ANTI-ADM ANTIBODY OR ANTI-ADM ANTIBODY FRAGMENTS OR ANTI-ADM NON-Ig STENT FOR THE TREATMENT OR PREVENTION OF SHOCKS

Abstract

The present application relates to 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 wherein the patient is characterized in that a dipeptidyl peptidase 3 (DPP3) level in a bodily fluid sample is below a threshold < = 40 ng / ml or in the range of 22 to 40 ng / ml, and the anti-ADM antibody or the anti-ADM fragment or the anti-ADM non-Ig scaffold is combined with an N-terminal part (amino acids at the positions 1 to 21) of ADM: YRQSMNNFQGLRSFGCRFGTC (SEQ ID No. 14).

Description

Technical Field

[0001] The subject matter of the present invention is an anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for treating or preventing shock in a patient, wherein the patient is characterized by a dipeptidyl peptidase 3 (DPP3) level in a body fluid sample that is below a threshold value, the threshold value being ≤ 40 ng / ml or within the range of 22 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 (amino acids 1-21) of ADM: YRQSMNNFQGLRSFGCRFGTC (SEQ ID No. 14). Background Art

[0002] Dipeptidyl peptidase 3, also known as dipeptidyl aminopeptidase III, dipeptidyl arylamidase III, dipeptidyl peptidase III, enkephalinase B or erythrocyte angiotensinase, abbreviated as DPP3, DPPIII, is a metallopeptidase that removes dipeptides from physiologically active peptides such as enkephalins and angiotensin. Ellis and Nuenke 1967 first identified DPP3 in purified bovine anterior pituitary extract and measured its activity. The enzyme, which is classified as EC 3.4.14.4, has a molecular mass of approximately 83 kDa and is highly conserved in prokaryotes and eukaryotes ( Prajapati and Chauhan 2011 The amino acid sequence of the human variant is described in SEQ ID NO 1. Dipeptidyl peptidase III is a ubiquitously expressed peptidase that is primarily in the cytosol. Despite the lack of a signal sequence, several studies have reported membrane activity ( Lee and Snyder 1982 ).

[0003] DPP3 is a zinc-dependent exopeptidase belonging to the peptidase family M49. It has a broad substrate specificity for oligopeptides of varying composition ranging from 3 / 4 amino acids to 10 amino acids and is also able to 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-enkephalins, endorphins 1 and 2. The metallopeptidase DPP3 has optimal activity at pH 8.0-9.0 and can be activated by the addition of divalent metal ions such as Co 2+ and Mg 2 + to activate.

[0004] Structural analysis of DPP3 revealed the catalytic motifs HELLGH (hDPP3 450-455) and EECRAE (hDPP3 507-512), as well as the following amino acids important for substrate binding and hydrolysis: Glu316, Tyr318, Asp366, Asn391, Asn394, His568, Arg572, Arg577, Lys666, and Arg669 ( Prajapati and Chauhan 2011; Kumar et al. 2016 ; numbering is for the sequence of human DPP3, see SEQ ID NO. 1). Taking into account 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 between amino acids 316 and 669.

[0005] The most prominent substrate of DPP3 is angiotensin II (Ang II), the major effector of the renin-angiotensin system (RAS). RAS plays an important role in cardiovascular disease ( 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 ( Corrêa et al., 2015. Crit Care 2015;19:98 ). Specifically, Ang II has been shown to regulate many cardiovascular functions, including the control of blood pressure and cardiac remodeling.

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

[0007] Circulating DPP3 levels have been shown to be elevated in patients with cardiogenic shock and are associated with an increased risk of short-term mortality and severe organ dysfunction ( Deaniau et al., 2019. Eur J Heart Fail. In press In addition, DPP3 measured at enrollment could distinguish patients with cardiogenic shock who developed refractory shock from those who did not, and DPP3 concentrations ≥59.1 ng / mL were associated with a higher risk of death ( Takagi et al., Eur J Heart Fail. In press ).

[0008] The peptide adrenomedullin (ADM) was first described in 1993 ( Kitamura et al., 1993. Biochem Biophys Res Comm 192 (2): 553-560) is a novel blood pressure lowering peptide comprising 52 amino acids, which has been isolated from a human pheochromocytoma cell line (SEQ ID No.: 20). In the same year, a cDNA encoding a precursor peptide comprising 185 amino acids and the complete amino acid sequence of the precursor peptide were also described. The precursor peptide, which contains, in particular, a 21-amino acid signal sequence at the N-terminus, is referred to as "adrenomedullin pre-peptide" (pre-proADM). In the present specification, all amino acid positions specified generally refer to the pre-proADM comprising 185 amino acids. Adrenomedullin (ADM) is a 52-amino acid peptide (SEQ ID NO: 20), and comprises amino acids 95 to 146 of pre-proADM, which is formed from pre-proADM by proteolytic cleavage. So far, only a few of the peptide fragments formed by cleavage of pre-proADM have been investigated more precisely, in particular the physiologically active peptides ADM and the "PAMPs", the latter being peptides comprising 20 amino acids (22-41) following the 21 amino acids of the signal peptide in pre-proADM. The discovery and characterization of ADM in 1993 triggered an intensive research campaign, the results of which have been summarized in various review articles, in the context of the present specification, specific reference may be made to the articles devoted specifically to ADM found in the publication "Peptides" ( Takahashi 2001. Peptides 22: 1691; Eto 2001. Peptides 22: 1693-1711 ). Another review is by Hinson et al., 2000 ( Hinson et al. 2000. Endocrine Reviews 21(2):138-167 In the scientific investigations to date, it has been found that ADM in particular can be regarded as a multifunctional regulatory peptide. It is released into the circulation 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 may also modulate the effects of ADM ( Pio et al., 2001. The Journal of Biological Chemistry 276 (15): 12292-12300 The most important physiological effect of ADM and PAMP investigated so far is the effect on blood pressure.

[0009] Thus, ADM is a potent vasodilator and the hypotensive effect can therefore be associated with a specific peptide segment in the C-terminal portion of ADM. Furthermore, it has been found that the above-mentioned physiologically active peptide PAMP formed from pre-proADM also exhibits a hypotensive effect, although it appears to have a different mechanism of action from ADM (in addition to the above-mentioned review article Eto et al., 2001 and Hinson et al., 2000 In addition, see Kuwasaki et al., 1997. FEBS Lett 414(1): 105-110; Kuwasaki et al., 1999. Ann. Clin. Biochem. 36: 622-628; Tsuruda et al., 2001 Life Sci. 69(2): 239-245 and EP-A2 0 622 458). It has also been found that the concentration of ADM, which can be measured in the circulation and other biological fluids, is significantly higher in many pathological conditions than that found in healthy control subjects. Thus, ADM levels are significantly increased in patients with congestive heart failure, myocardial infarction, kidney disease, hypertensive disorders, diabetes, in acute shock, and in sepsis and septic shock, although to varying degrees. PAMP concentrations are also increased in some of these pathological conditions, but the plasma levels are lower relative to ADM ( Eto 2001. Peptides 22: 1693-1711 Abnormally high concentrations of ADM have been reported in sepsis, with the highest concentrations being observed 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] Plasma concentrations of ADM are elevated in patients with heart failure and correlate with disease severity ( Hirayama et al., 1999. J Endocrinol 160: 297–303; Yu et al., 2001. Heart 86: 155–160 ). High plasma ADM was an independent negative prognostic indicator in these subjects ( Poyner et al., 2002. Pharmacol Rev 54: 233–246 ).

[0011] WO2004 / 097423 describes the use of antibodies to adrenomedullin for the diagnosis, prognosis and treatment of cardiovascular disorders. Treatment of diseases by blocking the ADM receptor has also been described in the art (e.g. WO2006 / 027147, PCT / EP2005 / 012844). The disease may be sepsis, septic shock, cardiovascular disease, infection, skin disease, endocrine disease, metabolic disease, gastrointestinal disease, cancer, inflammation, blood system disease, respiratory system disease, musculoskeletal disease, neurological disease, urinary system disease.

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

[0013] For other diseases, the blocking of ADM may be beneficial to a certain extent. However, if ADM is completely neutralized, it may also be harmful, because a certain amount of ADM may be required for several physiological functions. It is emphasized in many reports that the use of ADM may be beneficial in certain diseases. On the contrary, it is reported in other reports that ADM is life-threatening when used in certain diseases.

[0014] WO2013 / 072510 describes a non-neutralizing anti-ADM antibody for use in treating a serious chronic or acute disease or acute condition in a patient, for reducing the risk of death in said patient.

[0015] WO2013 / 072511 describes a non-neutralizing anti-ADM antibody for use in treating a chronic or acute disease or acute condition in a patient, for preventing or alleviating organ dysfunction or organ failure.

[0016] WO2013 / 072512 describes a non-neutralizing anti-ADM antibody that increases the half-life (t 1 / 2 The ADM stabilized antibody has a half retention time of 1:1. The ADM stabilized antibody blocks the biological activity of ADM by less than 80%.

[0017] WO2013 / 072513 describes a non-neutralizing anti-ADM antibody for use in treating an acute disease or condition in a patient for stabilizing circulation.

[0018] WO 2013 / 072514 describes a non-neutralizing anti-ADM antibody for use in regulating fluid balance in patients suffering from a chronic or acute disease or acute condition.

[0019] WO2017 / 182561 describes a method for determining the total amount or active DPP3 in a patient sample to diagnose a disease associated with a necrotic process. It also describes a method for treating a necrosis-related disease by an antibody against DPP3.

[0020] WO2021 / 170838 describes a method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients with shock and patients experiencing shock, the method comprising determining the DPP3 level and administering an anti-ADM antibody if the level is below a threshold of preferably 50 ng / ml.

[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 in the blood of shock patients is a factor contributing to the worsening of vascular tone in shock syndrome by inactivating the vasopressor effects 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 shock patients, the presence of endothelial dysfunction is detected by means of the biomarker bio-ADM, while cardiac dysfunction is detected by measuring DPP3. A DPP3 above the threshold has been used as an exclusion criterion for applying drugs directed at pathways different from DPP3 (such as anti-ADM antibodies, in particular the anti-ADM antibody Adrecizumab directed against the N-terminus of ADM). If patients with DPP3 above 50 ng / ml are excluded from treatment, the Adrecizumab therapy shows better efficacy. However, the surprising finding of the present invention is that if DPP3 is far below the threshold of 50 ng / ml, preferably below the threshold of 40 ng / ml or in the range of 22 ng / ml to 40 ng / ml, patients treated with the anti-ADM antibody Adrecizumab have a higher chance of benefiting from such treatment.

[0022] In other words, the surprising finding of the present invention is that in shock patients and patients suffering from shock, if the DPP3 level in a body fluid sample is below the threshold of 40 ng / ml or in the range of 22 ng / ml to 40 ng / ml, the DPP3 level in the body fluid sample will be used for therapy guidance and / or therapy monitoring and / or therapy stratification using an anti-ADM antibody and / or an anti-ADM antibody fragment and / or an anti-ADM non-Ig scaffold. Summary of the Invention

[0023] The subject matter of the present invention is an anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for treating or preventing shock in a patient, wherein the patient is characterized by a DPP3 level in a body fluid sample below a threshold, wherein the threshold of DPP3 < / = 40 ng / ml 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 (amino acids 1-21) of ADM: 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 treating or preventing shock in a patient, wherein the shock is selected from shock caused by hypovolemia, 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 treating or preventing shock in a patient, wherein:

[0026] ● In the case of cardiogenic shock, the patient may have had an acute coronary syndrome (e.g., acute myocardial infarction), or in which the patient has heart failure (e.g., acute decompensated heart failure), myocarditis, arrhythmias, cardiomyopathy, valvular heart disease, aortic dissection with acute aortic stenosis, traumatic chordae tendineae rupture, or massive pulmonary embolism, or

[0027] ● In the setting of hypovolemic shock, the patient may have had a bleeding disorder, including gastrointestinal bleeding, trauma, vascular causes (e.g., ruptured abdominal aortic aneurysm, tumor erosion into large vessels), and spontaneous bleeding in the setting of anticoagulant use, or a non-bleeding disorder, including vomiting, diarrhea, loss of renal function, skin losses / insensible losses (e.g., burns, heat stroke), or third space fluid losses in the setting of pancreatitis, cirrhosis, intestinal obstruction, trauma, or

[0028] ● In the case of obstructive shock, the patient may have cardiac tamponade, tension pneumothorax, pulmonary embolism, or aortic stenosis, or

[0029] • In the case of distributive shock, the patient may be suffering from septic shock, neurogenic shock, anaphylactic shock, or shock caused by adrenal crisis.

[0030] 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 treating or preventing shock in a patient, wherein the threshold value of DPP3 in the patient's body fluid sample is ≤ 30 ng / ml or is within the range of 22 to 30 ng / mL.

[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 treating or preventing shock in a patient, wherein the threshold value of DPP3 in the patient's body fluid sample is ≤ 25 ng / ml or is in the range of 22 to 25 ng / mL.

[0032] A specific embodiment of the present application relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for treating or preventing shock in a patient, wherein the DPP3 level is determined by contacting the body fluid sample with a capture binding agent that specifically binds to DPP3.

[0033] Another embodiment of the present application relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for treating or preventing shock in a patient, wherein any one of the DPP3 protein level and / or active DPP3 level is determined and compared with a threshold value.

[0034] One embodiment of the present application relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for treating or preventing shock in a patient, wherein another characteristic of the patient is that ADM-NH 2 Level is above threshold. Measure ADM-NH 2 Levels are intended to identify patients in shock.

[0035] 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 treating or preventing shock in a patient, wherein the ADM-NH 2 The threshold value is between 40 and 100 pg / mL, more preferably between 50 and 90 pg / mL, even more preferably between 60 and 80 pg / mL, most preferably the threshold value is 70 pg / mL.

[0036] Another embodiment of the present application relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for treating or preventing shock in a patient, wherein the ADM-NH 2 The level of ADM-NH 2 The assay is performed by contacting the capture binding agent with the capture binding agent.

[0037] 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 treating or preventing shock in a patient, wherein the patient's body fluid sample is selected from blood, serum, plasma, urine, cerebrospinal fluid (CSF) and saliva.

[0038] 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 treating or preventing shock in a patient, wherein the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold recognizes and binds to ADM-Gly and / or ADM-NH 2 The N-terminal end (amino acid position 1) of

[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 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, the C-terminal portion having the sequence of amino acids 43-52 of ADM: PRSKISPQGY-NH 2 (SEQ ID NO: 24).

[0040] One embodiment of the present application relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for treating or preventing shock in a patient, wherein the antibody or fragment or scaffold blocks no more than 80%, preferably no more than 50% of the biological activity of ADM.

[0041] Another embodiment of the present application relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for treating or preventing shock in a patient, wherein the antibody or fragment is a monoclonal antibody or fragment thereof that binds to ADM, wherein the heavy chain comprises the following sequence:

[0042] CDR1: SEQ ID NO: 1

[0043] GYTFSRYW

[0044] CDR2: SEQ ID NO: 2

[0045] ILPGSGST

[0046] CDR3: SEQ ID NO: 3

[0047] TEGYEYDGFDY

[0048] And wherein the light chain comprises the following sequence:

[0049] CDR1: SEQ ID NO: 4

[0050] QSIVYSNGNTY

[0051] CDR2:

[0052] RVS

[0053] CDR3: SEQ ID NO: 5

[0054] FQGSHIPYT.

[0055] Another embodiment of the present application relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for treating or preventing shock in a patient, wherein the antibody or fragment comprises a sequence selected from the following as the VH region:

[0056] SEQ ID NO: 6 (AM-VH-C)

[0057] QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK

[0058] SEQ ID NO:7(AM-VH1)

[0059] QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK

[0060] SEQ ID NO:8(AM-VH2-E40)

[0061] QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK

[0062] SEQ ID NO:9(AM-VH3-T26-E55)

[0063] QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK

[0064] SEQ ID NO: 10 (AM-VH4-T26-E40-E55)

[0065] QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK

[0066] and comprises a sequence selected from the following sequences as the VL region:

[0067] SEQ ID NO: 11 (AM-VL-C)

[0068] DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0069] SEQ ID NO: 12 (AM-VL1)

[0070] DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0071] SEQ ID NO: 13 (AM-VL2-E40)

[0072] DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0073] Another embodiment of the present application relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for treating or preventing shock in a patient, wherein the antibody or fragment comprises the following sequence or a sequence with identity > 95% thereof as a heavy chain:

[0074] SEQ ID NO: 32

[0075] QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGEILPGSGSTNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGT TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0076] and comprising as a light chain the following sequence or a sequence with > 95% identity thereto:

[0077] SEQ ID NO: 33

[0078] DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPRLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGGGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0079] The subject matter of the present application also relates to a pharmaceutical preparation for treating or preventing shock in a patient, which comprises an anti-ADM antibody or an anti-ADM antibody fragment or an anti-ADM non-Ig scaffold.

[0080] One embodiment of the present application relates to a pharmaceutical preparation for treating or preventing shock in a patient, wherein the pharmaceutical preparation is a solution, preferably a ready-to-use solution.

[0081] Another embodiment of the present application relates to a pharmaceutical preparation for treating or preventing shock in a patient, wherein the pharmaceutical preparation is in a lyophilized state.

[0082] One embodiment of the present application relates to a pharmaceutical preparation for treating or preventing shock in a patient, wherein the pharmaceutical preparation is administered intramuscularly.

[0083] A preferred embodiment of the present application relates to a pharmaceutical preparation for treating or preventing shock in a patient, wherein the pharmaceutical preparation is administered intravascularly.

[0084] Another embodiment of the present application relates to a pharmaceutical preparation for treating or preventing shock in a patient, wherein the pharmaceutical preparation is administered by infusion.

[0085] Another specific embodiment of the present application relates to a pharmaceutical preparation for treating or preventing shock in a patient, wherein the pharmaceutical preparation is administered systemically.

[0086] Another embodiment of the present application relates to a method for treating or preventing shock in a patient, the method comprising administering to the patient an anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment or an anti-ADM non-Ig scaffold, the method further comprising the following steps:

[0087] ● Measuring the level of DPP3 in a body fluid sample of the subject,

[0088] ● Comparing the measured DPP3 level with a predetermined threshold, and

[0089] wherein if the measured DPP3 level is lower than the predetermined threshold, the patient is treated, wherein the predetermined threshold of DPP3 is < / = 40 ng / ml or in the range of 22 ng / ml to 40 ng / ml, and

[0090] wherein the anti-ADM antibody or anti-ADM fragment or anti-ADM non-Ig scaffold binds to the N-terminal portion (aa 1-21) of ADM: YRQSMNNFQGLRSFGCRFGTC (SEQ ID No. 14).

[0091] Another specific embodiment of the present application relates to a method for treating or preventing shock in a patient, the method comprising administering to the patient an anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment or an anti-ADM non-Ig scaffold, wherein the method further comprises the following steps:

[0092] ● Measuring the ADM-NH 2 level,

[0093] ● Compare the ADM-NH 2 horizontally with a predetermined threshold,

[0094] wherein if the measured ADM-NH 2 level is higher than the predetermined threshold level, then treat the patient.

[0095] The subject matter of the present invention relates to an anti - adrenomedullin (ADM) antibody or anti - ADM antibody fragment or anti - ADM non - Ig scaffold for treating or preventing shock in a patient, wherein the patient is characterized in that the DPP3 level in a body fluid sample is lower than a threshold, wherein the threshold of DPP3 is < / = 40 ng / ml 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 (amino acids 1 - 21) of ADM: YRQSMNNFQGLRSFGCRFGTC (SEQ ID No. 14).

[0096] One embodiment of the present application relates to an anti - ADM antibody or anti - ADM antibody fragment or anti - ADM non - Ig scaffold for treating or preventing shock in a patient, wherein the shock is selected from shock caused by hypovolemia, cardiogenic shock, obstructive shock, and distributive shock, particularly cardiogenic shock or septic shock.

[0097] Another embodiment of the present application relates to an anti - ADM antibody or anti - ADM antibody fragment or anti - ADM non - Ig scaffold for treating or preventing shock in a patient, wherein:

[0098] ● In the case of cardiogenic shock, the patient may have had acute coronary syndrome (such as acute myocardial infarction) or wherein the patient has heart failure (such as acute decompensated heart failure), myocarditis, arrhythmia, cardiomyopathy, valvular heart disease, aortic dissection with acute aortic stenosis, traumatic chordae tendineae rupture, or massive pulmonary embolism, or

[0099] ● In the case of hypovolemic shock, the patient may have had a hemorrhagic disease, including gastrointestinal bleeding, trauma, vascular causes (such as rupture of abdominal aortic aneurysm, tumor erosion into large blood vessels), and spontaneous bleeding in the background of anticoagulant use, or non - hemorrhagic diseases, including vomiting, diarrhea, renal loss, skin defects / insensible water loss (such as burns, heat stroke), or third - space fluid loss in the background of pancreatitis, cirrhosis, intestinal obstruction, trauma, or

[0100] ● In the case of obstructive shock, the patient may have had cardiac tamponade, tension pneumothorax, pulmonary embolism, or aortic stenosis, or

[0101] • In the case of distributive shock, the patient may be suffering from septic shock, neurogenic shock, anaphylactic shock, or shock caused by adrenal crisis.

[0102] 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 treating or preventing shock in a patient, wherein the threshold value of DPP3 in the patient's body fluid sample is ≤ 30 ng / ml or is within the range of 22 to 30 ng / mL.

[0103] 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 treating or preventing shock in a patient, wherein the threshold value of DPP3 in the patient's body fluid sample is ≤ 25 ng / ml or is in the range of 22 to 25 ng / mL.

[0104] A specific embodiment of the present application relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for treating or preventing shock in a patient, wherein the DPP3 level is determined by contacting the body fluid sample with a capture binding agent that specifically binds to DPP3.

[0105] Another embodiment of the present application relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for treating or preventing shock in a patient, wherein any one of the DPP3 protein level and / or active DPP3 level is determined and compared with a predetermined threshold.

