RU2776811C2 - Therapy monitoring in treatment with binding agent against adrenomedullin (adm) - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, in particular to a method for therapy monitoring in a subject. The subject is undergoing treatment with an agent binding anti-adrenomedullin (hereinafter – ADM), selected from a group including an antibody, an antibody fragment and/or a non-Ig-frame binding to SEQ ID NO.1. A level of a fragment of pre-pro-adrenomedullin selected from mid-regional pro-adrenomedullin (MR-proADM), C-terminal pro-adrenomedullin (CT-proADM) and/or peptide of 20 N-terminal amino acids of pro-adrenomedullin (PAMP), or their fragments in biological fluid is determined. To determine the level of the fragment of pre-pro-adrenomedullin, at least one binding agent is bound to an area inside an amino acid sequence selected from SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, respectively. The level of the specified fragment of pre-pro-adrenomedullin is correlated with a clinical/medical condition of the subject and/or the risk of unfavorable outcome, and/or the need for regulation of therapeutic measures.

EFFECT: invention allows for monitoring of the risk of unfavorable outcome for a subject directly in the treatment with an antibody against ADM.

15 cl, 6 dwg, 2 tbl, 3 ex

Description

The technical field to which the invention belongs

The subject of the present invention is a method for monitoring therapy in a subject, wherein the subject is being treated with an anti-adrenomedullin (ADM) binder selected from the group consisting of an antibody, an antibody fragment, and/or a non-Ig scaffold, comprising determining the level of a pre-pro-adrenomedullin fragment, selected from the group consisting of mid-regional proadrenomedullin (MR-proADM), C-terminal proadrenomedullin (CT-proADM) and/or N-terminal 20 amino acid proadrenomedullin peptide (PAMP), or fragments thereof in a biological fluid obtained from said subject; and correlating the level of said pre-adrenomedullin fragment with the subject's clinical/medical condition and/or risk of adverse outcome and/or need to adjust therapeutic measures.

Description of the invention

ADM is a circulating peptide known to regulate vasodilation and vascular integrity. Elevated plasma concentrations of ADM have been described for several life-threatening conditions, including cardiovascular disease and septic shock. A method for detecting and quantifying biologically active ADM (bio-ADM) is described in WO2013072509. While monoclonal antibodies against the amidated C-terminus and the middle part of the bio-ADM were generated and used for immunoassay to quantify bioactive ADM in plasma.

In addition, it has been found that the administration of an anti-ADM antibody, or an anti-ADM antibody fragment, or an anti-ADM non-Ig scaffold that binds to ADM, can significantly reduce the risk of mortality in a patient having a severe acute illness or acute condition.

Recent studies have shown that induction of sepsis leads to an increase in plasma bio-ADM in animal experiments. Surprisingly, after administration of an anti-adrenomedullin (ADM) antibody, the apparent plasma concentration of ADM increased significantly faster and to a higher level than in control animals. Considering the huge molar excess of anti-ADM antibody compared to endogenous bio-ADM, it would be assumed that the measured plasma concentration of ADM after administration of anti-ADM antibody is actually mostly bio-ADM in complex with anti-ADM antibody. However, the disproportionate increase in plasma bio-ADM observed after anti-ADM antibody administration compared to control animals was not associated with worse outcome.

In fact, it has been found that, surprisingly, in anti-ADM antibody treatment, bio-ADM measurement is not suitable for monitoring the risk of adverse outcome for a subject. In contrast, measurement of fragments derived from an ADM precursor peptide to which said anti-ADM antibody does not bind is suitable for monitoring stimulation or downregulation of the ADM system under such conditions, since they are not directly affected by administration of the anti-ADM antibody, and such measurement time-dependently correlates with the clinical outcome of a patient who is on treatment with said anti-ADM antibody.

State of the art

The peptide adrenomedullin (ADM) was first described in 1993 ( Kitamura, K., et al. 1993. "Adrenomedullin: A Novel Hypotensive Peptide Isolated From Human Pheochromocytoma", Biochemical and Biophysical Research Communications, Vol. 192 (2), pp. .553-560 ) as a new antihypertensive peptide containing 52 amino acids, which was isolated from human pheochromocytoma; SEQ ID NO.1. In the same year, a cDNA encoding a 185 amino acid precursor peptide and the complete amino acid sequence of this precursor peptide was also described. The precursor peptide, which includes, inter alia, a 21 amino acid signal sequence at the N-terminus, is referred to as "preproadrenomedullin" (pre-proADM). In the present description, all amino acid positions indicated generally refer to pre-proADM, which contains 185 amino acids and has a sequence according to SEQ ID NO.2.

The mature adrenomedullin peptide is an amidated peptide (ADM-NH ₂ ) which contains 52 amino acids (SEQ ID NO.1) and which contains amino acids 95 to 146 of pre-proADM from which it is formed by proteolytic cleavage. Mature ADM, bio-ADM and ADM-NH ₂ are used as synonyms in this application and represent a molecule in accordance with SEQ ID NO.1.

To date, only a few fragments of the peptide fragments generated by the cleavage of pre-proADM have been studied substantially more precisely, in particular the physiologically active peptides adrenomedullin (ADM) and "PAMP", a peptide containing 20 amino acids (22-41) , which follow the 21 amino acid signal peptide in pre-proADM. The discovery and characterization of ADM in 1993 encouraged intense research activity, the results of which have been summarized in various review articles, and in the context of the present description, in particular, references are made to articles that can be found in the issue of Peptides, devoted in particular to , ADM ( Editorial, Takahashi, K. 2001. Peptides, Vol. 22: 1691 and Eto, T. 2001. Peptides Vol. 22: 1693-1711) . Another review is presented in ( Hinson, et al. 2000. Endocrine Reviews Vol. 21(2): 138-167) .

In addition, it has been found that the concentrations of ADM that can be measured in the bloodstream and other biological fluids in a number of pathological conditions are significantly higher than the concentrations found in healthy controls. Thus, the level of ADM in patients with congestive heart failure, myocardial infarction, kidney disease, hypertension, diabetes mellitus, in the acute phase of shock and in sepsis and septic shock is significantly increased, although to varying degrees. PAMP concentrations are also elevated in some of these pathological conditions, but plasma levels are lower compared to ADM ( Eto T. 2001. Peptides, Vol. 22: 1693-1711 ).

In addition, unusually high concentrations of ADM are known to occur in sepsis or in septic shock ( Eto et al. 2001. Peptides 22: 1693-1711; Hirata et al. 1996. Journal of Clinical Endocrinology and Metabolism 81 (4).) : 1449-1453; Ehlenz et al. 1997. Exp Clin Endocrinol Diabetes 105: 156-162; Tomoda et al. 2001. Peptides 22: 1783-1794; Ueda et al. 1999 Am. J. Respir. Crit. Care Med.160: 132-136; Wang et al. 2001. Peptides 22: 1835-1840 ). The data obtained are associated with typical hemodynamic changes, which are known to be typical events in the course of the disease in patients with sepsis and other severe syndromes, such as, for example, SIRS. Adrenomedullin plays a key role during the development of sepsis ( Wang, Shock 1998, 10(5):383-384; Wang et al. 1998. Archives of surgery 133(12): 1298-1304) and in numerous acute and chronic diseases ( Parlapiano et al. 1999. European Review for Medical and Pharmacological Sciences 3:53-61; Hinson et al. 2000 Endocrine Reviews 21(2):138-167 ).

In scientific studies carried out to date, it has been found, inter alia, that ADM can be considered as a multifunctional regulatory peptide. It is released into the bloodstream in part in an inactive, glycine-extended form ( Kitamura et al. 1998. Biochem. Biophys. Res. Commun. 244 (2): 551-555) . There is also a binding protein ( Pio et al. 2001. Journal of Biological Chemistry 276 (15): 12292-12300) that is specific for ADM and likely also modulates the effect of ADM.

In addition, the aforementioned another physiologically active PAMP peptide derived from pre-proADM was also found to exhibit an antihypertensive effect, even though it appears to have a different mechanism of action from that of ADM ( Eto et al. 2001. Peptides 22: 1693-1711 Hinson et al 2000 Endocrine Reviews 21 (2): 138-167 Kuwasako 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 Kangawa et al EP 0 622 458) .

Several methods have been described for measuring blood levels of ADM: ADM either directly or indirectly by determining a more stable fragment of its cognate precursor peptide. A method has recently been published describing an assay for measuring circulating mature ADM ( Marino et al. 2014. Crit Care 18: R34) .

Other methods for quantifying fragments derived from the ADM precursor have been described, such as MR-proADM measurement ( Morgenthaler et al. 2005. Clin Chem 51 (10): 1823-9) , PAMP ( Washimine et al. 1994. Biochem Biophys. Res Commun 202 (2): 1081-7) and CT-proADM ( EP 2 111 552) . A commercial time-resolved homogeneous fluoroimmunoassay is available for measuring MR-proADM in plasma on a fully automated system (BRAHMS MR-proADM KRYPTOR; BRAHMS GmbH, Hennigsdorf, Germany) ( Caruhel et al. 2009. Clin Biochem 42(7-8):725- 8) . Since these peptides are generated in stoichiometric ratio from the same precursor, their plasma levels correlate to a certain extent.

Plasma concentration of ADM is elevated in patients with heart failure and correlates with disease severity ( Hirayama et al. 1999. J Endocrinol 160: 297-303; Yu et al. 2001. Heart 86: 155-160) . High plasma ADM is an independent negative prognostic indicator in these subjects ( Poyner et al. 2002. Pharmacol Rev 54: 233-246) .

The role of MR-proADM in heart failure has been investigated in several studies. In the BACH study (Maisel et al. 2010. J. Am. Coll. cardiol. 55:2062-2076)MR-proADM had a high predictive value for death at 90 days, adding predictive value beyond natriuretic peptides. Follow-up data from the PRIDE study (Shah et al. 2012. EUR. Heart J. 33:2197-2205)confirmed the potential predictive role of MR-proADM; for patients, MR-proADM had the best area under the curve (AUC) for mortality at 1 year. Similarly, MR-proADM levels in patients with chronic heart failure (CHF) were highly correlated with disease severity, and elevated peptide levels were strongly associated with an increased risk of death at 12 months of follow-up (van Haehling et al. 2010. European Journal of Heart Failure 12: 484-491; Adlbrecht et al. 2009. European Journal of Heart Failure 11: 361-366).

MR-proADM was investigated during treatment in patients with acute decompensated heart failure ( Boyer et al. 2012. Congest Heart Fail 18(2):91-97) : in patients whose MR-proADM levels tended to increase during intensive therapy, signs associated with persistent congestion were found. Within 12-24 hours after therapy, patients with elevated levels of MR-proADM showed increased peripheral edema. Kaiser et al. measured MR-proADM in patients with univentricular hearts ( Kaiser et al. 2014. Europ J Heart Failure 16:1082-1088) . Levels in patients with Fontan circuit failure (showing ascites and peripheral edema) were significantly higher compared to patients without Fontan circuit failure. Moreover, Eisenhut has suggested that treatment that reduces adrenomedullin levels may reduce the severity and extent of alveolar edema in pneumonia and sepsis ( Eisenhut 2006. Crit Care 10: 418) .

The role of MR-proADM in the diagnosis and prognosis of sepsis has been explored in several studies. MR-proADM has been described as a biomarker to differentiate between septic and non-septic patients with SIRS ( Christ-Crain et al. 2005. Crit Care 9: R816-824; Angeletti et al. 2013. Clin Chem Lab Med 51: 1059-1067 ) . Moreover, several studies have reported that MR-proADM is a predictive biomarker in sepsis, severe sepsis, and septic shock ( Christ-Crain et al. 2005. Crit Care 9: R816-824; Suberviola et al. 2012. Swiss Med Wkly 142 : w13542; Guiginant et al. 2009. Intensive Care Med 35: 1859-1867; DE LA Torre-Prados et al. 2016. Minerva Anestesiol 82: 760-766; Andaluz-Ojeda et al. 2015. J Infect 71: 136- 139) .

WO-A1 2004/097423 describes the use of an anti-adrenomedullin antibody for the diagnosis, prognosis and treatment of cardiovascular disorders. Treatment of diseases by blocking the ADM receptor is also described in the art (e.g. WO-A1 2006/027147 , PCT/EP2005/012844), where said diseases can be sepsis, septic shock, cardiovascular diseases, infections, dermatological diseases, endocrinological diseases, metabolic diseases, gastroenterological diseases, cancer, inflammation, hematological diseases, respiratory diseases, musculoskeletal diseases, neurological diseases, urological diseases.

It has been found that administration of an anti-ADM antibody, or an anti-ADM antibody fragment, of a non-ADM Ig scaffold that binds to ADM, can significantly reduce the risk of mortality in a patient having a severe acute illness or condition ( WO2013/072510, WO2013072511, WO2013072512 , WO2013072513, WO2013072514) .

