NZ624876B2 - Anti-adrenomedullin (adm) antibody or anti-adm antibody fragment or anti-adm non-ig scaffold for use in therapy of an acute disease or acute condition of a patient for stabilizing the circulation - Google Patents

Abstract

Discloses use of an anti-Adrenomedullin (ADM) antibody or an anti-ADM antibody fragment binding to adrenomedullin or anti-ADM non-Ig scaffold binding to adrenomedullin in the manufacture of a medicament for use in therapy of an acute disease or acute condition of a patient for stabilizing the systemic circulation of said patient, and wherein said antibody or antibody fragment or non-Ig scaffold binds to a region of at least 4 amino acids within sequence of aa1-42 of mature human ADM (SEQ ID NO: 24), wherein the sequence is as defined in the complete specification. ic circulation of said patient, and wherein said antibody or antibody fragment or non-Ig scaffold binds to a region of at least 4 amino acids within sequence of aa1-42 of mature human ADM (SEQ ID NO: 24), wherein the sequence is as defined in the complete specification.

Description

Anti-Adrenomedullin (ADM) antibody or anti-ADM. antibody fragment or anti—ADM non-1g ld for use in therapy of an acute disease or acute condition of a patient for stabilizing the circulation Field of the invention Subject matter of the present invention is an anti—adrenomedullin (ADM) antibody or an antim adrenomedullin antibody fragment or anti—ADM non-1g scaffold for use in therapy of an acute disease or condition of a patient for stabilizing the circulation.

Background The peptide adrenomedullin (ADM) was described for the first time in 1993 (Kitamura, K., et 51]., ”Adrenomedullin: A Novel Hypotensive Peptide Isolated From Human Pheochromocytoma", Biochemical and Biophysical Research ications, Vol. 192 (2), pp. 553-560 (1993)) as a novel hypotensive peptide comprising 52 amino acids, which had been isolated from a human pheochromocytome; SEQ ID No.: 21. In the same year, cDNA coding for a precursor peptide comprising 185 amino acids and the complete amino acid sequence of this precursor peptide were also described. The sor peptide, which comprises, inter alia, a signal sequence of 21 amino acids at the N—terminus, is referred to as "preproadrenomedullin" (pre~proADM). In the t ption, all amino acid positions specified usually relate to the pre—proADM which ses the 185 amino acids. The e adrenomedullin (ADM) is a peptide which comprises 52 amino acids (SEQ ID NO: 21) and which comprises the amino acids 95 to 146 of pre—proADM, from which it is formed by proteolytic cleavage. To date, substantially only a few fragments of the peptide fragments formed in the ge of the pre-proADM have been more exactly characterized, in particular the physiologically active peptides adrenomedullin (ADM) and "PAMP", a peptide comprising 20 amino acids ) which s the 21 amino acids of the signal peptide in pre—prOADM. The discovery and characterization of - ADM in 1993 triggered intensive research activity, the results of which have been summarized in various review articles, in the context of the present description, nce being made in particular to the articles to be found in an issue of "Peptides" devoted to ADM in particular (Editorial, Takahashi, K., ”Adrenomedullin: from a romocytoma to the eyes", Peptides, Vol. 22, p. 1691 (2001)) and (Etc, T., "A review of the biological properties and al implications of adrenomedullin and proadrenomedullin N—terminal 20 peptide (PAMP), hypotensive and vasodilating peptides", Peptides, Vol. 22, pp. 1693—1711 (2001)). A further review is (Hinson, er al., "Adrenomednllin, a Multifunctional Regulatory Peptide“, ine Reviews, Vol. 21(2), pp. 138—167 (2000)). in the scientific investigations to date, it has been found, inter alia, that ADM may be regarded as a polyfunctional regulatory peptide. It is released into the circulation in an inactive form ed by glycine (Kitamura, K., at at, "The ediate form of glycine~extended adrenomedullin is the major circulating molecular form in human plasma“, Biochem. Biophys. Res. Commun., Vol. 244(2), pp. 551—555 . Abstract Only). There is also a binding protein (Pio, R., et at, ement Factor H is a Serum— binding n for adrenomedullin, and the Resulting Complex Modulates the Bioactivities of Both Partners", The Journal of Biological Chemistry, Vol. 276(15), pp. 12292-12300 (2001)) which is specific for ADM and probably se modulates the effect of ADM.

Those physiological effects of ADM as well as of PAMP which are of primary importance in the investigations to date were the effects cing blood pressure.

