57(8): 1236-59). This amidation reaction occurs in the lumen of the secretory granules prior to the exocytosis of the amidation product (Martinez et al. 1996. Am J Pathol 149(2): 707-16).

[0003] In humans, the PAM gene is located on chromosome 5q21.1, a 160kb long chromosome containing 25 known exons (Gaier et al. 2014. BMC Endocrine Disorders 14). At least six isoforms are known to be generated by alternative splicing (SEQ ID NOs. 39-44). The PAM enzyme is expressed at different concentrations in almost all mammalian cell types and has been found to be prominently expressed in airway epithelium, endothelial cells, ependymal cells in the brain, adult atria, brain, kidneys, 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] The longest known PAM isoform 1 (SEQ ID NO: 39) is the precursor protein (amino acids 1-973). The N-terminal signal sequence (amino acids 1-20) ensures that the developing PAM polypeptide is guided into the secretory lumen of the endoplasmic reticulum, where it is subsequently cleaved co-translationally. The PAM-pro-peptide is then processed by the same mechanism used in the biosynthesis of membrane-bound and secretory proteins, including cleavage of the pro region (amino acids 21-30) to ensure proper folding, disulfide bond formation, phosphorylation, and glycosylation (Bousquet-Moore et al. 2010. J Neurosci Res 88(12):2535-45). The PAM cDNA encodes two distinct enzymatic activities. The first enzymatic activity, named peptidylglycine α-hydroxylated monooxygenase (PHM; EC1.14.17.3), is an enzyme that catalyzes the conversion of the C-terminal glycine residue to α-hydroxyglycine. The second activity, named peptidyl α-hydroxyglycine α-amidate lyase (PAL; EC4.3.2.5), is an enzyme that catalyzes the conversion of α-hydroxyglycine to α-amide, subsequently releasing glyoxylic acid. The combined action of these separate enzymatic activities results in the overall peptidylglycine α-amidate activity. The first enzymatic activity (PHM) is located directly upstream of the pro region (within amino acids 31-494 of isoform 1 (SEQ ID NO: 45)). The second catalytic activity (PAL) is located after exon 16 in isoform 1 (within amino acids 495-817 (SEQ ID NO: 46)).

[0005] Both activities may be encoded together within one polypeptide as membrane-bound proteins (isoforms 1, 2, 5, and 6; corresponding to SEQ ID NOs. 39, 40, 43, and 44) ​​and within one polypeptide as soluble proteins lacking a transmembrane domain (TMD; isoforms 3 and 4; corresponding to SEQ ID NOs. 41 and 42). Isoforms 1, 2, 5, and 6 remain within the outer plasma membrane after fusion of secretory vesicles with the plasma membrane and subsequent intracellular uptake and regeneration or degradation, while the TMD-lacking soluble PAM isoforms (isoforms 3 and 4) (amino acids 864-887) are co-secreted with peptide hormones (Wand et al. 1985 Metabolism 34(11): 1044-52). Furthermore, prohormone-converting enzymes can convert membrane-bound PAM proteins to soluble PAM proteins by cleaving them within the flexible region (exons 25 / 26) that links PAL to TMD in the secretory pathway (Bousquet-Moore et al. 2010. J Neurosci Res 88(12):2535-45):2535-45). The PHM subunit may also be cleaved from soluble or membrane-bound PAM in the secretory pathway by prohormone-converting enzymes that process the double basal cleavage site in the exon 16 region. Furthermore, during intracellular uptake, full-length PAM proteins may also be converted to a soluble form by the action of α- and γ-secreting enzymes (Bousquet-Moore et al. 2010. J Neurosci Res 88(12):2535-45). Membrane-bound PAM derived from late endosomes may be further secreted in the form of exosomal vesicles.

[0006] The activity of PHM and PAL, as well as the activity of full-length PAM, has been determined in several human tissues and body fluids. However, the individual PHM and PAL activities in soluble forms lead to the formation of C-terminal α-amidate products from C-terminal glycinated substrates, provided that these individual reactions can be performed in the same compartment, body fluid, or in vitro experimental apparatus. How the transfer of PHM hydroxylated products to PAL occurs is not currently precisely understood. There is evidence that the hydroxylated products are released into solution and are not directly transferred from PHM to PAL (Yin et al. 2011. PLoS One 6(12):e28679). The source of PAM in the circulating system is also currently unknown.

[0007] PHM is a copper-dependent monooxygenase responsible for the stereospecific hydroxylation of C-terminal glycine at the α-carbon atom. In the hydroxylation reaction, ascorbate is considered to be a naturally occurring reducing agent, but it has been found that the oxygen in the newly formed hydroxyl group originates from molecular oxygen. The catalytic activity of PAL involves the abstraction of a proton from hydroxyglycine forming PHM by a base derived from the protein backbone, and nucleophilic attack of the oxygen of the hydroxyl group on a divalent metal, resulting in the cleavage of glyoxylic acid and the formation of the C-terminal amide.

[0008] Therefore, the terms “amidation activity,” “α-amidation activity,” “peptidylglycine α-amidation activity,” or “PAM activity” refer to the sequential enzymatic activity of PHM and PAL, but are independent of the splice variants or mixtures of splice variants of the present invention, or the post-translational modified PAM enzymes or the individual soluble PHM or PAL activity, or membrane-bound PAL or combinations thereof in all descriptive forms leading to the formation of peptide or non-peptide-like α-amidation products from soluble PHM and peptide or non-peptide-like glycinated substrates. In other words, the terms “amidation activity,” “α-amidation activity,” “peptidylglycine α-amidation activity,” or “PAM activity” may be described independently of the splice variants or mixtures of splice variants of the present invention as the sequential action of enzymatic activity located within amino acids 31-817 in the propeptide encoded by human PAM cDNA.

[0009] The discovery and characterization of ADM in 1993 spurred intensive research, the results of which are summarized in various review articles cited within the context of this specification, with particular reference to the "peptide" literature specifically on ADM (Takahashi 2001. Peptides 22: 1691; Eto 2001. Peptides 22: 1693-1711). Further reviews include those by Hinson et al. in 2000 (Hinson et al. 2000. Endocrine Reviews 21(2): 138-167). Scientific studies to date have shown that ADM may be considered a multifunctional regulatory peptide. As mentioned above, it is released into the circulating system in an inactive form amplified by glycine (Kitamura et al. 1998. Biochem Biophys Res Commun 244(2): 551-555). There are also binding proteins that are specific to ADM and are thought to similarly modulate the effects of ADM (Pio et al. 2001. The Journal of Biological Chemistry 276(15): 12292-12300). To date, the most important physiological effect of ADM and PAMP has been their effect on blood pressure.

[0010] Therefore, ADM is an effective vasodilator, and its antihypertensive effect can be attributed to a specific peptide compartment in the C-terminal region of ADM. Furthermore, the aforementioned bioactive peptide PAMP, generated from pre-pro-ADM, appears to have a different mechanism of action than ADM, but has been found to exhibit a similar antihypertensive effect (see also Eto et al. 2001 and Hinson et al. 2000, Kuwasaki et al. 1997. FEBS Lett 414(1): 105-110; Kuwasaki et al. 1999. Ann. Clin. Biochem. 36: 622-628; Tsuruda et al. 2001 Life Sci. 69(2): 239-245 and EP-A2 0 622 458 in addition to the review above). Furthermore, it has been found that ADM concentrations measurable in circulating fluids and other bodily fluids are significantly higher in some pathological conditions than in healthy control subjects. Therefore, ADM concentrations are significantly increased, to varying degrees, in patients with congestive heart failure, myocardial infarction, kidney disease, hypertensive disease, diabetes, the acute phase of shock, and sepsis and septic shock. PAMP concentrations are also increased in some of the aforementioned pathological conditions, although their plasma concentrations are lower compared to ADM (Eto 2001. Peptides 22: 1693-1711). High concentrations of ADM have been observed in sepsis, and the highest concentrations have been reported in septic shock (Eto 2001. Peptides 22: 1693-1711; Hirata et al. Journal of Clinical Endocrinology and Metabolism 81(4): 1449-1453; Ehlenz et al. 1997. Exp Clin Endocrinol Diabetes 105: 156-162; Tomoda et al. 2001. Peptides 22: 1783-1794; Ueda et al. 1999. Am. J. Respir. Crit. Care Med. 160: 132-136; Wang et al. 2001. Peptides 22: 1835-1840).Furthermore, plasma concentrations of ADM are elevated in heart failure patients and correlate with disease severity (Hirayama et al. 1999. J Endocrinol 160: 297-303; Yu et al. 2001. Heart 86: 155-160). High plasma ADM is an independent negative prognostic indicator in these patients (Poyner et al. 2002. Pharmacol Rev 54: 233-246).

[0011] Kitamura et al. reported that the concentrations of mature ADM and ADM-Gly were significantly elevated in the plasma of hypertensive patients compared to healthy volunteers (Kitamura et al. 1998. Biochem Biophys Res Comm 244(2): 551-5). In both groups, mature ADM was much lower than ADM-Gly. However, there was no significant difference in the ratio of mature ADM to ADM-Gly between hypertensive and non-hypertensive patients.

[0012] In the early stages of sepsis, ADM has been reported to improve cardiac function as well as blood supply to the liver, spleen, kidneys, and small intestine. Anti-ADM neutralizing antibodies neutralize these effects during the early stages of sepsis (Wang et al. 2001. Peptides 22: 1835-1840). In other diseases, inhibition of ADM may be beneficial to some extent. However, complete neutralization of ADM may be harmful because some physiological functions may require a certain amount of ADM. Many reports emphasize that ADM administration may be beneficial for certain diseases. In contrast, other reports state that ADM is life-threatening when administered under certain conditions.

[0013] WO2013 / 072510 discloses a non-neutralizing N-terminal anti-ADM antibody for use in the treatment of severe chronic or acute diseases or acute symptoms in patients to reduce the patient's risk of death.

[0014] WO2013 / 072511 discloses a non-neutralizing N-terminal anti-ADM antibody for use in the treatment of chronic or acute diseases or acute symptoms in patients for the prevention or reduction of organ dysfunction or organ failure.

[0015] WO2013 / 072513 discloses an N-terminal anti-ADM antibody used to treat acute illnesses or symptoms in patients in order to stabilize the circulatory system.

[0016] WO2013 / 072514 discloses an N-terminal anti-ADM antibody for regulating fluid balance in patients with chronic or acute diseases or acute symptoms.

[0017] WO2019 / 154900 discloses a non-neutralizing N-terminal anti-ADM antibody for use in the treatment and prevention of dementia. Furthermore, WO2019 / 154900 discloses a method for diagnosing dementia and monitoring its (preventive) treatment by measuring the ratio of the concentration of mature ADM to the concentration of proadrenomedullin or its fragments.

[0018] WO2013 / 072512 concerns the half-life (t) of adrenomedullin in serum, blood, and plasma. 1 / 2 This document discloses a non-neutralizing N-terminal anti-ADM antibody, which is an ADM-stabilizing antibody that enhances the half-retention time.

[0019] The efficacy of non-neutralizing antibodies targeting the N-terminus of ADM was investigated in survival studies of CLP-induced sepsis in mice. Preliminary treatment with non-neutralizing antibodies reduced catecholamine infusion, improved renal dysfunction, and improved overall survival (Struck et al. 2013. Intensive Care Med Exp 1(1):22; Wagner et al. 2013. Intensive Care Med Exp 1(1):21). Furthermore, antibodies against the central region of ADM (MR-ADM antibodies) also significantly improved survival in mice with CLP-induced sepsis, although to a lesser extent than N-terminal anti-ADM antibodies (Struck et al. 2013. Intensive Care Med Exp 1(1):22).

[0020] These positive results led to the development of a humanized N-terminal anti-ADM antibody, named adrecizumab, for further clinical application. The beneficial effects of adrecizumab on vascular barrier function and survival have recently been demonstrated in preclinical models of systemic inflammation and sepsis (Geven et al. 2018. Shock 50(6): 648-654). In this study, pretreatment with adrecizumab reduced renal vascular leakage in rats with endotoxemia and mice with CLP-induced sepsis, consistent with increased renal expression of the protective peptide Ang-1 and decreased expression of the harmful peptide vascular endothelial growth factor. Furthermore, pretreatment with adrecizumab improved the 7-day survival rate in mice with CLP-induced sepsis by 10–50% with a single dose and 0–40% with repeated doses. Furthermore, Phase I studies demonstrated excellent safety and tolerability as follows: no serious adverse events were observed, no signals of adverse events more frequently occurring in subjects treated with adrecizumab were detected, and no changes related to other safety parameters were found (Geven et al. 2017. Intensive Care Med Exp 5 (Suppl 2): ​​0427). Of particular interest is the disclosed mechanism of action of adrecizumab. Data from both animals and humans reveal a potent dose-dependent increase in circulating ADM after administration of this antibody. Based on pharmacokinetic data and the lack of increase in MR-proADM (an inactive peptide fragment derived from the same prohormone as ADM), the higher circulating ADM concentrations cannot be explained by increased production.

[0021] Although ADM is small enough to cross the endothelial barrier, its antibodies are not, so the mechanistic explanation for this increase may be that excess antibodies in the circulatory system may efflux ADM from the interstitium into the circulatory system (Geven et al. 2018. Shock. 50(2): 132-140). Furthermore, antibody binding to ADM leads to an extension of ADM's half-life. NT-ADM antibodies partially inhibit ADM-mediated signaling, but a significant increase in circulatory ADM leads to an overall "net" increase in ADM activity in blood compartments, where this increase has beneficial effects on endothelial cells (ECs; primarily barrier stabilization) while the harmful effects of ADM on interstitium vascular smooth muscle cells (VSMCs; vasodilatory effects) are reduced.

[0022] Vitamin C (ascorbic acid) is a water-soluble vitamin with various antioxidant, anti-inflammatory, and microvascular effects. Vitamin C levels are known to decrease in serious illnesses and are associated with disease severity. Vitamin C supplementation has shown promise in both animal models of sepsis and human trials in intensive care unit (ICU) settings, as follows: Early preclinical studies of sepsis have shown that vitamin C activates and isolates neutrophils in the lungs, thereby preventing the surge of sepsis-induced cytokines that damage alveolar capillaries (Fisher et a. 2012. Physiol Lung Cell Mol Physiol. 303(1): L20-L32; Fisher et al. 2011. Crit Care Med. 39 (6): 1454-1460). Vitamin C prevented the activation of neutrophil accumulation in the alveolar space, limited damage to alveolar epithelial waterways, and promoted increased expression of these vitamins, thereby increasing alveolar fluid drainage (Fisher et a. 2012. Physiol Lung Cell Mol Physiol. 303(1): L20-L32; Fisher et al. 2011. Crit Care Med. 39 (6): 1454-1460). Furthermore, vitamin C prevented the formation of extracellular trapping of neutrophils and the biological events of activated neutrophils that promote vascular damage (Mohammed et al. 2013 Nutrients 5(8): 3131-3151).

[0023] In a recent phase I study of 24 patients in the medical intensive care unit (ICU) with severe sepsis, high-dose administration of vitamin C reduced the degree of multiple organ dysfunction and decreased the concentration of circulatory system injury biomarkers (Fowler et al. 2014. J Transl Med 12: 32). In a double-blind randomized clinical trial enrolling 28 adult surgical patients with septic shock, compared with the placebo group, ascorbic acid treatment significantly reduced the required dose of vasopressors, accelerated vasopressor weaning, and significantly decreased 28-day mortality (Zhabet et al. 2016. Res Pharm Pract 5: 94-100). Furthermore, intravenous vitamin C therapy reduced 28-day mortality in patients with sepsis from 46% in the placebo group to approximately 30% in the vitamin C group (Fowler et al. 2019. JAMA 322(13):1261-1270).

[0024] Surprisingly, while the present invention demonstrates that the ratio of ADM-Gly to bioactive ADM is highly variable, current literature discloses that only 5-20% of ADM is ADM-Gly (Kitamura et al. 1998. Biochemical and Biophysical Research Communications. 244(2): 551-555). Furthermore, our data are the first to show that, despite the conventional explanation that peptide amidation, particularly of ADM, is an intracellular rather than intravascular process, the conversion from ADM-Gly to bioactive ADM also occurs within the circulatory system (Kumar, Mains, Eipper 2016. Journal of Molecular Endocrinology. 56(4): T63-T76). In addition to the effects described above, the present invention also clearly demonstrates that anti-ADM antibodies in the N-terminal and central regions promote the conversion of ADM-Gly to bioactive ADM in critically ill patients with acute diseases or conditions. Surprisingly, antibodies that bind to the central region of ADM exhibit a stronger effect than antibodies that bind to the N-terminal region of ADM and / or ADM-Gly. Furthermore, this invention clearly demonstrates that these N-terminal and central anti-ADM antibodies, combined with ascorbate, significantly increase the enzymatic amidation of glycinated adrenomedullin to mature ADM catalyzed by PAM in plasma. [Overview of the project]

[0025] Detailed description of the present invention The subject of the present application is an anti-adrenomedullin (ADM) antibody, an anti-adrenomedullin antibody fragment or an anti-ADM non-Ig scaffold for use in promoting the conversion of ADM-Gly to ADM-NH2 from circulating ADM-Gly in a severely ill patient suffering from an acute disease or an acute symptom selected from the group including severe infectious diseases (such as meningitis, systemic inflammatory response syndrome (SIRS), sepsis, etc.), shock (such as septic shock, cardiogenic shock, etc.), acute heart failure (including acute decompensated heart failure, chronic heart failure accompanied by worsening symptoms and signs), myocardial infarction, stroke, organ dysfunction (such as kidney, liver, heart, lung, etc.), or dementia, wherein the patient is characterized in that the ratio of pro-adrenomedullin or a fragment thereof to ADM-NH2 (SEQ ID NO: 20) in a body fluid sample exceeds a specific threshold, said pro-adrenomedullin or fragment thereof is selected from the group consisting of PAMP (SEQ ID NO: 32), MR-proADM (SEQ ID NO: 33), ADM-Gly (SEQ ID NO: 21), and CT-proADM (SEQ ID NO: 34), and said anti-ADM antibody, anti-ADM fragment or anti-ADM non-Ig scaffold binds to the N-terminal portion and / or the central region (amino acids 1 to 42): YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVA (SEQ ID NO: 23) of ADM-Gly and / or ADM-NH2.

[0026] In one embodiment, an anti-adrenomedullin (ADM) antibody, anti-adrenomedullin antibody fragment, or anti-ADM non-Ig scaffold is used to promote the conversion of ADM-Gly to ADM-NH2 in circulating ADM-Gly within the patient, in the treatment of a critically ill patient suffering from an acute disease or acute symptoms selected from the group including severe infection (meningitis, systemic inflammatory response syndrome (SIRS), sepsis, etc.), shock (septic shock, cardiogenic shock, etc.), acute heart failure (acute decompensated heart failure, chronic heart failure with worsening symptoms and signs, etc.), myocardial infarction, stroke, organ failure (kidney, liver, heart, lung, etc.), or dementia, wherein the ratio of pro-adrenomedullin or its fragment in a body fluid sample to ADM-NH2 (SEQ ID NO: 20) Characterized by exceeding a specific threshold, the pro-adrenomedullin or its fragment is selected from the group consisting of PAMP (SEQ ID NO: 32), MR-proADM (SEQ ID NO: 33), ADM-Gly (SEQ ID NO: 21), and CT-proADM (SEQ ID NO: 34), and the anti-ADM antibody or anti-ADM fragment or anti-ADM non-Ig scaffold binds to the N-terminal and / or central region (amino acids 1-42) of ADM-Gly and / or ADM-NH2: YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVA (SEQ ID NO: 23), and the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold is used in combination with L-ascorbic acid.

[0027] One embodiment of the present application relates to an anti-adrenomedullin (ADM) antibody, an anti-adrenomedullin antibody fragment, or an anti-ADM non-Ig scaffold used to promote the conversion of ADM-Gly to ADM-NH2 in circulating ADM-Gly within a critically ill patient suffering from an acute disease or acute symptoms selected from the group including severe infections (such as meningitis, systemic inflammatory response syndrome (SIRS), and sepsis), shock (such as septic shock and cardiogenic shock), acute heart failure (such as acute decompensated heart failure and chronic heart failure with exacerbation of symptoms and signs), myocardial infarction, stroke, and organ dysfunction (such as kidney, liver, heart, and lung), wherein the anti-ADM antibody, anti-ADM fragment, or anti-ADM non-Ig scaffold binds to the N-terminal portion (amino acids 1-21):YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO: 14) of ADM-Gly and / or ADM-NH2.

[0028] One embodiment of this application relates to the treatment of critically ill patients suffering from acute diseases or acute symptoms selected from the group including severe infections (such as meningitis, systemic inflammatory response syndrome (SIRS), and sepsis), shock (such as septic shock and cardiogenic shock), acute heart failure (such as acute decompensated heart failure and chronic heart failure with worsening symptoms and signs), myocardial infarction, stroke, and organ dysfunction (such as kidney, liver, heart, and lung), using an anti-adrenergic agent to promote the conversion of ADM-Gly to ADM-NH2 in circulating ADM-Gly within the patient. With respect to durin (ADM) antibodies, anti-adrenomedullin antibody fragments, or anti-ADM non-Ig scaffolds, the anti-ADM antibody, anti-ADM fragment, or anti-ADM non-Ig scaffold is bound to the N-terminal portion (amino acids 1-21) of ADM-Gly and / or ADM-NH2: YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO: 14), and is used in combination with L-ascorbic acid.

[0029] Another embodiment of the present application relates to an anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold used to promote the conversion of ADM-Gly to ADM-NH2 in circulating ADM-Gly within a critically ill patient suffering from an acute disease or acute symptoms selected from the group including severe infections (such as meningitis, systemic inflammatory response syndrome (SIRS), and sepsis), shock (such as septic shock and cardiogenic shock), acute heart failure (such as acute decompensated heart failure and chronic heart failure with exacerbation of symptoms and signs), myocardial infarction, stroke, and organ dysfunction (such as kidney, liver, heart, and lung), wherein the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold recognizes and binds to the N-terminus (amino acid 1) of ADM-Gly and / or ADM-NH2.

[0030] Another preferred embodiment of the present application relates to an anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold used to promote the conversion of ADM-Gly to ADM-NH2 in circulating ADM-Gly within a critically ill patient suffering from an acute disease or acute symptoms selected from the group including severe infections (such as meningitis, systemic inflammatory response syndrome (SIRS), and sepsis), shock (such as septic shock and cardiogenic shock), acute heart failure (such as acute decompensated heart failure and chronic heart failure with exacerbation of symptoms and signs), myocardial infarction, stroke, and organ dysfunction (such as kidney, liver, heart, and lung), wherein the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold binds to the central region (amino acids 21-42): CTVQKLAHQIYQFTDKDKDNVA (SEQ ID NO: 48) of ADM-Gly and / or ADM-NH2.

[0031] Another preferred embodiment of the present application relates to an anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold used to promote the conversion of ADM-Gly to ADM-NH2 in circulating ADM-Gly within a severely ill patient suffering from dementia, wherein the anti-ADM antibody or anti-ADM fragment or anti-ADM non-Ig scaffold binds to the central region (amino acids 21-42) of ADM-Gly and / or ADM-NH2: CTVQKLAHQIYQFTDKDKDNVA (SEQ ID NO: 48).