Detailed description of the invention

The subject of the present invention is a method for monitoring therapy in a subject, wherein the subject is being treated with a binder selected from the group consisting of an anti-adrenomedullin (ADM) antibody, an antibody fragment, and/or a non-Ig scaffold that binds to SEQ ID NO.1 (amino acid 1 -52), including:

- determination of the level of a fragment of pre-pro-adrenomedullin selected from the group including mid-regional pro-adrenomedullin (MR-proADM), C-terminal pro-adrenomedullin (CT-proADM) and/or a peptide of 20 N-terminal amino acids of pro-adrenomedullin (PAMP), or fragments of the specified , in the biological fluid obtained from the specified subject; and

- correlation of the specified level of the pre-adrenomedullin fragment, selected from the group including MR-proADM, CT-proADM and/or PAMP, with the clinical/medical condition of the subject and/or the risk of adverse outcome, and/or the need to adjust therapeutic measures, and

- at the same time, to determine the level of these fragments, at least one binding substance binds to a region within the amino acid sequence selected from the group consisting of SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, respectively.

As used herein, the term "subject" refers to a living organism, whether or not a human being, preferably, in the present description, the subject is a human, and said subject suffers from diseases or conditions, for example, a chronic or acute disease or an acute condition. Such a disease may be selected from the group consisting of severe infections such as, for example, meningitis, systemic inflammatory response syndrome (SIRS), sepsis; other diseases such as diabetes, cancer, acute and chronic vascular diseases such as heart failure, myocardial infarction, stroke, atherosclerosis; shock, eg septic shock and organ dysfunction, eg kidney dysfunction, liver dysfunction, burns, surgery, trauma.

As used herein, the term "PAMP" includes both circulating forms of PAMP, namely, biologically inactive PAMP with C-terminal glycine extension (PAMP-Gly) and biologically active PAMP with amidated C-terminus (PAMP-amide).

Throughout the specification, "anti-ADM antibodies" or "anti-ADM antibody fragments" or "non-Ig anti-ADM scaffolds" according to the invention are capable of binding ADM and are thus directed against ADM and thus may be referred to as "anti-ADM antibodies". ADM”, “anti-ADM antibody fragments”, or “anti-ADM non-Ig scaffolds”.

In accordance with the present invention, the administration of an anti-ADM antibody or an anti-ADM antibody fragment that binds to ADM or a non-ADM Ig framework that binds to ADM is preferably a systemic application.

In another embodiment of the present application, fragments of pre-adrenomedullin that can be determined in a biological fluid are selected from the group including:

SEQ ID NO.3 (N-20 terminal proadrenomedullin peptide, PAMP): amino acids 22-41 from preproADM

ARLDVASEF RKKWNKWALS R

SEQ ID NO.4 (Mid-regional proadrenomedullin, MR-proADM): amino acids 45-92 from preproADM

ELRMSS SYPTGLADVK AGPAQTLIRP QDMKGASRSP EDSSPDAARI RV

SEQ ID NO.5 (C-terminal proadrenomedullin, CT-proADM): amino acids 148-185 from preproADM

RRR RRSLPEAGPG RTLVSSKPQA HGAPAPPSGS APHFL

In another embodiment of the present application, said pre-proadrenomedullin fragment having at least 5 amino acids is selected from the group consisting of MR-proADM (SEQ ID NO.4), CT-proADM (SEQ ID NO.5) and/or PAMP ( SEQ ID NO.3).

In one embodiment of the present application, the level of pre-proADM fragments and/or fragments thereof is determined using at least one binder, said binder binding to a region contained in the MR-proADM sequence (SEQ ID NO.4).

In another embodiment of the present application, the level of pre-pro-ADM fragments and/or fragments thereof is determined using at least one binder, said binder binding to a region contained in the CT-proADM sequence (SEQ ID NO.5).

In another embodiment of the present application, the level of fragments of pre-proADM and/or their fragments is determined using at least one binder, and the specified binder binds to the region contained in the sequence of PAMP (SEQ ID NO.3).

The object of the invention in a specific embodiment of the present application is a method in which the specified fragment can be selected from MR-proADM according to SEQ ID NO.4 and/or CT-proADM according to SEQ ID NO.5 and/or PAMP according to SEQ ID NO.3.

Another embodiment of the present application relates to a method according to the preceding embodiments, wherein the anti-ADM antibody for treating a subject that binds to the N-terminus, amino acids 1-21, of adrenomedullin is a human CDR-grafted antibody, or a fragment thereof. an antibody that binds to ADM, wherein the CDR-grafted human antibody or fragment of this antibody contains an antibody heavy chain (H-chain) containing:

SEQ ID NO.6:

GYTFSRYW

SEQ ID NO.7:

ILPGSGST

and/or

SEQ ID NO.8:

TEGYEYDGFDY

and/or additionally contains an antibody light chain (L-chain) containing:

SEQ ID NO.9:

QSIVYSNGNTY

SEQ ID NO.28: (not mentioned in sequence listing due to 3 amino acid length)

RVS

and/or

SEQ ID NO.10:

FQGSHIPYT.

In another specific embodiment of the present application, the anti-ADM antibody for treating the subject is a human monoclonal antibody that binds to ADM, or a fragment of this antibody, wherein the heavy chain contains at least one CDR selected from the group consisting of:

SEQ ID NO.6:

GYTFSRYW

SEQ ID NO.7:

ILPGSGST

SEQ ID NO.8:

TEGYEYDGFDY

and wherein the light chain contains at least one CDR selected from the group consisting of:

SEQ ID NO.9:

QSIVYSNGNTY

SEQ ID NO.28:

RVS

SEQ ID NO.10:

FQGSHIPYT.

In another embodiment of the present application, the anti-ADM antibody for treating the subject is a human monoclonal antibody that binds to ADM, or a fragment of this antibody, wherein the heavy chain contains the sequences

SEQ ID NO.6:

GYTFSRYW

SEQ ID NO.7:

ILPGSGST

SEQ ID NO.8:

TEGYEYDGFDY

and the light chain contains the sequences

SEQ ID NO.9:

QSIVYSNGNTY

SEQ ID NO.28:

RVS

SEQ ID NO.10:

FQGSHIPYT.

Another embodiment of the present application relates to the method of the previous embodiment, wherein said antibody or fragment for treatment is a human monoclonal antibody or a fragment thereof that binds to ADM, or a fragment of this antibody, wherein the heavy chain contains the sequences

CDR1: SEQ ID NO.6:

GYTFSRYW

CDR2: SEQ ID NO.7:

ILPGSGST

CDR3: SEQ ID NO.8:

TEGYEYDGFDY

and the light chain contains the sequences

CDR1: SEQ ID NO.9:

QSIVYSNGNTY

CDR2: SEQ ID NO.28:

RVS

CDR3: SEQ ID NO.10:

FQGSHIPYT.

Another embodiment of the present application relates to the method of the previous embodiment, wherein said antibody or fragment for treatment contains the following sequences as a VH region:

SEQ ID NO.11 (AM-VH-C):

QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILP

GSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFD

YWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN

SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKR

VEPC

SEQ ID NO.12 (AM-VH1):

QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRIL

PGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFD

YWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN

SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKR

VEPC

SEQ ID NO.13 (AM-VH2-E40):

QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRIL

PGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFD

YWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN

SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKR

VEPC

SEQ ID NO.14 (AM-VH3-T26-E55):

QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEIL

PGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFD

YWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN

SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKR

VEPC; or

SEQ ID NO.15 (AM-VH4-T26-E40-E55):

QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEIL

PGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFD

YWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN

SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKR

VEPC;

and contains the following sequence as a VL region:

SEQ ID NO.16 (AM-VL-C):

DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYR

VSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKLE

IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS

QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE

FROM

SEQ ID NO.17 (AM-VL1):

DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYR

VSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKL

EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN

SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG

EU

SEQ ID NO.18 (AM-VL2-E40):

DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRVSNR DSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSPVTYSLSSTLTLSSYKADYEKVYYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFP

Another embodiment of the present application relates to the method of the previous embodiment, wherein said antibody or fragment for treatment comprises the following sequence as a heavy chain:

SEQ ID NO.26:

QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGEILPGSGST NYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSAS TKGPSVFPLAPSSKSTSGGT AALGCLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK

and contains the following sequence as a light chain:

SEQ ID NO.27

DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPRLLIYRVSNRF SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKVHSSY

It should be understood that the level of a pre-adrenomedullin fragment selected from the group consisting of MR-proADM (SEQ ID NO.4), CT-proADM (SEQ ID NO.5) and/or PAMP (SEQ ID NO.3), also encompasses fragments thereof lacking at least one amino acid and said fragments being at least 5 amino acids in length, more preferably at least 10 amino acids, most preferably at least 15 amino acids in length.

One embodiment of the present application relates to the method of the previous embodiment, wherein the level of pre-adrenomedullin fragments (of at least 5 amino acids) is determined using a binder for said fragments (of at least 5 amino acids).

Another embodiment of the present application relates to the method of the previous embodiment, wherein the binder is selected from the group consisting of an antibody, an antibody fragment or a non-Ig scaffold that binds to fragments of pre-adrenomedullin, or fragments of the specified (at least 5 amino acids).

The biological fluid in accordance with the present application is a blood sample. The blood sample may be selected from the group consisting of whole blood, serum and plasma. In one embodiment of the present application, said sample is selected from the group consisting of citrated plasma, heparinized plasma, and EDTA-treated human plasma.

An antibody of the present invention is a protein containing one or more polypeptides substantially encoded by immunoglobulin genes that specifically bind an antigen. Recognized immunoglobulin genes also include the 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 a variety of immunoglobulin variable region genes. Full length immunoglobulin light chains are typically about 25 kDa or 214 amino acids in length. Full length immunoglobulin heavy chains are typically about 50 kDa or 446 amino acids in length. Light chains are encoded by a variable region gene at the NH ₂ terminus (about 110 amino acids long) and a kappa or lambda constant region gene at the COOH terminus. Heavy chains are similarly encoded by the variable region gene (about 116 amino acids long) and one of the other constant region genes.

The basic structural unit of an antibody is usually a tetramer, which consists of two identical pairs of immunoglobulin chains, each with one light and one heavy chain. In each pair, the light and heavy chain variable regions bind to the antigen, and the constant regions provide 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 (eg, Lanzavecchia et al. 1987. Eur. J. Immunol. 17: 105; Huston et al 1988 Proc Natl Acad Sci USA 85: 5879-5883 Bird et al 1988 Science 242: 423-426 Hood et al 1984 Immunology, Benjamin, NY, 2nd ed.; Hunkapiller and Hood 1986. Nature 323: 15-16) . The variable region of an immunoglobulin light or heavy chain comprises 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., US Department of Health and Human Services, 1983 ). As noted above, CDRs are primarily responsible for binding to an antigen epitope. An immune complex is an antibody, such as a monoclonal antibody, a chimeric antibody, a humanized or human antibody, or a functional antibody fragment specifically associated with an antigen.

Chimeric antibodies are antibodies whose light and heavy chain genes have been constructed, typically through genetic engineering, from immunoglobulin variable and constant region genes belonging to different species. For example, variable gene segments from a mouse monoclonal antibody can be fused to human constant segments such as kappa and gamma 1 or gamma 3. In one example, the therapeutic chimeric antibody is thus a fusion 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 be used, or the variable region can be generated by molecular methods. Methods for making chimeric antibodies are well known in the art, for example, see US Pat. No. 5,807,715. A "humanized" immunoglobulin is an immunoglobulin comprising a human framework region and one or more CDRs of a non-human immunoglobulin (such as murine, rat, or synthetic). A non-human immunoglobulin providing a CDR is referred to as a "donor", and a human immunoglobulin providing a scaffold is referred to as an "acceptor". In one embodiment, all CDRs in the humanized immunoglobulin are from a donor immunoglobulin. The constant regions need not be present, but if they are, they must be substantially identical to the constant regions of human immunoglobulin, i.e. must be identical, at least about 85-90%, for example, about 95% or more. Therefore, all parts of the humanized immunoglobulin, with the possible exception of the CDR, are essentially identical to the corresponding parts of the sequences of natural human immunoglobulin. A "humanized antibody" is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. The humanized antibody binds to the same antigen as the donor antibody that provides the CDR. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of amino acid substitutions taken from the donor framework. Humanized or other monoclonal antibodies may have additional conservative amino acid substitutions that do not substantially affect antigen binding or other immunoglobulin functions. Examples of conservative substitutions are gly, ala; val, ile, leu; asp, glu; asn, glin; ser, thr; lys, arg; and phe, tyr. Humanized immunoglobulins can be created using genetic engineering (for example, see US patent No. 5580089). A human antibody is an antibody in which the light and heavy chain genes are of human origin. Human antibodies can be obtained using methods known in the art. Human antibodies can be obtained by immortalizing a human B cell secreting the antibody of interest. Immortalization can be achieved, for example, by EBV infection or by fusing a human B cell with a myeloma or hybridoma cell to produce a trioma cell. Human antibodies can also be generated by phage display methods (see, for example, Doweret al., PCT Publication WO91/17271; McCaffertyet al., PCT Publication No. WO92/001047; and Winter, PCT Publication WO92/20791), or selected from a combinatorial library of human monoclonal antibodies (see the Morphosys website). Human antibodies can also be generated using transgenic animals carrying the human immunoglobulin gene (for example, see Lonberget al., PCT Publication WO93/12227; and Kucherlapati, PCT Publication WO91/10741).

