Anti - adrenomedullin (ADM) antibody or anti - ADM antibody fragment or anti - ADM non - Ig scaffold for use in the treatment or prevention of shock

Abstract

This application relates to an anti - adrenomedullin (ADM) antibody or anti - ADM antibody fragment or anti - ADM non - Ig scaffold for use in treating or preventing shock in a patient, where the patient is characterized by having a level of dipeptidyl peptidase 3 (DPP3) in a body fluid sample that is below a threshold value, the threshold value of the DPP3 being 40 ng / ml or less or in the range of 22 - 40 ng / ml, and the anti - ADM antibody or anti - ADM fragment or anti - ADM non - Ig scaffold binds to the N - terminal portion of ADM (amino acids 1 - 21): YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO: 14).

Description

Technical Field

[0001] The subject of the present invention is an anti - adrenomedullin (ADM) antibody or an anti - ADM antibody fragment or an anti - ADM non - Ig scaffold for use in the treatment or prevention of shock in a patient, where the patient is characterized in that the level of dipeptidyl peptidase 3 (DPP3) in a sample of body fluid is less than a threshold value, the threshold value of DPP3 is 40 ng / ml or less or in the range of 22 - 40 ng / ml, and the anti - ADM antibody or anti - ADM fragment or anti - ADM non - Ig scaffold binds to the N - terminal portion of ADM (amino acids 1 - 21): YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO: 14).

Background Art

[0002] Dipeptidyl peptidase 3, - dipeptidyl aminopeptidase III, dipeptidyl arylamidase III, dipeptidyl peptidase III, enkephalinase B or erythrocyte angiotensinase, also known as DPP3, DPPIII for short, is a metallopeptidase that removes dipeptides from bioactive peptides such as enkephalin and angiotensin. DPP3 was first identified and its activity measured in an extract of bovine anterior pituitary purified by Ellis & Nuenke in 1967. This enzyme, described as EC 3.4.14.4, has a molecular weight of approximately 83 kDa and is highly conserved in prokaryotes and eukaryotes (Prajapati & Chauhan 2011). The amino acid sequence of the human variant is shown in SEQ ID NO: 1. Dipeptidyl peptidase III is mainly a cytosolic peptidase and is ubiquitously expressed. Despite lacking a signal sequence, membrane activity has been reported in some studies (Lee & Snyder 1982).

[0003] DPP3 is a zinc-dependent exopeptidase belonging to the peptidase family M49. It has a broad substrate specificity for oligopeptides of 3 / 4 to 10 amino acids of various compositions and can also cleave after proline. DPP3 is known to hydrolyze dipeptides from the N-terminus of its substrates, including angiotensin II, III, and IV, Leu- and Met-enkephalin, endomorphin 1 and 2. The metallopeptidase DPP3 has an optimal activity pH of 8.0 - 9.0 and can be activated by the addition of divalent metal ions such as Co 2+ and Mg 2+ etc.

[0004] Structural analysis of DPP3 has revealed the catalytic motifs HELLGH (hDPP3 450 - 455) and EECRAE (hDPP3 507 - 512), and the following amino acids important for substrate binding and hydrolysis: Glu316, Tyr318, Asp366, Asn391, Asn394, His568, Arg572, Arg577, Lys666, and Arg669 (Prajapati & Chauhan 2011, Kumar et al. 2016; the numbering refers to the sequence of human DPP3 (see SEQ ID NO: 1)). Considering all known amino acids or sequence regions involved in substrate binding and hydrolysis, the active site of human DPP3 can be defined as the region of amino acids 316 - 669.

[0005] The most prominent substrate of DPP3 is angiotensin II (Ang II), a major effector of the renin-angiotensin system (RAS). The RAS is activated in cardiovascular diseases (Dostal et al. 1997. J Mol Cell Cardiol; 29: 2893 - 902, Roks et al. 1997. Heart Vessels. Suppl 12: 119 - 24), sepsis, and septic shock (Correa et al. 2015. Crit Care 2015; 19: 98). In particular, Ang II has been shown to regulate many cardiovascular functions, including the control of blood pressure and cardiac remodeling.

[0006] Recently, to specifically detect DPP3 in human body fluids (e.g., blood, plasma, serum), the following two assays have been created, characterized, and validated: a luminescence immunoassay (LIA) to detect DPP3 protein concentration, and an enzyme capture activity assay (ECA) to detect specific DPP3 activity (Rehfeld et al. 2019. JALM 3(6):943-953). The washing step removes all interfering substances before the actual detection of DPP3 activity. Both methods are highly specific and enable reproducible detection of DPP3 in blood samples.

[0007] Blood circulation DPP3 levels have been shown to increase in patients with cardiogenic shock and are associated with an increased risk of short-term death and severe organ dysfunction (Deaniau et al. 2019. Eur J Heart Fail. in press). Furthermore, DPP3 measured at the time of incorporation discriminated between cardiogenic shock patients who developed refractory shock and those who did not, and a DPP3 concentration of 59.1 ng / mL or higher was associated with a greater risk of death (Takagi et al. Eur J Heart Fail. in press).

[0008] Peptide adrenomedullin (ADM) was first reported in 1993 as a novel hypotensive peptide containing 52 amino acids (SEQ ID NO: 20) isolated from a human pheochromocytoma cell line (Kitamura et al., 1993. Biochem Biophys Res Comm 192(2):553-560). In the same year, the cDNA encoding the precursor peptide containing 185 amino acids and the complete amino acid sequence of this precursor peptide were also reported. 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 amino acid positions specified usually relate to pre-proADM containing 185 amino acids. Peptide adrenomedullin (ADM) contains 52 amino acids (SEQ ID NO: 20) and is a peptide containing amino acids 95-146 of pre-proADM, which is formed by proteolytic cleavage from pre-proADM. To date, substantially only a very small number of peptide fragments formed in the cleavage of pre-proADM, particularly the bioactive peptides ADM and "PAMP", that is, the 20 amino acids (22-41) following the 21 amino acids of the signal peptide in pre-proADM, have been more precisely investigated. The discovery and characterization of ADM in 1993 initiated intensive research activities, and the results have been summarized in various review articles, and in the context of this specification, particularly the articles published in the ADM special issue of "Peptides" (Takahashi 2001. Peptides 22:1691, Eto 2001. Peptides 22:1693-1711) are referred to. A further review is Hinson et al. 2000 (Hinson et al. 2000. Endocrine Reviews 21(2):138-167). In scientific investigations to date, it has been found that, among other things, ADM can be regarded as a multifunctional regulatory peptide. It is released into the bloodstream in an inactive form extended by glycine (Kitamura et al. 1998. Biochem Biophys Res Commun 244(2):551-555).There are also binding proteins that are specific for ADM and perhaps similarly regulate the action of ADM (Pio et al. 2001. The Journal of Biological Chemistry 276(15):12292-12300). In the investigations to date, the most important physiological actions of ADM and PAMP were those that affected blood pressure.

[0009] Therefore, ADM is a potent vasodilator, and thus, the hypotensive effect can be associated with specific peptide segments of the C-terminal portion of ADM. Furthermore, the above-described bioactive peptide PAMP formed from pre-proADM has been found to exhibit a hypotensive effect as well, even when it is thought to have a mechanism of action different from that of ADM (in addition to the above-mentioned review papers Eto et al. 2001 and Hinson et al. 2000, see also 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 European Patent Application Publication No. 0622458 (A2)). Furthermore, it has been found that the concentrations of ADM that can be measured in the blood circulation and other biological fluids are significantly higher in some pathological conditions than in healthy control subjects. Therefore, the ADM levels in patients with congestive heart failure, myocardial infarction, renal disease, hypertensive disorders, patients with type 2 diabetes, the acute phase of shock, and patients with sepsis and septic shock are significantly increased, although to varying degrees. The PAMP concentration also increases in some of the above pathological conditions, but the plasma level is decreased compared to ADM (Eto 2001. Peptides 22:1693-1711). Abnormally high concentrations of ADM have been observed in sepsis and reported to reach the highest concentration 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 and Wang et al. 2001. Peptides 22:1835-1840).

[0010] The plasma concentration of ADM is elevated in patients with heart failure and correlates with the severity of the disease (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 subjects (Poyner et al. 2002. Pharmacol Rev 54:233 - 246).

[0011] WO 2004 / 097423 describes the use of antibodies against adrenomedullin for the diagnosis, prognosis and treatment of cardiovascular disorders. Treatment of diseases by blocking ADM receptors has also been described in the art (for example, WO 2006 / 027147, PCT / EP2005 / 012844), and the diseases can be sepsis, septic shock, cardiovascular diseases, infectious diseases, skin diseases, endocrine diseases, metabolic diseases, gastrointestinal diseases, cancer, inflammation, blood diseases, respiratory diseases, musculoskeletal diseases, neurological diseases, urological diseases.

[0012] In the early stage of sepsis, ADM has been reported to improve cardiac function and blood supply in the liver, spleen, kidney and small intestine. Anti - ADM neutralizing antibodies neutralize the aforementioned effects during the early stage of sepsis (Wang et al. 2001. Peptides 22:1835 - 1840).

[0013] For other diseases, blockade of ADM may be beneficial to some extent. However, since a certain amount of ADM may be required for some physiological functions, complete neutralization of ADM can also be harmful. In many reports, it has been emphasized that administration of ADM can be beneficial in certain diseases. In contrast, in other reports, ADM has been reported to be life - threatening when administered in certain conditions.

[0014] WO 2013 / 072510 describes non - neutralizing anti - ADM antibodies for use in the treatment of severe chronic or acute diseases or acute conditions of a patient to reduce the patient's risk of death.

[0015] International Publication No. 2013 / 072511 describes a non-neutralizing anti-ADM antibody for use in the treatment of chronic or acute diseases or acute conditions of a patient for the prevention or alleviation of organ dysfunction or organ failure.

[0016] International Publication No. 2013 / 072512 describes a non-neutralizing anti-ADM antibody that is an ADM-stabilizing antibody that extends the half-life (t 1 / 2 half retention time) of adrenomedullin in serum, blood, and plasma. This ADM-stabilizing antibody blocks the biological activity of ADM to less than 80%.

[0017] International Publication No. 2013 / 072513 describes a non-neutralizing anti-ADM antibody for use in the treatment of acute diseases or conditions of a patient for stabilizing blood circulation.

[0018] International Publication No. 2013 / 072514 describes a non-neutralizing anti-ADM antibody for regulating fluid balance in a patient having a chronic or acute disease or acute condition.

[0019] International Publication No. 2017 / 182561 describes a method for determining the total amount of active DPP3 in a patient's sample for the diagnosis of diseases associated with a necrotic process. This further describes a method for treating necrotic-related diseases with an antibody against DPP3.

[0020] International Publication No. 2021 / 170838 describes a method for guiding and / or monitoring and / or stratifying treatment in a patient having shock and a patient at risk of shock by determining the level of DPP3 and, when the level is preferably less than the threshold of 50 ng / ml, administering an anti-ADM antibody.

[0021] High DPP3 blood levels are associated with higher organ dysfunction scores, the need for cardiovascular support, and the development of myocardial dysfunction, refractory shock, acute kidney injury, and increased short-term mortality. Based on these clinical associations, it is reasonable to assume that active DPP3 released into the blood of shock patients contributes to the reduction of vasoconstriction in shock syndrome by nullifying the pressor effect of endogenous Ang II (Malovan G, Hierzberger B, Suraci S, Schaefer M, Santos K, Jha S, Macheroux P. FEBS J. 2022 Mar 12. doi: 10.1111 / febs.16429.). In patients with shock, the presence of endothelial dysfunction is detected using the biomarker bio-ADM, while myocardial dysfunction is detected by measuring DPP3. DPP3 above the threshold is used as an exclusion criterion for the application of pharmaceuticals that address different pathways than DPP3, such as anti-ADM antibodies, particularly the anti-ADM antibody atacicept that targets the N-terminus of ADM. Atacicept therapy showed better efficacy when patients with DPP3 above 50 ng / ml were excluded from treatment. However, the surprising discovery of the present invention is that patients treated with the anti-ADM antibody atacicept have a higher likelihood of benefiting from this treatment when the level of DPP3 in the body fluid sample is well below the 50 ng / ml threshold, preferably below the 40 ng / ml threshold, or in the range of 22 ng / ml to 40 ng / ml.

[0022] In other words, the surprising discovery of the present invention is that in patients with shock and patients who have fallen into a shock state, when the level of DPP3 in the body fluid sample is below the 40 ng / ml threshold or in the range of 22 ng / ml to 40 ng / ml, the level of DPP3 in the body fluid sample is used for treatment guidance and / or treatment monitoring and / or treatment stratification by anti-ADM antibodies and / or anti-ADM antibody fragments and / or anti-ADM non-Ig scaffolds.

Summary of the Invention

[0023] The subject of the present invention is an anti - adrenomedullin (ADM) antibody or anti - ADM antibody fragment or anti - ADM non - Ig scaffold for use in the treatment or prevention of shock in a patient, wherein the patient is characterized in that the level of DPP3 in a sample of body fluid is less than a threshold value, the threshold value of DPP3 is 40 ng / ml or less or in the range of 22 ng / ml to 40 ng / ml, and the anti - ADM antibody or anti - ADM fragment or anti - ADM non - Ig scaffold binds to the N - terminal portion of ADM (amino acids 1 - 21): YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO: 14).

[0024] One 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 or prevention of shock in a patient, wherein the shock is selected from the group including shock due to decreased circulating blood volume, cardiogenic shock, obstructive shock, and distributive shock, particularly cardiogenic shock or septic shock.

[0025] 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 or prevention of shock in a patient, wherein ● in the case of cardiogenic shock, the patient may be suffering from acute coronary syndrome (e.g., acute myocardial infarction), or the patient has heart failure (e.g., acute decompensated heart failure), myocarditis, arrhythmia, cardiomyopathy, valvular heart disease, aortic dissection with acute aortic valve stenosis, traumatic chordae tendineae rupture or massive pulmonary embolism, or ● in the case of shock due to decreased circulating blood volume, the patient may be suffering from a hemorrhagic disease including gastrointestinal bleeding, trauma, vascular etiology (e.g., ruptured abdominal aortic aneurysm, tumor eroding major blood vessels) and spontaneous bleeding in the context of anticoagulant use, or a non - hemorrhagic disease including vomiting, diarrhea, renal loss, cutaneous loss / insensible loss (e.g., burns, heat stroke) or loss to the third space in the context of pancreatitis, cirrhosis, intestinal obstruction, trauma, or ● In the case of obstructive shock, the patient may be suffering from cardiac tamponade, tension pneumothorax, pulmonary embolism or aortic valve stenosis, or ● In the case of distributive shock, the patient may have septic shock, neurogenic shock, anaphylactic shock or shock due to adrenal crisis.

[0026] A preferred embodiment of the present application relates to an anti-ADM antibody or an anti-ADM antibody fragment or an anti-ADM non-Ig scaffold for use in the treatment or prevention of shock in a patient, wherein the threshold value of the DPP3 in a sample of the body fluid of the patient is 30 ng / ml or less or in the range of 22 to 30 ng / mL.

[0027] A preferred embodiment of the present application relates to an anti-ADM antibody or an anti-ADM antibody fragment or an anti-ADM non-Ig scaffold for use in the treatment or prevention of shock in a patient, wherein the threshold value of the DPP3 in a sample of the body fluid of the patient is 25 ng / ml or less or in the range of 22 to 25 ng / ml.

[0028] A specific embodiment of the present application relates to an anti-ADM antibody or an anti-ADM antibody fragment or an anti-ADM non-Ig scaffold for use in the treatment or prevention of shock in a patient, wherein the level of the DPP3 is determined by contacting the sample of the body fluid with a capture binder that specifically binds to the DPP3.

[0029] Another embodiment of the present application relates to an anti-ADM antibody or an anti-ADM antibody fragment or an anti-ADM non-Ig scaffold for use in the treatment or prevention of shock in a patient, wherein either the level of the DPP3 protein and / or the level of the active DPP3 is determined and compared with a threshold value.

[0030] One 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 or prevention of shock in a patient, wherein the patient is further characterized by having a level of ADM-NH2 that exceeds a threshold value. To identify a patient with shock, the level of ADM-NH2 is measured.

[0031] A preferred 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 or prevention of shock in a patient, wherein the threshold value of the ADM-NH2 in a sample of the patient's body fluid is 40 to 100 pg / mL, more preferably 50 to 90 pg / mL, even more preferably 60 to 80 pg / mL, and most preferably, the threshold value is 70 pg / mL.

[0032] 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 or prevention of shock in a patient, wherein the level of the ADM-NH2 is determined by contacting the sample of the body fluid with a capture binder that specifically binds to ADM-NH2.

[0033] Another preferred 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 or prevention of shock in a patient, wherein the sample of the patient's body fluid is selected from the group consisting of blood, serum, plasma, urine, cerebrospinal fluid (CSF), and saliva.

[0034] Another specific 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 or prevention of shock in a patient, 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.

[0035] A further 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 or prevention of shock in a patient, wherein the antibody, antibody fragment or non-Ig scaffold does not bind to the C-terminal portion of ADM having the amino acids 43-52 of the ADM sequence: PRSKISPQGY-NH2 (SEQ ID NO: 24).

[0036] One 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 or prevention of shock in a patient, wherein the antibody or fragment or scaffold blocks the biological activity of ADM by 80% or less, preferably 50% or less.

[0037] 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 or prevention of shock in a patient, wherein the antibody or fragment is a monoclonal antibody or fragment or antibody fragment thereof that binds to ADM, and the heavy chain has the sequence: CDR1: SEQ ID NO: 1 GYTFSRYW CDR2: SEQ ID NO: 2 ILPGSGST CDR3: SEQ ID NO: 3 including TEGYEYDGFDY, and the light chain has the sequence: CDR1: SEQ ID NO: 4 QSIVYSNGNTY CDR2: RVS CDR3: SEQ ID NO: 5 including FQGSHIPYT.

[0038] 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 or prevention of shock in a patient, wherein the antibody or fragment is as the VH region: SEQ ID NO: 6 (AM-VH-C) QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK Sequence number 7 (AM-VH1) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK Sequence number 8 (AM-VH2-E40) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK Sequence number 9 (AM-VH3-T26-E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK Sequence number 10 (AM-VH4-T26-E40-E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK comprising a sequence selected from the group consisting of as the VL region the following sequence: Sequence number 11 (AM-VL-C) DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Sequence number 12 (AM-VL1) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Sequence number 13 (AM-VL2-E40)[[ID=*3]] It includes a sequence selected from the group consisting of DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0039] 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 or prevention of shock in a patient, wherein the antibody or fragment has the following sequence as the heavy chain: Sequence number 32 Note: In the translation of line 4, the original text seems to have a minor difference in "YLNWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC" compared to line 0. It is translated based on the text provided in line 4. Also, the " " in line 3 is marked with an asterisk as it seems to be an oddity in the original text structure and might be a misplacement or something to be further clarified in the original context. The translation attempts to maintain the integrity of the provided text as much as possible.QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGEILPGSGSTNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK or a sequence that is more than 95% identical thereto, and as the light chain the following sequence: SEQ ID NO: 33 DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPRLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC or a sequence that is more than 95% identical thereto.

[0040] The subject matter of the present application is also a pharmaceutical preparation for use in the treatment or prevention of shock in a patient, comprising an anti-ADM antibody or an anti-ADM antibody fragment or an anti-ADM non-Ig scaffold.

[0041] One embodiment of the present application relates to a pharmaceutical preparation for use in the treatment or prevention of shock in a patient, wherein the pharmaceutical preparation is a solution, preferably a ready-to-use solution.

[0042] Another embodiment of the present application relates to a pharmaceutical preparation for use in the treatment or prevention of shock in a patient, wherein the pharmaceutical preparation is in a lyophilized state.

[0043] One embodiment of the present application relates to a pharmaceutical preparation for use in the treatment or prevention of shock in a patient, wherein the pharmaceutical preparation is administered intramuscularly.

[0044] One embodiment of the present application relates to a pharmaceutical preparation for use in the treatment or prevention of shock in a patient, wherein the pharmaceutical preparation is administered intravascularly.

[0045] Another embodiment of the present application relates to a pharmaceutical preparation for use in the treatment or prevention of shock in a patient, wherein the pharmaceutical preparation is administered via infusion.

[0046] Another specific embodiment of the present application relates to a pharmaceutical preparation for use in the treatment or prevention of shock in a patient, wherein the pharmaceutical preparation is administered systemically.

[0047] Another embodiment of the present application relates to a method for treating or preventing shock in a patient, wherein the method comprises administering to the patient an anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment or an anti-ADM non-Ig scaffold, and the method ● determining the level of DPP3 in a sample of the subject's body fluid; ● comparing the determined level of DPP3 with a predetermined threshold, and further comprising, if the determined level of DPP3 is less than the predetermined threshold, the patient is treated, and the predetermined threshold of DPP3 is 40 ng / ml or less or in the range of 22 ng / ml to 40 ng / ml, When the anti-ADM antibody or anti-ADM fragment or anti-ADM non-Ig scaffold binds to the N-terminal portion of ADM (aa 1-21): YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO: 14).

[0048] Another specific embodiment of the present application relates to a method for treating or preventing shock in a patient, wherein the method comprises administering to the patient an anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold, and the method ● determining the level of ADM-NH2 in a sample of the subject's body fluid; ● further comprising comparing the level of the ADM-NH2 with a predetermined threshold value; The patient is treated when the determined level of ADM-NH2 exceeds the predetermined threshold level.

[0049] The subject of the present invention is an anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in treating or preventing shock in a patient, wherein the patient is characterized in that the level of DPP3 in a sample of body fluid is less than a threshold value, the threshold value of DPP3 is 40 ng / ml or less or in the range of 22 ng / ml to 40 ng / ml, and the anti-ADM antibody or anti-ADM fragment or anti-ADM non-Ig scaffold binds to the N-terminal portion of ADM (amino acids 1-21): YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO: 14).

[0050] One 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 treating or preventing shock in a patient, wherein the shock is selected from the group comprising shock due to decreased circulating blood volume, cardiogenic shock, obstructive shock, and distributive shock, particularly cardiogenic shock or septic shock.