[0032] Another embodiment of this application relates to an anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold used to promote the conversion of ADM-Gly to ADM-NH2 in circulating ADM-Gly within a critically ill patient suffering from an acute disease or acute symptoms selected from the group including severe infections (such as meningitis, systemic inflammatory response syndrome (SIRS), and sepsis), shock (such as septic shock and cardiogenic shock), acute heart failure (such as acute decompensated heart failure and chronic heart failure with exacerbation of symptoms and signs), myocardial infarction, stroke, and organ dysfunction (such as kidney, liver, heart, and lung), wherein the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold binds to the central region (amino acids 21-32):CTVQKLAHQIYQ (SEQ ID NO: 15) of ADM-Gly and / or ADM-NH2.

[0033] Another embodiment of this application relates to an anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold used to promote the conversion of ADM-Gly to ADM-NH2 in circulating ADM-Gly within a severely ill patient suffering from dementia, wherein the anti-ADM antibody or anti-ADM fragment or anti-ADM non-Ig scaffold binds to the central region (amino acids 21-32):CTVQKLAHQIYQ (SEQ ID NO: 15) of ADM-Gly and / or ADM-NH2.

[0034] Another embodiment of the present application relates to an anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold used to promote the conversion of ADM-Gly to ADM-NH2 in circulating ADM-Gly within a critically ill patient suffering from an acute disease or acute symptoms selected from the group including severe infections (such as meningitis, systemic inflammatory response syndrome (SIRS), and sepsis), shock (such as septic shock and cardiogenic shock), acute heart failure (such as acute decompensated heart failure and chronic heart failure with exacerbation of symptoms and signs), myocardial infarction, stroke, and organ dysfunction (such as kidney, liver, heart, and lung), wherein the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold binds to the central region (amino acids 27-39): AHQIYQFTDKDKD (SEQ ID NO: 49) of ADM-Gly and / or ADM-NH2.

[0035] Another embodiment of this application relates to an anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold used to promote the conversion of circulating ADM-Gly to ADM-NH2 in a severely ill patient suffering from dementia, wherein the anti-ADM antibody or anti-ADM fragment or anti-ADM non-Ig scaffold binds to the central region (amino acids 27-39): AHQIYQFTDKDKD (SEQ ID NO: 49) of ADM-Gly and / or ADM-NH2.

[0036] One embodiment of the present application relates to an anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold used to promote the conversion of ADM-Gly to ADM-NH2 in circulating ADM-Gly within a critically ill patient suffering from an acute disease or acute symptoms selected from the group including severe infection (meningitis, systemic inflammatory response syndrome (SIRS), sepsis, etc.), shock (septic shock, cardiogenic shock, etc.), acute heart failure (acute decompensated heart failure, chronic heart failure with exacerbation of symptoms and signs, etc.), myocardial infarction, stroke, organ dysfunction (kidney, liver, heart, lung, etc.), or dementia, wherein the concentration of proadrenomedullin or its fragments, consisting of the group PAMP (SEQ ID NO: 32), MR-proADM (SEQ ID NO: 33), ADM-Gly (SEQ ID NO: 21), and CT-proADM (SEQ ID NO: 34), and the concentration of ADM-NH2 (SEQ ID NO: 20) are measured in a body fluid sample of the patient.

[0037] A preferred embodiment of this application is for the treatment of critically ill patients suffering from acute diseases or acute symptoms selected from the group including severe infections (such as meningitis, systemic inflammatory response syndrome (SIRS), and sepsis), shock (such as septic shock and cardiogenic shock), acute heart failure (such as acute decompensated heart failure and chronic heart failure with exacerbation of symptoms and signs), myocardial infarction, stroke, organ dysfunction (such as kidney, liver, heart, and lung), or dementia, wherein the conversion of ADM-Gly to ADM-NH2 in circulating ADM-Gly within the patient is promoted. With respect to anti-adrenomedullin (ADM) antibodies, anti-ADM antibody fragments, or anti-ADM non-Ig scaffolds used for this purpose, the concentration ratio of ADM-Gly (SEQ ID NO: 21) to ADM-NH2 (SEQ ID NO: 20) is measured in a body fluid sample from the patient, and if the ADM-Gly / ADM-NH2 ratio exceeds a specific threshold of 1 to 10, preferably 1.5 to 7.5, preferably 2 to 5, and most preferably 2.5, the patient is treated with the anti-ADM antibody, anti-ADM antibody fragment, or anti-ADM non-Ig scaffold.

[0038] Another embodiment of the present application relates to an anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold used to promote the conversion of ADM-Gly to ADM-NH2 in circulating ADM-Gly within the patient, for the treatment of a critically ill patient suffering from an acute disease or acute symptoms selected from the group including severe infection (meningitis, systemic inflammatory response syndrome (SIRS), sepsis, etc.), shock (septic shock, cardiogenic shock, etc.), acute heart failure (acute decompensated heart failure, chronic heart failure with exacerbation of symptoms and signs, etc.), myocardial infarction, stroke, organ failure (kidney, liver, heart, lung, etc.), or dementia, wherein the patient's body fluid sample is selected from the group including blood, serum, plasma, urine, cerebrospinal fluid (CSF), and saliva.

[0039] Further embodiments of this application relate to anti-adrenomedullin (ADM) antibodies or anti-ADM antibody fragments or anti-ADM non-Ig scaffolds used to promote the conversion of ADM-Gly to ADM-NH2 in circulating ADM-Gly within the patient, in the treatment of critically ill patients suffering from acute diseases or acute symptoms selected from the group including severe infections (such as meningitis, systemic inflammatory response syndrome (SIRS), and sepsis), shock (such as septic shock and cardiogenic shock), acute heart failure (such as acute decompensated heart failure and chronic heart failure with exacerbation of symptoms and signs), myocardial infarction, stroke, organ dysfunction (such as kidney, liver, heart, and lung), or dementia, wherein the sample is selected from the group including human citrate plasma, heparin plasma, and EDTA plasma.

[0040] Another embodiment of this application relates to an anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold used to promote the conversion of ADM-Gly to ADM-NH2 in circulating ADM-Gly within a critically ill patient suffering from an acute disease or acute symptoms selected from the group including severe infection (meningitis, systemic inflammatory response syndrome (SIRS), sepsis, etc.), shock (septic shock, cardiogenic shock, etc.), acute heart failure (acute decompensated heart failure, chronic heart failure with exacerbation of symptoms and signs, etc.), myocardial infarction, stroke, organ dysfunction (kidney, liver, heart, lung, etc.), or dementia, wherein the ratio of pro-adrenomedullin or its fragment to ADM-NH2 in the patient is measured. In the most preferred embodiment, the ratio is measured using an immunoassay.

[0041] Another embodiment of this application relates to the treatment of critically ill patients suffering from acute diseases or acute symptoms selected from the group including severe infections (such as meningitis, systemic inflammatory response syndrome (SIRS), and sepsis), shock (such as septic shock and cardiogenic shock), acute heart failure (such as acute decompensated heart failure and chronic heart failure with exacerbation of symptoms and signs), myocardial infarction, stroke, organ dysfunction (such as kidney, liver, heart, and lung), or dementia, wherein the conversion of ADM-Gly to ADM-NH2 in circulating ADM-Gly within the patient is being addressed. With respect to anti-adrenomedullin (ADM) antibodies, anti-ADM antibody fragments, or anti-ADM non-Ig scaffolds used to promote conversion, an immunoassay is used to measure the ratio of proadrenomedullin or its fragment to ADM-NH2, and the proadrenomedullin or its fragment is selected from the group consisting of PAMP (SEQ ID NO: 32), MR-proADM (SEQ ID NO: 33), ADM-Gly (SEQ ID NO: 21), and CT-proADM (SEQ ID NO: 34).

[0042] Another embodiment of this application relates to an anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold used to promote the conversion of ADM-Gly to ADM-NH2 in circulating ADM-Gly within a critically ill patient suffering from an acute disease or acute symptoms selected from the group including severe infection (meningitis, systemic inflammatory response syndrome (SIRS), sepsis, etc.), shock (septic shock, cardiogenic shock, etc.), acute heart failure (acute decompensated heart failure, chronic heart failure with exacerbation of symptoms and signs, etc.), myocardial infarction, stroke, organ dysfunction (kidney, liver, heart, lung, etc.), or dementia, wherein the immunoassay is a sandwich immunoassay, preferably a fully automated immunoassay.

[0043] One embodiment of the present application relates to an anti-adrenomedullin (ADM) antibody, an anti-ADM antibody fragment, or an anti-ADM non-Ig scaffold used to promote the conversion of ADM-Gly to ADM-NH2 in circulating ADM-Gly within a critically ill patient suffering from an acute disease or acute symptoms selected from the group including severe infections (such as meningitis, systemic inflammatory response syndrome (SIRS), and sepsis), shock (such as septic shock and cardiogenic shock), acute heart failure (such as acute decompensated heart failure and chronic heart failure with exacerbation of symptoms and signs), myocardial infarction, stroke, organ dysfunction (such as kidney, liver, heart, and lung), or dementia, wherein the measurement sensitivity of the assay for detecting ADM-NH2 allows for the quantification of ADM-NH2 in a healthy subject, with a sensitivity of less than 70 pg / mL, preferably less than 40 pg / mL, and more preferably less than 10 pg / mL.

[0044] Another embodiment of the present application relates to an anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold used to promote the conversion of ADM-Gly to ADM-NH2 in circulating ADM-Gly within a critically ill patient suffering from an acute disease or acute symptoms selected from the group including severe infection (meningitis, systemic inflammatory response syndrome (SIRS), sepsis, etc.), shock (septic shock, cardiogenic shock, etc.), acute heart failure (acute decompensated heart failure, chronic heart failure with exacerbation of symptoms and signs, etc.), myocardial infarction, stroke, organ dysfunction (kidney, liver, heart, lung, etc.), or dementia, wherein the measurement sensitivity of the assay for ADM-Gly allows for the quantification of ADM-Gly in a healthy subject, the sensitivity of which is 20 pg / mL, preferably 15 pg / mL, more preferably 10 pg / mL.

[0045] One embodiment of the present application relates to an anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold used to promote the conversion of ADM-Gly to ADM-NH2 in circulating ADM-Gly within a critically ill patient suffering from an acute disease or acute symptoms selected from the group including severe infections (such as meningitis, systemic inflammatory response syndrome (SIRS), and sepsis), shock (such as septic shock and cardiogenic shock), acute heart failure (such as acute decompensated heart failure and chronic heart failure with exacerbation of symptoms and signs), myocardial infarction, stroke, organ dysfunction (such as kidney, liver, heart, and lung), or dementia, wherein the measurement sensitivity of the assay for detecting MR-proADM allows for the quantification of MR-proADM in a healthy subject, with a sensitivity of less than 0.5 nmol / L, preferably less than 0.4 nmol / L, and more preferably less than 0.2 nmol / L.

[0046] Another embodiment of the present application relates to an anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold used to promote the conversion of ADM-Gly to ADM-NH2 in circulating ADM-Gly within a critically ill patient suffering from an acute disease or acute symptoms selected from the group including severe infection (meningitis, systemic inflammatory response syndrome (SIRS), sepsis, etc.), shock (septic shock, cardiogenic shock, etc.), acute heart failure (acute decompensated heart failure, chronic heart failure with exacerbation of symptoms and signs, etc.), myocardial infarction, stroke, organ dysfunction (kidney, liver, heart, lung, etc.), or dementia, wherein the measurement sensitivity of the said assay method for PAMP allows for the quantification of PAMP in a healthy subject, the sensitivity of which is less than 0.5 pmol / L, preferably less than 0.25 pmol / L, and more preferably less than 0.1 pmol / L.

[0047] One embodiment of the present application relates to an anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold used to promote the conversion of ADM-Gly to ADM-NH2 in circulating ADM-Gly within a critically ill patient suffering from an acute disease or acute symptoms selected from the group including severe infections (such as meningitis, systemic inflammatory response syndrome (SIRS), and sepsis), shock (such as septic shock and cardiogenic shock), acute heart failure (such as acute decompensated heart failure and chronic heart failure with exacerbation of symptoms and signs), myocardial infarction, stroke, organ dysfunction (such as kidney, liver, heart, and lung), or dementia, wherein the measurement sensitivity of the measurement method for detecting CT-proADM allows for the quantification of CT-proADM in a healthy subject, the sensitivity of which is less than 100 pmol / L, preferably less than 75 pmol / L, and more preferably less than 50 pmol / L.

[0048] Further embodiments of this application relate to anti-adrenomedullin (ADM) antibodies or anti-ADM antibody fragments or anti-ADM non-Ig scaffolds used to promote the conversion of ADM-Gly to ADM-NH2 in circulating ADM-Gly within a critically ill patient suffering from an acute disease or acute symptoms selected from the group including severe infections (such as meningitis, systemic inflammatory response syndrome (SIRS), and sepsis), shock (such as septic shock and cardiogenic shock), acute heart failure (such as acute decompensated heart failure and chronic heart failure with exacerbation of symptoms and signs), myocardial infarction, stroke, organ dysfunction (such as kidney, liver, heart, and lung), or dementia. The concentrations of nomedullin or its fragment and ADM-NH2 (SEQ ID NO: 20) are measured using a first binder for proadrenomedullin and a second binder for ADM-NH2 (SEQ ID NO: 20), wherein the proadrenomedullin or its fragment is selected from the group consisting of PAMP (SEQ ID NO: 32), MR-proADM (SEQ ID NO: 33), ADM-Gly (SEQ ID NO: 21), and CT-proADM (SEQ ID NO: 34), and both binders are selected from the group consisting of antibodies, antibody fragments, or non-Ig scaffolds that bind to the proadrenomedullin or its fragment and ADM-NH2.

[0049] Another embodiment of this application relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in the treatment of a patient, wherein the anti-ADM antibody or anti-ADM fragment or anti-ADM non-Ig scaffold is conjugated to the N-terminal and / or central region (amino acids 1-42) of ADM-Gly and / or ADM-NH2 (SEQ ID NO: 23), and the levels of PAM and / or its isoform and / or fragment are measured in a body fluid sample of the patient, and if the level of PAM is below a threshold, the patient is treated with the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold.

[0050] Another embodiment of the present application relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in the treatment of a patient, wherein the level of the PAM and / or its isoform and / or fragment is the total concentration of the PAM and / or its isoform and / or fragment having at least 12 amino acids, or the activity of the PAM and / or its isoform and / or fragment containing the sequences of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47.

[0051] As is known to those skilled in the art, the PAM isoform sequences (SEQ ID NOs. 39-44) shown in the sequence listing include an N-terminal signal sequence (amino acids 1-20) that is cleaved before the protein is secreted. Therefore, in a preferred embodiment, the PAM isoform sequences (SEQ ID NOs. 39-44) do not include an N-terminal signal sequence.

[0052] Another embodiment of this application relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in the treatment of a patient, wherein the anti-ADM antibody or anti-ADM fragment or anti-ADM non-Ig scaffold is conjugated to the N-terminal and / or central region (amino acids 1-42) of ADM-Gly and / or ADM-NH2: YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVA (SEQ ID NO: 23) and used in combination with L-ascorbic acid.

[0053] In a preferred embodiment, an anti-adrenomedullin (ADM) antibody, anti-ADM antibody fragment, or anti-ADM non-Ig scaffold combined with L-ascorbic acid is intended for the following purposes: a. To be used in the treatment of an acute disease or acute condition in a patient in order to stabilize the patient's systemic circulatory system. Here, the patient needs to have their systemic circulatory system stabilized and has a heart rate greater than 100 beats / min and / or a mean arterial pressure less than 65 mmHg, and systemic circulatory system stabilization means raising the mean arterial pressure above 65 mmHg. Or b. To be used in patients with acute illness or acute condition to prevent an increase in heart rate to more than 100 beats / min and / or a decrease in mean arterial pressure to less than 65 mmHg. Or c. To be used to treat acute illnesses or acute symptoms in patients suffering from chronic and / or acute illnesses or acute symptoms, for the prevention or reduction of organ dysfunction in patients or for the prevention of organ failure. Here, the organs are selected from the group including the heart, kidneys, liver, lungs, pancreas, small intestine, and spleen. Or d. To be used to treat or prevent shock in patients such as SIRS, meningitis, sepsis, or septic shock. e. To reduce the risk of death in patients suffering from shock, such as SIRS, meningitis, sepsis, and septic shock. Here, the anti-ADM antibody, anti-ADM fragment, or anti-ADM non-Ig scaffold binds to the N-terminal and / or central region (amino acids 1-42) of ADM-Gly and / or ADM-NH2: YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVA (SEQ ID NO: 23).

[0054] Another preferred embodiment of the present application relates to the use of an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in patient intervention and treatment, and the use thereof in combination with L-ascorbic acid, wherein L-ascorbic acid is a single enantiomer, a mixture of enantiomers, a mixture of diastereomers, or a pharmaceutically acceptable salt thereof, solvate, hydrate, or prodrug.

[0055] According to the present invention, it has been found that administering an anti-ADM antibody, an anti-ADM antibody fragment, or an anti-ADM non-Ig scaffold to a patient can promote the conversion from ADM-Gly to ADM-NH2, wherein the anti-ADM antibody, anti-ADM fragment, or anti-ADM non-Ig scaffold binds to the N-terminal and / or central region (amino acids 1-42) of ADM-Gly and / or ADM-NH2: YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVA (SEQ ID NO: 23).

[0056] Throughout this specification, the “antibody,” “antibody fragment,” or “non-Ig scaffold” according to the present invention can bind to ADM and thus be directed toward ADM, and thus can be referred to as “anti-ADM antibody,” “anti-ADM antibody fragment,” or “anti-ADM non-Ig scaffold.”

[0057] In one embodiment, the ascorbic acid compound is L-ascorbic acid or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate or hydrate thereof. L-ascorbic acid is also known as vitamin C, L-xyloascorbic acid, 3-oxo-L-globulanolactone (enol type), L-3-ketotreohexuronate lactone, anti-scurvy vitamin, cevitanic acid, adenex, allercorb, ascorline, ascortil, ascorbit, cantan, cantaxin, catavin C, sevicure, sebion, secon, sediolan, cerascon, serine, ceneton, seleon, cergona, sescorbat, cetami Also known as D, Setabe, Setemikan, Sevalin, Sebatin, Sebex, Sebimin, Sebisol, Sebitan, Sevitex, Sewin, Ciamin, Shipka, Consemin, C-Bin, Damiamon C, Duoscorb, Hybrin, Laroscorbin, Remascorb, Pranavit C, Proscorbin, Redoxone, Livena, Scorbacid, Scorb C, Testa Alcobic Acid, Biserato, VitaC, Vitacimin, Vitacin, Vitacorbol, and Xixitic.

[0058] In one embodiment, the ascorbic acid compound is L-ascorbic acid. In another embodiment, the ascorbic acid compound is a pharmaceutically acceptable salt of L-ascorbic acid, or a pharmaceutically acceptable solvate or hydrate thereof.

[0059] Suitable bases for producing pharmaceutically acceptable salts of L-ascorbic acid include, but are not limited to, inorganic bases such as magnesium hydroxide, calcium hydroxide, potassium hydroxide, zinc hydroxide, and sodium hydroxide, and, but are not limited to, L-arginine, benesamin, benzathine, choline, deanol, diethanolamine, diethylamine, dimethylamine, dipropylamine, diisopropylamine, 2-(diethylamino)-ethanol, ethanolamine, ethylamine, ethylenediamine, isopropylamine, and N-methylglucamine. This includes organic bases such as primary, secondary, tertiary, and quaternary aliphatic and aromatic amines, including hydravamin, 1H-imidazole, L-lysine, morpholine, 4-(2-hydroxyethyl)-morpholine, methylamine, piperidine, piperazine, propylamine, pyrrolidine, 1-(2-hydroxyethyl)-pyrrolidine, pyridine, quinuclidine, quinoline, isoquinoline, triethanolamine, trimethylamine, triethylamine, N-methyl-D-glucamine, 2-amino-2-(hydroxymethyl)-1,3-propanediol, and tromethamine.

[0060] In one embodiment, the ascorbic acid compound is an alkali metal salt or alkaline earth metal salt of L-ascorbic acid, or a pharmaceutically acceptable solvate or hydrate thereof. In another embodiment, the ascorbic acid compound is sodium L-ascorbate, potassium, calcium, or magnesium, or a pharmaceutically acceptable solvate or hydrate thereof. In yet another embodiment, the ascorbic acid compound is sodium L-ascorbate, or a pharmaceutically acceptable solvate or hydrate thereof. In yet another embodiment, the ascorbic acid compound is sodium L-ascorbate, also known as vitamin C sodium, ascorbin, sodacorbate, natrascorb, senolate, ascorbicin, or cevicate. In yet another embodiment, the ascorbic acid compound is potassium L-ascorbate, or a pharmaceutically acceptable solvate or hydrate thereof. In yet another embodiment, the ascorbic acid compound is calcium L-ascorbate, or a pharmaceutically acceptable solvate or hydrate thereof. In yet another embodiment, the ascorbic acid compound is calcium L-ascorbate. In yet another embodiment, the ascorbic acid compound is magnesium L-ascorbate, or a pharmaceutically acceptable solvate or hydrate thereof. In yet another embodiment, the ascorbic acid compound is magnesium L-ascorbate.

[0061] In certain embodiments, the ascorbic acid compound is D-ascorbic acid, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate or hydrate thereof.

[0062] In one embodiment, the ascorbic acid compound is D-ascorbic acid. In another embodiment, the ascorbic acid compound is a pharmaceutically acceptable salt of D-ascorbic acid, or a pharmaceutically acceptable solvate or hydrate thereof.

[0063] Suitable bases for producing pharmaceutically acceptable salts of D-ascorbic acid include, but are not limited to, inorganic bases such as magnesium hydroxide, calcium hydroxide, potassium hydroxide, zinc hydroxide, and sodium hydroxide, and, but are not limited to, L-arginine, benesamine, benzathine, choline, denol, diethanolamine, diethylamine, dimethylamine, dipropylamine, diisopropylamine, 2-(diethylamino)-ethanol, ethanolamine, ethylamine, ethylenediamine, isopropylamine, and N-methyl-glucamine. This includes organic bases such as primary, secondary, tertiary, and quaternary aliphatic and aromatic amines, including hydravamin, 1H-imidazole, L-lysine, morpholine, 4-(2-hydroxyethyl)-morpholine, methylamine, piperidine, piperazine, propylamine, pyrrolidine, 1-(2-hydroxyethyl)-pyrrolidine, pyridine, quinuclidine, quinoline, isoquinoline, triethanolamine, trimethylamine, triethylamine, N-methyl-D-glucamine, 2-amino-2-(hydroxymethyl)-1,3-propanediol, and tromethamine.

[0064] In one embodiment, the ascorbic acid compound is an alkali metal salt or alkaline earth metal salt of D-ascorbic acid, or a pharmaceutically acceptable solvate or hydrate thereof. In another embodiment, the ascorbic acid compound is sodium D-ascorbate, potassium, calcium, or magnesium, or a pharmaceutically acceptable solvate or hydrate thereof. In yet another embodiment, the ascorbic acid compound is sodium D-ascorbate, or a pharmaceutically acceptable solvate or hydrate thereof. In yet another embodiment, the ascorbic acid compound is sodium D-ascorbate, also known as vitamin C sodium, ascorbin, sodacorbate, natrascorb, senolate, ascorbicin, or cevicate. In yet another embodiment, the ascorbic acid compound is potassium D-ascorbate, or a pharmaceutically acceptable solvate or hydrate thereof. In yet another embodiment, the ascorbic acid compound is calcium D-ascorbate, or a pharmaceutically acceptable solvate or hydrate thereof. In yet another embodiment, the ascorbic acid compound is calcium D-ascorbate. In yet another embodiment, the ascorbic acid compound is magnesium D-ascorbate, or a pharmaceutically acceptable solvate or hydrate thereof. In yet another embodiment, the ascorbic acid compound is magnesium D-ascorbate.