Thus, the antibody may be in formats known in the art. Examples are human antibodies, monoclonal antibodies, humanized antibodies, chimeric antibodies, CDR-grafted antibodies. In a preferred embodiment, the antibodies of the present invention are recombinantly produced antibodies, such as, for example, IgG, a typical full-length immunoglobulin, or antibody fragments containing at least a heavy and/or light chain F-variable domain, such as, for example, chemically linked antibodies (antigen-binding fragments), including, but not limited to, Fab fragments, including Fab mini-antibodies, single-chain Fab antibodies, monovalent Fab antibodies with epitope tags, for example, Fab-V5Sx2; a divalent Fab (mini-antibody) dimerized with a CH3 domain; divalent Fab or polyvalent Fab, for example,formed by multimerization with a heterologous domain, for example, by dimerization of dHLX domains, eg Fab-dHLX-FSx2; F(ab')₂-fragments, scFv fragments, multimerized multivalent and/or multispecific scFv fragments, bivalent and/or bispecific diabodies, BITE^®(bispecific T-cell activator), trifunctional antibodies, polyvalent antibodies, e.g.,from a class other than G; single-domain antibodies, for example, nanobodies derived from camel or fish immunoglobulins, and many others.

In addition to antibodies, other biopolymer scaffolds for creating a target molecule complex are well known in the art and have been used to generate target-specific biopolymers. Examples are aptamers, spiegelmers, anticalins and conotoxins.

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

Non-Ig scaffolds can be protein scaffolds and can be used as antibody mimics because they are able to bind to ligands or antigens. The non-Ig scaffolds may be selected from the group consisting of tetranectin-based non-Ig scaffolds (e.g.,described in US 2010/0028995), fibronectin scaffolds (for example,described in EP 1266025; scaffolds based on lipocalin (eg., described in WO 2011/154420); ubiquitin scaffolds (eg., described in WO 2011/073214), transfer scaffolds (for example,described in US 2004/0023334), protein A frameworks (for example, described in EP 2231860), scaffolds based on ankyrin repeat (for example,described in WO 2010/060748), scaffolds based on microproteins, preferably cystine knot-forming microproteins) (for example,described in EP 2314308), Fyn SH3 domain-based frameworks (for example,described in WO 2011/023685), EGFR-A domain-based frameworks (for example,described in WO 2005/040229) and frameworks based on Kunitz domains (for example, described in EP 1941867).

One embodiment of the present application relates to the method of the previous embodiment, wherein the binder for treating the subject is an anti-ADM antibody, or an anti-adrenomedullin antibody fragment, or a non-Ig anti-ADM protein scaffold, wherein said antibody, or fragment thereof , or scaffold binds to the N-terminus, amino acids 1-21, adrenomedullin:

YRQSMNNFQGLRSFGCRFGTC; SEQ ID NO.19

Another embodiment of the present application relates to the method of the previous embodiment, wherein the binder for treating the subject is an anti-ADM antibody, or an anti-adrenomedullin antibody fragment, or a non-Ig anti-ADM protein scaffold, wherein said antibody, or fragment thereof, or the scaffold binds to the C-terminus, amino acids 42-52-amide, adrenomedullin:

APRSKISPQGY-NH ₂ ; SEQ ID NO.20

Another embodiment of the present application relates to the method of the previous embodiment, wherein the binding agent for treating the subject is an anti-ADM antibody, or an anti-adrenomedullin antibody fragment, or a non-Ig anti-ADM protein backbone, wherein said antibody or fragment backbone binds to mid-regional part, amino acids 21-42, adrenomedullin:

CTVQKLAHQIYQFTDKDKDNVA; SEQ ID NO.21

Another embodiment of the present application relates to the method according to the previous embodiments, in which the determination of the level of said fragments is performed at least once.

Another embodiment of the present application relates to a method in accordance with the preceding embodiments, in which the determination of the level of these fragments is performed at least once after the start of treatment with an anti-ADM antibody, or an anti-ADM antibody fragment, or a non-Ig anti-ADM protein scaffold. .

Another embodiment of the present application relates to a method in accordance with the preceding embodiments, in which the determination of the level of these fragments is performed more than once in one patient after the start of treatment with an anti-ADM antibody, or an anti-adrenomedullin antibody fragment, or a non-Ig anti-ADM protein scaffold.

Another embodiment of the present application relates to a method in accordance with the preceding embodiments, in which the determination of the level of these fragments is performed more than twice in one patient after the start of treatment with an anti-ADM antibody, or an anti-adrenomedullin antibody fragment, or a non-Ig anti-ADM protein scaffold.

Another embodiment of the present application relates to a method in accordance with the previous embodiments, in which the determination of the level of these fragments is carried out more than three times after the start of treatment with an anti-ADM antibody, or an anti-adrenomedullin antibody fragment, or a non-Ig anti-ADM protein framework.

Another embodiment of the present application relates to the method according to the previous embodiments, wherein the adverse outcome is selected from the group consisting of clinical deterioration, such as deterioration in organ function, and mortality.

The term "clinical deterioration" as used herein refers to worsening of symptoms (e.g., change in clinical parameters that determine the progression of the disease), need for hospitalization, or death, and can be assessed by a medical indicator (e.g., a system for assessing acute functional and chronic changes health status (APACHE, APACHE II)).

The term "organ function deterioration" according to the present invention includes deterioration in kidney function (WRF), deterioration in cardiovascular function, deterioration in liver function, and deterioration in respiratory function, and deterioration can be assessed by dynamic organ failure assessment (SOFA), multiple organ dysfunction assessment (MODS) or simplified assessment of acute physiological changes (SAPS, SAPS II).

Another embodiment of the present application relates to the method according to the previous embodiments, wherein said subject is suffering from sepsis or septic shock.

The clinical criteria for systemic inflammatory response syndrome (SIRS), sepsis and septic shock will be defined below:

1) Systemic inflammatory response syndrome (SIRS) characterized by at least two of the following symptoms:

- patients have hypotension (mean arterial pressure <65 mmHg)

- elevated serum lactate level >4 mmol/l

- blood glucose >7.7 mmol/l (in the absence of diabetes)

- Central venous pressure is not within 8-12 mm Hg. Art.

- urine output <0.5 ml × kg ^-1 × h ^-1

- central venous (superior vena cava) oxygen saturation <70%, or mixed venous <65%

- heart rate >90 bpm

- temperature <36°C or >38°C

- respiratory rate >20/min

- the number of white blood cells <4 or >12×10 ^9/ l (leukocytes); >10% young neutrophils.

2) Sepsis

- the presence of at least two of the symptoms referred to in point 1) and, in addition, the clinical suspicion of a new infection:

- patients have hypotension (mean arterial pressure <65 mmHg)

- elevated serum lactate level >4 mmol/l

- blood glucose >7.7 mmol/l (in the absence of diabetes)

- Central venous pressure is not within 8-12 mm Hg.

- urine output <0.5 ml × kg ^-1 × h ^-1

- central venous (superior vena cava) oxygen saturation <70%, or mixed venous <65%

- heart rate >90 bpm

- temperature <36°C or >38°C

- respiratory rate >20/min

- the number of white blood cells <4 or >12×10 ^9/ l (leukocytes); >10% young neutrophils,

and additionally clinical suspicion of a new infection:

- cough/phlegm/chest pain

- abdominal pain/bloating/diarrhea

- infection through a central catheter

- endocarditis

- dysuria

- headache with neck stiffness

- cellulitis/wound/infection of the joints

- positive microbiological tests for any infection.

3) Severe sepsis

Provided that sepsis is manifested in the patient and, in addition, there is a clinical suspicion of organ dysfunction, namely:

- systolic blood pressure <90/mean; <65 mmHg

- lactate >2 mmol/l

- bilirubin >34 µmol/l

- urine output <0.5 ml/kg/hour for 2 hours

- creatinine >177 µmol/l

- platelets <100×10 ^9/ l

- SpO ₂ >90% if O is not given.

4) Septic shock

There is at least one sign of target organ dysfunction as mentioned in 3). Septic shock is defined in the presence of refractory hypotension that does not respond to treatment, and when intravenous fluid alone is not enough to maintain the patient's blood pressure at a level that does not allow hypotension.

More recently, the definitions of sepsis and septic shock have been revised and updated by the Sepsis Definition Task Force ( Singer et al. 2016. JAMA 315 (8): 801-810) , which is incorporated herein by reference. Sepsis is defined as life-threatening organ dysfunction caused by an uncontrolled host response to infection, where the host's response to infection damages its own tissues and organs. Organ dysfunction can be identified as an acute change in total SOFA score ≥2 as a result of infection. The baseline SOFA score can be assumed to be zero in patients with no previously identified organ dysfunction. The SOFA score ≥2 reflects an overall risk of mortality of approximately 10% in the general population with suspected infection. Even in patients with moderate dysfunction, there may be further deterioration, highlighting the seriousness of the condition and the need for prompt and appropriate intervention if not already initiated.

Septic shock is a type of sepsis in which the underlying circulatory and cellular/metabolic disturbances are profound enough to significantly increase mortality. Patients with septic shock can be identified by the clinical design of sepsis with persistent hypotension requiring vasopressors to maintain MAP ≥65 mmHg. and having a lactate level >2 mmol/L (18 mg/dL) despite adequate volume replacement.

Table 1: Sequential (sepsis-related) scoring system for organ failure

System/Score 0 one 2 3 four Respiration PaO ₂ /FiO _2, mm Hg (kPa) ≥400 (53.3) <400 (53.3) <300 (40) <200 (26.7) with assisted ventilation <100 (13.3) with assisted ventilation Coagulation Platelets, ×10 ³ /µl ≥150 <150 <100 <50 <20 Liver Bilirubin, mg/dL (µmol/L) <1.2 (20) 1.2-1.9 (20-32) 2.0-5.9 (33-101) 6.0-11.9 (102-204) >12.0 (204) Cardiovascular parameters MAP≥ 70 mmHg MAP < 70 mmHg Dopamine<5 or dobutamine (any dose) ^b Dopamine 5.1-14, or epinephrine≤
0.1, or norepinephrine ≤ 0.1 ^b Dopamine>15, or epinephrine>
0.1, or norepinephrine>0.1 ^b central nervous system Glasgow Coma Scale ^c fifteen 13-14 10-12 6-9 <6 Renal parameters Creatine mg/dL (µmol/L) <1.2 (110) 1.2-1.9 (110-170) 2.0-3.4 (171-299) 3.5-4.9 (300-440) >5.0 (440) Urine output, ml/day <500 <200

Abbreviations: FIO ₂ , fraction of oxygen in inhaled air; MAP, mean arterial pressure; PaO ₂ , oxygen partial pressure.

^a adapted from Vincent et al. ²⁷

^b The dose of catecholamines is given as mcg/kg/min for at least 1 hour.

^c Glasgow Coma Scale score ranges from 3 to 15; the higher the score, the better the neurological function.

APACHE II ("Acute Functional and Chronic Health Change Assessment System II") is a disease severity classification system ( Knaus et al. 1985. Crit Care Med 13 (10): 818-29) , one of several for the intensive care unit. It is applied within 24 hours of the patient's admission to the intensive care unit: an integer from 0 to 71 is calculated based on several measurements; higher scores correspond to more severe illness and a higher risk of death.

SAPS II is a disease severity classification system ( Le Gall et al. 1993. JAMA 270: 2957-2963) . Standing for Simplified Assessment of Acute Physiological Changes, it is one of several Intensive Care Unit (ICU) assessment systems and was developed to measure disease severity in patients admitted to the ICU.

Another embodiment of the present application relates to the method according to the preceding embodiments, wherein said therapeutic measures are selected from the group consisting of fluid resuscitation, vasopressors/inotropes, renal replacement therapy, antibiotics, hydrocortisone, insulin, enteral nutrition/parenteral nutrition.

Another embodiment of the present application relates to a method according to the preceding embodiments, wherein said anti-ADM antibody or antibody fragment or non-Ig scaffold does not bind to a pre-pro-adrenomedullin fragment selected from the group consisting of MR-proADM, CT-proADM and/or PAMP.

Another embodiment of the present application relates to a method according to the preceding embodiments, wherein said pre-adrenomedullin fragment selected from the group consisting of MR-pro ADM, CT-proADM and/or PAMP is at least 5 amino acids in length. , respectively.

Another embodiment of the present application relates to a method in accordance with the previous embodiments, in which the level of the specified fragment of pre-adrenomedullin selected from the group including MR-proADM, CT-proADM and/or PAMP or their fragments of at least , 5 amino acids, determined by immunoassay using at least one binding agent selected from the group consisting of a binding agent for MR-proADM or a fragment thereof and/or for CT-proADM or a fragment thereof and/or for PAMP or a fragment thereof, respectively.

In a specific embodiment of the present application, an immunoassay is used to determine the level of MR-proADM and/or its fragments (having at least 5 amino acids), such immunoassay being a sandwich assay, preferably a fully automated assay.

In a specific embodiment, the present application immunoassay is used to determine the level of CT-proADM and/or its fragments (having at least 5 amino acids), while such an immunoassay is a sandwich assay, preferably a fully automated assay.

In a specific embodiment, the present application immunoassay is used to determine the level of PAMP and/or its fragments (having at least 5 amino acids), while such an immunoassay is a sandwich assay, preferably a fully automated assay.