[0106] One embodiment of the present application relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for treating or preventing shock in a patient, wherein another characteristic of the patient is that ADM-NH 2 Level is above threshold.

[0107] 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 treating or preventing shock in a patient, wherein the ADM-NH 2 The threshold value is between 40 and 100 pg / mL, more preferably between 50 and 90 pg / mL, even more preferably between 60 and 80 pg / mL, most preferably the threshold value is 70 pg / mL.

[0108] Another embodiment of the present application relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for treating or preventing shock in a patient, wherein the ADM-NH 2 The level of ADM-NH2 The assay is performed by contacting the capture binding agent with the capture binding agent.

[0109] 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 treating or preventing shock in a patient, wherein the patient's body fluid sample is selected from blood, serum, plasma, urine, cerebrospinal fluid (CSF) and saliva.

[0110] In a preferred embodiment, the bio-ADM measures from plasma. However, in the technological life cycle of analyte measurements, there is often the possibility to measure such analytes in other at least blood-based matrices rather than just plasma. For example, in the case of bio-ADM, another technology using whole (EDTA-) blood has been developed, known as the IB10 sphingotest® bio-ADM ( https: / / www.nexus-dx.com / wp-content / uploads / 2020 / 07 / bio-ADM-IFU-REV-A.pdf The IB10 sphingotest® bio-ADM® is a rapid point-of-care (POC) immunoassay for the in vitro quantitative determination of human amidated adrenomedullin peptide (1-52), hereinafter referred to as bioactive adrenomedullin (bio-ADM), in human EDTA whole blood and plasma.

[0111] 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 treating or preventing shock in a patient, wherein the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold recognizes and binds to ADM-Gly and / or ADM-NH 2 The N-terminal end (amino acid position 1) of

[0112] Another embodiment of the present application relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for 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, the C-terminal portion having the sequence of amino acids 43-52 of ADM: PRSKISPQGY-NH 2 (SEQ ID NO: 24).

[0113] One embodiment of the present application relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for treating or preventing shock in a patient, wherein the antibody or fragment or scaffold blocks no more than 80%, preferably no more than 50% of the biological activity of ADM.

[0114] Another embodiment of the present application relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for treating or preventing shock in a patient, wherein the antibody or fragment is a monoclonal antibody or fragment thereof that binds to ADM, wherein the heavy chain comprises the following sequence:

[0115] CDR1: SEQ ID NO: 1

[0116] GYTFSRYW

[0117] CDR2: SEQ ID NO: 2

[0118] ILPGSGST

[0119] CDR3: SEQ ID NO: 3

[0120] TEGYEYDGFDY

[0121] And wherein the light chain comprises the following sequence:

[0122] CDR1: SEQ ID NO: 4

[0123] QSIVYSNGNTY

[0124] CDR2:

[0125] RVS

[0126] CDR3: SEQ ID NO: 5

[0127] FQGSHIPYT.

[0128] Another embodiment of the present application relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for treating or preventing shock in a patient, wherein the antibody or fragment comprises a sequence selected from the following as the VH region:

[0129] SEQ ID NO: 6 (AM-VH-C)

[0130] QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYWG QGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK

[0131] SEQ ID NO:7(AM-VH1)

[0132] QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK

[0133] SEQ ID NO:8(AM-VH2-E40)

[0134] QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK

[0135] SEQ ID NO:9(AM-VH3-T26-E55)

[0136] QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK

[0137] SEQ ID NO:10(AM-VH4-T26-E40-E55)

[0138] QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK

[0139] and comprises a sequence selected from the following sequences as the VL region:

[0140] SEQ ID NO: 11 (AM-VL-C)

[0141] DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0142] SEQ ID NO: 12 (AM-VL1)

[0143] DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0144] SEQ ID NO: 13 (AM-VL2-E40)

[0145] DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0146] Another embodiment of the present application relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for treating or preventing shock in a patient, wherein the antibody or fragment comprises the following sequence or a sequence with identity > 95% thereof as a heavy chain:

[0147] SEQ ID NO: 32

[0148] QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGEILPGSGSTNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGT TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0149] and comprising as a light chain the following sequence or a sequence with > 95% identity thereto:

[0150] SEQ ID NO: 33

[0151] DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPRLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGGGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0152] 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 treating or preventing shock in a patient, wherein the anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold is bound to the N-terminal portion (amino acids 1-10) of ADM: YRQSMNNFQG (SEQ ID No. 26).

[0153] 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 therapy, wherein the antibody or fragment or scaffold exhibits at least 10% affinity for ADM when measured by label-free surface plasmon resonance using a Biacore 2000 system. -7 The binding affinity of M.

[0154] 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 treating or preventing shock in a patient, wherein the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold exhibits 1 x 10 -9 Up to 3 x 10 -9 Affinity between M.

[0155] 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 treating or preventing shock in a patient, wherein the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold is an IgG1 antibody.

[0156] The subject matter of the present application also relates to a pharmaceutical preparation for treating or preventing shock in a patient, which comprises an anti-ADM antibody or an anti-ADM antibody fragment or an anti-ADM non-Ig scaffold.

[0157] One embodiment of the present application relates to a pharmaceutical preparation for treating or preventing shock in a patient, wherein the pharmaceutical preparation is a solution, preferably a ready-to-use solution.

[0158] Another embodiment of the present application relates to a pharmaceutical preparation for treating or preventing shock in a patient, wherein the pharmaceutical preparation is in a lyophilized state.

[0159] One embodiment of the present application relates to a pharmaceutical preparation for treating or preventing shock in a patient, wherein the pharmaceutical preparation is administered intramuscularly.

[0160] A preferred embodiment of the present application relates to a pharmaceutical preparation for treating or preventing shock in a patient, wherein the pharmaceutical preparation is administered intravascularly.

[0161] Another embodiment of the present application relates to a pharmaceutical preparation for treating or preventing shock in a patient, wherein the pharmaceutical preparation is administered by infusion.

[0162] Another specific embodiment of the present application relates to a pharmaceutical preparation for treating or preventing shock in a patient, wherein the pharmaceutical preparation is administered systemically.

[0163] Another embodiment of the present application relates to a method for treating or preventing shock in a patient, the method comprising administering to the patient an anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment or an anti-ADM non-Ig scaffold, the method further comprising the following steps:

[0164] ● Measuring the DPP3 level in a body fluid sample of the subject,

[0165] ● Comparing the measured DPP3 level with a predetermined threshold, and

[0166] wherein if the measured DPP3 level is lower than the predetermined threshold, the patient is treated, wherein the predetermined threshold of DPP3 < / = 40 ng / ml or in the range of 22 ng / ml to 40 ng / ml, and

[0167] wherein the anti-ADM antibody or anti-ADM fragment or anti-ADM non-Ig scaffold binds to the N-terminal portion (aa 1-21) of ADM: YRQSMNNFQGLRSFGCRFGTC (SEQ ID No. 14).

[0168] Another specific embodiment of the present application relates to a method for treating or preventing shock in a patient, the method comprising administering to the patient an anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment or an anti-ADM non-Ig scaffold, wherein the method further comprises the following steps:

[0169] ● Determination of ADM-NH in the body fluid sample of the subject 2 level,

[0170] ● The ADM-NH 2 The level is compared with a predetermined threshold,

[0171] If the ADM-NH 2 If the level is above the predetermined threshold level, the patient is treated.

[0172] In one embodiment of the invention, any one of the DPP3 protein level and / or the active DPP3 level is determined and compared to a threshold level.

[0173] In a specific embodiment of the present invention, the threshold value of DPP3 in the patient's body fluid sample is ≤ 30 ng / ml or is in the range of 22 to 30 ng / mL.

[0174] In another specific embodiment of the present invention, the threshold value of DPP3 in the patient's body fluid sample is ≤ 25 ng / ml or is in the range of 22 to 25 ng / mL.

[0175] The DPP3 level, which is the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of the subject, can be determined by different methods, such as immunoassay, activity assay, mass spectrometry, and the like.

[0176] According to the present invention, the level of DPP3 in the sample can be determined using any type of binding assay (immunoassays and similar assays using other types of antigen-specific binding agents instead of antibodies) and b) DPP3 enzyme activity assays (which are specific for DPP3 by specifically capturing DPP3 from the sample using a specific binding agent (anti-DPP3 antibodies or other types of binding agents) prior to determining the enzyme activity).

[0177] DPP3 activity can be measured by detecting the cleavage products of DPP3-specific substrates. Known peptide hormone substrates include Leu-enkephalin, Met-enkephalin, endorphins 1 and 2, valorphin, β-casomorphin, dynorphin, protoenterin, ACTH (adrenocorticotropic hormone), and MSH (melanocyte stimulating hormone; Abramić et al., 2000, Baršun et al., 2007, Dhanda et al. 2008 The above peptide hormones and other untagged oligopeptides (e.g. Ala-Ala-Ala-Ala, Dhanda et al., 2008 ) can be monitored by detecting the corresponding cleavage products. Detection methods include but are not limited to HPLC analysis (e.g. Lee and Snyder 1982 ), mass spectrometry (e.g. Abramić et al., 2000), H1-NMR analysis (e.g. Vandenberg et al., 1985 ), capillary zone electrophoresis (CE; e.g. Baršun et al., 2007 ), thin layer chromatography (e.g. Dhanda et al., 2008 ) or reverse phase chromatography (e.g. Mazocco et al., 2006 ).

[0178] Detection of fluorescence generated by hydrolysis of fluorogenic substrates by DPP3 is a standard procedure for monitoring DPP3 activity. Those substrates are specific dipeptides or tripeptides (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) coupled to fluorophores. 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; Abramić et al., 2000, Ohkubo et al., 1999 ). Cleavage of these fluorogenic substrates results in the release of fluorescent β-naphthylamine or 7-amino-4-methylcoumarin, respectively. In a liquid phase assay or ECA, the substrate and DPP3 are incubated in, for example, a 96-well plate format, and fluorescence is measured using a fluorescence detector ( Ellis & Nuenke 1967 In addition, samples with DPP3 can be immobilized and separated on a gel by electrophoresis, the gel can be stained with a fluorogenic substrate (e.g., Arg-Arg-βNA) and Fast Garnet GBC, and the fluorescent protein bands can be detected by a fluorescence reader ( Ohkubo et al., 1999 The same peptide (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 coupled to a chromophore such as p-nitroaniline diacetic acid. Detection of the color change caused by hydrolysis of the chromogenic substrate can be used to monitor DPP3 activity.

[0179] Another option for detecting DPP3 activity is Protease-Glo TMAssay (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 coupled to aminoluciferin. The aminoluciferin is released after being cleaved by DPP3 and serves as a substrate for a coupled luciferase reaction that emits detectable luminescence.

[0180] In a preferred embodiment, DPP3 activity is measured by adding the fluorogenic substrate Arg-Arg-βNA and monitoring fluorescence in real time.

[0181] In one embodiment of the method for determining active DPP3 in a body fluid sample of a subject, the capture binding agent reactive with DPP3 is immobilized on a solid phase.

[0182] The test sample is passed over an immobilized binding agent, and DPP3, if present, binds to the binding agent and itself becomes immobilized, ready for detection. A substrate may then be added, and the reaction product may be detected to indicate the presence or amount of DPP3 in the test sample. For the purposes of this specification, the term "solid phase" may be used to include any material or container in or on which an assay may 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 from ThermoFisher Scientific), TM or Pierce TM Magnetic beads), etc.

[0183] In another embodiment of the present invention, the DPP3 level is determined by contacting the body fluid sample with a capture binding agent that specifically binds to DPP3.

[0184] In another preferred embodiment of the present invention, the capture binding agent used to determine the level of DPP3 may be selected from antibodies, antibody fragments or non-IgG scaffolds.

[0185] In one embodiment of the present invention, the capture binding agent is an antibody.

[0186] The amount of DPP3 protein and / or DPP3 activity in the subject's body fluid sample can be determined, for example, by one of the following methods:

[0187] 1. Luminescent Immunoassay (LIA) for Quantification of DPP3 Protein Concentration ( Rehfeld et al., 2019 JALM 3 (6):943-953 )

[0188] The LIA is a one-step chemiluminescent sandwich immunoassay using white high-binding polystyrene microtiter plates as the solid phase. The plates were coated with monoclonal anti-DPP3 antibody AK2555 (capture antibody). The tracer anti-DPP3 antibody AK2553 was labeled with MA70-acridine-NHS-ester and used at a concentration of 20 ng per well. 20 microliters of sample (e.g., serum, heparin plasma, citrate plasma, or EDTA plasma derived from patient blood) and calibrants were drawn into the coated white microtiter plates. After adding the tracer antibody AK2553, the microtiter plates were incubated at room temperature and 600 rpm for 3 h. Unbound tracers were then removed by 4 washing steps (350 μL per well). The remaining chemiluminescence of each well was measured for 1 s using a microtiter plate luminometer. The concentration of DPP3 was determined using a 6-point calibration curve. Calibrators and samples are preferably run in duplicate.

[0189] 2. Enzyme capture activity assay (ECA) for quantification of DPP3 activity ( Rehfeld et al., 2019 JALM 3(6): 943-953 )

[0190] The ECA is a DPP3-specific activity assay using black high-binding polystyrene microtiter plates as the solid phase. The plates are coated with the monoclonal anti-DPP3 antibody AK2555 (capture antibody). 20 microliters of sample (e.g., serum, heparinized plasma, citrated plasma, cerebrospinal fluid, and urine) and calibrants are pipetted into the coated black microtiter plates. After adding the assay buffer (200 μL), the microtiter plates are incubated at 22°C and 600 rpm for 2 h. DPP3 present in the sample is immobilized by binding to the capture antibody. Unbound sample components are removed by 4 washing steps (350 μL per well). The specific activity of the immobilized DPP3 is determined by adding the fluorogenic substrate Arg-Arg-β-naphthylamide (Arg2-βNA) to the reaction buffer followed by incubation at 37°C for 1 h. DPP3 specifically cleaves Arg2-βNA into Arg-Arg dipeptide and fluorescent β-naphthylamine. Fluorescence was measured using a fluorimeter using an excitation wavelength of 340 nm and emission detected at 410 nm. The activity of DPP3 was determined using a 6-point calibration curve. Calibrators and samples were preferably run in duplicate.

[0191] 3. Liquid phase assay (LAA) for quantification of DPP3 activity (from Jones et al., Analytical Biochemistry, 1982 Revise)

[0192] The LAA is a liquid phase assay that uses a black non-binding polystyrene microtiter plate to measure DPP3 activity. 20 microliters of sample (e.g., serum, heparin plasma, citrate plasma) and calibrant are drawn into a non-binding black microtiter plate. After adding the fluorogenic substrate Arg2-βNA in the assay buffer (200 μL), the initial βNA fluorescence (T=0) is measured using an excitation wavelength of 340 nm in a fluorometer, and emission is detected at 410 nm. The plate is then incubated at 37°C for 1 hour. The final fluorescence of (T=60) is measured. The difference between the final and initial fluorescence is calculated. The activity of DPP3 is determined using a 6-point calibration curve. Calibrators and samples are preferably run in duplicate.

[0193] In a specific embodiment, the DPP3 level is determined using an assay, wherein the assay sensitivity of the assay is capable of quantifying DPP3 in healthy subjects and is < 20 ng / ml, preferably < 30 ng / ml, more preferably < 40 ng / ml.

[0194] 4. Another immunoassay for measuring DPP3 in plasma from whole blood samples is available, the IB10sphingotest® DPP3 ( https: / / www.nexus-dx.com / wp-content / uploads / 2020 / 11 / DPP3- 022-00072-IFU-REV-B_8x11.pdf )

[0195] The IB10 sphingotest® DPP3 is a rapid point-of-care (POC) immunoassay for the in vitro quantitative determination of dipeptidyl peptidase 3 (DPP3) in human EDTA whole blood and plasma. The Nexus IB10 immunochemistry system combines chemistry with microfluidics and centrifugal flow to rapidly prepare cell-free plasma from whole blood, which can then be moved through channels for rehydration, dissolution, and mixing with freeze-dried immunoconjugates.

[0196] In one embodiment, the binding agent exhibits at least 10 7 M -1 , preferably 10 8 M -1 More preferably, the affinity is higher than 10 9 M -1 , most preferably above 10 10 M -1 A person skilled in the art will appreciate that it may be considered to compensate for the lower affinity by applying a higher dose of the compound and that such measures would not lead to exceeding the scope of the present invention.

[0197] In another embodiment of the present invention, the body fluid sample is selected from the group consisting of whole blood, plasma and serum.

[0198] Throughout this application, mature ADM, bio-ADM and ADM-NH 2 are used synonymously and are the molecule according to SEQ ID No.: 20.

[0199] In a particular embodiment, the body fluid according to the present invention is a blood sample. The blood sample can be selected from whole blood, serum and plasma. In a specific embodiment of the method, the sample is selected from human citrate plasma, heparin plasma and EDTA plasma.

[0200] In one embodiment, an assay is used to measure ADM-NH 2 The assay has a sensitivity capable of quantifying mature ADM-NH in healthy subjects. 2 , and is < 70 pg / ml, preferably < 40 pg / ml, more preferably < 10 pg / ml.

[0201] In one embodiment of the present invention, ADM-NH 2 The threshold value is between 40 and 100 pg / mL, more preferably between 50 and 90 pg / mL, even more preferably between 60 and 80, most preferably a threshold value of 70 pg / ml is applied.

[0202] In one embodiment of the present invention, plasma ADM-NH 2 The threshold value 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.

[0203] In one embodiment, the binding agent is ADM-NH 2 Show at least 10 7 M -1 , preferably 10 8 M -1 The binding affinity is preferably greater than 10 9 M -1 , most preferably above 10 10 M -1 A person skilled in the art will appreciate that it is conceivable to compensate for the lower affinity by applying a higher dosage of the compound, and such measures do not result in exceeding the scope of the present invention.

[0204] To determine the affinity of the antibodies for adrenomedullin, the kinetics of adrenomedullin binding to the immobilized antibodies was determined using label-free surface plasmon resonance using a Biacore 2000 system (GE Healthcare Europe GmbH, Freiburg, Germany). Reversible immobilization of the antibodies was performed using anti-mouse Fc antibodies covalently coupled at high density to the surface of a CM5 sensor according to the manufacturer's instructions (Mouse Antibody Capture Kit; GE Healthcare). Lorenz et al., 2011. Antimicrob Agents Chemother. 55 (1): 165–173 ).

[0205] In one embodiment, the binding agent is selected from ADM-NH 2 Conjugated antibodies or antibody fragments or non-Ig scaffolds.

[0206] In one embodiment, an assay is used to measure ADM-NH 2 level, wherein such assay is a sandwich assay, preferably a fully automated assay.

[0207] In one embodiment, such assays are used to determine biomarkers (DPP3 and / or ADM-NH 2 ) level is a sandwich immunoassay using any type of detection technology including but not limited to enzyme markers, chemiluminescent markers, electrochemiluminescent markers, preferably a fully automated assay. In one embodiment of the diagnostic method, the 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®.

[0208] A variety of different immunoassays are known and can be used in the assays and methods of the present invention, including: radioimmunoassay ("RIA"), homogeneous enzyme amplified immunoassay ("EMIT"), enzyme-linked immunosorbent assay ("ELISA"), enzyme protein reactivation immunoassay ("ARIS"), test strip immunoassays, and immunochromatographic assays.

[0209] In one embodiment of the invention, the assay is a sandwich assay using any type of detection technology including but not limited to enzyme labels, chemiluminescent labels, electrochemiluminescent labels, preferably a fully automated assay. In one embodiment of the invention, the assay is an enzyme-labeled sandwich assay. Examples of automated or fully automated assays include assays that can be used in one of the following systems: Roche Elecsys®, Abbott Architect®, Siemens Centauer®, Brahms Kryptor®, Biomerieux Vidas®, Alere Triage®.

[0210] In one embodiment of the invention, it may be a so-called POC test (point-of-care), i.e. a testing technology that does not require a fully automated assay system, allowing the test to be performed near the patient in less than an hour. An example of such a technology is the immunochromatographic testing technology.

[0211] In a preferred embodiment, the label is selected from chemiluminescent labels, enzyme labels, fluorescent labels, and radioactive iodine labels.

[0212] The assay may be a homogeneous or heterogeneous assay, a competitive or non-competitive assay. In one embodiment, the assay takes the form of a sandwich assay, which is a non-competitive immunoassay, in which the molecule to be detected and / or quantified is bound to a first antibody and a second antibody. The first antibody may be bound to a solid phase such as a bead, a surface of a well or other container, a chip or a test strip, and the second antibody is an antibody labeled, for example, with a dye, a radioisotope or a reactive or catalytically active component. The amount of labeled antibody bound to the analyte is then measured by a suitable method. The general compositions and procedures associated with "sandwich assays" are well established and known to the skilled person ( "waived The Immunoassay Handbook, edited by David Wild, Elsevier LTD, Oxford; 3rd edition (May 2005), ISBN-13: 978-0080445267; Hultschig C et al., Curr Opin Chem Biol. 2006 Feb;10(1):4-10. PMID: 16376134 ).

[0213] In another embodiment, the assay comprises two capture molecules, preferably antibodies, both present as a dispersed system in a liquid reaction mixture, wherein a first labeling component is attached to the first capture molecule, wherein the first labeling component is part of a labeling system based on fluorescence- or chemiluminescence-quenching or amplification, and a second labeling component of the labeling system is attached to a second capture molecule, such that a measurable signal is generated upon binding of the two capture molecules to the analyte, allowing detection of the formed sandwich complex in a solution containing the sample.

[0214] In another embodiment, the labeling system comprises a combination of a rare earth cryptate or a rare earth chelate and a fluorescent dye or a chemiluminescent dye, in particular a cyanine dye.