[0051] 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 treating or preventing shock in a patient, wherein ● In the case of cardiogenic shock, the patient may be suffering from acute coronary syndrome (e.g., acute myocardial infarction), or the patient has heart failure (e.g., acute decompensated heart failure), myocarditis, arrhythmia, cardiomyopathy, valvular heart disease, aortic dissection with acute aortic valve stenosis, traumatic chordae tendineae rupture or extensive pulmonary embolism, or ● In the case of hypovolemic shock, the patient may be suffering from a hemorrhagic disease including gastrointestinal bleeding, trauma, vascular etiology (e.g., ruptured abdominal aortic aneurysm, tumor eroding major blood vessels) and spontaneous bleeding in the context of anticoagulant use, or a non-hemorrhagic disease including vomiting, diarrhea, renal loss, cutaneous loss / insensible loss (e.g., burns, heat stroke) or loss to the third space in the context of pancreatitis, cirrhosis, intestinal obstruction, trauma, or ● In the case of obstructive shock, the patient may be suffering from cardiac tamponade, tension pneumothorax, pulmonary embolism or aortic valve stenosis, or ● In the case of distributive shock, the patient may have septic shock, neurogenic shock, anaphylactic shock or shock due to adrenal crisis.

[0052] A preferred 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 treating or preventing shock in a patient, wherein the threshold value of the DPP3 in a sample of the patient's body fluid is 30 ng / ml or less or in the range of 22 - 30 ng / mL.

[0053] A preferred 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 treating or preventing shock in a patient, wherein the threshold value of the DPP3 in a sample of the patient's body fluid is 25 ng / ml or less or in the range of 22 - 25 ng / mL.

[0054] One specific embodiment of the present application relates to an anti-ADM antibody or an anti-ADM antibody fragment or an anti-ADM non-Ig scaffold for use in treating or preventing shock in a patient, wherein the level of DPP3 is determined by contacting a sample of the body fluid with a capture binder that specifically binds to DPP3.

[0055] Another embodiment of the present application relates to an anti-ADM antibody or an anti-ADM antibody fragment or an anti-ADM non-Ig scaffold for use in treating or preventing shock in a patient, wherein either the level of DPP3 protein and / or the level of active DPP3 is determined and compared with a predetermined threshold.

[0056] One embodiment of the present application relates to an anti-ADM antibody or an anti-ADM antibody fragment or an anti-ADM non-Ig scaffold for use in treating or preventing shock in a patient, which is further characterized in that the patient has a level of ADM-NH2 exceeding the threshold.

[0057] A preferred embodiment of the present application relates to an anti-ADM antibody or an anti-ADM antibody fragment or an anti-ADM non-Ig scaffold for use in treating or preventing shock in a patient, wherein the threshold of ADM-NH2 in a sample of the patient's body fluid is 40 to 100 pg / mL, more preferably 50 to 90 pg / mL, even more preferably 60 to 80 pg / mL, and most preferably the threshold is 70 pg / mL.

[0058] Another embodiment of the present application relates to an anti-ADM antibody or an anti-ADM antibody fragment or an anti-ADM non-Ig scaffold for use in treating or preventing shock in a patient, wherein the level of ADM-NH2 is determined by contacting a sample of the body fluid with a capture binder that specifically binds to ADM-NH2.

[0059] Another preferred 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 treating or preventing shock in a patient, where a sample of the patient's body fluid is selected from the group consisting of blood, serum, plasma, urine, cerebrospinal fluid (CSF), and saliva.

[0060] In a preferred embodiment, bio-ADM is measured from plasma. However, in improving the technical life cycle of the measurement of an analyte, it is typical that there is a possibility of measuring such an analyte not only in plasma but also in other (at least blood-based) matrices. For example, in the case of bio-ADM, another technique has been developed, which uses whole (EDTA-) blood called IB10 sphingotest® bio-ADM (https: / / www.nexus-dx.com / wp-content / uploads / 2020 / 07 / bio-ADM-IFU-REV-A.pdf). IB10 sphingotest® bio-ADM® is a rapid point-of-care (POC) immunoassay for the in vitro quantitative measurement of human amidated adrenomedullin peptide (1-52) (hereinafter referred to as bioactive adrenomedullin (bio-ADM)) in human EDTA whole blood and plasma.

[0061] Another specific 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 treating or preventing shock in a patient, where 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.

[0062] A further 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 treating or preventing shock in a patient, wherein the antibody, antibody fragment or non-Ig scaffold does not bind to the C-terminal portion of ADM having the amino acids 43-52 of the ADM sequence: PRSKISPQGY-NH2 (SEQ ID NO: 24).

[0063] One 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 treating or preventing shock in a patient, wherein the antibody or fragment or scaffold blocks 80% or less, preferably 50% or less, of the biological activity of ADM.

[0064] 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 treating or preventing shock in a patient, wherein the antibody or fragment is a monoclonal antibody or fragment or antibody fragment thereof that binds to ADM, and the heavy chain has the sequence: CDR1: SEQ ID NO: 1 GYTFSRYW CDR2: SEQ ID NO: 2 ILPGSGST CDR3: SEQ ID NO: 3 including TEGYEYDGFDY, and the light chain has the sequence: CDR1: SEQ ID NO: 4 QSIVYSNGNTY CDR2: RVS CDR3: SEQ ID NO: 5 including FQGSHIPYT.

[0065] 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 treating or preventing shock in a patient, wherein the antibody or fragment is as the VH region: SEQ ID NO: 6 (AM-VH-C) QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK Sequence number 7 (AM-VH1) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK Sequence number 8 (AM-VH2-E40) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK Sequence number 9 (AM-VH3-T26-E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SEQ ID NO: 10 (AM-VH4-T26-E40-E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK comprising an array selected from the group consisting of as the VL region, the following array: SEQ ID NO: 11 (AM-VL-C) DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 12 (AM-VL1) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Sequence number 13 (AM-VL2-E40) It includes a sequence selected from the group consisting of DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0066] 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 treating or preventing shock in a patient, wherein the antibody or fragment has the following sequence as the heavy chain: Sequence number 32 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGEILPGSGSTNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK or a sequence that is more than 95% identical thereto, comprising as the light chain the following sequence: SEQ ID NO: 33 DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPRLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC or a sequence that is more than 95% identical thereto.

[0067] Another embodiment of the present application relates to an anti - adrenomedullin (ADM) antibody or anti - ADM antibody fragment or anti - ADM non - Ig scaffold for use in the treatment or prevention of shock in a patient, wherein the anti - adrenomedullin (ADM) antibody or anti - ADM antibody fragment or anti - ADM non - Ig scaffold binds to the N - terminal portion of ADM (amino acids 1 - 10): YRQSMNNFQG (SEQ ID NO: 26).

[0068] Another embodiment of the present application relates to an anti - adrenomedullin (ADM) antibody or anti - adrenomedullin antibody fragment or anti - ADM non - Ig scaffold for use in treatment, wherein the antibody or fragment or scaffold exhibits a binding affinity of at least 10 -7 M for ADM by label - free surface plasmon resonance using a Biacore 2000 system.

[0069] Another embodiment of the present application relates to an anti - adrenomedullin (ADM) antibody or anti - ADM antibody fragment or anti - ADM non - Ig scaffold for use in the treatment or prevention of shock in a patient, wherein the anti - ADM antibody or anti - ADM antibody fragment or anti - ADM non - Ig scaffold exhibits an affinity of 1×10 -9 ~3×10 -9 M for human ADM by label - free surface plasmon resonance using a Biacore 2000 system.

[0070] Another embodiment of the present application relates to an anti - adrenomedullin (ADM) antibody or anti - ADM antibody fragment or anti - ADM non - Ig scaffold for use in the treatment or prevention of shock in a patient, wherein the anti - ADM antibody or anti - ADM antibody fragment or anti - ADM non - Ig scaffold is an IgG1 antibody.

[0071] The subject matter of the present application also relates to a pharmaceutical formulation for use in the treatment or prevention of shock in a patient, comprising an anti - ADM antibody or anti - ADM antibody fragment or anti - ADM non - Ig scaffold.

[0072] One embodiment of the present application relates to a pharmaceutical preparation for use in treating or preventing shock in a patient, wherein the pharmaceutical preparation is a solution, preferably a ready-to-use solution.

[0073] Another embodiment of the present application relates to a pharmaceutical preparation for use in treating or preventing shock in a patient, wherein the pharmaceutical preparation is in a lyophilized state.

[0074] One embodiment of the present application relates to a pharmaceutical preparation for use in treating or preventing shock in a patient, wherein the pharmaceutical preparation is administered intramuscularly.

[0075] A preferred embodiment of the present application relates to a pharmaceutical preparation for use in treating or preventing shock in a patient, wherein the pharmaceutical preparation is administered intravascularly.

[0076] Another embodiment of the present application relates to a pharmaceutical preparation for use in treating or preventing shock in a patient, wherein the pharmaceutical preparation is administered via infusion.

[0077] Another specific embodiment of the present application relates to a pharmaceutical preparation for use in treating or preventing shock in a patient, wherein the pharmaceutical preparation is administered systemically.

[0078] Another embodiment of the present application relates to a method for treating or preventing shock in a patient, wherein the method comprises administering to the patient an anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment or an anti-ADM non-Ig scaffold, and the method ● determining the level of DPP3 in a sample of the subject's body fluid; ● comparing the determined level of DPP3 with a predetermined threshold, and further comprising, when the determined level of DPP3 is less than the predetermined threshold, the patient is treated, and the predetermined threshold of DPP3 is 40 ng / ml or less or in the range of 22 ng / ml to 40 ng / ml, When the anti-ADM antibody or anti-ADM fragment or anti-ADM non-Ig scaffold binds to the N-terminal portion of ADM (aa 1-21): YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO: 14).

[0079] Another specific embodiment of the present application relates to a method for treating or preventing shock in a patient, where the method includes administering to the patient an anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold, and the method ● determining the level of ADM-NH2 in a sample of the subject's body fluid; ● further including comparing the level of the ADM-NH2 with a predetermined threshold value; When the determined level of ADM-NH2 exceeds the predetermined threshold level, the patient is treated.

[0080] In one embodiment of the present invention, either the level of DPP3 protein and / or the level of active DPP3 is determined and compared with a threshold level.

[0081] In a specific embodiment of the present invention, the threshold value of DPP3 in a sample of the patient's body fluid is 30 ng / ml or less or in the range of 22-30 ng / mL.

[0082] In another specific embodiment of the present invention, the threshold value of DPP3 in a sample of the patient's body fluid is 25 ng / ml or less or in the range of 22-25 ng / mL.

[0083] The amount of DPP3 protein and / or the level of DPP3 as DPP3 activity in a sample of the subject's body fluid can be determined by different methods, such as immunoassay, activity assay, mass spectrometry, etc.

[0084] According to the present invention, any type of binding assay (immunoassays and similar assays that use other types of antigen-specific binding agents instead of antibodies) and b) a DPP3 enzyme activity assay (specific for DPP3 by specifically capturing DPP3 from a sample using a specific binding agent (anti-DPP3 antibody or other type of binding agent) prior to determination of enzyme activity) may be used to determine the level of DPP3 in the sample.

[0085] DPP3 activity can be measured by detection of cleavage products of a DPP3-specific substrate. Known peptide hormone substrates include Leu-enkephalin, Met-enkephalin, endomorphin 1 and 2, barorphin, β-casomorphin, dynorphin, proctolin, ACTH (adrenocorticotropic hormone), and MSH (melanocyte-stimulating hormone; Abramic et al. 2000, Barsun et al. 2007, Dhanda et al. 2008). Cleavage of the peptide hormones and other untagged oligopeptides mentioned (e.g., Ala-Ala-Ala-Ala, Dhanda et al. 2008) can be monitored by detection of the respective cleavage products. Detection methods include, but are not limited to, HPLC analysis (e.g., Lee & Snyder 1982), mass spectrometry (e.g., Abramic et al. 2000), H1-NMR analysis (e.g., Vandenberg et al. 1985), capillary zone electrophoresis (CE; e.g., Barsun et al. 2007), thin layer chromatography (e.g., Dhanda et al. 2008), or reverse phase chromatography (e.g., Mazocco et al. 2006).

[0086] Detection of fluorescence by hydrolysis of a fluorescence-generating substrate by DPP3 is a standard procedure for monitoring DPP3 activity. These substrates are specific di- or tri-peptides (Arg-Arg, Ala-Ala, Ala-Arg, Ala-Phe, Asp-Arg, Gly-Ala, Gly-Arg, Gly-Phe, Leu-Ala, Leu-Gly, Lys-Ala, Phe-Arg, Suc-Ala-Ala-Phe) conjugated to a fluorophore. Fluorophores include, but are not limited to, β-naphthylamide (2-naphthylamide, βNA, 2NA), 4-methoxy-β-naphthylamide (4-methoxy-2-naphthylamide), and 7-amido-4-methylcoumarin (AMC, MCA; Abramic et al. 2000, Ohkubo et al. 1999). Cleavage of these fluorescence-generating substrates results in the release of fluorescent β-naphthylamine or 7-amino-4-methylcoumarin, respectively. In a liquid-phase assay, the ECA substrate and DPP3 are incubated, for example, in a 96-well plate format, and fluorescence is measured using a fluorescence detector (Ellis & Nuenke 1967). Additionally, DPP3-bearing samples can be immobilized, separated on a gel by electrophoresis, and the gel stained with a fluorescence-generating substrate (e.g., Arg-Arg-βNA) and Fast Garnet GBC, and fluorescent protein bands can be detected by a fluorescence reader (Ohkubo et al. 1999). The same peptides (Arg-Arg, Ala-Ala, Ala-Arg, Ala-Phe, Asp-Arg, Gly-Ala, Gly-Arg, Gly-Phe, Leu-Ala, Leu-Gly, Lys-Ala, Phe-Arg, Suc-Ala-Ala-Phe) can be conjugated to a chromophore, such as p-nitroanilide diacetate. Detection of a color change due to hydrolysis of the chromogenic substrate can be used to monitor DPP3 activity.

[0087] Another option for detecting DPP3 activity is the Protease-Glo™ Assay (commercially available from Promega). In this embodiment of the method, a DPP3-specific dipeptide or tripeptide (Arg-Arg, Ala-Ala, Ala-Arg, Ala-Phe, Asp-Arg, Gly-Ala, Gly-Arg, Gly-Phe, Leu-Ala, Leu-Gly, Lys-Ala, Phe-Arg, Suc-Ala-Ala-Phe) is conjugated to aminoluciferin. When cleaved by DPP3, aminoluciferin is released and serves as a substrate for a coupled luciferase reaction that emits detectable light.

[0088] In a preferred embodiment, DPP3 activity is measured by the addition of the fluorogenic substrate Arg-Arg-βNA and real-time monitoring of fluorescence.

[0089] In a specific embodiment of the method for determining active DPP3 in a sample of a subject's body fluid, the capture binding agent reactive with DPP3 is immobilized on a solid phase.

[0090] The test sample is passed over the immobilized binding agent, and if DPP3 is present, DPP3 binds to the binding agent and becomes immobilized for detection. A substrate is then added and the reaction product detected to indicate the presence or amount of DPP3 in the test sample. For the purposes of this specification, the term "solid phase" can be used to include any material or container on which an assay can be performed, and includes, but is not limited to, porous materials, non-porous materials, test tubes, wells, slides, agarose resins (e.g., Sepharose from GE Healthcare Life Sciences), magnetic particles (e.g., Dynabeads™ or Pierce™ magnetic beads from Thermo Fisher Scientific).

[0091] In another embodiment of the invention, the level of DPP3 is determined by contacting a sample of the body fluid with a capture binding agent that specifically binds to DPP3.

[0092] In another preferred embodiment of the present invention, the capture binder for determining the level of DPP3 can be selected from the group consisting of an antibody, an antibody fragment, or a non-IgG scaffold.

[0093] In a specific embodiment of the present invention, the capture binder is an antibody.

[0094] The amount and / or DPP3 activity of DPP3 protein in a sample of the subject's body fluid can be determined, for example, by one of the following methods: 1. Luminescence immunoassay (LIA) for quantification of DPP3 protein concentration (Rehfeld et al., 2019 JALM 3(6):943-953).

[0095] LIA is a one-step chemiluminescent sandwich immunoassay using white high-binding polystyrene microtiter plates as the solid phase. These plates are coated with the monoclonal anti-DPP3 antibody AK2555 (capture antibody). The tracer anti-DPP3 antibody AK2553 is labeled with MA70-acridinium-NHS-ester and used at a concentration of 20 ng / well. 20 microliters of sample (e.g., serum, heparin plasma, citrate plasma, or EDTA plasma derived from a patient's blood) and calibrator are pipetted into the coated white microtiter plates. After adding the tracer antibody AK2553, the microtiter plates are incubated at room temperature and 600 rpm for 3 hours. Unbound tracer is then removed by four washing steps (350 μL / well). The remaining chemiluminescence is measured for 1 second per well using a microtiter plate luminometer. The concentration of DPP3 is determined using a 6-point calibration curve. Calibrator and sample are preferably run in duplicate.

[0096] 2. Enzyme capture activity assay (ECA) for quantification of DPP3 activity (Rehfeld et al., 2019 JALM 3(6):943-953).

[0097] The ECA is a DPP3-specific activity assay that uses a black high-binding polystyrene microtiter plate as the solid phase. These plates are coated with the monoclonal anti-DPP3 antibody AK2555 (capture antibody). 20 microliters of sample (e.g., serum, heparin plasma, citrate plasma, EDTA plasma, cerebrospinal fluid, and urine) and calibrator are pipetted into the coated black microtiter plate. After adding assay buffer (200 μL), the microtiter plate is incubated at 22 °C and 600 rpm for 2 hours. DPP3 present in the sample is immobilized by binding to the capture antibody. Unbound sample components are removed by four washing steps (350 μL / well). The specific activity of the immobilized DPP3 is measured by adding the fluorogenic substrate Arg-Arg-β-naphthylamide (Arg2-βNA) into the reaction buffer, followed by incubation at 37 °C for 1 hour. DPP3 specifically cleaves Arg2-βNA into the Arg-Arg dipeptide and the fluorescent β-naphthylamine. Fluorescence is measured with a fluorometer using an excitation wavelength of 340 nm, and emission is detected at 410 nm. The activity of DPP3 is determined using a six-point calibration curve. Calibrator and sample are preferably run in duplicate.

[0098] 3. Liquid-phase assay (LAA) for the quantification of DPP3 activity (modified from Jones et al., Analytical Biochemistry, 1982).

[0099] The LAA is a liquid-phase assay that measures DPP3 activity using a black non-binding polystyrene microtiter plate. 20 microliters of sample (e.g., serum, heparin plasma, citrate plasma) and calibrator are pipetted into a non-binding black microtiter plate. After adding the fluorogenic substrate Arg2-βNA into the assay buffer (200 μL), the initial βNA fluorescence (T = 0) is measured with a fluorometer using an excitation wavelength of 340 nm, and the emission is detected at 410 nm. Then, the plate is incubated at 37 °C for 1 hour. The final fluorescence at (T = 60) is measured. The difference between the final fluorescence and the initial fluorescence is calculated. The activity of DPP3 is determined using a 6-point calibration curve. The calibrator and the sample are preferably run in duplicate.

[0100] In a specific embodiment, the assay is used to determine the level of DPP3, and the assay sensitivity of the assay is such that it can quantify DPP3 in healthy subjects and is less than 20 ng / ml, preferably less than 30 ng / ml, more preferably less than 40 ng / ml.

[0101] 4. Another immunoassay method for measuring DPP3 from plasma of whole blood samples, the IB10 sphingotest® DPP3 (https: / / www.nexus-dx.com / wp-content / uploads / 2020 / 11 / DPP3-022-00072-IFU-REV-B_8x11.pdf) is available.

[0102] The IB10 sphingotest® DPP3 is a rapid point-of-care (POC) immunoassay for the in vitro quantitative measurement of dipeptidyl peptidase 3 (DPP3) in human EDTA whole blood and plasma. The Nexus IB10 immunochemical system can rapidly prepare cell-free plasma from whole blood by combining chemicals with a microfluidic device and a centrifugal flow, and then move this through channels to rehydrate, solubilize, and mix the lyophilized immunoconjugate. In a specific embodiment, the binder has a binding affinity for DPP3 of at least 10 7 M -1 , preferably 10 8 M -1 , and a more preferred affinity is greater than 10 9 M -1 , most preferably greater than 10 10 M -1 . Those skilled in the art know that it may be considered possible to compensate for a lower affinity by applying a higher dose of the compound, and this measure is not outside the scope of the present invention.

[0103] In another embodiment of the present invention, the sample of the body fluid is selected from the group consisting of whole blood, plasma, and serum.

[0104] Mature ADM, bio-ADM, and ADM-NH2 are used synonymously throughout this application and are the molecules described in SEQ ID NO: 20.

[0105] In a specific embodiment of the present invention, the body fluid is, in a particular embodiment, a blood sample. The blood sample can be selected from the group comprising whole blood, serum, and plasma. In a specific embodiment of the method, the sample is selected from the group comprising human citrate plasma, heparin plasma, and EDTA plasma.

[0106] In a specific embodiment, the assay is used to determine the level of ADM-NH2, and the assay sensitivity of the assay is such that it can quantify mature ADM-NH2 in healthy subjects and is less than 70 pg / ml, preferably less than 40 pg / ml, more preferably less than 10 pg / ml.

[0107] In a specific embodiment of the present invention, the threshold value of ADM-NH2 is 40 - 100 pg / mL, more preferably 50 - 90 pg / mL, even more preferably 60 - 80, and the most preferred threshold value of 70 pg / ml is applied.

[0108] In a specific embodiment of the present invention, the threshold value of plasma ADM-NH2 is 5 times the median concentration of a normal healthy population, preferably 4 times the median concentration, more preferably 3 times the median concentration, and most preferably 2 times the median concentration.

[0109] In a specific embodiment, the binder has a binding affinity for ADM-NH2 of at least 10 7 M -1 , preferably 10 8 M -1 . A preferred affinity is greater than 10 9 M -1 , and most preferably greater than 10 10 M -1 . Those skilled in the art know that it may be considered possible to compensate for a lower affinity by applying a higher dose of the compound, and this measure does not fall outside the scope of the present invention.

[0110] To determine the affinity of the antibody for adrenomedullin, the binding kinetics of adrenomedullin to the immobilized antibody were determined by label-free surface plasmon resonance using a Biacore 2000 system (GE Healthcare Europe GmbH, Freiburg, Germany). Reversible immobilization of the antibody was performed according to the manufacturer's instructions using an anti-mouse Fc antibody covalently bound at high density to the CM5 sensor surface (mouse antibody capture kit; GE Healthcare), (Lorenz et al. 2011. Antimicrob Agents Chemother. 55 (1): 165-173).

[0111] In a specific embodiment, the binder is selected from the group comprising an antibody or antibody fragment that binds to ADM-NH2 or a non-Ig scaffold.