In one of the embodiments of the application, the immunoassay that is used to determine the level of MR-proADM and/or CT-proADM and/or PAMP, respectively, may be a so-called POC test (at the point of treatment), which is a testing technology that allows perform testing in less than 1 hour next to the patient, and does not require a fully automated analysis system. One example of this technology is the immunochromatographic test technology.

In one application, such an immunoassay is a sandwich immunoassay using any type of detection technology, including, but not limited to, an enzyme label, a chemiluminescent label, an electrochemiluminescent label, preferably a fully automated assay.

In one embodiment, such an immunoassay is an enzyme-labeled sandwich assay. Examples of automated or fully automated assays include assays that can be used on one of the following systems: Roche Elecsys®, Abbott Architect®, Siemens Centauer®, Brahms Kryptor®, BiomerieuxVidas®, Alere Triage®.

A variety of immunoassays are known and can be used for the assays and methods of the present invention, these include: radioimmunoassays ("RIA"), homogeneous enzymatically enhanced immunoassays ("EMIT"), enzyme-linked immunosorbent assays ("ELISA"), apoenzyme reactivation immunoassay ("ARIS"), immunoassays using test strips (dipstick) and immunochromatographic assays.

In a specific embodiment of the invention, at least one of these two binders is labeled for detection.

Preferred detection methods include immunoassays in various formats such as, for example, radioimmunoassay (RIA), chemiluminescent and fluorescent immunoassay, enzyme-linked immunosorbent assay (ELISA), Luminex-based bead kits, protein microarrays, and rapid test formats such as, for example, tests using immunochromatographic strips.

In a preferred embodiment, said label is selected from the group consisting of a chemiluminescent label, an enzyme label, a fluorescent label, a radioactive iodine label.

The assays may be homogeneous or heterogeneous assays, competitive or non-competitive assays. In one embodiment, the assay is carried out in a sandwich assay format, 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 associated with a solid phase, such as a bead, the surface of a well or other container, a chip, or a strip, and the second antibody is an antibody that is labeled with, for example, a dye, a radioisotope, or a reactive or catalytic fragment. The amount of labeled antibody bound to the analyte is then measured using a suitable method. The usual compositions and procedures used in such "sandwich assays" are well established and known to those skilled in the art ( The Immunoassay Handbook, Ed. David Wild, Elsevier LTD, Oxford; 3rd ed. (May 2005), ISBN-13: 978-0080445267; Hultschig C et al., Curr Opin Chem Biol 2006 Feb;10(1):4-10 PMID: 16376134) .

In another embodiment, the assay uses two capture molecules, preferably antibodies, both of which are present as dispersions in a liquid reaction mixture, wherein the first labeling component is attached to the first capture molecule, said first labeling component being part of the system. labeling based on quenching or amplification of fluorescence or luminescence, and the second labeling component of said marker system is attached to a second capture molecule, due to which, when both capture molecules bind to the analyte, a measurable signal is generated that allows detection of the resulting sandwich complexes in a solution containing sample.

In another embodiment of the invention, said labeling system comprises rare earth metal cryptates or rare earth metal chelates in combination with a fluorescent dye or a chemiluminescent dye, in particular a cyanine type dye.

In the context of the present invention, a fluorescence-based assay comprises the use of dyes which can be selected, for example, from the group consisting of 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'-dimethodifluorescein (JOE), N,N,N',N'-tetramethyl-6-carboxyrodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 5-carboxyrodamine-60 ( R6G5), 6-carboxyrodamine-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, acridinium dyes, carbazole dyes, phenoxazine dyes, porphyrin dyes, polymethine dyes, and the like.

In the context of the present invention, chemiluminescence-based assays include the use of dyes based on the physical principles described for chemiluminescent materials in ( Kirk-Othmer, Encyclopedia of chemical technology, 4th ed., executive editor, JI Kroschwitz; editor, M. Howe-Grant , John Wiley & Sons, 1993, vol.15, pp. 518-562, incorporated herein by reference, including citations on pages 551-562) . Preferred chemiluminescent dyes are acridinium esters.

As indicated in the present description, "analysis" or "diagnostic analysis" can be any type of analysis that can be used in the field of diagnostics. Such an assay may be based on the binding of the analyte to be detected to one or more capture probes having a certain affinity. From the point of view of the interaction between the exciting molecules and target molecules or molecules of interest, the affinity constant should preferably exceed 10 ⁸ M ^-1 .

In the context of the present invention, "binding molecules" are molecules that can be used to bind to target molecules or molecules of interest, i.e. analytes present in the sample. Thus, binding molecules must have an adequate shape, both in terms of spatial and surface characteristics, such as surface charge, hydrophobicity, hydrophilicity, the presence or absence of Lewis donors and/or acceptors, in order to specifically bind to molecules. targets or molecules of interest. In this case, the binding may be mediated, for example, by ionic, van der Waals, pi-pi, sigma-pi, hydrophobic interactions or hydrogen bonds, or a combination of two or more of the above interactions between exciting molecules and target molecules or molecules of interest. In the context of the present invention, binding molecules can be selected, for example, from the group consisting of a nucleic acid molecule, a carbohydrate molecule, a PNA molecule, a protein, an antibody, a peptide or a glycoprotein. Preferably, the binding molecules are antibodies, including fragments thereof, having sufficient affinity for the target or molecule of interest, and including recombinant antibodies or recombinant antibody fragments, as well as chemically and/or biochemically modified derivatives of these antibodies or fragments derived from the variable chain , which are at least 12 amino acids long.

The chemiluminescent label may be an acridinium ester label, steroid labels including isoluminol labels, and the like.

Enzyme labels can be lactate dehydrogenase (LDH), creatine kinase (CPK), alkaline phosphatase, aspartate aminotransferase (AST), alanine aminotransferase (ALT), acid phosphatase, glucose-6-phosphate dehydrogenase, and so on.

In a specific embodiment, the cut-off is within the cut-off range for plasma MR-proADM, which is 0.5 to 1.5 nmol/L, preferably 0.7 to 1 nmol/L, most preferably the cut-off is applied. 0.8 nmol/l.

In a specific embodiment, the threshold is within the threshold range for plasma CT-proADM, which is 85 to 350 pmol/L, preferably 100 to 250 pmol/L, most preferably a cut-off value of 150 pmol/L is used.

In a specific embodiment of the invention, the cut-off value for PAMP-amide in plasma is 0.3 to 1.2 pmol/l, preferably 0.4 to 1.0 pmol/l, most preferably a cut-off value of 0.8 pmol/l is used. / l.

In a specific embodiment of the invention, a cut-off value for PAMP-glycine in plasma is applied, which is from 0.5 to 2.0 pmol/l, preferably from 0.7 to 1.8 pmol/l, most preferably, a cut-off value of 1 is applied. .5 pmol/l.

Another embodiment of the present application relates to a method according to the preceding embodiments, wherein the binder is selected from the group consisting of an antibody, an antibody fragment, or a non-Ig scaffold that binds to MR-proADM or a fragment thereof and/or to CT-proADM or a fragment thereof and/or PAMP or a fragment thereof, respectively.

Another embodiment of the present application relates to a method according to the preceding embodiments, wherein said anti-adrenomedullin (ADM) antibody or adrenomedullin-binding anti-ADM antibody fragment or adrenomedullin-binding non-Ig anti-ADM protein scaffold that binds adrenomedullin is monospecific.

Another embodiment of the present application relates to a method according to the preceding embodiments, wherein said antibody, or fragment or framework thereof, exhibits a binding affinity for ADM of at least 10 ^-7 M.

Another embodiment of the present application relates to a method according to the preceding embodiments, wherein said anti-adrenomedullin (ADM) antibody, or anti-ADM antibody fragment, or non-Ig anti-ADM protein scaffold binding to adrenomedullin, exhibits binding affinity for ADM, component of at least 10 ^-7 M, where the specified binding affinity is determined by surface plasmon resonance without labels using the Biacore 2000 system.

Another embodiment of the present application relates to a method in accordance with the previous embodiments, in which, for correlation, an increased level of the specified fragment of pre-adrenomedullin selected from the group including MR-proADM, CT-proADM and/or PAMP, or fragments thereof, exceeding a certain threshold is predictive of an increased risk of adverse outcome, and/or a level of said pre-pro-adrenomedullin fragment or fragments thereof below a certain threshold is predictive of a reduced risk of adverse outcome.

Used in the present description, the term "elevated level" means a level above a certain threshold level.

Another embodiment of the present application relates to the method in accordance with the previous embodiments, in which the specified threshold is the upper concentration determined for a healthy control population, such as the 90th, 95th or 99th percentile.

Another embodiment of the present application relates to a method in accordance with the previous embodiments, in which, in order to correlate the level of a fragment of pre-adrenomedullin, the level determination is performed at least two times, and at the same time a decrease in the second measured level of said fragment compared to the first the measured level of said fragment is predictive of a reduced risk of adverse outcome.

Another embodiment of the present application relates to a method in accordance with the previous embodiments, in which, in order to correlate the level of a fragment of pre-adrenomedullin, the level determination is performed at least two times, and at the same time an increase in the second measured level of said fragment compared to the first the measured level of said fragment is predictive of an increased risk of poor outcome.

As used herein, the term "risk" refers to the likelihood of an undesirable event or effect (eg, disease) occurring.

Another embodiment of the present application relates to the method according to the preceding embodiments, in which a decrease in the level of a fragment of pre-pro-adrenomedullin selected from the group including MR-proADM, CT-proADM and/or PAMP, or fragments thereof, is a predictor of reduced risk unfavorable outcome.

Another embodiment of the present application relates to the method according to the previous embodiments, in which an increase in the level of a fragment of pre-pro-adrenomedullin selected from the group including MR-proADM, CT-proADM and/or PAMP, or fragments thereof, is a predictor of increased risk unfavorable outcome.

Another embodiment of the present application relates to the method according to the previous embodiments, wherein said reduction is characterized by an improvement in the clinical/medical condition of the patient and/or a halving of the concentration of said pre-pro-adrenomedullin fragment.

Another embodiment of the present application relates to the method according to the previous embodiments, wherein said increase is characterized by a worsening of the clinical/medical condition of the patient and/or a doubling of the concentration of said pre-adrenomedullin fragment.

Another embodiment of the present application relates to a method in accordance with the previous embodiments, in which the assay is used to determine the level of MR-proADM, while the sensitivity of the assay for the specified analysis allows to quantify MR-proADM in healthy subjects and is <0.5 nmol / l, preferably <0.4 nmol/l and more preferably <0.2 nmol/l.

Another embodiment of the present application relates to a method in accordance with the previous embodiments, in which the assay is used to determine the level of CT-proADM, while the sensitivity of the assay for this assay allows for the quantitative determination of CT-proADM in healthy subjects and is <100 pmol/l, preferably <75 pmol/l and more preferably <50 pmol/l.

Another embodiment of the present application relates to a method in accordance with the previous embodiments, in which the assay is used to determine the level of PAMP-amide, while the sensitivity of the analysis for the specified analysis allows the quantitative determination of PAMP-amide in healthy subjects and is <0.3 pmol/ l, preferably <0.2 pmol/l and more preferably <0.1 pmol/l.

Another embodiment of the present application relates to a method in accordance with the previous embodiments, in which the assay is used to determine the level of PAMP-glycine, while the sensitivity of the assay for the specified analysis allows to quantify PAMP-glycine in healthy subjects and is <0.5 pmol/ l, preferably <0.25 pmol/l and more preferably <0.1 pmol/l.

Another embodiment of the present application relates to a method in accordance with the previous embodiments, in which the assay is used to determine the level of MR-proADM and/or CT-proADM and/or PAMP, and the sensitivity of the assay is <0.5 nmol/l, preferably <0.4 nmol/l and more preferably <0.2 nmol/l for MR-proADM and/or <100 pmol/l, preferably <75 pmol/l and more preferably <50 pmol/l l for CT-proADM, and/or <0.3 pmol/l, preferably <0.2 pmol/l and more preferably <0.1 pmol/l for PAMP.

The levels of proADM or its fragments of the present invention were determined using the described assays, as shown in the examples. The thresholds mentioned above may differ in other assays if they were calibrated differently than in the analytical systems used in the present invention. Therefore, the aforementioned cut-offs indicated above should be applied to such otherwise calibrated assays in accordance with differences in calibration. One way to quantify the difference in calibration is to compare (correlate) the assay method in question with the corresponding biomarker assay used in the present invention by measuring the appropriate biomarker (eg, bio-ADM) in samples using both methods. Another possibility is to use the test assay, provided that the assay has sufficient analytical sensitivity, to determine the average level of biomarkers of a representative normal population, compare the results with the average levels of biomarkers described in the literature, and recalculate the calibration based on the difference obtained from this comparison. Using the calibration used in the present invention, samples from normal (healthy) subjects were measured: median plasma level of bio-ADM (mature ADM-NH₂) was 24.7 pg/ml, the lowest value was 11 pg/ml and the 99th percentile was 43 pg/ml. Alternatively, commercially available controls can be used to set up different calibrations (eg, ICI Diagnostics, Berlin, Germany).

The median plasma concentration of MR-proADM in normal (healthy) subjects was 0.41 (interquartile range 0.23-0.64) nmol/L (Smith et al. 2009. Clin Chem 55: 1593-1595) using automatic fluorescence sandwich assay for the detection of MR-proADM as described in Caruhel et al. ( Caruhel et al. 2009. Clin Biochem 42: 725-8) .