[0065] The term “solvate” refers to a complex or aggregate formed by one or more solute molecules, e.g., the compounds provided herein, and one or more solvent molecules, present in stoichiometric or non-stoichiometric amounts. Suitable solvents include, but are not limited to, water, methanol, ethanol, 1-propanol, 2-propanol, and acetic acid. In certain embodiments, the solvent is pharmaceutically acceptable. In one embodiment, the complex or aggregate is in crystalline form. In another embodiment, the complex or aggregate is in amorphous form. When the solvent is water, the solvate is a hydrate. Examples of hydrates include, but are not limited to, hemihydrate, monohydrate, dihydrate, trihydrate, tetrahydrate, and pentahydrate.

[0066] In one embodiment, the ascorbic acid compound in each pharmaceutical composition is independently L-ascorbic acid or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate or hydrate thereof. In another embodiment, the ascorbic acid compound in each pharmaceutical composition is independently an alkali metal salt or alkaline earth metal salt of L-ascorbic acid, or a pharmaceutically acceptable solvate or hydrate thereof, or a mixture thereof. In yet another embodiment, the ascorbic acid compound in each pharmaceutical composition is independently a sodium salt, potassium salt, calcium salt, or magnesium salt of L-ascorbic acid, or a pharmaceutically acceptable solvate or hydrate thereof, or a mixture thereof. In yet another embodiment, the ascorbic acid compound in each pharmaceutical composition is independently L-sodium ascorbate. In yet another embodiment, the ascorbic acid compound in each pharmaceutical composition is independently L-calcium ascorbate. In yet another embodiment, the ascorbic acid compound in each pharmaceutical composition is independently L-magnesium ascorbate. In yet another embodiment, the ascorbic acid compound in each pharmaceutical composition is independently a mixture of two or three of the following: sodium L-ascorbate, calcium L-ascorbate, and magnesium L-ascorbate.

[0067] In certain embodiments, the patient may be administered the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold that binds to ADM, with the help of a diagnostic method, to promote the conversion from ADM-Gly to ADM-NH2. The diagnostic method is described below.

[0068] Mature ADM, bio-ADM, and ADM-NH2 are used synonymously throughout this application and are molecules according to Sequence ID No. 20.

[0069] The aforementioned concentration ratio is measured as the ratio of the concentration of proadrenomedullin or a fragment thereof to the concentration of ADM-NH2 (SEQ ID NO: 20). The proadrenomedullin or a fragment thereof is selected from the group consisting of PAMP (SEQ ID NO: 32), MR-proADM (SEQ ID NO: 33), ADM-Gly (SEQ ID NO: 21), and CT-proADM (SEQ ID NO: 34) in the subject's body fluid sample. The ratio of the concentration of bio-ADM measured in the subject's body fluid sample to the concentration of the marker is compared to a threshold ratio. If the concentration ratio of proadrenomedullin or a fragment thereof to ADM-NH2 exceeds a specific threshold, an anti-ADM antibody or anti-ADM antibody fragment that binds to ADM, or an anti-ADM non-Ig scaffold that binds to ADM, is administered to the patient as treatment or intervention.

[0070] This means that in a particular embodiment of the present invention, the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold that binds to ADM is used in a patient to promote the conversion from ADM-Gly to ADM-NH2, wherein a body fluid sample taken from the patient shows an increase in the concentration ratio of proadrenomedullin or its fragment to ADM-NH2 above a certain threshold, where the proadrenomedullin or its fragment is selected from the group consisting of PAMP (SEQ ID NO: 32), MR-proADM (SEQ ID NO: 33), ADM-Gly (SEQ ID NO: 21), and CT-proADM (SEQ ID NO: 34).

[0071] The subject of this invention is a diagnostic method for measuring the concentrations of ADM-Gly and ADM-NH2 using a binder for ADM-Gly and ADM-NH2.

[0072] The subject of the present invention is a diagnostic method in which the binder is selected from the group comprising an antibody, antibody fragment, or non-Ig scaffold that binds to proadrenomedullin or a fragment thereof, and ADM-NH2, wherein the proadrenomedullin or a fragment thereof is selected from the group consisting of PAMP (SEQ ID NO: 32), MR-proADM (SEQ ID NO: 33), ADM-Gly (SEQ ID NO: 21), and CT-proADM (SEQ ID NO: 34).

[0073] As used herein, the term "sample" refers to a fluid sample obtained for the purpose of diagnosis, prognosis, or evaluation of a target subject, such as a patient. Preferred test samples include blood, serum, and plasma. Furthermore, those skilled in the art will know that some test samples can be analyzed more easily after fractionation or purification procedures, for example, after separating whole blood into serum or plasma components.

[0074] In one particular embodiment of the present invention, the body fluid is a blood sample. The blood sample may be selected from the group including whole blood, serum, and plasma. In one particular embodiment of the diagnostic method, the sample is selected from the group including human citrate plasma, heparin plasma, and EDTA plasma.

[0075] In a particular embodiment of the present invention, a measurement method is used to measure the ratio of proadrenomedullin or a fragment thereof to ADM-NH2, wherein the concentration of proadrenomedullin or a fragment thereof is selected from the group consisting of PAMP (SEQ ID NO: 32), MR-proADM (SEQ ID NO: 33), ADM-Gly (SEQ ID NO: 21), and CT-proADM (SEQ ID NO: 34), and the measurement sensitivity of the measurement method for ADM-NH2 allows for the quantification of mature ADM-NH2 in healthy subjects, the sensitivity being less than 70 pg / mL, preferably less than 40 pg / mL, and more preferably less than 10 pg / mL.

[0076] In a particular embodiment of the present invention, a measurement method is used to measure the ratio of proadrenomedullin or a fragment thereof to ADM-NH2, wherein the concentration of proadrenomedullin or a fragment thereof is ADM-Gly (SEQ ID NO: 21), and the measurement sensitivity of the measurement method for ADM-Gly allows for the quantification of ADM-Gly in a healthy subject, with a sensitivity of 20 pg / mL, preferably 15 pg / mL, and more preferably 10 pg / mL.

[0077] In a particular embodiment of the present invention, a measurement method is used to measure the ratio of proadrenomedullin or a fragment thereof to ADM-NH2, wherein the concentration of proadrenomedullin or a fragment thereof is MR-proADM (SEQ ID NO: 33), and the measurement sensitivity of the measurement method allows for the quantification of MR-proADM in healthy subjects, with a sensitivity of less than 0.5 nmol / L, preferably less than 0.4 nmol / L, and more preferably less than 0.2 nmol / L.

[0078] In a particular embodiment of the present invention, a measurement method is used to measure the ratio of proadrenomedullin or a fragment thereof to ADM-NH2, wherein the concentration of proadrenomedullin or a fragment thereof is CT-proADM (SEQ ID NO: 34), and the measurement sensitivity of the measurement method allows for the quantification of CT-proADM in healthy subjects, with a sensitivity of less than 100 pmol / L, preferably less than 75 pmol / L, and more preferably less than 50 pmol / L.

[0079] In a particular embodiment of the present invention, a measurement method is used to measure the ratio of proadrenomedullin or a fragment thereof to ADM-NH2, wherein the concentration of proadrenomedullin or a fragment thereof is PAMP (SEQ ID NO: 34), and the measurement sensitivity of the measurement method allows for the quantification of PAMP in a healthy subject, with a sensitivity of less than 0.5 pmol / L, preferably less than 0.25 pmol / L, and more preferably less than 0.1 pmol / L.

[0080] In one embodiment of the subject matter of the present invention, the threshold ratio of ADM-Gly to ADM-NH2 is in the range of 1 to 10, preferably 1.5 to 7.5, preferably 2 to 5, and most preferably 2.5.

[0081] For the purpose of calculating the ratio, the concentrations of the two markers should preferably be expressed in the same unit (e.g., pg / mL).

[0082] Using the concentrations of both markers, a calculation is performed which may be a ratio of both markers (e.g., the ratio between ADM-Gly and ADM-NH2 or the ratio between ADM-NH2 and ADM-Gly), a formula into which both markers are introduced, or a mathematical algorithm into which both markers are introduced. The result of such a ratio, formula, or mathematical algorithm can then be a numerical value to be compared to a predetermined threshold, and this comparison result is then used in the patient to promote the conversion from ADM-Gly to ADM-NH2 by an anti-ADM antibody or anti-ADM antibody fragment that binds to ADM, or an anti-ADM non-Ig scaffold that binds to ADM.

[0083] Threshold concentrations can be obtained, for example, from Kaplan-Meier analysis, where the incidence of disease correlates with the quartile of the marker ratio in the population. According to this analysis, for example, subjects with a marker ratio above the 75th percentile have a significantly higher risk of developing, for example, the disease according to the present invention, or of experiencing adverse events (e.g., mortality).

[0084] Other preferred thresholds include, for example, the 90th, 95th, or 99th percentile of the normal population. Using a percentile higher than the 75th percentile can reduce the number of false positives identified, but increases the risk of missing moderately risky cases. Therefore, the threshold may be chosen depending on whether it is considered more appropriate to identify the majority of risky cases at the expense of identifying "false positives," or whether it is considered more appropriate to identify primarily high-risk cases at the expense of missing some moderately risky cases.

[0085] The above-mentioned threshold may differ when another measurement method is used, if said measurement method is calibrated in a different way from the measurement system used in the present invention. Therefore, the aforementioned threshold should be adjusted accordingly for such differently calibrated measurement methods, taking into account the difference in calibration. One possibility for quantifying the calibration difference is a method of performing comparative analysis (correlation) of the assay in question (e.g., a bio-ADM assay) for the respective biomarker (e.g., bio-ADM) in a sample using both methods. Another possibility is, assuming that this assay has sufficient analytical sensitivity, comparing the median biomarker concentration of a representative normal population with the median biomarker concentration described in the literature (e.g., Weber et al. 2017. JALM 2(2): 222-233), and recalculating the calibration based on the difference obtained from this comparison to determine the assay in question. Using the calibration employed in the present invention, when samples from normal (healthy) subjects were measured, the median concentration of plasma bio-ADM (mature ADM-NH2) was 13.7 pg / mL (interquartile range [IQR]: 9.6 to 18.7 pg / mL) (Weber et al. 2017. JALM 2(2): 222-233).

[0086] In a specific embodiment of the diagnostic method, the binding agent has at least 10 for pro-adrenomedullin (which is not ADM-NH2 according to SEQ ID NO: 20) or a fragment thereof and ADM-NH2 7 M -1 , preferably 10 8 M -1 , preferably 10 9 M -1 or more, most preferably 10 10 M -1 or higher binding affinity. A person skilled in the art would appreciate that lower affinity can be compensated for by using higher doses of the compound, and it is known that this measure does not fall outside the scope of the claims of the present invention.

[0087] To measure the affinity of antibodies to adrenomedullin, the binding dynamics of adrenomedullin to immobilized antibodies were measured by label-free surface plasmon resonance using a Biacore2000 system (GE Healthcare Europe GmbH, Freiburg, Germany). Reversible antibody immobilization was performed using anti-mouse Fc antibodies covalently bound to the CM5 sensor surface at high density, according to the manufacturer's instructions (mouse antibody capture kit; GE Healthcare) (Lorenz et al. 2011. Antimicrob Agents Chemother. 55 (1): 165-173).

[0088] In a particular embodiment of the diagnostic method, one assay is used to determine the concentration of proadrenomedullin or its fragments and ADM-NH2, wherein the concentration of proadrenomedullin or its fragments is selected from the group consisting of PAMP (SEQ ID NO: 32), MR-proADM (SEQ ID NO: 33), ADM-Gly (SEQ ID NO: 21), and CT-proADM (SEQ ID NO: 34), and such assay is a sandwich assay, preferably a fully automated assay.

[0089] In one embodiment of the present invention, the measurement method may be a so-called POC (point-of-care) test, which is a testing technique that can be performed within one hour near the patient without requiring a fully automated measurement system. An example of this technique is immunochromatography.

[0090] In one embodiment of the diagnostic method, the measurement is a sandwich immunoassay, preferably a fully automated assay, using any type of detection technique, including, but not limited to, enzyme labeling, chemiluminescence labeling, and electrochemiluminescence labeling. In one embodiment of the diagnostic method, the measurement is an enzyme-labeled sandwich assay. Examples of automated or fully automated assays include assays that may use one of the systems of Roche Elecsys, Abbott Architect, Siemens Centauer, Brahms Kryptor, BiomerieuxVidas, and Alere Triage (all registered trademarks).

[0091] Various immunoassay methods are known and may be used in the assay methods and procedures of the present invention, including radioimmunoassay ("RIA"), homogeneous enzyme amplification immunoassay ("EMIT"), enzyme-linked immunosorbent assay ("ELISA"), apoenzyme reactivation immunoassay ("ARIS"), weighing rod immunoassay, and immunochromatography.

[0092] In a particular embodiment of the diagnostic method, at least one of the two binders is labeled for detection.

[0093] The subject of the present invention is an anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold for use in a patient to promote the conversion from ADM-Gly to ADM-NH2, wherein the patient is a critically ill patient suffering from an acute disease or acute symptoms selected from the group including severe infection (meningitis, systemic inflammatory response syndrome (SIRS), sepsis, etc.), shock (septic shock, cardiogenic shock, etc.), acute heart failure (acute decompensated heart failure, chronic heart failure with exacerbation of symptoms and signs, etc.), myocardial infarction, stroke, organ failure (kidney, liver, heart, lung, etc.), or dementia.

[0094] Heart failure (HF) is a cardiac condition that occurs when problems with the structure or function of the heart impair its ability to supply enough blood flow to meet the body's needs. This heart failure can cause a variety of symptoms, including shortness of breath (SOB), especially at rest or during exercise, symptoms of fluid retention such as pulmonary congestion or ankle swelling, and objective evidence of abnormalities in the structure or function of the heart at rest.

[0095] Heart failure is a clinical syndrome characterized by a set of symptoms and signs caused by cardiac dysfunction. It is one of the leading causes of morbidity and mortality in developed countries, with a prevalence of 1-2%. Heart failure can be classified into chronic heart failure and acute heart failure. Patients with chronic heart failure can be classified into stable chronic heart failure, worsening signs and symptoms of chronic heart failure, and acute decompensation of chronic heart failure. Acute heart failure (AHF) is defined as a condition in which signs and symptoms of heart failure appear rapidly, requiring emergency treatment and hospitalization. AHF can present as acute neonatal heart failure (new onset of AHF in patients with no prior cardiac dysfunction) or as acute decompensation of chronic heart failure. AHF is a leading cause of hospitalization in adults aged 65 and older. Despite the remarkable improvement in the prognosis of chronic heart failure patients, primarily due to advances in treatment over the past few decades, once a patient is hospitalized with decompensated heart failure, both short-term and long-term outcomes remain very poor. Nearly 25% of patients hospitalized for AHF require readmission within 30 days of discharge, and less than 50% survive more than five years after hospitalization.

[0096] Heart failure encompasses a wide range of patients, from those with normal left ventricular ejection fraction (LVEF), typically considered to be 50% or more, also known as heart failure with preserved ejection fraction (HFpEF), to those with reduced ejection fraction (HFrEF), typically considered to be less than 40%. Patients with an LVEF in the 40-49% range are represented as the "gray area" and are defined as moderate heart failure (HFmrEF) (Ponikowski et al. 2016. European Heart Journal 18(8): 891-97).

[0097] Symptoms / signs of left-sided congestion are defined as orthopnea, paroxysmal nocturnal dyspnea, pulmonary Russell sounds (bilateral), and peripheral edema (bilateral). Symptoms / signs of right-sided congestion are defined as jugular vein dilation, peripheral edema (bilateral), congestive hepatomegaly, hepatojugular reflux, ascites, and symptoms of intestinal congestion (see Table 12.2 of Ponikowski et al. 2016. Eur Heart J. ehw128 for a review).

[0098] Edema is the accumulation of fluid within intercellular tissues due to an abnormal expansion of interstitial fluid volume. The fluid between the interstitium and intravascular spaces is regulated by the hydrostatic gradient of the capillaries and the colloid osmotic gradient across the capillaries (Trayes et al. 2013. Am Fam Physician 88(2):102-110). Fluid accumulation occurs when a local or systemic pathological condition disrupts this balance, leading to increased capillary hydrostatic pressure, increased plasma volume, decreased plasma colloid osmotic pressure (hypoalbuminemia), increased capillary permeability, or lymphatic obstruction.

[0099] Clinically, edema manifests as swelling, and the amount of interstitial fluid is determined by the balance of fluid homeostasis. Increased secretion of fluid into the interstitium or impaired fluid removal can cause edema. Elevated hydrostatic pressure occurs in heart failure. Causes of systemic edema can include edema in multiple organs and peripheral areas. For example, severe heart failure can cause pulmonary edema, pleural effusion, ascites, and peripheral edema.

[0100] Pulmonary edema is the accumulation of fluid in the air spaces and parenchymal tissues of the lungs. Pulmonary edema can lead to impaired gas exchange and respiratory failure. Pulmonary edema can result from either a defect in the left ventricle of the heart that prevents sufficient blood from being drawn from the pulmonary circulation ("cardiogenic pulmonary edema") or damage to the parenchymal tissues or pulmonary vascular system ("non-cardiogenic pulmonary edema") (Ware and Matthay 2005. N. Engl. J. Med. 353 (26): 2788-96). Treatment focuses on three aspects: first, improving respiratory function; second, treating the underlying cause; and third, preventing further damage to the lungs. Acute pulmonary edema, in particular, can lead to fatal respiratory distress or cardiac arrest due to hypoxia, a fundamental characteristic of congestive heart failure.

[0101] The term "acute" means a rapid onset and is used to describe an exacerbation or decompensation of heart failure, referring to an event characterized by a patient having signs and changes in symptoms of heart failure that necessitate emergency treatment or hospitalization.

[0102] The term "chronic" refers to a condition that persists for a long period of time. Chronic heart failure is a long-term condition, but it is usually kept stable through symptomatic treatment (stable chronic heart failure).

[0103] Stable chronic heart failure is characterized by the presence of structural or functional impairment of the heart that impairs the heart's ability to supply sufficient blood flow to meet the body's needs, the absence of volume overload (manifested by pulmonary congestion and / or systemic congestion) and / or severe reduction in cardiac output (manifested by hypotension, renal failure, and / or shock syndrome), and the patient does not require emergency treatment or optimization of treatment and does not require hospitalization.

[0104] Chronic heart failure with worsening signs and symptoms is characterized by the presence of structural or functional impairment of the heart that impairs the heart's ability to supply sufficient blood flow to meet the body's needs, volume overload (manifested by pulmonary congestion and / or systemic congestion) and / or severe reduction in cardiac output (manifested by hypotension, renal failure, and / or shock syndrome). While this patient does not require emergency treatment or hospitalization, optimization of treatment is necessary.

[0105] Chronic heart failure is also a form of decompensation (also called acute decompensated heart failure or acute decompensated chronic heart failure), which is most commonly a result of comorbidities (such as pneumonia), myocardial infarction, arrhythmias, uncontrolled hypertension, or patient failure to adhere to fluid restriction, dietary restrictions, or medication. After treatment, patients with acute decompensated chronic heart failure may revert to a stable chronic compensated state (stable chronic heart failure).

[0106] Newly developed acute heart failure and acute decompensated chronic heart failure are characterized by the presence of structural or functional impairment of the heart that impairs its ability to supply sufficient blood flow to meet the body's needs, volume overload (manifested by pulmonary congestion and / or systemic congestion) and / or severe reduction in cardiac output (manifested by hypotension, renal failure and / or shock syndrome), and the patient requires urgent treatment or optimization of treatment and hospitalization.

[0107] Sepsis is defined as a life-threatening organ failure caused by dysregulation of the host response to infection (see Singer et al. 2016. JAMA 315(8): 801-810). Organ failure can be identified as a rapid change in the total SOFA score of 2 or more due to infection. The baseline SOFA score can be assumed to be zero in patients who are not known to have pre-existing organ failure. An SOFA score of 2 or more reflects an overall mortality risk of approximately 10% in the general hospital population suspected of infection. Even patients with mild dysfunction may worsen further, highlighting the seriousness of this condition and the need for prompt and appropriate intervention if not already implemented. Sepsis is a life-threatening condition that occurs when the body's response to infection damages its own tissues and organs. Patients suspected of infection who are likely to require a prolonged stay in the ICU or die in the hospital may be rapidly identified at the bedside by qSOFA, i.e., by changes in mental state, systolic blood pressure below 100 mmHg, or respiratory rate above 22 breaths / min.

[0108] Septic shock is a subtype of sepsis in which underlying circulatory and cellular / metabolic abnormalities are so severe that they substantially increase mortality. Patients with septic shock can be identified by the clinical composition of sepsis, which includes persistent hypotension requiring vasopressors to maintain mean arterial pressure (MAP) above 65 mmHg, and serum lactate levels exceeding 2 mmol / L (18 mg / dL) despite adequate resuscitation. In these cases, the hospital mortality rate exceeds 40%.

[0109] Dementia is a clinical syndrome characterized by a set of symptoms and signs that manifest as memory problems, speech disorders, psychological and psychiatric changes, and impairments in daily living activities. Various predisposing factors (sometimes called subtypes) of dementia syndrome include Alzheimer's disease (about 50% of cases), vascular dementia (about 25%), mixed forms of Alzheimer's disease and vascular dementia (25% included in the above), Lewy body dementia (15%), and others (totaling about 5%), including frontotemporal dementia, focal dementia (such as progressive aphasia), subcortical dementia (such as Parkinson's disease dementia), and secondary predisposing factors of dementia syndrome (such as intracranial lesions).

[0110] Alzheimer's disease (AD) is the most common form of dementia. With the aging of the global population, the prevalence of AD is rapidly increasing, and more people are entering a critical period of this age-related disorder. The number of victims in the United States is projected to increase from 5.3 million currently affected to over 13 million by 2050, and the total number of affected individuals worldwide is projected to rise to a staggering 100 million (Alzheimer's Association. 2015 Alzheimer's disease facts and figures. Alzheimers Dement 11: 332-84). Key molecular mechanisms and histopathological features of AD in the brain include the pathological amyloidogenesis of amyloid precursor protein (APP) cleavage and amyloid-beta peptide (Aβ). 1-42 This includes the formation of various β-amyloid species, including its dimers, trimers, oligomers, and subsequent amyloid aggregation and deposition in plaques, abnormal hyperphosphorylation and aggregation of τ proteins, progressive intracellular neurofibrillary degeneration, changes within the congenital immune system, and inflammation, as well as a dynamic chain of biochemical events.

[0111] Approximately 5% of patients develop symptoms before the age of 65 and are characterized as having "early-onset (young-onset) Alzheimer's disease" (EOAD). While the majority of these patients have sporadic episodes, 10–15% have a genetic variant that is generally inherited in an autosomal dominant pattern. Three genes—presenilin 1 and 2 and the amyloid precursor protein (APP) gene—have been suggested to be involved in the development of EOAD. Other candidate genes are under investigation. While the genetic variant tends to begin at age 30 or 40 and follow a rapid course, sporadic EOAD tends to begin after age 50 and generally has a temporal characteristic similar to that of patients with "late-onset Alzheimer's disease" (LOAD).