[0112] In a specific embodiment, the assay is used to determine the level of ADM-NH2, and such an assay is a sandwich assay, preferably a fully automated assay.

[0113] In one embodiment, such an assay for determining the level of a biomarker (DPP3 and / or ADM-NH2) is a sandwich immunoassay using any type of detection technique including, but not limited to, enzyme labeling, chemiluminescent labeling, and electrochemiluminescent labeling, and is preferably a fully automated assay. In one embodiment of the diagnostic method, such an assay is an enzyme-labeled sandwich assay. Examples of automated or fully automated assays include assays that can be used in one of the following systems: Roche Elecsys®, Abbott Architect®, Siemens Centauer®, Brahms Kryptor®, Biomerieux Vidas®, Alere Triage®.

[0114] Various immunoassays are known and can be used in the assays and methods of the present invention. These include radioimmunoassay ("RIA"), homogeneous enzyme immunoassay ("EMIT"), enzyme-linked immunosorbent assay ("ELISA"), apoenzyme reactivation immunoassay ("ARIS"), dipstick immunoassay, and immunochromatography assay.

[0115] In one embodiment of the present invention, such an assay is a sandwich immunoassay using any type of detection technique including, but not limited to, enzyme labeling, chemiluminescent labeling, and electrochemiluminescent labeling, and is preferably a fully automated assay. In one embodiment of the present invention, such an assay is an enzyme-labeled sandwich assay. Examples of automated or fully automated assays include assays that can be used in one of the following systems: Roche Elecsys®, Abbott Architect®, Siemens Centauer®, Brahms Kryptor®, Biomerieux Vidas®, Alere Triage®.

[0116] In one embodiment of the present invention, it may be a so-called POC test (point of care), which is a testing technology that enables a test to be performed within less than one hour near a patient without the need for a fully automated assay system. An example of this technology is immunochromatography testing technology.

[0117] In a preferred embodiment, the label is selected from the group comprising chemiluminescent labels, enzyme labels, fluorescent labels, and radioiodine labels.

[0118] The assay may be a homogeneous or heterogeneous assay, a competitive and non-competitive assay. In one embodiment, the assay is in the form of a sandwich assay which is a non-competitive immunoassay, and the molecule to be detected and / or quantified is bound to a first antibody and a second antibody. The first antibody may be bound to a solid phase, such as the surface of beads, wells or other containers, chips or strips, and the second antibody is an antibody labeled, for example, with a dye, a radioisotope, or a reactive or catalytically active moiety. Then, the amount of the labeled antibody bound to the analyte is measured by an appropriate method. The general compositions and procedures involved in "sandwich assays" are well established and known to those skilled in the art (The Immunoassay Handbook, Ed. David Wild, Elsevier LTD, Oxford; 3rd ed. (May 2005), ISBN-13: 978-0080445267, Hultschig C et al., Curr Opin Chem Biol. 2006 Feb; 10(1): 4-10. PMID: 16376134).

[0119] In another embodiment, the assay comprises two capture molecules, preferably both antibodies present as a dispersion in a liquid reaction mixture, a first label component bound to the first capture molecule, the first label component being part of a labeling system based on fluorescence or chemiluminescence quenching or amplification, a second label component of the marking system being bound to the second capture molecule, such that when both capture molecules bind to the analyte, a measurable signal is generated that enables detection of the sandwich complex formed in the solution containing the sample.

[0120] In another embodiment, the labeling system comprises a rare earth cryptate or chelate in combination with a fluorescent dye or chemiluminescent dye, particularly a cyanine-type dye.

[0121] In the context of the present invention, a fluorescence-based assay involves the use of a dye which may be selected from the group comprising, for example, FAM (5- or 6-carboxyfluorescein), VIC, NED, fluorescein, fluorescein isothiocyanate (FITC), IRD-700 / 800, cyanine dyes such as CY3, CY5, CY3.5, CY5.5, Cy7, xanthene, 6-carboxy-2’,4’,7’,4,7-hexachlorofluorescein (HEX), TET, 6-carboxy-4’,5’-dichloro-2’,7’-dimethodyfluorescein (JOE), N,N,N’,N’-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 5-carboxyrhodamine-6G (R6G5), 6-carboxyrhodamine-6G (RG6), rhodamine, rhodamine green, rhodamine red, rhodamine 110, BODIPY dyes such as BODIPY TMR, Oregon Green, coumarin such as umbelliferone, benzimidazole such as Hoechst 33258; phenanthridine such as Texas Red, Yakima Yellow, Alexa Fluor, PET, ethidium bromide, acridinium dyes, carbazole dyes, phenoxazine dyes, porphyrin dyes, polymethine dyes and the like.

[0122] In the context of the present invention, an assay based on chemiluminescence involves the use of dyes based on the physical principles described below for chemiluminescent materials (Kirk-Othmer, Encyclopedia of chemical technology, 4th ed., executive editor, J.I. Kroschwitz; editor, M. Howe-Grant, John Wiley & Sons, 1993, vol. 15, p. 518-562, including the citation on pages 551-562, incorporated herein by reference). Preferred chemiluminescent dyes are acridinium esters.

[0123] As referred to herein, an "assay" or "diagnostic assay" can be of any type applicable in the field of diagnostics. Such an assay can be based on the binding of an analyte to be detected to one or more capture probes having a specific affinity. With respect to the interaction between a capture molecule and a target molecule or molecule of interest, the affinity constant is preferably greater than 10 8 M -1 greater.

[0124] In a specific embodiment, at least one of the two binding agents is labeled for detection.

[0125] The ADM-NH2 level of the present invention was determined using the described ADM-NH2 assay (Weber et al. 2017. JALM 2(2):1-4). The DPP3 level of the present invention was determined using the described DPP3 assay as outlined in the Examples (Rehfeld et al. 2019. JALM 3(6):943-953). The above-mentioned thresholds may be different in other assays if the other assays are calibrated in a different way from the assay system used in the present invention. Therefore, the above-mentioned cut-off values shall be applied to assays calibrated in such different ways, taking into account the differences in calibration accordingly. One possibility to quantify the differences in calibration is the comparative analysis (correlation) method between the assay in question and each biomarker assay used in the present invention, which is done by measuring each biomarker (e.g., bio-ADM, DPP3) in the sample using both methods. Another possibility is to use the assay to determine the median of the biomarker levels in a representative normal population, assuming that this test has sufficient analytical sensitivity, compare the results with the median of the biomarker levels as described in the literature, and recalculate the calibration based on the differences obtained from this comparison. Using the calibration used in the present invention, samples from normal (healthy) subjects were measured: the median of plasma bio-ADM (mature ADM-NH2) was 24.7 pg / ml, the minimum value was 11 pg / ml, and the 99th percentile was 43 pg / ml (Marino et al. 2014. Critical Care 18:R34). Using the calibration used in the present invention, samples from 5,400 normal (healthy) subjects (Swedish single-center prospective population-based study (MPP-RES)) were measured: the median (interquartile range) of plasma DPP3 was 14.5 ng / ml (11.3 ng / ml - 19 ng / ml). The DPP3 concentration has been shown to strongly correlate with the DPP3 activity in the blood (Rehfeld et al. 2019. J Appl Lab Med 3:943-953, Deniau et al. 2019. Eur J Heart Fail 22:290-299).As a result, the level of active DPP3 can be determined using respective thresholds and threshold ranges corresponding to the thresholds and threshold ranges used to determine the level of the DPP3 protein.

[0126] Therefore, in the present invention, the level of ADM-NH2 is measured to identify patients at high risk of falling into a shock state.

[0127] In another specific embodiment of the present invention, the shock is selected from the group including shock due to decreased circulating blood volume, cardiogenic shock, obstructive shock, and distributive shock, particularly cardiogenic or septic shock.

[0128] In a specific embodiment of the present invention, the shock is selected from the group including the following cases: ● In the case of cardiogenic shock, the patient has acute coronary syndrome (e.g., acute myocardial infarction), or has heart failure (e.g., acute decompensated heart failure), myocarditis, arrhythmia, cardiomyopathy, valvular heart disease, aortic dissection with acute aortic valve stenosis, traumatic chordae tendineae rupture, or massive pulmonary embolism, or ● In the case of hypovolemic shock, the patient may have a hemorrhagic disease including gastrointestinal bleeding, trauma, vascular etiology (e.g., ruptured abdominal aortic aneurysm, tumor eroding major blood vessels), and spontaneous bleeding in the context of anticoagulant use, or a non-hemorrhagic disease including vomiting, diarrhea, renal loss, skin loss / insensible loss (e.g., burns, heat stroke), or loss to the third space in the context of pancreatitis, cirrhosis, intestinal obstruction, trauma, or ● In the case of obstructive shock, the patient may have cardiac tamponade, tension pneumothorax, pulmonary embolism, or aortic valve stenosis, or ● In the case of distributive shock, the patient has septic shock, neurogenic shock, anaphylactic shock, or shock due to adrenal crisis.

[0129] Shock is characterized by inadequate oxygen utilization that results in decreased oxygen delivery and / or increased oxygen consumption or hypoxia of cells and tissues. It is a life-threatening state of circulatory failure and most commonly presents as hypotension (systolic blood pressure less than 90 mmHg or mean arterial pressure less than 65 mmHg). Shock is classified into four major types based on the underlying cause: hypovolemic shock, cardiogenic shock, obstructive shock, and distributive shock (Vincent and De Backer 2014. N. Engl. J. Med. 370(6):583).

[0130] Hypovolemic shock is characterized by a decrease in intravascular volume and can be divided into two broad subtypes: hemorrhagic and non-hemorrhagic. Common causes of hemorrhagic hypovolemic shock include gastrointestinal bleeding, trauma, vascular etiologies (e.g., ruptured abdominal aortic aneurysm, tumors eroding major vessels), and spontaneous bleeding in the setting of anticoagulant use. Common causes of non-hemorrhagic hypovolemic shock include vomiting, diarrhea, renal losses, cutaneous losses / insensible losses (e.g., burns, heatstroke), or third-space losses in the setting of pancreatitis, cirrhosis, intestinal obstruction, trauma. See Koya and Paul 2018. Shock. StatPearls [Internet]. Treasure Island(FL): StatPearls Publishing; 2019-2018 Oct 27 for a review.

[0131] Cardiogenic shock (CS) is defined as a state of severe end-organ hypoperfusion due to reduced cardiac output. In particular, CS forms a spectrum ranging from mild hypoperfusion to profound shock. Established criteria for the diagnosis of CS are: (i) systolic blood pressure ≤90 mmHg for over 30 minutes, or the need for vasopressors to achieve a blood pressure ≥90 mmHg, (ii) pulmonary congestion or elevated left ventricular filling pressure, (iii) signs of organ perfusion impairment by at least one of the following criteria: (a) change in mental status, (b) cold skin, (c) oliguria (<0.5 mL / kg / hour or <30 mL / hour), (d) increased serum lactate (Reynolds and Hochman 2008. Circulation 117:686-697). Subsequent acute myocardial infarction (AMI) with ventricular dysfunction is the most frequent cause of CS, accounting for approximately 80% of cases. Mechanical complications such as ventricular septal rupture (4%) or free wall rupture (2%), and acute severe mitral regurgitation (7%) are less frequent causes of CS after AMI. (Hochman et al. 2000. J Am Coll Cardiol 36:1063-1070). Non-AMI-related CS may be caused by decompensated valvular heart disease, acute myocarditis, arrhythmias, etc., and treatment options are diverse. This translates to 40,000 - 50,000 patients per year in the United States and 60,000 - 70,000 patients per year in Europe. Despite advances in treatment mainly due to early revascularization and subsequent reduction in mortality, CS remains the leading cause of death in AMI, and according to recent registries and randomized trials, the mortality rate remains close to 40 - 50% (Goldberg et al. 2009. Circulation 119:1211-1219).

[0132] Obstructive shock is due to physical obstruction of the great vessels or the heart itself. Several conditions can result in this form of shock (e.g., cardiac tamponade, tension pneumothorax, pulmonary embolism, aortic stenosis). For a review, see Koya and Paul 2018. Shock. StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2019 - 2018 Oct 27.

[0133] According to the cause, there are four types of distributive shock: neurogenic shock (decreased sympathetic stimulation leading to decreased vasoconstriction), anaphylactic shock, septic shock, and shock due to adrenal crisis. In addition to sepsis, distributive shock can be caused by systemic inflammatory response syndrome (SIRS) due to conditions other than infections such as pancreatitis, burns, or trauma. Other causes include toxic shock syndrome (TSS), anaphylaxis (a sudden severe allergic reaction), adrenal insufficiency (acute exacerbation of chronic adrenal insufficiency, destruction or removal of the adrenal glands, suppression of adrenal function by exogenous steroids, hypopituitarism, and metabolic disorders of hormone production), reactions to drugs or toxins, heavy metal poisoning, liver (hepatic) failure, and damage to the central nervous system. For a review, see Koya and Paul 2018. Shock. StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2019 - 2018 Oct 27.

[0134] Refractory shock is defined as the requirement for norepinephrine infusion at a rate exceeding 0.5 μg / kg / min despite adequate fluid resuscitation. The mortality rate in these patients can be as high as 94%, and the assessment and management of these patients require a much more aggressive approach for survival. The term "refractory shock" is used when tissue perfusion cannot be restored with the initial corrective measures (e.g., vasopressors) used and can thus be referred to as "high vasopressor dependence" or "vasopressor-resistant" shock (Udupa and Shetty 2018. Indian J Respir Care 7:67-72). Patients with refractory shock may have features of inadequate perfusion such as hypotension (mean arterial pressure <65 mmHg), tachycardia, cold extremities, prolonged capillary refill time, and tachypnea resulting from hypoxemia and acidosis. Fever may be seen in septic shock. Other signs of hypoperfusion such as altered sensorium, hyperlactatemia, and oliguria may also be seen. These well-known signs of shock are not useful in differentiating whether the problem lies with the pump (heart) or the circuit (blood vessels and tissues). Different types of shock can coexist, and all forms of shock can potentially become refractory, as evidenced by non-responsiveness to high doses of vasopressors (Udupa and Shetty 2018. Indian J Respir Care 7:67-72).

[0135] Septic shock is a life-threatening medical condition that occurs when sepsis, an organ dysfunction or injury in response to infection, leads to dangerously low blood pressure and abnormal cellular metabolism. The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3) defines septic shock as a subset of sepsis in which particularly severe circulatory, cellular, and metabolic abnormalities are associated with a higher risk of death than sepsis alone. Patients with septic shock can be clinically identified by the need for vasopressors to maintain a mean arterial pressure of 65 mmHg or more in the absence of reduced circulating blood volume and a serum lactate level greater than 2 mmol / L (greater than 18 mg / dL). This combination is associated with an in-hospital mortality rate of more than 40% (Singer et al. 2016. JAMA. 315 (8):801-10). The primary infection is most commonly caused by bacteria but can also be caused by fungi, viruses, or parasites. It can be located in any part of the body but is most commonly located in the lungs, brain, urinary tract, skin, or abdominal organs. This can lead to multiple organ dysfunction syndrome (formerly known as multiple organ failure) and death. In many cases, patients with septic shock are treated in an intensive care unit. Children, immunocompromised individuals, and the elderly are most commonly affected because their immune systems are not as effective at combating infections as those of healthy adults. The mortality rate due to septic shock is approximately 25-50%.

[0136] As used herein, the term "prevention" or any grammatical variation thereof (e.g., prevent, preventing, and prevention) includes, but is not limited to, delaying the onset of symptoms, preventing recurrence of a disease, prolonging the latency between symptomatic episodes, or combinations thereof. Prevention, as used herein, does not require the complete absence of symptoms.

[0137] The efficacy of a non-neutralizing antibody targeted against the N-terminus of ADM was investigated in a survival study in CLP-induced sepsis in mice. Pretreatment with the non-neutralizing antibody resulted in a decreased catecholamine infusion rate, renal dysfunction, and ultimately improved survival (Struck et al. 2013. Intensive Care Med Exp 1(1):22, Wagner et al. 2013. Intensive Care Med Exp 1(1):21).

[0138] Based on these positive results, a humanized version of the N-terminal anti-ADM antibody, called adrenocizumab, has been developed for further clinical development. The beneficial effects of adrenocizumab 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 adrenocizumab attenuated renal vascular leakage in endotoxemic rats and mice with CLP-induced sepsis, which was accompanied by increased renal expression of the protective peptide Ang-1 and decreased expression of the detrimental peptide vascular endothelial growth factor. Also, pretreatment with adrenocizumab improved the 7-day survival rate in CLP-induced sepsis in mice, from 10 to 50% for single-dose administration and from 0 to 40% for repeated-dose administration. Furthermore, excellent safety and tolerability were demonstrated in a Phase I study (see Example 6): no severe adverse events were observed, no signals of adverse events occurring more frequently in the adrenocizumab-treated subjects were detected, and no relevant changes in other safety parameters were found (Geven et al. 2017. Intensive Care Med Exp 5 (Suppl 2):0427). Of particular interest is the proposed mechanism of action of adrenocizumab. Data from both animal and human studies have revealed a strong dose-dependent increase in circulating ADM after administration of this antibody. Based on pharmacokinetic data and the lack of an increase in MR-proADM (an inactive peptide fragment derived from the same prohormone as ADM), the higher circulating ADM levels cannot be explained by increased production.

[0139] A mechanistic explanation for this increase could be that ADM is small enough to pass through the endothelial barrier, while antibodies are not, so that excess antibodies in the blood circulation can expel ADM from the interstitial space back into the blood circulation (Geven et al. 2018. Shock. 50(2):132-140 and Voors et al (J. Eur J Heart Fail. 2019 Feb;21(2):163-171)). Furthermore, the binding of the antibody to ADM results in an extended half-life of ADM. Even if the NT-ADM antibody partially inhibits ADM-mediated signaling, the substantial increase in circulating ADM results in an overall "net" increase in ADM activity in the blood compartment, exerting a beneficial effect (mainly barrier stabilization) on ECs, while the detrimental effect of ADM on VSMCs in the interstitial space (vasodilation) is reduced.

[0140] The present invention is not particularly limited to the use of adrecizumab. There is no reason to doubt that what applies to adrecizumab also applies to antibodies sharing the major essential features (especially affinity and epitope specificity). Antibodies targeting the same region should be expected to have the same technical effect, provided they have the same affinity and the same or very similar structural features (size, shape, etc.).

[0141] Throughout this specification, an "antibody" or "antibody fragment" or "non-Ig scaffold" according to the present invention can bind to ADM, and thus targets ADM, and can therefore be referred to as an "anti-ADM antibody", "anti-ADM antibody fragment", or "anti-ADM non-Ig scaffold".

[0142] The term "antibody" generally includes monoclonal antibodies and polyclonal antibodies and their binding fragments, particularly Fc fragments, as well as so-called "single-chain antibodies" (Bird et al. 1988), chimeric antibodies, humanized antibodies, particularly CDR-grafted antibodies, and diabodies or tetrabodies (Holliger et al. 1993). For example, also included are immunoglobulin-like proteins selected via techniques including phage display that specifically bind to a molecule of interest contained in a sample. In this context, the term "specific binding" refers to an antibody raised against a molecule of interest or a fragment thereof. An antibody is considered specific if its affinity for the molecule of interest or the aforementioned fragment thereof is at least preferably 50-fold higher, more preferably 100-fold higher, and most preferably at least 1000-fold higher than its affinity for other molecules contained in a sample containing the molecule of interest. Methods for making antibodies and selecting antibodies with a given specificity are well known in the art.

[0143] In one embodiment of the invention, an anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold is monospecific.

[0144] A monospecific anti-adrenomedullin (ADM) antibody or monospecific anti-adrenomedullin antibody fragment or monospecific anti-ADM non-Ig scaffold means that the antibody or antibody fragment or non-Ig scaffold binds to one specific region encompassing at least 5 amino acids within the target ADM. A monospecific anti-adrenomedullin (ADM) antibody or monospecific anti-adrenomedullin antibody fragment or monospecific anti-ADM non-Ig scaffold are all anti-adrenomedullin (ADM) antibodies or anti-adrenomedullin antibody fragments or anti-ADM non-Ig scaffolds having the same affinity for the same antigen. Monoclonal antibodies are monospecific, but monospecific antibodies can also be made by means other than producing them from a common germ cell.

[0145] The anti-ADM antibody or antibody fragment that binds to ADM or the non-Ig scaffold that binds to ADM can be a non-neutralizing anti-ADM antibody or antibody fragment or a non-neutralizing non-Ig scaffold that binds to ADM.

[0146] In a specific embodiment, the anti-ADM antibody, anti-ADM antibody fragment or anti-ADM non-Ig scaffold is a non-neutralizing antibody, fragment or non-Ig scaffold. A neutralizing anti-ADM antibody, anti-ADM antibody fragment or anti-ADM non-Ig scaffold blocks the biological activity of ADM by up to almost 100%, at least more than 90%, preferably at least more than 95%.

[0147] In contrast, a non-neutralizing anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold blocks the biological activity of ADM to less than 100%, preferably less than 95%, preferably less than 90%, more preferably less than 80%, even more preferably less than 50%. This means that the biological activity of ADM is reduced to less than 100%, not more than 95% but 95% or less, not more than 90% but 90% or less, not more than 80% but 80% or less, not more than 50% but 50% or less. This means that the remaining biological activity of ADM bound to a non-neutralizing anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold can be more than 0%, preferably more than 5%, preferably more than 10%, more preferably more than 20%, even more preferably more than 50%.

[0148] In this context, (a) a molecule that has "non-neutralizing anti-ADM activity" and is generically referred to herein as a "non-neutralizing" anti-ADM antibody, antibody fragment or non-Ig scaffold that blocks the biological activity of ADM, for example, to less than 80%, is defined as follows. One or more molecules that bind to ADM and, when added to a culture of a eukaryotic cell line expressing a functional human recombinant ADM receptor composed of CRLR (calcitonin receptor like receptor) and RAMP3 (receptor-activity modifying protein 3), reduce the amount of cAMP produced by the cell line via the action of concurrently added human synthetic ADM peptide, wherein the added human synthetic ADM is added in an amount that provides a maximal half-maximal stimulation of cAMP synthesis in the absence of the non-neutralizing antibody being assayed, and the decrease in cAMP by the molecule(s) that bind to ADM occurs to an extent of 80% or less even when the non-neutralizing molecule(s) that bind to the ADM being assayed are added in an amount more than 10-fold the amount required to obtain the maximal decrease in cAMP synthesis obtained with the non-neutralizing antibody being assayed.