The median plasma concentration of CT-proADM in normal healthy subjects (n=200) was 77.6 pmol/L (min 46.6 pmol/L, max 136.2 pmol/L) and the 95% percentile was 113. 8 pmol/l (EP 2 111 552 B1).

The plasma concentration of PAMP-amide in normal healthy individuals (n=51) was 0.51±0.19 pmol/L (mean±SD) ( Hashida et al. 2004. Clin Biochem 37: 14-21) .

The plasma concentration of PAMP-glycine in normal healthy individuals (n=51) was 1.15±0.38 pmol/L (mean±SD) ( Hashida et al. 2004. Clin Biochem 37: 14-21) .

Another embodiment of the present application relates to the method according to the previous embodiments, wherein said biological fluid may be selected from the group consisting of blood, serum, plasma, urine, cerebrospinal fluid (CSF) and saliva.

Another embodiment of the present application relates to a method in accordance with the previous embodiments, in which at least one clinical parameter is additionally determined and parameters selected from the group including age, gender, SOFA score (or its subclasses) are additionally taken into account in correlations , SAPSII, BUN, sodium, potassium, creatinine, bilirubin, platelet count, arterial pH, hematocrit, leukocyte, HCO ^3- , invasive mechanical ventilation/non-invasive mechanical ventilation, hemodynamic characteristics (including blood pressure, systolic and diastolic, mean arterial pressure, central venous pressure, heart rate), fluid balance, urine output, base excess, chloride, CRP, PCT, BNP or NT-proBNP, troponin T or troponin I, pro-enkephalin, hemoglobin, glucose, lactate, INR, alkaline phosphatase, AST, ALT, Gamma GT, total protein, albumin, temperature, respiratory rate, PaO ₂ and FiO ₂ , therapeutic measures (fluid resuscitation, vasopressors/inotropes, renal replacement therapy, antibiotics, hydrocortisone, insulin, enteral/parenteral nutrition, comorbidities, chronic medications.

Another embodiment of the present application relates to the method according to the previous embodiments for assigning said subjects to risk groups.

Another embodiment of the present application relates to the method according to the previous embodiments, in which the specified subject is assigned to a group of patients, wherein one group includes patients in need of therapy, and the other group includes patients who do not need therapy.

Additional embodiments within the scope of the present invention are set forth below:

1. A method for monitoring therapy in a subject, wherein the subject is being treated with a binder selected from the group consisting of an anti-adrenomedullin (ADM) antibody, an antibody fragment, and/or a non-Ig framework that binds to SEQ ID NO.1 (amino acid 1-52 ), including:

- determination of the level of a fragment of pre-pro-adrenomedullin, selected from the group including mid-regional pro-adrenomedullin (MR-proADM), C-terminal pro-adrenomedullin (CT-proADM) and/or a peptide of 20 N-terminal amino acids of pro-adrenomedullin (PAMP), or fragments of the specified , in the biological fluid obtained from the specified subject; and

- correlation of the specified level of the pre-adrenomedullin fragment, selected from the group including MR-proADM, CT-proADM and/or PAMP, with the clinical/medical condition of the subject and/or the risk of adverse outcome, and/or the need to adjust therapeutic measures, and

- at the same time, to determine the level of these fragments, at least one binding substance binds to a region within the amino acid sequence selected from the group consisting of SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, respectively.

2. The method according to Embodiment 1, wherein the treatment binder is an anti-ADM antibody or an anti-adrenomedullin antibody fragment or a non-Ig anti-ADM protein framework fragment, wherein said antibody or fragment or framework binds to N -terminal part, amino acids 1-21, adrenomedullin:

YRQSMNNFQGLRSFGCRFGTC; SEQ ID NO.19

3. The method according to embodiment 1-2, wherein the treatment binder is an anti-ADM antibody or an anti-adrenomedullin antibody fragment or a non-Ig anti-ADM protein scaffold, wherein said antibody or fragment or scaffold thereof binds to the C-terminal part, amino acids 42-52-amide, adrenomedullin:

APRSKISPQGY-NH ₂ ; SEQ ID NO.20

4. The method according to embodiment 1-3, wherein the treatment binder is an anti-ADM antibody, or an anti-adrenomedullin antibody fragment, or a non-Ig anti-ADM protein scaffold, wherein said antibody or fragment scaffold binds to the mid-regional portion , amino acids 21-42, adrenomedullin:

CTVQKLAHQIYQFTDKDKDNVA; SEQ ID NO.21

5. The method according to any of the preceding embodiments, wherein the determination of the level of said fragments is performed at least once.

6. The method of any of the preceding embodiments, wherein the adverse outcome is selected from the group consisting of clinical deterioration such as organ failure and mortality.

7. The method of any of the preceding embodiments, wherein clinical deterioration refers to worsening of symptoms (e.g., change in clinical parameters that determine disease progression), need for hospitalization, or death, and can be assessed by a medical indicator (e.g., an acute scoring system). functional and chronic health changes (APACHE, APACHE II)).

8. The method according to Embodiments 1 to 6, wherein the deterioration of organ function includes deterioration of kidney function (WRF), deterioration of cardiovascular function, deterioration of liver function, and deterioration of respiratory function, and can be assessed by dynamic organ failure assessment (SOFA ), assessment of multiple organ dysfunction (MODS) or simplified assessment of acute physiological changes (SAPS, SAPS II).

9. The method according to any of the preceding embodiments, wherein said subject is suffering from a disease or condition, such as a chronic or acute disease, or an acute condition.

10. The method according to any of the preceding embodiments, wherein the disease the subject suffers from can be selected from the group consisting of severe infections such as, for example, meningitis, systemic inflammatory response syndrome (SIRS), sepsis; other diseases such as diabetes, cancer, acute and chronic vascular diseases such as heart failure, myocardial infarction, stroke, atherosclerosis; shock, eg septic shock and organ dysfunction, eg kidney dysfunction, liver dysfunction, burns, surgery, trauma.

11. The method of any of the preceding embodiments, wherein said therapeutic measures are selected from the group consisting of fluid resuscitation, vasopressors/inotropes, renal replacement therapy, antibiotics, hydrocortisone, insulin, enteral nutrition/parenteral nutrition.

12. A method according to any of the preceding embodiments, wherein the level of said pre-pro-adrenomedullin fragment selected from the group consisting of MR-proADM, CT-proADM and/or PAMP or fragments thereof at least 5 amino acids in length is determined by immunoassay using at least one binding agent selected from the group consisting of a binding agent for MR-proADM or a fragment thereof and/or for CT-proADM or a fragment thereof and/or for PAMP or a fragment thereof, respectively.

13. The method according to any of the preceding embodiments, wherein, for correlation, an elevated level of said pre-adrenomedullin fragment selected from the group consisting of MR-proADM, CT-proADM and/or PAMP, or fragments thereof, exceeding a certain threshold, is predictive factor for an increased risk of adverse outcome, and/or the level of the specified fragment of pre-adrenomedullin or its fragments below a certain threshold is a predictive factor for a reduced risk of adverse outcome.

14. The method of any of the preceding embodiments, wherein said threshold is an upper concentration determined for a healthy control population, such as the 90th, 95th, or 99th percentile.

15. The method according to any of the preceding embodiments, wherein in order to correlate the level of the pre-adrenomedullin fragment, the level determination is performed at least two times, and wherein the decrease in the second measured level of said fragment compared to the first measured level of said fragment is predictive factor for a reduced risk of adverse outcome.

16. The method according to any of the preceding embodiments, wherein in order to correlate the level of the pre-adrenomedullin fragment, the level determination is performed at least two times, and the increase in the second measured level of said fragment compared to the first measured level of said fragment is predictive factor for an increased risk of poor outcome.

17. The method according to any of the preceding embodiments, wherein the assay is used to determine the level of MR-proADM and/or CT-proADM and/or PAMP and the sensitivity of the assay is <0.5 nmol/l, preferably <0, 4 nmol/l and more preferably <0.2 nmol/l for MR-pro ADM and/or <100 pmol/l, preferably <75 pmol/l and more preferably <50 pmol/l for CT- proADM, and/or <0.3 pmol/l, preferably <0.2 pmol/l and more preferably <0.1 pmol/l for PAMP.

18. The method of any of the preceding embodiments, wherein said biological fluid may be selected from the group consisting of blood, serum, plasma, urine, cerebrospinal fluid (CSF), and saliva.

19. The method of any of the preceding embodiments, further comprising determining at least one clinical parameter and further accounting for correlations with parameters selected from the group consisting of age, gender, SOFA score (or subclasses thereof), SAPSII score, BUN , sodium, potassium, creatinine, bilirubin, platelet count, arterial pH, hematocrit, leukocyte, HCO ^3- , invasive mechanical ventilation / non-invasive mechanical ventilation of the lungs, hemodynamic characteristics (including blood pressure, systolic and diastolic, mean arterial pressure, central venous pressure , heart rate), fluid balance, urine output, base excess, chloride, CRP, PCT, BNP or NT-proBNP, troponin T or troponin I, pro-enkephalin, hemoglobin, glucose, lactate, INR, alkaline phosphatase, AST, ALT, Gamma GT, total protein, albumin, temperature, respiratory rate, PaO ₂ and FiO ₂ , therapeutic measures (fluid resuscitation, vasopressor s / inotropes, renal replacement therapy, antibiotics, hydrocortisone, insulin, enteral nutrition / parenteral nutrition, comorbidities, chronic drugs.

20. A method according to any of the preceding embodiments for assigning said subjects to risk groups.

Example 1

Creation of antibodies and determination of their affinity constants

Several human and mouse antibodies were generated and their affinity constants determined (see Table 2).

Peptides/conjugates for immunization:

Peptides for immunization were synthesized, see table 2 (JPT Technologies, Berlin, Germany) with the inclusion of an additional N-terminal cysteine residue for conjugation of the peptides with bovine serum albumin (BSA). The peptides were covalently linked to BSA using a Sulfolink binding gel (Perbio-science, Bonn, Germany). The coupling procedure was carried out according to the Perbio manual.

Mouse antibodies were obtained according to the following method:

BALB/c mice were immunized with 100 μg of peptide-BSA conjugate on days 0 and 14 (emulsified in 100 μl of complete Freund's adjuvant) and 50 μg on days 21 and 28 (in 100 μl of incomplete Freund's adjuvant). Three days prior to the fusion experiment, the animal was given 50 μg of the conjugate dissolved in 100 μg of saline by one intraperitoneal and one intravenous injection.

Splenocytes from an immunized mouse were fused with SP2/0 myeloma cells using 1 ml of 50% polyethylene glycol for 30 seconds at 37°C. After washing, the cells were plated in 96-well cell culture plates. Hybrid clones were selected by growing in HAT medium [RPMI 1640 medium supplemented with 20% fetal bovine serum and HAT supplement]. Two weeks later, HAT medium was replaced with HT medium and after three passages returned to standard cell culture medium.

Three weeks after the fusion, a primary screening of cell culture supernatants was performed for antigen-specific IgG-type antibodies. Microcultures positive for these tests were transferred to 24-well multiplication plates. After retesting, selected cultures were cloned and recloned using the serial dilution method and isotyped (see also Lane, RD 1985. J. Immunol. Meth. 81: 223-228; Ziegler et al. 1996. Horm. Metab Res. 28:11-15) .

Obtaining mouse monoclonal antibodies:

Antibodies were prepared using standard antibody production methods ( Marx et al., 1997. Monoclonal Antibody Production, ATLA 25, 1997, p. 121) and purified on protein A. Antibody purity was >95% by gel electrophoresis analysis in the presence of DSN.

human antibodies

Human antibodies were obtained using phage display according to the following procedure:

To isolate recombinant single-chain fragments of the variable region (scFv) to the adrenomedullin peptide, libraries of human naive HAL7/8 antibody genes were used. The antibody gene libraries were screened using a panning strategy using peptides that contained a biotin tag linked via two different spacers to the adrenomedullin peptide sequence. To minimize the background of non-specific binding agents, a combination of panning cycles using a non-specifically bound antigen and a streptavidin-bound antigen was used. Phages eluted after the third round of panning were used to generate monoclonal scFv-expressing E. coli strains. Supernatants obtained after culturing the indicated clonal strains were used directly for antigen evaluation by ELISA (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 an ELISA signal positive for antigen and negative for streptavidin-sensitized microtiter plates. For further characterization, the scFv open reading frame was cloned into the expression plasmid pORE107 (Hust et al., J. Biotechn. 2011) , isolated from the culture supernatant by immobilized metal ion affinity chromatography, and purified by gel filtration.

Affinity constants:

To determine the affinity of antibodies to adrenomedullin, the kinetics of adrenomedullin binding to the immobilized antibody was evaluated using the label-free surface plasmon resonance method using the Biacore 2000 system (GE Healthcare Europe GmbH, Freiburg, Germany). For reversible antibody immobilization, an anti-mouse Fc antibody covalently bound at high density to a CM5 sensor chip according to the manufacturer's instructions (mouse antibody immobilization kit; GE Healthcare) was used ( Lorenz et al. 2011. Antimicrob Agents Chemother. 55(1): 165-173) .