[0215] In the context of the present invention, fluorescence-based assays involve the use of dyes which may be, for example, selected from FAM (5- or 6-carboxyfluorescein), VIC, NED, fluorescein, fluorescein isothiocyanate (FITC), IRD-700 / 800, cyanine dyes such as CY3, CY5, CY3.5, CY5.5, Cy7, xanthene, 6-carboxy-2',4',7',4,7-hexachlorofluorescein (HEX), TET, 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (JOE), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 5-carboxyrhodamine-6G (R6G5), 6-carboxyrhodamine-6G (RG6), rhodamine, rhodamine green, rhodamine red, rhodamine 110, BODIPY dyes such as BODIPY TMR, Oregon Green, coumarins such as umbelliferone, benzimides such as Hoechst 33258, phenanthridines such as Texas Red, Yakima Yellow, Alexa Fluor, PET, ethidium bromide, acridine dyes, carbazole dyes, phenoxazine dyes, porphyrin dyes, polymethine dyes, etc.

[0216] In the context of the present invention, chemiluminescence-based assays involve the use of dyes based on the physical principles described for chemiluminescent materials in the literature ( Kirk-Othmer, Encyclopedia of Chemical Technology Chemical Technology), 4th edition, Executive Editor JI Kroschwitz, Editor-in-Chief M. Howe-Grant, John Wiley & Sons, 1993, vol. 15, p. 518-562, incorporated herein by reference, including citations on pp. 551-562. arts ). Preferred chemiluminescent dyes are acridinium esters.

[0217] As referred to herein, an "assay" or "diagnostic assay" may be any type of assay used in the field of diagnostics. Such an assay may be based on the binding of an analyte to be detected to one or more capture probes with a certain 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 .

[0218] In one embodiment, at least one of the two binding agents is labeled for detection.

[0219] The ADM-NH of the present invention 2 The levels have been described using ADM-NH 2 Assay method Weber et al., 2017. JALM 2(2):1-4The DPP3 levels of the present invention have been determined using the described DPP3 assay, as outlined in the Examples ( Rehfeld et al., 2019. JALM 3(6):943-953 ). The values ​​of the threshold values ​​mentioned above may be different in other assays if the calibration of these assays is different from the assay system used in the present invention. Therefore, the above-mentioned cut-off values ​​should be applied accordingly to such differently calibrated assays taking into account the differences in calibration. One possibility to quantify the calibration differences is to perform a method comparison analysis (correlation) between the assay in question and the corresponding biomarker assay used in the present invention by measuring the corresponding biomarker (e.g. bio-ADM, DPP3) in the sample using both methods. Another possibility is to determine the median biomarker level of a representative normal population with the assay in question (if such a test has sufficient analytical sensitivity), compare the results with the median biomarker levels described in the literature, and recalculate the calibration based on the differences obtained by this comparison. Using the calibration used in the present invention, samples from normal (healthy) subjects were measured: median plasma bio-ADM (mature ADM-NH 2 ) was 24.7 pg / ml, the lowest 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 Study (MPP-RES)) were measured: median (interquartile range) plasma DPP3 was 14.5 ng / ml (11.3 ng / ml – 19 ng / ml). It has been demonstrated that DPP3 concentrations strongly correlate with DPP3 activity in blood ( Rehfeld et al., 2019. J Appl Lab Med 3: 943–953; Deniau et al., 2019. Eur J Heart Fail 22: 290–299 ). Thus, the level of active DPP3 can be determined using corresponding threshold values ​​and threshold ranges that correspond to the threshold values ​​and threshold ranges used to determine the level of DPP3 protein.

[0220] Therefore, in the present invention, ADM-NH 2 levels in order to identify patients at increased risk of developing shock.

[0221] In another specific embodiment of the present invention, the shock is selected from shock caused by hypovolemia, cardiogenic shock, obstructive shock and distributive shock, in particular cardiogenic or septic shock.

[0222] In a specific embodiment of the present invention, the shock is selected from:

[0223] ● In the case of cardiogenic shock, the patient has had an acute coronary syndrome (e.g., acute myocardial infarction) or has heart failure (e.g., acute decompensated heart failure), myocarditis, arrhythmias, cardiomyopathy, valvular heart disease, aortic dissection with acute aortic stenosis, traumatic chordae tendineae rupture, or massive pulmonary embolism, or

[0224] ● In the setting of hypovolemic shock, the patient may have had a bleeding disorder, including spontaneous bleeding in the setting of gastrointestinal bleeding, trauma, vascular etiologies (e.g., ruptured abdominal aortic aneurysm, tumor erosion into large vessels), and anticoagulant use, or a non-bleeding disorder, including vomiting, diarrhea, loss of renal function, skin loss / silent dehydration (e.g., burns, heat stroke), or third space fluid loss in the setting of pancreatitis, cirrhosis, intestinal obstruction, trauma, or

[0225] ● In the case of obstructive shock, the patient may have cardiac tamponade, tension pneumothorax, pulmonary embolism, or aortic stenosis, or

[0226] • In cases of distributive shock, the patient is suffering from septic shock, neurogenic shock, anaphylactic shock, or shock caused by adrenal crisis.

[0227] Shock is characterized by decreased oxygen delivery and / or increased oxygen consumption or inadequate oxygen utilization, resulting in cellular and tissue hypoxia. It is a life-threatening condition of circulatory failure, most commonly manifested by hypotension (systolic blood pressure less than 90 mmHg or MAP less than 65 mmHg). Shock is divided into four main types based on the underlying etiology: hypovolemic, cardiogenic, obstructive, and distributive shock ( Vincent and De Backer 2014. N. Engl. J. Med. 370(6): 583 ).

[0228] Hypovolemic shock is characterized by a decrease in intravascular volume and can be divided into two major subtypes: hemorrhagic and nonhemorrhagic. Common causes of hemorrhagic hypovolemic shock include gastrointestinal bleeding, trauma, vascular etiologies (eg, ruptured abdominal aortic aneurysm, tumor erosion into large vessels), and spontaneous bleeding in the setting of anticoagulant use. Common causes of nonhemorrhagic hypovolemic shock include vomiting, diarrhea, loss of renal function, skin defects / silent dehydration (eg, burns, heat shock), or third space fluid loss in the setting of pancreatitis, cirrhosis, intestinal obstruction, trauma, etc. For review, see Koya and Paul 2018. Shock. StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2019-2018 Oct 27 .

[0229] Cardiogenic shock (CS) is defined as a state of hypoperfusion of critical end organs due to decreased cardiac output. Notably, CS forms a spectrum of disease ranging from mild hypoperfusion to profound shock. Established criteria for the diagnosis of CS are: (i) systolic blood pressure ≤90 mmHg for >30 minutes or the need for vasopressors to achieve a blood pressure ≥90 mmHg; (ii) pulmonary congestion or elevated left ventricular filling pressures; (iii) signs of impaired organ perfusion with at least one of the following criteria: (a) altered mental status; (b) cool, clammy skin; (c) oliguria (< 0.5 mL / kg / h or < 30 mL / h); (d) elevated serum lactate ( Reynolds and Hochman 2008. Circulation 117: 686–697 Ventricular dysfunction secondary to acute myocardial infarction (AMI) is the most common cause of CS, accounting for approximately 80% of cases. Mechanical complications such as ventricular septal (4%) or free wall rupture (2%) and acute severe mitral regurgitation (7%) are less common 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., with different treatment options. This represents 40 000 to 50 000 patients per year in the United States and 60 000 to 70 000 patients in Europe. Despite therapeutic advances, mainly through early revascularization and subsequent reductions in mortality, CS remains the leading cause of death in AMI, with a mortality rate still approaching 40-50% according to recent registries and randomized trials (Goldberg et al., 2009. Circulation 119: 1211–1219).

[0230] Obstructive shock results from a physical blockage of a large blood vessel or the heart itself. Several conditions can cause this form of shock (eg, cardiac tamponade, tension pneumothorax, pulmonary embolism, aortic stenosis). Koya and Paul 2018. Shock. StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2019-2018 Oct 27 .

[0231] There are four types of distributive shock, based on etiology: neurogenic shock (decreased sympathetic nerve stimulation leading to decreased vascular tone), anaphylactic shock, septic shock, and shock due to adrenal crisis. In addition to sepsis, distributive shock can also be caused by conditions other than infection, such as systemic inflammatory response syndrome (SIRS) due to pancreatitis, burns, or trauma. Other causes include toxic shock syndrome (TSS), anaphylaxis (sudden, severe allergic reaction), adrenal insufficiency (acute exacerbation of chronic adrenal insufficiency, destruction or removal of the adrenal glands, suppression of adrenal function due to exogenous steroids, hypopituitarism, and metabolic disorders of hormone production), reactions to drugs or toxins, heavy metal poisoning, hepatic insufficiency, and central nervous system injury. For a review, see Koya and Paul 2018. Shock. StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2019-2018 Oct 27 .

[0232] Refractory shock is defined as the requirement for >0.5 μg / kg / min of norepinephrine infusion despite adequate volume resuscitation. Mortality in these patients can be as high as 94%, and evaluation and management of these patients require a much more aggressive approach in order to survive. The term "refractory shock" is used in situations where tissue perfusion cannot be restored with initially used corrective measures (e.g., vasopressors), and thus may be referred to as "high vasopressor-dependent" or "vasopressor-resistant" shock ( Udupa and Shetty 2018. Indian J Respir Care 7: 67-72 ). Patients with refractory shock may have features of hypoperfusion such as hypotension (mean arterial pressure <65 mmHg), tachycardia, peripheral coldness, prolonged capillary refill time, and tachypnea caused by hypoxia and acidosis. Fever may be seen in septic shock. Other signs of hypoperfusion may also be seen, such as altered sensorium, hyperlactatemia, and oliguria. These well-known signs of shock do not help identify whether the problem lies with the pump (heart) or the circuit (vascular and tissue). Different types of shock can coexist, and all forms of shock can become refractory, as evidenced by lack of response to high doses of vasopressors ( Udupa and Shetty 2018. Indian J Respir Care 7: 67-72 ).

[0233] Septic shock is a potentially fatal medical condition that occurs when sepsis (which is organ injury or damage in response to infection) leads to dangerous hypotension and abnormalities in cellular metabolism. The Third International Consensus Definition of Sepsis and Septic Shock (Sepsis-3) defines septic shock as a subtype of sepsis in which particularly severe circulatory, cellular, and metabolic abnormalities are associated with a greater risk of mortality than sepsis alone. Patients with septic shock can be clinically identified by the need for vasopressors to maintain a mean arterial pressure of 65 mm Hg or higher in the absence of hypovolemia and by serum lactate levels above 2 mmol / L (>18 mg / dL). This combination is associated with a hospital mortality rate of more than 40% ( Singer et al., 2016. JAMA. 315(8): 801–10. ). The primary infection is most often caused by bacteria, but can also be caused by fungi, viruses, or parasites. It can be located anywhere in the body, but is most often in the lungs, brain, urinary tract, skin, or abdominal organs. It can cause multiple organ dysfunction syndrome (formerly known as multiple organ failure) and death. Often, people with septic shock are cared for in an intensive care unit. It most often affects children, immunocompromised individuals, and the elderly because their immune systems cannot respond to infection as effectively as healthy adults. The mortality rate for septic shock is about 25-50%.

[0234] As used herein, the term "prevent" or any grammatical variation thereof includes, but is not limited to, delaying the onset of symptoms, preventing disease recurrence, increasing the latency period between symptomatic episodes, or a combination thereof. Preventing, as used herein, does not require the complete absence of symptoms.

[0235] The efficacy of a non-neutralizing antibody targeting 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 decrease in 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 ).

[0236] As a result of these positive results, a humanized version of the N-terminal anti-ADM antibody, called aderezumab, was developed for further clinical development. The beneficial effects of aderezumab on vascular barrier function and survival were recently demonstrated in preclinical models of systemic inflammation and sepsis ( Geven et al., 2018. Shock 50(6):648-654 ). In this study, pretreatment with adeleizumab reduced renal vascular leakage in endotoxemic rats and CLP-induced septic mice, which was consistent with increased renal expression of the protective peptide Ang-1 and decreased expression of the harmful peptide vascular endothelial growth factor. In addition, pretreatment with adeleizumab increased the 7-day survival rate in CLP-induced septic mice from 10% to 50% for a single dose and from 0 to 40% for repeated doses. In addition, in the Phase I study, excellent safety and tolerability were confirmed (see Example 6): no serious adverse events were observed, no signals of more frequent adverse events were detected in subjects treated with adeleizumab, 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 aderezumab. Both animal and human data show a potent, dose-dependent increase in circulating ADM following administration of this antibody. Based on pharmacokinetic data and the absence 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.

[0237] A mechanistic explanation for this increase could be that excess antibody in the circulation may expel ADM from the interstitium into the circulation, since ADM is small enough to cross the endothelial barrier, whereas antibodies cannot (Geven et al., 2018. Shock. 50(2):132-140 and Voors et al., (J. Eur J Heart Fail. 2019 Feb;21(2):163-171)). In addition, binding of the antibody to ADM results in an increased half-life of ADM. Although NT-ADM antibodies partially inhibit ADM-mediated signaling, the large increase in circulating ADM results in an overall “net” increase in ADM activity in the blood compartment, where it exerts beneficial effects on ECs (mainly barrier stabilization) while reducing the deleterious effects of ADM on VSMCs in the interstitium (vasodilation).

[0238] The present invention is not limited to the specific use of Adrezumab. There is no reason to doubt that what is true for Adrezumab also applies to antibodies that share the main essential characteristics (in particular affinity and epitope specificity). Antibodies targeting the same region must be expected to have the same technical effect, provided they have the same affinity and the same or very comparable structural features (size, shape, etc.).

[0239] Throughout this specification, an "antibody" or "antibody fragment" or "non-Ig scaffold" according to the present invention is capable of binding to ADM and is therefore directed against ADM, and may therefore be referred to as an "anti-ADM antibody", "anti-ADM antibody fragment" or "anti-ADM non-Ig scaffold".

[0240] The term "antibody" generally comprises monoclonal and polyclonal antibodies and binding fragments thereof, in particular Fc-fragments, as well as so-called "single-chain antibodies" (Bird et al., 1988), chimeric, humanized, in particular CDR-grafted antibodies and diabodies or tetrabodies (Holliger et al., 1993). Also included are immunoglobulin-like proteins that specifically bind to a molecule of interest contained in a sample, selected by techniques including, for example, phage display. In this context, the term "specific binding" refers to antibodies produced against a molecule of interest or a fragment thereof. If the antibody has an affinity for the molecule of interest or its above-mentioned fragments than for other molecules contained in a sample containing the molecule of interest, preferably 50 times, more preferably 100 times, and most preferably at least 1000 times, the antibody is considered specific. How to make antibodies and select antibodies with a given specificity is well known in the art.

[0241] In one embodiment of the present invention, the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold is monospecific.

[0242] 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 a specific region within the target ADM covering at least 5 amino acids. A monospecific anti-Adrenomedullin (ADM) antibody or monospecific anti-Adrenomedullin antibody fragment or monospecific anti-ADM non-Ig scaffold is an anti-Adrenomedullin (ADM) antibody or anti-Adrenomedullin antibody fragment or anti-ADM non-Ig scaffold that all have affinity for the same antigen. Monoclonal antibodies are monospecific, but monospecific antibodies can also be produced by other means than producing them from a common germ cell.

[0243] The anti-ADM antibody or antibody fragment binding to ADM or non-Ig scaffold binding to ADM may be a non-neutralizing anti-ADM antibody or antibody fragment binding to ADM or non-Ig scaffold binding to ADM.

[0244] 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. Neutralizing anti-ADM antibody, anti-ADM antibody fragment or anti-ADM non-Ig scaffold blocks the biological activity of ADM by nearly 100%, at least more than 90%, preferably at least more than 95%.

[0245] In contrast, the non-neutralizing anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold blocks the biological activity of ADM by less than 100%, preferably less than 95%, preferably less than 90%, more preferably less than 80%, and even more preferably less than 50%. This means that the biological activity of ADM is reduced by less than 100%, 95% or less but not more, 90% or less but not more, 80% or less but not more, 50% or less but not more. This means that the residual biological activity of ADM bound to the non-neutralizing anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold will be higher than 0%, preferably higher than 5%, preferably higher than 10%, more preferably higher than 20%, and more preferably higher than 50%.

[0246] In this context, a molecule that blocks the biological activity of ADM by less than 80% is an antibody or antibody fragment or non-Ig scaffold having "non-neutralizing anti-ADM activity", collectively referred to herein for simplicity as a "non-neutralizing" anti-ADM antibody, antibody fragment or non-Ig scaffold, which is defined as:

[0247] - one or more molecules that bind to ADM and which, after addition to a culture of a eukaryotic cell line expressing a functional recombinant human ADM receptor composed of CRLR (calcitonin receptor-like receptor) and RAMP3 (receptor activity modifying protein 3), reduce the amount of cAMP produced by said cell line through the action of a human synthetic ADM peptide added in parallel, wherein said added human synthetic ADM is added in an amount that causes a half-maximal stimulation of cAMP synthesis in the absence of said non-neutralizing antibody to be analyzed, wherein the reduction of cAMP caused by said molecule that binds to ADM occurs to an extent of no more than 80%, even when said non-neutralizing molecule that binds to ADM to be analyzed is added in an amount that is 10 times higher than the amount required to obtain the maximal reduction of cAMP synthesis obtainable using said non-neutralizing antibody to be analyzed.

[0248] The same definition applies to other ranges: 95%, 90%, 50%, etc.

[0249] The antibodies or fragments according to the present invention are proteins comprising one or more polypeptides substantially encoded by immunoglobulin genes and specifically binding to an antigen. The recognized immunoglobulin genes include kappa, lambda, alpha (IgA), gamma (IgG 1 IgG 2 IgG 3 IgG 4 ), delta (IgD), epsilon (IgE), and mu (IgM) constant region genes, as well as numerous immunoglobulin variable region genes. Full-length immunoglobulin light chains are typically about 25Kd or 214 amino acids in length.

[0250] A full-length immunoglobulin heavy chain is usually about 50 Kd or 446 amino acids in length. 2 The heavy chain is encoded by a variable region gene (about 110 amino acids in length) at the COOH end and by a kappa or lambda constant region gene at the COOH end. The heavy chain is also encoded by a variable region gene (about 116 amino acids in length) and one of the remaining constant region genes.

[0251] The basic structural unit of an antibody is usually a tetramer composed of two identical pairs of immunoglobulin chains, each pair having one light chain and one heavy chain. In each pair, the light and heavy chain variable regions bind to the antigen, and the constant regions mediate effector functions. Immunoglobulins also exist in a variety of other forms, including, for example, Fv, Fab, and (Fab') 2 , as well as bifunctional hybrid antibodies and single chains (e.g. Lanzavecchia et al., 1987. Eur. J. Immunol. 17:105; Huston et al., 1988. Proc. Natl. Acad. Sci. USA, 85:5879-5883; Bird et al., 1988. Science 242:423-426; Hood et al., 1984, Immunology, Benjamin, NY, 2nd ed.; Hunkapille and Hood 1986. Nature 323:15-16 The variable region of an immunoglobulin light or heavy chain consists of a framework region interrupted by three hypervariable regions, also called complementarity determining regions (CDRs) (see Sequences of Proteins of Immunological Interest, E. Kabat et al., 1983, US Department of Health and Human Services ). As noted above, the CDRs are primarily responsible for binding to the epitope of the antigen. An immune complex is an antibody, such as a monoclonal antibody, a chimeric antibody, a humanized antibody or a human antibody or a functional antibody fragment, that specifically binds to the antigen.

[0252] Chimeric antibodies are antibodies whose light chain and heavy chain genes are usually constructed from immunoglobulin variable and constant region genes belonging to different species by genetic engineering. For example, the variable segment of the gene from a mouse monoclonal antibody can be connected to a human constant segment such as κ and γ1 or γ3. Therefore, in one example, a therapeutic chimeric antibody is a hybrid protein consisting of a variable or antigen-binding domain from a mouse antibody and a constant or effector domain from a human antibody, although other mammalian species can also be used, or the variable region can be produced by molecular technology. Methods for making chimeric antibodies are well known in the art, for example, see U.S. Patent No. 5,807,715. "Humanized" immunoglobulins are immunoglobulins comprising human framework regions and one or more CDRs from non-human (e.g., mouse, rat or synthetic) immunoglobulins. The non-human immunoglobulins providing CDRs are referred to as "donors", and the human immunoglobulins providing frameworks are referred to as "acceptors". In one embodiment, in humanized immunoglobulins, all CDRs are from donor immunoglobulins. Constant regions do not necessarily exist, but if they do, they must be substantially identical to human immunoglobulin constant regions, i.e., the identity is at least about 85-90%, such as about 95% or higher. Thus, all parts of humanized immunoglobulins, except possibly CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences. "Humanized antibodies" are antibodies comprising humanized light chains and humanized heavy chain immunoglobulins. Humanized antibodies bind to the same antigen as the donor antibody providing the CDRs. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of amino acid substitutions obtained from the donor framework. Humanized or other monoclonal antibodies may have other conservative amino acid substitutions that do not substantially affect antigen binding or other immunoglobulin functions. Exemplary conservative substitutions are, for example: gly, ala; val, ile, leu; asp, glu; asn, gln; ser, thr; lys, arg; and phe, tyr. Humanized immunoglobulins can be constructed using genetic engineering (see, for example, U.S. Patent No. 5,585,089). Human antibodies are antibodies in which the light and heavy chain genes are of human origin. Human antibodies can be produced using methods known in the art. Human antibodies can be produced by immortalizing human B cells that secrete the antibody of interest. Immortality can be achieved, for example, by EBV infection or by fusing human B cells with myeloma or hybridoma cells to produce tri-hybridoma cells. Human antibodies can also be produced by phage display methods (see, e.g., WO91 / 17271; WO92 / 001047; WO92 / 20791), or selected from a human combinatorial monoclonal antibody library (see the Morphosys website). Human antibodies can also be produced using transgenic animals carrying human immunoglobulin genes (see, for example, WO93 / 12227; WO91 / 10741 ).