[0149] Other ranges; the same definition applies for 95%, 90%, 50%, etc.

[0150] An antibody or fragment according to the 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 kappa, lambda, alpha (IgA), gamma (IgG1, IgG2, IgG3, IgG4), delta (IgD), epsilon (IgE) and mu (IgM) constant region genes, as well as innumerable immunoglobulin variable region genes. A full-length immunoglobulin light chain is generally about 25 Kd or 214 amino acids in length.

[0151] A full-length immunoglobulin heavy chain is generally about 50 Kd or 446 amino acids in length. The light chain is encoded by an NH2-terminal variable region gene (about 110 amino acids in length) and a COOH-terminal kappa or lambda constant region gene. The heavy chain is similarly encoded by a variable region gene (about 116 amino acids in length) and one of the other constant region genes.

[0152] The basic structural unit of an antibody generally consists of a tetramer composed of two identical pairs of immunoglobulin chains, with 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, and the constant regions mediate effector functions. Immunoglobulins also exist in various other forms, such as Fv, Fab, and (Fab’)2, as well as 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. U.S.A., 85:5879-5883, Bird et al. 1988. Science 242:423-426, Hood et al. 1984, Immunology, Benjamin, N.Y., 2nd ed., Hunkapiller and Hood 1986. Nature 323:15-16). The variable region of an immunoglobulin light or heavy chain contains framework regions interrupted by three hypervariable regions, also called complementarity determining regions (CDRs) (see Sequences of Proteins of Immunological Interest, E. Kabat et al. 1983, U.S. Department of Health and Human Services). As noted above, the CDRs are mainly involved in binding to the epitope of the antigen. An immune complex is an antibody, such as a monoclonal antibody, chimeric antibody, humanized antibody, or human antibody, or a functional antibody fragment, that specifically binds to an antigen.

[0153] A chimeric antibody is an antibody in which the light chain gene and the heavy chain gene are typically constructed from immunoglobulin variable region genes and constant region genes belonging to different species by genetic engineering. For example, the variable segments of genes derived from a mouse monoclonal antibody can be linked to human constant segments, such as kappa and gamma 1 or gamma 3. Thus, in one example, a therapeutic chimeric antibody is a hybrid protein composed of a variable domain or antigen-binding domain derived from a mouse antibody and a constant domain or effector domain derived from a human antibody, although other mammalian species can also be used, or the variable region can be produced by molecular techniques. Methods for producing chimeric antibodies are well known in the art; see, for example, U.S. Patent No. 5,807,715. A "humanized" immunoglobulin is an immunoglobulin that contains a human framework region and one or more CDRs derived from a non-human (e.g., mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin that provides the CDRs is called the "donor," and the human immunoglobulin that provides the framework is called the "acceptor." In one embodiment, all of the CDRs in the humanized immunoglobulin are derived from the donor immunoglobulin. The constant regions need not be present, but if present, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, e.g., about 95% or more identical. Thus, in some cases, all parts of the humanized immunoglobulin, except for the CDRs, are substantially identical to the corresponding parts of the native human immunoglobulin sequence. A "humanized antibody" is an antibody that includes humanized light and heavy chain immunoglobulins. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized immunoglobulin or antibody can have a limited number of substitutions with amino acids taken from the donor framework. A humanized or other monoclonal antibody can have additional conservative amino acid substitutions that do not substantially affect antigen binding or other immunoglobulin functions. Exemplary conservative substitutions are substitutions such as 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, e.g., U.S. Patent No. 5,585,089). Human antibodies are antibodies in which the light and heavy chain genes are of human origin. Human antibodies can be produced using methods known in the art. Human antibodies can be prepared by immortalizing human B cells that secrete the antibody of interest. Immortalization can be achieved, for example, by EBV infection or by fusing human B cells with myeloma or hybridoma cells to produce trioma cells. Human antibodies can also be produced by phage display methods (see, e.g., WO 91 / 17271, WO 92 / 001047, WO 92 / 20791) or can be selected from human combinatorial monoclonal antibody libraries (see the Morphosys website). Human antibodies can also be prepared by using transgenic animals that carry human immunoglobulin genes (see, e.g., WO 93 / 12227, WO 91 / 10741).

[0154] Accordingly, the anti-ADM antibody can have a format known in the art. Examples are human antibodies, monoclonal antibodies, humanized antibodies, chimeric antibodies, CDR-grafted antibodies. In a preferred embodiment, the antibody according to the invention is an antibody recombinantly produced, for example, as an IgG, a typical full-length immunoglobulin, or an antibody fragment containing at least the F variable domains of the heavy and / or light chains, for example, as a chemically conjugated antibody (fragment antigen binding), including Fab minibodies, single-chain Fab antibodies, monovalent Fab antibodies having an epitope tag, for example, Fab fragments including Fab-V5Sx2; bivalent Fab (minibodies) dimerized with the CH3 domain; bivalent or multivalent Fab formed, for example, via multimerization using a heterologous domain, for example, via dimerization of the dHLX domain, for example, Fab-dHLX-FSx2; F(ab’)2 fragments, scFv fragments, multimerized multivalent and / or multispecific scFv fragments, bivalent and / or bispecific diabodies, BITE® (bispecific T cell engagers), trifunctional antibodies, multivalent antibodies, for example, multivalent antibodies derived from classes different from G; single-domain antibodies, for example, nanobodies derived from camelid or fish immunoglobulins, and the like, but not limited thereto.

[0155] In addition to anti-ADM antibodies, other biopolymer scaffolds are well known in the art for complexing target molecules and are used for the generation of highly target-specific biopolymers. Examples are aptamers, Spiegelmers, anticalins, and conotoxins. See FIGS. 1a, 1b, and 1c for illustrations of antibody formats.

[0156] In a preferred embodiment, the anti-ADM antibody format is selected from the group consisting of an Fv fragment, a scFv fragment, a Fab fragment, a scFv fragment, an F(ab)2 fragment, and an scFv-Fc fusion protein. In another preferred embodiment, the antibody format is selected from the group consisting of an scFab fragment, a Fab fragment, an scFv fragment, and their bioavailability-optimized conjugates, such as PEGylated fragments. One of the most preferred formats is the scFab format.

[0157] The non-Ig scaffold may be a protein scaffold and can bind to a ligand or antigen, so it can be used as an antibody mimic. The non-Ig scaffold can be selected from the group consisting of a non-Ig scaffold based on tetranectin (e.g., described in US Patent Application Publication No. 2010 / 0028995), a fibronectin scaffold (e.g., described in European Patent No. 1266025); a scaffold based on lipocalin (e.g., described in International Publication No. 2011 / 154420); a ubiquitin scaffold (e.g., described in International Publication No. 2011 / 073214), a transferrin scaffold (e.g., described in US Patent Application Publication No. 2004 / 0023334), a protein A scaffold (e.g., described in European Patent No. 2231860), a scaffold based on ankyrin repeat (e.g., described in International Publication No. 2010 / 060748), a microprotein, preferably a microprotein forming a cysteine knot) scaffold (e.g., described in European Patent No. 2314308), a scaffold based on the Fyn SH3 domain (e.g., described in International Publication No. 2011 / 023685), a scaffold based on the EGFR-A domain (e.g., described in International Publication No. 2005 / 040229), and a scaffold based on the Kunitz domain (e.g., described in European Patent No. 1941867).

[0158] In one embodiment of the present invention, the anti-ADM antibody according to the present invention can be prepared by synthesizing a fragment of ADM as an antigen, as outlined in Example 1. Thereafter, binders to the fragment are identified using the methods described below or other methods known in the art.

[0159] Humanization of mouse antibodies can be performed according to the following procedure. For humanization of antibodies of mouse origin, the antibody sequences are analyzed for the framework region (FR), complementary determining region (CDR), and structural interactions with the antigen. Based on the structural modeling, appropriate FRs of human origin are selected and the mouse CDR sequences are transplanted into the human FRs. Mutations in the amino acid sequences of the CDRs or FRs can be introduced to restore the structural interactions lost due to the species switch in the FR sequences. This restoration of structural interactions can be achieved via a random approach using a phage display library or a directed approach guided by molecular modeling (Almagro and Fransson 2008. Humanization of antibodies. Front Biosci. 2008 Jan 1;13:1619-33).

[0160] In a preferred embodiment, the ADM antibody format is selected from the group consisting of Fv fragment, scFv fragment, Fab fragment, scFab fragment, F(ab)2 fragment, and scFv-Fc fusion protein. In another preferred embodiment, the antibody format is selected from the group consisting of scFab fragment, Fab fragment, scFv fragment, and their bioavailability-optimized conjugates, such as PEGylated fragments. One of the most preferred formats is the scFab format.

[0161] 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.

[0162] In a preferred embodiment, the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold is directed against and can bind to an epitope at least 5 amino acids in length contained in ADM.

[0163] In a more preferred embodiment, the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold is directed against and can bind to an epitope at least 4 amino acids in length contained in ADM.

[0164] In a specific embodiment of the present invention, there is provided an anti-ADM antibody or anti-ADM antibody fragment that binds to adrenomedullin or an anti-ADM non-Ig scaffold that binds to adrenomedullin for use in the treatment or prevention of shock in a patient, wherein the antibody or fragment or scaffold is not adrenomedullin-binding protein 1 (complement factor H).

[0165] In a specific embodiment of the present invention, there is provided an anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment that binds to adrenomedullin or an anti-ADM non-Ig scaffold that binds to adrenomedullin for use in the treatment or prevention of shock in a patient, wherein the antibody or fragment or scaffold binds to a region or epitope of at least 4, or at least 5 amino acids within the sequence of amino acids 1-21: YRQSMNNFQGLRSFGCRFGTC SEQ ID NO: 22 of mature human ADM.

[0166] In a preferred embodiment of the present invention, the anti-ADM antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold binds to a region or epitope of ADM located in the N-terminal portion (amino acids 1-21) of adrenomedullin.

[0167] In another preferred embodiment, the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold recognizes and binds to a region or epitope within amino acids 1-14 of adrenomedullin: YRQSMNNFQGLRSF (SEQ ID NO: 25), which represents the N-terminal portion (amino acids 1-14) of adrenomedullin.

[0168] In another preferred embodiment, the anti-ADM antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold recognizes and binds to a region or epitope within amino acids 1-10 of adrenomedullin: YRQSMNNFQG (SEQ ID NO: 26), which represents the N-terminal portion (amino acids 1-10) of adrenomedullin.

[0169] In another preferred embodiment, the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold recognizes and binds to a region or epitope within amino acids 1-6 of adrenomedullin: YRQSMN (SEQ ID NO: 27), which represents the N-terminal portion (amino acids 1-6) of adrenomedullin. As described above, the region or epitope preferably comprises at least 4 or at least 5 amino acids in length.

[0170] In another preferred embodiment, 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 adrenomedullin. The N-terminus means amino acid 1, which is "Y" in SEQ ID NOs: 20, 22 or 23 respectively, and is essential for binding. The antibody or fragment or scaffold does not bind to N-terminally extended or N-terminally modified adrenomedullin, nor to N-terminally degraded adrenomedullin. This means that in another preferred embodiment, the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold binds only to the region within the sequence of mature ADM when the N-terminus of ADM is free. In this embodiment, the anti-ADM antibody or anti-ADM antibody fragment or non-Ig scaffold does not bind to the region within the sequence when the sequence of mature ADM is contained, for example, within pro-ADM.

[0171] For the sake of clarity, it is understood by those skilled in the art that the N-terminal portion of ADM, such as the "N-terminal portion (amino acids 1 to 21)", consists of amino acids 1 to 21 of the mature ADM sequence, based on the numbers within the parentheses for the specific region of ADM.

[0172] In another specific embodiment according to the present invention, the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold provided herein does not bind to the C-terminal portion of ADM, i.e., amino acids 43 to 52 of ADM: PRSKISPQGY-NH2 (SEQ ID NO: 24).

[0173] An epitope, also known as an antigenic determinant, is a part of an antigen that is recognized by the immune system, particularly by an antibody. For example, an epitope is a specific piece of an antigen to which an antibody binds. The part of the antibody that binds to the epitope is called a paratope. Epitopes of protein antigens are classified into two categories, conformational epitopes and linear epitopes, based on their structure and interaction with the paratope.

[0174] Conformational epitopes and linear epitopes interact with the paratope based on the 3-D conformation adopted by the epitope, which is determined by the surface features of the epitope residues involved and the shape or tertiary structure of other segments of the antigen. Conformational epitopes are formed by the 3-D conformation adopted by the interaction of discontinuous amino acid residues. A linear epitope or continuous epitope is an epitope that is recognized by an antibody based on its linear sequence or primary structure of amino acids and is formed by the 3-D conformation adopted by the interaction of adjacent amino acid residues.

[0175] In a specific embodiment, it is preferable to use an anti-ADM antibody or an anti-ADM antibody fragment or an anti-ADM non-Ig scaffold according to the present invention, wherein the anti-ADM antibody or the anti-ADM antibody fragment or the anti-ADM non-Ig scaffold results in an increase of at least 10%, preferably at least 50%, more preferably more than 50%, and most preferably more than 100% in the ADM level or ADM immunoreactivity in serum, blood, or plasma.

[0176] In a specific embodiment, it is preferable to use an anti-ADM antibody or an anti-ADM antibody fragment or an anti-ADM non-Ig scaffold according to the present invention, wherein the anti-ADM antibody or the anti-ADM antibody fragment or the anti-ADM non-Ig scaffold is an ADM-stabilizing antibody or an ADM-stabilizing antibody fragment or an ADM-stabilizing non-Ig scaffold that extends the half-life (t1 / 2; half-value retention time) of adrenomedullin in serum, blood, or plasma by at least 10%, preferably at least 50%, more preferably more than 50%, and most preferably more than 100%.

[0177] The half-life (half-value retention time) of ADM can be determined in human serum, blood, or plasma in the absence and presence of an ADM-stabilizing antibody or an ADM-stabilizing antibody fragment or an ADM-stabilizing non-Ig scaffold, respectively, using an immunoassay for the quantification of ADM.

[0178] The following steps can be performed: - ADM can be diluted in human citrated plasma in the absence and presence of an ADM-stabilizing antibody or an adrenomedullin-stabilizing antibody fragment or an adrenomedullin-stabilizing non-Ig scaffold, respectively, and can be incubated at 24°C.

[0179] - An aliquot can be taken at a selected time point (e.g., within 24 hours) and frozen at -20°C to stop the degradation of ADM in the aliquot.

[0180] - When the amount of ADM is not affected by the stabilizing antibody in the selected assay, it can be directly determined by the hADM immunoassay. Alternatively, an aliquot may be treated with a denaturing agent (e.g., HCl), the sample may be clarified (e.g., by centrifugation), the pH neutralized, and ADM quantified by the ADM immunoassay. Alternatively, non-immunoassay techniques (e.g., RP-HPLC) can be used for ADM quantification.

[0181] Calculate the half-life of ADM for ADM incubated in the absence and presence of an ADM stabilizing antibody or adrenomedullin stabilizing antibody fragment or adrenomedullin stabilizing non-Ig scaffold, respectively.

[0182] The extension of the half-life for stabilized ADM is calculated by comparison with ADM incubated in the absence of an ADM stabilizing antibody or adrenomedullin stabilizing antibody fragment or adrenomedullin stabilizing non-Ig scaffold.

[0183] A two-fold increase in the half-life of ADM is a 100% extension of the half-life.

[0184] The half-life (half-value holding time) is defined as the period it takes for the concentration of a particular chemical or drug to decrease to half of its baseline concentration in a particular fluid or blood.

[0185] Assays that can be used for determination of the half-life (half-value holding time) of adrenomedullin in serum, blood, plasma are described in Example 3.

[0186] In a preferred embodiment, the anti-ADM antibody, anti-ADM antibody fragment or anti-ADM non-Ig scaffold is a non-neutralizing antibody, fragment or scaffold. A neutralizing anti-ADM antibody, anti-ADM antibody fragment or anti-ADM non-Ig scaffold blocks the biological activity of ADM by almost 100%, at least more than 90%, preferably at least more than 95%. In other words, this means that the non-neutralizing anti-ADM antibody, anti-ADM antibody fragment or anti-ADM non-Ig scaffold blocks the biological activity of ADM to less than 100%, preferably less than 95%, preferably less than 90%. In an embodiment where the non-neutralizing anti-ADM antibody, anti-ADM antibody fragment or anti-ADM non-Ig scaffold blocks the biological activity of ADM to less than 95%, an anti-ADM antibody, anti-ADM antibody fragment or anti-ADM non-Ig scaffold that blocks the biological activity of ADM to more than 95% may be outside the scope of this embodiment. This means that in one embodiment, the biological activity is reduced to 95% or less, preferably 90% or less, more preferably 80% or less, more preferably 50% or less, rather than 95% or more, 90% or more, 80% or more, 50% or more.

[0187] In one embodiment of the present invention, the non-neutralizing antibody is an antibody that binds to a region of at least 5 amino acids within the sequence of amino acids 1 to 21 (SEQ ID NO: 14) of mature human ADM, or an antibody that binds to a region of at least 5 amino acids within the sequence of amino acids 1 to 19 (SEQ ID NO: 17) of mature mouse ADM.

[0188] In another preferred embodiment of the present invention, the non-neutralizing antibody is an antibody that binds to a region of at least 4 amino acids within the sequence of amino acids 1 to 21 (SEQ ID NO: 14) of mature human ADM, or an antibody that binds to a region of at least 5 amino acids within the sequence of amino acids 1 to 19 (SEQ ID NO: 17) of mature mouse ADM.

[0189] In a specific embodiment according to the present invention, a non-neutralizing anti-ADM antibody or anti-ADM antibody fragment or ADM non-Ig scaffold is used, wherein the anti-ADM antibody or anti-ADM antibody fragment blocks the biological activity of ADM to less than 80%, preferably less than 50% (of the baseline value). It should be understood that this limited blockade of the biological activity of ADM (which means reduction of biological activity) occurs even when the antibody, fragment or scaffold is in excess concentration with respect to ADM, i.e., the antibody, fragment or scaffold is in excess of ADM. This limited blockade is an inherent property of the ADM binder itself in this specific embodiment. This means that the antibody, fragment or scaffold has a maximum inhibition of 80% or 50% respectively. In a preferred embodiment, the anti-ADM antibody, anti-ADM antibody fragment or anti-ADM non-Ig scaffold blocks / reduces the biological activity of anti-ADM to at least 5%. The above description means that there remains approximately 20% or 50% or even 95% of the residual ADM biological activity respectively.

[0190] Therefore, according to the present invention, the provided anti-ADM antibodies, anti-ADM antibody fragments, and anti-ADM non-Ig scaffolds do not neutralize the respective ADM biological activities.

[0191] Biological activity is defined as the effect exhibited by a substance on a living organism or tissue or organ or functional unit in vivo or in vitro (e.g., in an assay) after their interaction. In the case of ADM biological activity, this can be the effect of ADM in a human recombinant ADM receptor cAMP function assay. Therefore, according to the present invention, biological activity is defined by the ADM receptor cAMP function assay. The following steps can be carried out to determine the biological activity of ADM in such an assay. - Set a dose-response curve using ADM in the human recombinant ADM receptor cAMP function assay.

[0192] - The ADM concentration for half-maximal cAMP stimulation can be calculated.

[0193] - At a certain half-maximal cAMP-stimulating ADM concentration, a dose-response curve (final concentration up to 100 μg / ml maximum) is set by each of an ADM-stabilizing antibody or an ADM-stabilizing antibody fragment or an ADM-stabilizing non-Ig scaffold.

[0194] 50% maximal inhibition in the ADM bioassay means that the anti-ADM antibody or the anti-ADM antibody fragment or the anti-ADM non-Ig scaffold, respectively, blocks the biological activity of ADM to 50% of the baseline value. 80% maximal inhibition in the ADM bioassay means that the anti-ADM antibody or the anti-adrenomedullin antibody fragment or the anti-adrenomedullin non-Ig scaffold, respectively, blocks the biological activity of ADM to 80%. This means blocking the biological activity of ADM to 80% or less. This means that approximately 20% of the residual ADM biological activity remains.

[0195] However, in the context of this specification and the above, the expression "block the biological activity of ADM" with respect to the anti-ADM antibodies, anti-ADM antibody fragments, and anti-ADM non-Ig scaffolds disclosed herein is to be understood as simply reducing the biological activity of ADM from 100% to a maximum of 20% remaining ADM biological activity, preferably reducing the biological activity of ADM from 100% to 50% remaining ADM biological activity. However, in any case, the ADM biological activity as determined as detailed above remains.

[0196] The biological activity of ADM can be determined in a human recombinant adrenomedullin receptor cAMP functional assay (adrenomedullin bioassay) according to Example 2.

[0197] In a preferred embodiment, a modulating anti-ADM antibody or a modulating anti-ADM antibody fragment or a modulating anti-ADM non-Ig scaffold is used for the treatment or prevention of shock in a patient.

[0198] A "modulating" anti-ADM antibody or a modulating anti-ADM antibody fragment or a modulating anti-ADM non-Ig scaffold is an antibody or antibody fragment or non-Ig scaffold that extends the half-life (t 1 / 2 half-life retention time) of adrenomedullin in serum, blood, plasma by at least 10%, preferably at least 50%, more preferably more than 50%, most preferably more than 100%, and blocks the biological activity of ADM to less than 80%, preferably less than 50%. The anti-ADM antibody, anti-ADM antibody fragment or anti-ADM non-Ig scaffold can block the biological activity of ADM by at least 5%. These values related to the blockade of half-life and biological activity should be understood in relation to the aforementioned assays for determining these values. This means blockade of ADM biological activity of 80% or less or 50% or less, respectively.

[0199] Such a modulating anti-ADM antibody or a modulating anti-ADM antibody fragment or a modulating anti-ADM non-Ig scaffold provides the advantage of easier administration of the drug. The combination of partial blockade or partial reduction of ADM biological activity and extension of the in vivo half-life (increase in ADM biological activity) results in a beneficial simplification of anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold dosing. In the situation of excessive endogenous ADM (maximum stimulation, late septic phase, shock, hemodynamic decline phase), the activity-lowering effect is the main effect brought about by the antibody or fragment or scaffold, limiting the (negative) action of ADM. In the case of low or normal endogenous ADM concentrations, the biological effect of the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold is a combination of decreasing (by partially blocking) and increasing by extending the ADM half-life. Therefore, non-neutralizing and modulating anti-ADM antibodies or anti-ADM antibody fragments or anti-ADM non-Ig scaffolds act like ADM biological activity buffers to maintain the biological activity of ADM within a specific physiological range.