Received monoclonal antibodies to the following areas of ADM human and mouse ADM, respectively. The table below shows the antibodies selected from the obtained antibodies, which were used in subsequent experiments. The selection was based on the target area:

Table 2:

Sequence number Antigen/Immunogen ADM area Designation Affinity constants
Kd (M) SEQ ID: 19 YRQSMNNFQGLRSFGCRFGTC 1-21 NT-N 5.9×10 ^-9 SEQ ID: 24 CTVQKLAHQIYQ 21-32 MR-N 2×10 ^-9 SEQ ID: 20 CAPRSKISPQGY-NH ₂ С-42-52 CT-H 1.1×10 ^-9 SEQ ID: 25 YRQSMNQGSRSNGCRFGTC 1-19 NT-M 3.9×10 ^-9

Obtaining antibody fragments by enzymatic cleavage:

To obtain Fab - and F(ab) ₂ -fragments used enzymatic cleavage of murine full-length antibodies NT-M. The NT-M antibody was digested using a) a pepsin-based F(ab) ₂ kit (Pierce, 44988) and b) a papain-based Fab kit (Pierce, 44985). The fragmentation procedures were carried out according to the instructions provided by the supplier. In the case of F(ab) ₂ fragmentation, the procedure was carried out for 8 h at 37°C. Cleavage to obtain Fab fragments was carried out for 16 hours, respectively.

Procedure for obtaining and cleaning Fab:

The immobilized papain was equilibrated by washing the resin with 0.5 ml of digestion buffer and centrifugation on a column at 5000×g for 1 min. After that, the buffer was discarded. A desalting column was prepared by removing the solution in which it was stored and washing it with a digestion buffer, followed by centrifuging it each time at 1000×g for 2 minutes. 0.5 ml of the resulting IgG sample was added to the spinning tube containing the equilibrated immobilized papain. The split reaction mixture was incubated for 16 h on a laboratory shaker at 37°C. The column was centrifuged at 5000×g for 1 min to separate the cleavage product from the immobilized papain. The resin was then washed with 0.5 ml PBS and centrifuged at 5000×g for 1 min. The fraction obtained by washing was added to the digested antibody, resulting in a total sample volume of 1.0 ml. The column loaded with Nab protein A was equilibrated with PBS and IgG elution buffer at room temperature. The column was centrifuged for 1 min to remove the storage solution (containing 0.02% sodium azide) and equilibrated by adding 2 ml of PBS, centrifuged again for 1 min and the eluate passed through the column was discarded. The sample was applied to the column and resuspended by inversion. Incubation was carried out at room temperature, mixing by inversion for 10 minutes. The column was centrifuged for 1 min, obtaining an eluate containing Fab fragments. (References: Coulter and Harris 1983. J. Immunol. Meth. 59, 199-203; Lindner et al. 2010. Cancer Res. 70, 277-87; Kaufmann et al. 2010. PNAS. 107, 18950-5. ; Chen et al 2010 PNAS 107, 14727-32 Uysal et al 2009 J Exp Med 206 449-62 Thomas et al 2009 J Exp Med 206 1913-27 Kong et al 2009 J. Cell Biol 185, 1275-840) .

Procedure for obtaining and purifying F(ab') ₂ fragments:

The immobilized pepsin was equilibrated by washing the resin with 0.5 ml of digestion buffer and centrifuged on a column at 5000×g for 1 min. After that, the buffer was discarded. A desalting column was prepared by removing the solution in which it had been stored and washing it with digestion buffer, followed by centrifugation at 1000×g for 2 minutes each time. 0.5 ml of the resulting IgG sample was added to the spinning tube containing the equilibrated immobilized pepsin. The split reaction mixture was incubated for 16 h on a laboratory shaker at 37°C. The column was centrifuged at 5000×g for 1 min to separate the cleavage product from the immobilized pepsin. The resin was then washed with 0.5 ml PBS and centrifuged at 5000×g for 1 min. The fraction obtained by washing was added to the digested antibody, resulting in a total sample volume of 1.0 ml. The column loaded with Nab protein A was equilibrated with PBS and IgG elution buffer at room temperature. The column was centrifuged for 1 min to remove the storage solution (containing 0.02% sodium azide) and equilibrated by adding 2 ml of PBS, centrifuged again for 1 min and the eluate passed through the column was discarded. The sample was applied to the column and resuspended by inversion. Incubation was carried out at room temperature, mixing by inversion for 10 minutes. The column was centrifuged for 1 min, obtaining an eluate containing Fab fragments. (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) .

Humanization of the NT-H antibody fragment

The antibody fragment was humanized by CDR transplantation ( Jones et al. 1986. Nature 321, 522-525) .

To obtain a humanized sequence, the following steps were carried out:

Extraction of total RNA: total RNA was extracted from NT-H hybridomas using a Qiagen kit.

First RT-PCR cycle: QIAGEN® kit was used OneStep RT-PCR (catalog #210210). RT-PCR was performed using a set of primers specific for heavy and light chains. For each RNA sample, RT-PCR reactions were performed for 12 individual heavy chains and 11 light chains using a mixture of degenerate "forward" primers spanning the leader sequences of the variable regions. "Reverse" primers were localized in the constant regions of the heavy and light chains. No restriction sites were inserted into the primers.

Reaction kit: 5×buffer from QIAGEN® kit OneStep RT-PCR, 5.0 µl; dNTP mix (containing 10 mM of each dNTP), 0.8 µl; primer set, 0.5 µl; enzyme mixture from the QIAGEN®OneStep RT-PCR kit, 0.8 µl; messenger RNA, 2.0 μl; RNase-free water up to 20.0 µl; total volume 20.0 μl. PCR conditions: reverse transcription: 50°C, 30 min; initial PCR activation: 95°C, 15 min; Cycle program: 20 cycles at 94°C, 25 s; 54°C, 30 s; 72°C, 30 s; final elongation: 72°C, 10 min. Second round using semi-nested PCR: The RT-PCR products from the first round of reactions were further amplified using a second round of PCR. RT-PCR reactions were performed for 12 individual heavy chains and 11 light chains using "semi-nested" primer sets specific for antibody variable regions.

Reaction kit: 2×PCR mix, 10 µl; primer set, 2 µl; product obtained in the first PCR cycle, 8 µl; total volume 20 μl; cloning report obtained using hybridoma antibodies (Hybridoma Antibody Cloning Report). PCR conditions: initial denaturation for 5 min at 95°C; 25 cycles at 95°C for 25 s, 57°C for 30 s, 68°C for 30 s; final elongation for 10 min at 68°C.

After completion of the PCR, the samples obtained by PCR reactions were analyzed in an agarose gel to visualize the amplified DNA fragments. After sequencing more than 15 cloned DNA fragments amplified by nested RT-PCR, several mouse antibody heavy and light chains were cloned and found to be correct. One heavy chain and one light chain were identified by protein sequence comparison and CDR analysis. After comparison with homologous human framework sequences, the humanized heavy chain variable region sequence was determined as shown in Figure 5. Since the amino acids at positions 26, 40 and 55 in the heavy chain variable region and the amino acid at position 40 in the light chain variable region critical for binding ability, they can be converted back to the original mouse amino acids. The resulting candidates are presented below ( Padlan 1991. Mol. Immunol. 28, 489-498; Harris and Bajorath. 1995. Protein Sci. 4, 306-310) .

Annotation to the sequences of fragments of antibodies (SEQ ID NO.11-18 and 26-27): highlighted in bold and underlined CDR 1, 2, 3, arranged in chronological order; italics indicate constant regions; hinge regions are in bold and the histidine label is in bold and italics

SEQ ID NO.11 (AM-VH-C):

QVQLQQSGAELMKPGASVKISCKAT GYTFSRYW IEWVKQRPGHGLEWIGE ILPGSGS T NYNEKFKGKATITADTSS NTAYMQLSSLTSEDSAVYYC TEGYEYDGFDY WGQGTTLTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLGTQTYICNVNHKPSNTKVDKRV EPK HHHHHH

SEQ ID NO.12 (AM-VH1):

QVQLVQSGAEVKKPGSSVKVSCKAS GYTFSRYW ISWVRQAPGQGLEWMGR ILPGSG ST NYAQKFQGRVTITADE STSTAYMELSSLRSEDTAVYYC TEGYEYDGFDY WGQGTTVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLGTQTYICNVNHKPSNTKVDKRV EPK HHHHHH

SEQ ID NO.13 (AM-VH2-E40):

QVQLVQSGAEVKKPGSSVKVSCKAS GYTFSRYW IEWVRQAPGQGLEWMGR ILPGSG ST NYAQKFQGRVTITADE STSTAYMELSSLRSEDTAVYYC TEGYEYDGFDY WGQGTTVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLGTQTYICNVNHKPSNTKVDKRV EPK HHHHHH

SEQ ID NO.14 (AM-VH3-T26-E55):

QVQLVQSGAEVKKPGSSVKVSCKAT GYTFSRYW ISWVRQAPGQGLEWMGE ILPGSG ST NYAQKFQGRVTITADE STSTAYMELSSLRSEDTAVYYC TEGYEYDGFDY WGQGTTVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLGTQTYICNVNHKPSNTKVDKRV EPK HHHHHH

SEQ ID NO.15 (AM-VH4-T26-E40-E55):

QVQLVQSGAEVKKPGSSVKVSCKAT GYTFSRYW IEWVRQAPGQGLEWMGE ILPGSG ST NYAQKFQGRVTITADE STSTAYMELSSLRSEDTAVYYC TEGYEYDGFDY WGQGTTVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLGTQTYICNVNHKPSNTKVDKRV EPK HHHHHH

SEQ ID NO.16 (AM-VL-C):

FGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC FQGSHIPYT FGGGTKLEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKVDSTYADSLSSTKHTLK

SEQ ID NO.17 (AM-VL1):

DVVMTQSPLSLPVTLGQPASISCRSS QSIVYSNGNTY LNWFQQRPGQSPRRLIY RVS NR DSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC

SEQ ID NO.18 (AM-VL2-E40):

DVVMTQSPLSLPVTLGQPASISCRSS QSIVYSNGNTY LEWFQQRPGQSPRRLIY RVS NR DSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC

SEQ ID NO.26 (heavy chain HAM8101):

QVQLVQSGAEVKKPGSSVKVSCKAS GYTFSRYW IEWVRQAPGQGLEWIGE ILPGSGS T NYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYC TEGYEYDGFDY WGQGTTVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO.27 (light chain HAM8101):

FGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC FQGSHIPYT FGGGTKLEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKVDSTYADSLSSTKHTLK

Example 2

The effect of the anti-NT-H-adrenomedullin antibody HAM8101 (adrecizumab) was studied in clinical and laboratory parameters of septic pigs in a two-intervention model. The first intervention was hemorrhagic shock and the second intervention was induction of sepsis by application of an E. coli fibrin clot (peritoneal contamination and infection; PCI). HAM8101 was administered at the time when sepsis was induced.

Materials and methods

Animal strain: Sus scrofa domestica (Deutsche Landrasse) 14-16 weeks, 30-35 kg.

Group size: 6

Groups:

a) PCI+carrier

b) PCI+HAM8101

Tested materials

HAM8101 (adrecizumab) Lot #: HAM-160714-FiB in 20 mM His/HCl pH 6.0

Carrier: 20 mM His/HCl pH 6.0

Execution of the study

Animals

We anesthetized and ventilated 16 female German Landrace pigs (n=16; mean ± standard deviation (SD) of 33 ± 1.5 kg body weight (BW)) and followed standard procedures for the care of laboratory animals. This study was approved by the institutional and local committee for the care and use of animals (Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen, Germany, 84-02.04.2015.A037).

General anesthesia and catheterization

Animals were pretreated with azaperone (1-2 mg/kg bw) and ketamine (10 mg/kg bw), and general anesthesia was induced by intravenous injection of propofol (1-2 mg/kg bw). Animals were orally intubated and placed in the supine position. General anesthesia was maintained with propofol and fentanyl infusions. The pressure controlled ventilation mode was chosen to ventilate animals with an inspiratory oxygen fraction of 0.5, an inhalation/expiration ratio of 1:1.5, PEEP set at 5 cm H ₂ O, and a tidal volume of 8-10 ml/kg body weight. . The respiratory rate was set to maintain a PaCO ₂ pressure of 3.5-4.5 kPa. Core body temperature was maintained above 37.5° C. with a warming blanket. Two central venous catheters were inserted into the external jugular vein and femoral vein, and a PICCO arterial catheter was inserted into the femoral artery by percutaneous puncture.

At the end of the study, the animals were euthanized in the presence of a veterinarian with a lethal dose of Narcoren® (Merial, Hallbergmoos, Germany) while still under deep anesthesia.

Fibrin clot Escherichia coli

In this model, we used a fibrin clotE. coliWith 7-9×10^elevencolony forming units (CFU) per kg/body weight, to induce septic shock.

Hemodynamic measurements

All intravascular pressure measurements were referenced to the mid-chest level and values were taken at the end of exhalation. Heart rate, mean arterial pressure (MAP), central venous pressure (CVP), and systolic flow volume variation (SVV) were recorded continuously. Cardiac output was measured using transpulmonary thermodilution (PICCO, Puls medical systems, Feldkirchen, Germany). Extravascular lung water (EVLW), intrathoracic blood volume (ITBV), and global end-diastolic volume (GEDV) were calculated using a standard formula.