[0253] Therefore, the anti-ADM antibody may have a form 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 a recombinantly produced antibody such as IgG, a typical full-length immunoglobulin or an antibody fragment containing at least the F-variable domain of a heavy chain and / or a light chain, such as a chemically coupled antibody (antigen binding fragment), including but not limited to a Fab fragment, including a Fab miniantibody, a single-chain Fab antibody, a monovalent Fab antibody with an epitope tag such as Fab-V5Sx2; a divalent Fab (miniantibody) dimerized with a CH3 domain; a divalent Fab or a multivalent Fab, for example, formed by multimerization with the help of a heterologous domain, for example, by dimerization of a dHLX domain, such as Fab-dHLX-FSx2; a 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, e.g. from classes other than G; single domain antibodies, e.g. nanobodies derived from camel or fish immunoglobulins, etc.

[0254] In addition to anti-ADM antibodies, other biopolymer scaffolds are known in the art to complex with target molecules and have been used to generate highly target-specific biopolymers. Examples are aptamers, spiegelmers, anticalins, and conotoxins. For illustrations of antibody formats, see Figures 1a, 1b, and 1c.

[0255] In a preferred embodiment, the anti-ADM antibody form is selected from the group consisting of Fv fragment, scFv fragment, Fab fragment, scFab fragment, F(ab) 2 In another preferred embodiment, the antibody form is selected from scFab fragments, Fab fragments, scFv fragments and bioavailability-optimized conjugates thereof, such as PEGylated fragments. A most preferred form is the scFab form.

[0256] The non-Ig scaffolds may be protein scaffolds and may be used as antibody mimics because they are able to bind to ligands or antigens. The non-Ig scaffolds may be selected from tetranectin-based non-Ig scaffolds (e.g., US 2010 / 0028995 ), fibronectin scaffolds (e.g., EP 1 266 025described in ), apolipoprotein-based scaffolds (e.g., WO 2011 / 154420 ), ubiquitin scaffolds (e.g., WO 2011 / 073214 ), transferrin scaffolds (e.g., US 2004 / 0023334 ), Protein A scaffolds (e.g., EP 2 231 860 ), ankyrin repeat-based scaffolds (e.g., WO 2010 / 060748 ), mini-protein (preferably cysteine ​​knot-forming mini-protein) scaffolds (e.g. EP 2314308 ), Fyn SH3 domain-based scaffolds (e.g. WO 2011 / 023685 ), EGFR-A domain-based scaffolds (e.g., WO 2005 / 040229 ) and Kunitz domain-based scaffolds (e.g., in EP 1 941 867 described in ).

[0257] In one embodiment of the invention, anti-ADM antibodies according to the invention can be produced by synthesizing ADM fragments as antigens as outlined in Example 1. Subsequently, binding agents of the fragments are identified using the methods described below or other methods known in the art.

[0258] Humanization of murine antibodies can be performed according to the following procedure:

[0259] To humanize a murine antibody, the antibody sequence is analyzed to understand the structural interactions of the framework region (FR) with the complementarity determining region (CDR) and the antigen. Suitable human-derived FRs are selected on the basis of structural modeling, and the murine CDR sequences are transplanted into the human FRs. Mutations can be introduced in the amino acid sequence of the CDR or FR to regain structural interactions that were abolished by species switching of the FR sequence. This restoration of structural interactions can be achieved by random approaches using phage display libraries or by directed approaches guided by molecular modeling ( Almagro and Fransson 2008. Humanization of antibodies, Front Biosci. 2008 Jan 1;13:1619-33 ).

[0260] In a preferred embodiment, the ADM antibody form is selected from the group consisting of Fv fragment, scFv fragment, Fab fragment, scFab fragment, F(ab) 2 In another preferred embodiment, the antibody form is selected from scFab fragments, Fab fragments, scFv fragments and bioavailability-optimized conjugates thereof, such as PEGylated fragments. A most preferred form is the scFab form.

[0261] 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.

[0262] 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 of at least 5 amino acids in length contained in ADM.

[0263] 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 of at least 4 amino acids in length contained in ADM.

[0264] In one embodiment of the present invention, the anti-ADM antibody or anti-ADM antibody fragment that binds to adrenomedullin or anti-ADM non-Ig scaffold that binds to adrenomedullin is provided for treating or preventing shock in a patient, wherein the antibody or fragment or scaffold is not ADM binding protein-1 (complement factor H).

[0265] In a specific embodiment of the present invention, the anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment binding to adrenomedullin or anti-ADM non-Ig scaffold binding to adrenomedullin is provided for treating or preventing shock in a patient, wherein the antibody or antibody fragment or non-Ig scaffold binds to a region of preferably at least 4 or at least 5 amino acids within the amino acid sequence of positions 1-21 of mature human ADM: YRQSMNNFQGLRSFGCRFGTC SEQ ID No.: 22.

[0266] 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.

[0267] 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 means binding to the N-terminal part of adrenomedullin (amino acids 1-14).

[0268] 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 (SEQID No.: 26); this means binding to the N-terminal portion of adrenomedullin (amino acids 1-10).

[0269] 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); this means binding to the N-terminal portion of adrenomedullin (amino acids 1-6). As described above, the region or epitope preferably comprises a length of at least 4 or at least 5 amino acids.

[0270] 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-terminal end (amino acid position 1) of adrenomedullin. The N-terminal end means the amino acid position 1, which is "Y" of SEQ ID No. 20, 22 or 23, which is mandatory for binding. The antibody or fragment or scaffold binds neither to N-terminally extended or N-terminally modified adrenomedullin nor to N-terminally degraded adrenomedullin. In another preferred embodiment, this means that the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold only binds to a region within the mature ADM sequence if the N-terminal end of ADM is free. In the embodiment, the anti-ADM antibody or anti-ADM antibody fragment or non-Ig scaffold does not bind to a region within the mature ADM sequence if the sequence is, for example, contained in pro-ADM.

[0271] For the sake of clarity, the numbers in brackets for specific regions of ADM such as "N-terminal portion (amino acids 1-21)" are understood by those skilled in the art to mean that the N-terminal portion of ADM consists of amino acids 1-21 of the mature ADM sequence.

[0272] In another 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 43-52 of ADM: PRSKISPQGY-NH 2 (SEQ ID No.: 24).

[0273] Epitopes, also known as antigenic determinants, are the parts of an antigen that are recognized by the immune system, especially antibodies. For example, an epitope is a specific segment of an antigen that binds to an antibody. The part of an antibody that binds to an epitope is called a paratope. Epitopes of protein antigens are divided into two categories: conformational epitopes and linear epitopes, depending on their structure and interaction with the paratope.

[0274] Conformational and linear epitopes are based on the 3-D conformation adopted by the epitope and the interaction of the complement, 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. Linear epitopes or continuous epitopes are epitopes recognized by antibodies by their linear sequence or primary structure of amino acids and are formed by the 3-D conformation adopted by the interaction of continuous amino acid residues.

[0275] In one embodiment, it is preferred 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 causes an increase in ADM levels or ADM immunoreactivity in serum, blood, or plasma by at least 10%, preferably at least 50%, more preferably >50%, and most preferably >100%.

[0276] In one embodiment, it is preferred 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 stabilized antibody or an ADM stabilized antibody fragment or an ADM stabilized non-Ig scaffold, which reduces the half-life (t 1 / 2 ; half retention time) is increased by at least 10%, preferably at least 50%, more preferably >50%, most preferably >100%.

[0277] The half-life (half-retention time) of ADM can be determined in human serum, blood or plasma in the absence and presence of ADM-stabilized antibodies or ADM-stabilized antibody fragments or ADM-stabilized non-Ig scaffolds, respectively, using an immunoassay for ADM quantification.

[0278] The following steps can be performed:

[0279] - ADM can be diluted in human citrated plasma in the absence and presence of ADM stabilizing antibodies or adrenomedullin stabilizing antibody fragments or adrenomedullin stabilizing non-Ig scaffolds, respectively, and can be incubated at 24°C.

[0280] - Aliquots are taken at selected time points (e.g. within 24 hours) and degradation of ADM in the aliquots can be stopped by freezing at -20°C.

[0281] - If the chosen assay is not affected by the stabilizing antibody, the amount of ADM can be determined directly by a hADM immunoassay. Alternatively, the aliquot can be treated with a denaturing agent such as HCl and after clarifying the sample (e.g. by centrifugation), the pH can be neutralized and ADM quantified by an ADM immunoassay. Alternatively, non-immunoassay techniques such as RP-HPLC can be used for ADM quantification.

[0282] - The half-life of ADM was calculated for ADM incubated in the absence and presence of ADM stabilizing antibodies or adrenomedullin stabilizing antibody fragments or adrenomedullin stabilizing non-Ig scaffolds, respectively.

[0283] - Calculate the increase in half-life of stabilized ADM compared to ADM incubated in the absence of ADM stabilizing antibody or adrenomedullin stabilizing antibody fragment or adrenomedullin stabilizing non-Ig scaffold.

[0284] A two-fold increase in the half-life of ADM is a 100% increase in half-life.

[0285] Half-life (half retention time) is defined as the length of time it takes for the concentration of a given chemical or drug in a given fluid or blood to decrease to half of its baseline concentration.

[0286] An assay that can be used to determine the half-life (half-retention time) of adrenomedullin in serum, blood, or plasma is described in Example 3.

[0287] 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. The 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 by less than 100%, preferably less than 95%, preferably less than 90%. In embodiments where the non-neutralizing anti-ADM antibody, anti-ADM antibody fragment or anti-ADM non-Ig scaffold blocks the biological activity of ADM by less than 95%, the anti-ADM antibody, anti-ADM antibody fragment or anti-ADM non-Ig scaffold that blocks the biological activity of ADM by more than 95% is outside the scope of the embodiment. In one embodiment, this means that the biological activity is reduced by 95% or less but not more, preferably 90% or less, more preferably 80% or less, more preferably 50% or less but not more.

[0288] 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 amino acid sequence of positions 1-21 of mature human ADM (SEQ ID No.: 14), or an antibody that binds to a region of at least 5 amino acids within the amino acid sequence of positions 1-19 of mature murine ADM (SEQ ID No.: 17).

[0289] 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 amino acid sequence of positions 1-21 of mature human ADM (SEQ ID No.: 14), or an antibody that binds to a region of at least 5 amino acids within the amino acid sequence of positions 1-19 of mature murine ADM (SEQ ID No.: 17).

[0290] 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 by less than 80%, preferably less than 50% (baseline value). It must be understood that the limited blocking of the biological activity of ADM (meaning the reduction of biological activity) occurs even at an excess concentration of the antibody, fragment or scaffold, meaning that the antibody, fragment or scaffold is in excess relative to ADM. The limited blocking is an inherent property of the ADM binder itself in the 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 the biological activity of ADM / reduces the biological activity of ADM by at least 5%. The above statements mean that there is still about 20% or 50% or even 95% residual ADM biological activity, respectively.

[0291] 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 corresponding ADM biological activity.

[0292] Described biological activity is defined as the effect that material presents to living organism or tissue or organ or functional unit after it interacts with living organism or tissue or organ or functional unit in vivo or in vitro (for example in assay method).Under the situation of ADM biological activity, this may be the effect of ADM in human recombinant ADM receptor cAMP functional assay method.Therefore, according to the present invention, biological activity is defined by ADM receptor cAMP functional assay method.In order to measure the biological activity of ADM in this assay method, following steps can be carried out:

[0293] - Dose response curves were performed using ADM in the human recombinant ADM receptor cAMP functional assay.

[0294] - The ADM concentration that gives half-maximal cAMP stimulation can be calculated.

[0295] - Dose response curves were performed with ADM-stabilized antibodies or ADM-stabilized antibody fragments or ADM-stabilized non-Ig scaffolds at a constant ADM concentration that stimulates half-maximal cAMP (up to a final concentration of 100 µg / ml).

[0296] The maximum inhibition of 50% in the ADM bioassay means that the anti-ADM antibody or the anti-ADM antibody fragment or the anti-ADM non-Ig scaffold blocks the biological activity of ADM to 50% of the baseline value, respectively. The maximum inhibition of 80% in the ADM bioassay means that the anti-ADM antibody or the anti-adrenomedullin antibody fragment or the anti-adrenomedullin non-Ig scaffold blocks the biological activity of ADM by 80%, respectively. This is within the meaning of blocking the biological activity of ADM by no more than 80%. This means that there is still about 20% residual ADM biological activity.

[0297] However, throughout this specification and hereinabove, for the anti-ADM antibodies, anti-ADM antibody fragments and anti-ADM non-Ig scaffolds disclosed herein, the expression "blocking the biological activity of ADM" should be understood as at most only reducing the biological activity of ADM from 100% to 20% of the residual ADM biological activity, preferably reducing the ADM biological activity from 100% to 50% of the residual ADM biological activity; but in any case there is residual ADM biological activity that can be determined as detailed above.

[0298] The biological activity of the ADM can be determined according to Example 2 in a human recombinant adrenomedullin receptor cAMP functional assay (adrenomedullin bioassay).

[0299] In a preferred embodiment, the regulatory anti-ADM antibody or regulatory anti-ADM antibody fragment or regulatory anti-ADM non-Ig scaffold is used to treat or prevent shock in a patient.

[0300] A "regulatory" anti-ADM antibody or a regulatory anti-ADM antibody fragment or a regulatory anti-ADM non-Ig scaffold is a protein that converts the half-life (t 1 / 2 The half-retention time (half retention time) of ADM is increased by at least 10%, preferably by at least 50%, more preferably by >50%, and most preferably by >100%, and the biological activity of ADM is blocked by less than 80%, preferably by less than 50%, and the anti-ADM antibody, anti-ADM antibody fragment or non-Ig scaffold blocks the biological activity of ADM by at least 5%. These values ​​related to half-life and blocking of biological activity must be understood in conjunction with the aforementioned assays in order to determine these values. This is within the meaning of blocking the biological activity of ADM by no more than 80% or no more than 50%, respectively.

[0301] Such regulatory anti-ADM antibodies or regulatory anti-ADM antibody fragments or regulatory anti-ADM non-Ig scaffolds provide the advantage of being easy to administer quantitatively. The combination of partially blocking or partially reducing the biological activity of ADM and increasing the half-life in vivo (increasing the biological activity of ADM) leads to a beneficial simplification of the dosage of anti-ADM antibodies or anti-ADM antibody fragments or anti-ADM non-Ig scaffolds. In the case of excess endogenous ADM (maximum stimulation, late sepsis, shock, debilitation), the activity reduction effect is the main effect of the antibody or fragment or scaffold, limiting the (negative) effect of ADM. In the case of low or normal endogenous ADM concentrations, the biological effect of anti-ADM antibodies or anti-ADM antibody fragments or anti-ADM non-Ig scaffolds is a combination of reduction (by partial blocking) and improvement by increasing the half-life of ADM. Therefore, the non-neutralizing and regulatory anti-ADM antibodies or anti-ADM antibody fragments or anti-ADM non-Ig scaffolds act as a buffer similar to the biological activity of ADM, so as to keep the biological activity of ADM within a certain physiological range.

[0302] In one 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 or humanized antibody or a fragment derived therefrom. In one embodiment, one or more (murine) CDRs are transplanted into a human antibody or antibody fragment.

[0303] In one aspect, the subject matter of the present invention is a human or humanized CDR-grafted antibody or antibody fragment thereof that binds to ADM, wherein the human or humanized CDR-grafted antibody or antibody fragment thereof comprises an antibody heavy chain (H chain) comprising the following sequence:

[0304] GYTFSRYW (SEQ ID No.: 1),

[0305] ILPGSGST (SEQ ID No.: 2) and / or

[0306] TEGYEYDGFDY (SEQ ID No.: 3)

[0307] And / or further comprises an antibody light chain (L chain) comprising the following sequence:

[0308] QSIVYSNGNTY (SEQ ID No.: 4),

[0309] RVS (not part of the sequence listing) and / or

[0310] FQGSHIPYT (SEQ ID No.: 5).

[0311] In one embodiment of the present invention, the subject matter of the present invention is a human or humanized monoclonal antibody or an antibody fragment thereof that binds to ADM, wherein the heavy chain comprises at least one CDR selected from the following sequences:

[0312] GYTFSRYW (SEQ ID No.: 1),

[0313] ILPGSGST (SEQ ID No.: 2),

[0314] TEGYEYDGFDY (SEQ ID No.: 3)

[0315] And wherein the light chain comprises at least one CDR selected from the following sequences:

[0316] QSIVYSNGNTY (SEQ ID No.: 4),

[0317] RVS (not part of the sequence listing),

[0318] FQGSHIPYT (SEQ ID No.: 5).

[0319] In a more specific embodiment of the present invention, the subject matter of the present invention is a human monoclonal antibody or an antibody fragment thereof that binds to ADM, wherein the heavy chain comprises the following sequence:

[0320] GYTFSRYW (SEQ ID No.: 1),

[0321] ILPGSGST (SEQ ID No.: 2),

[0322] TEGYEYDGFDY (SEQ ID No.: 3)

[0323] And wherein the light chain comprises the following sequence:

[0324] QSIVYSNGNTY (SEQ ID No.: 4),

[0325] RVS (not part of the sequence listing),

[0326] FQGSHIPYT (SEQ ID No.: 5).

[0327] In a very specific embodiment, the anti-ADM antibody has a sequence selected from the group consisting of SEQ ID No. 6, 7, 8, 9, 10, 11, 12, 13, 32 and 33.

[0328] The anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold according to the present invention exhibits a certain affinity for human ADM, such that the affinity constant is greater than 10 -7 M, preferably greater than 10 -8 M, preferably with an affinity greater than 10 -9 M, most preferably higher than 10 -10 M. Those skilled in the art will appreciate that it may be considered to compensate for the lower affinity by administering a higher dose of the compound, and such measures will not lead to exceeding the scope of the present invention. The affinity constant may be determined according to the method described in Example 1.

[0329] The subject matter of the present invention is a human or humanized monoclonal antibody or fragment thereof which binds to ADM for use in treating or preventing shock in a patient according to the present invention, wherein the antibody or fragment comprises a sequence selected from the group consisting of:

[0330] SEQ ID NO: 6 (AM-VH-C)

[0331] QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYWG QGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK

[0332] SEQ ID NO: 7 (AM-VH1)

[0333] QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWG QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK

[0334] SEQ ID NO: 8 (AM-VH2-E40)

[0335] QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK

[0336] SEQ ID NO:9(AM-VH3-T26-E55)

[0337] QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK

[0338] SEQ ID NO:10(AM-VH4-T26-E40-E55)

[0339] QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK

[0340] SEQ ID NO:11(AM-VL-C)

[0341] DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0342] SEQ ID NO: 12 (AM-VL1)

[0343] DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0344] SEQ ID NO: 13 (AM-VL2-E40)

[0345] DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0346] Another embodiment of the present invention relates to a human or humanized monoclonal antibody or fragment thereof that binds to ADM for use in treating or preventing shock in a patient, wherein the antibody or fragment comprises the following sequence as a heavy chain:

[0347] SEQ ID NO: 32

[0348] QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGEILPGSGSTNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGT TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFScSVMHEALHNHYTQKSLSLSPGK

[0349] and contains the following sequence as a light chain:

[0350] SEQ ID NO: 33

[0351] DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPRLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGGGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0352] In a specific embodiment of the invention, the antibody comprises the following sequence or a sequence with an identity of > 95%, preferably > 98%, preferably > 99% thereof as a heavy chain:

[0353] SEQ ID NO: 32

[0354] QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGEILPGSGSTNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGT TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0355] and comprises the following sequence or a sequence with an identity > 95%, preferably > 98%, preferably > 99% thereof as a light chain:

[0356] SEQ ID NO: 33

[0357] DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPRLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGGGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0358] To assess the identity between two amino acid sequences, a pairwise alignment is performed. Identity is defined as the percentage of directly matching amino acids in the alignment.

[0359] In a specific embodiment of the invention, the antibody comprises the following sequence or comprises 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 > 95%, preferably > 98%, preferably > 99% identical to SEQ ID NO: 32 as a heavy chain:

[0360] SEQ ID NO: 32

[0361] QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGEILPGSGSTNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGT TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0362] and comprising the following sequence or a sequence comprising a CDR sequence that is 100% identical to SEQ ID No.: 4 and / or SEQ ID No.: 5 and a sequence that is > 95%, preferably > 98%, preferably > 99% identical to SEQ ID NO: 33 as a light chain:

[0363] SEQ ID NO: 33

[0364] DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPRLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGGGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0365] In an embodiment of the present invention, the anti-ADM antibody or anti-ADM antibody fragment for treating or preventing shock in a patient can be administered at a dose of at least 0.5 mg / kg body weight, particularly at least 1.0 mg / kg body weight, more particularly 1.0 to 20.0 mg / kg body weight, for example 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.

[0366] As used herein, the term "pharmaceutical formulation" refers to a preparation that is in a form that permits the biological activity of the active ingredients contained therein to be effective, and that contains no additional components that are unacceptably toxic to a subject to which the formulation is to be administered.

[0367] The present invention also relates to a pharmaceutical preparation comprising a therapeutically effective dose of the active ingredient in combination with at least one pharmaceutically acceptable excipient.

[0368] "Pharmaceutically acceptable excipient" refers to an excipient that does not produce adverse, allergic or other untoward reactions when administered to a subject. In addition to the therapeutic protein, it includes carriers, various diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials known in the art. The characteristics of the carrier will depend on the route of administration.

[0369] In the above background, the following consecutively numbered embodiments provide other specific aspects of the present invention:

[0370] 1. An anti - adrenomedullin (ADM) antibody or anti - ADM antibody fragment or anti - ADM non - Ig scaffold for treating or preventing shock in a patient, wherein the patient is characterized by a dipeptidyl peptidase 3 (DPP3) level in a body fluid sample that is below a threshold, where the threshold of DPP3 is < / = 40 ng / ml 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 (amino acids 1 - 21) of ADM: YRQSMNNFQGLRSFGCRFGTC (SEQ ID No. 14).

[0371] 2. The anti - adrenomedullin (ADM) antibody or anti - ADM antibody fragment or anti - ADM non - Ig scaffold for treating or preventing shock in a patient according to embodiment 1, wherein the shock is selected from shock caused by hypovolemia, cardiogenic shock, obstructive shock, and distributive shock, particularly cardiogenic shock or septic shock.