[0200] In a specific embodiment 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 is derived therefrom. In a specific embodiment, one or more (mouse) CDRs are transplanted into a human antibody or antibody fragment.

[0201] In one aspect, the subject matter of the present invention is a human or humanized CDR-grafted antibody or an antibody fragment thereof that binds to ADM, wherein the human or humanized CDR-grafted antibody or an antibody fragment thereof comprises an antibody heavy chain (H chain) comprising: GYTFSRYW (SEQ ID NO: 1), ILPGSGST (SEQ ID NO: 2) and / or TEGYEYDGFDY (SEQ ID NO: 3) and / or an antibody light chain (L chain) comprising: QSIVYSNGNTY (SEQ ID NO: 4), RVS (not part of the sequence listing) and / or further comprising FQGSHIPYT (SEQ ID NO: 5).

[0202] In a specific embodiment of the present invention, the subject matter of the present invention is a human or humanized monoclonal antibody that binds to ADM or an antibody fragment thereof that binds to ADM, wherein the heavy chain comprises GYTFSRYW (SEQ ID NO: 1), ILPGSGST (SEQ ID NO: 2), TEGYEYDGFDY (SEQ ID NO: 3) comprises at least one CDR selected from the group consisting of, and the light chain comprises QSIVYSNGNTY (SEQ ID NO: 4), RVS (not part of the sequence listing), comprises at least one CDR selected from the group consisting of FQGSHIPYT (SEQ ID NO: 5).

[0203] In a more specific embodiment of the present invention, the subject of the present invention is a human monoclonal antibody that binds to ADM or an antibody fragment thereof that binds to ADM, wherein the heavy chain has the sequence: GYTFSRYW (SEQ ID NO: 1), ILPGSGST (SEQ ID NO: 2), TEGYEYDGFDY (SEQ ID NO: 3) and the light chain has the sequence: QSIVYSNGNTY (SEQ ID NO: 4), RVS (not part of the sequence listing), including FQGSHIPYT (SEQ ID NO: 5).

[0204] In a very specific embodiment, the anti-ADM antibody has a sequence selected from the group consisting of SEQ ID NOs: 6, 7, 8, 9, 10, 11, 12, 13, 32, and 33.

[0205] The anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold according to the present invention has an affinity constant greater than 10 -7 M for human ADM, preferably 10 -8 M, and a preferred affinity greater than 10 -9 M, most preferably greater than 10 -10 M. Those skilled in the art know that it may be considered possible to compensate for a lower affinity by applying a higher dose of the compound, and this measure is not outside the scope of the present invention. The affinity constant can be determined according to the method described in Example 1.

[0206] The subject of the present invention is a human or humanized monoclonal antibody or fragment or an antibody fragment thereof that binds to ADM for use in the treatment or prevention of shock in a patient according to the present invention, wherein the antibody or fragment SEQ ID NO: 6 (AM-VH-C) QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK Sequence number 7 (AM-VH1) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK Sequence number 8 (AM-VH2-E40) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK Sequence number 9 (AM-VH3-T26-E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK Sequence number 10 (AM-VH4-T26-E40-E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK Sequence number 11 (AM-VL-C) DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Sequence number 12 (AM-VL1) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Sequence number 13 (AM-VL2-E40) It includes a sequence selected from the group consisting of DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0207] Another embodiment of the present invention relates to a human or humanized monoclonal antibody or fragment or an antibody fragment thereof that binds to ADM for use in the treatment or prevention of shock in a patient, wherein the antibody or fragment has the following sequence as the heavy chain: Sequence number 32 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGEILPGSGSTNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFScSVMHEALHNHYTQKSLSLSPGK comprising, as the light chain, the following sequence: SEQ ID NO: 33 DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPRLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0208] In a specific embodiment of the present invention, the antibody has, as the heavy chain, the following sequence: SEQ ID NO: 32 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGEILPGSGSTNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK or a sequence that is more than 95%, preferably more than 98%, preferably more than 99% identical thereto, and as the light chain the following sequence: SEQ ID NO: 33 DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPRLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC or a sequence that is more than 95%, preferably more than 98%, preferably more than 99% identical thereto.

[0209] To assess identity between two amino acid sequences, a pairwise alignment is performed. Identity defines the percentage of amino acids that directly match in the alignment.

[0210] In a specific embodiment of the present invention, the antibody has the following sequence as the heavy chain: SEQ ID NO: 32 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGEILPGSGSTNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Or it contains a CDR sequence that is 100% identical to SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3, and a sequence that is more than 95%, preferably more than 98%, preferably more than 99% identical to SEQ ID NO: 32, and has the following sequence as the light chain: SEQ ID NO: 33 DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPRLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Or it contains a CDR sequence that is 100% identical to SEQ ID NO: 4 and / or SEQ ID NO: 5, and a sequence that is more than 95%, preferably more than 98%, preferably more than 99% identical to SEQ ID NO: 33.

[0211] In an embodiment of the present invention, the anti-ADM antibody or anti-ADM antibody fragment for use in treating or preventing shock in a patient can be administered at a dose of at least 0.5 mg / kg body weight, specifically at least 1.0 mg / kg body weight, more specifically 1.0 to 20.0 mg / kg body weight, such as 2.0 to 10 mg / kg body weight, 2.0 to 8.0 mg / kg body weight, or 2.0 to 5.0 mg / kg body weight.

[0212] The term "pharmaceutical preparation" as used herein refers to a preparation in a form that enables the biological activity of the active ingredient contained therein to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the preparation is administered.

[0213] The present invention also relates to a pharmaceutical preparation comprising a therapeutically effective dose of an active ingredient in combination with at least one pharmaceutically acceptable excipient.

[0214] "Pharmaceutically acceptable excipient" refers to an excipient that does not cause harmful reactions, allergic reactions, or other adverse reactions when administered to a subject. It includes carriers, various diluents, fillers, salts, buffers, stabilizers, solubilizers, and other substances well known in the art, in addition to therapeutic proteins. The characteristics of the carrier vary depending on the route of administration.

[0215] Based on the above context, the following successively numbered embodiments provide further specific aspects of the present invention. 1. An anti - adrenomedullin (ADM) antibody or anti - ADM antibody fragment or anti - ADM non - Ig scaffold for use in the treatment or prevention of shock in a patient, wherein the patient is characterized in that the level of dipeptidyl peptidase 3 (DPP3) in a sample of body fluid is less than a threshold value, the threshold value of DPP3 is 40 ng / ml or less or in the range of 22 ng / ml to 40 ng / ml, and the anti - ADM antibody or anti - ADM fragment or anti - ADM non - Ig scaffold binds to the N - terminal portion of ADM (amino acids 1 - 21): YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO: 14).

[0216] 2. The anti - adrenomedullin (ADM) antibody or anti - ADM antibody fragment or anti - ADM non - Ig scaffold for use in the treatment or prevention of shock in a patient according to embodiment 1, wherein the shock is selected from the group consisting of shock due to decreased circulating blood volume, cardiogenic shock, obstructive shock, and distributive shock, particularly cardiogenic shock or septic shock.

[0217] 3. ● In the case of cardiogenic shock, the patient may be suffering from acute coronary syndrome (e.g., acute myocardial infarction), or the patient has heart failure (e.g., acute decompensated heart failure), myocarditis, arrhythmia, cardiomyopathy, valvular heart disease, aortic dissection with acute aortic valve stenosis, traumatic chordae tendineae rupture, or massive pulmonary embolism, or ● In the case of shock due to decreased circulating blood volume, the patient may be suffering from a hemorrhagic disease including gastrointestinal bleeding, trauma, vascular etiology (e.g., ruptured abdominal aortic aneurysm, tumor eroding major blood vessels) and spontaneous bleeding in the context of anticoagulant use, or a non - hemorrhagic disease including vomiting, diarrhea, renal loss, cutaneous loss / insensible loss (e.g., burns, heat stroke), or loss to the third space in the context of pancreatitis, cirrhosis, intestinal obstruction, trauma, or ● In the case of obstructive shock, the patient may be suffering from cardiac tamponade, tension pneumothorax, pulmonary embolism, or aortic valve stenosis, or ● In the case of distributive shock, for use in the treatment or prevention of shock in a patient according to Embodiment 1 or 2, where the patient may have septic shock, neurogenic shock, anaphylactic shock, or shock due to adrenal crisis, an anti - adrenomedullin (ADM) antibody or an anti - ADM antibody fragment or an anti - ADM non - Ig scaffold.

[0218] 4. For use in the treatment or prevention of shock in a patient according to Embodiments 1 - 3, where the threshold value of DPP3 in a sample of the patient's body fluid is 30 ng / ml or less or in the range of 22 - 30 ng / mL, an anti - adrenomedullin (ADM) antibody or an anti - ADM antibody fragment or an anti - ADM non - Ig scaffold.

[0219] 5. For use in the treatment or prevention of shock in a patient according to Embodiments 1 - 4, where the threshold value of DPP3 in a sample of the patient's body fluid is 25 ng / ml or less or in the range of 22 - 25 ng / mL, an anti - adrenomedullin (ADM) antibody or an anti - ADM antibody fragment or an anti - ADM non - Ig scaffold.

[0220] 6. For use in the treatment or prevention of shock in a patient according to Embodiments 1 - 5, where the level of DPP3 is determined by contacting the sample of the body fluid with a capture binder that specifically binds to DPP3, an anti - adrenomedullin (ADM) antibody or an anti - ADM antibody fragment or an anti - ADM non - Ig scaffold.

[0221] 7. For use in the treatment or prevention of shock in a patient according to Embodiment 6, where the capture binder is an antibody, an anti - adrenomedullin (ADM) antibody or an anti - ADM antibody fragment or an anti - ADM non - Ig scaffold.

[0222] 8. For use in the treatment or prevention of shock in a patient according to Embodiments 1 - 7, where either the level of DPP3 protein and / or the level of active DPP3 is determined and compared with a predetermined threshold value, an anti - adrenomedullin (ADM) antibody or an anti - ADM antibody fragment or an anti - ADM non - Ig scaffold.

[0223] 9. When the level of DPP3 is for use in the treatment or prevention of shock in a patient according to Embodiments 1 to 8, as determined by an immunoassay, particularly a sandwich immunoassay, an anti - adrenomedullin (ADM) antibody or an anti - ADM antibody fragment or an anti - ADM non - Ig scaffold.

[0224] 10. When the level of DPP3 is DPP3 activity, and the method for determining DPP3 activity in a body fluid sample of the subject is ● a step of contacting the sample with a capture binder that specifically binds to full - length DPP3, ● a step of separating DPP3 bound to the capture binder, ● a step of adding a substrate of DPP3 to the separated DPP3, ● a step of quantifying the DPP3 activity by measuring and quantifying the conversion of the substrate of DPP3, and is for use in the treatment or prevention of shock in a patient according to Embodiments 1 to 8, an anti - adrenomedullin (ADM) antibody or an anti - ADM antibody fragment or an anti - ADM non - Ig scaffold.

[0225] 11. Further characterized in that the patient has a level of ADM - NH2 exceeding a threshold value, an anti - adrenomedullin (ADM) antibody or an anti - ADM antibody fragment or an anti - ADM non - Ig scaffold for use in the treatment or prevention of shock in a patient according to Embodiments 1 to 10.

[0226] 12. The threshold value of ADM - NH2 in a sample of the patient's body fluid is 40 - 100 pg / mL, more preferably 50 - 90 pg / mL, even more preferably 60 - 80 pg / mL, and most preferably the threshold value is 70 pg / mL, an anti - adrenomedullin (ADM) antibody or an anti - ADM antibody fragment or an anti - ADM non - Ig scaffold for use in the treatment or prevention of shock in a patient according to Embodiment 11.

[0227] 13. An anti - adrenomedullin (ADM) antibody or anti - ADM antibody fragment or anti - ADM non - Ig scaffold for use in the treatment or prevention of shock in a patient according to embodiment 12, wherein the threshold value of the ADM - NH2 in a sample of the patient's body fluid is 70 pg / mL.

[0228] 14. An anti - adrenomedullin (ADM) antibody or anti - ADM antibody fragment or anti - ADM non - Ig scaffold for use in the treatment or prevention of shock in a patient according to embodiments 1 to 13, wherein the body fluid is selected from whole blood, plasma, and serum.

[0229] 15. An anti - adrenomedullin (ADM) antibody or anti - ADM antibody fragment or anti - ADM non - Ig scaffold for use in the treatment or prevention of shock in a patient according to embodiment 14, wherein the body fluid is plasma.

[0230] 16. An anti - adrenomedullin (ADM) antibody or anti - ADM antibody fragment or anti - ADM non - Ig scaffold for use in the treatment or prevention of shock in a patient according to embodiments 11 to 15, wherein the level of the ADM - NH2 is determined by contacting a sample of the body fluid with a capture binder that specifically binds to ADM - NH2.

[0231] 17. An anti - adrenomedullin (ADM) antibody or anti - ADM antibody fragment or anti - ADM non - Ig scaffold for use in the treatment or prevention of shock in a patient according to embodiment 16, wherein the capture binder is an antibody.

[0232] 18. An anti - adrenomedullin (ADM) antibody or anti - ADM antibody fragment or anti - ADM non - Ig scaffold for use in the treatment or prevention of shock in a patient according to embodiments 1 to 17, 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.

[0233] 19. An anti - adrenomedullin (ADM) antibody, anti - ADM antibody fragment, or anti - ADM non - Ig scaffold for use in the treatment or prevention of shock in a patient according to embodiments 1 - 18, wherein the antibody, antibody fragment, or non - Ig scaffold does not bind to the C - terminal portion of ADM having the amino acids 43 - 52 of the ADM sequence: PRSKISPQGY - NH2 (SEQ ID NO: 24).

[0234] 20. An anti - adrenomedullin (ADM) antibody, anti - ADM antibody fragment, or anti - ADM non - Ig scaffold for use in the treatment or prevention of shock in a patient according to embodiments 1 - 19, wherein the antibody, fragment, or scaffold blocks 80% or less, preferably 50% or less, of the biological activity of ADM.

[0235] 21. The antibody or fragment or scaffold is a monoclonal antibody or fragment or antibody fragment thereof that binds to ADM, and the heavy chain has the sequence: CDR1: SEQ ID NO: 1 GYTFSRYW CDR2: SEQ ID NO: 2 ILPGSGST CDR3: SEQ ID NO: 3 including TEGYEYDGFDY, and the light chain has the sequence: CDR1: SEQ ID NO: 4 QSIVYSNGNTY CDR2: RVS CDR3: SEQ ID NO: 5 including FQGSHIPYT. An anti - adrenomedullin (ADM) antibody, anti - ADM antibody fragment, or anti - ADM non - Ig scaffold for use in the treatment or prevention of shock in a patient according to embodiments 1 - 20.

[0236] 22. The antibody or fragment is As the VH region: SEQ ID NO: 6 (AM - VH - C) QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK Sequence number 7 (AM-VH1) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK Sequence number 8 (AM-VH2-E40) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK Sequence number 9 (AM-VH3-T26-E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SEQ ID NO: 10 (AM-VH4-T26-E40-E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK comprising a sequence selected from the group consisting of as the VL region, the following sequence: SEQ ID NO: 11 (AM-VL-C) DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 12 (AM-VL1) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Sequence number 13 (AM-VL2-E40) An anti - adrenomedullin (ADM) antibody or anti - ADM antibody fragment or anti - ADM non - Ig scaffold for use in the treatment or prevention of shock in a patient according to embodiments 1 - 21, comprising a sequence selected from the group consisting of DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0237] 23. The antibody or fragment has, as the heavy chain, the following sequence: Sequence number 32 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGEILPGSGSTNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK or a sequence that is more than 95% identical thereto, as the light chain, the following sequence: SEQ ID NO: 33 DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPRLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC or a sequence that is more than 95% identical thereto, for use in the treatment or prevention of shock in a patient according to any one of embodiments 1 to 22, an anti - adrenomedullin (ADM) antibody or an anti - ADM antibody fragment or an anti - ADM non - Ig scaffold.

[0238] 24. An anti - adrenomedullin (ADM) antibody or anti - ADM antibody fragment or anti - ADM non - Ig scaffold, which binds to the N - terminal portion of ADM (amino acids 1 - 10): YRQSMNNFQG (SEQ ID NO: 26), for use in the treatment or prevention of shock in a patient according to any one of embodiments 1 - 23.

[0239] 25. An anti - adrenomedullin (ADM) antibody or anti - ADM antibody fragment or anti - ADM non - Ig scaffold for use in the treatment according to any one of embodiments 1 - 24, wherein the antibody or fragment or scaffold exhibits a binding affinity of at least 10 -7 M for ADM as determined by label - free surface plasmon resonance using a Biacore 2000 system.

[0240] 26. An anti - adrenomedullin (ADM) antibody or anti - ADM antibody fragment or anti - ADM non - Ig scaffold for use in the treatment or prevention of shock in a patient according to embodiment 25, wherein the anti - adrenomedullin (ADM) antibody or anti - ADM antibody fragment or anti - ADM non - Ig scaffold exhibits an affinity of 1×10 -9 ~3×10 -9 M for human ADM as determined by label - free surface plasmon resonance using a Biacore 2000 system.

[0241] 27. An anti - adrenomedullin (ADM) antibody or anti - ADM antibody fragment or anti - ADM non - Ig scaffold for use in the treatment or prevention of shock in a patient according to any one of embodiments 1 - 26, wherein the anti - adrenomedullin (ADM) antibody or anti - ADM antibody fragment is an IgG1 antibody.

[0242] 28. A pharmaceutical preparation for use in the treatment or prevention of shock in a patient, comprising an anti - adrenomedullin (ADM) antibody or anti - ADM antibody fragment or anti - ADM non - Ig scaffold according to any one of embodiments 1 - 27.

[0243] 29. A pharmaceutical preparation for use in the treatment or prevention of shock in a patient according to embodiment 28, wherein the pharmaceutical preparation is a solution, preferably a ready-to-use solution.

[0244] 30. A pharmaceutical preparation for use in the treatment or prevention of shock in a patient according to embodiment 28 or 29, wherein the pharmaceutical preparation is in a lyophilized state.

[0245] 31. A pharmaceutical preparation for use in the treatment or prevention of shock in a patient according to embodiment 28 or 30, wherein the pharmaceutical preparation is administered intramuscularly.

[0246] 32. A pharmaceutical preparation for use in the treatment or prevention of shock in a patient according to embodiment 28 or 31, wherein the pharmaceutical preparation is administered intravascularly.

[0247] 33. A pharmaceutical preparation for use in the treatment or prevention of shock in a patient according to embodiment 28 or 32, wherein the pharmaceutical preparation is administered via infusion.

[0248] 34. A pharmaceutical preparation for use in the treatment or prevention of shock in a patient according to embodiment 28 or 33, wherein the pharmaceutical preparation is administered systemically.

[0249] Method for obtaining a monoclonal antibody In all of the following embodiments, the term monoclonal antibody means a monoclonal antibody and fragments of monoclonal antibodies such as those detailed herein, more particularly including monoclonal antibodies.

[0250] Hybridoma In a further aspect, the antibody according to the invention is a monoclonal antibody that can be obtained by a method comprising: i) fusing antibody-secreting cells derived from an animal pre-immunized with an antigen with myeloma cells to obtain a number of hybridomas; ii) isolating from the number of hybridomas a hybridoma that produces the desired monoclonal antibody.

[0251] In certain embodiments, an antibody according to the present invention is a monoclonal antibody that can be obtained by isolating a hybridoma that produces the desired monoclonal antibody from a number of hybridomas, where the number of hybridomas is prepared by fusing antibody-secreting cells derived from an animal pre-immunized with an antigen with myeloma cells to obtain a number of hybridomas.

[0252] In particular, the desired monoclonal antibody is a monoclonal antibody that binds to an antigen with a binding affinity of at least 10 7 M -1 , preferably 10 8 M -1 or greater, more preferably a binding affinity greater than 10 9 M -1 , and most preferably greater than 10 10 M -1 .

[0253] In certain embodiments of the method for obtaining an antibody, in step i), the animal is a mammal, specifically a rabbit, mouse or rat, more specifically a mouse, and more specifically a Balb / c mouse.

[0254] In certain embodiments of the method for obtaining an antibody, in step i), the antibody-secreting cells are spleen cells, more specifically activated B cells.

[0255] In certain embodiments of the method for obtaining an antibody, in step i), the fusion involves the use of polyethylene glycol.

[0256] In certain embodiments of the method for obtaining an antibody, in step i), the myeloma is derived from a mammal, and in certain embodiments, it is derived from the same species of mammal from which a number of antibody-secreting cells are obtained. In certain specific embodiments of the method for obtaining an antibody, in step i), the myeloma cells are of the cell line SP2 / 0.

[0257] In certain embodiments of the method for obtaining an antibody, the fusing in step i) includes PEG-mediated fusion, Sendai virus-mediated fusion, or electric current-mediated fusion.

[0258] In certain embodiments of the method for obtaining an antibody, the isolating in step ii) includes performing an antibody capture assay, an antigen capture assay, and / or a functional screening.

[0259] In certain embodiments of the method for obtaining an antibody, isolating a hybridoma that produces a desired monoclonal antibody in step ii) can involve cloning and recloning the hybridoma using the limiting dilution method.

[0260] In one embodiment, the antibody capture assay includes the following: a) Binding an antigen to a substrate, particularly a solid substrate; b) Binding the produced antibody to the antigen; c) Removing unbound antibodies by washing; d) Detecting the bound antibody.

[0261] In one embodiment, the antigen capture assay includes the following: a) Binding the produced antibody to a substrate, particularly a solid substrate; b) Binding the antigen to the antibody; c) Removing unbound antigen by washing; d) Detecting the bound antigen; Or the antigen capture assay includes the following: a) Binding an antigen to the produced antibody to form an antibody-antigen complex; b) Binding the antibody-antigen complex to a substrate, particularly a solid substrate; c) Removing unbound antigen by washing; d) Detecting the bound antigen.

[0262] In one embodiment, said isolating in step ii) includes performing an enzyme-linked immunosorbent assay, fluorescence-activated cell sorting, cell staining, immunoprecipitation, and / or Western blot.

[0263] In one embodiment, said detecting of the antibody or antigen is achieved using an immunoassay.

[0264] In one embodiment, the animal is a transgenic animal, particularly a transgenic mouse (especially where the mouse immunoglobulin (Ig) locus in the transgenic animal genome has been replaced by a human locus), such as a HuMabMouse or XenoMouse.