[0112] Mental state tests assess memory, the ability to solve simple problems, and other cognitive skills. These tests provide an overall assessment of a person's ability to recognize symptoms, know the date, time, and location, remember short lists of words, and perform simple calculations according to instructions. The Mini-Mental State Examination (MMSE) and the Mini-Cognitive Examination (mini-cog) are two commonly used tests. The MMSE, or Folstein test, is a 30-point questionnaire widely used in clinical and research settings to assess cognitive impairment (Pangman, et al. 2000. Applied Nursing Research 13 (4): 209-213; Folstein et al. 1975. Journal of Psychiatric Research. 12 (3): 189-98). The MMSE consists of a series of questions designed to test a patient's various everyday mental skills. The maximum score for the MMSE is 30. A score of 20-24 indicates mild dementia, 13-20 indicates moderate dementia, and less than 12 indicates severe dementia. On average, the MMSE score of Alzheimer's disease patients decreases by approximately 2-4 points each year. The advantages of the MMSE include not requiring special equipment or training to administer, and it is both valid and reliable for the diagnosis and longitudinal assessment of Alzheimer's disease. In the mini-cog, subjects are asked to complete two tasks: memorizing the names of three common objects and repeating them a few minutes later, and drawing a clock face with all 12 digits and a time specified by the examiner in the correct place. The results of this simple test help physicians determine whether further evaluation is necessary.Other testing methods, such as computerized tests like Hodkinson's simplified mental test score (Hodkinson 1972. Age and ageing. 1 (4): 233-8) or the General Practitioner Assessment of Cognition (CoP) and Mental Attributes Profiling System, as well as longer-term, formal testing methods that provide a deeper analysis of specific deficiencies, are also used.

[0113] Mild cognitive impairment (MCI) is a heterogeneous clinical condition with several underlying contributing factors. However, the majority of MCI cases represent a transitional state between healthy aging and very mild Alzheimer's disease (AD) (DeCarli 2003. Lancet Neurol. 2: 15-21). Studies suggest that approximately 10% to 15% of MCI patients are clinically almost certain to progress to AD each year (Markesbery 2010. J Alzheimers Dis. 19: 221-228).

[0114] Alzheimer's disease is typically diagnosed based on the patient's medical history, the medical history of close relatives, and behavioral observations. It is also determined by the presence of characteristic neurological and neuropsychological features and the absence of other cognitive impairments. Advanced medical imaging techniques, such as computed tomography (CT) or magnetic resonance imaging (MRI), and single-photon emission computed tomography (SPECT) or positron emission tomography (PET), can help rule out other brain lesions or subtypes of dementia. Furthermore, these imaging techniques can predict the progression from the prodromal stage (mild cognitive impairment) to Alzheimer's disease. Assessments of intellectual function, including memory tests, can further characterize the disease state. Healthcare institutions have developed diagnostic criteria to facilitate and standardize the physician's diagnostic process. This diagnosis can be confirmed with very high accuracy by autopsy, if brain tissue is available and histologically examineable.

[0115] To date, only symptomatic treatments exist for this disease, and all therapies attempt to balance neurotransmitter dysfunction. Three cholinesterase inhibitors are currently available and approved for the treatment of mild to moderate AD. A further treatment option available for moderate to severe AD is memantine, a non-competitive antagonist of the N-methyl-D-aspartate receptor. Therapies that can halt or at least effectively modify the progression of AD, known as "disease-modifying drugs," are still under extensive research.

[0116] There is an urgent need for new therapies to treat affected patients, prevent, delay, slow the onset of Alzheimer's disease (AD), or improve its symptoms. It is estimated that delaying the onset of the disease by five years would reduce the overall incidence of the disease by almost 50%. Symptomatic treatments are drugs aimed at improving cognitive function or controlling neuropsychiatric symptoms, typically acting through neurotransmitter mechanisms, while disease modification therapies or treatments (DMTs) are drugs that target the underlying pathophysiological mechanisms of AD to prevent, delay, or slow its progression. Currently, there are more than 100 drugs in the supply chain for the development of AD treatments (Cummings et al. 2017. Alzheimer's & Dementia: Translational Research & Clinical Interventions 3: 367-384).

[0117] Lewy body dementia (DLB) is a type of dementia that worsens over time. Further symptoms may include attentional instability, hallucinations, sluggishness, gait disturbance, and rigidity. DLB is the third most common cause of dementia, after Alzheimer's disease and vascular dementia. It typically develops after the age of 50. Approximately 0.1% of people over 65 are affected. Men are generally more susceptible than women. The underlying mechanism involves the formation of Lewy bodies, which are made up of alpha-synuclein protein, in nerve cells. Diagnosis may be suspected based on symptoms, and blood tests and medical imaging are performed to rule out other possible causes. Currently, there is no cure for DLB. Treatment is supportive and attempts to alleviate some of the motor and psychological symptoms associated with the disease. Acetylcholinesterase inhibitors, such as donepezil, may provide some relief. Some motor impairments may improve with levodopa. For a review, see McKeith et al. 2017. Neurology 89: 88-100.

[0118] Vascular dementia (VaD), also known as multiple infarct dementia (MID) and vascular cognitive impairment (VCI), is a type of dementia resulting from problems with blood supply to the brain, typically a series of mild strokes, which leads to a gradual decline in cognitive function. As the term suggests, it refers to a syndrome that results from a complex interaction of risk factors leading to cerebrovascular disease and structural changes in the brain due to stroke or lesions, resulting in cognitive changes. The temporal relationship between stroke and cognitive impairment is necessary for diagnosis. Differentiating between various dementia syndromes can be difficult due to the high frequency and overlap of clinical features and the associated underlying pathology. Alzheimer's disease, in particular, often co-occurs with vascular dementia. Individuals with vascular dementia often present with acute or subacute progressive cognitive impairment, such as gradual mild cognitive impairment, following multiple cerebrovascular events (strokes). For a review, see Venkat et al. 2015. Exp Neurol 272: 97-108.

[0119] Frontotemporal dementia (FTD) is a clinical manifestation of frontotemporal lobar degeneration, characterized by progressive neuronal loss, primarily involving the frontal or temporal lobes, and a typical loss of over 70% of spindle-shaped neurons, while other neuronal types remain intact. FTD accounts for 20% of cases of early-onset dementia. Signs and symptoms typically appear in late adulthood, more commonly between the ages of 55 and 65, and affect men and women almost equally. Common signs and symptoms include significant changes in social and personal behavior, apathy, emotional blunting, and deficits in both expressive and receptive language. Currently, there is no cure for FTD, but there are therapies that can help alleviate symptoms. For a review, see Bott et al. 2014. Neurodegener Dis Manag 4(6): 439-454.

[0120] In one particular embodiment, the anti-ADM antibody or anti-ADM fragment or anti-ADM non-Ig scaffold is bound to the N-terminal portion (amino acids 1-21) of ADM-Gly and / or ADM-NH2:YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO: 14), where the patient does not have dementia or Alzheimer's disease.

[0121] In another specific embodiment, the present application relates to the treatment of critically ill patients suffering from acute diseases or acute symptoms selected from the group including severe infections (such as meningitis, systemic inflammatory response syndrome (SIRS), and sepsis), shock (such as septic shock and cardiogenic shock), acute heart failure (such as acute decompensated heart failure and chronic heart failure with worsening symptoms and signs), myocardial infarction, stroke, and organ dysfunction (such as kidney, liver, heart, and lung failure), wherein ADM-Gly in the circulating ADM-Gly within the patient is converted to A With respect to an anti-adrenomedullin (ADM) antibody, anti-ADM antibody fragment, or anti-ADM non-Ig scaffold used to promote conversion to DM-NH2, the anti-ADM antibody, anti-ADM fragment, or anti-ADM non-Ig scaffold binds to the N-terminal portion (amino acids 1-21) of ADM-Gly and / or ADM-NH2: YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO: 14), where the disease or condition is not dementia or Alzheimer's disease.

[0122] As used herein, organ dysfunction means a medical condition or health state in which an organ fails to perform its expected function. “Organ dysfunction” means organ dysfunction to the extent that normal homeostasis cannot be maintained without external clinical intervention. Such organ dysfunction may relate to organs selected from the group including the kidneys, liver, heart, lungs, and nervous system. In contrast, organ function refers to the expected function of each organ within its physiological range. Those skilled in the art will know the function of each organ in a health examination.

[0123] Organ dysfunction may be defined by a set of organ dysfunction assessment scores (SOFA scores) or their components. The SOFA score, formerly known as the sepsis-associated organ dysfunction assessment score (Singer et al. 2016. JAMA 315(8): 801-10), is used to track the condition of subjects staying in the intensive care unit (ICU) and to determine the degree or rate of organ dysfunction in the subject. The score is based on six different scores, increasing from 0 to 4 for each of the respiratory, cardiovascular, hepatic, coagulation, renal, and nervous systems, reflecting the worsening of organ dysfunction. The evaluation criteria for the SOFA score are described, for example, by Lambden et al. (see Lambden et al. 2019. Critical Care 23: 374 for a review). The SOFA score may traditionally be calculated upon admission to the ICU and every 24 hours thereafter. In particular, the aforementioned organ dysfunction is selected from a group that includes renal impairment, cardiac dysfunction, hepatic dysfunction, or airway dysfunction.

[0124] The rapid SOFA score (quickSOFA or qSOFA) was introduced in February 2016 by the Sepsis-3 group as a simplified version of the SOFA score, and the first method to identify patients at high risk of poor outcomes due to infection (Angus et al. 2016. Critical Care Medicine. 44 (3): e113-e121). qSOFA dramatically simplifies the SOFA score by including only three clinical criteria and including "any change in mental state" instead of requiring a GCS score of less than 15. qSOFA can be concise, rapid, and repeated sequentially for each patient. Scores range from 0 to 3 points. 1 point is awarded for hypotension (SBP ≤ 100 mmHg), high respiratory rate (≥ 22 breaths / min), and change in mental state (GCS ≤ 15). The presence of qSOFA scores of 2 or higher close to the onset of infection was associated with a higher risk of death or prolonged stay in the intensive care unit. These outcomes are more common in infected patients who may have sepsis than in infected patients without complications. Based on these findings, the Third International Consensus Definitions for Sepsis recommends qSOFA as a concise input request to identify infected patients likely to be sepsis outside the ICU (Seymour et al. 2016. JAMA 315(8): 762-774).

[0125] In this application, the term “promoting conversion” is defined as an increase in the conversion of glycinated adrenomedullin (ADM-Gly) to mature ADM (ADM-NH2) within a given period of time, in the presence of an anti-ADM antibody or anti-adrenomedullin antibody fragment or an anti-ADM non-Ig scaffold.

[0126] Furthermore, in one embodiment of the present invention, the anti-adrenomedullin (ADM) antibody, anti-adrenomedullin antibody fragment, or anti-ADM non-Ig scaffold is monospecific.

[0127] Monospecificity means that the antibody, antibody fragment, or non-Ig scaffold binds to one specific region within the target ADM that comprises at least four amino acids. The monospecific antibody, fragment, or non-Ig scaffold according to the present invention is an antibody, fragment, or non-Ig scaffold that all have affinity for the same antigen. While monoclonal antibodies are monospecific, monospecific antibodies may also be produced by methods other than generating them from common germ cells.

[0128] The anti-ADM antibody or antibody fragment that binds to ADM, or the non-Ig scaffold that binds to ADM, may be a non-neutralizing anti-ADM antibody or antibody fragment that binds to ADM, or a non-neutralizing non-Ig scaffold that binds to ADM.

[0129] The antibody or fragment according to the present invention is a protein comprising one or more polypeptides substantially encoded by an immunoglobulin gene that specifically binds to an antigen. Recognized immunoglobulin genes include the constant region genes of κ, λ, α (IgA), γ (IgG1, IgG2, IgG3, IgG4), δ (IgD), ε (IgE), and μ (IgM), as well as numerous immunoglobulin variable region genes. Full-length immunoglobulin light chains are generally about 25 kd or 214 amino acids in length.

[0130] Full-length immunoglobulin heavy chains are generally about 50 kd or 446 amino acids long. The light chain is encoded by a variable region gene at the NH2 terminus (about 110 amino acids long) and a constant region gene at the COOH terminus (κ or λ). The heavy chain is similarly encoded by a variable region gene (about 116 amino acids long) and one of the other constant region genes.

[0131] The basic structural unit of an antibody is generally a tetramer consisting of two identical pairs of immunoglobulin chains, each pair having one light chain and one heavy chain. In each pair, the variable regions of the light and heavy chains bind to the antigen, while the constant region mediates effector function. Immunoglobulins also exist in various other forms, such as Fv, Fab, and (Fab')2, as well as in bifunctional hybrid antibodies and single chains (e.g., Lanzavecchia et al. 1987. Eur. J. Immunol. 17:105; Huston et al. 1988. Proc. Natl. Acad. Sci. USA, 85: 5879-5883; Bird et al. 1988. Science 242: 423-426; Hood et al. 1984, Immunology, Benjamin, NY, 2nd ed.; Hunkapiller and Hood 1986. Nature 323: 15-16). The variable regions of the light or heavy chains of immunoglobulins include a framework region interrupted by three hypervariable regions, also known as complementarity-determining regions (CDRs) (see Sequences of Proteins of Immunological Interest, E. Kabat et al. 1983, US Department of Health and Human Services). As mentioned above, CDRs are primarily responsible for binding to the antigen's epitope. Immune complexes are antibodies such as monoclonal antibodies, chimeric antibodies, humanized antibodies or human antibodies, or functional antibody fragments that are specifically bound to an antigen.

[0132] Chimeric antibodies are antibodies in which the light and heavy chain genes are typically constructed by genetic engineering from the variable and constant regions of immunoglobulins belonging to various species. For example, the variable compartment of a gene derived from a mouse monoclonal antibody can bind to human constant compartments such as κ and γ1 or γ3. Thus, in one example, a therapeutic chimeric antibody is a variable or antigen-binding domain derived from a mouse antibody and a constant or effector domain derived from a human antibody, but other mammalian species may be used, or the variable region may be generated by molecular technology. Methods for generating chimeric antibodies are well known in the art; see, for example, US Patent No. 5,807,715. "Humanized" immunoglobulins are immunoglobulins containing a human framework region and one or more CDRs derived from non-human immunoglobulins (such as mouse, rat, or synthetic). The non-human immunoglobulin providing the CDRs is called the "donor," and the human immunoglobulin providing the framework is called the "receptor." In one embodiment, all CDRs are derived from donor immunoglobulins in the humanized immunoglobulin. The presence of a constant region is not essential, but if present, it must be substantially identical to the constant region of human immunoglobulin, i.e., at least about 85-90%, e.g., about 95% or more identical. Therefore, perhaps all parts of humanized immunoglobulin, except for the CDR, are substantially identical to the corresponding parts of the native human immunoglobulin sequence. A "humanized antibody" is an antibody containing humanized light chain and humanized heavy chain immunoglobulins. A humanized antibody binds to the same antigen as the donor antibody that provides the CDR. The receptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions by amino acids taken from the donor framework. A humanized antibody or other monoclonal antibody may have additional conserved amino acid substitutions that do not substantially affect antigen binding or other immunoglobulin functions. Exemplary conserved substitutions include those of gly, ala, val, ile, leu; asp, glu; asn, gln; ser, thr; lys, arg; and phe, tyr. Humanized immunoglobulins can be constructed by genetic engineering (see, for example, US5,585,089).Human antibodies are antibodies whose light and heavy chain genes are derived from humans. Human antibodies may be produced using methods known in the art. Human antibodies may also 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 cells or fusion cell tumor cells to produce trioma cells. Human antibodies may also be produced by phage display (see, e.g., WO91 / 17271; WO92 / 001047; WO92 / 20791) or selected from a human combined monoclonal antibody library (see Morphosys website). Human antibodies may also be produced using genetically modified animals possessing human immunoglobulin genes (see, e.g., WO93 / 12227; WO91 / 10741).

[0133] Therefore, anti-ADM antibodies may have forms known in the art. Examples include human antibodies, monoclonal antibodies, humanized antibodies, chimeric antibodies, and CDR-transplanted antibodies. In preferred embodiments, the antibodies according to the present invention are antibody fragments comprising at least the F variable domain of a heavy chain and / or light chain, such as recombinantly produced antibodies like IgG, which is a typical full-length immunoglobulin, or chemically bound antibodies (fragment antigen binding), and these antibodies include, but are not limited to, Fab minibodies, single-chain Fab antibodies, monovalent Fab antibodies with epitope tags such as Fab-V5Sx2; bivalent Fab (mini-antibodies) dimerized with a CH3 domain; bivalent or polyvalent Fab formed via multimerization with the help of heterologous domains, such as dimerization of a dHLX domain such as Fab-dHLX-FSx2; F(ab')2 fragments, scFv fragments, multimerized polyvalent and / or polyspecific scFv fragments, bivalent and / or bispecific antibodies, BITE® (bispecific T cell engager), trifunctional antibodies, polyvalent antibodies; and single-domain antibodies, such as immunoglobulins from camelid or fish and antibodies from numerous other species.

[0134] In addition to anti-ADM antibodies, other biopolymer scaffolds that form complexes with target molecules are well known in the art and have been used to produce highly target-specific biopolymers. Examples include aptamers, spiegelmers, antikalins, and conotoxins. See Figures 1a, 1b, and 1c for descriptions of antibody forms.

[0135] In a preferred embodiment, the anti-ADM antibody form is selected from the group comprising Fv fragment, scFv fragment, Fab fragment, scFab fragment, F(ab)2 fragment, and scFv-Fc fusion protein. In another preferred embodiment, the antibody form is selected from the group comprising bioavailability-optimized complexes such as scFab fragment, Fab fragment, scFv fragment, and PEGylated fragment. One of the most preferred forms is the scFab form.

[0136] Non-Ig scaffolds may also be protein scaffolds, and since these scaffolds can bind to ligands or antigens, they may be used as antibody mimetic materials. Non-Ig scaffolds include tetranectin-based non-Ig scaffolds (e.g., disclosed in US2010 / 0028995), fibronectin scaffolds (e.g., disclosed in EP 1 266 025); lipocalin-based scaffolds (e.g., disclosed in WO2011 / 154420); ubiquitin scaffolds (e.g., described in WO2011 / 073214); transferrin scaffolds (e.g., disclosed in US2004 / 0023334); protein A scaffolds (e.g., disclosed in EP2 231 860); ankyrin repeat-based scaffolds (e.g., disclosed in WO2010 / 060748); microprotein scaffolds, preferably microprotein scaffolds that form cysteine ​​knots (e.g., disclosed in EP2314308); Fyn The group may be selected from the SH3 domain-based scaffolding (e.g., disclosed in WO2011 / 023685), the EGFR-A domain-based scaffolding (e.g., disclosed in WO2005 / 040229), and the Knitz domain-based scaffolding (e.g., disclosed in EP 1 941 867).

[0137] In one embodiment of the present invention, the anti-ADM antibody according to the present invention may be produced by synthesizing a fragment of ADM as an antigen, as outlined in Example 1. Subsequently, a binder to the fragment is identified using the method described below or other methods known in the art.

[0138] Humanization of mouse antibodies may be carried out according to the following procedure: For humanization of mouse-derived antibodies, the antibody sequence is analyzed for structural interactions between the complementarity-determining region (CDR) and the antigen-framework region (FR). Based on structural modeling, an appropriate human-derived FR is selected, and the mouse CDR sequence is transplanted into the human FR. Amino acid sequence mutations of the CDR or FR may be introduced to restore the structural interactions abolished by seed switching of the FR sequence. This restoration of structural interactions may be achieved by a random method using a phage display library or by a direct method induced by molecular modeling (Almagro and Fransson 2008. Front Biosci. 13: 1619-33).

[0139] In another preferred embodiment, the anti-ADM antibody, anti-ADM antibody fragment, or anti-ADM non-Ig scaffold is a full-length antibody, antibody fragment, or non-Ig scaffold.

[0140] In a preferred embodiment, an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold can be directed to and bound to an epitope of at least four or at least five amino acids in the N-terminal and / or central region (amino acids 1-42) of ADM-Gly and / or ADM-NH2 (SEQ ID NO: 23).

[0141] Epitopes, also known as antigenic determinants, are parts of antigens (such as peptides and proteins) that are recognized by the immune system, particularly by antibodies. For example, an epitope is a specific part of an antigen to which an antibody binds. Some antibodies that bind to an epitope are called paratopes. Protein antigen epitopes are classified into two types based on their structure and interaction with paratopes: structural epitopes and linear epitopes.

[0142] Chain-like, or continuous, epitopes are epitopes recognized by antibodies due to their chain-like sequence of amino acids, i.e., their primary structure, and are formed by a three-dimensional structure adopted through the interaction of continuous amino acid residues. Structural epitopes and chain-like epitopes interact with paratopes based on the three-dimensional structure adopted by the epitope, which is determined by the surface features of the epitope residues involved and the shape or tertiary structure of other compartments of the antigen. Structural epitopes are formed by a three-dimensional structure adopted through the interaction of discontinuous amino acid residues.

[0143] In certain embodiments, an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold is directed to and can bind to at least four or at least five amino acids within the N-terminal and / or central region (amino acids 1-42) of ADM-Gly and / or ADM-NH2: YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVA (SEQ ID NO: 23), and binding requires the free N-terminus (amino acid 1) of ADM and / or ADM-Gly.

[0144] In another specific embodiment of the present invention, an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold is directed to and can bind to at least four or at least five amino acids in the N-terminal and / or central region (amino acids 1-42) of ADM-Gly and / or ADM-NH2: YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVA (SEQ ID NO: 23), but not to the free N-terminus (amino acid 1) of ADM and / or ADM-Gly.

[0145] In a particular embodiment of the present invention, an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold can be directed to and bound to at least four or at least five amino acids in the N-terminal portion (amino acids 1-21) of ADM-Gly and / or ADM-NH2:YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO: 14).

[0146] In a particular embodiment of the present invention, an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold is directed to and can bind to at least four or at least five amino acids in the N-terminus (amino acids 1-21) of ADM-Gly and / or ADM-NH2 (SEQ ID NO: 14), and binding requires the free N-terminus (amino acid 1) of ADM and / or ADM-Gly.

[0147] In a particular embodiment of the present invention, an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold is directed to and can bind to at least four or at least five amino acids in the N-terminus (amino acids 1-21) of ADM-Gly and / or ADM-NH2 (SEQ ID NO: 14), but does not bind to the free N-terminus (amino acid 1) of ADM and / or ADM-Gly.

[0148] In another preferred embodiment, the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold is directed to and can bind to at least four or at least five amino acids in the N-terminal region (amino acids 1-14) of ADM-Gly and / or ADM-NH2 (SEQ ID NO: 25).

[0149] In another preferred embodiment, the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold is directed to and can bind to at least four or at least five amino acids in the N-terminus (amino acids 1-14):YRQSMNNFQGLRSF (SEQ ID NO: 25) of ADM-Gly and / or ADM-NH2, and binding requires the free N-terminus (amino acid 1) of ADM and / or ADM-Gly.

[0150] In another preferred embodiment, the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold is directed to and can bind to at least four or at least five amino acids in the N-terminus (amino acids 1-14) of ADM-Gly and / or ADM-NH2:YRQSMNNFQGLRSF (SEQ ID NO: 25), but does not bind to the free N-terminus (amino acid 1) of ADM and / or ADM-Gly.

[0151] In another preferred embodiment, the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold is directed to and can bind to at least four or at least five amino acids in the N-terminal region (amino acids 1-10) of ADM-Gly and / or ADM-NH2 (SEQ ID NO: 26).

[0152] In another preferred embodiment, the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold is directed to and can bind to preferably at least four or at least five amino acids in the N-terminus (amino acids 1-10):YRQSMNNFQG (SEQ ID NO: 26) of ADM-Gly and / or ADM-NH2, and binding requires the free N-terminus (amino acid 1) of ADM and / or ADM-Gly.