Hence, ADM is an effective vasodilator, and it is possible to associate the hypotensive effect with the particular peptide segments in the C-terminal part of ADM. It has rmore been found that the mentioned further physiologically active peptide PAMP formed from pre—proADM likewise exhibits a hypotensive effect, even if it appears to have an action mechanism differing from that ofADM (of. in addition to the abovementioned review articles (Eto, T., ”A review of the biological properties and clinical implications of adrenomedullin and proadrenomedullin N-terminal 20 peptide (PAMP), hypotensive and lating peptides", Peptides, Vol. 22, pp. 1693-1711 (2001)) and (Hinson, er of, “Adrenomeduliin, a Multifunctional Regulatory Peptide”, Endocrine Reviews, Vol. 21(2), pp. 138-167 (2000)) also (Kuwasako, K., et at, "Purification and characterization of PAMP—12 (PAMP—20) in porcine adrenal medulla as a major endogenous biologically active peptide", FEBS Lett, Vol. 414(1), pp. 105-110 (1997). Abstract only), (Kuwasaki, K., at at, "increased plasma proadrenomedullin N—tenninal 20 peptide in patients with essential hypertension", Ann. Clin. m, Vol. 36 (Pt. 5), pp. 622628 (1999). Abstract only) or (Tsuruda, T., et all, "Secretion of proadrenomedullin Nntermina120 peptide from ed neonatal rat cardiac cells", Life Sci, Vol. 69(2), pp. 23 9—245 (2001). Abstract only) and EDP-AZ 0 622 458). It has furthermore been found that the trations of ADM which can be measured in the circulation and other biological liquids are, in a number of pathological states, significantly above the concentrations to be found in y control persons. Thus, the ADM level in ts with congestive heart failure, myocardial infarction, kidney diseases, hypeitensive disorders, Diabetes mellitus, in the acute phase of shock and in sepsis and septic shock are significantly increased, although to different extents. The PAMP concentrations are also increased in some of said ogical states, but the plasma levels are lower relative to ADM ((Eto, T., ”A review of the biological properties and clinical implications of adrenornedullin and proadrenomedullin N—tenninal 20 peptide (PAMP), hypotensive and vasodilating peptides", Peptides, Vol. 22, pp. 711 (2001)); page 1702). It is fiirthcnnore known that unusually high concentrations of ADM are to be observed in , and the highest concentrations in septic shock (cf. (Eto, T., "A review of the ical properties and clinical implications of adrenomedullin and proadrenomedullin inal 20 peptide (PAMP), hypotensive and lating peptides", Peptides, Vol. 22, pp. 1693—1711 (2001)) and (Hirata, at 611., "Increased Circulating medullin, a Novel Vasodilatory Peptide, in Sepsis", Journal of Clinical Endocrinology and lism, Vol. 81(4), pp. 1449—1453 (1996)), (Ehlenz, K., at at, ”High levels of ating adrenoniedullin in severe illness: Correlation with C—reactive n and evidence against the adrenal medulla as site of origin“, Exp Clin Endocrinol Diabetes, Vol. 105, pp. 156—162 (l997)), (Tomoda, Y., et at, "Regulation of adrenomedullin secretion from cultured cells", Peptides, Vol. 22, pp. 17834794 (2001)), (Ueda, S., et 01., ased Plasma Levels of Adrenomedullin in Patients with Systemic Inflammatory Response Syndrome", Am. J. Respir. Crit. Care Med, Vol. 160, pp. 132-136 (1999)) and (Wang, P., ”Adrenomedullin and vascular responses in ", Peptides, Vol. 22, pp. 1835—1840 (2001)).

Known in the art is further a method for identifying adrenomedullin immunoreactivity in biological s for diagnostic purposes and, in particular within the scope of sepsis diagnosis, cardiac diagnosis and cancer diagnosis. According to the invention, the midregional partial peptide of the proadrenomedullin, which contains amino acids (45—92) of the entire preproadrenomedullin, is measured, in particular, with an immunoassay which works with at least one labeled antibody that specifically recognizes a sequence of the mid- proADM (W02004/090546).

WO—Al 2004/097423 describes the use of an antibody against adrenomedullin for diagnosis, prognosis, and treatment of cardiovascular disorders. Treatment of diseases by blocking the ADM or are also described in the art, (a.g. WO—Al 2006/027147, ) said diseases may be sepsis, septic shock, cardiovascular diseases, infections, dermatological WO 72513 diseases, endocrinological diseases, metabolic diseases, gastroenterological diseases, cancer, inflammation, hematological diseases, atory diseases, muscle skeleton diseases, neurological diseases, urological diseases.