[0372] 3. The anti - adrenomedullin (ADM) antibody or anti - ADM antibody fragment or anti - ADM non - Ig scaffold for treating or preventing shock in a patient according to embodiments 1 and 2, wherein:

[0373] ● in the case of cardiogenic shock, the patient may have had acute coronary syndrome (such as acute myocardial infarction), or wherein the patient has heart failure (such as acute decompensated heart failure), myocarditis, arrhythmia, cardiomyopathy, valvular heart disease, aortic dissection with acute aortic stenosis, traumatic chordae tendineae rupture, or massive pulmonary embolism, or

[0374] ● in the case of hypovolemic shock, the patient may have had a hemorrhagic disease, including gastrointestinal bleeding, trauma, vascular etiologies (such as rupture of abdominal aortic aneurysm, tumor erosion into large blood vessels), and spontaneous bleeding in the background of anticoagulant use, or non - hemorrhagic diseases, including vomiting, diarrhea, renal loss, skin defects / insensible water loss (such as burns, heat stroke), or third - space fluid loss in the background of pancreatitis, cirrhosis, intestinal obstruction, trauma, or

[0375] ● in the case of obstructive shock, the patient may have had cardiac tamponade, tension pneumothorax, pulmonary embolism, or aortic stenosis, or

[0376] ● in the case of distributive shock, the patient may have septic shock, neurogenic shock, anaphylactic shock, or shock caused by adrenal crisis.

[0377] 4. An anti-adrenomedullin (ADM) antibody or an anti-ADM antibody fragment or an anti-ADM non-Ig scaffold for treating or preventing shock in a patient according to embodiments 1 to 3, wherein the threshold value of DPP3 in the patient's body fluid sample is ≤ 30 ng / ml or is in the range of 22 to 30 ng / mL.

[0378] 5. An anti-adrenomedullin (ADM) antibody or an anti-ADM antibody fragment or an anti-ADM non-Ig scaffold for treating or preventing shock in a patient according to embodiment 4, wherein the threshold value of DPP3 in the patient's body fluid sample is ≤25 ng / ml or is within the range of 22 to 25 ng / mL.

[0379] 6. An anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for treating or preventing shock in a patient according to embodiments 1 to 5, wherein the DPP3 level is determined by contacting the body fluid sample with a capture binding agent that specifically binds to DPP3.

[0380] 7. An anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for treating or preventing shock in a patient according to embodiment 6, wherein the capture binding agent is an antibody.

[0381] 8. An anti-adrenomedullin (ADM) antibody or an anti-ADM antibody fragment or an anti-ADM non-Ig scaffold for treating or preventing shock in a patient according to embodiments 1 to 7, wherein the DPP3 protein level and / or active DPP3 level is determined and compared with a predetermined threshold value.

[0382] 9. An anti-adrenomedullin (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 according to embodiments 1 to 8, wherein the DPP3 level is determined using an immunoassay, in particular a sandwich immunoassay.

[0383] 10. An anti-adrenomedullin (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 according to embodiments 1 to 8, wherein the DPP3 level is DPP3 activity, wherein the method for determining DPP3 activity in a body fluid sample of the subject comprises the following steps:

[0384] ● contacting the sample with a capture binding agent that specifically binds to full-length DPP3,

[0385] ● isolating DPP3 bound to the capture binder,

[0386] ● adding a substrate of DPP3 to the isolated DPP3,

[0387] • Quantification of the DPP3 activity by measuring and quantifying the conversion of a DPP3 substrate.

[0388] 11. 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 according to embodiments 1 to 10, wherein the patient is further characterized by ADM-NH 2 Level is above threshold.

[0389] 12. The anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for treating or preventing shock in a patient according to embodiment 11, wherein the ADM-NH 2 The threshold value is between 40 and 100 pg / mL, more preferably between 50 and 90 pg / mL, even more preferably between 60 and 80 pg / mL, most preferably the threshold value is 70 pg / mL.

[0390] 13. The anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for treating or preventing shock in a patient according to embodiment 12, wherein the ADM-NH 2 The threshold for chromatin is 70 pg / mL.

[0391] 14. An anti-adrenomedullin (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 according to embodiments 1 to 13, wherein the body fluid is selected from whole blood, plasma and serum.

[0392] 15. An anti-adrenomedullin (ADM) antibody or an anti-ADM antibody fragment or an anti-ADM non-Ig scaffold for treating or preventing shock in a patient according to embodiment 14, wherein the body fluid is plasma.

[0393] 16. An anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for treating or preventing shock in a patient according to embodiments 11-15, wherein the ADM-NH 2 The level of ADM-NH 2 The assay is performed by contacting the capture binding agent with the capture binding agent.

[0394] 17. An anti-adrenomedullin (ADM) antibody or an anti-ADM antibody fragment or an anti-ADM non-Ig scaffold for treating or preventing shock in a patient according to embodiment 16, wherein the capture binding agent is an antibody.

[0395] 18. An anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for treating or preventing shock in a patient according to embodiments 1 to 17, wherein the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold recognizes and binds to ADM-Gly and / or ADM-NH 2 The N-terminal end (amino acid position 1).

[0396] 19. An anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for treating or preventing shock in a patient according to embodiments 1 to 18, wherein the antibody, antibody fragment or non-Ig scaffold does not bind to the C-terminal portion of ADM, the C-terminal portion having the sequence of amino acids 43-52 of ADM: PRSKISPQGY-NH 2 (SEQ ID NO: 24).

[0397] 20. 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 according to embodiments 1 to 19, wherein the antibody or fragment or scaffold blocks no more than 80%, preferably no more than 50% of the biological activity of ADM.

[0398] 21. 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 according to embodiments 1 to 20, wherein the antibody or fragment is a monoclonal antibody or fragment thereof that binds to ADM, wherein the heavy chain comprises the following sequence:

[0399] CDR1: SEQ ID NO: 1

[0400] GYTFSRYW

[0401] CDR2: SEQ ID NO: 2

[0402] ILPGSGST

[0403] CDR3: SEQ ID NO: 3

[0404] TEGYEYDGFDY

[0405] And wherein the light chain comprises the following sequence:

[0406] CDR1: SEQ ID NO: 4

[0407] QSIVYSNGNTY

[0408] CDR2:

[0409] RVS

[0410] CDR3: SEQ ID NO: 5

[0411] FQGSHIPYT.

[0412] 22. An anti-adrenomedullin (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 according to embodiments 1 to 21, wherein the antibody or fragment comprises as VH region a sequence selected from the group consisting of:

[0413] SEQ ID NO: 6 (AM-VH-C)

[0414] QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYWG QGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK

[0415] SEQ ID NO: 7 (AM-VH1)

[0416] QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWG QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK

[0417] SEQ ID NO: 8 (AM-VH2-E40)

[0418] QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK

[0419] SEQ ID NO: 9 (AM-VH3-T26-E55)

[0420] QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK

[0421] SEQ ID NO: 10 (AM-VH4-T26-E40-E55)

[0422] QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK

[0423] and includes a sequence selected from the following sequences as the VL region:

[0424] SEQ ID NO: 11 (AM-VL-C)

[0425] DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0426] SEQ ID NO: 12 (AM-VL1)

[0427] DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0428] SEQ ID NO: 13 (AM-VL2-E40)

[0429] DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0430] 23. An anti-adrenomedullin (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 according to embodiments 1 to 22, wherein the antibody or fragment comprises as a heavy chain the following sequence or a sequence with > 95% identity thereto:

[0431] SEQ ID NO: 32

[0432] QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGEILPGSGSTNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGT TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0433] and comprising as a light chain the following sequence or a sequence with > 95% identity thereto:

[0434] SEQ ID NO: 33

[0435] DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPRLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGGGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0436] 24. An anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for treating or preventing shock in a patient according to embodiments 1-23, wherein the anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold is bound to the N-terminal portion (amino acids 1-10) of ADM: YRQSMNNFQG (SEQID No. 26).

[0437] 25. An anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold for use in therapy according to any one of embodiments 1-24, wherein the antibody or fragment or scaffold exhibits at least 10% affinity for ADM when measured by label-free surface plasmon resonance using a Biacore 2000 system. -7 The binding affinity of M.

[0438] 26. An anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for treating or preventing shock in a patient according to embodiment 25, wherein the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold exhibits 1 x 10 -9 Up to 3 x 10 -9 Affinity between M.

[0439] 27. 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 according to embodiments 1-26, wherein the anti-ADM antibody or anti-ADM antibody fragment is an IgG1 antibody.

[0440] 28. A pharmaceutical preparation for treating or preventing shock in a patient, comprising the anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold according to any one of embodiments 1-27.

[0441] 29. A pharmaceutical preparation for treating or preventing shock in a patient according to embodiment 28, wherein the pharmaceutical preparation is a solution, preferably a ready-to-use solution.

[0442] 30. The pharmaceutical preparation for treating or preventing shock in a patient according to embodiments 28 and 29, wherein the pharmaceutical preparation is in a freeze-dried state.

[0443] 31. A pharmaceutical formulation for treating or preventing shock in a patient according to embodiments 28 and 30, wherein the pharmaceutical formulation is administered intramuscularly.

[0444] 32. The pharmaceutical formulation for treating or preventing shock in a patient according to embodiments 28 and 31, wherein the pharmaceutical formulation is administered intravascularly.

[0445] 33. The pharmaceutical formulation for treating or preventing shock in a patient according to embodiments 28 and 32, wherein the pharmaceutical formulation is administered by infusion.

[0446] 34. A pharmaceutical formulation for treating or preventing shock in a patient according to embodiments 28 and 33, wherein the pharmaceutical formulation is administered systemically.

[0447] Methods for obtaining monoclonal antibodies

[0448] In all of the following embodiments, the term monoclonal antibody is meant to include monoclonal antibodies as well as fragments of monoclonal antibodies, such as the fragments described in detail herein, and more specifically monoclonal antibodies.

[0449] Hybridoma

[0450] In another aspect, the antibody according to the present invention is a monoclonal antibody obtainable by a method comprising the steps of:

[0451] i) fusing antibody-secreting cells from an animal previously immunized with the antigen with myeloma cells to obtain a large number of hybridomas,

[0452] ii) isolating hybridomas producing the desired monoclonal antibody from the plurality of hybridomas.

[0453] In certain embodiments, the antibodies according to the present invention are monoclonal antibodies that can be obtained by isolating a hybridoma that produces the desired monoclonal antibody from a large number of hybridomas, wherein the large number of hybridomas are produced by fusing antibody-secreting cells from an animal previously immunized with an antigen with myeloma cells to obtain a large number of hybridomas.

[0454] The desired monoclonal antibody is in particular a monoclonal antibody that binds to the antigen, in particular with a binding affinity of at least 10 7 M -1 , preferably 10 8 M -1 , preferably with an affinity greater than 10 9 M -1 , most preferably greater than 10 10 M -1 .

[0455] In certain embodiments of the method for obtaining an antibody, in step i), the animal is a mammal, particularly a rabbit, a mouse or a rat, more particularly a mouse, more particularly a Balb / c mouse.

[0456] In certain embodiments of the method for obtaining antibodies, in step i), the antibody-secreting cells are spleen cells, more particularly activated B cells.

[0457] In certain embodiments of the method for obtaining an antibody, in step i), the fusion comprises the use of polyethylene glycol.

[0458] In certain embodiments of the method for obtaining an antibody, in step i), the myeloma is derived from a mammal, in certain embodiments derived from a mammal of the same species from which the plurality of antibody-secreting cells are obtained. In certain specific embodiments of the method for obtaining an antibody, in step i), the myeloma cell is a cell line SP2 / 0.

[0459] In certain embodiments of the method for obtaining an antibody, the fusion in step i) comprises PEG-assisted fusion, Sendai virus-assisted fusion or electric current-assisted fusion.

[0460] In certain embodiments of the method for obtaining antibodies, the isolating in step ii) comprises performing an antibody capture assay, an antigen capture assay and / or a functional screening.

[0461] In certain embodiments of the method for obtaining an antibody, in step ii), isolating a hybridoma producing a desired monoclonal antibody may include cloning and re-cloning the hybridoma using a limiting dilution technique.

[0462] In one embodiment, the antibody capture assay comprises

[0463] a) binding the antigen to a substrate, in particular a solid substrate,

[0464] b) allowing the generated antibodies to bind to said antigen,

[0465] c) removing unbound antibodies by washing,

[0466] d) Detection of bound antibodies.

[0467] In one embodiment, the antigen capture assay comprises

[0468] a) binding the produced antibody to a substrate, in particular a solid substrate,

[0469] b) allowing the antigen to bind to said antibody,

[0470] c) remove unbound antigen by washing,

[0471] d) detecting bound antigen;

[0472] Alternatively, the antigen capture assay comprises

[0473] a) allowing the antigen to bind to the produced antibody to form an antibody-antigen complex,

[0474] b) binding the antibody-antigen complex to a substrate, in particular a solid substrate,

[0475] c) remove unbound antigen by washing,

[0476] d) Detection of bound antigen.

[0477] In one embodiment, the separation in step ii) comprises performing enzyme-linked immunosorbent assay, fluorescence activated cell sorting, cell staining, immunoprecipitation and / or western blotting.

[0478] In one embodiment, detection of the antibody or antigen is accomplished using an immunoassay.

[0479] In one embodiment, the animal is a transgenic animal, particularly a transgenic mouse (in which the mouse immunoglobulin (Ig) loci have been replaced by human loci, particularly within the genome of the transgenic animal), such as a HuMabMouse or a XenoMouse.

[0480] In one embodiment, the antigen comprises a peptide as described in Table 1 or Table 6 herein, respectively, which in certain embodiments (particularly for immunization) may be coupled to a protein, particularly a serum protein, more particularly serum albumin, more particularly BSA.

[0481] In a preferred embodiment, the antibody according to the present invention is a monoclonal antibody obtainable by a method comprising the following steps:

[0482] i) fusing spleen cells from Balb / c mice previously immunized with the peptides described in Table 1 or Table 6 herein with SP2 / 0 myeloma cells using polyethylene glycol to obtain a large number of hybridomas,

[0483] ii) isolating hybridomas producing the desired monoclonal antibody from the plurality of hybridomas;

[0484] More preferably, the method comprises

[0485] ● Grow the hybridomas in HAT medium [RPMI 1640 medium supplemented with 20% fetal bovine serum and HAT supplement] for a first period of time (typically 2 weeks)

[0486] ● Subsequent passages (especially 3 times) were performed using HT medium instead of HAT medium.

[0487] ● Then return to normal cell culture medium for a second period of time, typically until the end of three weeks post-confluency

[0488] ● Preliminary screening of antigen-specific IgG antibodies in cell culture supernatant

[0489] ● Propagate microcultures of cells that tested positive in 4)

[0490] ● Retest cell culture supernatants from microcultures for antigen-specific IgG antibodies

[0491] ● Use limiting dilution techniques to clone and reclon the cultures that tested positive in 6)

[0492] ● Optionally determine the isotype of the clones obtained from 7)

[0493] ● Optionally purify the antibody by protein A

[0494] Phage display

[0495] In another aspect, the antibody according to the present invention is a monoclonal antibody obtainable by a method comprising the steps of:

[0496] i) isolating at least one antibody having affinity for the antigen from the antibody gene library;

[0497] ii) producing at least one cell line expressing said at least one antibody;

[0498] iii) isolating the at least one antibody from the culture of the at least one cell line obtained in step ii).

[0499] Specifically, an antibody having an affinity for an antigen has a binding affinity of at least 10 7 M -1 , preferably 10 8 M -1 Antibodies with an affinity greater than 10 9 M -1 , most preferably greater than 10 10 M -1 .

[0500] In a certain embodiment, the antibody according to the present invention is a monoclonal antibody obtainable by isolating at least one antibody from a culture derived from at least one cell line expressing at least one antibody having affinity for an antigen from an antibody gene library.

[0501] In one embodiment, the antigen comprises a peptide as described in Table 1 or Table 6, respectively, herein, which in certain embodiments may be bound to a solid phase.

[0502] In certain embodiments of the method for obtaining antibodies, in step i), the antibody gene library is a naive antibody gene library, in particular a human naive antibody gene library, more specifically, in the library, the antibodies are presented by phage display, i.e., presented on phages comprising nucleotide sequences encoding such corresponding antibodies; more specifically, it is library HAL 7, HAL 8 or HAL 9, more specifically, it is a library comprising human naive antibody gene library HAL7 / 8.

[0503] In certain embodiments of the method for obtaining antibodies, in step i), the screening comprises the use of an antigen, in particular an antigen containing a tag, more particularly a biotin tag, attached thereto via two different spacers. In a particular embodiment, this panning strategy comprises a mixture of panning rounds using non-specifically bound antigens and antigens specifically bound via the tag (in the case of a biotin tag, to streptavidin). In this way, the background of non-specific binders can be minimized.

[0504] 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 phage presenting the antibody (and comprising a nucleotide sequence encoding the antibody).

[0505] In certain embodiments of the method for obtaining an antibody, in step ii), the cell strain is produced by introducing a nucleotide sequence encoding the antibody; in embodiments where the library in step i) is a phage display library, the phage isolated from step i) can be used to produce a bacterial strain, such as an Escherichia coli strain, that expresses the antibody.

[0506] In certain embodiments of the method for obtaining antibodies, in step iv), in embodiments wherein the library in step i) is a phage display library and wherein a bacterial strain is produced in step ii), antibodies may be isolated from the supernatant of the culture.

[0507] It should be understood that when used in describing a method for obtaining an antibody, the term "an antibody" in the expression "at least one antibody" may specifically include more than one antibody molecule of an antibody having the same amino acid sequence. This understanding also applies, mutatis mutandis, to the term "a cell line".

[0508] In certain embodiments of the method for obtaining antibodies, more than one antibody (referring to a plurality of antibodies having different amino acid sequences, respectively) is isolated in step i), and thus more than one cell line is produced in step ii). This method may include selecting clones that are positive for binding to the antigen, for example, by selecting clones that are positive for binding to the antigen through a binding assay, such as an ELISA assay involving the antigen, and cells that are positive for binding to the antigen may be isolated to produce a monoclonal cell line.

[0509] In a preferred embodiment, the antibody according to the present invention is a monoclonal antibody obtainable by a method comprising the following steps:

[0510] i) isolating at least one of the antibodies from an antibody gene library comprising a human naive antibody gene library HAL7 / 8 by eluting phages carrying antibodies having affinity for an antigen from the library;

[0511] ii) producing at least one E. coli cell strain expressing said at least one antibody;

[0512] iii) isolating the at least one antibody from the culture supernatant of the at least one E. coli cell strain obtained in step ii).

[0513] In yet another aspect, antibody fragment according to the present invention is produced by a method comprising enzymatic digestion of antibody. In some embodiments, this method produces for example Fab or F (ab) 2 antibody fragment. In some embodiments, this method comprises digestion with pepsin or papain, and the enzyme is optionally fixed on the surface.

[0514] In certain embodiments, antibodies can be humanized by CDR grafting, in particular by a method comprising the following steps:

[0515] - extracting RNA from a hybridoma expressing an antibody of interest (e.g. obtained by the methods described herein);

[0516] - amplifying the extracted RNA by RT-PCR, in particular using primer sets specific for the heavy and light chains of the antibody of interest, to obtain a DNA product;

[0517] - further amplifying said DNA product by PCR, in particular using a semi-nested primer set specific for the antibody variable region;

[0518] - determining the sequence of the DNA product;

[0519] - Aligning the sequences with homologous human framework sequences to determine the humanized sequences for the variable heavy and variable light chain sequences (of the desired antibody).

[0520] In certain embodiments, the antibody can be humanized by the following method: the sequence of the DNA product obtained by RT-PCR amplification (particularly using a primer set specific for the heavy chain and light chain of the antibody of interest) of RNA extracted from a hybridoma expressing the antibody of interest and further amplification by PCR (particularly using a semi-nested primer set specific for the variable region of the antibody) is compared with the homologous human framework sequence to determine the humanized sequence for the variable heavy chain and variable light chain sequences (of the desired antibody).

[0521] In certain embodiments, antibodies can be humanized by:

[0522] - determination of the complementarity determining regions (CDRs), which can be achieved by analyzing the structural interactions of the framework regions (FRs) with the CDRs and the antigen;

[0523] - Grafting the CDR sequences into human framework regions.

[0524] In certain embodiments, antibodies can be humanized by grafting CDR sequences, which can preferably be determined by analyzing the structural interactions of the framework region (FR) with the complementarity determining region (CDR) and antigen, into human framework regions.

[0525] In certain embodiments, variations can be introduced in the amino acid sequence of a CDR or FR to maintain structural interactions with the antigen that might otherwise be abolished by the introduction of the human FR sequence, for example, by random approaches using phage display libraries or by directed approaches guided by molecular modeling.

[0526] The DNA sequences encoding the antibodies identified as described in detail herein can be transferred into cells by known genetic engineering techniques and used to produce the antibodies.

[0527] Producing antibodies

[0528] In another aspect, the antibody according to the invention is a monoclonal antibody obtainable by the method described herein, which is prepared by a method comprising the steps of:

[0529] - cultivating a cell line comprising a nucleotide sequence encoding the antibody;

[0530] - isolating said antibody from said culture.

[0531] In another specific aspect, the antibody according to the invention is a monoclonal antibody obtainable by the method described herein, which is produced by isolating said antibody from a culture of a cell line comprising a nucleotide sequence encoding said antibody.

[0532] In certain embodiments of the method, the cell strain is generated as described above and can include bacterial cells, such as Gram-negative bacteria such as Escherichia coli, Proteus mirabilis or Pseudomonas putidas, Gram-positive bacteria such as Bacillus brevis, Bacillus subtilis, Bacillus megaterium, Lactobacillus such as Lactobacillus zeae / casei or Lactobacillus paracasei, or Streptomyces such as Streptomyces lividans; eukaryotic cells, such as yeast such as Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, Schizosaccharomyces pombe. pombe, Schwanniomyces occidentalis, Kluyveromyces lactis or Yarrowia lipolytica; fungi, for example filamentous fungi, for example Trichoderma or Aspergillus, for example A. niger (e.g. subgenus A. awamori) and Aspergillus oryzae, Trichoderma reesei, Chrysosporium, for example C. lucknowense; protozoa, for example Leishmania, for example L. tarentolae; insect cells, for example insect cells transfected with a baculovirus, for example AcNPV, for example insect cell lines from Spodoptera frugiperda, for example Sf-9 or Sf-21, Drosophila melanogaster, for example DS2, or Trichopulsia ni ni) such as High Five cells (BTI-TN-5B1-4); mammalian cells, such as hamster cells, such as Chinese hamster ovary such as K1-, DukX B11-, DG44, Lec13 or BHK cells, mouse cells, such as mouse myeloma such as NS0 cells, Homo sapiens cells, such as Per.C6, AGE1.HN, HEK293 cells.