[0265] In one embodiment, the antigen comprises the peptides described in Table 1 or Table 6 herein, which in certain embodiments (especially for immunization) can be conjugated to a protein, particularly a serum protein, more particularly serum albumin, and more particularly BSA.

[0266] In a preferred embodiment, the antibody according to the invention is a monoclonal antibody that can be obtained by a method comprising: i) Using polyethylene glycol to fuse spleen cells from Balb / c mice pre-immunized with the peptides described in Table 1 or 6 herein with SP2 / 0 myeloma cells to obtain a number of hybridomas; ii) Isolating from said number of hybridomas the hybridomas that produce the desired monoclonal antibody. More preferably, the method comprises: ● Growing the hybridomas in HAT medium [RPMI1640 culture medium supplemented with 20% fetal bovine serum and HAT supplement] for a first period (particularly 2 weeks); ● Subsequently replacing the HAT medium with HT medium for multiple passages (particularly 3 passages); ● Subsequently returning to normal cell culture medium until a second period, particularly until 3 weeks have elapsed since the fusion; ●Perform primary screening of the cell culture supernatant for antigen-specific IgG antibodies, ●Grow the microcultures of the cells that were positive in the test in 4), ●Retest the cell culture supernatant of the microcultures for antigen-specific IgG antibodies, ●Using the limiting dilution method, clone and reclone the cultures that were positive in the test in 6), ●Optionally, determine the isotype of the clones obtained from 7), ●Optionally, purify the antibody via Protein A. Phage display In a further aspect, the antibody according to the invention is a monoclonal antibody that can be obtained by a method comprising: i) Isolating at least one antibody having affinity for the antigen from an antibody gene library, ii) Generating at least one cell line expressing the at least one antibody, iii) Isolating at least one antibody from the culture of the at least one cell line obtained in step ii).

[0267] Antibodies having affinity for the antigen are, in particular, antibodies having a binding affinity of at least 10 7 M -1 , preferably 10 8 M -1 , more preferably an affinity greater than 10 9 M -1 , most preferably greater than 10 10 M -1 .

[0268] In certain embodiments, the antibody according to the invention is a monoclonal antibody that can be obtained by isolating at least one antibody from a culture derived from at least one cell line expressing at least one antibody having affinity for the antigen from an antibody gene library.

[0269] In one embodiment, the antigen comprises the peptides described in Table 1 or Table 6 herein, respectively, which may be bound to a solid phase in certain embodiments.

[0270] In certain embodiments of the method for obtaining an antibody, in step i), the antibody gene library is a naive antibody gene library, specifically a human naive antibody gene library, and more specifically in said library, the antibody is presented via phage display, i.e., on phages containing the nucleotide sequences encoding such respective antibodies; more specifically, the antibody gene library is library HAL7, HAL8, or HAL9, and more specifically, a library containing the human naive antibody gene library HAL7 / 8.

[0271] In certain embodiments of the method for obtaining an antibody, in step i), the screening involves the use of an antigen, specifically an antigen containing a tag linked thereto via two different spacers, and more specifically a biotin tag. In certain embodiments, such a panning strategy involves a mixture of panning rounds of non-specifically bound antigen and specifically bound antigen via the tag (in the case of a biotin tag, bound to streptavidin). In this way, the background of non-specific binders can be minimized.

[0272] In certain embodiments of the method for obtaining an antibody, in step i), in embodiments where the library is a phage display library, the antibody is isolated by isolating the phage presenting the antibody (and containing the nucleotide sequence encoding the antibody).

[0273] In certain embodiments of the method for obtaining an antibody, in step ii), the cell line is generated via introduction of a nucleotide sequence encoding the antibody, and in embodiments where the library in step i) is a phage display library, the phage isolated from step i) can be used to generate a cell strain expressing the antibody, such as an E. coli strain.

[0274] In certain embodiments of the method for obtaining an antibody, in step iv), in embodiments where the library in step i) is a phage display library and the cell strain is generated in step ii), the antibody can be isolated from the supernatant of the culture.

[0275] When used in describing the method for obtaining an antibody, it is understood that the term "one antibody" in the expression "at least one antibody" can particularly include two or more antibody molecules having the same amino acid sequence. This understanding applies, with necessary modifications, to the term "one cell line".

[0276] In certain embodiments of the method for obtaining an antibody, two or more antibodies (referring to a number of antibodies each having a distinct amino acid sequence) are isolated in step i), and thus two or more cell lines are generated in step ii). Such a method can involve, for example, the selection of clones that are positive for binding to an antigen via a binding assay, such as an ELISA assay involving the antigen, and the cells that are positive for binding to the antigen can be isolated to generate monoclonal cell lines.

[0277] In a preferred embodiment, the antibody according to the invention is a monoclonal antibody that can be obtained by a method comprising: i) isolating at least one antibody having affinity for an antigen from an antibody gene library comprising the human naive antibody gene library HAL7 / 8 by eluting phage carrying the antibody from the library; ii) generating at least one Escherichia coli cell line expressing the at least one antibody; iii) isolating at least one antibody from the supernatant of a culture of the at least one Escherichia coli cell line obtained in step ii).

[0278] In a further aspect, the antibody fragments according to the invention are produced by a method involving enzymatic digestion of an antibody. In certain embodiments, this method produces, for example, Fab or F(ab')2 antibody fragments. In certain embodiments, this method involves digestion with pepsin or papain, optionally immobilized on a surface.

[0279] In certain embodiments, the antibody can be humanized by CDR grafting, in particular by a method comprising the following steps: - extracting RNA from a hybridoma expressing the antibody of interest (e.g., obtained by the methods described herein); - amplifying the extracted RNA via RT-PCR to obtain a DNA product, in particular using a primer set specific for the heavy and light chains of the antibody of interest; - further amplifying the DNA product via PCR, in particular using a semi-nested primer set specific for the antibody variable regions; - determining the sequence of the DNA product; - aligning the sequence with homologous human framework sequences to determine the humanized sequences for the variable heavy and variable light chain sequences (of the desired antibody).

[0280] In certain embodiments, the antibody can be humanized by aligning the DNA products obtained by amplifying RNA extracted from a hybridoma expressing the antibody of interest via RT-PCR, using primer sets specific for the heavy and light chains of the antibody of interest in particular, and further amplifying the DNA obtained therefrom via PCR, using a semi-nested primer set specific for the antibody variable regions in particular, with a homologous human framework sequence, to determine the humanized sequences for the variable heavy and variable light chain sequences (of the desired antibody).

[0281] In certain embodiments, the antibody can be humanized by: - determining the complementarity-determining regions (CDRs), which can be achieved by analyzing the structural interactions of the framework regions (FRs) with the CDRs and the antigen, - transplanting the CDR sequences into human framework regions.

[0282] In certain embodiments, the antibody can be humanized by transplanting CDR sequences, which can preferably be determined by analyzing the structural interactions of the framework regions (FRs) with the CDRs and the antigen, into human framework regions.

[0283] In certain embodiments, mutations in the amino acid sequences of the CDRs or FRs can be introduced to maintain the structural interaction with the antigen, for example, via a random approach using a phage display library or a directed approach guided by molecular modeling (this structural interaction can otherwise be lost by introducing the human FR sequences).

[0284] The DNA sequences encoding the antibodies determined as detailed herein can be introduced into cells by known genetic engineering techniques and used for the production of antibodies.

[0285] Production of Antibodies In a further aspect, the antibody according to the invention is a monoclonal antibody obtainable by the methods described herein, which is produced by a method comprising: - culturing a cell line comprising a nucleotide sequence encoding the antibody, - isolating the antibody from the culture.

[0286] In a further specific aspect, the antibody according to the invention is a monoclonal antibody obtainable by the methods described herein, which is produced by isolating the antibody from a culture of a cell line comprising a nucleotide sequence encoding the antibody.

[0287] In certain embodiments of the method, the cell line is produced as described above herein and is a bacterial cell such as a Gram-negative bacterium, e.g., Escherichia coli, Proteus mirabilis, or Pseudomonas putida, a Gram-positive bacterium, e.g., Bacillus brevis, Bacillus subtilis, Bacillus megaterium, Lactobacilli such as Lactobacillus zeae / casei or Lactobacillus paracasei, or Streptomyces such as Streptomyces lividans; a eukaryotic cell such as a yeast, e.g., Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, Schizosaccharomyces pombe, Schwanniomyces occidentalis, Kluyveromyces lactis, or Yarrowia lipolytica; a fungus such as a filamentous fungus of the genus Trichoderma, Aspergillus such as A. niger (e.g., A. awamori subgenus) and Aspergillus oryzae, Trichoderma reesei, Chrysosporium such as C. lucknowense; Leishmania, e.g., L.protozoa such as *tarentolae*; insect cells transfected with a baculovirus, such as an AcNPV, such as insect cell lines derived from *Spodoptera frugiperda*, such as Sf-9 or Sf-21, insect cell lines derived from *Drosophila melanogaster*, such as DS2, or insect cell lines derived from *Trichopulsia ni*, such as High Five cells (BTI-TN-5B1-4); hamster cells, such as Chinese hamster ovary cells, such as K1-, DukX B11-, DG44, Lec13, or BHK cells, mouse cells, such as mouse myeloma cells, such as NS0 cells, mammalian cells such as *Homo sapiens* cells, such as Per.C6 cells, AGE1.HN cells, HEK293 cells, etc., may be included.

[0288] In certain embodiments of the method, the cells can be, for example, hybridoma cells as described herein.

[0289] In certain embodiments of the method, culturing can be performed in static suspension culture, stirred suspension culture, membrane-based culture, matrix-based culture, or a high cell density bioreactor. Containers for such culture can be selected from the group including T-flasks, roller culture, spinner culture, stirred tank bioreactors, air-lift bioreactors, static membrane-based or matrix-based culture systems, suspension bioreactors, fluidized bed bioreactors, ceramic bioreactors, perfusion systems, hollow fiber bioreactors.

[0290] In certain embodiments of the method, the cells can be immobilized on a matrix.

[0291] A high cell density bioreactor is a culture system that can achieve a cell density of more than 10 8 cells / ml.

[0292] In a further aspect, the antibody according to the invention is a monoclonal antibody obtainable by the methods described herein, which is produced by a method comprising: - generating a transgenic plant or animal comprising a nucleotide sequence encoding the antibody; - isolating the antibody from the plant or animal, or a secretion or product of the plant or animal.

[0293] In certain further aspects, the antibody according to the invention is a monoclonal antibody obtainable by the methods described herein, produced by isolating the antibody from a transgenic plant or transgenic animal comprising a nucleotide sequence encoding the antibody, or a secretion or product of a transgenic plant or transgenic animal.

[0294] The animal can be selected, for example, from chicken, mouse, rat, rabbit, cow, goat, sheep, pig. The secretion or product can be, for example, milk or eggs. The plant can be selected, for example, from tobacco (N. tabacum, or Nicotiana benthamiana), duckweed (Lemna minor), Chlamydomonas reinhardtii, rice, Arabidopsis thaliana, alfalfa (Medicago sativa), lettuce, maize.

[0295] In certain embodiments, the antibody can be isolated by physicochemical fractionation, for example, size exclusion chromatography, precipitation using ammonium sulfate, ion exchange chromatography, immobilized metal chelate chromatography, gel filtration, zone electrophoresis; based on their classification, for example, binding to bacterial protein A, G, or L, jacalin; antigen-specific affinity purification using an immobilized ligand / antigen, and if necessary, low molecular weight components can be removed by methods such as dialysis, desalting, and diafiltration.

[0296] In some embodiments, the antibody is encoded by a nucleotide sequence that is the reverse transcription of an amino acid sequence from an antibody made by one of the methods described herein.

Brief Description of the Drawings

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Example

[0319] Example 1 - Generation of Antibodies and Determination of Their Affinity Constants Several human and mouse antibodies were prepared and their affinity constants were determined (see Tables 1 and 2). It should be emphasized that the antibodies, antibody fragments, and non-Ig scaffolds in the example section according to the present invention bind to ADM and should therefore be regarded as anti-ADM antibodies / antibody fragments / non-Ig scaffolds.

[0320] Peptide / Conjugate for Immunization Peptides for immunization were synthesized using an additional N-terminal cysteine residue (when no cysteine was present within the selected ADM sequence) for conjugation of the peptide to bovine serum albumin (BSA) (see Table 1) (JPT Technologies, Berlin, Germany). The peptide was covalently linked to BSA by using Sulfolink coupling gel (Perbio-science, Bonn, Germany). The coupling procedure was carried out according to the manual of Perbio.

[0321] Production of Mouse Monoclonal Antibodies Balb / c mice were immunized on days 0 and 14 with 100 μg of peptide-BSA conjugate (emulsified in 100 μl of complete Freund's adjuvant) and on days 21 and 28 with 50 μg of peptide-BSA conjugate (in 100 μl of incomplete Freund's adjuvant). Three days before performing the fusion experiment, the animals were administered 50 μg of conjugate dissolved in 100 μl of physiological saline, which was administered as one intraperitoneal injection and one intravenous injection. Cells from immunized mouse spleens and the myeloma cell line SP2 / 0 were fused at 37°C for 30 seconds with 1 ml of 50% polyethylene glycol. After washing, the cells were seeded into 96-well cell culture plates. Hybrid clones were selected by growing them in HAT medium (RPMI 1640 culture medium supplemented with 20% fetal bovine serum and HAT supplement). After 2 weeks, the HAT medium was replaced with HT medium and subcultured three times, and then returned to normal cell culture medium. The cell culture supernatant was initially screened for antigen-specific IgG antibodies 3 weeks after fusion. Microcultures with positive test results were transferred to 24-well plates for growth. After retesting, the selected cultures were cloned and recloned using the limiting dilution method, and the isotype was determined (see Lane, R.D. 1985. J. Immunol. Meth. 81:223-228, Ziegler et al. 1996. Horm. Metab. Res. 28:11-15).

[0322] Antibodies were prepared by standard antibody production methods (Marx et al, 1997. Monoclonal Antibody Production, ATLA 25, 121) and purified by Protein A. The antibody purity exceeded 95% based on SDS gel electrophoresis analysis.

[0323] Human antibodies: Human antibodies were prepared by phage display according to the following procedure: The human naive antibody gene library HAL7 / 8 was used for the isolation of recombinant single-chain F variable domains (scFv) against adrenomedullin peptides. The antibody gene library was screened using a panning strategy that included the use of a peptide containing a biotin tag linked to the adrenomedullin peptide sequence via two different spacers. The background of non-specific binders was minimized using a mixture of panning rounds using non-specific binding antigens and streptavidin-binding antigens. Phage eluted from the third panning was used for the generation of monoclonal scFv-expressing E. coli strains. The supernatants from the cultures of these clone strains were used directly in antigen ELISA tests (see also Hust et al. 2011. Journal of Biotechnology 152, 159 - 170, Schutte et al. 2009. PLoS One 4, e6625). Positive clones were selected based on a positive ELISA signal against the antigen and a negative ELISA signal against streptavidin-coated microtiter plates. For further characterization, the scFv open reading frame was cloned into the expression plasmid pOPE107 (Hust et al., J. Biotechn. 2011), captured from the culture supernatant via immobilized metal ion affinity chromatography, and purified by size exclusion chromatography.

[0324] Affinity constant: To determine the affinity of the antibody for ADM, the binding kinetics of ADM to the immobilized antibody were determined by label-free surface plasmon resonance using a Biacore 2000 system (GE Healthcare Europe GmbH, Freiburg, Germany). Reversible immobilization of the antibody was performed according to the manufacturer's instructions using an anti-mouse Fc antibody covalently bound at high density to the CM5 sensor surface (mouse antibody capture kit; GE Healthcare). (Lorenz et al. 2011. Antimicrob Agents Chemother. 55(1):165-173).

[0325] Monoclonal antibodies were raised against the following ADM regions of human and mouse ADM, respectively. The following table represents the selected, obtained antibodies used in further experiments. Selection was based on the target region.

Table 1

Table 2

[0326] Fab Generation and Purification Procedure: The immobilized papain was equilibrated by washing the resin with 0.5 ml of digestion buffer and centrifuging the column at 5000×g for 1 minute. Then, the buffer was discarded. The desalting column was prepared by removing the storage solution, washing it with digestion buffer, and then centrifuging it at 1000×g for 2 minutes each time. 0.5 ml of the prepared IgG sample was added to a spin column tube containing the equilibrated immobilized papain. The incubation time for the digestion reaction was carried out at 37°C for 16 hours on a tabletop rocker. The column was centrifuged at 5000×g for 1 minute to separate the digest from the immobilized papain. Then, the resin was washed with 0.5 ml of PBS and centrifuged at 5000×g for 1 minute. The wash fraction was added to the digested antibody to make the total sample volume 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), equilibrated by adding 2 ml of PBS, centrifuged again for 1 minute, and the flow-through was discarded. The sample was applied to the column and resuspended by inversion. Incubation was carried out at room temperature with inversion mixing for 10 minutes. The column was centrifuged for 1 minute, and the flow-through containing the Fab fragment was saved. (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).

[0327] Procedure for the production and purification of F(ab’)2 fragment: The immobilized pepsin was equilibrated by washing the resin with 0.5 ml of digestion buffer and centrifuging the column at 5000×g for 1 minute. Then, the buffer was discarded. The desalting column was prepared by removing the storage solution, washing with digestion buffer, and then centrifuging at 1000×g for 2 minutes each time. 0.5 ml of the prepared IgG sample was added to a spin column tube containing the equilibrated immobilized pepsin. The incubation time for the digestion reaction was carried out at 37 °C for 16 hours on a desktop rocker. The column was centrifuged at 5000×g for 1 minute to separate the digest from the immobilized papain. Then, the resin was washed with 0.5 mL of PBS and centrifuged at 5000×g for 1 minute. The wash fraction was added to the digested antibody to make the total sample volume 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), equilibrated by adding 2 mL of PBS, centrifuged again for 1 minute, and the flow-through was discarded. The sample was applied to the column and resuspended by inversion. Incubation was carried out at room temperature with inversion mixing for 10 minutes. The column was centrifuged for 1 minute, and the flow-through containing the Fab fragment was saved.(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).

[0328] Humanization of the NT - H antibody fragment: The antibody fragment was humanized by CDR grafting (Jones et al. 1986. Nature 321,522 - 525).

[0329] The following steps were performed to obtain the humanized sequence. Total RNA extraction: Total RNA was extracted from NT - H hybridoma using a Qiagen kit. First - round RT - PCR: The QIAGEN® OneStep RT - PCR Kit (Catalog number 210210) was used. RT - PCR was performed using primer sets specific for the heavy and light chains. For each RNA sample, 12 individual heavy - chain RT - PCR reactions and 11 light - chain RT - PCR reactions were set up using a degenerate forward primer mixture covering the leader sequence of the variable region. The reverse primers were located in the constant regions of the heavy and light chains. No restriction sites were introduced into the primers.

[0330] Reaction setup: 5.0 μl of 5× QIAGEN® OneStep RT-PCR Buffer, 0.8 μl of dNTP mix (containing 10 mM of each dNTP), 0.5 μl of primer set, 0.8 μl of QIAGEN® OneStep RT-PCR Enzyme Mix, 2.0 μl of template RNA, up to 20.0 μl with RNase-free water, total volume 20.0 μl. PCR conditions: Reverse transcription: 50 °C, 30 min; Initial PCR activation: 95 °C, 15 min; Cycling: 94 °C, 25 s; 54 °C, 30 s; 72 °C, 30 s for 20 cycles; Final extension: 72 °C, 10 min. Second-round semi-nested PCR: The RT-PCR products from the first-round reaction were further amplified in the second-round PCR. Twelve individual heavy-chain RT-PCR reactions and eleven light-chain RT-PCR reactions were set up using semi-nested primer sets specific for the antibody variable regions.

[0331] Reaction setup: 10 μl of 2× PCR mix; 2 μl of primer set; 8 μl of first-round PCR product; Total volume: 20 μl; Hybridoma antibody cloning report. PCR conditions: Initial denaturation at 95 °C for 5 min; 25 cycles of 95 °C, 25 s; 57 °C, 30 s; 68 °C, 30 s; Final extension at 68 °C for 10 min.

[0332] After PCR was completed, the PCR reaction sample was run on an agarose gel to visualize the amplified DNA fragments. After sequencing more than 15 cloned DNA fragments amplified by nested RT-PCR, several mouse antibody heavy and light chains were cloned and considered to be accurate. Protein sequence alignment and CDR analysis identified one heavy chain and one light chain. After alignment with the homologous human framework sequence, the humanized sequences obtained for the variable heavy chain are as follows. See Figure 5. Amino acids 26, 40, and 55 of the variable heavy chain and amino acid 40 of the variable light chain are important for binding properties and may be reverted to their mouse origin. The candidates obtained are shown below. (Padlan 1991. Mol. Immunol. 28, 489-498, Harris and Bajorath. 1995. Protein Sci. 4, 306-310).

[0333] Annotation of antibody fragment sequences (SEQ ID NOs: 6-13, 32, and 33): Bold and underlined are CDR1, 2, 3, and italicized is the constant region. The hinge region is emphasized in bold. Framework point mutations are shown with a gray text background.

[0334] SEQ ID NO: 6 (AM-VH-C) [Table 3] SEQ ID NO: 7 (AM-VH1) [Table 4] SEQ ID NO: 8 (AM-VH2-E40) [Table 5] SEQ ID NO: 9 (AM-VH3-T26-E55) [Table 6] SEQ ID NO: 10 (AM-VH4-T26-E40-E55)

Table 7

Table 8

Table 9

Table 10

Table 11

Table 12

[0335] Example 2 - Effect of selected anti-ADM antibodies on anti-ADM biological activity The effect of selected ADM antibodies on ADM biological activity was tested in a human recombinant adrenomedullin receptor cAMP function assay (adrenomedullin bioassay).

[0336] Testing of antibodies targeting human or mouse adrenomedullin in a human recombinant adrenomedullin receptor cAMP function assay (adrenomedullin bioassay) Materials: Cell line CHO-K1, receptor adrenomedullin (CRLR+RAMP3), receptor accession number cell line: CRLR: U17473; RAMP3: AJ001016 CHO-K1 cells expressing human recombinant adrenomedullin receptor (FAST-027C), which had been grown prior to testing in an antibiotic-free medium, were detached by gently flowing PBS-EDTA (5 mM EDTA) over them, collected by centrifugation, and resuspended in assay 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).

[0337] Dose-response curves were established in parallel with a reference agonist (hADM or mADM).