It is reported for the early phase of sepsis that ADM improves heart on and the blood supply in liver, spleen, kidney and small intestine. ADM—neutralizing antibodies neutralize the before mentioned effects during the early phase of sepsis (Wang, P., "Adrenomedullin and cardiovascular responses in sepsis", Peptides, Vol. 22, pp. 840 (2001).

In the later phase of sepsis, the hypodynamical phase of sepsis, ADM tutes a risk factor that is strongly associated with the mortality of patients in septic shock. (Schiitz et at, “Circulating Precursor levels of elin-1 and adrenomedullin, two endothelium—derived, counteracting substances, in sepsis”, Endothelium, 14:345‘351, (2007)). Methods for the diagnOSis and treatment of critically ill patients, eg. in the very late phases of , and the use of endothelin and endothelin agonists with vasoconstrictor activity for the preparation of medicaments for the treatment of critically ill patients have been described in WO—Al 20077062676. It is r described in WO—Al 2007/062676 to use, in place of endothelin and/or endothelin agonists, or in combination therewith, adrenomedullin antagonists, 1'.e. molecules which prevent or attenuate the vasodilating action of adrenomedullin, egg. by blocking its relevant receptors, or substances preventing the binding of adrenomedullin to its or (ag. specific binders as rag. antibodies binding to adrenomedullin and blocking its receptor bindings sites; “immunological neutralization“). Such use, or combined use, including a subsequent or preceding separate use, has been described in certain cases to be desirable for example to improve the eutic success, or to avoid undesirable physiological stress or side effects. Thus, it is ed that neutralizing ADM antibodies may be used for the treatment of sepsis in the late stage of .

Administration of ADM in combination with ADM—binding—Protein~l is described for treatment of sepsis and septic shock in the art. It is d that treatment of septic animals with ADM and ADM-binding-Protein-l prevents transition to the late phase of sepsis. It has to be noted that in a living organism ADM binding protein (complement factor H) is present in the circulation of said organism in high concentrations (Pic et at: Identification, characterization, and physiological actions of factor H as an Adrenomedullin g Protein present in Human ; Microscopy Res. and Technique, 55:23-27 (2002) and Martinez et (Followed by page 5A) In one particular aspect the present invention es use of an anti-Adrenomedullin (ADM) antibody or an anti-ADM antibody fragment binding to adrenomedullin or anti-ADM non-Ig scaffold binding to adrenomedullin in the manufacture of a medicament for use in therapy of an acute disease or acute condition of a patient for stabilizing the systemic circulation of said t, and wherein said antibody or antibody fragment or non-Ig scaffold binds to a region of at least 4 amino acids within sequence of aa1-42 of mature human ADM (SEQ ID NO: 24).

In another aspect the invention provides a pharmaceutical formulation comprising an anti- medullin (ADM) antibody or an anti-ADM antibody nt binding to adrenomedullin or anti-ADM non-Ig scaffold binding to adrenomedullin, wherein said antibody or dy fragment or non-Ig scaffold binds to a region of at least 4 amino acids within sequence of aa1-42 of mature human ADM (SEQ ID NO: 24).

In a still further aspect the present invention es the use of catecholamine, ADM binding protein or intravenous fluids, in the manufacture of a medicament, for use in therapy (Followed by page 6) of an acute disease or acute condition of a patient for stabilizing the systemic circulation of said patient, wherein said therapy comprises use in ation with an anti-Adrenomedullin (ADM) antibody or an anti-ADM antibody fragment binding to medullin or anti-ADM non-Ig scaffold binding to adrenomedullin, wherein said antibody or antibody fragment or non-Ig scaffold binds to a region of at least 4 amino acids within sequence of aa1-42 of mature human ADM (SEQ ID NO: 24). parameters see above). A patient in need for stabilizing the circulation may be, thus a patient that exhibits a heart rate of > 100 beats fmin and or < 65 mm Hg mean arterial pressure. If the circulation is stabilized by the administration of an anti-Adrencmedullin (ADM) dy or by an anti-ADM antibody fragment binding to adrenomeduliin or an DM non—lg scaffold g to adrenomedullin, this can be measured and is characterized by an increase of the mean al pressure over 65 mm Hg and/or an decrease of heart rate under 100 beats/min.