Laboratory

Blood gas analysis was performed using a standard blood gas oximetry system (ABL 800; Radiometer, Copenhagen, Denmark) with a co-oximeter. Blood formula, electrolytes, creatinine, urea and liver enzymes were determined using standard laboratory methods. We measured neutrophil gelatinase-associated lipofalin (NGAL) by enzyme immunoassay using a commercially available kit (Pig NGAL ELISA Kit, BioPorto, Hellerup, Denmark). Creatinine clearance was measured as an estimate of glomerular filtration rate (ClCrea=Ucrea×Uvol/Pcrea×collection period duration; Ucrea=urinary creatinine concentration; Uvol=urine volume during collection period; Pcrea=serum creatinine concentration). We measured the body weight of the animals before and after the experiment. The moisture/dryness ratio of the lungs was determined by weighing a part of the left upper lobe immediately after the experiment and after 5 days. Blood coagulation was analyzed using rotational thromboelastometry (ROTEM delta, TEM international, Basel, Switzerland). Plasma levels of cytokines (IL-6 and TNF-α) were assessed by enzyme immunoassay using commercially available porcine-specific kits.

Bioactive adrenomedullin (bio-ADM) was measured using a novel immunochemical immunoassay provided by Sphingotec GmbH (Hennigsdorf, Germany) as previously described ( Marino et al. 2014. Crit Care 18: R34) . Briefly, a one-step chemiluminescent sandwich immunoassay based on NHS-acridinium ester labeling to detect biologically active ADM in raw, pure plasma uses two mouse monoclonal antibodies, one directed against the middle region (solid phase) and the other directed against amidated C-terminal fragment of ADM (labeled antibody). The assay uses 50 µl of plasma samples/calibrators and 200 µl of labeled detection antibody. The sensitivity of the analytical assay is 2 pg/mL. The assay is suitable for measuring bio-ADM in numerous mammalian species, including humans and pigs, and detects both free bio-ADM and bio-ADM when HAM 8101 (adrecizumab) is bound to it ( Weber et al. 2017. J Appl Lab Medicine, in press) .

Plasma MR-proADM was measured using the KRYPTOR B·R·A·H·M·S MR-proADM assay according to the manufacturer's instructions (Thermo Fisher, Hennigsdorf, Germany).

Experiment Protocol

During catheterization, the animals received 10 ml/kg body weight/hour of a balanced crystalloid solution. Hemorrhagic shock was induced by inducing bleeding in animals through a femoral vein catheter. Animals were bled until half of the original MAP was reached. Hemorrhagic shock persisted for 45 minutes, after which fluid resuscitation was performed using a balanced crystalloid solution to restore the initial mean arterial pressure. 2 hours after the hemorrhagic shock, the blood collected during the hemorrhagic shock was re-transfused. As a second intervention, sepsis was induced using a clot containing a large amount of E. coli placed in the abdominal cavity 6 hours after hemorrhagic shock. Animals were randomized to receive either anti-adrenomedullin antibody or vehicle solution. The solutions were delivered in neutral sachets and the groups were not known to the investigators and were labeled A or B. Antibody or vehicle solution therapy started immediately after sepsis induction. 2 mg/kg body weight of antibody/vehicle solution was administered over 30 minutes. 4 hours after the onset of sepsis, therapy for septic shock was started with balanced crystalloids and norepinephrine on demand. Volume replacement and the vasopressor were titrated to maintain central venous pressure at 8-12 mmHg and mean arterial pressure above 65 mmHg. and central venous oxygen saturation of 70% as recommended in the Surviving Sepsis Campaign. Sepsis therapy was continued for another 8 hours. Measurements were taken before hemorrhagic shock, before sepsis induction, and 1, 2, 3, 4, 6, 8, 10, and 12 hours after sepsis induction. Hemorrhagic and septic shock was not induced in the SHAM animals, but in addition they received the same treatment, including all intravascular catheters, midline laparotomy, and blind application of antibody/vehicle solution.

Results:

As expected, the induction of sepsis resulted in an increase in plasma bio-ADM in carrier animals. Surprisingly, after administration of HAM 8101, the apparent plasma concentration of bio-ADM increased significantly faster and to higher levels than in vehicle-treated animals (FIG. 2A). Given the huge molar excess of HAM 8101 compared to endogenous bio-ADM, it should be assumed that the measured plasma concentration of ADM after administration of HAM 8101 is actually mainly bio-ADM in complex with the HAM 8101 antibody. To find out if the observed HAM8101 induced faster and more pronounced increase in plasma bio-ADM due to increased expression of the ADM gene and/or release of ADM gene products, plasma concentrations of another peptide derived from the ADM precursor peptide, namely MR-proADM (mid-regional pro-adrenomedullin ). As shown in FIG. 2B, plasma levels of MR-proADM increased in a similar manner upon induction of sepsis, whether animals were treated with HAM8101 or vehicle. Thus, the HAM8101-induced faster and more pronounced increase in plasma ADM does not appear to be associated with increased ADM gene expression and/or release of ADM gene products.

The overproportionate increase in plasma bio-ADM observed after HAM 8101 administration compared to vehicle-treated animals was not associated with worse outcome:

Animals treated with HAM 8101 required less volumetric resuscitation to achieve the target mean arterial pressure than those treated with vehicle (FIG. 3).

Only a third of the animals treated with HAM 8101 required norepinephrine in addition to volumetric resuscitation to achieve the target mean arterial pressure, while all animals treated with vehicle required norepinephrine (Fig. 4).

As shown in FIG. 4, among the animals treated with HAM 8101, one third required norepinephrine, while two thirds did not. The two groups differed in plasma concentrations of MR-proADM (FIG. 5). In those animals that developed shock, for example requiring norepinephrine in addition to fluid resuscitation, MR-proADM concentrations were significantly higher than in other animals. Lastly, the MR-proADM concentration was lower in the successfully treated animals than in the vehicle group.

Taken together, the data demonstrate that - surprisingly - in the treatment of HAM 8101, the measurement of bio-ADM is not suitable for monitoring the risk of adverse outcome for the subject. In contrast, measurement of MR-proADM, as an example of another fragment derived from an ADM precursor peptide to which HAM 8101 does not bind, is suitable for monitoring stimulation or suppression of the ADM system under such conditions and is not immediately affected by administration of HAM 8101. and it correlates with clinical outcome over time.

Example 3

The effect of a monoclonal antibody against C-terminal adrenomedullin generated against SEQ ID NO.20 (HAM23 02) on ADM levels in male Wistar rats (Charles River, Sulzfeld) was studied.

Blood sampling (K ₃ -EDTA-plasma ≤ 150 μl or 3-4 ml in terminal bleeding (-80°C)) was carried out at time points -3 d, 12 min, 1 h, 3 h, 6 h, 24 h , 48, 4 d, 7 d, 10 d. Animals were treated with a single intravenous injection (5 ml/kg body weight).

Injection and sampling were successful in all animals. Animals treated with the antibody showed no obvious signs of toxic effects.

Treatment Dose Number of animals HAM 2302 20 mg/kg 6 PBS 0 mg/kg 6

Measurement of ADM and free HAM2302 in plasma

tADM analysis was used to measure adrenomedullin in plasma samples containing HAM2302. This assay uses an N-terminal anti-ADM antibody generated against SEQ ID NO.25 as solid phase (HAM 1112) and a mid-regional anti-ADM antibody generated against amino acids 27 to 39 of ADM (AHQIYQFTDK DKD; SEQ ID NO.22 ) (HAM 2903) as an indicator. Calibrators derived from synthetic rat bio-ADM (1-50)-H ₂ (YRQSMNQGSRSTGCRFGTCTMQKLAHQIYQFTDKDKDGMAPRNKISPQGY-NH ₂ , SEQ ID NO.23) were used to quantify ADM in the sample. Unlike the bio-ADM assay mentioned above, the tADM assay does not specifically measure the amidated form of ADM, but can detect all forms of ADM.

Purified monoclonal antibody HAM 2903 (1 g/l) was labeled by incubation in 10% labeling buffer (500 mmol/l sodium phosphate, pH 8.0) with a ratio of 1:5 mol/l acridinium MACN-NHS-ester ( 1 g)/l, InVent GmbH) for 30 min at 22°C in the dark. After adding 5% 1 mol/l Tris-HCl, pH 8.0 for 10 minutes, the HAM 2903 antibody was separated from the free label through CentriPure P5 columns (emp Biotech GmbH) and KW-803 protein size exclusion HPLC (Shodex, Showa Denko Europe).

White polystyrene microtiter plates (Greiner Bio-One International AG) were coated (18 h at 22°C) with HAM 1112 monoclonal antibody (1.5 μg/0.2 ml per well 50 mmol/l Tris-HCl, 100 mmol/ l NaCl, pH 7.8). After washing and blocking with 30 g/l carion, 5 g/l bovine serum albumin (without protease), 6.5 mmol/l monopotassium phosphate, 3.5 mmol/l sodium dihydrogen phosphate (pH 6.5) for 1, The plates were vacuum dried for 5 hours.

Synthetic rat ADM (rADM) (Peptides & elephants) was serially diluted using 20 mmol/L potassium hydrogen phosphate, 0.5 g/L bovine serum albumin (BSA), 6 mmol/L sodium EDTA, 50 µmol/L amastatin, 100 µmol /l leupeptin; pH 8.

Fifty µl of samples/calibrators were pipetted into coated microtiter plates. After adding 150 μl of labeled HAM 2302 C-terminal antibody, microtiter plates were incubated for 20 hours at 2-8° C. with shaking. Unbound indicator was removed by washing five times (350 μl/well each) with wash solution (400 mmol/L Tris, 1 g/L Tween 20, 3 mol/L NaCL, pH 7.5). Chemiluminescence measurement in binding wells was performed for 1 second per well using a Centro LB 960 microtiter plate luminescence reader (Berthold Technologies).

Adrenomab-1 assay was used to determine free HAM2302. An antibody against mid-regional ADM generated against amino acids 21-32 (SEQ ID NO.24) functions as a solid phase. MACN-tagged C-terminal antibody (HAM 2302) serves as an indicator. A competitive assay scheme is used to quantify HAM 2302 in a sample. Calibrators made from unlabeled HAM 2302, together with a constant concentration of hADM (10 ng/mL), are used to generate a standard curve for determining concentrations in unknown samples. As the concentration of calibrator/antibody in the sample increases, the measured light signal decreases because less indicator can bind to adrenomedullin.

Purified monoclonal HAM 2302 (1 g/l) was labeled by incubation in 10% labeling buffer (500 mmol/l sodium phosphate, pH 8.0) with a MACN-NHS-acridinium ester ratio of 1:4.5 mol/l (1 g/l), InVent GmbH) for 30 min at 22°C in the dark. After addition of 5% 1 mol/l Tris-HCl, pH 8.0, over 10 minutes, HAM 2302 was separated from the free label through CentriPure P10 columns (emp Biotech GmbH) and KW-803 protein size exclusion HPLC (Shodex, Showa Denko-Europe).

Microtiter plates made of white polystyrene (Greiner Bio-One International AG) were coated (18 h at 20°C) with a monoclonal mid-regional antibody against ADM amino acids 21-32 (1 μg/0.2 ml per well, 50 mmol/l Tris-HCl, 100 mmol/l NaCl, pH 7.8). After blocking with 30 g/l carion, 5 g/l BSA (no protease), 6.5 mmol/l monopotassium phosphate, 3.5 mmol/l sodium dihydrogen phosphate (pH 6.5) for 1.5 hours, the plates were exposed to vacuum drying.

A serial dilution of HAM 2302 prepared with phosphate buffered saline (PBS), 2.5 g/l bovine serum albumin, pH 7.4 was used.

Fifty µl of samples/calibrators and 100 µl of 10 ng/ml hADM in phosphate buffered saline (PBS), 2.5 g/l bovine serum albumin pH 7.4 were pipetted into coated microtiter plates. After a one hour incubation at 2-8° C. with stirring, 100 μl of labeled HAM 2302 was pipetted into coated microtiter plates. After incubating plates for a further 2.5 hours at 2-8°C with shaking, unbound indicator was removed by washing five times (350 µl/well each) with wash solution (20 mmol/L PBS, 1 g/L Triton X-100, pH 7.4). Chemiluminescence measurement in binding wells was performed for 1 second per well using a Centro LB 960 microtiter plate luminescence reader (Berthold Technologies).

Results:

In samples from animals treated with HAM2302, plasma concentrations of free HAM2302 measured 3 hours after administration were 481.9 μg/mL (±46.8 μg/mL). The mean concentration of ADM in animals treated with the vehicle was 14.6±3.75 pg/ml. The concentration of adrenomedullin in animals treated with HAM2302 was approximately 100 times higher (1411±67 pg/ml) compared to animals that were injected with the vehicle (Fig. 6).