[0533] In certain embodiments of the methods, the cell can be a hybridoma cell, such as a hybridoma cell described herein.

[0534] In certain embodiments of the method, the culturing can be carried out in static suspension culture, stirred suspension culture, membrane-based culture, matrix-based culture or high cell density bioreactor; the container for such culturing can be selected from T-flasks, roller cultures, spinner cultures, stirred tank bioreactors, airlift bioreactors, static membrane-based or matrix-based culture systems, suspension bioreactors, fluidized bed bioreactors, ceramic bioreactors, perfusion systems and hollow fiber bioreactors.

[0535] In certain embodiments of the methods, the cells may be immobilized on a matrix.

[0536] A high cell density bioreactor is a bioreactor that can produce more than 10 8 The cell density of the culture system is 10 cells / ml.

[0537] In another aspect, the antibody according to the invention is a monoclonal antibody obtainable by the method described herein, which is prepared by a method comprising the steps of:

[0538] - producing transgenic plants or animals comprising a nucleotide sequence encoding said antibody;

[0539] - isolating said antibody from said plant or animal or a secretion or product of said plant or animal.

[0540] In another specific aspect, the antibody according to the present invention is a monoclonal antibody obtainable by the method described herein, which is produced by isolating the antibody from a transgenic plant or transgenic animal having a nucleotide sequence encoding the antibody or a secretion or product of the transgenic plant or transgenic animal.

[0541] The animal may be, for example, selected from chicken, mouse, rat, rabbit, cow, goat, sheep, pig; the secretion or product may be, for example, milk or eggs. The plant may be, for example, selected from tobacco (N. tabacum or N. benthamiana), duckweed (Lemna minor), Chlamydomonas reinhardtii, rice, Arabidopsis thaliana, alfalfa (Medicago sativa), lettuce, corn.

[0542] In certain embodiments, the antibodies can be isolated by: physicochemical fractionation, such as size exclusion chromatography, precipitation (e.g., using ammonium sulfate), ion exchange chromatography, immobilized metal chelate chromatography gel filtration, zone electrophoresis; based on their classification, such as binding to bacterial proteins A, G or L, jacalin; antigen-specific affinity purification by immobilized ligand / antigen; if necessary, low molecular weight components can be removed by methods such as dialysis, desalting and diafiltration.

[0543] In some embodiments, the antibody is encoded by a nucleotide sequence, wherein the nucleotide sequence is a reverse transcription of an amino acid sequence from an antibody produced by one of the methods described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0544] Figure 1a: Illustration of antibody formats - Fv and scFv-variants.

[0545] Figure 1b: Illustration of antibody formats - heterologous fusions and bifunctional antibodies.

[0546] Figure 1c: Illustration of antibody formats - diabodies and bispecific antibodies.

[0547] Figure 2: Dose response and dose inhibition curves

[0548] a: Dose response curve of human ADM. Maximum cAMP stimulation was adjusted to 100% activation.

[0549] b: Dose / inhibition curve of human ADM 22-52 (ADM receptor antagonist) in the presence of 5.63 nM hADM.

[0550] c: Dose / inhibition curve of CT-H in the presence of 5.63 nM hADM.

[0551] d: Dose / inhibition curve of MR-H in the presence of 5.63 nM hADM.

[0552] e: Dose / inhibition curve of NT-H in the presence of 5.63 nM hADM.

[0553] f: Dose-response curve of mouse ADM. Maximum cAMP stimulation was adjusted to 100% activation.

[0554] g: Dose / inhibition curve of human ADM 22-52 (ADM receptor antagonist) in the presence of 0,67 nM mADM.

[0555] h: Dose / inhibition curve of CT-M in the presence of 0,67 nM mADM.

[0556] i: Dose / inhibition curve of MR-M in the presence of 0,67 nM mADM.

[0557] j: Dose / inhibition curve of NT-M in the presence of 0,67 nM mADM.

[0558] k: shows the inhibition of ADM by F(ab)2 NT-M and Fab NT-M.

[0559] 1: shows the inhibition of ADM by F(ab)2 NT-M and Fab NT-M.

[0560] Figure 3 : The figure shows a typical hADM dose / signal curve and the dose-signal curve of hADM in the presence of 100 µg / mL antibody NT-H.

[0561] Figure 4 : This figure shows the stability of hADM in human plasma (citrate) in the absence and presence of NT-H antibody.

[0562] Figure 5 : Alignment of Fab with homologous human framework sequences.

[0563] Figure 6 : ADM concentrations in healthy human subjects up to 60 days after administration of NT-H at different doses.

[0564] Figure 7 : Kaplan-Meier survival plots relative to low (< 40.5 ng / mL) and high (≥ 40.5 ng / mL) DPP3 concentrations. (A) 7-day survival rate of sepsis patients relative to DPP3 plasma concentration; (B) 7-day survival rate of cardiogenic shock patients relative to DPP3 plasma concentration; (C) 7-day survival rate of septic shock patients relative to DPP3 plasma concentration.

[0565] Figure 8 : Kaplan-Meier survival plot for all patients (14-day mortality in patients treated with placebo (Plac) or the N-terminal ADM antibody aderizumab (Adz))

[0566] Fig. 9 : Kaplan-Meier survival plot of patients with DPP3 < 50 ng / mL (14-day mortality in patients treated with placebo (Plac) or the N-terminal ADM antibody aderizumab (Adz))

[0567] Fig.10: Kaplan-Meier survival plot of patients with DPP3 > 50 ng / mL (14-day mortality in patients treated with placebo (Plac) or the N-terminal ADM antibody aderizumab (Adz))

[0568] Figure 11: Kaplan Meier plot of the effect of aderezumab treatment on 28-day mortality in patients with high and low pre-dose bio-ADM concentrations. Investigation population: PP and PP / DPP3≤70. Figure 11a shows patients with PP / DPP3≤70 -bio-ADM between 70 and 182 pg / mL (below the median), Figure 11b shows patients with bio-ADM above 182 pg / mL (above the median). The calculated HRs and log-rank p-values ​​(two-sided) with 95% CIs were as follows: PP below the median: HR=0.630 [0.276 -1.44], log-rank p=0.270; PP above the median: HR=0.870 [0.499 -1.52], log-rank p=0.634; PP / DPP3≤70 below the median: HR=0.607 [0.242 -1.52], log-rank p=0.284; PP / DPP3≤70 above the median: HR=0.623 [0.327-1.19], log-rank p=0.151.

[0569] Figure 12: Efficacy of aderezumab treatment using different DPP3 thresholds: Kaplan-Meier survival plots (28-day mortality) of patients treated with placebo or the N-terminal ADM antibody aderezumab, including patients with (A) DPP3 < 50 ng / mL, (B) DPP3 < 40 ng / mL, (C) DPP3 < 30 ng / mL, and (D) DPP3 < 22 ng / mL, respectively. Example

[0570] Example 1 – Antibody production and determination of affinity constants

[0571] Several human and murine antibodies were generated and their affinity constants determined (see Tables 1 and 2). It should be emphasized that the antibodies, antibody fragments and non-Ig scaffolds according to the Examples section of the present invention bind to ADM and should therefore be considered as anti-ADM antibodies / antibody fragments / non-Ig scaffolds.

[0572] Peptides / conjugates for immunization:

[0573] Peptides for immunization were synthesized, see Table 1 (JPT Technologies, Berlin, Germany), with an additional N-terminal cysteine ​​(if cysteine ​​is not present in the selected ADM sequence) residue for coupling the peptide to bovine serum albumin (BSA). The peptide was covalently linked to BSA using Sulfolink coupling gel (Perbio-science, Bonn, Germany). The coupling procedure was performed according to the manual of Perbio.

[0574] Mouse monoclonal antibody production:

[0575] Balb / c mice were immunized with 100 µg of peptide-BSA conjugate (emulsified in 100 µl Freund's complete adjuvant) on days 0 and 14 and 50 µg (in 100 µl Freund's incomplete adjuvant) on days 21 and 28. Three days before fusion experiments, the animals received 50 µg of the conjugate dissolved in 100 µl saline, given as one intraperitoneal and one intravenous injection. Splenocytes from immunized mice were fused with cells of the myeloma cell line SP2 / 0 with 1 ml of 50% polyethylene glycol at 37°C for 30 s. After washing, cells were seeded in 96-well cell culture plates. Hybrid clones were selected by growth in HAT medium [RPMI 1640 medium supplemented with 20% fetal bovine serum and HAT supplement]. After two weeks, the HAT medium was replaced with HT medium for three passages and then returned to normal cell culture medium. Cell culture supernatants were screened for antigen-specific IgG antibodies three weeks after fusion. Microcultures that tested positive were transferred to 24-well plates for propagation. After retesting, selected cultures were cloned and recloned using limiting dilution techniques and isotyped (see also ). Lane, RD 1985. J. Immunol. Meth. 81: 223-228; Ziegler et al., 1996. Horm. Metab. Res. 28: 11-15 ).

[0576] Antibodies were produced by standard antibody production methods ( Marx et al., 1997. Monoclonal Antibody Production, ATLA 25, 121 ) and purified by protein A. Based on SDS gel electrophoresis analysis, the antibody purity was > 95%.

[0577] Human Antibodies:

[0578] Human antibodies were produced using phage display according to the following procedure: A human naive antibody gene library HAL7 / 8 was used to isolate recombinant single chain F-variable domains (scFv) against the peptide adrenomedullin. The antibody gene library was screened using a panning strategy involving the use of peptides containing a biotin tag linked to the adrenomedullin peptide sequence via two different spacers. A mixture of panning rounds using nonspecifically bound antigen and streptavidin-bound antigen was used to minimize the background of nonspecific binders. Phage eluted from the third round of panning was used to generate E. coli strains expressing monoclonal scFvs. Supernatants from the cultures of these clonal strains were used directly for antigen ELISA testing (see also Hust et al. 2011. Journal of Biotechnology 152, 159–170; Schütte et al., 2009. PLoS One 4, e6625 Positive clones were selected on the basis of positive ELISA signals to the antigen and negative on streptavidin-coated microtiter plates. For further characterization, the open reading frame of the scFv was cloned into the expression plasmid pOPE107 ( Hust et al., J. Biotechn. 2011 ), captured from culture supernatant by immobilized metal ion affinity chromatography and purified by size exclusion chromatography.

[0579] Affinity constant: To determine the affinity of the antibodies for ADM, the binding kinetics of ADM to the immobilized antibodies were measured using label-free surface plasmon resonance using a Biacore 2000 system (GE Healthcare Europe GmbH, Freiburg, Germany). Reversible immobilization of the antibodies was performed using anti-mouse Fc antibodies covalently coupled at high density to the surface of a CM5 sensor according to the manufacturer's instructions (Mouse Antibody Capture Kit; GE Healthcare). Lorenz et al. 2011. Antimicrob Agents Chemother. 55(1): 165–173 ).

[0580] Monoclonal antibodies were generated against the ADM region of the human and murine ADM depicted below, respectively. The following table represents a selected set of resulting antibodies used in further experiments. The selection was based on the target region:

[0581]

[0582] Below is a list of further available monoclonal antibodies:

[0583]

[0584] Generation of antibody fragments by enzymatic digestion: Fab and F(ab) 2Fragment generation was performed by enzymatic digestion of the full-length murine antibody NT-M. Antibody NT-M was treated with a) pepsin-based F(ab) 2 Preparation kit (Pierce 44988) and b) papain-based Fab preparation kit (Pierce 44985). The fragmentation procedure was performed according to the instructions provided by the supplier. 2 In the case of fragmentation, digestion was performed at 37°C for 8 h. Digestion for Fab fragmentation was performed accordingly for 16 h.

[0585] Procedure for the production and purification of Fab: The immobilized papain was balanced by washing the resin with 0.5 ml digestion buffer and centrifuging the column at 5000 x g for 1 minute. The buffer was then discarded. The desalting column was prepared by removing the storage solution and washing it with digestion buffer, then centrifuging it at 1000 x g for 2 minutes each time. 0.5 ml of the prepared IgG sample was added to the spin column tube containing the balanced immobilized papain. The incubation time of the digestion reaction was 16 h on a table shaker at 37 ° C. The column was centrifuged at 5000 × g for 1 minute to separate the digestion solution from the immobilized papain. The resin was then washed with 0.5 ml PBS and centrifuged at 5000 × g for 1 minute. The wash fraction was added to the digested antibody, and the total sample volume was 1.0 ml. The NAb protein A column was balanced 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), and balanced by adding 2 ml PBS, centrifuged again for 1 minute, and the flow-through was discarded. The sample was applied to the column and resuspended by flipping. The incubation was carried out for 10 minutes at room temperature with mixing by inversion. The column was centrifuged for 1 minute and the flow-through containing the Fab fragment was saved. (Reference: 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 ).

[0586] For F(ab´) 2 Procedure for fragment generation and purification:The immobilized pepsin was equilibrated by washing the resin with 0.5 ml digestion buffer and centrifuging the column at 5000 x g for 1 minute. The buffer was then discarded. The desalting column was prepared by removing the storage solution and washing it with digestion buffer, followed by centrifugation at 1000 x g for 2 minutes each time. 0.5 ml of the prepared IgG sample was added to the spin column tube containing the equilibrated immobilized pepsin. The incubation time of the digestion reaction was 16 h on a benchtop shaker at 37 ° C. The column was centrifuged at 5000 × g for 1 minute to separate the digestion solution from the immobilized pepsin. The resin was then washed with 0.5 ml PBS and centrifuged at 5000 × g for 1 minute. The wash fraction was added to the digested antibody, with a total sample volume of 1.0 ml. The NAb protein A column was equilibrated with PBS and IgG elution buffer at room temperature. The column was centrifuged for 1 minute to remove the storage solution (containing 0.02% sodium azide) and equilibrated by adding 2 ml PBS, centrifuged again for 1 minute, and the flow-through was discarded. The sample was applied to the column and resuspended by flipping. Incubate at room temperature with mixing by inversion for 10 minutes. Centrifuge the column for 1 minute to save the F(ab´) 2 The flowthrough of the fragment. (References: Mariani et al., 1991. Mol. Immunol. 28: 69-77; Beale 1987. Exp Comp Immunol 11:287-96; Ellerson et al., 1972. FEBS Letters 24(3):318-22; Kerbel and Elliot 1983. Meth Enzymol 93:113-147; Kulkarni et al., 1985. Cancer Immunol Immunotherapy 19:211-4; Lamoyi 1986. Meth Enzymol 121:652-663; Parham et al., 1982. J Immunol Meth 53:133-73; Raychaudhuri et al. 1985. Mol Immunol 22(9):1009-19; Rousseaux et al., 1980. Mol Immunol 17:469-82; Rousseaux et al., 1983. J Immunol Meth 64:141-6; Wilson et al., 1991. J Immunol Meth 138: 111-9 ).

[0587] NT-H- Antibody fragment humanization: Antibody fragments are humanized by CDR grafting method ( Jones et al., 1986. Nature 321, 522–525 ).

[0588] Perform the following steps to obtain the humanized sequence:

[0589] Total RNA extraction: Total RNA was extracted from NT-H hybridoma using Qiagen kit. First round RT-PCR: Total RNA was extracted from NT-H hybridoma using Qiagen kit. ® OneStep RT-PCR Kit (Catalog No. 210210). RT-PCR was performed using primer sets specific for heavy and light chains. For each RNA sample, 12 separate heavy chain and 11 light chain RT-PCR reactions were set up using a degenerate forward primer mix covering the leader sequence of the variable region. The reverse primers were located in the constant region of the heavy and light chains. No restriction sites were engineered in the primers.

[0590] Reaction setup: 5x QIAGEN ®OneStep RT-PCR buffer 5.0 μl, dNTP mix (containing 10 mM of each dNTP) 0.8 μl, primer set 0.5 μl, QIAGEN ® OneStep RT-PCR enzyme mix 0.8 μl, template RNA 2.0 μl, add RNase-free water to 20.0 μl, total volume 20.0 μl. PCR conditions: reverse transcription: 50℃, 30 min; initial PCR activation: 95℃, 15 min; cycle: 94℃, 25 sec; 54℃, 30 sec; 72℃, 30 sec for 20 cycles; final extension: 72℃, 10 min. Second round of semi-nested PCR: RT-PCR products from the first round of reaction were further amplified in the second round of PCR. 12 separate heavy chain and 11 light chain RT-PCR reactions were set up using semi-nested primer sets specific for the variable region of the antibody.

[0591] Reaction setup: 2x PCR mix 10 μl; primer set 2 μl; first round PCR product 8 μl; total volume 20 μl; hybridoma antibody clone reporter PCR conditions: initial denaturation at 95°C for 5 min; 25 cycles of 95°C for 25 sec, 57°C for 30 sec, and 68°C for 30 sec; final extension at 68°C for 10 min.

[0592] After PCR was completed, the PCR reaction samples were run on an agarose gel to visualize the amplified DNA fragments. After sequencing the DNA fragments of more than 15 clones amplified by nested RT-PCR, several mouse antibody heavy and light chains were cloned and appeared correct. Protein sequence alignment and CDR analysis identified one heavy chain and one light chain. After alignment with the homologous human framework sequence, the resulting humanized sequence of the variable heavy chain is as follows: Figure 5 Since the amino acids at positions 26, 40, and 55 in the variable heavy chain and at position 40 in the variable light chain are critical for binding properties, they can be restored to their murine origin. The resulting candidates are depicted below. ( Padlan 1991. Mol. Immunol. 28, 489–498; Harris and Bajorath. 1995. Protein Sci. 4, 306–310 ).

[0593] Annotation of antibody fragment sequences (SEQ ID No.: 6-13, 32 and 33): CDRs 1, 2, 3 are in bold and underline; constant regions are in italics; hinge regions are emphasized with bold letters; framework point mutations have a grey letter background.

[0594] SEQ ID No.: 6 (AM-VH-C)

[0595] QVQLQQSGAELMKPGASVKISCKAT GYTFSRYW IEWVKQRPGHGLEWIGE ILPGSGST NYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYC TEGYEYDGFDY WGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK

[0596] SEQ ID No.:7(AM-VH1)

[0597] QVQLVQSGAEVKKPGSSVKVSCKAS GYTFSRYW ISWVRQAPGQGLEWMGR ILPGSGST NYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYC TEGYEYDGFDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK

[0598] SEQ ID No.:8(AM-VH2-E40)

[0599] QVQLVQSGAEVKKPGSSVKVSCKAS GYTFSRYW IEWVRQAPGQGLEWMGR ILPGSGST NYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYC TEGYEYDGFDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK

[0600] SEQ ID No.:9(AM-VH3-T26-E55)

[0601] QVQLVQSGAEVKKPGSSVKVSCKAT GYTFSRYW ISWVRQAPGQGLEWMGE ILPGSGST NYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYC TEGYEYDGFDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK

[0602] SEQ ID No.:10(AM-VH4-T26-E40-E55)

[0603] QVQLVQSGAEVKKPGSSVKVSCKAT GYTFSRYW IEWVRQAPGQGLEWMGE ILPGSGST NYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYC TEGYEYDGFDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK

[0604] SEQ ID No.:11(AM-VL-C)

[0605] DVLLSQTPLSLPVSLGDQATISCRSS QSIVYSNGNTY LEWYLQKPGQSPKLLIY RVS NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC FQGSHIPYT FGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0606] SEQ ID No.:12(AM-VL1)

[0607] DVVMTQSPLSLPVTLGQPASISCRSS QSIVYSNGNTY LNWFQQRPGQSPRRLIY RVS NRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC FQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0608] SEQ ID No.: 13 (AM-VL2-E40)

[0609] DVVMTQSPLSLPVTLGQPASISCRSS QSIVYSNGNTY LEWFQQRPGQSPRRLIY RVS NRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC FQGSHIPYT FGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0610] SEQ ID NO: 32 (Adelizumab heavy chain)

[0611] QVQLVQSGAEVKKPGSSVKVSCKAS GYTFSRYW IEWVRQAPGQGLEWIGE ILPGSGST NYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYC TEGYEYDGFDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0612] SEQ ID NO: 33 (Adrelizumab light chain)

[0613] DVVLTQSPLSLPVTLGQPASISCRSS QSIVYSNGNTY LEWYLQRPGQSPRLLIY RVS NRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC FQGSHIPYT FGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0614] Example 2 – Effect of selected anti-ADM antibodies on anti-ADM biological activity

[0615] Selected ADM antibodies were tested for their effects on ADM bioactivity in a human recombinant adrenomedullin receptor cAMP functional assay (adrenomedullin bioassay).

[0616] Targeting in a human recombinant adrenomedullin receptor cAMP functional assay (adrenomedullin bioassay) Testing for antibodies to human or mouse adrenomedullin

[0617] Material: Cell line CHO-K1, receptor adrenomedullin (CRLR + RAMP3) receptor cell line accession number: CRLR: U17473; RAMP3: AJ001016

[0618] CHO-K1 cells expressing human recombinant adrenomedullin receptor (FAST-027C) grown in antibiotic-free medium prior to the assay were detached by gentle washing with PBS-EDTA (5 mM EDTA), recovered by centrifugation, and resuspended in assay buffer (KRH: 5 mM KCl, 1.25 mM MgSO 4 , 124 mM NaCl, 25 mM HEPES, 13.3 mM glucose, 1.25 mM KH 2 PO 4 , 1.45 mM CaCl 2 , 0.5 g / l BSA).

[0619] Dose response curves were performed in parallel with the reference agonist (hADM or mADM).