It should be emphasized that the ed anti—adrenomedullin (ADM) antibody or an anti- adrenomedullin antibody fragment or anti—ADM non—1g scaffold are intended by the present invention to be applied for sake of stabilizing the systemic circulation, and thus are not necessarily intended for any methods of primary treatment or first line treatment to the acute disease or acute condition itself that has to be ered as underlying e(s). This means the present invention do not provide for a therapy of healing/curing rag. cancer, diabetes, meningitis, pelytrauma, and the like. Accordingly, the therapy for an acute disease or acute condition of a patient within the scope of the invention is related to any kind of systemic circulatory insufficiency, or poor ic circulation of the blood as an acute event.

Acute disease or acute ions may be selected from the group but are not limited to the group comprising severe infections as ag. meningitis, Systemic inflammatory Response— Syndrem (SIRS), or sepsis; other diseases as diabetes, cancer, acute and chronic vascular diseases as rag. heart failure, myocardial infarction, , atherosclerosis; shock as ag. septic shock and organ dysfunction as ag. kidney dysfunction, liver dysfunction, burnings, surgery, traumata, poisoning, damages induced by chemotherapy. Especially useful is the antibody or fragment or scaffold ing to the present invention for reducing the risk of mortality during sepsis and septic shock, 1'. 9. late phases of .

In the following clinical criteria for SIRS, sepsis, severe sepsis, septic shock will be defined. 1) Systemic atory host response (SIRS) characterized by at least two of the following symptoms 0 patients exhibit hypotension (mean arterial pressure is < 65 mm Hg) - elevated serum lactate level being > 4 mmol/L a blood giucose > 7.7 mmol/L (in absence of es) 0 central venous pressure is not within the range 8—12 mm Hg - urine output is < 0.5 mL X kg'1 2: hr'1 9 l venous (superior vena cava) oxygen saturation is < 70% or mixed venous < 0 heart rate is > 90 beats/min 0 temperature < 36°C or > 38°C - respiratory rate > ZO/min a White cell count < 4 or > lZXlOg/L (leucocytes); > 10% immature neutrophiis 2) Sepsis Following at least two of the symptoms mentioned under 1), and additionally a ciinical suspicion of new infection, being: o cough/sputum/chest pain a abdominal pain/distension/diarrhoea 0 line infection 0 endocarditis o dysuria o headache with neck stiffness I itis/wound/joint infection 0 positive iology for any infection 3) Severe sepsis Provided that sepsis is manifested in t, and additionally a clinical suspicion of any organ dysfunction, being: 0 blood pressure systolic < 90/mean; < 65mmHG o lactate > 2 mmoi/L o Bilirubine > 34amol/L o urine output < 0.5 mL/kg/h fer 2h o creatinine > 177 amok/L o platelets < 100x109/L o SpOg > 90% unless 02 given 4) Septic shock At least One sign of end—organ ction as mentioned under 3) is sted. Septic shock is indicated, if there is refractory hypotension that does not respond to treatment and intravenous fluid administration alone is insufficient to maintain a patient‘s blood pressure from becoming hypotensive also provides for an administration of an anti—ADM antibody or an anti—ADM antibody fragment or an anti—ADM non—1g ld in accordance with the t invention. in one embodiment of the present invention the patient is not suffering from SIRS, a severe infection, sepsis, shock as ag. septic shock. Said severe infection denotes ag. meningitis, Systemic inflammatory Response-Syndrome , sepsis, severe sepsis, and shock as eg. septic shock. in this regard, a severe sepsis is characterized in that sepsis is manifested in said patient, and additionally a clinical suspicion of any organ dysfunction is present, being it: - blood pressure systolic < 90/mean; < 65mmHG a lactate > 2 mmol/L e Bilirubine > /L o urine output < 0.5 mL/kg/h for 2h I creatinine >3 77 nmol/L o ets < IOOXIOg/L 0 81302 > 90% unless 02 given In another embodiment said acute disease or acute condition is not sepsis, or not severe sepsis, or not SIRS, or not shock, or not septic shock.

In another embodiment said acute e or acute condition is not sepsis.

In another embodiment said acute e or acute condition is selected from the group ed meningitis, diabetes, cancer, acute and chronic vascular diseases as tag. heart e, myocardial infarction, stroke, atherosclerosis; shock as ag. septic shock and organ dysfunction as ag. kidney dysfunction, liver dysfunction, burnings, surgery, traumata, poisoning, damages induced by chemotherapy.