[0153] In another preferred embodiment, the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold is directed to and can bind to at least four or at least five amino acids in the N-terminus (amino acids 1-10):YRQSMNNFQG (SEQ ID NO: 26) of ADM-Gly and / or ADM-NH2, but does not bind to the free N-terminus (amino acid 1) of ADM and / or ADM-Gly.

[0154] In a very specific embodiment, the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold is directed to and can bind to at least four or at least five amino acids in the N-terminus (amino acids 1-6):YRQSMN (SEQ ID NO: 27) of ADM-Gly and / or ADM-NH2, and binding requires the free N-terminus (amino acid 1) of ADM and / or ADM-Gly.

[0155] In another, very specific embodiment of the present invention, an anti-ADM antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold recognizes and binds to the N-terminus (amino acid 1) of ADM-Gly and / or ADM-NH2. The N-terminus means that amino acid 1, i.e., "Y" in SEQ ID NOs. 14, 20, 22, 23, 25, 26, and 27, is essential for antibody binding. This antibody or fragment or scaffold does not bind to N-terminally extended ADM, N-terminally modified ADM, or N-terminally degraded ADM-Gly and / or ADM-NH2. This means that when the N-terminus of ADM is free, the anti-ADM antibody or anti-ADM antibody fragment or non-Ig scaffold binds only to regions within the sequence of ADM-Gly and / or ADM-NH2. Anti-ADM antibodies, anti-ADM antibody fragments, or anti-ADM non-Ig scaffolds do not appear to bind to regions within the ADM-Gly and / or ADM-NH2 sequences if the sequence is included, for example, within pro-ADM.

[0156] For clarity, the numbers in parentheses in a specific region of ADM, such as "N-terminus (amino acids 1-21)," indicate that the N-terminus of ADM consists of amino acids 1-21 of the ADM-Gly and / or ADM-NH2 sequence, as will be known to those skilled in the art.

[0157] In another specific embodiment, the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold can be directed to and bound to at least four or at least five amino acids within the central region (amino acids 21-42) of ADM-Gly and / or ADM-NH2.

[0158] In another specific embodiment, the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold can be directed to and bound to at least four or at least five amino acids in the central region (amino acids 21-32) of ADM-Gly and / or ADM-NH2: CTVQKLAHQIYQ (SEQ ID NO: 15).

[0159] In another specific embodiment, the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold can be directed to and bound to at least four or at least five amino acids in the central region (amino acids 27-39):AHQIYQFTDKDKD (SEQ ID NO: 49) of ADM-Gly and / or ADM-NH2.

[0160] In another specific embodiment of the present invention, the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold presented herein does not bind to the C-terminal portion of ADM, i.e., amino acids 43-52 (SEQ ID NO: 24) of ADM.

[0161] In a particular embodiment, it is preferable to use an anti-ADM antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold according to the present invention, wherein the anti-adrenomedullin antibody or anti-adrenomedullin antibody fragment or non-Ig scaffold increases the ADM-NH2 concentration or ADM-NH2 immunoreactivity in serum, blood, or plasma by at least 10%, preferably at least 50%, more preferably more than 50%, and most preferably more than 100%.

[0162] A measurement method that can be used to measure the half-life (half-retention time) of adremedullin in serum, blood, and plasma is described in Example 3.

[0163] In certain embodiments of the present invention, the antibody is a monoclonal antibody or a fragment thereof. In one embodiment of the present invention, the anti-ADM antibody or anti-ADM antibody fragment is a human antibody or a humanized antibody, or derived therefrom. In one particular embodiment, one or more (mouse) CDRs are transplanted onto a human antibody or antibody fragment ("humanized").

[0164] In one aspect, the subject of the present invention is a humanized CDR implantation antibody or antibody fragment thereof, wherein the antibody recognizes or binds to the N-terminus of ADM-Gly and / or ADM-NH2, and in patient use to promote the conversion from ADM-Gly to ADM-NH2, the free N-terminus (amino acid 1) of ADM-Gly and / or ADM-NH2 is required for binding, and here the humanized CDR implantation antibody or antibody fragment comprises an antibody heavy chain (H chain) having the following sequence: Sequence ID 1: GYTFSRYW Sequence ID 2: ILPGSGST and / or Sequence ID 3: TEGYEYDGFDY, and / or further comprising an antibody light chain (L chain) containing the following sequence: Sequence ID 4: QSIVYSNGNTY Array "RVS" (not part of the array list): RVS and / or Sequence ID 5: FQGSHIPYT.

[0165] A particular embodiment of the present invention recognizes or binds to the N-terminal region (amino acids 1-21) of ADM-Gly and / or ADM-NH2:YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO: 14) and, for use in patients to promote the conversion of ADM-Gly to ADM-NH2, requires the free N-terminal region (amino acid 1) of ADM-Gly and / or ADM-NH2 for binding, where the heavy chain comprises at least one CDR selected from the group including the following sequences: Sequence ID 1: GYTFSRYW Sequence ID 2: ILPGSGST Sequence ID 3: TEGYEYDGFDY, Furthermore, the light chain contains at least one CDR selected from the group including the following sequences: Sequence ID 4: QSIVYSNGNTY Array "RVS" (not part of the array list): RVS Sequence ID 5 FQGSHIPYT.

[0166] In more specific embodiments of the present invention, the subject of the present invention is a humanized and / or human monoclonal antibody or an antibody fragment thereof, wherein the antibody recognizes or binds to the N-terminal portion (amino acids 1-21) of ADM-Gly and / or ADM-NH2: YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO: 14), and in patient use to promote the conversion of ADM-Gly to ADM-NH2, the free N-terminal portion (amino acid 1) of ADM-Gly and / or ADM-NH2 is required for binding, and the heavy chain comprises the following sequence: Sequence ID 1: GYTFSRYW Sequence ID 2: ILPGSGST Sequence ID 3: TEGYEYDGFDY, Furthermore, the light chain contains the following sequence: Sequence ID 4: QSIVYSNGNTY Array "RVS" (not part of the array list): RVS Sequence ID 5: FQGSHIPYT.

[0167] In a very specific embodiment, the anti-ADM antibody has a sequence selected from the group including SEQ ID NOs: 6, 7, 8, 9, 10, 11, 12, 13, 35, and 36.

[0168] The anti-ADM antibody, anti-ADM antibody fragment, or anti-ADM non-Ig scaffold according to the present invention has an affinity constant of 10 -7 M or more, preferably 10 -8 M more, more 10 -9 M or more, most preferably 10 -10It exhibits affinity for human ADM-Gly and / or ADM-NH2 such that the affinity constant is greater than M. Those skilled in the art will know that administering higher doses of the compound may compensate for lower affinity, and that this measure is not outside the scope of the claims of the present invention. This affinity constant can be measured according to the method described in Example 1.

[0169] The subject of the present invention is a human or humanized monoclonal antibody or fragment that binds to ADM-Gly and / or ADM-NH2, wherein the antibody or fragment binds to the N-terminus (amino acids 1-21) of ADM-Gly and / or ADM-NH2: YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO: 14), and for use in patients to promote conversion from ADM-Gly to ADM-NH2, the free N-terminus (amino acid 1) of ADM-Gly and / or ADM-NH2 is required for binding, wherein the antibody or fragment comprises a sequence selected from the group comprising the following sequences: Sequence number 6 (AM-VH-C): [ka] Sequence ID 7 (AM-VH1): [ka] Sequence ID 8 (AM-VH2-E40): [ka] Sequence ID 9 (AM-VH3-T26-E55): [ka] Sequence ID 10 (AM-VH4-T26-E40-E55): [ka] Sequence ID 11(AM-VL-C): [ka] Sequence ID 12 (AM-VL1): [ka] Sequence ID 13 (AM-VL2-E40): [ka] Sequence ID 35 (heavy chain HAM8101): [ka] Sequence ID 36 (Light Chain HAM8101) [ka]

[0170] The subject of the present invention is further a human and / or humanized monoclonal antibody or fragment that binds to ADM-Gly and / or ADM-NH2, wherein the antibody or fragment binds to the N-terminus (amino acids 1-21) of ADM-Gly and / or ADM-NH2: YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO: 14), and for use in patients to promote conversion from ADM-Gly to ADM-NH2, the free N-terminus (amino acid 1) of ADM-Gly and / or ADM-NH2 is required for binding, where the antibody or fragment comprises the following sequence as a heavy chain: Sequence ID 35: [ka] Furthermore, the light chain contains the following sequence. Sequence ID 36: [ka]

[0171] In a particular embodiment of the present invention, the antibody comprises the following sequence as a heavy chain: Sequence ID 35: [ka] Alternatively, the above sequence may contain a sequence having more than 95%, preferably more than 98%, preferably more than 99% identity, and may also contain the following sequence as a light chain: Sequence ID 36: [ka] Alternatively, the above sequence may include a sequence having more than 95%, preferably more than 98%, and preferably more than 99% identity.

[0172] A pairwise sorting test was performed to evaluate the identity between two amino acid sequences. Identity is defined as the percentage of amino acids that directly match in the sorting.

[0173] The subject of the present invention is a pharmaceutical formulation comprising an antibody, fragment, or scaffold according to the present invention, for use in the intervention and treatment of congestion in patients.

[0174] The subject of the present invention is a pharmaceutical formulation comprising an antibody, fragment, or scaffold according to the present invention for use in a patient to promote the conversion from ADM-Gly to ADM-NH2, the patient being a critically ill patient suffering from an acute disease or acute condition. The acute disease or condition is selected from the group including severe infections (such as meningitis, systemic inflammatory response syndrome (SIRS), and sepsis), shock (such as septic shock and cardiogenic shock), acute heart failure (such as acute decompensated heart failure and chronic heart failure with exacerbation of symptoms and signs), myocardial infarction, stroke, organ failure (such as kidney, liver, heart, and lung), or dementia.

[0175] The subject of the present invention is a pharmaceutical formulation for use in the intervention and treatment of congestion in patients according to the present invention, the pharmaceutical formulation being a solution, preferably a ready-to-use solution.

[0176] The subject of this invention is a pharmaceutical formulation used for intervention and treatment of congestion in patients according to the present invention, the pharmaceutical formulation being in a lyophilized state.

[0177] The subject of this invention is a pharmaceutical formulation used for intervention and treatment of congestion in patients according to the present invention, the pharmaceutical formulation being administered intramuscularly.

[0178] The subject of this invention is a pharmaceutical formulation used for intervention and treatment of congestion in patients according to the present invention, the pharmaceutical formulation being administered intravascularly.

[0179] The subject of this invention is a pharmaceutical formulation used for intervention and treatment of congestion in patients according to the present invention, the pharmaceutical formulation being administered by infusion.

[0180] The subject of the present invention is a pharmaceutical formulation for use in the intervention and treatment of congestion in patients according to the present invention, the pharmaceutical formulation being administered systemically.

[0181] In addition to the above description, the following sequentially numbered embodiments provide further specific aspects of the present invention: 1. In the treatment of a critically ill patient suffering from an acute disease or acute symptoms selected from the group including severe infection (meningitis, systemic inflammatory response syndrome (SIRS), sepsis, etc.), shock (septic shock, cardiogenic shock, etc.), acute heart failure (acute decompensated heart failure, chronic heart failure with worsening symptoms and signs, etc.), myocardial infarction, stroke, organ failure (kidney, liver, heart, lung, etc.), or dementia, an anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold used to promote the conversion of ADM-Gly to ADM-NH2 in circulating ADM-Gly within the patient, wherein the patient has proadrenomedullin or in a body fluid sample. The pro-adrenomedullin or its fragment is characterized by a ratio of the fragment to ADM-NH2 (SEQ ID NO: 20) exceeding a specific threshold, and the pro-adrenomedullin or its fragment is selected from the group consisting of PAMP (SEQ ID NO: 32), MR-proADM (SEQ ID NO: 33), ADM-Gly (SEQ ID NO: 21), and CT-proADM (SEQ ID NO: 34), and the anti-ADM antibody or anti-ADM fragment or anti-ADM non-Ig scaffold binds to the N-terminal and / or central region (amino acids 1-42) of ADM-Gly and / or ADM-NH2: YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVA (SEQ ID NO: 23).

[0182] 2. An anti-adrenomedullin (ADM) antibody, anti-ADM antibody fragment, or anti-ADM non-Ig scaffold used to promote the conversion of ADM-Gly to ADM-NH2 in circulating ADM-Gly within a critically ill patient suffering from an acute disease or acute symptoms selected from the group including severe infections (such as meningitis, systemic inflammatory response syndrome (SIRS), and sepsis), shock (such as septic shock and cardiogenic shock), acute heart failure (such as acute decompensated heart failure and chronic heart failure with worsening of symptoms and signs), myocardial infarction, stroke, organ dysfunction (such as kidney, liver, heart, and lung), or dementia, as described in Embodiment 1, wherein the anti-adrenomedullin (ADM) antibody, anti-ADM antibody fragment, or anti-ADM non-Ig scaffold binds to the N-terminal portion (amino acids 1-21):YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO: 14) of ADM-Gly and / or ADM-NH2.

[0183] 3. An anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold used to promote the conversion of ADM-Gly to ADM-NH2 in circulating ADM-Gly within a critically ill patient suffering from an acute disease or acute symptoms selected from the group including severe infections (such as meningitis, systemic inflammatory response syndrome (SIRS), and sepsis) as described in Embodiments 1 and 2, shock (such as septic shock and cardiogenic shock), acute heart failure (such as acute decompensated heart failure and chronic heart failure with exacerbation of symptoms and signs), myocardial infarction, stroke, organ dysfunction (such as kidney, liver, heart, and lung), or dementia, wherein the anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold recognizes and binds to the N-terminus (amino acid 1) of ADM-Gly and / or ADM-NH2.

[0184] 4. An anti-adrenomedullin (ADM) antibody, anti-ADM antibody fragment, or anti-ADM non-Ig scaffold used to promote the conversion of ADM-Gly to ADM-NH2 in circulating ADM-Gly within a critically ill patient suffering from an acute disease or acute symptoms selected from the group including severe infections (such as meningitis, systemic inflammatory response syndrome (SIRS), and sepsis), shock (such as septic shock and cardiogenic shock), acute heart failure (such as acute decompensated heart failure and chronic heart failure with exacerbation of symptoms and signs), myocardial infarction, stroke, organ dysfunction (such as kidney, liver, heart, and lung), or dementia, as described in Embodiment 1, wherein the anti-adrenomedullin (ADM) antibody, anti-ADM antibody fragment, or anti-ADM non-Ig scaffold binds to the central region (amino acids 21-42): CTVQKLAHQIYQFTDKDKDNVA (SEQ ID NO: 48) of ADM-Gly and / or ADM-NH2.

[0185] 5. An anti-adrenomedullin (ADM) antibody, anti-ADM antibody fragment, or anti-ADM non-Ig scaffold used to promote the conversion of ADM-Gly to ADM-NH2 in circulating ADM-Gly within a critically ill patient suffering from an acute disease or acute symptoms selected from the group including severe infections (such as meningitis, systemic inflammatory response syndrome (SIRS), and sepsis), shock (such as septic shock and cardiogenic shock), acute heart failure (such as acute decompensated heart failure and chronic heart failure with exacerbation of symptoms and signs), myocardial infarction, stroke, organ dysfunction (such as kidney, liver, heart, and lung), or dementia, as described in Embodiment 4, wherein the anti-adrenomedullin (ADM) antibody, anti-ADM antibody fragment, or anti-ADM non-Ig scaffold binds to the central region (amino acids 21-32):CTVQKLAHQIYQ (SEQ ID NO: 15) of ADM-Gly and / or ADM-NH2.

[0186] 6. An anti-adrenomedullin (ADM) antibody, an anti-ADM antibody fragment, or an anti-ADM non-Ig scaffold used to promote the conversion of circulating ADM-Gly to ADM-NH2 in a severely ill patient suffering from dementia as described in Embodiment 1, wherein the anti-ADM antibody, anti-ADM fragment, or anti-ADM non-Ig scaffold binds to the central region (amino acids 21-42) of ADM-Gly and / or ADM-NH2: CTVQKLAHQIYQFTDKDKDNVA (SEQ ID NO: 48).

[0187] 7. An anti-adrenomedullin (ADM) antibody, an anti-ADM antibody fragment, or an anti-ADM non-Ig scaffold used to promote the conversion of circulating ADM-Gly to ADM-NH2 in a severely ill patient suffering from dementia as described in Embodiment 6, wherein the anti-ADM antibody, anti-ADM fragment, or anti-ADM non-Ig scaffold binds to the central region (amino acids 21-32):CTVQKLAHQIYQ (SEQ ID NO: 15) of ADM-Gly and / or ADM-NH2.

[0188] 8. An anti-adrenomedullin (ADM) antibody, an anti-ADM antibody fragment, or an anti-ADM non-Ig scaffold used to promote the conversion of circulating ADM-Gly to ADM-NH2 in a severely ill patient suffering from dementia as described in Embodiment 6, wherein the anti-ADM antibody, anti-ADM fragment, or anti-ADM non-Ig scaffold binds to the central region (amino acids 27-39): AHQIYQFTDKDKD (SEQ ID NO: 49) of ADM-Gly and / or ADM-NH2.

[0189] 9. In the treatment of a critically ill patient suffering from an acute disease or acute symptoms selected from the group including severe infections (meningitis, systemic inflammatory response syndrome (SIRS), sepsis, etc.), shock (septic shock, cardiogenic shock, etc.), acute heart failure (acute decompensated heart failure, chronic heart failure with worsening of symptoms and signs, etc.), myocardial infarction, stroke, organ dysfunction (kidney, liver, heart, lung, etc.), or dementia as described in Embodiments 1 to 8, an anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold used to promote the conversion of ADM-Gly to ADM-NH2 in circulating ADM-Gly within the patient, wherein the concentration of pro-adrenomedullin or its fragments, consisting of PAMP (SEQ ID NO: 32), MR-proADM (SEQ ID NO: 33), ADM-Gly (SEQ ID NO: 21), and CT-proADM (SEQ ID NO: 34), and the concentration of ADM-NH2 (SEQ ID NO: 20) are measured in a body fluid sample of the patient.

[0190] 10. Treatment of critically ill patients suffering from acute diseases or acute symptoms selected from the group including severe infections (such as meningitis, systemic inflammatory response syndrome (SIRS), and sepsis), shock (such as septic shock and cardiogenic shock), acute heart failure (such as acute decompensated heart failure and chronic heart failure with worsening of symptoms and signs), myocardial infarction, stroke, organ dysfunction (such as kidney, liver, heart, and lung), or dementia, as described in Embodiments 1 to 9, wherein the conversion of ADM-Gly to ADM-NH2 in circulating ADM-Gly within the patient is promoted. An anti-adrenomedullin (ADM) antibody, anti-ADM antibody fragment, or anti-ADM non-Ig scaffold is used for the purpose of treating the patient with the anti-ADM antibody, anti-ADM antibody fragment, or anti-ADM non-Ig scaffold if the concentration ratio of ADM-Gly (SEQ ID NO: 21) to ADM-NH2 (SEQ ID NO: 20) is measured in a body fluid sample of the patient and the ADM-Gly / ADM-NH2 ratio exceeds a specific threshold of 1 to 10, preferably 1.5 to 7.5, preferably 2 to 5, and most preferably 2.5.

[0191] 11. An anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold used to promote the conversion of ADM-Gly to ADM-NH2 in circulating ADM-Gly within the patient, for the treatment of a critically ill patient suffering from an acute disease or acute symptoms selected from the group including severe infections (such as meningitis, systemic inflammatory response syndrome (SIRS), sepsis), shock (such as septic shock, cardiogenic shock), acute heart failure (such as acute decompensated heart failure, chronic heart failure with worsening symptoms and signs), myocardial infarction, stroke, organ dysfunction (such as kidney, liver, heart, lung), or dementia, wherein the patient's body fluid sample is selected from the group including blood, serum, plasma, urine, cerebrospinal fluid (CSF), and saliva.

[0192] 12. An anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold used to promote the conversion of ADM-Gly to ADM-NH2 in circulating ADM-Gly within a critically ill patient suffering from an acute disease or acute symptoms selected from the group including severe infections (such as meningitis, systemic inflammatory response syndrome (SIRS), and sepsis) as described in Embodiment 11, shock (such as septic shock and cardiogenic shock), acute heart failure (such as acute decompensated heart failure and chronic heart failure with exacerbation of symptoms and signs), myocardial infarction, stroke, organ dysfunction (such as kidney, liver, heart, and lung), or dementia, wherein the sample is selected from the group including human citrate plasma, heparin plasma, and EDTA plasma.

[0193] 13. Treatment of a critically ill patient suffering from an acute disease or acute symptoms selected from the group including severe infections (meningitis, systemic inflammatory response syndrome (SIRS), sepsis, etc.), shock (septic shock, cardiogenic shock, etc.), acute heart failure (acute decompensated heart failure, chronic heart failure with worsening of symptoms and signs, etc.), myocardial infarction, stroke, organ dysfunction (kidney, liver, heart, lung, etc.), or dementia, as described in Embodiments 1 to 12, wherein the patient undergoes a transformation from ADM-Gly to ADM-NH2 in circulating ADM-Gly within the patient. An anti-adrenomedullin (ADM) antibody, an anti-ADM antibody fragment, or an anti-ADM non-Ig scaffold used to promote conversion, wherein an immunoassay is used to measure the ratio of proadrenomedullin or its fragment to ADM-NH2, and the proadrenomedullin or its fragment is selected from the group consisting of PAMP (SEQ ID NO: 32), MR-proADM (SEQ ID NO: 33), ADM-Gly (SEQ ID NO: 21), and CT-proADM (SEQ ID NO: 34).

[0194] 14. An anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold used to promote the conversion of ADM-Gly to ADM-NH2 in circulating ADM-Gly within a critically ill patient suffering from an acute disease or acute symptoms selected from the group including severe infections (such as meningitis, systemic inflammatory response syndrome (SIRS), and sepsis) as described in Embodiment 13, shock (such as septic shock and cardiogenic shock), acute heart failure (such as acute decompensated heart failure and chronic heart failure with exacerbation of symptoms and signs), myocardial infarction, stroke, organ dysfunction (such as kidney, liver, heart, and lung), or dementia, wherein the immunoassay method is a sandwich immunoassay method, preferably a fully automated immunoassay method.

[0195] 15. An anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold used to promote the conversion of ADM-Gly to ADM-NH2 in circulating ADM-Gly within a critically ill patient suffering from an acute disease or acute symptoms selected from the group including severe infections (meningitis, systemic inflammatory response syndrome (SIRS), sepsis, etc.), shock (septic shock, cardiogenic shock, etc.), acute heart failure (acute decompensated heart failure, chronic heart failure with worsening of symptoms and signs, etc.), myocardial infarction, stroke, organ dysfunction (kidney, liver, heart, lung, etc.), or dementia, as described in Embodiments 1 to 14, wherein the ADM-NH2 in a healthy subject can be quantified by the measurement sensitivity of the measurement method for detecting ADM-NH2, the sensitivity of which is less than 70 pg / mL, preferably less than 40 pg / mL, and more preferably less than 10 pg / mL.

[0196] 16. An anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold used to promote the conversion of ADM-Gly to ADM-NH2 in circulating ADM-Gly within a critically ill patient suffering from an acute disease or acute symptoms selected from the group including severe infections (meningitis, systemic inflammatory response syndrome (SIRS), sepsis, etc.), shock (septic shock, cardiogenic shock, etc.), acute heart failure (acute decompensated heart failure, chronic heart failure with worsening of symptoms and signs, etc.), myocardial infarction, stroke, organ dysfunction (kidney, liver, heart, lung, etc.), or dementia, as described in Embodiments 1 to 14, wherein the ADM-Gly in a healthy subject can be quantified by the measurement sensitivity of the measurement method for ADM-Gly, the sensitivity of which is 20 pg / mL, preferably 15 pg / mL, more preferably 10 pg / mL.