[0620] Antagonist assay (96-well):For antagonist assays, 6 μl of reference agonist (human (5.63 nM) or mouse (0.67 nM) adrenomedullin) was mixed with 6 μl of test sample at different antagonist dilutions; or with 6 μl of buffer. After 60 min at room temperature, 12 μl of cells (2,500 cells / well) were added. The plate was incubated at room temperature for 30 min. After adding lysis buffer, the percentage of ΔF was estimated using the HTRF kit from Cis-Bio International (Cat. No. 62AM2PEB) according to the manufacturer's instructions, using hADM 22-52 as the reference antagonist.

[0621] Antibody testing for cAMP-HTRF assay

[0622] Antagonist activity of anti-h-ADM antibodies (NT-H, MR-H, CT-H) was tested in the presence of 5.63 nM human ADM1-52 in a human recombinant adrenomedullin receptor (FAST-027C) cAMP functional assay using the following final antibody concentrations: 100 µg / ml, 20 µg / ml, 4 µg / ml, 0.8 µg / ml, 0.16 µg / ml.

[0623] Antagonist activity of anti-m-ADM antibodies (NT-M, MR-M, CT-M) was tested in the presence of 0.67 nM mouse ADM 1-50 in a human recombinant ADM receptor (FAST-027C) cAMP functional assay using the following final antibody concentrations: 100 µg / ml, 20 µg / ml, 4 µg / ml, 0.8 µg / ml, 0.16 µg / ml. Data for relative inhibition were plotted against antagonist concentration (see Figures 2 a to 2 l). The maximum inhibition caused by each antibody is given in Table 3.

[0624]

[0625] Example 3 - Stabilization of hADM by anti-ADM antibodies

[0626] The hADM immunoassay was used to test the stabilizing effect of human ADM antibodies on human ADM.

[0627] Immunoassay for the Quantification of Human Adrenomedullin

[0628] The technique used was an acridinium ester-labeled sandwich-coated tube luminescent immunoassay.

[0629] Labeled compound (tracer):100 µg (100 µl) of CT-H (1 mg / ml in PBS, pH 7.4, AdrenoMed AG Germany) was mixed with 10 µl of acridine NHS ester (1 mg / ml in acetonitrile, InVent GmbH, Germany) (EP 0353971) and incubated at room temperature for 20 min. The labeled CT-H was incubated in Bio-Sil ® Purification was performed by gel filtration HPLC on a SEC 400-5 (Bio-Rad Laboratories, Inc., USA). The purified CT-H was diluted in (300 mmol / L potassium phosphate, 100 mmol / L NaCl, 10 mmol / L Na-EDTA, 5 g / L bovine serum albumin, pH 7.0). The final concentration was about 800.000 relative light units (RLU) of labeled compound (about 20 ng labeled antibody) per 200 µL. Acridinium ester chemiluminescence was measured using an AutoLumat LB 953 (Berthold Technologies GmbH & Co. KG).

[0630] 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 h at room temperature. After blocking with 5% bovine serum albumin, the tubes were washed with PBS, pH 7.4, and dried under vacuum.

[0631] calibration: The assay was calibrated using dilutions of hADM (BACHEM AG, Switzerland) in 250 mmol / L NaCl, 2 g / L Triton X-100, 50 g / L bovine serum albumin, 20 tablets / L protease inhibitor cocktail (Roche Diagnostics AG, Switzerland).

[0632] hADM immunoassay: 50 µl of sample (or calibrator) was pipetted into the coated tube and after adding labeled CT-H (200 µl), the tube was incubated for 4 h at 4°C. Unbound tracer was removed by washing 5 times (1 ml each) with washing solution (20 mM PBS, pH 7.4, 0.1% Triton X-100).

[0633] The chemiluminescence bound to the tube is measured using LB 953: Figure 3 A typical hADM dose / signal curve and a hADM dose-signal curve in the presence of 100 µg / mL antibody NT-H are shown. NT-H did not affect the hADM immunoassay described.

[0634] 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). The remaining hADM was quantified using the hADM immunoassay described above.

[0635] Figure 4 The stability of hADM in human plasma (citrate) in the absence and presence of NT-H antibody is shown. The half-life of hADM alone is 7.8 h, in the presence of NT-H the half-life is 18.3 h (2.3-fold higher stability).

[0636] Example 4 – Sepsis mortality

[0637] a) Early treatment of sepsis

[0638] Animal model: Male C57Bl / 6 mice (Charles River Laboratories, Germany) aged 12-15 weeks were used for the study. Peritonitis was induced surgically under light isoflurane anesthesia. An incision was made in the left upper quadrant of the abdominal cavity (normal location of the cecum). The cecum was exposed and a tight ligature was placed around the cecum, with the suture away from the access to the small intestine. A puncture wound was made in the cecum with a 24-gauge needle, and a small amount of cecal contents was squeezed out through the wound. The cecum was replaced into the abdominal cavity and the laparotomy site was closed. Finally, the animals were returned to their cages with free access to food and water. 500 μl of saline was given subcutaneously as rehydration.

[0639] Administration and Dosage of Compounds (NT-M, MR-M, CT-M): Mice were treated immediately after CLP (early treatment). CLP is the abbreviation for cecal ligation and puncture (CLP).

[0640] Research Group: The three compounds were tested against vehicle and against control compound treatment. Each group contained 5 mice for blood draw after 1 day for BUN (serum urea nitrogen test). An additional 10 mice per group were followed over a 4-day period.

[0641] Group Treatment (10µl / g body weight) Dosage / Follow-up:

[0642] 1 NT-M, 0.2 mg / ml, survival rate within 4 days

[0643] 2 MR-M, 0.2 mg / ml, survival rate within 4 days

[0644] 3 CT-M, 0.2 mg / ml, survival rate within 4 days

[0645] 4 Non-specific mouse IgG, 0.2 mg / ml, survival rate within 4 days

[0646] 5 Control – PBS, 10 μl / g body weight, survival rate within 4 days

[0647] Clinical Chemistry: Blood urea nitrogen (BUN) concentration was measured at baseline and on day 1 after CLP to assess renal function. Blood samples were obtained from the cavernous sinus with a capillary tube under light ether anesthesia. Measurements were performed using an AU 400 Olympus multifunction analyzer. The 4-day mortality and mean BUN concentrations are given in Table 4.

[0648]

[0649] As can be seen from Table 4, NT-M antibody greatly reduced mortality. When treated with NT-M antibody, 70% of the mice survived after 4 days. When treated with MR-M antibody, 30% of the animals survived after 4 days, and when treated with CT-M antibody, 10% of the animals survived after 4 days. In contrast, when treated with nonspecific mouse IgG, all mice died after 4 days. The same results were obtained in a control group in which PBS (phosphate buffered saline) was administered to mice. Renal function is assessed using a blood urea nitrogen or BUN test to help diagnose kidney disease and monitor patients with acute or chronic renal dysfunction or failure. The results of the S-BUN test revealed that NT-M antibody was most effective in protecting the kidneys.

[0650] b) Late treatment of sepsis

[0651] Animal Model: Male C57Bl / 6 mice (Charles River Laboratories, Germany) aged 12-15 weeks were used for the study. Peritonitis was induced surgically under light isoflurane anesthesia. An incision was made in the left upper quadrant of the abdominal cavity (normal location of the cecum). The cecum was exposed and a tight ligature was placed around the cecum, with the suture away from the access to the small intestine. A puncture wound was made in the cecum with a 24-gauge needle, and a small amount of cecal contents was squeezed out through the wound. The cecum was replaced into the abdominal cavity and the laparotomy site was closed. Finally, the animals were returned to their cages with free access to food and water. 500 μl of saline was given subcutaneously as rehydration.

[0652] Administration and Dosage of Compound (NT-M FAB2): NT-M FAB2 was tested against vehicle and against control compound treatment. Treatment was performed after sepsis had fully developed 6 hours after CLP (late treatment). Each group contained 4 mice and was followed over a 4-day period.

[0653] Group Treatment (10µl / g body weight) Dosage / Follow-up:

[0654] 1 NT-M, FAB2 0.2 mg / ml, survival rate within 4 days

[0655] 2 Control nonspecific mouse IgG, 0.2 mg / ml, survival rate within 4 days

[0656] 3 Carrier: - PBS, 10 μl / g body weight, survival rate within 4 days

[0657]

[0658] As can be seen from Table 5, NT-M FAB 2 antibody greatly reduced the mortality rate. When treated with NT-M FAB 2 antibody, 75% of the mice survived after 4 days. In contrast, when treated with nonspecific mouse IgG, all mice died after 4 days. The same results were obtained in a control group in which PBS (phosphate buffered saline) was administered to mice.

[0659] Example 5 - Administration of NT-H in healthy subjects

[0660] The study was conducted as a randomized, double-blind, placebo-controlled study in healthy male subjects using a single escalating dose of NT-H antibody (0,5 mg / kg in group 1, 2 mg / kg in group 2, 8 mg / kg in group 3) administered as intravenous (iv) infusion in 3 consecutive groups of 8 healthy male subjects per group (n=6 active per group, n=2 placebo). The main inclusion criteria were written informed consent, age 18-35 years, agreement to use a reliable form of contraception and BMI between 18 and 30 kg / m². Subjects received a single dose of iv NT-H antibody (0.5 mg / kg; 2 mg / kg; 8 mg / kg) or placebo by slow infusion over a 1 hour period in the study unit. There were no differences in baseline ADM values ​​among the 4 groups. The median ADM value 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 in healthy individuals, the ADM value increased rapidly within the first 1.5 hours after NT-H antibody administration, then reached a plateau and slowly decreased ( Figure 6 ).

[0661] Example 6 - Methods for measuring DPP3 protein and DPP3 activity

[0662] Antibody Generation and Determination of DPP3 Binding Ability: Several murine antibodies were generated and screened for their ability to bind human DPP3 in a specific binding assay (see Table 6).

[0663] Peptides / conjugates for immunization: See Table 6. DPP3 peptides for immunization were synthesized (JPT Technologies, Berlin, Germany) with an additional N-terminal cysteine ​​residue (if cysteine ​​is not present in the selected DPP3 sequence) for coupling the peptide to bovine serum albumin (BSA). The peptide was covalently linked to BSA using Sulfolink coupling gel (Perbio-science, Bonn, Germany). The coupling procedure was performed according to the manual of Perbio. Recombinant GST-hDPP3 was produced by USBio (United States Biological, Salem, MA, USA).

[0664] Immunization of mice, immune cell fusion and screening:Balb / c mice were injected intraperitoneally (ip) with 84 µg GST-hDPP3 or 100 µg DPP3 peptide-BSA-conjugate (emulsified in TiterMax Gold adjuvant) on day 0, 84 µg or 100 µg (emulsified in Freund's complete adjuvant) on day 14, and 42 µg or 50 µg (in Freund's incomplete adjuvant) on days 21 and 28. On day 49, the animals received an intravenous (iv) injection of 42 µg GST-hDPP3 or 50 µg DPP3 peptide-BSA-conjugate dissolved in saline. Three days later, the mice were sacrificed and immune cell fusion was performed.

[0665] Splenocytes from immunized mice were fused with cells of the myeloma cell line SP2 / 0 using 1 ml of 50% polyethylene glycol at 37°C for 30 s. After washing, cells were seeded in 96-well culture plates. Hybrid clones were selected by growth in HAT medium [RPMI 1640 medium supplemented with 20% fetal bovine serum and HAT supplement]. One week later, the HAT medium was replaced with HT medium, passaged three times, and then returned to normal cell culture medium. Two weeks after fusion, cell culture supernatants were first screened for recombinant IgG antibodies that bind to DPP3. Therefore, 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 for 2 h at room temperature. After washing, 50 µl / well of POD-rabbit anti-mouse IgG antibody was added and incubated for 1 h at RT. After the next wash step, 50 µl of chromogen solution (3.7 mM o-phenylenediamine, 0.012% H 2 O 2 ), incubate at RT for 15 min and stop the color reaction by adding 50 µl 4N sulfuric acid. Absorbance is measured at 490 nm.

[0666] Microcultures that tested positive were transferred to 24-well plates for propagation. After retesting, selected cultures were cloned and recloned using limiting dilution techniques and subtyped.

[0667] Mouse monoclonal antibody production

[0668] Antibodies raised against GST-tagged human DPP3 or DPP3 peptide were generated by standard antibody generation methods ( Marx et al., 1997 ) and purified by protein A. The antibody purity was ≥ 90% according to SDS gel electrophoresis analysis.

[0669] Characterization of antibodies – binding to hDPP3 and / or immunizing peptides

[0670] To analyze the binding capacity of different antibodies and antibody clones to DPP3 / immunizing peptides, binding assays were performed:

[0671] 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 a high-binding microtiter plate (96-well polystyrene microplate, Greiner Bio-Oneinternational AG, Austria, 1 µg / well in coupling buffer [50 mM Tris, 100 mM NaCl, pH 7,8], at RT for 1 h). After blocking with 5% bovine serum albumin, the microplate was vacuum dried.

[0672] Labeling procedure (tracer): 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 acridine NHS ester (1 mg / ml in acetonitrile; InVent GmbH, Germany; EP 0353 971) and incubated for 30 min at room temperature. The labeled anti-DPP3 antibodies were purified by gel filtration HPLC on Shodex Protein 5 µm KW-803 (Showa Denko, Japan). The purified labeled antibodies were lysed in assay buffer (50 mmol / l potassium phosphate, 100 mmol / l NaCl, 10 mmol / l Na 2 -EDTA, 5 g / l bovine serum albumin, 1 g / l mouse IgG, 1 g / l bovine IgG, 50 µmol / l aminopeptidase inhibitor, 100 µmol / l leupeptin, pH 7.4). The final concentration is about 5-7*10 per 200 µl. 6 Relative light units (RLU) of labeled compounds (approximately 20 ng labeled antibody). Acridinium ester chemiluminescence was measured using a Centro LB 960 luminometer (Berthold Technologies GmbH & Co. KG).

[0673] hDPP3 binding assay: 200 µl of labeled and diluted detection antibody (tracer) was loaded into the plate and incubated for 2-4 h at 2-8°C. Unbound tracer was removed by washing 4 times with 350 µl wash solution (20 mM PBS, pH 7.4, 0.1% Triton X-100). Well-bound chemiluminescence was measured using a Centro LB 960 luminometer (Berthold Technologies GmbH & Co. KG).

[0674] Characterization of antibodies – hDPP3 inhibition assay

[0675] To analyze the DPP3 inhibitory capacity of different antibodies and antibody clones, a DPP3 activity assay was performed using known procedures (Jones et al., 1982). Recombinant GST-tagged hDPP3 was incubated in assay buffer (25 ng / ml GST-DPP3 in 50 mM Tris-HCl, pH 7,5, and 100 µM ZnCl 2 ) and 200 µl of this solution was incubated with 10 µg of the corresponding antibody at room temperature. After a pre-incubation of 1 h, the fluorogenic substrate Arg-Arg-βNA (20 µl, 2 mM) was added to the solution and the generation of free βNA was monitored over time at 37 °C using a Twinkle LB 970 microplate fluorimeter (Berthold Technologies GmbH & Co. KG). The fluorescence of βNA was detected by excitation at 340 nm and measurement of emission at 410 nm. The slope of the fluorescence increase (in RFU / min) was calculated for the different samples. The slope of GST-hDPP3 using the buffer control was assigned to 100% activity. The inhibitory capacity of a possible capture binder was defined as the reduction in GST-hDPP3 activity caused by incubation with the capture binder, expressed in percentage.

[0676] The following table presents a portion of the obtained antibodies and their binding rate expressed in relative light units (RLU) and their relative inhibitory capacity (%; Table 6). Monoclonal antibodies generated against the following DPP3 regions were selected for their ability to bind recombinant DPP3 and / or immunizing peptides and for their inhibitory potential.

[0677] All antibodies raised against the full-length form of GST-tagged recombinant hDPP3 showed strong binding to immobilized GST-tagged hDPP3. Antibodies raised against the SEQ ID NO.: 35 peptide also bound to GST-hDPP3. The SEQ ID NO.: 35 antibody also bound strongly to the immunizing peptide.

[0678]

[0679] The development of a luminescent immunoassay for quantifying DPP3 protein concentration (DPP3-LIA) as well as an enzyme capture activity assay for quantifying DPP3 activity (DPP3-ECA) has been recently described ( Rehfeld et al., 2019. JALM 3(6):943- 953 ), which is incorporated herein by reference in its entirety.

[0680] Example 7 - DPP3 in septic shock and cardiogenic shock

[0681] DPP3 concentrations were measured in the plasma of patients with sepsis / septic shock and cardiogenic shock and were correlated with their short-term mortality.

[0682] a) Study Group – Sepsis / Septic Shock

[0683] DPP3 was measured in 574 plasma samples from patients in the Adrenomedullin and Outcomes in Severe Sepsis and Septic Shock (AdrenOSS-1) study. AdrenOSS-1 was 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). Septic shock was diagnosed in 292 patients.

[0684] b) Study Group – Cardiogenic Shock

[0685] Plasma samples from 108 patients diagnosed with cardiogenic shock were screened for DPP3. Blood was drawn within 6 hours of detection of cardiogenic shock. Mortality was followed for 7 days.

[0686] hDPP3 Immunoassay: Immunoassays (LIA) or enzymatic activity assays (ECA) that detect the amount of human DPP3 (LIA) or the activity of human DPP3 (ECA), respectively, are used to measure DPP3 levels in patient plasma. Antibody immobilization, labeling, and incubation as described by Rehfeld et al. ( Rehfeld et al., 2019. JALM 3(6):943-953 ) as described in .

[0687] result: Short-term patient survival in patients with sepsis was associated with DPP3 plasma concentrations at admission. Patients with DPP3 plasma concentrations above 40.5 ng / mL (third quartile) had an increased risk of death compared with patients with DPP3 plasma concentrations below this threshold ( Figure 7 A). Applying this cutoff value to a subgroup of patients with septic shock revealed an even more significant short-term mortality risk associated with high DPP3 plasma concentrations ( Figure 7B). When the same cutoff value was used for patients with cardiogenic shock, an increased risk of short-term mortality within 7 days was also observed in patients with high DPP3 ( Figure 7 C).

[0688] Example 8 – NT-ADM antibodies in patients with septic shock (AdrenOSS-2)

[0689] AdrenOSS-2 is a double-blind, placebo-controlled, randomized, multicenter, proof-of-concept and dose-finding Phase II clinical trial to investigate the safety, tolerability, and efficacy of an N-terminal ADM antibody called aderizumab 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 bio-ADM concentrations > 70 pg / mL were randomized (2:1:1) to placebo (n=152), aderezumab 2 ng / kg (n=72), or aderezumab 4 ng / kg (n=77) using a single intravenous infusion over approximately 1 hour. The all-cause mortality rate within 28 (90) days of enrollment was 25.8% (34.8%). The mean age was 68.4 years, and 61% were male. For the per-protocol analysis, n=294 patients remained eligible, and the 14-day all-cause mortality rate was 18.5%.

[0690] A trend toward lower short-term mortality (14 days after admission) was observed in patients treated with aderezumab (both doses combined, per-protocol population) compared with placebo (hazard ratio (HR) 0.701 [0.408-1.21], p=0.100) ( Figure 8 Surprisingly, the treatment effect was more significant in patients whose DPP3 concentration was less than 50 ng / mL at admission (n=244, HR 0.426, p=0.007) ( Fig. 9 ), while in patients with elevated DPP3 (greater than 50 ng / mL, n=44), results were comparable between adeleizumab and placebo (HR 1.69, p=0.209) ( Fig.10 ).

[0691] The treatment effects (14-day mortality) for different DPP3 thresholds are summarized in Table 7.

[0692]

[0693] Example 9 : Therapeutic effect in patients with bio-ADM below 70 pg / mL

[0694] In order to show that anti-ADM treatment is also effective in patients with bio-ADM levels below 70 pg / mL, it was investigated in the AdrenOSS-2 study cohort whether patients in the included patient population (all with bio-ADM >70 pg / mL) showed different treatment effects according to their pre-dose bio-ADM concentrations. No such interaction was detected, in other words, meaning that for all patients, a beneficial treatment effect could be detected regardless of their pre-dose bio-ADM concentrations. This finding strongly suggests that treatment is also effective in patients with bio-ADM levels below 70 pg / mL, with the possible (and acceptable) limitation that this effect might not be expected in patients with bio-ADM concentrations within the normal healthy range.

[0695] P-values ​​for the interaction between treatment and pre-dose bio-ADM:

[0696] • Per-protocol population: p=0.279

[0697] • Per regimen population / pre-dose DPP3 < 70 ng / mL: p = 0.150

[0698] This is further illustrated in the Kaplan Meier plots, where the patient populations were separated according to their pre-dose bio-ADM concentrations (below / above the median bio-ADM value) (see Figures 11a and 11b).

[0699] Example 10 - Low DPP3 Threshold in Sepsis and Septic Shock (AdrenOSS-1)

[0700] The AdrenOSS-1 study as described in Example 7 was used to analyze DPP3 levels well below 50 ng / ml at baseline. The patients were followed to see whether they showed an increase in DPP3 plasma concentrations above 50 ng / ml in the following days.

[0701] result:

[0702] A statistical analysis of DPP3 plasma levels at baseline (day 1, DPP3.d1) in patients with septic shock (n=292) was performed to determine the threshold in which patients showed an increase in DPP3 plasma concentrations above 50 ng / ml on subsequent days. A DPP3 concentration of 50 ng / ml reflects a threshold above which patients a) exceed the upper limit of normal for DPP3 and b) have a high risk of organ dysfunction and mortality ( Blet et al., 2021. Crit Care 25(1): 61 ), and c) have been shown to have a lower therapeutic effect on the N-terminal ADM antibody aderizumab ( WO2021 / 170838 ).