Sequence list

SEQ ID NO.1 (mature adrenomedullin (mature ADM); amidated ADM; bio-ADM): amino acids 95-146-CONH ₂ from preproADM

YRQSMN NFQGLRSFGC RFGTCTVQKL AHQIYQFTDK DKDNVAPRSK ISPQGY - CONH ₂

SEQ ID NO.2 (pre-pro-adrenomedullin (pre-proADM)): amino acids 1-185

MKLVSVALMYLGSLAFLGADTARLDVASEFRKKWNKWALSRGKRELRMSSSYPTGL ADVKAGPAQTLIRPQDMKGASRSPEDSSPDAARIRVKRYRQSMNNFQGLRSFGCRFGTCTVQ KLAHQIYQFTDKDKDNVAPRSKISPQGYGRRRRRSLPEAGPGRTLVSSKPQAHGAPAPPSGS APHFL

SEQ ID NO.3 (N-20 terminal proadrenomedullin peptide, PAMP): amino acids 22-41 from preproADM

ARLDVASEF RKKWNKWALS R

SEQ ID NO.4 (Mid-regional proadrenomedullin, MR-proADM): amino acids 45-92 from preproADM

ELRMSS SYPTGLADVK AGPAQTLIRP QDMKGASRSP EDSSPDAARI RV

SEQ ID NO.5 (C-terminal proadrenomedullin, CT-proADM): amino acids 148-185 from preproADM

RRR RRSLPEAGPG RTLVSSKPQA HGAPAPPSGS APHFL

SEQ ID NO.6:

GYTFSRYW

SEQ ID NO.7:

ILPGSGST

SEQ ID NO.8:

TEGYEYDGFDY

SEQ ID NO.9:

QSIVYSNGNTY

SEQ ID NO.10:

FQGSHIPYT.

SEQ ID NO.11 (AM-VH-C):

QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILP

GSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFD

YWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN

SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKR

VEPC

SEQ ID NO.12 (AM-VH1):

QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRIL

PGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFD

YWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN

SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKR

VEPC

SEQ ID NO.13 (AM-VH2-E40):

QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRIL

PGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFD

YWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN

SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKR

VEPC

SEQ ID NO.14 (AM-VH3-T26-E55):

QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEIL

PGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFD

YWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN

SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKR

VEPC

SEQ ID NO.15 (AM-VH4-T26-E40-E55):

QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEIL

PGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFD

YWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN

SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKR

VEPC

SEQ ID NO.16 (AM-VL-C):

DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYR

VSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKLE

IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS

QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE

FROM

SEQ ID NO.17 (AM-VL1):

DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYR

VSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKL

EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN

SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG

EU

SEQ ID NO.18 (AM-VL2-E40):

DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRVSNR DSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSPVKVDNALQSGNSQESVTEQDSKDSPVKHSFK

SEQ ID NO.19 (N-terminal, amino acids 1-21, adrenomedullin)

YRQSMNNFQGLRSFGCRFGTC

SEQ ID NO.20 (C-terminal, amino acids 42-52, adrenomedullin)

APRSKISPQGY-NH ₂

SEQ ID NO.21 (middle part, adrenomedullin amino acids 21-42)

CTVQKLAHQIYQFTDKDKDNVA

SEQ ID NO.22 (middle part, adrenomedullin amino acids 27-39)

AHQIYQFTDK DKD

SEQ ID NO.23 (rat adrenomedullin, amino acids 1-50)

YRQSMNQGSRSTGCRFGTCTMQKLAHQIYQFTDKDKDGMAPRNKISPQGY-NH ₂

SEQ ID NO.24 (mid-regional part of adrenomedullin, amino acids 21-32)

CTVQKLAHQIYQ

SEQ ID NO.25 (N-terminal portion of mouse adrenomedullin, amino acids 1-19)

YRQSMNQGSRSNGCRFGTC

SEQ ID NO.28: (not listed in sequence listing due to 3 amino acid length)

RVS

SEQ ID NO.26 (heavy chain HAM8101)

QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGEILPGSGST NYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSAS TKGPSVFPLAPSSKSTSGGT AALGCLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO.27 (light chain HAM8101)

DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPRLLIYRVSNRF SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKVHSSY

Description of drawings

Fig. 1: Schedule of the study in the pig model with two interventions

Fig. 2: bio-ADM (A) and MR-proADM (B) in plasma: Means±SEM are shown for both groups. For bio-ADM (A), interaction (7 h - 19 h, multivar. time * group): 0.003

Fig. 3: Fluid resuscitation: Means±SEM are shown for both groups.

Fig. 4: Frequency of need for norepinephrine. The percentage of animals requiring norepinephrine in addition to fluid resuscitation is indicated to achieve the target MAP per group. Chi-square test (19 h): 0.014.

Fig. 5: Plasma MR-proADM versus treatment success: Mean±SEM values are shown for three groups: HAM 8101 treatment requiring norepinephrine (shock), HAM 8101 treatment not requiring norepinephrine (no shock), vehicle (all requiring norepinephrine) .

Fig. 6: Comparison of adrenomedullin concentrations 3 hours after injection of 20 mg/kg HAM2302 (n=3). Mean antibody concentrations for treated animals are indicated below the graph.

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SEQUENCE LIST

<110> Adrenomed AG

<120> MONITORING OF THERAPY IN TREATMENT WITH ANTI-BINDING

ADRENOMEDULIN (ADM)

<130> A75177WO

<150>EP17197177.3

<151> 2017-10-18

<160> 27

<170> PatentIn version 3.5

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Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys

210 215

<210> 14

<211> 219

<212> PRT

<213> Homo sapiens

<400> 14

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Arg Tyr

20 25 30

Trp Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Leu Pro Gly Ser Gly Ser Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Glu Gly Tyr Glu Tyr Asp Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys

210 215

<210> 15

<211> 219

<212> PRT

<213> Homo sapiens

<400> 15

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Arg Tyr

20 25 30

Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Leu Pro Gly Ser Gly Ser Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Glu Gly Tyr Glu Tyr Asp Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys

210 215

<210> 16

<211> 219

<212> PRT

<213> Homo sapiens

<400> 16

Asp Val Leu Leu Ser Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Thr Ile Ser Cys Arg Ser Ser Gln Ser Ile Val Tyr Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Ile Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 17

<211> 219

<212> PRT

<213> Homo sapiens

<400> 17

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val Tyr Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Arg Leu Ile Tyr Arg Val Ser Asn Arg Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Ile Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 18

<211> 219

<212> PRT

<213> Homo sapiens

<400> 18

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val Tyr Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Phe Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Arg Leu Ile Tyr Arg Val Ser Asn Arg Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Ile Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 19

<211> 21

<212> PRT

<213> Homo sapiens

<400> 19

Tyr Arg Gln Ser Met Asn Asn Phe Gln Gly Leu Arg Ser Phe Gly Cys

1 5 10 15

Arg Phe Gly Thr Cys

twenty

<210> 20

<211> 11

<212> PRT

<213> Homo sapiens

<400> 20

Ala Pro Arg Ser Lys Ile Ser Pro Gln Gly Tyr

1 5 10

<210> 21

<211> 22

<212> PRT

<213> Homo sapiens

<400> 21

Cys Thr Val Gln Lys Leu Ala His Gln Ile Tyr Gln Phe Thr Asp Lys

1 5 10 15

Asp Lys Asp Asn Val Ala

twenty

<210> 22

<211> 13

<212> PRT

<213> Homo sapiens

<400> 22

Ala His Gln Ile Tyr Gln Phe Thr Asp Lys Asp Lys Asp

1 5 10

<210> 23

<211> 50

<212> PRT

<213> Homo sapiens

<400> 23

Tyr Arg Gln Ser Met Asn Gln Gly Ser Arg Ser Thr Gly Cys Arg Phe

1 5 10 15

Gly Thr Cys Thr Met Gln Lys Leu Ala His Gln Ile Tyr Gln Phe Thr

20 25 30

Asp Lys Asp Lys Asp Gly Met Ala Pro Arg Asn Lys Ile Ser Pro Gln

35 40 45

Gly Tyr

fifty

<210> 24

<211> 12

<212> PRT

<213> Homo sapiens

<400> 24

Cys Thr Val Gln Lys Leu Ala His Gln Ile Tyr Gln

1 5 10

<210> 25

<211> 19

<212> PRT

<213> Mouse

<400> 25

Tyr Arg Gln Ser Met Asn Gln Gly Ser Arg Ser Asn Gly Cys Arg Phe

1 5 10 15

Gly Thr Cys

<210> 26

<211> 448

<212> PRT

<213> Humanized mouse

<400> 1

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Arg Tyr

20 25 30

Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Leu Pro Gly Ser Gly Ser Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Glu Gly Tyr Glu Tyr Asp Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 27

<211> 219

<212> PRT

<213> Humanized mouse

<400> 2

Asp Val Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val Tyr Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Ile Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<---

Claims (18)

1. A method for monitoring therapy in a subject, wherein the subject is being treated with a binder selected from the group consisting of an anti-adrenomedullin (ADM) antibody, an antibody fragment, and/or a non-Ig framework that binds to SEQ ID NO.1 (amino acid 1-52 ), including: - determination of the level of a fragment of pre-pro-adrenomedullin selected from the group including mid-regional pro-adrenomedullin (MR-proADM), C-terminal pro-adrenomedullin (CT-proADM) and/or a peptide of 20 N-terminal amino acids of pro-adrenomedullin (PAMP), or fragments of the specified , in the biological fluid obtained from the specified subject; and - correlation of the specified level of the pre-adrenomedullin fragment, selected from the group including MR-proADM, CT-proADM and/or PAMP, with the clinical/medical condition of the subject and/or the risk of adverse outcome, and/or the need to adjust therapeutic measures, and - at the same time, to determine the level of these fragments, at least one binding substance binds to a region within the amino acid sequence selected from the group consisting of SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, respectively. 2. The method of claim 1, wherein the treatment binder is an anti-ADM antibody or an anti-adrenomedullin antibody fragment or a non-Ig anti-ADM protein framework fragment, wherein said antibody, or fragment, or framework binds to the N-terminal part, amino acids 1-21, adrenomedullin: YRQSMNNFQGLRSFGCRFGTC - SEQ ID NO.19. 3. The method according to claim 1 or 2, wherein the treatment binder is an anti-ADM antibody or an anti-adrenomedullin antibody fragment or a non-Ig anti-ADM protein scaffold, wherein said antibody, or fragment or scaffold thereof binds to C-terminal, amino acids 42-52-amide, adrenomedullin: APRSKISPQGY-NH ₂ - SEQ ID NO.20. 4. The method according to any one of paragraphs. 1-3, wherein the treatment binder is an anti-ADM antibody, or an anti-adrenomedullin antibody fragment, or a non-Ig anti-ADM protein backbone, wherein said antibody or fragment backbone binds to the mid-regional portion, amino acids 21-42, of adrenomedullin: CTVQKLAHQIYQFTDKDKDNVA - SEQ ID NO.21. 5. The method according to any one of the preceding claims, wherein the determination of the level of said fragments is performed at least once. 6. The method of any one of the preceding claims, wherein the adverse outcome is selected from the group consisting of clinical deterioration such as organ failure and mortality. 7. A method according to any one of the preceding claims, wherein said subject suffers from a disease or condition, such as a chronic or acute disease, or an acute condition. 8. The method according to any one of the preceding paragraphs, in which the disease from which the subject suffers can be selected from the group including severe infections, such as, for example, meningitis, systemic inflammatory response syndrome (SIRS), sepsis; other diseases such as diabetes, cancer, acute and chronic vascular diseases such as heart failure, myocardial infarction, stroke, atherosclerosis; shock, eg septic shock and organ dysfunction, eg kidney dysfunction, liver dysfunction, burns, surgery, trauma. 9. A method according to any one of the preceding claims, wherein said therapeutic measures are selected from the group consisting of fluid resuscitation, vasopressors/inotropes, renal replacement therapy, antibiotics, hydrocortisone, insulin, enteral nutrition/parenteral nutrition. 10. The method according to any one of the preceding claims, wherein the level of said pre-adrenomedullin fragment selected from the group consisting of MR-proADM, CT-proADM and/or PAMP or fragments thereof at least 5 amino acids in length is determined by immunoassay using at least one binder selected from the group consisting of a binder for MR-proADM or a fragment thereof and/or for CT-proADM or a fragment thereof and/or for PAMP or a fragment thereof, respectively. 11. The method according to any of the preceding claims, wherein for correlation, an elevated level of said pre-adrenomedullin fragment selected from the group consisting of MR-proADM, CT-proADM and/or PAMP, or fragments thereof, exceeding a certain threshold, is predictive a factor for an increased risk of an adverse outcome, and/or a level of said fragment of pre-adrenomedullin or fragments thereof below a certain threshold is a predictive factor for a reduced risk of an adverse outcome. 12. The method according to any one of the preceding claims, wherein, in order to correlate the level of the pre-adrenomedullin fragment, the level determination is performed at least two times, and the decrease in the second measured level of said fragment compared to the first measured level of said fragment is predictive factor for a reduced risk of poor outcome. 13. The method according to any one of the preceding claims, wherein, in order to correlate the level of the pre-adrenomedullin fragment, the level determination is performed at least two times, and the increase in the second measured level of the specified fragment compared to the first measured level of the specified fragment is predictive factor for an increased risk of poor outcome. 14. The method of any one of the preceding claims, wherein said biological fluid may be selected from the group consisting of blood, serum, plasma, urine, cerebrospinal fluid (CSF), and saliva. 15. The method according to any one of the preceding paragraphs for classifying said subjects as risk groups.

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