[0197] 17. An anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold used to promote the conversion of ADM-Gly to ADM-NH2 in circulating ADM-Gly within the patient, in the treatment of a critically ill patient suffering from an acute disease or acute symptoms selected from the group including severe infections (such as meningitis, systemic inflammatory response syndrome (SIRS), sepsis), shock (such as septic shock, cardiogenic shock), acute heart failure (such as acute decompensated heart failure, chronic heart failure with worsening symptoms and signs), myocardial infarction, stroke, organ dysfunction (such as kidney, liver, heart, lung), or dementia, as described in Embodiments 1 to 16, wherein pro-adrenomedullin The concentrations of phosphorus or its fragment and ADM-NH2 (SEQ ID NO: 20) are measured using a first binder for proadrenomedullin or its fragment and a second binder for ADM-NH2 (SEQ ID NO: 20), wherein the proadrenomedullin or its fragment is selected from the group consisting of PAMP (SEQ ID NO: 32), MR-proADM (SEQ ID NO: 33), ADM-Gly (SEQ ID NO: 21), and CT-proADM (SEQ ID NO: 34), and both binders are selected from the group consisting of antibodies, antibody fragments, or non-Ig scaffolds that bind to the proadrenomedullin or its fragment and ADM-NH2.

[0198] 18. An anti-ADM antibody, anti-ADM antibody fragment, or anti-ADM non-Ig scaffold for use in the treatment of a patient, wherein the anti-ADM antibody, anti-ADM fragment, or anti-ADM non-Ig scaffold is bound to the N-terminal and / or central region (amino acids 1-42) of ADM-Gly and / or ADM-NH2: YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVA (SEQ ID NO: 23), and the levels of peptidylglycine α-amidate monooxygenase (PAM) and / or its isoform and / or fragment are measured in a body fluid sample of the patient, and if the level of peptidylglycine α-amidate monooxygenase (PAM) is below a threshold, the patient is treated with the anti-ADM antibody, anti-ADM antibody fragment, or anti-ADM non-Ig scaffold.

[0199] 19. An anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in the treatment of a patient as described in Embodiment 19, wherein the level of the PAM and / or its isoform and / or its fragment is the total concentration of the PAM and / or its isoform and / or its fragment having at least 12 amino acids, or the activity of the PAM and / or its isoform and / or its fragment containing the sequences of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47.

[0200] 20. An anti-ADM antibody, anti-ADM antibody fragment, or anti-ADM non-Ig scaffold for use in the treatment of a patient as described in any of Embodiments 1 to 19, wherein the anti-adrenomedullin (ADM) antibody, anti-adrenomedullin antibody fragment, or anti-ADM non-Ig scaffold is used in combination with L-ascorbic acid.

[0201] 21. Anti-adrenomedullin (ADM) antibodies, anti-ADM antibody fragments, or anti-ADM non-Ig scaffolds combined with L-ascorbic acid are intended for the following purposes: a. To be used in the treatment of an acute disease or acute condition in a patient in order to stabilize the patient's systemic circulatory system, where the patient requires systemic circulatory system stabilization and exhibits a heart rate greater than 100 beats / min and / or a mean arterial pressure less than 65 mmHg, and systemic circulatory system stabilization means raising the mean arterial pressure above 65 mmHg; or b. In patients suffering from an acute disease or acute condition, use to prevent an increase in heart rate to more than 100 beats / min and / or a decrease in mean arterial pressure to less than 65 mmHg; or c. For the prevention or reduction of organ dysfunction in patients, or for the prevention of organ failure, to be used in the treatment of acute illnesses or acute symptoms in patients suffering from chronic and / or acute illnesses or acute symptoms, wherein the organs are selected from the group including the heart, kidneys, liver, lungs, pancreas, small intestine, and spleen; or d. To use in the treatment or prevention of shock such as SIRS, meningitis, sepsis, or septic shock in patients; or e. To reduce the risk of death in patients suffering from shock such as SIRS, meningitis, sepsis, and septic shock. Here, the anti-ADM antibody, anti-ADM fragment, or anti-ADM non-Ig scaffold binds to the N-terminal and / or central region (amino acids 1-42) of ADM-Gly and / or ADM-NH2: YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVA (SEQ ID NO: 23).

[0202] 22. An anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold combined with L-ascorbic acid as described in Embodiment 21, wherein the L-ascorbic acid is a single enantiomer, a mixture of enantiomers, a mixture of diastereomers, or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof. [Brief explanation of the drawing]

[0203] [Figure 1a] Figure 1a is an explanatory diagram of antibody form:Fv isomers and scFv variants. [Figure 1b] Figure 1b is an explanatory diagram of antibody types: heterogeneous fusion antibodies and bifunctional antibodies. [Figure 1c] Figure 1c is an explanatory diagram of antibody types: bivalent antibodies and bispecific antibodies. [Figure 2ab] Figure 2 shows the following: a: Dose-response curve for human ADM. Maximum cAMP stimulation was adjusted to 100% activity. b: Dose / inhibition rate curve for human ADM 22-52 (ADM receptor antagonist) in the presence of 5.63 nM hADM. [Figure 2cd] c: Shows the dose / inhibition rate curve for CT-H in the presence of 5.63 nM hADM. d: Shows the dose / inhibition rate curve for MR-H in the presence of 5.63 nM hADM. [Figure 2ef]e: Shows the dose / inhibition rate curve of NT-H in the presence of 5.63 nM hADM. f: Shows the dose-response curve of mouse ADM. Maximum cAMP stimulation was adjusted to 100% activity. [Figure 2gh] g: Dose / inhibition rate curves for human ADM 22-52 (ADM receptor antagonists) in the presence of 0.67 nM mADM. h: Dose / inhibition rate curves for CT-M in the presence of 0.67 nM mADM. [Figure 2ij] i: Shows the dose / inhibition rate curve for MR-M in the presence of 0.67 nM mADM. j: Shows the dose / inhibition rate curve for NT-M in the presence of 0.67 nM mADM. [Figure 2kl] k: Shows the inhibition rate of ADM by F(ab)2 NT-M and Fab NT-M. l: Shows the inhibition rate of ADM by F(ab)2 NT-M and Fab NT-M. [Figure 3] Figure 3 shows a typical hADM dose / signal curve, as well as the hADM dose / signal curve in the presence of 100 μg / mL NT-H antibody. [Figure 4] Figure 4 shows the stability of hADM in human plasma (citrate) with and without NT-H antibody. [Figure 5] Figure 5 shows the alignment of Fabs having homologous human framework sequences. [Figure 6] Figure 6 shows a typical dose / signal curve for ADM-Gly. [Figure 7] Figure 7 shows the effect of HAM8101 on the bio-ADM generation rate by human recombinant PAM. The change in bio-ADM signal value per minute (RLU / min) is shown as a percentage. The relative bio-ADM signal value for an antibody concentration of 0 μg / mL was set to 100%. [Figure 8] Figure 8 shows the effect of HAM8101 on the bio-ADM production rate by human natural PAM (with and without the use of exogenous ADM-Gly as a substrate). [Figure 9] Figure 9 shows the effects of NT- and MR- anti-ADM antibodies on human natural adrenomedullin maturation activity (AMA). [Figure 10] Figure 10 shows ADM concentrations in healthy human subjects after administration of NT-H at various doses for up to 60 days. [Figure 11] Figure 11 shows the ADM maturation activity (AMA) in subjects before and after administration of HAM8101 (average activity from n=3 samples). [Figure 12] Figure 12 shows the ADM-Gly bio-ADM ratio in Phase 1A clinical trial samples at various time points (before administration and 1, 4, and 24 hours after HAM8101 administration). [Figure 13] Figure 13 shows the correlation between bio-ADM and the ADM-Gly / bio-ADM ratio in patients with sepsis and septic shock (AdrenOSS-1) (r=0.21, p=0.003). [Figure 14] Figure 14 shows a Kaplan-Meier plot of the ADM-Gly / bioADM ratio for 28-day survival outcomes in patients with sepsis and septic shock (AdrenOSS-1). [Figure 15] Figure 15 shows the ADM maturation activity (AMA) in plasma containing HAM8101 and ascorbates at various concentrations. [Examples]

[0204] It is important to emphasize that, in accordance with the present invention, some of the antibodies, antibody fragments, and non-Ig scaffolds in the examples bind to ADM, and therefore should be considered as anti-ADM antibodies / antibody fragments / non-Ig scaffolds.

[0205] Example 1 - Antibody Production and Measurement of its Affinity Constant Several anti-human and anti-mouse ADM antibodies were generated, and their affinity constants were measured (see Tables 1 and 2).

[0206] Immunotherapy peptides / complexes: As shown in Table 1, an immunization peptide (JPT Technologies, Berlin, Germany) was synthesized using an additional N-terminal cysteine ​​residue to bind the peptide to bovine serum albumin (BSA) (if cysteine ​​was not present in the selected ADM sequence). The peptide was covalently bound to BSA using a Sulfolink-binding gel (Perbio-science, Bonn, Germany). The binding procedure was carried out according to Perbio's instructions.

[0207] Mouse monoclonal antibody production: Balb / c mice were immunized with 100 μg of peptide-BSA conjugate (emulsified in 100 μL of complete Freund's retrieval) on days 0 and 14, and with 50 μg of the same conjugate (emulsified in 100 μL of incomplete Freund's retrieval) on days 21 and 28. Three days before performing the fusion test, the animals were administered 50 μg of the conjugate dissolved in 100 μL of saline via a single intraperitoneal injection and a single intravenous injection. Splenocytes and myeloma cell lineage SP2 / 0 cells from the immunized mice were fused with 1 mL of 50% polyethylene glycol at 37°C for 30 seconds. After washing, these cells were seeded in 96-well cell culture plates. Hybrid clones were selected by growing them in HAT medium [RPMI1640 culture medium supplemented with 20% fetal bovine serum and HAT supplement]. After two weeks, the HAT medium was replaced with HT medium and the cells were subcultured three times, after which the cells were returned to the standard cell culture medium.

[0208] Cell culture supernatants were primary screened for antigen-specific IgG antibodies three weeks after fusion. Tested positive microcultures were transferred to 24-well plates for growth. After retesting, selected cultures were cloned and re-cloned using limiting dilution to determine isotype (see Lane, RD 1985. J. Immunol. Meth. 81: 223-228; Ziegler et al. 1996. Horm. Metab. Res. 28: 11-15).

[0209] Antibodies were produced using a standard antibody production method (Marx et al, 1997. Monoclonal Antibody Production, ATLA 25, 121) and purified with Protein A. Antibody purity was over 95% based on SDS gel electrophoresis analysis.

[0210] Human antibody production via phage display: The human naive antibody gene library HAL7 / 8 was used to isolate recombinant single-chain F variable domains (scFv) against the adrenomedullin peptide. The antibody gene library was sorted using a panning method that included the use of peptides containing biotin tags linked to the adrenomedullin peptide sequence via two different spacers. Background from nonspecific binders was minimized using a combination of panning cycles with nonspecific binding antigens and streptavidin-binding antigens. Phages eluted from the third panning cycle were used to generate monoclonal scFv expressing the E. coli strain. The supernatant from the cultures of these clones was used directly for antigen ELISA (see Hust et al. 2011. Journal of Biotechnology 152, 159-170; Schutte et al. 2009. PLoS One 4, e6625).

[0211] Positive clones were selected based on positive ELISA signals to the antigen and negative ELISA signals to streptavidin-coated microtiter plates. For further characterization, scFv open reading frames were cloned into the expression plasmid pOPE107 (Hust et al., J. Biotechn. 2011), captured from the culture supernatant by fixed metal ion affinity chromatography, and purified by size exclusion chromatography.

[0212] Affinity constant: To determine the affinity of the antibody for adremedullin, the binding dynamics of adremedullin to the immobilized antibody were measured by label-free surface plasmon resonance using a Biacore 2000 system (GE Healthcare Europe GmbH, Freiburg, Germany). Reversible antibody immobilization was performed using high-density covalently bound anti-mouse Fc antibody on the CM5 sensor surface, according to the manufacturer's instructions (mouse antibody capture kit; GE Healthcare). (Lorenz et al. 2011. Antimicrob Agents Chemother. 55(1): 165-173).

[0213] Monoclonal antibodies were prepared targeting the following ADM regions of human ADM and mouse ADM, respectively. The table below shows the selection of isolated antibodies for further testing. The selection is based on the target region. [Table 1]

[0214] The following is a list of the monoclonal antibodies that were further collected. [Table 2-1] [Table 2-2]

[0215] Production of antibody fragments by enzymatic digestion: Fab and F(ab)2 fragments were generated by enzymatic digestion of the full-length mouse antibody NT-M. Antibody NT-M was digested using a) a pepsin-based F(ab)2 preparation kit (Pierce 44988) and b) a papain-based Fab preparation kit (Pierce 44985). The fragmentation procedure was carried out according to the instructions provided by the supplier. For F(ab)2 fragmentation, digestion was carried out at 37°C for 8 hours, and for Fab fragmentation, digestion was carried out for 16 hours.

[0216] Procedure for generating and purifying Fab: The fixed papain was washed with 0.5 mL of digestion buffer, and the column was equilibrated by centrifugation at 5000 x g for 1 minute. The buffer was then discarded. A desalted column was prepared by removing the storage solution, washing with digestion buffer, and then centrifugation at 1000 x g for 2 minutes each time. 0.5 mL of the prepared IgG sample was added to a spin column tube containing equilibrated fixed papain. The culture time for the digestion reaction was 16 hours at 37°C on a benchtop oscillating apparatus. This column was centrifuged at 5000 x g for 1 minute to separate the digest from the fixed papain. The resin was then washed with 0.5 mL of PBS and centrifuged at 5000 x g for 1 minute. The washed fraction was added to the digested antibody to make a total sample volume of 1.0 mL. The Nab protein A column was equilibrated at room temperature with PBS and IgG elution buffer. This column was centrifuged for 1 minute to remove the storage solution (containing 0.02% sodium azide), 2 mL of PBS was added to equilibrate, and the column was centrifuged again for 1 minute to discard the pass-through fraction. The sample was applied to the column and resuspended by inversion. The culture was performed at room temperature for 10 minutes by inversion mixing. The column was centrifuged for 1 minute, and the circulating fraction was stored together with the Fab fragment. (References: Coulter and Harris 1983. J. Immunol. Meth. 59, 199-203.;Lindner et al. 2010. Cancer Res. 70, 277-87;Kaufmann et al. 2010. PNAS. 107, 18950-5.;Chen et al. 2010. PNAS. 107, 14727-32;Uysal et al. 2009 J. Exp. Med. 206, 449-62;Thomas et al. 2009. J. Exp. Med. 206, 1913-27;Kong et al. 2009 J. Cell Biol. 185, 1275-840).

[0217] Procedure for generating and purifying the F(ab')2 fragment: The fixed pepsin was washed with 0.5 mL of digestion buffer, and the column was equilibrated by centrifugation at 5000 x g for 1 minute. The buffer was then discarded. Desalting columns were prepared by removing the storage solution, washing with digestion buffer, and then centrifugation at 1000 x g for 2 minutes each time. 0.5 mL of the prepared IgG sample was added to a spin column tube containing equilibrated fixed pepsin. The digestion reaction culture time was 16 hours at 37°C on a benchtop shaking apparatus. This column was centrifuged at 5000 x g for 1 minute to separate the digest from the fixed papain. The resin was then washed with 0.5 mL of PBS and centrifuged at 5000 x g for 1 minute. The washed fraction was added to the digested antibody to make a total sample volume of 1.0 mL. The Nab protein A column was equilibrated at room temperature with PBS and IgG elution buffer. The column was centrifuged for 1 minute to remove the storage solution (containing 0.02% sodium azide), 2 mL of PBS was added to equilibrate, and the column was centrifuged again for 1 minute to discard the passed fraction. The sample was applied to the column and resuspended by inversion. The culture was carried out at room temperature for 10 minutes by inversion mixing. The column was centrifuged for 1 minute, and the passed fraction was stored together with the Fab fragment.(References:Mariani et al. 1991. Mol. Immunol. 28: 69-77;Beale 1987. Exp Comp Immunol 11: 287-96;Ellerson et al. 1972. FEBS Letters 24(3): 318-22;Kerbel and Elliot 1983. Meth Enzymol 93: 113-147;Kulkarni et al. 1985. Cancer Immunol Immunotherapy 19:211-4;Lamoyi 1986. Meth Enzymol 121: 652-663;Parham et al. 1982. J Immunol Meth 53: 133-73;Raychaudhuri et al. 1985. Mol Immunol 22(9): 1009-19;Rousseaux et al. 1980. Mol Immunol 17: 469-82;Rousseaux et al. 1983. J Immunol Meth 64: 141-6;Wilson et al. 1991. J Immunol Meth 138: 111-9).

[0218] Humanization of NT-H antibody fragments: Antibody fragments were humanized by CDR transplantation (Jones et al. 1986. Nature 321, 522-525). To achieve the humanized sequence, the following steps were performed: Total RNA was extracted from NT-H fusion cell tumors using the Qiagen kit. For the first RT-PCR, the QIAGEN® one-step RT-PCR kit (catalog number 210210) was used. RT-PCR was performed using heavy and light chain specific primer sets. For each RNA sample, RT-PCR reactions for 12 heavy chains and 11 light chains were set up using a degenerate forward primer mixture containing variable region leader sequences. Reverse primers were positioned within the constant regions of the heavy and light chains. Restriction sites were not designed within the primers.

[0219] The reaction setup was as follows: 5.0 μL of 5x QIAGEN® one-step RT-PCR buffer, 0.8 μL of dNTP mixture (containing 10 mM of each dNTP), 0.5 μL of primer set, 0.8 μL of QIAGEN® one-step RT-PCR enzyme mixture, 2.0 μL of template RNA, and 20.0 μL of RNA-degrading enzyme-free solution, for a total volume of 20.0 μL. The PCR conditions were: reverse transcription at 50°C for 30 minutes; initial PCR activation at 95°C for 15 minutes; 20 cycles at 94°C for 25 seconds; 54°C for 30 seconds; 72°C for 30 seconds; and final extension at 72°C for 10 minutes. Second semi-nested PCR: The RT-PCR product from the first reaction was further amplified in the second PCR. The RT-PCR reactions for 12 heavy chains and 11 light chains were set up using semi-nested primer sets specific to the antibody variable region.

[0220] The reaction setup was as follows: 10 μL of double PCR mixture; 2 μL of primer set; 8 μL of first PCR product; 20 μL total volume; PCR conditions for the antibody cloning report of fusion cell tumor: 5 min at 95°C, 25 cycles of 25 seconds at 95°C, 30 seconds at 57°C, 30 seconds at 68°C, and a final extension of 10 minutes.

[0221] After PCR was completed, the PCR reaction sample was run on an agarose gel to visualize the amplified DNA fragments. Following sequencing of more than 15 cloned DNA fragments amplified by nested RT-PCR, the heavy and light chains of several mouse antibodies were cloned and accurately displayed. Protein sequence alignment and CDR analysis identified one heavy chain and one light chain. After alignment to a homologous human framework sequence, the resulting humanized sequence of the variable heavy chain is shown in Figure 5. The amino acids at positions 26, 40, and 55 in the variable heavy chain and the amino acid at position 40 in the variable light chain are important for binding properties, and these amino acids may be returned to the original mouse sequence. The resulting candidates are shown below. (Padlan 1991. Mol. Immunol. 28: 489-498; Harris and Bajorath 1995. Protein Sci. 4: 306-310).

[0222] As a note, antibody fragment sequences (SEQ ID NOs: 7 to 13, 35, and 36) are shown below, wherein the bold and underlined sequences are CDR1, 2, and 3 arranged sequentially. SEQ ID NO: 6 (AM-VH-C)

Chemical Formula

Chemical Formula

Chemical Formula

Chemical Formula

Chemical Formula

Chemical Formula

Chemical Formula

Chemical Formula

Chemical Formula

Chemical Formula

[0223] Example 2 - Effect of selected anti-ADM antibodies on anti-ADM bioactivity The effect of selected ADM antibodies on ADM bioactivity was tested using the human recombinant adrenomedullin receptor cAMP function assay (adrenomedullin bioassay). The following materials were used: cell line CHO-K1, adrenomedullin receptor (CRLR+RAMP3); receptor cell line accession numbers (CRLR:U17473; RAMP3:AJ001016). CHO-K1 cells expressing human recombinant adrenomedullin receptor (FAST-027C), grown in antibiotic-free medium prior to the test, were separated by slow flow with PBS-EDTA (5 mM EDTA), recovered by centrifugation, and resuspended in the measurement buffer (KRH: 5 mM KCl, 1.25 mM MgSO4, 124 mM NaCl, 25 mM HEPES, 13.3 mM glucose, 1.25 mM KH2PO4, 1.45 mM CaCl2, 0.5 g / L BSA). Dose-response curves were measured together with a control agent (hADM or mADM).

[0224] Antagonist test (96 wells): For antagonist testing, 6 μL of control agonist (human (5.63 nM) or mouse (0.67 nM) adrenomedullin) was mixed with 6 μL of test sample or 6 μL of buffer in various antagonist dilutions. After incubation at room temperature for 60 minutes, 12 μL of cells (2,500 cells / well) were added. The plates were incubated at room temperature for 30 minutes. After adding lysis buffer, the DeltaF percentage was estimated using the Cis-BioInternational HTRF kit (catalog number: 62AM2PEB) according to the manufacturer's specifications. hADM22-52 were used as control antagonists.

[0225] Antibodies used to test the cAMP-HTRF assay: Anti-h-ADM antibodies (NT-H, MR-H, CT-H) were tested for antagonist activity in human recombinant adrenomedullin receptor (FAST-027C) cAMP function measurement in the presence of 5.63 nM human ADM1-52 (SEQ ID NO: 20) at final antibody concentrations of 100 μg / mL, 20 μg / mL, 4 μg / mL, 0.8 μg / mL, and 0.16 μg / mL. Anti-m-ADM antibodies (NT-M, MR-M, CT-M) were tested for antagonist activity in human recombinant adrenomedullin receptor (FAST-027C) cAMP function measurement in the presence of 0.67 nM mouse ADM1-50 (SEQ ID NO: 22) at final antibody concentrations of 100 μg / mL, 20 μg / mL, 4 μg / mL, 0.8 μg / mL, and 0.16 μg / mL. The data plots the relative inhibition rate against the antagonist concentration (see Figures 2a-2l). The maximum inhibition rates for each antibody are shown in Table 3.

[0226] [Table 3]

[0227] Example 3 - Stabilization of hADM with anti-ADM antibody The stabilizing effect of human ADM antibodies on human ADM was tested using hADM immunoassay. The technique used was a sandwich tube luminescence immunoassay based on acridinium ester labeling.

[0228] Labeled compound (tracer): 100 μg (100 μL) of CT-H (1 mg / mL in PBS, pH 7.4, AdrenoMed AG Germany) was mixed with 10 μL of acridinium NHS ester (1 mg / mL in acetonitrile, InVent GmbH, Germany) (EP0353971), and incubated at room temperature for 20 minutes. Labeled CT-H was purified by gel filtration HPLC on Bio-Sil® SEC 400-5 (Bio-Rad Laboratories, Inc., USA). The purified CT-H was diluted in (300 mmol / L potassium phosphate, 100 mmol / L NaCl, 10 mmol / L Na-EDTA, 5 g / L bovine serum albumin, pH 7.0). The final concentration was approximately 800,000 relative light units (RLU) of labeled compound (approximately 20 ng of labeled antibody) per 200 μL. Chemiluminescence of the acridinium ester was measured using an AutoLumat LB953 (Berthold Technologies GmbH&Co.KG).