[0338] Antagonist assay (96-well): For the antagonist assay, 6 μl of reference agonist (human (5.63 nM) or mouse (0.67 nM) adrenomedullin) was mixed with 6 μl of test sample with antagonist at different dilution factors, or 6 μl of buffer. After incubation at room temperature for 60 minutes, 12 μl of cells (2500 cells / well) was added. The plate was incubated at room temperature for 30 minutes. After addition of lysis buffer, the percentage of DeltaF was estimated using a HTRF kit (catalog number 62AM2 PEB) from Cis-Bio International according to the manufacturer's specifications. hADM 22-52 was used as the reference antagonist.

[0339] Antibody test cAMP-HTRF assay In a cAMP functional assay of human recombinant adrenomedullin receptor (FAST-027C), the antagonist activity of anti-h-ADM antibodies (NT-H, MR-H, CT-H) was tested at the following final antibody concentrations: 100 μg / ml, 20 μg / ml, 4 μg / ml, 0.8 μg / ml, 0.16 μg / ml in the presence of 5.63 nM human ADM 1-52.

[0340] In the cAMP functional assay of human recombinant ADM receptor (FAST-027C), in the presence of 0.67 nM mouse ADM 1-50, the antagonist activity of anti-m-ADM antibodies (NT-M, MR-M, CT-M) was tested at the following final antibody concentrations: 100 μg / ml, 20 μg / ml, 4 μg / ml, 0.8 μg / ml, 0.16 μg / ml. The data were plotted as relative inhibition vs antagonist concentration (see Figures 2a-2l). The maximum inhibition by each antibody is shown in Table 3.

[0341]

Table 13

[0342] Example 3 - Stabilization of hADM by Anti-ADM Antibodies The stabilizing effect of human ADM antibodies on human ADM was tested using an hADM immunoassay.

[0343] Immunoassay for the Quantification of Human Adrenomedullin The technology of a sandwich-coated tube luminescence immunoassay based on acridinium ester labeling was used.

[0344] Labeling 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) (European Patent No. 0353971) and incubated for 20 minutes at room temperature. The labeled CT-H was purified by gel filtration HPLC on Bio-Sil® SEC400-5 (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 the labeling compound (approximately 20 ng of the labeled antibody) per 200 μL. Acridinium ester chemiluminescence was measured by using an AutoLumat LB953 (Berthold Technologies GmbH & Co. KG).

[0345] 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 100 mmol / L NaCl, 50 mmol / L TRIS / HCl, pH 7.8) (for 18 hours at room temperature). After blocking with 5% bovine serum albumin, the tubes were washed with PBS (pH 7.4) and vacuum dried.

[0346] Calibration: The assay was calibrated with dilutions of hADM (BACHEM AG, Switzerland) in 250 mmol / L NaCl, 2 g / L Triton X-100, 50 g / L bovine serum albumin, 20 tablets / L protease inhibitor cocktail (Roche Diagnostics AG, Switzerland).

[0347] hADM Immunoassay: 50 μl of sample (or calibrator) was pipetted into a coated tube, labeled CT-H (200 μl) was added, and then the tube was incubated at 4 °C for 4 hours. Unbound tracer was removed by washing 5 times (1 ml each) with a washing solution (20 mM PBS, pH 7.4, 0.1% Triton X-100).

[0348] Tube-bound chemiluminescence was measured by using LB953: Figure 3 shows a typical hADM dose / signal curve. hADM dose-signal curve in the presence of 100 μg / mL of antibody NT-H. NT-H did not affect the described hADM immunoassay.

[0349] Stability of human adrenomedullin: Human ADM was diluted in human citrated plasma (final concentration 10 nM) and incubated at 24 °C. At selected time points, degradation of hADM was stopped by freezing at -20 °C. Incubations were performed in the absence and presence of NT-H (100 μg / ml). Residual hADM was quantified by using the hADM immunoassay described above.

[0350] Figure 4 shows the stability of hADM in human plasma (citrate) in the absence and presence of the NT-H antibody. The half-life of hADM alone was 7.8 hours, and in the presence of NT-H, the half-life was 18.3 hours. (2.3-fold higher stability).

[0351] Example 4 - Septic Mortality a) Early treatment of sepsis Animal model: Male C57Bl / 6 mice, 12 - 15 weeks old (Charles River Laboratories, Germany) were used in the study. Peritonitis was surgically induced under light isoflurane anesthesia. The upper left abdomen of the peritoneal cavity (normal position of the cecum) was incised. The cecum was exposed and ligated firmly distal to the insertion of the small intestine using a suture thread. One puncture wound was made in the cecum using a 24 - gauge needle, and a small amount of cecal contents was extruded through the wound. The cecum was returned to the abdominal cavity, and the laparotomy site was closed. Finally, the animals were returned to their cages and allowed free access to food and water. 500 μl of physiological saline was administered subcutaneously as a fluid supplement.

[0352] Application and dosage of compounds (NT - M, MR - M, CT - M): Mice were treated immediately after CLP (initial treatment). CLP is the abbreviation for cecal ligation and puncture.

[0353] Study groups: Three compounds were tested against vehicle treatment and a control compound treatment. Each group included 5 mice for blood sampling 1 day later for BUN (blood urea nitrogen test) measurement. An additional 10 mice per group were followed for 4 days.

[0354] Group treatment (10 μl / g body weight) dosage / follow - up investigation: 1 NT - M, 0.2 mg / ml Survival over 4 days 2 MR - M, 0.2 mg / ml Survival over 4 days 3 CT - M, 0.2 mg / ml Survival over 4 days 4 Nonspecific mouse IgG, 0.2 mg / ml Survival over 4 days 5 Control - PBS 10 μl / g body weight Survival over 4 days

[0355] Clinical chemistry: Blood urea nitrogen (BUN) concentrations regarding renal function were measured at baseline and on day 1 after CLP. Under light ether anesthesia, blood samples were obtained from the cavernous sinus of the sponge using a capillary. The measurements were performed using an AU 400 Olympus Multianalyser. The 4-day mortality and mean BUN concentrations are shown in Table 4.

[0356]

Table 14

[0357] b) Late treatment of sepsis Animal model: Male C57Bl / 6 mice, 12 - 15 weeks old (Charles River Laboratories, Germany), were used in the study. Peritonitis was surgically induced under light isoflurane anesthesia. The upper left abdomen of the peritoneal cavity (the normal position of the cecum) was incised. The cecum was exposed and ligated firmly distal to the insertion of the small intestine using a suture. One puncture wound was made in the cecum using a 24-gauge needle, and a small amount of cecal contents was extruded through the wound. The cecum was returned to the abdominal cavity, and the laparotomy site was closed. Finally, the animals were returned to their cages and allowed free access to food and water. 500 μl of physiological saline was administered subcutaneously as a fluid replacement.

[0358] Application and dosage of compound (NT-M FAB2): NT-M FAB2 was tested against vehicle treatment and control compound treatment. Treatment was carried out 6 hours after CLP (late treatment) after sepsis had fully developed. Each group included 4 mice and was followed up for 4 days.

[0359] Group treatment (10 μl / g body weight) dosage / follow-up investigation: 1 NT-M, FAB2 0.2 mg / ml Survival over 4 days 2 Control non-specific mouse IgG, 0.2 mg / ml Survival over 4 days 3 Vehicle: PBS 10 μl / g body weight Survival over 4 days

Table 15

[0360] Example 5 - Administration of NT-H in healthy humans This study was conducted in healthy male subjects as a randomized double-blind placebo-controlled study, and single escalating doses of the NT-H antibody were administered as intravenous (i.v.) infusions to 3 consecutive groups of healthy male subjects consisting of 8 healthy male subjects each (the first group was 0.5 mg / kg, the second group was 2 mg / kg, and the third group was 8 mg / kg). The main inclusion criteria were written informed consent, age 18 - 35 years, consent to use reliable contraception, and 18 - 30 kg / m 2had a BMI. Subjects were administered a single i.v. dose of NT-H antibody (0.5 mg / kg, 2 mg / kg, 8 mg / kg) or placebo by slow infusion over 1 hour in the research unit. The baseline ADM values in the four groups were not different. The median ADM was 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 the ADM values increased rapidly within the first 1.5 hours after administration of the NT-H antibody in healthy human individuals, then reached a plateau and decreased slowly (Figure 6).

[0361] Example 6 - Method for Measuring DPP3 Protein and DPP3 Activity Generation of Antibodies and Determination of DPP3 Binding Ability: Several mouse antibodies were prepared and screened by their ability to bind to human DPP3 in a specific binding assay (see Table 6).

[0362] Peptide / Conjugate for Immunization: The DPP3 peptide for immunization was synthesized using an additional N-terminal cysteine residue (when no cysteine was present within the selected DPP3 sequence) for binding of the peptide to bovine serum albumin (BSA) (see Table 6) (JPT Technologies, Berlin, Germany). The peptide was covalently bound to BSA by using Sulfolink coupling gel (Perbio-science, Bonn, Germany). The coupling procedure was carried out according to the Perbio manual. Recombinant GST-hDPP3 was prepared by USBio (United States Biological, Salem, MA, USA).

[0363] Immunization, immunocyte fusion and screening of mice: Balb / c mice were intraperitoneally (i.p.) injected on day 0 with 84 μg of GST-hDPP3 or 100 μg of DPP3-peptide-BSA conjugate (emulsified in TiterMax Gold Adjuvant), on day 14 with 84 μg of GST-hDPP3 or 100 μg of DPP3-peptide-BSA conjugate (emulsified in complete Freund's adjuvant), and on days 21 and 28 with 42 μg of GST-hDPP3 or 50 μg of DPP3-peptide-BSA conjugate (in incomplete Freund's adjuvant). On day 49, the animals were administered an intravenous (i.v.) injection of 42 μg of GST-hDPP3 or 50 μg of DPP3-peptide-BSA conjugate dissolved in physiological saline. Three days later, the mice were sacrificed and immunocyte fusion was performed.

[0364] Cells of splenocytes derived from immunized mice and the myeloma cell line SP2 / 0 were fused with 1 ml of 50% polyethylene glycol at 37 °C for 30 seconds. After washing, the cells were seeded into 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 1 week, the HAT medium was replaced with HT medium and subcultured three times, and then returned to normal cell culture medium. The cell culture supernatant was initially screened for recombinant DPP3-binding IgG antibodies 2 weeks after fusion. Thus, recombinant GST-tagged hDPP3 (USBiologicals, Salem, USA) was immobilized in 96-well plates (100 ng / well) and incubated with 50 μl of cell culture supernatant per well at room temperature for 2 hours. After washing the plates, 50 μl / well of POD-rabbit anti-mouse IgG was added and incubated at room temperature for 1 hour. After the next washing step, 50 μl of chromogen solution (3.7 mM o-phenylenediamine, 0.012% H2O2 in citrate / phosphate buffer) was added to each well, incubated at room temperature for 15 minutes, and the color reaction was stopped by adding 50 μl of 4N sulfuric acid. Absorbance at 490 nm was detected.

[0365] The microcultures with positive test results were transferred to 24-well plates for growth. After retesting, the selected cultures were cloned and recloned using the limiting dilution method, and the isotype was determined.

[0366] Production of mouse monoclonal antibodies Antibodies generated against GST-tagged human DPP3 or DPP3-peptide were produced by standard antibody production methods (Marx et al. 1997) and purified by protein A. The antibody purity was over 90% based on SDS gel electrophoresis analysis.

[0367] Characterization of antibodies - binding to hDPP3 and / or immunizing peptides Binding assays were performed to analyze the ability of different antibodies and antibody clones to bind DPP3 / immunizing peptides. Solid phase: Recombinant GST-tagged hDPP3 (SEQ ID NO: 34) or DPP3 peptide (immunizing peptide, SEQ ID NO: 35) was immobilized on the surface of high-binding microtiter plates (96-well polystyrene microplates, Greiner Bio-One international AG, Austria, 1 μg / well in coupling buffer [50 mM Tris, 100 mM NaCl, pH 7.8], 1 hour at room temperature). After blocking with 5% bovine serum albumin, the microplates were vacuum dried.

[0368] Labeling procedure (tracers): 100 μg (100 μl) of different anti-DPP3 antibodies (detection antibodies, 1 mg / ml in PBS, pH 7.4) were mixed with 10 μl of acridinium NHS-ester (1 mg / ml in acetonitrile, InVent GmbH, Germany, European Patent No. 0353971) and incubated at room temperature for 30 minutes. The labeled anti-DPP3 antibodies were purified by gel filtration HPLC using Shodex Protein 5 μm KW-803 (Showa Denko, Japan). The purified labeled antibodies were diluted in assay buffer (50 mmol / l potassium phosphate, 100 mmol / l NaCl, 10 mmol / l Na2-EDTA, 5 g / l bovine serum albumin, 1 g / l mouse IgG, 1 g / l bovine IgG, 50 μmol / l amastatin, 100 μmol / l leupeptin, pH 7.4). The final concentration was approximately 5 - 7×10 6 relative light units (RLU) of the labeled compound (approximately 20 ng of the labeled antibody). Acridinium ester chemiluminescence was measured by using a Centro LB960 luminometer (Berthold Technologies GmbH&Co.KG).

[0369] hDPP3 binding assay: Plates were filled with 200 μl of the labeled and diluted detection antibody (tracer) and incubated at 2 - 8°C for 2 - 4 hours. Unbound tracer was removed by washing 4 times with 350 μl of wash solution (20 mM PBS, pH 7.4, 0.1% Triton X-100). Chemiluminescence bound to the wells was measured using a Centro LB960 luminometer (Berthold Technologies GmbH&Co.KG).

[0370] Characterization of antibodies - Analysis of hDPP3 inhibition To analyze the ability of different antibodies and antibody clones to inhibit DPP3, a DPP3 activity assay was performed according to a known procedure (Jones et al., 1982). Recombinant GST-tagged hDPP3 was diluted in assay buffer (25 ng / ml GST-DPP3 in 50 mM Tris-HCl, pH 7.5 and 100 μM ZnCl2), and 200 μl of this solution was incubated with 10 μg of each antibody at room temperature. After a 1-hour pre-incubation, the fluorogenic substrate Arg-Arg-βNA (20 μl, 2 mM) was added to the solution, and the generation of free βNA over time was monitored at 37 °C using a Twinkle LB970 microplate fluorometer (Berthold Technologies GmbH & Co. KG). The fluorescence of βNA was detected by exciting at 340 nm and measuring the emission at 410 nm. The increasing fluorescence gradient (RFU / min) of different samples was calculated. The gradient of GST-hDPP3 using a buffer control was designated as 100% activity. The inhibitory ability of a possible capture binder was defined as the decrease (%) in GST-hDPP3 activity by incubation with the capture binder.

[0371] The following table represents the selected obtained antibodies and their binding rates in relative light units (RLU), as well as their relative inhibitory abilities (%, Table 6). Monoclonal antibodies generated against the DPP3 regions shown below were selected by their ability to bind to recombinant DPP3 and / or immunizing peptides, as well as by their inhibitory ability.

[0372] All antibodies generated against the full-length form of recombinant hDPP3 with a GST tag showed strong binding to immobilized GST-tagged hDPP3. Antibodies generated against the peptide of SEQ ID NO: 35 also bound to GST-hDPP3. The antibody of SEQ ID NO: 35 also strongly bound to the immunizing peptide.

[0373]

Table 16

[0374] The development of a luminescence immunoassay (DPP3-LIA) for the quantification of DPP3 protein concentration and an enzyme capture activity assay (DPP3-ECA) for the quantification of DPP3 activity has been recently described (Rehfeld et al. 2019. JALM 3(6):943-953), the entire content of which is incorporated herein by reference.

[0375] Example 7 - DPP3 in Septic and Cardiogenic Shock The DPP3 concentration in plasma from patients with sepsis / septic shock and cardiogenic shock was determined and correlated with the short-term mortality of the patients.

[0376] a) Study Cohort - Sepsis / Septic Shock DPP3 was measured in 574 plasma samples from patients in the Adrenomedullin and Outcome in Severe Sepsis and Septic Shock (AdrenOSS-1) study. AdrenOSS-1 is a prospective observational, multinational study that included 583 patients admitted to the intensive care unit with sepsis or septic shock (Hollinger et al., 2018 Aug 22;3(6):1424-1433). 292 patients were diagnosed with septic shock.

[0377] b) Study Cohort - Cardiogenic Shock Plasma samples from 108 patients diagnosed with cardiogenic shock were screened for DPP3. Blood samples were taken within 6 hours of the detection of cardiogenic shock. Mortality was followed up for 7 days.

[0378] hDPP3 immunoassay: To determine the DPP3 level in the plasma of patients, an immunoassay (LIA) or an enzyme activity assay (ECA) that detects the amount (LIA) or the activity (ECA) of human DPP3, respectively, was used. Antibody immobilization, labeling, and incubation were performed as described by Rehfeld et al. (Rehfeld et al. 2019. JALM 3(6):943-953).

[0379] Results: The short-term patient survival rate in septic patients was related to the DPP3 plasma concentration at admission. Patients with a DPP3 plasma concentration exceeding 40.5 ng / mL (third quartile) had an increased risk of death compared to patients with a DPP3 plasma concentration below this threshold (Figure 7A). Applying this cut-off to a subcohort of patients with septic shock revealed an even more pronounced risk of short-term mortality associated with high DPP3 plasma concentrations (Figure 7B). When the same cut-off was applied to patients with cardiogenic shock, an increased risk of short-term death within 7 days was also observed in patients with high DPP3 (Figure 7C).

[0380] Example 8 - NT-ADM antibody (AdrenOSS-2) in patients with septic shock AdrenOSS-2 is a double-blind, placebo-controlled, randomized, multi-center, proof-of-concept and dose-ranging Phase II clinical trial to investigate the safety, tolerability and efficacy of an N-terminal ADM antibody named adrenocizumab in patients with septic shock and elevated adrenomedullin (Geven et al. BMJ Open 2019;9:e024475). A total of 301 patients with septic shock and a bio-ADM concentration of more than 70 pg / mL were randomized (2:1:1) to treatment with either placebo (n = 152), adrenocizumab 2 ng / kg (n = 72) or adrenocizumab 4 ng / kg (n = 77) by a single intravenous infusion over approximately 1 hour. The all-cause mortality within 28 (90) days after inclusion was 25.8% (34.8%). The mean age was 68.4 years and 61% were male. In the per-protocol analysis, n = 294 patients remained eligible and the 14-day all-cause mortality was 18.5%.

[0381] In patients treated with adrenocizumab (combination of both doses, per-protocol population), a trend towards a reduction in short-term mortality (14 days after admission) compared to placebo was observed (hazard ratio (HR) 0.701 [0.408 - 1.21], p = 0.100) (Figure 8). Surprisingly, in patients with a DPP3 concentration of less than 50 ng / mL at admission, the treatment effect was more pronounced (n = 244, HR 0.426, p = 0.007) (Figure 9), while in patients with elevated DPP3 (more than 50 ng / mL, n = 44), the outcome was equivalent between adrenocizumab and placebo (HR 1.69, p = 0.209) (Figure 10).

[0382] The treatment effects (14-day mortality) for different DPP3 thresholds are summarized in Table 7.

[0383]

Table 17

[0384] Example 9: Treatment Effect in Patients with Bio-ADM Less than 70 pg / mL To show that anti-ADM treatment also functions in patients with bio-ADM levels less than 70 pg / mL, in the AdrenOSS-2 study cohort, it was investigated whether patients in the included patient population (all having bio-ADM > 70 pg / mL) showed different treatment effects according to pre-dose bio-ADM concentration. No such interaction was detected. In other words, this means that a beneficial treatment effect was detectable for all patients regardless of pre-dose bio-ADM concentration. This finding strongly suggests that treatment also functions in patients with bio-ADM levels less than 70 pg / mL, with the appropriate (and acceptable) limitation that such an effect may not be expected in patients with bio-ADM concentrations within the healthy normal range.

[0385] P-value for the interaction term between treatment and pre-dose bio-ADM: · Protocol-compliant population: p = 0.279 · Protocol-compliant population / pre-dose DPP3 < 70 ng / mL: p = 0.150 This is further illustrated in Kaplan-Meier plots dividing the patient population by pre-dose bio-ADM concentration (below / above the bio-ADM median) (see Figures 11a and 11b).

[0386] Example 10 - Low DPP3 Threshold in Sepsis and Septic Shock (AdrenOSS-1) Using the AdrenOSS-1 study described in Example 7, it was analyzed whether patients with DPP3 levels well below 50 ng / ml at baseline showed an increase in DPP3 plasma concentration above 50 ng / ml in the following days.

[0387] Results: The DPP3 plasma levels in septic shock patients (n = 292) at baseline (day 1, DPP3.d1) were statistically analyzed to determine the threshold above which patients showed an increase in DPP3 plasma concentration exceeding 50 ng / ml a few days later. A DPP3 concentration of 50 ng / ml reflects the threshold above which patients a) exceed the normal upper limit of DPP3, b) have high organ dysfunction and mortality (Blet et al. 2021. Crit Care 25(1):61), and c) show a reduced treatment effect against the N-terminal ADM antibody adrecizumab (International Publication No. WO 2021 / 170838).

[0388]

Table 18

[0389] The different DPP3 thresholds at baseline (d1) were analyzed for the percentage of patients whose DPP3 plasma concentration increased above 50 ng / ml in the following few days (day 2 and day 3). Table 8 shows that the lower the DPP3 plasma concentration at baseline (DPP3.d1), the lower the percentage of patients with an increase in DPP3 exceeding 50 ng / ml in the following few days. In this septic shock population, 223 and 156 patients had DPP3 concentrations of less than 50 ng / ml or less than 30 ng / ml at baseline, respectively. For the 156 patients with less than 30 ng / ml at baseline, 7 patients (4%) showed an increase in DPP3 concentration exceeding the 50 ng / ml threshold in the following few days. On the other hand, among the 67 septic shock patients with a DPP3 plasma concentration of 30 - 50 ng / ml at baseline, 15 patients (22.4%) had an increase in DPP3 plasma level above 50 ng / ml in the following few days. As a result, a low DPP3 threshold (well below 50 ng / ml) is suitable for indicating that DPP3 levels increase during follow-up. Since patients with DPP3 concentrations above 50 ng / ml were shown to have significantly reduced efficacy for treatment with anti-ADM antibody (adrecizumab), these results suggest that a lower threshold level for DPP3 can be used for treatment decisions regarding anti-ADM antibody (adrecizumab) therapy at baseline in patients with septic shock. The low DPP3 concentration threshold at baseline (d1) supports the fact that the DPP3 pathological pathway (associated with high short-term organ dysfunction and mortality) is not the dominant pathway in the selected septic shock population. Therefore, this septic shock population with a DPP3 concentration below the above-mentioned threshold range at baseline may have a higher treatment effect, for example, from anti-ADM antibody therapy (adrecizumab).