[0703]

[0704] Different DPP3 thresholds at baseline (d1) were analyzed to determine the percentage of patients whose DPP3 plasma concentrations increased to above 50 ng / ml on the following days (days 2 and 3). Table 8 shows that the lower the DPP3 plasma concentration at baseline (DPP3.d1), the lower the percentage of patients whose DPP3 increased to above 50 ng / ml on the following days. In this septic shock population, 223 and 156 patients had DPP3 concentrations <50 or <30 ng / ml, respectively, at baseline. For the 156 patients with less than 30 ng / ml at baseline, 7 (4%) patients had an increase in DPP3 concentrations above the DPP3 threshold of 50 ng / ml on the following days. On the other hand, among the 67 septic shock patients with DPP3 plasma concentrations between 30 and 50 ng / ml at baseline, 15 (22.4%) patients had an increase in DPP3 plasma levels above 50 ng / ml on the following days. Therefore, a low DPP3 threshold (well below 50 ng / ml) is appropriate to show that DPP3 levels will increase during follow-up. Since patients with DPP3 concentrations above 50 ng / ml have been shown to have significantly reduced efficacy when treated with anti-ADM antibody (adrezumab), these results suggest that a lower threshold level of DPP3 at baseline can be used for treatment decisions for anti-ADM antibody (adrezumab) therapy in patients with septic shock. The low DPP3 concentration threshold at baseline (d1) ensures that the DPP3 pathological pathway (which is associated with high short-term organ dysfunction and mortality) is not the main pathway in the selected septic shock population. Therefore, this septic shock population with DPP3 concentrations below the above threshold range at baseline may have a higher treatment effect from, for example, anti-ADM antibody therapy (adrezumab).

[0705] In a second step, the septic shock population with bio-ADM plasma concentrations above 70 pg / ml was analyzed. Bio-ADM concentrations above 70 pg / ml have been associated with greater severity of sepsis and the development of organ dysfunction, including vasopressor / inotrope 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: 354Different DPP3 thresholds at baseline (d1) were analyzed to determine the percentage of patients whose DPP3 plasma concentrations increased to above 50 ng / ml on the following days (days 2 and 3). Table 9 again shows that the lower the DPP3 plasma concentration at baseline (DPP3.d1), the lower the percentage of patients with septic shock with high bio-ADM who had an increase in DPP3 above 50 ng / ml on the following days. In this septic shock and high bio-ADM population, 154 and 100 patients had DPP3 concentrations <50 or <30 ng / ml at baseline, respectively. For the 100 patients with less than 30 ng / ml at baseline, 4 (4%) patients had an increase in DPP3 concentrations above the DPP3 threshold of 50 ng / ml within the next 2 days. On the other hand, among 54 patients with septic shock whose baseline DPP3 plasma concentrations were between 30 and 50 ng / ml, 13 (24.1%) patients had an increase in DPP3 plasma levels above 50 ng / ml in the subsequent days.

[0706]

[0707] Example 11 - Lower DPP3 threshold in AdrenOSS-2 and efficacy of NT-ADM antibody therapy

[0708] To validate the findings from Example 10 and to prove the concept, different lower thresholds of DPP3 at baseline (Day 1) were evaluated in the septic shock population with high bio-ADM from the AdrenOSS-2 study cohort to evaluate the efficacy of anti-ADM antibody (adrezumab) therapy, and patients with DPP3 plasma values ​​above the different lower thresholds were excluded from the analysis. The efficacy of the anti-bio-ADM antibody therapy was specifically evaluated for the mortality endpoint in the placebo and treatment groups.

[0709] result:

[0710] A statistical analysis of DPP3 plasma levels in septic shock patients (n=298) with high bio-ADM (above 70 pg / ml) at baseline and within 144 h of follow-up was performed to determine the DPP3 threshold at baseline to assess the efficacy of anti-ADM antibody therapy. Patients above the corresponding DPP3 plasma threshold were excluded from the analysis. Different DPP3 plasma concentration thresholds were applied to the 28-day all-cause mortality assessment using Kaplan-Meier plots to compare 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 risk reduction of death caused by anti-ADM antibody therapy relative to placebo.

[0711] The baseline DPP3 plasma concentration thresholds used were 50 ng / ml, 40 ng / ml, 30 ng / ml, and 22 ng / ml. For each threshold, the number of patients excluded from the analysis of all-cause mortality was determined. For the thresholds of 50, 40, 30, and 22 ng / ml, 16%, 24%, 35%, and 51% of the patients were excluded from the analysis, respectively.

[0712] The HR for each DPP3 plasma concentration threshold at baseline was also determined to estimate the reduction in mortality with anti-ADM antibody therapy relative to placebo. The HRs were 0.606, 0.568, 0.309, and 0.258 for thresholds of 50, 40, 30, and 22 ng / ml, respectively. This analysis showed that the lower the DPP3 plasma concentration threshold, the greater the reduction in mortality in the treatment group. Similarly, the all-cause mortality analysis using Kaplan-Meier showed that the lower the DPP3 plasma concentration threshold, the more obvious and significant the reduction in mortality in the treatment group ( Fig. 12A -D).

[0713] For each DPP3 plasma concentration threshold at baseline, the percentage of patients who showed that DPP3 plasma concentration increased to above 50 ng / ml in the following 144h was also estimated. Similar to the results from AdrenOSS-1 research in Example 10, the results show that the lower the DPP3 plasma concentration threshold at baseline, the lower the percentage of patients who showed that DPP3 plasma levels were above 50 ng / ml thresholds in the following days. In this septic shock and high bio-ADM population, 249 and 195 patients had DPP3 concentrations at baseline respectively <50 or <30 ng / ml. For 195 patients who were lower than 30 ng / ml at baseline, 16 (8%) patients showed that DPP3 concentrations increased to above 50 ng / ml DPP3 thresholds in the following 6 days. On the other hand, among 54 patients with septic shock whose baseline DPP3 plasma concentrations were between 30 and 50 ng / ml, 11 (20.4%) patients had an increase in DPP3 plasma levels above 50 ng / ml in the following days. These results suggest that a lower DPP3 threshold (well below 50 ng / ml) is appropriate for guiding the use of anti-ADM antibody therapy and for selecting patients who are likely to benefit from such therapy.

[0714] Different thresholds of 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 and placebo groups to compare anti-ADM antibody therapy (adrezumab) with placebo. The baseline DPP3 plasma concentration thresholds used were 50 ng / ml and 30 ng / ml.

[0715] When all-cause mortality was assessed in the septic shock population (n=195) with DPP3 levels below the 30 ng / ml threshold, there was a surprisingly significant reduction in mortality in the anti-ADM antibody (adrezumab) treatment group compared with the placebo group ( Fig. 12C ). When only patients with DPP3 values ​​below 50 ng / ml were included, the same results were observed, with lower mortality in the treatment group than in the placebo group. Finally, when all-cause mortality from admission to 28 days was assessed in septic shock patients whose DPP3 levels were below 50 ng / ml at baseline and whose DPP3 levels remained low (<50 ng / ml) over the next 144 h, mortality was significantly lower in the treatment group than in the placebo group (Figure 13). Therefore, a low DPP3 threshold at baseline (well below 50 ng / ml) is most suitable for selecting patients who will benefit from the anti-ADM antibody therapy.

[0716] In summary, patients with DPP3 levels above the 30 ng / ml threshold at baseline have a higher chance of showing an increase in DPP3 plasma concentrations in the following days. Subsequent increases in DPP3 plasma concentrations above 50 ng / ml are associated with a lower efficacy of anti-ADM antibody therapy. Therefore, to stratify patients for anti-ADM antibody therapy, a lower threshold should be used, much lower than 50 ng / ml, preferably ≤ 40 ng / ml or in the range of 22 to 40 ng / ml, with a threshold of 30 ng / ml being the most preferred.

[0717] sequence

[0718] SEQ ID No.:1

[0719] GYTFSRYW

[0720] SEQ ID No.:2

[0721] ILPGSGST

[0722] SEQ ID No.:3

[0723] TEGYEYDGFDY

[0724] SEQ ID No.:4

[0725] QSIVYSNGNTY

[0726] Sequence "RVS" (not part of the sequence listing):

[0727] RVS

[0728] SEQ ID No.:5

[0729] FQGSHIPYT

[0730] SEQ ID No.:6(AM-VH-C)

[0731] QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK

[0732] SEQ ID No.:7(AM-VH1)

[0733] QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK

[0734] SEQ ID No.:8(AM-VH2-E40)

[0735] QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK

[0736] SEQ ID No.:9(AM-VH3-T26-E55)

[0737] QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK

[0738] SEQ ID No.:10(AM-VH4-T26-E40-E55)

[0739] QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK

[0740] SEQ ID No.:11(AM-VL-C)

[0741] DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0742] SEQ ID No.:12(AM-VL1)

[0743] DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0744] SEQ ID No.: 13 (AM-VL2-E40)

[0745] DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0746] SEQ ID No.: 14 (human ADM 1-21)

[0747] YRQSMNNFQGLRSFGCRFGTC

[0748] SEQ ID No.: 15 (human ADM 21-32)

[0749] CTVQKLAHQIYQ

[0750] SEQ ID No.: 16 (human ADM C-42-52)

[0751] CAPRSKISPQGY-CONH 2

[0752] SEQ ID No.: 17 (murine ADM 1-19)

[0753] YRQSMNQGSRSNGCRFGTC

[0754] SEQ ID No.: 18 (murine ADM 19-31)

[0755] CTFQKLAHQIYQ

[0756] SEQ ID No.: 19 (murine ADM C-40-50)

[0757] CAPRNKISPQGY-CONH 2

[0758] SEQ ID No.: 20 (mature human adrenomedullin (mature ADM); amidated ADM; bio-ADM): amino acids 1-52 or amino acids 95-146 of pro-ADM

[0759] YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVAPRSKISPQGY-CONH 2

[0760] SEQ ID No.: 21 (murine ADM 1-50)

[0761] YRQSMNQGSRSNGCRFGTCTFQKLAHQIYQLTDKDKDGMAPRNKISPQGY-CONH 2

[0762] SEQ ID No.: 22 (1-21 of human ADM):

[0763] YRQSMNNFQGLRSFGCRFGTC

[0764] SEQ ID No.: 23 (1-42 of human ADM):

[0765] YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVA

[0766] SEQ ID No.: 24 (aa 43 – 52 of human ADM)

[0767] PRSKISPQGY-NH2

[0768] SEQ ID No.: 25 (aa 1-14 of human ADM)

[0769] YRQSMNNFQGLRSF

[0770] SEQ ID No.: 26 (aa 1-10 of human ADM)

[0771] YQS

[0772] SEQ ID No.: 27 (aa 1-6 of human ADM)

[0773] YQ

[0774] SEQ ID No.: 28 (aa 1-32 of human ADM)

[0775] YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQ

[0776] SEQ ID No.: 29 (aa 1-40 of murine ADM)

[0777] YRQSMNQGSRSNGCRFGTCTFQKLAHQIYQLTDKDKDGMA

[0778] SEQ ID No.: 30 (aa 1-31 of murine ADM)

[0779] YRQSMNQGSRSNGCRFGTCTFQKLAHQIYQL

[0780] SEQ ID No.: 31 (proADM: 164 amino acids (22 – 185 of preproADM))

[0781] APHFL

[0782] SEQ ID NO: 32 (Adrelizumab heavy chain)

[0783] QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGEILPGSGSTNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0784] SEQ ID NO: 33 (Adelizumab light chain)

[0785] DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPRLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0786] SEQ ID No. 34 - Human DPP3 (amino acids 1 - 737)

[0787] MADTQYILPNDIGVSSLDCREAFRLLSPTERLYAYHLSRAAWYGGLAVLLQTSPEAPYIYALLSRLFRAQDPDQLRQHALAEGLTEEEYQAFLVYAAGVYSNMGNYKSFGDTKFVPNLPKEKLERVILGSEAAQQHPEEVRGLWQTCGELMFSLEPRLRHLGLGKEGITTYFSGNCTMEDAKLAQDFLDSQNLSAYNTRLFKEVDGEGKPYYEVRLASVLGSEPSLDSEVTSKLKSYEFRGSPFQVTRGDYAPILQKVVEQLEKAKAYAANSHQGQMLAQYIESFTQGSIEAHKRGSRFWIQDKGPIVESYIGFIESYRDPFGSRGEFEGFVAVVNKAMSAKFERLVASAEQLLKELPWPPTFEKDKFLTPDFTSLDVLTFAGSGIPAGINIPNYDDLRQTEGFKNVSLGNVLAVAYATQREKLTFLEEDDKDLYILWKGPSFDVQVGLHELLGHGSGKLFVQDEKGAFNFDQETVINPETGEQIQSWYRSGETWDSKFSTIASSYEECRAESVGLYLCLHPQVLEIFGFEGADAEDVIYVNWLNMVRAGLLALEFYTPEAFNWRQAHMQARFVILRVLLEAGEGLVTITPTTGSDGRPDARVRLDRSKIRSVGKPALERFLRRLQVLKSTGDVAGGRALYEGYATVTDAPPECFLTLRDTVLLRKESRKLIVQPNTRLEGSDVQLLEYEASAAGLIRSFSERFPEDGPELEEILTQLATADARFWKGPSEAPSGQA

[0788] SEQ ID No. 35 - Human DPP3 (amino acids 474 - 493 (N - Cys)) - Immunizing peptide with an additional N - terminal cysteine

[0789] CETVINPETGEQIQSWYRSGE

[0790] SEQ ID No. 36 - IGHV1 - 69*11

[0791] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARYYYYYGMDVWGQGTTVTVSS

[0792] SEQ ID No. 37 - HB3

[0793] QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYWGQGTTLTVSS

[0794] For the avoidance of doubt, in the amino acid sequences shown herein, the C-terminal groups -NH2 and -CONH2 refer equally to the C-terminal amide group (ie, a C-terminally amidated polypeptide).

Claims

1. An anti - adrenomedullin (ADM) antibody or anti - ADM antibody fragment or anti - ADM non - Ig scaffold for treating or preventing shock in a patient, wherein the patient is characterized by a dipeptidyl peptidase 3 (DPP3) level in a body fluid sample that is lower than a threshold, wherein the threshold of DPP3 is < / = 40 ng / ml 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 (amino acids 1 - 21) of ADM: YRQSMNNFQGLRSFGCRFGTC (SEQ ID No. 14).

2. The anti - adrenomedullin (ADM) antibody or anti - ADM antibody fragment or anti - ADM non - Ig scaffold for treating or preventing shock in a patient according to claim 1, wherein the shock is selected from shock caused by hypovolemia, cardiogenic shock, obstructive shock, and distributive shock, particularly cardiogenic shock or septic shock.

3. The anti - adrenomedullin (ADM) antibody or anti - ADM antibody fragment or anti - ADM non - Ig scaffold for treating or preventing shock in a patient according to claims 1 and 2, wherein: ● in the case of cardiogenic shock, the patient may have had acute coronary syndrome (such as acute myocardial infarction), or wherein the patient has heart failure (such as acute decompensated heart failure), myocarditis, arrhythmia, cardiomyopathy, valvular heart disease, aortic dissection with acute aortic stenosis, traumatic chordae tendineae rupture, or massive pulmonary embolism, or ● in the case of hypovolemic shock, the patient may have had a bleeding disorder, including gastrointestinal bleeding, trauma, vascular etiologies (such as rupture of abdominal aortic aneurysm, tumor erosion into large blood vessels), and spontaneous bleeding in the background of anticoagulant use, or non - bleeding disorders, including vomiting, diarrhea, renal loss, cutaneous deficit / insensible water loss (such as burns, heat stroke), or third - space fluid loss in the background of pancreatitis, cirrhosis, intestinal obstruction, trauma, or ● in the case of obstructive shock, the patient may have had cardiac tamponade, tension pneumothorax, pulmonary embolism, or aortic stenosis, or ● in the case of distributive shock, the patient may have septic shock, neurogenic shock, anaphylactic shock, or shock caused by adrenal crisis.

4. The anti - adrenomedullin (ADM) antibody or anti - ADM antibody fragment or anti - ADM non - Ig scaffold for treating or preventing shock in a patient according to claims 1 to 3, wherein the threshold of DPP3 in the body fluid sample of the patient is ≤ 30 ng / ml or in the range of 22 to 30 ng / mL.

5. The anti - adrenomedullin (ADM) antibody or anti - ADM antibody fragment or anti - ADM non - Ig scaffold for treating or preventing shock in a patient according to claim 4, wherein the threshold of DPP3 in the body fluid sample of the patient is ≤ 25 ng / ml or in the range of 22 to 25 ng / mL.

6. 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 according to claims 1 to 5, wherein the DPP3 level is determined by contacting the body fluid sample with a capture binding agent that specifically binds to DPP3.

7. 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 according to claim 6, wherein the capture binding agent is an antibody.

8. 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 according to claims 1 to 7, wherein the DPP3 protein level and / or active DPP3 level is determined and compared to a predetermined threshold value.

9. An anti-Adrenomedullin (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 according to claims 1 to 8, wherein the DPP3 level is determined using an immunoassay, in particular a sandwich immunoassay.

10. An anti-adrenomedullin (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 according to claims 1 to 8, wherein the DPP3 level is DPP3 activity, wherein the method for determining DPP3 activity in a body fluid sample of the subject comprises the steps of: ● contacting the sample with a capture binding agent that specifically binds to full-length DPP3, ● isolating DPP3 bound to the capture binder, ● adding a substrate of DPP3 to the isolated DPP3, • Quantification of the DPP3 activity by measuring and quantifying the conversion of a DPP3 substrate.

11. 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 according to claims 1 to 10, wherein the patient is further characterized by an ADM-NH2 level above a threshold.

12. An anti-adrenomedullin (ADM) antibody or an anti-ADM antibody fragment or an anti-ADM non-Ig scaffold for treating or preventing shock in a patient according to claim 11, wherein the threshold value of the ADM-NH2 in the patient's body fluid sample is between 40 and 100 pg / mL, more preferably between 50 and 90 pg / mL, even more preferably between 60 and 80 pg / mL, and most preferably the threshold value is 70 pg / mL.

13. The anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for treating or preventing shock in a patient according to claim 12, wherein the threshold value of the ADM-NH2 in the body fluid sample of the patient is 70 pg / mL.

14. 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 according to claims 1 to 13, wherein the body fluid is selected from whole blood, plasma and serum.

15. 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 according to claim 14, wherein the body fluid is plasma.

16. An anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for treating or preventing shock in a patient according to claims 11-15, wherein the ADM-NH2 level is determined by contacting the body fluid sample with a capture binder that specifically binds to ADM-NH2.

17. 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 according to claim 16, wherein the capture binding agent is an antibody.

18. 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 according to claims 1 to 17, wherein the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold recognizes and binds to the N-terminal end (amino acid 1) of ADM-Gly and / or ADM-NH2.

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

20. 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 according to claims 1 to 19, wherein the antibody or fragment or scaffold blocks no more than 80%, preferably no more than 50% of the biological activity of ADM.

21. 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 according to claims 1 to 20, wherein the antibody or fragment is a monoclonal antibody or fragment thereof that binds to ADM, wherein the heavy chain comprises the following sequence: CDR1: SEQ ID NO: 1 GYTFSRYW CDR2: SEQ ID NO: 2 ILPGSGST CDR3: SEQ ID NO: 3 TEGYEYDGFDY And wherein the light chain comprises the following sequence: CDR1: SEQ ID NO: 4 QSIVYSNGNTY CDR2: RVS CDR3: SEQ ID NO: 5 FQGSHIPYT.

22. 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 according to claims 1 to 21, wherein the antibody or fragment comprises as VH region a sequence selected from the group consisting of: SEQ ID NO: 6 (AM-VH-C) QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYWG QGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SEQ ID NO: 7 (AM-VH1) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWG QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SEQ ID NO: 8 (AM-VH2-E40) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWG QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SEQ ID NO: 9 (AM-VH3-T26-E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SEQ ID NO: 10 (AM-VH4-T26-E40-E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK and comprises a sequence selected from the following sequences as the VL region: SEQ ID NO: 11 (AM-VL-C) DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 12 (AM-VL1) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 13 (AM-VL2-E40) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

23. An anti-adrenomedullin (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 according to claims 1 to 22, wherein the antibody or fragment comprises as a heavy chain the following sequence or a sequence with > 95% identity thereto: SEQ ID NO: 32 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGEILPGSGSTNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGT TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK and comprising as a light chain the following sequence or a sequence with > 95% identity thereto: SEQ ID NO: 33 DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPRLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGGGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

24. An anti-Adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for treating or preventing shock in a patient according to claims 1-23, wherein the anti-Adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold is conjugated to the N-terminal portion (amino acids 1-10) of ADM: YRQSMNNFQG (SEQ ID No. 26).

25. An anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold for use in therapy according to any one of claims 1 to 24, wherein the antibody or fragment or scaffold exhibits at least 10% affinity for ADM when measured by label-free surface plasmon resonance using a Biacore 2000 system. -7 The binding affinity of M.

26. An anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for treating or preventing shock in a patient according to claim 25, wherein the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold exhibits 1 x 10 -9 Up to 3 x 10 -9 Affinity between M.

27. 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 according to claims 1-26, wherein the anti-ADM antibody or anti-ADM antibody fragment is an IgG1 antibody.

28. A pharmaceutical preparation for treating or preventing shock in a patient, comprising the anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold according to any one of claims 1 to 27.

29. The pharmaceutical preparation for treating or preventing shock in a patient according to claim 28, wherein the pharmaceutical preparation is a solution, preferably a ready-to-use solution.

30. The pharmaceutical preparation for treating or preventing shock in a patient according to claims 28 and 29, wherein the pharmaceutical preparation is in a freeze-dried state.

31. A pharmaceutical preparation for treating or preventing shock in a patient according to claims 28 and 30, wherein the pharmaceutical preparation is administered intramuscularly.

32. A pharmaceutical preparation for treating or preventing shock in a patient according to claims 28 and 31, wherein the pharmaceutical preparation is administered intravascularly.

33. A pharmaceutical preparation for use in treating or preventing shock in a patient according to claims 28 and 32, wherein the pharmaceutical preparation is administered by infusion.

34. A pharmaceutical preparation for treating or preventing shock in a patient according to claims 28 and 33, wherein the pharmaceutical preparation is administered systemically.

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