[0229] Solid phase: Polystyrene tubes (Greiner Bio-One International AG, Austria) were coated with MR-H (AdrenoMed AG, Germany) (1.5 μg MR-H / 0.3 mL in 100 mmol / L NaCl, 50 mmol / L TRIS / HCl, pH 7.8) at room temperature for 18 hours. After blocking with 5% bovine serum albumin, the tubes were washed with PBS, pH 7.4, and vacuum dried.

[0230] Calibration: The assay was calibrated using dilutions of hADM (BACHEM AG, Switzerland) in 250 mmol / L NaCl, 2 g / L Triton X-100, 50 g / L bovine serum albumin, and 20 tablets / L proteinase inhibitor cocktail (Roche Diagnostics AG, Switzerland).

[0231] hADM immunoassay: 50 μL of sample (or calibrator) was pipetteed into a coated tube, labeled CT-H (200 μL) was added, and the tube was incubated at 4°C for 4 hours. Unbound tracers were removed by washing five times (1 mL each) with a washing solution (20 mM PBS, pH 7.4, 0.1% Triton X-100). Chemiluminescence bound to the tube was measured using LB953 (Berthold, Germany). Figure 3 shows a typical hADM dose / signal curve and the hADM dose / signal curve in the presence of 100 μg / mL NT-H antibody. NT-H did not affect the hADM immunoassay described above.

[0232] Stability of human adrenomedullin: Human adrenomedullin (HDM) was diluted in human citrate plasma (final concentration 10 nM) and cultured at 24°C. At predetermined time points, the degradation of hADM was stopped by freezing at -20°C. This culture was performed both with and without NT-H (100 μg / mL). The residual hADM was quantified using the hADM immunoassay method described above. Figure 4 shows the stability of hADM in human plasma (citrate) with and without NT-H antibody. The half-life of hADM alone was 7.8 hours, but in the presence of NT-H, the half-life was 18.3 hours (2.3 times higher stability).

[0233] Example 4 - Immunoassay method for detecting ADM-Gly Based on Weber et al. (Weber et al. 2017. JALM 2(2): 222-233), bioactive ADM-Gly was quantified by the following modification: A tracer antibody labeled with MACN-acridinium-NHS was directed to the C-terminal glycine of ADM-Gly. This assay was calibrated with synthetic ADM-Gly. The limit of detection (LOD) was 10 pg / mL of ADM-Gly. Cross-reactivity between the antibody directed to the C-terminal glycine of ADM and bio-ADM ranged from 6% to 50% depending on the concentration. All measured ADM-Gly concentrations were corrected for cross-reactivity as follows: For each quantification of ADM-Gly, further quantification of bio-ADM in the corresponding sample was performed using the sphingotest® bio-ADM immunoassay. Using the corresponding bio-ADM values, the signal (RLU) generated by the antibody directed to the C-terminal glycine of ADM on the bio-ADM calibration curve was measured. Using the measured signal (RLU), the false-positive ADM-Gly concentration (pg / mL) was calculated using the ADM-Gly calibration curve. This concentration was subtracted from the initially measured ADM-Gly concentration. A typical standard curve is shown in Figure 6.

[0234] Example 5 - Effects of NT-ADM antibody and MR-ADM antibody on the conversion of ADM-Gly to bio-ADM (in vitro) a) Recombinant human PAM We investigated the generation of bioadrenomedullin (ADM) from c-terminally glycinated 1-53 adrenomedullin (ADM-Gly) by recombinant human PAM, and the effect of an N-terminal anti-adrenomedullin antibody on the generation of bioadrenomedullin from ADM-Gly by PAM.

[0235] In the first step, ADM-Gly (50 ng / mL) dissolved in buffer (300 mM potassium phosphate, 100 mM NaCl, 10 mM Na-EDTA, 5 g / L BSA, pH 7.0) was added to microtiter plate wells pre-coated with anti-MR-ADM antibody at a rate of 200 μL per well, and cultured at 22°C for 1 hour with stirring (600 rpm). Unbound material was removed by washing. In the second step, one of the following (0-100 μg / mL) concentrations of N-terminal antibody (HAM8101), a C-terminal anti-adrenomedullin antibody that specifically inhibits the glycinated C-terminus of ADM-Gly, or a non-specific antibody (control antibody) was added to the wells (200 μL per well) with stirring (600 rpm) for 1 hour at 22°C. As described above, each antibody was diluted with buffer. Unbound antibody was removed by washing. The amidation reaction was initiated by adding a PAM reaction buffer (100 mM Tris-HCl, 5 μM CuSO4, 2 mM L-ascorbate, 50 μM astatin, and 200 μM leupeptin) containing a 50 μg / mL recombinant human PAM-containing protein solution (In Vivo Biotech Services GmbH, Hennigsdorf). The amidation reaction was carried out at 37°C and stopped at 0 minutes (t=0) and 40 minutes (t=40) by adding EDTA (final concentration 10 mM). The plate was washed again, and a labeled anti-ADM antibody specific to the amidated C-terminus of ADM was added as a tracer. The plate was incubated at 22°C and 600 rpm for 1 hour. After the final washing step, residual chemiluminescence was measured per well for 1 second using a CentroLB960 microtiter plate luminescence reader (Berthold Technologies). To evaluate the rate of the enzyme PAM, the bio-ADM signal measured at t=0 min was subtracted from the bio-ADM signal at t=40 min for each antibody concentration. The signals for each antibody concentration (t40-t0) were normalized against the signal without antibody addition (t40-t0), which was set to 100%.

[0236] As shown in Figure 7, control antibodies that are not specific to adrenomedullin did not affect the activity of the PAM enzyme. The C-terminal anti-adrenomedullin antibody, which specifically recognizes ADM-Gly, inhibits the C-terminal glycine residue, a substrate of the PAM enzyme, and thus inhibits the PAM enzyme reaction in a concentration-dependent manner. Surprisingly, the N-terminal anti-ADM antibody (HAM8101) significantly promotes the conversion of ADM-Gly to bio-ADM catalyzed by PAM in a concentration-dependent manner. The PAM-promoting effect of HAM8101 was detected at a maximum concentration of 10 μg / mL. At concentrations above 10 μg / mL, PAM activity did not increase further. The PAM activity of HAM8101 at a concentration of 10 μg / mL was 233% compared to the reaction without antibody.

[0237] b) Natural Human PAM In further tests, the inventors investigated the generation of bio-ADM from ADM-Gly using natural human plasma PAM, and the effects of anti-adrenomedullin antibodies in the N-terminal and central regions.

[0238] For the testing of the N-terminal anti-ADM antibody HAM8101, the test procedure was set up as follows: Human Li-heparinized plasma (accumulating 3 samples) was used as the source of natural human PAM. The amidation reaction was carried out at 37°C in a total volume of 120 μL. HAM8101 (final concentration 375 μg / mL) or ADM-Gly (final concentration 5 ng / mL), or both, were rapidly added to 96 μL of plasma. As a control, an equal volume of 100 mM Tris-HCl, pH 7.5 was added to untreated plasma. The prepared samples were cooled at room temperature for 15 minutes. The amidation reaction was initiated by adding 24 μL of PAM reaction buffer with final concentrations of 2 mM L-ascorbic acid and 5 μM CuSO4, respectively. The final concentrations of HAM8101 and ADM-Gly were 300 μg / mL and 4 ng / mL, respectively. The reaction was allowed to proceed at 37°C for 90 minutes. The reaction was stopped by adding 20 mM EDTA (to a final concentration) at 0, 30, 60, and 90 minutes of incubation. The concentration of bio-ADM in the reaction sample was quantified using the sphingotest® bio-ADM immunoassay method, as reported in recent studies (Weber et al. 2017. JALM 2(2): 222-233).

[0239] In the testing of central region anti-ADM antibodies against HAM8101 and four additional N-terminal anti-ADM antibodies, the experimental method was set up as follows: Human serum was used as the source of human natural PAM. Each sample (20 μL) was diluted 2-fold in 100 mM Tris-HCl. The amidation reaction was initiated by adding 160 μL of NT-ADM antibody (HAM8101, AK1373, AK1388, AK1398, or AK1434) or MR-ADM antibody (ADM43, ADM38, ADM41, ADM2901, ADM2902, and ADM2903) to a 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 astatin, 250 μM leupeptin, and 36 ng / mL synthetic 1-53 adrenomedullin-Gly as a substrate). The final antibody concentration was 100 μg / mL in each case. Subsequently, 100 μL of each reaction mixture from the replicated samples were combined and transferred to 20 μL of 200 mM EDTA to terminate the amidation reaction, which was set as reaction time t=0 minutes. The mixture was then incubated at 37°C for 40 minutes. The unterminated reaction was then stopped with 10 μL of 200 mM EDTA. To measure PAM activity in the NT-ADM antibody-containing samples, bio-ADM was quantified in each reaction mixture using the sphingotest® bio-ADM immunoassay (Weber et al. 2017, supra). The MR-ADM antibody-containing reaction mixture was transferred to another bio-ADM assay (described below). Both bio-ADM assays were used to measure the antibody-free control reaction mixture. For each sample, the difference in bio-ADM concentrations at t=40 minutes and t=0 minutes was calculated. PAM activity was expressed as bio-ADM (in ng units) produced per hour and per L of sample, normalized to the antibody-free reaction mixture, which was set to 100%.

[0240] Another bio-ADM assay method: All components and conditions were as described by Weber et al. in 2017, with the N-terminal anti-ADM antibody used as a solid-phase capture antibody instead of the central-region anti-ADM antibody.

[0241] In samples without the addition of exogenous ADM-Gly, no change in bio-ADM concentration was detected, regardless of the presence or absence of HAM8101. When ADM-Gly was added to the sample, chain formation of bio-ADM was detected within 90 minutes. When HAM8101 was present in the reaction solution in addition to ADM-Gly, chain formation of bio-ADM was detected within 90 minutes and increased up to four times after 90 minutes compared to the reaction without HAM8101 (Figure 8). All test antibodies that bound to the N-terminus of ADM increased amidation activity, but HAM8101 showed the strongest effect (Figure 9). All test antibodies that bound to the central region of ADM (ADM38, ADM41, and ADM43 directed to SEQ ID NO: 15, which contains amino acids 21-32 of ADM, or ADM2901, ADM2902, and ADM2903 directed to SEQ ID NO: 49, which contains amino acids 27-39 of ADM) increased amidation activity, but ADM43 showed the strongest effect. Furthermore, the test antibody ADM43 that bound to MR-ADM significantly increased bio-ADM production more than the NT-ADM antibody. This clearly indicates that the conversion of ADM-Gly to bio-ADM by human natural PAM is significantly increased by anti-ADM antibodies of the N-terminal and central regions.

[0242] Example 6 - Administration of NT-H in healthy humans and its in vivo effects on the conversion of ADM-Gly to bio-ADM This study is described by Geven et al. (2017. Intensive Care Med Exp 5 (Suppl 2): ​​0427). In short, this study was conducted as a randomized, double-blind, placebo-controlled study in healthy men, in which a single escalating dose of the NT-H antibody (HAM8101) was administered intravenously (iv) to three consecutive groups of eight healthy men. These groups were administered to healthy men (n=6 in each group receiving the active drug and n=2 receiving the placebo) at different doses (Group 1: 0.5 mg / kg, Group 2: 2 mg / kg, Group 3: 8 mg / kg). The main selection criteria were informed consent, i.e., age 18-35 years, agreement to use reliable contraception, and a BMI of 18-30 kg / m². 2 The study group received a single intravenous injection of either NT-H antibody (HAM8101) (0.5 mg / kg; 2 mg / kg; 8 mg / kg) or a placebo over one hour per study unit.

[0243] There were no differences in the reference ADM values ​​among the four groups. The median ADM values ​​were 7.1 pg / mL in the placebo group, 6.8 pg / mL in the first treatment group (0.5 mg / kg), 5.5 pg / mL in the second treatment group (2 mg / kg), and 7.1 pg / mL in the third treatment group (8 mg / mL). The results showed that ADM values ​​rapidly increased within the first 1.5 hours after administration of NT-H antibody (HAM8101) to healthy human subjects, then reached a plateau and gradually decreased (Figure 10). Administration of NT-H antibody (HAM8101) was safe because the antibody did not affect heart rate, mean arterial pressure, peripheral oxygen saturation, or body temperature. Furthermore, there were no significant differences between the groups in standard hematological and biochemical safety tests.

[0244] We investigated the generation of bio-ADM from ADM-Gly using recombinant human PAM enzyme, and the in vivo effects of HAM8101, an N-terminal anti-adrenomedullin antibody, by measuring bio-ADM maturation activity (AMA) in samples from healthy subjects administered HAM8101.

[0245] Samples were taken from three subjects in each dose group before and 1 hour after administration of HAM8101. Activity was measured as described for serum samples in Example 5b). The measurement method was calibrated using recombinant human PAM (InVivo Biotech Services GmbH, Hennigsdorf) of known activity. Calibration materials, control materials, and samples were processed in the same manner. Bio-ADM generated in the reaction samples was quantified using the sphingotest® bio-ADM assay (Weber et al., 2017). For each sample, the difference in signal (RLU, relative light units) at t=40 min and t=0 min was calculated, and the AMA in the tested serum sample was determined using the antibiotic signal (RLU(t40-t0 min)). AMA is expressed as bio-ADM in ng units (ng / [h*L]) generated per hour and per L of sample.

[0246] Figures 11A and 11B show the mean bio-ADM maturation activity (AMA) before HAM8101 administration and at 1, 4, and 24 hours after administration, as values ​​from n=3 samples. In all three groups, AMA with ADM-Gly as a substrate increased after HAM8101 administration compared to the activity before administration of HAM8101, which is an N-terminal anti-ADM antibody (Figure 11A). In one-way ANOVA, the difference in group 1 (0.5 mg / kg) was not significant, but the differences in activity in group 2 (2 mg / kg) and group 3 (8 mg / kg) were significant (p<0.05 for groups 2 and 3, respectively). In group 1, AMA increased by approximately 60%, 40%, and 52% at 1, 4, and 24 hours, respectively. In group 2, AMA increased by approximately 27%, 25%, and 40% at 1, 4, and 24 hours, respectively. In group 3, AMA increased by approximately 65%, 48%, and 60% after 1 hour, 4 hours, and 24 hours, respectively (Figure 11B). These results clearly demonstrate that the conversion of ADM-Gly to bio-ADM by human natural PAM is significantly increased by the presence of HAM8101, a circulating N-terminal anti-ADM antibody. To further demonstrate the improved conversion of ADM-Gly to bio-ADM by human natural PAM, the inventors measured the concentrations of bioADM and ADM-Gly in the aforementioned samples. The ADM-Gly / bio-ADM ratio was greater than 1 in each group before HAM8101 administration, but surprisingly, it decreased to approximately 0.75 in group 1, approximately 0.63 in group 2, and approximately 0.43 in group 3 after 1 hour (Figure 12). Four hours after HAM8101 administration, the ratio further decreased to approximately 0.38, 0.25, and 0.38 in groups 1, 2, and 3, respectively. No further changes in the ratio were detected in groups 1 and 2 after 24 hours, but a slight increase to approximately 0.5 was detected in group 3. These results clearly demonstrate that the increased conversion of ADM-Gly to bio-ADM by natural human PAM due to HAM8101 administration shifts the ADM-Gly / bio-ADM ratio toward the bio-ADM side.

[0247] Example 7 - Adrenomedullin and Outcomes in Sepsis and Septic Shock 1 (AdrenOSS-1) Studies AdrenOSS-1 is a promising observational study in Europe. Twenty-four research sites in five countries (France, Belgium, the Netherlands, Italy, and Germany) contributed to the completion of the trial with 583 enrolled patients (recruited from June 2015 to May 2016). The study plan was approved by local ethics committees and conducted in accordance with the Declaration of Helsinki. This study enrolled patients aged 18 years or older who (1) were admitted to the ICU with sepsis or septic shock, or (2) were transferred from another ICU within 24 hours of admission in a state of sepsis or septic shock. Participating patients were stratified into severe sepsis and septic shock based on the 2001 definitions of sepsis and organ failure (Levy et al. 2003. 2001 SCCM / ESICM / ACCP / ATS / SIS International Sepsis Definitions Conference. Crit Care Med. 31(4):1250-6). The term "sepsis" refers to the latest definition in "Sepsis-3" (Singer et al. 2016 The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). JAMA. 315(8):801-10). Patients were treated according to clinical practice, and the treatment and procedures were documented. The primary outcome measure was mortality at day 28. Secondary outcomes included organ failure and organ support (defined by the Continuing Organ Failure Assessment [SOFA] score), use of vasopressors / cardiotonic agents, fluid balance, and use of renal replacement therapy (RRT).

[0248] Upon admission, personal statistics (age, sex), body mass index, presence of septic shock, type of ICU admission, organ dysfunction scores (SOFA, Acute Physiological Assessment and Chronic Health Assessment II [APACHE II]), origin of sepsis, pre-existing complications (i.e., cases treated within the past year), medical history, laboratory values, and organ support were recorded, and blood was collected for measurement of bioADM and other markers. After patient enrollment, the following data were collected daily during the first week: SOFA score, antimicrobial therapy, fluid balance, respiratory status, Glasgow Coma Scale score, central venous pressure, need for RRT, invasive procedures for sepsis control, and vasopressor / inotropic agent therapy. In addition, discharge status and mortality were recorded on day 28 after ICU admission. Blood for the central laboratory was collected within 24 hours of ICU admission and two days after the first sample (mean: 47 hours, standard deviation: 9 hours). Samples were then processed and stored at -80°C.

[0249] Bio-ADM was measured using a recently developed immunoassay method described by Weber et al. (Weber et al. 2017. JALM 2(2): 222-233). ADM-Gly was measured in a randomly selected subset of available samples (n=170) as described in Example 4. Bio-ADM and the ADM-Gly / bio-ADM ratio were significantly correlated (r=0.25, p=0.0011) (Figure 13).

[0250] In the aforementioned patient population (AdrenOSS-1 patients with sepsis, severe sepsis, or septic shock), the ratio of plasma ADM-Gly to bio-ADM on day 1 post-admission was measured as described above. Using the median ratio of 3.01 as a simple cutoff value, the population was divided into two groups (above and below 3.01), and the corresponding 28-day survival rates were shown in a Kaplan-Meier plot (Figure 14). The survival rate for patients with an ADM-Gly / bio-ADM ratio of less than 3.01 on admission was high at 84.7% (95% CI: 77.4-92.7), but the survival rate was low at 71.8% (95% CI: 62.8-82) when the ratio exceeded 3.01. Survival probabilities could be stratified using two groups with different ADM-Gly / bio-ADM ratios. The hazard ratio (HR) was significantly higher in the group with an ADM-Gly / bio-ADM ratio above the median compared to the group with a ratio below the median (HR = 2.04).

[0251] Example 8 - Comparison of ADM-Gly / Bio-ADM ratios in healthy patients, healthy treated patients, and critically ill patients (survivors and non-survivors). Severely ill subjects were from the AdrenOSS-1 population described in Example 7. Healthy subjects and healthy HAM8101-treated subjects were from the study population described in Example 6 (Geven et al. 2017. Intensive Care Med Exp 5 (Suppl 2): ​​0427). Bio-ADM was measured as described by Weber et al. in 2017. ADM-Gly was measured as described in Example 4. [Table 4]

[0252] Explanation of results: The ADM-Gly / bio-ADM ratio in critically ill patients who died within 28 days of ICU admission was 5.2. The ADM-Gly / bio-ADM ratio in critically ill patients who survived 28 days after ICU admission was surprisingly statistically low at 3.9 (p=0.0116). In healthy subjects, the ADM-Gly / bio-ADM ratio was even more significantly lower at 1.29 compared to critically ill subjects (p<0.05). Surprisingly, in healthy subjects administered HAM8101, the ADM-Gly / bio-ADM ratio was even more significantly lower one hour after HAM8101 administration (p=0.0002). Four hours after HAM8101 administration, this ratio further decreased to 0.33 (p=0.0354), thus demonstrating a direct effect of HAM8101 on the conversion from ADM-Gly to bio-ADM, as expressed by the ADM-Gly / bio-ADM ratio.

[0253] Example 9 - Comparison of MR-proADM / bioADM ratios in healthy patients, healthy treated patients, and critically ill patients (survivors and non-survivors) The same patients described in Example 8 were also analyzed for their MR-proADM / ADM-NH2 ratio. [Table 5]

[0254] Explanation of results: In critically ill patients who died within 28 days of ICU admission, the MR-proADM / bioADM ratio was 293.2, while in critically ill subjects who were still alive at 28 days after ICU admission, the ratio was lower at 283.9. Compared to critically ill subjects, the MR-proADM / bioADM ratio decreased to 239.4 in healthy subjects. Surprisingly, in healthy subjects administered HAM8101, the MR-proADM / bioADM ratio decreased even more significantly one hour after HAM8101 administration (p<0.0001). Four hours after HAM8101 administration, the ratio did not decrease any further.

[0255] Example 10: Effects of N-terminal anti-adrenomedullin antibody and L-ascorbate on the bio-ADM production rate from exogenous ADM-Gly using PAM. Serial antibody dilutions with concentrations of 0, 5, 10, 50, 100, 500, and 3000 μg / mL (in 20 μL) per well were pipetteed into a 96-well microtiter plate. Then, 160 μL of PAM reaction buffer (100 mM Tris-HCl, 6.25 μM CuSO4, 62.5 μM amastatin, 250 μM leupeptin, 36 ng / mL ADM-Gly) with various L-ascorbic acid concentrations were added to each well and incubated for 15 minutes. Subsequently, 20 μL of Li-heparinized plasma containing natural PAM was added to initiate the reaction. Immediately after plasma addition, 100 μL was taken from each well, and EDTA was added (final concentration: 20 mM) to inactivate the reaction point at t=0 minutes. Inactivated and uninactivated samples were incubated at 37°C for 40 minutes to stop the reaction in the uninactivated sample as described above. The generated ADM-NH2 was quantified using the sphingotest® Bio-ADM Kit (Weber et al. 2017) according to the manufacturer's instructions. ADM maturation activity (AMA) was calculated according to Formula 1 (DF: dilution factor of the sample mother liquor).

number

[0256] Increasing the ascorbate concentration in the range of 0.4–2 mM increased ADM maturation activity (Figure 15). The overall ascorbate dependence of the amidation reaction did not change with the addition of HAM8101; that is, the optimal activity remained at ascorbate concentrations of 0.8–2 mM. However, the presence of HAM8101 in the amidation assay showed a positive concentration-dependent effect on amidation for each measured ascorbate concentration. The presence of 100 μg / mL of HAM8101 in the amidation assay increased bio-ADM production by PAM by 40%. These results clearly demonstrate that the combination of N-terminal anti-ADM antibody with ascorbate significantly enhances the activity of PAM enzymes and bio-ADM production in plasma.