[0390] In the second step, a septic shock population with a bio-ADM plasma concentration exceeding 70 pg / ml was analyzed. A Bio-ADM concentration exceeding 70 pg / ml has been associated with the development of sepsis severity and organ dysfunction, including vasopressor / circulatory agent dependence (Marino et al. 2014. Critical Care 18:R34, Caironi et al. 2017. Chest 152(2):312 - 320, Mebazaa et al. 2018. Crit Care 22:354). Different DPP3 thresholds at baseline (d1) were analyzed for the percentage of patients whose DPP3 plasma concentration increased above 50 ng / ml in the following days (day 2 and day 3). Table 9 shows again that the lower the DPP3 plasma concentration at baseline (DPP3.d1), the lower the percentage of septic shock patients with high bio-ADM who had an increase in DPP3 above 50 ng / ml in the following days. In this septic shock and high bio-ADM population, 154 and 100 patients had DPP3 concentrations below 50 ng / ml or below 30 ng / ml at baseline, respectively. Among the 100 patients with a DPP3 concentration below 30 ng / ml at baseline, 4 (4%) patients showed an increase in DPP3 concentration exceeding the 50 ng / ml threshold within 2 days. On the other hand, among 54 septic shock patients with a DPP3 plasma concentration of 30 - 50 ng / ml at baseline, 13 (24.1%) patients increased their DPP3 plasma levels above 50 ng / ml in the following days.

[0391]

Table 19

[0392] Lower DPP3 Threshold and Efficacy of NT-ADM Antibody Therapy in Example 11 - AdrenOSS-2 To verify the findings from Example 10 and for proof-of-concept, different lower thresholds for DPP3 at baseline (day 1) were evaluated for the effectiveness of anti-ADM antibody (adrecizumab) therapy in a septic shock population with high bio-ADM from the AdrenOSS-2 study cohort, and patients with DPP3 plasma values exceeding the different lower thresholds were excluded from the analysis. The effectiveness of anti-bio-ADM antibody therapy was specifically evaluated against the death endpoint in the placebo group and the treatment group.

[0393] Results: To determine the DPP3 threshold at baseline for evaluating the effectiveness of anti-ADM antibody therapy, the DPP3 plasma levels in septic shock patients (n = 298) with high bio-ADM (above 70 pg / ml) at baseline and at 144 hours thereafter were statistically analyzed. Patients exceeding each DPP3 plasma threshold were excluded from the analysis. Different DPP3 plasma concentration thresholds were applied to the 28-day all-cause mortality assessment using a Kaplan–Meier plot comparing anti-ADM antibody therapy with placebo. The log-rank test was selected to show the difference in mortality between treatment groups. The hazard ratio (HR) was calculated for each DPP3 plasma concentration threshold to estimate the reduction in the risk of death provided by anti-ADM antibody therapy relative to placebo.

[0394] The DPP3 plasma concentration thresholds at baseline used were 50 ng / ml, 40 ng / ml, 30 ng / ml, and 22 ng / ml, respectively. For each threshold, the number of patients excluded from the all-cause mortality analysis was determined. For the 50, 40, 30, and 22 ng / ml thresholds, 16%, 24%, 35%, and 51% of the patients, respectively, were excluded from the analysis.

[0395] HRs were also determined for each DPP3 plasma concentration threshold at baseline to estimate the reduction in mortality due to anti-ADM antibody therapy relative to placebo. For thresholds of 50, 40, 30, and 22 ng / ml, the HRs were 0.606, 0.568, 0.309, and 0.258, respectively. This analysis indicates that the lower the DPP3 plasma concentration threshold, the greater the reduction in mortality in the treatment group. Similarly, a Kaplan–Meier all-cause mortality analysis indicates that the lower the DPP3 plasma concentration threshold, the more pronounced and significant the reduction in mortality in the treatment group (Figures 12A–12D).

[0396] For each DPP3 plasma concentration threshold at baseline, the percentage of patients showing an increase in DPP3 plasma concentration above 50 ng / ml over the subsequent 144 hours was also estimated. Similar to the results from the AdrenOSS-1 study in Example 10, it was shown that the lower the DPP3 plasma concentration threshold at baseline, the lower the percentage of patients showing an increase in DPP3 plasma levels above the 50 ng / ml threshold over the subsequent few days. In this septic shock and high bio-ADM cohort, 249 and 195 patients had DPP3 concentrations below 50 ng / ml or below 30 ng / ml at baseline, respectively. For the 195 patients with a DPP3 concentration below 30 ng / ml at baseline, 16 (8%) patients showed an increase in DPP3 concentration above the 50 ng / ml threshold at 6 days later. On the other hand, of the 54 septic shock patients with a DPP3 plasma concentration of 30–50 ng / ml at baseline, 11 (20.4%) patients increased their DPP3 plasma levels above 50 ng / ml over the subsequent few days. These results indicate that a lower DPP3 threshold (well below 50 ng / ml) is suitable for guiding the use of anti-ADM antibody therapy and selecting patients who tend to benefit from anti-ADM antibody therapy.

[0397] The different thresholds for DPP3 levels at baseline (day 1) were further used for subgroup analysis for 28-day all-cause mortality assessment using Kaplan-Meier plots in the treatment group comparing anti-ADM antibody therapy (adrecizumab) with placebo and the placebo group. The DPP3 plasma concentration thresholds at baseline used were 50 ng / ml and 30 ng / ml.

[0398] When evaluating all-cause mortality in a septic shock population (n = 195) with DPP3 levels below the 30 ng / ml threshold, the mortality rate in the anti-ADM antibody (adrecizumab) therapy group was strikingly significantly lower compared to the placebo group (Figure 12C). The same result was observed when only including patients with DPP3 values below 50 ng / ml, and the mortality rate in the treatment group was lower than that in the placebo group. Finally, when evaluating all-cause mortality from admission to 28 days in septic shock patients with DPP3 levels below 50 ng / ml at baseline and continuously low (below 50 ng / ml) DPP3 levels during the next 144 hours, the mortality rate in the treatment group was significantly lower than that in the placebo group (Figure 13). As a result, a low DPP3 threshold at baseline (well below 50 ng / ml) is most suitable for selecting patients who benefit from anti-ADM antibody therapy.

[0399] In summary, patients with DPP3 levels exceeding the 30 ng / ml threshold at baseline are more likely to show an increase in DPP3 plasma concentration in the following days. The subsequent increase in DPP3 plasma concentration above 50 ng / ml is associated with a decrease in the effectiveness of anti-ADM antibody therapy. Therefore, a lower threshold, well below 50 ng / ml, preferably 40 ng / ml or less, or in the range of 22 - 40 ng / ml, most preferably a threshold of 30 ng / ml, should be used to stratify patients for anti-ADM antibody therapy.

[0400] Sequence SEQ ID NO:1 GYTFSRYW SEQ ID NO:2 ILPGSGST Sequence number 3 TEGYEYDGFDY Sequence number 4 QSIVYSNGNTY Sequence "RVS" (not part of the sequence listing): RVS Sequence number 5 FQGSHIPYT Sequence number 6 (AM-VH-C) QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK Sequence number 7 (AM-VH1) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK Sequence number 8 (AM-VH2-E40) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK Sequence number 9 (AM-VH3-T26-E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK Sequence number 10 (AM-VH4-T26-E40-E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK Sequence number 11 (AM-VL-C) DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Sequence number 12 (AM-VL1) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Sequence number 13 (AM-VL2-E40) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Sequence number 14 (human ADM 1-21) YRQSMNNFQGLRSFGCRFGTC Sequence number 15 (human ADM 21-32) CTVQKLAHQIYQ Sequence number 16 (human ADM C-42-52) CAPRSKISPQGY-CONH2 Sequence number 17 (mouse ADM 1-19) YRQSMNQGSRSNGCRFGTC Sequence number 18 (mouse ADM 19-31) CTFQKLAHQIYQ Sequence number 19 (mouse ADM C-40-50) CAPRNKISPQGY-CONH2 Sequence number 20 (mature human adrenomedullin (mature ADM); amidated ADM; bio-ADM): amino acids 1-52 or amino acids 95-146 of pro-ADM YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVAPRSKISPQGY-CONH2 SEQ ID NO: 21 (Mouse ADM 1-50) YRQSMNQGSRSNGCRFGTCTFQKLAHQIYQLTDKDKDGMAPRNKISPQGY-CONH2 SEQ ID NO: 22 (Human ADM 1-21): YRQSMNNFQGLRSFGCRFGTC SEQ ID NO: 23 (Human ADM 1-42): YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVA SEQ ID NO: 24 (Human ADM aa 43-52) PRSKISPQGY-NH2 SEQ ID NO: 25 (Human ADM aa 1-14) YRQSMNNFQGLRSF SEQ ID NO: 26 (Human ADM aa 1-10) YRQSMNNFQG SEQ ID NO: 27 (Human ADM aa 1-6) YRQSMN SEQ ID NO: 28 (Human ADM aa 1-32) YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQ SEQ ID NO: 29 (Mouse ADM aa 1-40) YRQSMNQGSRSNGCRFGTCTFQKLAHQIYQLTDKDKDGMA SEQ ID NO: 30 (Mouse ADM aa 1-31) YRQSMNQGSRSNGCRFGTCTFQKLAHQIYQL SEQ ID NO: 31 (proADM: 164 amino acids (22-185 of preproADM)) ARLDVASEF RKKWNKWALS RGKRELRMSS SYPTGLADVK AGPAQTLIRP QDMKGASRSP EDSSPDAARI RVKRYRQSMN NFQGLRSFGC RFGTCTVQKL AHQIYQFTDK DKDNVAPRSK ISPQGYGRRR RRSLPEAGPG RTLVSSKPQA HGAPAPPSGS APHFL Sequence number 32 (Adalimumab heavy chain) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGEILPGSGSTNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence number 33 (Adalimumab light chain) DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPRLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Sequence number 34 - Human DPP3 (amino acids 1 to 737) MADTQYILPNDIGVSSLDCREAFRLLSPTERLYAYHLSRAAWYGGLAVLLQTSPEAPYIYALLSRLFRAQDPDQLRQHALAEGLTEEEYQAFLVYAAGVYSNMGNYKSFGDTKFVPNLPKEKLERVILGSEAAQQHPEEVRGLWQTCGELMFSLEPRLRHLGLGKEGITTYFSGNCTMEDAKLAQDFLDSQNLSAYNTRLFKEVDGEGKPYYEVRLASVLGSEPSLDSEVTSKLKSYEFRGSPFQVTRGDYAPILQKVVEQLEKAKAYAANSHQGQMLAQYIESFTQGSIEAHKRGSRFWIQDKGPIVESYIGFIESYRDPFGSRGEFEGFVAVVNKAMSAKFERLVASAEQLLKELPWPPTFEKDKFLTPDFTSLDVLTFAGSGIPAGINIPNYDDLRQTEGFKNVSLGNVLAVAYATQREKLTFLEEDDKDLYILWKGPSFDVQVGLHELLGHGSGKLFVQDEKGAFNFDQETVINPETGEQIQSWYRSGETWDSKFSTIASSYEECRAESVGLYLCLHPQVLEIFGFEGADAEDVIYVNWLNMVRAGLLALEFYTPEAFNWRQAHMQARFVILRVLLEAGEGLVTITPTTGSDGRPDARVRLDRSKIRSVGKPALERFLRRLQVLKSTGDVAGGRALYEGYATVTDAPPECFLTLRDTVLLRKESRKLIVQPNTRLEGSDVQLLEYEASAAGLIRSFSERFPEDGPELEEILTQLATADARFWKGPSEAPSGQA Array No. 35 - Immunizing peptide with additional N-terminal cysteine for human DPP3 (amino acids 474 - 493 (N-Cys)) CETVINPETGEQIQSWYRSGE Array No. 36 - IGHV1-69*11 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARYYYYYGMDVWGQGTTVTVSS Sequence number 37-HB3 QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYWGQGTTLTVSS To avoid misunderstanding, in the amino acid sequences shown in this specification, the C-terminal groups -NH2 and -CONH2 similarly refer to the C-terminal amide group (i.e., the C-terminal amidated polypeptide).

Claims

1. A therapeutic or prophylactic agent for shock in patients, comprising an anti-adrenomedullin (ADM) antibody, an anti-ADM antibody fragment, or an anti-ADM non-Ig scaffold, The aforementioned patient is characterized by having a level of dipeptidyl peptidase 3 (DPP3) below the threshold in a body fluid sample, The threshold for DPP3 is 40 ng / mL or less, or in the range of 22 ng / mL to 40 ng / mL. A therapeutic or prophylactic agent comprising the anti-ADM antibody, anti-ADM antibody fragment, or anti-ADM non-Ig scaffold that binds to the N-terminal portion (amino acids 1-21) of ADM: YRQSMNNNFQGLRSFGCRFGTC (SEQ ID NO: 14).

2. The therapeutic or prophylactic agent according to claim 1, wherein the shock is selected from the group including hypovolemic shock, cardiogenic shock, obstructive shock, and distributive shock, particularly cardiogenic shock or septic shock.

3. The therapeutic or prophylactic agent according to claim 1 or 2, which is any of the following: ● In cases of cardiogenic shock, the patient may have acute coronary syndrome, such as acute myocardial infarction, or the patient may have heart failure, such as acute decompensated heart failure, myocarditis, arrhythmia, cardiomyopathy, valvular heart disease, aortic dissection with acute aortic stenosis, traumatic chordal rupture, or extensive pulmonary embolism; ●In cases of hypovolemic shock, the patient may have a hemorrhagic disorder including gastrointestinal bleeding, trauma, vascular etiology such as ruptured abdominal aortic aneurysm or tumors eroding major blood vessels, and spontaneous bleeding under anticoagulant use, or a non-hemorrhagic disorder including vomiting, diarrhea, renal loss, skin loss or insensitivity loss such as burns or heatstroke, or loss to the third space associated with pancreatitis, cirrhosis, intestinal obstruction, or trauma; ●In the case of obstructive shock, the patient may have cardiac tamponade, tension pneumothorax, pulmonary embolism, or aortic stenosis; or ●In the case of distributive shock, the patient has septic shock, neurogenic shock, anaphylactic shock, or shock due to adrenal crisis.

4. The therapeutic or prophylactic agent according to claim 1 or 2, wherein the threshold value of DPP3 in a sample of the patient's body fluid is 30 ng / mL or less, or in the range of 22 to 30 ng / mL.

5. The therapeutic or prophylactic agent according to claim 4, wherein the threshold value of DPP3 in a sample of the patient's body fluid is 25 ng / mL or less or in the range of 22 to 25 ng / mL.

6. The therapeutic or prophylactic agent according to claim 1 or 2, wherein the level of DPP3 is determined by contacting the body fluid sample with a scavenging agent that specifically binds to DPP3.

7. The therapeutic or prophylactic agent according to claim 6, wherein the aforementioned capture and binding agent is an antibody.

8. The therapeutic or prophylactic agent according to claim 1 or 2, wherein the level of DPP3 protein and / or the level of active DPP3 is determined and compared with a predetermined threshold.

9. The therapeutic or prophylactic agent according to claim 1 or 2, wherein the level of DPP3 is determined by an immunoassay, particularly a sandwich immunoassay.

10. The level of DPP3 is DPP3 activity, and the method for determining the DPP3 activity in the target body fluid sample is: ● A step of contacting the sample with a scavenging agent that specifically binds to full-length DPP3; ● A step of separating the DPP3 bound to the aforementioned scavenging agent; ● A step of adding a substrate of DPP3 to the separated DPP3; and ● A process for quantifying DPP3 activity by measuring and quantifying the substrate conversion of DPP3. A therapeutic or preventive agent according to claim 1 or 2, comprising:

11. The aforementioned patient had ADM-NH exceeding the threshold. ２ The therapeutic or preventive agent according to claim 1 or 2, further characterized by having a level of [a certain level].

12. The ADM-NH in the sample of the patient's bodily fluids ２ The therapeutic or prophylactic agent according to claim 11, wherein the threshold is 40 to 100 pg / mL, more preferably 50 to 90 pg / mL, even more preferably 60 to 80 pg / mL, and most preferably 70 pg / mL.

13. The ADM-NH in the sample of the patient's bodily fluids ２ The therapeutic or prophylactic agent according to claim 12, wherein the threshold is 70 pg / mL.

14. The therapeutic or prophylactic agent according to claim 1 or 2, wherein the body fluid is selected from whole blood, plasma, and serum.

15. The therapeutic or prophylactic agent according to claim 14, wherein the body fluid is plasma.

16. Said ADM-NH ２ The level of the bodily fluid sample is ADM-NH ２ The therapeutic or prophylactic agent according to claim 11, determined by contacting it with a scavenging agent that specifically binds to it.

17. The therapeutic or prophylactic agent according to claim 16, wherein the aforementioned capture and binding agent is an antibody.

18. The anti-ADM antibody, anti-ADM antibody fragment, or anti-ADM non-Ig scaffold is ADM-Gly and / or ADM-NH ２ A therapeutic or prophylactic agent according to claim 1 or 2, which recognizes the N-terminus (amino acid 1) of and binds thereto.

19. The antibody, antibody fragment, or non-Ig scaffold is the sequence of amino acids 43-52 of ADM: PRSKISPQGY-NH ２ The therapeutic or prophylactic agent according to claim 1 or 2, which does not bind to the C-terminal portion of ADM having (Sequence ID 24).

20. The therapeutic or prophylactic agent according to claim 1 or 2, wherein the antibody, antibody fragment, or non-Ig scaffold blocks 80% or less, preferably 50% or less, the biological activity of ADM.

21. The antibody or antibody fragment is a monoclonal antibody or a fragment thereof that binds to ADM, Heavy chain, arrangement: CDR1: Sequence ID 1 GYTFSRYW CDR2: Sequence ID 2 ILPGSGST CDR3: Sequence ID 3 Contains TEGYEYDGFDY, Includes, Light chains, arrangement: CDR1: Sequence ID 4 QSIVYSNGNTY CDR2: RVS CDR3: Sequence ID 5 Includes FQGSHIPYT A therapeutic or prophylactic agent according to claim 1 or 2, comprising:

22. The antibody or antibody fragment is As a VH area: Sequence number 6 (AM-VH-C) QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQ RPGHGLEWIGEILPGSGSTNYNEKFKGKATITADTSSNTA YMQLSSLTSEDSAVYYCTEGYEYDGFDYWGQGTTLTVSSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKRVEPK Sequence ID 7 (AM-VH1) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQ APGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTA YMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKRVEPK Sequence ID 8 (AM-VH2-E40) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQ APGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTA YMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKRVEPK Sequence ID 9 (AM-VH3-T26-E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQ APGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTA YMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKRVEPK Sequence ID 10 (AM-VH4-T26-E40-E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQ APGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTA YMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKRVEPK Includes sequences selected from the group including, The following sequence is used for the VL region: Sequence ID 11 (AM-VL-C) DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLE WYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLK ISRVEAEDLGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPS VFIFPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC EVTHQGLSSSPVTKSFNRGEC Sequence ID 12 (AM-VL1) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLN WFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLK ISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPS VFIFPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC EVTHQGLSSSPVTKSFNRGEC Sequence ID 13 (AM-VL2-E40) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLE WFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLK ISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPS VFIFPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC Includes sequences selected from the group including EVTHQGLSSPVTKSFNRGEEC, A therapeutic or prophylactic agent according to claim 1 or 2.

23. The antibody or antibody fragment is The heavy chain consists of the following sequence: Sequence ID 32 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQ APGQGLEWIGEILPGSGSTNYNQKFQGRVTITADTSTSTA YMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK or containing sequences that are identical to it by more than 95%, The light chain has the following sequence: Sequence ID 33 DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLE WYLQRPGQSPRLLIYRVSNRFSGVPDRFSGSGSGTDFTLK ISRVEAEDVGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPS VFIFPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC EVTHQGLSSSPVTKSFNRGEC or containing sequences that are more than 95% identical to it, The therapeutic or preventive agent according to claim 1 or 2.

24. The anti-ADM antibody, anti-ADM antibody fragment, or anti-ADM non-Ig scaffold binds to the N-terminal portion (amino acids 1-10) of ADM: YRQSMNNNFQG (SEQ ID NO: 26). The therapeutic or preventive agent according to claim 1 or 2.

25. When the antibody, antibody fragment, or non-Ig scaffold is measured by label-free surface plasmon resonance using the Biacore2000 system, it is found to have at least 10% of the ADM. －７ A therapeutic or prophylactic agent according to claim 1 or 2, exhibiting binding affinity for M.

26. When the anti-ADM antibody, anti-ADM antibody fragment or anti-ADM non-Ig scaffold is measured by label-free surface plasmon resonance using a Biacore 2000 system, it has an affinity for human ADM of 1×10 －９ to 3×10 －９ M. The therapeutic or prophylactic agent according to claim 25, which exhibits such affinity.

27. The therapeutic or prophylactic agent according to claim 1 or 2, wherein the anti-ADM antibody or anti-ADM antibody fragment is an IgG1 antibody.

28. A pharmaceutical formulation for the treatment or prevention of shock in a patient, comprising an anti-adrenomedullin (ADM) antibody, an anti-ADM antibody fragment, or an anti-ADM non-Ig scaffold, The aforementioned patient is characterized by having a level of dipeptidyl peptidase 3 (DPP3) below the threshold in a body fluid sample, The threshold for DPP3 is 40 ng / mL or less, or in the range of 22 ng / mL to 40 ng / mL. A pharmaceutical formulation comprising the anti-ADM antibody, anti-ADM antibody fragment, or anti-ADM non-Ig scaffold, which binds to the N-terminal portion (amino acids 1-21) of ADM: YRQSMNNNFQGLRSFGCRFGTC (SEQ ID NO: 14).

29. The pharmaceutical formulation according to claim 28, wherein the pharmaceutical formulation is a solution, preferably a ready-to-use solution.

30. The pharmaceutical preparation according to claim 28, wherein the pharmaceutical preparation is in a freeze-dried state.

31. The pharmaceutical preparation according to claim 28, wherein the pharmaceutical preparation is administered intramuscularly.

32. The pharmaceutical preparation according to claim 28, wherein the pharmaceutical preparation is administered intravascularly.

33. The pharmaceutical preparation according to claim 28, wherein the pharmaceutical preparation is administered by injection.

34. The pharmaceutical preparation according to claim 28, wherein the pharmaceutical preparation is administered systemically.

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