[0257] array Sequence ID 1: GYTFSRYW Sequence ID 2: ILPGSGST Sequence ID 3: TEGYEYDGFDY Sequence ID 4: QSIVYSNGNTY Array "RVS" (not part of an array list): RVS Sequence ID 5: FQGSHIPYT Sequence number 6 (AM-VH-C): [ka] Sequence ID 7 (AM-VH1): [ka] Sequence ID 8 (AM-VH2-E40): [ka] Sequence ID 9 (AM-VH3-T26-E55): [ka] Sequence ID 10 (AM-VH4-T26-E40-E55): [ka] Sequence ID 11(AM-VL-C): [ka] Sequence ID 12 (AM-VL1): [ka] Sequence ID 13 (AM-VL2-E40): [ka] Sequence ID 14 (Human ADM 1-21): YRQSMNNFQGLRSFGCRFGTC Sequence ID 15 (Human ADM 21-32): CTVQKLAHQIYQ Sequence ID 16 (Human ADM C-42~52): CAPRSKISPQGY-CONH2 Sequence ID 17 (Mouse ADM 1-19): YRQSMNQGSRSNGCRFGTC Sequence ID 18 (Mouse ADM 19-31): CTFQKLAHQIYQ Sequence ID 19 (Mouse ADM C40~50): CAPRNKISPQGY-CONH2 Sequence ID No. 20 (Mature Human Adrenomedullin (Mature ADM); Amidated ADM; Bio-ADM): Amino acids 1-52 or 95-146 of pro-ADM: [ka] SEQ ID NO: 21 (Adrenomedullin 1-52-Gly (ADM 1-52-Gly): Amino acids 95-147 of prepro-ADM: [ka] Sequence ID 22 (Mouse ADM 1-50): [ka] Sequence ID 23 (Human ADM 1-42): [ka] Sequence ID No. 24 (Amino acids 43-52 of human ADM): PRSKISPQGY-NH2 Sequence ID No. 25 (Amino acids 1-14 of human ADM): YRQSMNNFQGLRSF Sequence ID 26 (Amino acids 1-10 of human ADM): YRQSMNNFQG Sequence ID 27 (Amino acids 1-6 of human ADM): YRQSMN Sequence ID No. 28 (Amino acids 1-32 of human ADM): [ka] Sequence ID 29 (Amino acids 1-40 of mouse ADM): [ka] Sequence ID 30 (Amino acids 1-31 of mouse ADM): [ka] Sequence ID 31 (pro-ADM; 164 amino acids (22-185 of prepro-ADM)): [ka] Sequence ID 32 (Proadrenomedullin N-20 terminal peptide, PAMP: amino acids 22-41 of prepro-ADM): ARLDVASEF RKKWNKWALS R SEQ ID NO: 33 (Central region proadrenomedullin, MR-proADM: amino acids 45-92 of prepro-ADM): [ka] SEQ ID NO: 34 (C-terminal proadrenomedullin, CT-proADM: amino acids 148-185 of prepro-ADM): [ka] Sequence ID 35 (heavy chain, HAM8101): [ka] Sequence ID 36 (light chain, HAM8101): [ka] Sequence ID 37-IGHV1-69*11: [ka] Sequence ID 38-HB3: [ka] Sequence ID 39-Prepro-PAM isoform 1 AS 1~973: [ka] Sequence ID 40-Prepro-PAM isoform 2 AS 1~868: [ka] SEQ ID NO: 41-Prepro-PAM isoform 3 AS (missing amino acids 829-896 of SEQ ID NO: 1): [ka] SEQ ID NO: 42-Prepro-PAM isoform 4 (missing amino acids 829-914 of SEQ ID NO: 1): [ka] SEQ ID NO: 43-Prepro-PAM isoform 5 (Isoform 1 with an additional amino acid at position 896): [ka] SEQ ID NO: 44-Prepro-PAM isoform 6 (missing amino acids 897-914 of SEQ ID NO: 1): [ka] PHM subunit of sequence number 45-PAM: [ka] PAL subunit of sequence number 46-PAM: [ka] Sequence ID 47 - Recombinant Human PAM sequence: [ka] Sequence ID 48-MR-ADM 21~42: CTVQKLAHQIYQFTDKDKDNVA Sequence ID 49-MR-ADM 27~39: AHQIYQFTDKDKD

Claims

1. In the treatment of critically ill patients suffering from acute diseases or acute symptoms selected from the group including severe infections (such as meningitis, systemic inflammatory response syndrome (SIRS), and sepsis), shock (such as septic shock and cardiogenic shock), acute heart failure (including acute decompensated heart failure and chronic heart failure with worsening symptoms and signs), myocardial infarction, stroke, organ dysfunction (such as kidney, liver, heart, and lung), or dementia, ADM-NH is derived from ADM-Gly in the circulating ADM-Gly within the patient. 2 An anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold used to promote conversion to ADM-NH, wherein the patient has proadrenomedullin or its fragment in a body fluid sample and ADM-NH 2 The ratio to (SEQ ID NO: 20) exceeds a certain threshold, and the pro-adrenomedullin or its fragment is selected from the group consisting of PAMP (SEQ ID NO: 32), MR-proADM (SEQ ID NO: 33), ADM-Gly (SEQ ID NO: 21), and CT-proADM (SEQ ID NO: 34), and the anti-ADM antibody or anti-ADM fragment or anti-ADM non-Ig scaffold is ADM-Gly and / or ADM-NH 2 The N-terminal and / or central region (amino acids 1-42) of YRQSMNNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVA (SEQ ID NO: 23) is bound to an anti-adrenomedullin (ADM) antibody, an anti-ADM antibody fragment, or an anti-ADM non-Ig scaffold.

2. The anti-ADM antibody, anti-ADM fragment, or anti-ADM non-Ig scaffold is ADM-Gly and / or ADM-NH 2 In the treatment of a critically ill patient suffering from an acute disease or acute symptoms selected from the group including severe infections (meningitis, systemic inflammatory response syndrome (SIRS), sepsis, etc.), shock (septic shock, cardiogenic shock, etc.), acute heart failure (including acute decompensated heart failure, chronic heart failure with worsening of symptoms and signs), myocardial infarction, stroke, and organ dysfunction (kidney, liver, heart, lung, etc.), the N-terminal portion (amino acids 1-21) of YRQSMNNNFQGLRSFGCRFGTC (SEQ ID NO: 14), the N-terminal portion 2 Anti-adrenomedullin (ADM) antibodies, anti-ADM antibody fragments, or anti-ADM non-Ig scaffolds used to promote conversion to [a specific type of antibody].

3. The anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold is ADM-Gly and / or ADM-NH 2 In the treatment of a critically ill patient suffering from an acute disease or acute symptoms selected from the group including severe infections (such as meningitis, systemic inflammatory response syndrome (SIRS), and sepsis), shock (such as septic shock and cardiogenic shock), acute heart failure (including acute decompensated heart failure and chronic heart failure with worsening of symptoms and signs), myocardial infarction, stroke, and organ dysfunction (such as kidney, liver, heart, and lung), the N-terminus (amino acid 1) of is recognized and bound to ADM-NH in the circulating ADM-Gly within the patient. 2 Anti-adrenomedullin (ADM) antibodies, anti-ADM antibody fragments, or anti-ADM non-Ig scaffolds used to promote conversion to [a specific type of antibody].

4. The said anti-ADM antibody, anti-ADM fragment or anti-ADM non-Ig scaffold binds to the central region (amino acids 21 to 42) of ADM-Gly and / or ADM-NH 2 : CTVQKLAHQIYQFTDKDKDNVA (SEQ ID NO: 48), for use in the treatment of a critically ill patient suffering from an acute disease or acute symptom selected from the group comprising severe infectious diseases (such as meningitis, systemic inflammatory response syndrome (SIRS), sepsis, etc.), shock (such as septic shock, cardiogenic shock, etc.), acute heart failure (including acute decompensated heart failure, chronic heart failure with worsening symptoms and signs), myocardial infarction, stroke, organ dysfunction (such as kidney, liver, heart, lung, etc.), or dementia, wherein said use is for promoting the conversion of ADM-Gly to ADM-NH in circulating ADM-Gly within said patient 2 An anti-adrenomedullin (ADM) antibody, anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use as described above.

5. The anti-ADM antibody, anti-ADM fragment, or anti-ADM non-Ig scaffold is ADM-Gly and / or ADM-NH 2 In the treatment of a critically ill patient suffering from an acute disease or acute symptoms selected from the group including severe infection (meningitis, systemic inflammatory response syndrome (SIRS), sepsis, etc.), shock (septic shock, cardiogenic shock, etc.), acute heart failure (including acute decompensated heart failure, chronic heart failure with worsening of symptoms and signs), myocardial infarction, stroke, organ dysfunction (kidney, liver, heart, lung, etc.), or dementia, the central region (amino acids 21-32) of CTVQKLAHQIYQ (SEQ ID NO: 15) is bound to CTVQKLAHQIYQ as described in claim 4, wherein ADM-NH is converted from ADM-Gly in the circulating ADM-Gly within the patient. 2 Anti-adrenomedullin (ADM) antibodies, anti-ADM antibody fragments, or anti-ADM non-Ig scaffolds used to promote conversion to [a specific type of antibody].

6. The anti-ADM antibody, anti-ADM fragment, or anti-ADM non-Ig scaffold is ADM-Gly and / or ADM-NH 2 The central region (amino acids 21-42): CTVQKLAHQIYQFTDKDKDNVA (SEQ ID NO: 48) is bound to the treatment of a severely ill patient suffering from dementia according to claim 1, wherein ADM-Gly in the circulating ADM-Gly within the patient is converted to ADM-NH 2 Anti-adrenomedullin (ADM) antibodies, anti-ADM antibody fragments, or anti-ADM non-Ig scaffolds used to promote conversion to [a specific type of antibody].

7. The anti-ADM antibody, anti-ADM fragment, or anti-ADM non-Ig scaffold is ADM-Gly and / or ADM-NH 2 The central region (amino acids 21-32): CTVQKLAHQIYQ (SEQ ID NO: 15) is bound to the treatment of a severely ill patient suffering from dementia according to claim 6, wherein ADM-Gly in the circulating ADM-Gly within the patient is converted to ADM-NH 2 Anti-adrenomedullin (ADM) antibodies, anti-ADM antibody fragments, or anti-ADM non-Ig scaffolds used to promote conversion to [a specific type of antibody].

8. The anti-ADM antibody, anti-ADM fragment, or anti-ADM non-Ig scaffold is ADM-Gly and / or ADM-NH 2 In the treatment of a severely ill patient suffering from dementia according to claim 6, the central region (amino acids 27-39) of AHQIYQFTDKDKD (SEQ ID NO: 49) is bound to ADM-Gly in the circulating ADM-Gly within the patient, and ADM-NH 2 Anti-adrenomedullin (ADM) antibodies, anti-ADM antibody fragments, or anti-ADM non-Ig scaffolds used to promote conversion to [a specific type of antibody].

9. In the patient's body fluid sample, the levels of proadrenomedullin or its fragments, consisting of PAMP (SEQ ID NO: 32), MR-proADM (SEQ ID NO: 33), ADM-Gly (SEQ ID NO: 21), and CT-proADM (SEQ ID NO: 34), and ADM-NH 2 In the treatment of a critically ill patient suffering from an acute disease or acute symptoms selected from the group including severe infection (such as meningitis, systemic inflammatory response syndrome (SIRS), sepsis), shock (such as septic shock, cardiogenic shock), acute heart failure (including acute decompensated heart failure, chronic heart failure with worsening of symptoms and signs), myocardial infarction, stroke, organ dysfunction (such as kidney, liver, heart, lung), or dementia, the level of (SEQ ID NO: 20) is measured, wherein the level of ADM-Gly in the circulating ADM-Gly in the patient is measured from ADM-NH 2 Anti-adrenomedullin (ADM) antibodies, anti-ADM antibody fragments, or anti-ADM non-Ig scaffolds used to promote conversion to [a specific type of antibody].

10. In the body fluid sample from the aforementioned patient, ADM-Gly (SEQ ID NO: 21) and ADM-NH 2 If the level ratio with (SEQ ID NO: 20) is measured and exceeds a certain threshold, the patient is treated with the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold, where ADM-Gly / ADM-NH 2 In the treatment of a critically ill patient suffering from an acute disease or acute symptoms selected from the group including severe infection (meningitis, systemic inflammatory response syndrome (SIRS), sepsis, etc.), shock (septic shock, cardiogenic shock, etc.), acute heart failure (including acute decompensated heart failure, chronic heart failure with worsening of symptoms and signs), myocardial infarction, stroke, organ dysfunction (kidney, liver, heart, lung, etc.), or dementia, wherein the ratio is 1 to 10, preferably 1.5 to 7.5, preferably 2 to 5, most preferably 2.5, in which the ratio is 1 to 10, preferably 1.5 to 7.5, preferably 2 to 5, most preferably 2.5 2 Anti-adrenomedullin (ADM) antibodies, anti-ADM antibody fragments, or anti-ADM non-Ig scaffolds used to promote conversion to [a specific type of antibody].

11. In the treatment of a critically ill patient suffering from an acute disease or acute symptoms selected from the group including severe infection (such as meningitis, systemic inflammatory response syndrome (SIRS), sepsis), shock (such as septic shock, cardiogenic shock), acute heart failure (including acute decompensated heart failure, chronic heart failure with worsening of symptoms and signs), myocardial infarction, stroke, organ dysfunction (such as kidney, liver, heart, lung), or dementia, the patient's body fluid sample is selected from the group including blood, serum, plasma, urine, cerebrospinal fluid (CSF), and saliva, in which case the patient's body fluid sample is selected from the group including blood, serum, plasma, urine, cerebrospinal fluid (CSF), and saliva, the patient's body fluid sample is selected from the group including blood, serum, plasma, urine, cerebrospinal fluid (CSF), and saliva, in which case the patient's body fluid sample is selected from the group including severe infection (such as meningitis, systemic inflammatory response syndrome (SIRS), sepsis), shock (such as septic shock, cardiogenic shock), acute heart failure (including acute decompensated heart failure, chronic heart failure with worsening of symptoms and signs), myocardial infarction, stroke, organ dysfunction (such as kidney, liver, heart, lung), or dementia, the patient's body fluid sample is selected from the group including blood, serum, plasma, urine, cerebrospinal fluid (CSF), and saliva, in which case ADM-Gly, ADM-NH 2 Anti-adrenomedullin (ADM) antibodies, anti-ADM antibody fragments, or anti-ADM non-Ig scaffolds used to promote conversion to [a specific type of antibody].

12. In the treatment of a critically ill patient suffering from an acute disease or acute symptoms selected from the group including severe infection (meningitis, systemic inflammatory response syndrome (SIRS), sepsis, etc.), shock (septic shock, cardiogenic shock, etc.), acute heart failure (including acute decompensated heart failure, chronic heart failure with worsening of symptoms and signs), myocardial infarction, stroke, organ dysfunction (kidney, liver, heart, lung, etc.), or dementia, the sample is selected from the group including human citrate plasma, heparin plasma, and EDTA plasma, wherein the sample is selected from the group including human citrate plasma, heparin plasma, and EDTA plasma, and 2 Anti-adrenomedullin (ADM) antibodies, anti-ADM antibody fragments, or anti-ADM non-Ig scaffolds used to promote conversion to [a specific type of antibody].

13. Proadrenomedullin or its fragments and ADM-NH 2 An immunoassay method is used to measure the ratio of proadrenomedullin or its fragments, and the proadrenomedullin or its fragments are selected from the group consisting of PAMP (SEQ ID NO: 32), MR-proADM (SEQ ID NO: 33), ADM-Gly (SEQ ID NO: 21), and CT-proADM (SEQ ID NO: 34), in the treatment of a critically ill patient suffering from an acute disease or acute symptoms selected from the group including severe infection (meningitis, systemic inflammatory response syndrome (SIRS), sepsis, etc.), shock (septic shock, cardiogenic shock, etc.), acute heart failure (including acute decompensated heart failure, chronic heart failure with worsening of symptoms and signs), myocardial infarction, stroke, organ dysfunction (kidney, liver, heart, lung, etc.), or dementia, in which an immunoassay method is used to measure the ratio of ADM-NH to ADM-Gly in the circulating ADM-Gly in the patient. 2 Anti-adrenomedullin (ADM) antibodies, anti-ADM antibody fragments, or anti-ADM non-Ig scaffolds used to promote conversion to [a specific type of antibody].

14. The immunoassay method is a sandwich immunoassay method, preferably a fully automated immunoassay method, and in the treatment of a critically ill patient suffering from an acute disease or acute symptoms selected from the group including severe infection (meningitis, systemic inflammatory response syndrome (SIRS), sepsis, etc.), shock (septic shock, cardiogenic shock, etc.), acute heart failure (including acute decompensated heart failure, chronic heart failure with worsening of symptoms and signs), myocardial infarction, stroke, organ dysfunction (kidney, liver, heart, lung, etc.), or dementia, the immunoassay method is a sandwich immunoassay method, preferably a fully automated immunoassay method, and the immunoassay method is described in claim 13, and the immunoassay method is selected from the group including severe infection (meningitis, systemic inflammatory response syndrome (SIRS), sepsis, etc.), shock (septic shock, cardiogenic shock, etc.), acute heart failure (including acute decompensated heart failure, chronic heart failure with worsening of symptoms and signs), myocardial infarction, stroke, organ dysfunction (kidney, liver, heart, lung, etc.), or dementia, in which ADM-NH is extracted from ADM-Gly in the circulating ADM-Gly within the patient. 2 Anti-adrenomedullin (ADM) antibodies, anti-ADM antibody fragments, or anti-ADM non-Ig scaffolds used to promote conversion to [a specific type of antibody].

15. ADM-NH 2 The measurement sensitivity of the measurement method for detecting ADM-NH in healthy subjects is 2 In the treatment of a critically ill patient suffering from an acute disease or acute symptoms selected from the group including severe infection (meningitis, systemic inflammatory response syndrome (SIRS), sepsis, etc.), shock (septic shock, cardiogenic shock, etc.), acute heart failure (including acute decompensated heart failure, chronic heart failure with worsening of symptoms and signs), myocardial infarction, stroke, organ dysfunction (kidney, liver, heart, lung, etc.), or dementia, the ability to quantify ADM-NH from ADM-Gly in the circulating ADM-Gly within the patient, wherein the sensitivity is less than 70 pg / mL, preferably less than 40 pg / mL, more preferably less than 10 pg / mL, according to any one of claims 1 to 14, wherein the sensitivity is less than 70 pg / mL, preferably less than 40 pg / mL, more preferably less than 10 pg / mL 2 Anti-adrenomedullin (ADM) antibodies, anti-ADM antibody fragments, or anti-ADM non-Ig scaffolds used to promote conversion to [a specific type of antibody].

16. The measurement sensitivity of the measurement method for ADM-Gly is such that it can quantify ADM-Gly in a healthy subject, and the sensitivity is 20 pg / mL, preferably 15 pg / mL, more preferably 10 pg / mL, as described in any one of claims 1 to 14, wherein in the treatment of a critically ill patient suffering from an acute disease or acute symptoms selected from the group including severe infection (meningitis, systemic inflammatory response syndrome (SIRS), sepsis, etc.), shock (septic shock, cardiogenic shock, etc.), acute heart failure (including acute decompensated heart failure, chronic heart failure with exacerbation of symptoms and signs), myocardial infarction, stroke, organ dysfunction (kidney, liver, heart, lung, etc.), or dementia, the measurement method for ADM-Gly is such that it can quantify ADM-Gly in a healthy subject, and the sensitivity is 20 pg / mL, preferably 15 pg / mL, more preferably 10 pg / mL, the measurement sensitivity of the measurement method for ADM-Gly is such that it can quantify ADM-Gly in a healthy subject, and the sensitivity is 20 pg / mL, preferably 15 pg / mL, more preferably 10 pg / mL, the measurement sensitivity of the measurement method for ADM-Gly is such that it can quantify ADM-Gly in a healthy subject, and the measurement 2 Anti-adrenomedullin (ADM) antibodies, anti-ADM antibody fragments, or anti-ADM non-Ig scaffolds used to promote conversion to [a specific type of antibody].

17. Proadrenomedullin or its fragments and ADM-NH 2 The level of (SEQ ID NO: 20) is the first binder for the proadrenomedullin or its fragment and ADM-NH 2 The measurement was performed using a second binder for (SEQ ID NO: 20), and the proadrenomedullin or its fragment was selected from the group consisting of PAMP (SEQ ID NO: 32), MR-proADM (SEQ ID NO: 33), ADM-Gly (SEQ ID NO: 21), and CT-proADM (SEQ ID NO: 34), and both binders were the proadrenomedullin or its fragment and ADM-NH 2 In the treatment of a critically ill patient suffering from an acute disease or acute symptoms selected from the group including severe infections (such as meningitis, systemic inflammatory response syndrome (SIRS), sepsis, etc.), shock (such as septic shock, cardiogenic shock, etc.), acute heart failure (including acute decompensated heart failure, chronic heart failure with worsening of symptoms and signs), myocardial infarction, stroke, organ dysfunction (such as kidney, liver, heart, lung, etc.), or dementia, selected from the group comprising antibodies, antibody fragments, or non-Ig scaffolds that bind to ADM-NH 2 Anti-adrenomedullin (ADM) antibodies, anti-ADM antibody fragments, or anti-ADM non-Ig scaffolds used to promote conversion to [a specific type of antibody].

18. An anti-adrenomedullin (ADM) antibody, anti-ADM antibody fragment, or anti-ADM non-Ig scaffold for use in the treatment of a patient, wherein the anti-ADM antibody, ADM fragment, or anti-ADM non-Ig scaffold is ADM-Gly and / or ADM-NH 2 The N-terminal and / or central region (amino acids 1-42) of YRQSMNNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVA (SEQ ID NO: 23) is bound to the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold, and the levels of peptidylglycine α-amidate monooxygenase (PAM) and / or its isoform and / or fragment are measured in a body fluid sample of the patient, and if the level of peptidylglycine α-amidate monooxygenase (PAM) is below a threshold, the patient is treated with an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold.

19. The level of the PAM and / or its isoform and / or its fragment is the total concentration of the PAM and / or its isoform and / or its fragment having at least 12 amino acids, or the activity of the PAM and / or its isoform and / or its fragment containing the sequences of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47, an anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in the treatment of a patient according to claim 18.

20. An anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold combined with L-ascorbic acid, a. To be used in the treatment of an acute disease or acute condition in a patient in order to stabilize the patient's systemic circulatory system, where the patient requires stabilization of the systemic circulatory system and exhibits a heart rate greater than 100 beats / min and / or a mean arterial pressure less than 65 mmHg, and stabilization of the systemic circulatory system means raising the mean arterial pressure above 65 mmHg; or b. To be used in patients suffering from acute illness or acute condition to prevent an increase in heart rate to more than 100 beats / min and / or a decrease in mean arterial pressure to less than 65 mmHg; or c. For the prevention or reduction of organ dysfunction in the aforementioned patient, or for the prevention of organ failure, to be used in the treatment of acute illnesses or acute symptoms in patients suffering from chronic and / or acute illnesses or acute symptoms, wherein the organs are selected from the group including the heart, kidneys, liver, lungs, pancreas, small intestine, and spleen; or d. To use in the treatment or prevention of shock such as SIRS, meningitis, sepsis, or septic shock in patients; or e. To reduce the risk of death in patients suffering from shock such as SIRS, meningitis, sepsis, and septic shock. Here, the anti-ADM antibody, anti-ADM fragment, or anti-ADM non-Ig scaffold is ADM-Gly and / or ADM-NH 2 The N-terminal and / or central region (amino acids 1-42) of YRQSMNNNFQGLRSFGCRFGTCTTVQKLAHQIYQFTDKDKDNVA (SEQ ID NO: 23) is conjugated to an anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold combined with L-ascorbic acid.

21. An anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold combined with L-ascorbic acid according to claim 20, wherein the L-ascorbic acid is a single enantiomer, a mixture of enantiomers, a mixture of diastereomers, or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof.

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