In all of the above mentioned acute diseases and conditions there might be the need for stabilizing the circulation of a patient by the administration of an anti-adrenomedullin (ADM) antibody or an antinadrenomedullin dy fragment or anti-ADM nonulg scaffold. an anti- adrenomedullin (ADM) antibody or an anti—adrenomedullin antibody nt or anti—ADM nonmlg scaffold may also be administered preventively in patients having an acute disease or condition in order to prevent that the heart rate increases of > 100 beats lmin and/ or mean arterial pressure decreases to < 65 mm Hg.

Anti~Adrenomedullin (ADM) antibody is an antibody that binds specifically to ADM, Anti~ adrenomedullin antibody fragment is a fragment of an anti—ADM antibody, wherein said fragment binds specifically to ADM. An antiuADM non—lg scaffold is a non-lg scaffold that binds specifically to ADM. Specifically binding to ADM allows binding to other antigens as well. This means, this specificity would not exclude that the antibody may cross—react with other polypeptides than that against it has been raised.

In one embodiment the anti—ADM antibody or the anti—adrenomedullin dy fragment or anti—ADM nonwlg scaffold according to the present invention reduces the vasopressor-agents requirement, eg. catecholamine requirement, of said patient. The vasopressonagents ement, e.g catecholamine requirement of a patient is an indicator for the condition of the circulation of said t. Thus, the DM antibody or the anti~adrenomedullin antibody fragment or anti—ADM non-lg scaffold may be administered at a point of time when the patient is in need of a essor agent, ag. catecholamine.

In one ment of the invention said patient is a patient in need of increasing the blood pressure.

A patient in need of stabilizing the circulation may a patient with low cardiac output and /or a low blood pressure (hypotension). This may be a patient with a heart rate > 100 beats/min.

This may be a patient with mean al pressures < 65 mmHg or even with < 60 mmHg.

Mean al re is defined as MAP 2 (COXSVR)+CVP where CO is cardiac output; SVR is systemic ar resistance and CV1J is central venous pressure and y small enough to be neglected in this formula. A patient in need of stabilizing the circulation may be also a t having in addition to the above symptoms a respiratory rate > 20 /min.

In a specific ment of the invention a t in need of stabilizing the circulation may be a patient with low cardiac output and /or a low blood pressure (hypotension). This may be a patient with a heart rate > 90 beats/min. This may be a patient with mean arterial pressures < 65 mmHg or even with < 60 mmHg.

Some patients with sepsis—induced hypofusion may remain hypotensive despite adequate fluid replacement. In these cases vasopressor agents are needed to increase MAP. Thus, in one embodiment of the invention the patient having a chronic or acute disease or acute condition is a patient in need of vasopressor agents to se MAP. Catecholamines such as ne, epinephrine (adrenaline), norepinephrine (noradrenaline), and phenylephrine have been traditionally used to raise blood pressure in patients with eg. septic shock. Recently also vasopressin has been suggested as potential vasopressor in patients with a c or acute disease or acute condition in need for stabilizing the ation. 2O Vasopressor agents as catecholamine may stabilize the circulation of a patient having a chronic or acute disease or acute condition. In case the condition of the patient (low blood pressure) is very critical, vasopressor agents administration, e.g. catecholamine administration, alone may not prevent the break—dOWn of the circulation. The additional administration of antian dy or the anti—ADM antibody fragment or anti—ADM non- Ig scaffold together with administration of ag. catecholamine may help to stabilize the circulation of a patient whose condition is so critical that catecholamine administration t administration of anti—ADM antibody or anti—ADM antibody fragment or anti—ADM non—1g scaffold would not be ent in order to stabilize the circulation of said patient.

Further, vasopressors may have serious side effects. Dopamine stimulates D1 receptors in the renal regional circulation, producing vasodilation and increases blood flow. This is one of the reasons why clinicians have utilized low doses of dopamine to protect kidney function. Also for other vasopressors it has been suggested that increasing the blood pressure with certain drugs, despite its intuitive appeal as something beneficial, can be associated with worse Outcomes.

Thus, subject of the invention is an anti—adrenoniedullin (ADM) dy or an anti— adrenomedullin antibody fragment or DM non-1g scaffold for use in y of an acute disease or acute condition of a patient in order to replace the administration of a vasopressor totally or partially. This means the patient ing to the present invention may be a patient being in need of or treatment with vasopressors or a patient receiving a treatment with VflSOpI‘fiSSOTS .

The anti—ADM antibody or the anti—ADM antibody fragment or anti-ADM non—lg ld may be also administered preventively before the patient exhibits any signs of Serious circulation problems. This might be the case if the patient has a chronic or acute disease or acute condition where circulation problems may be expected, comprising severe ions as ag. meningitis, Systemic inflammatory Response—Syndrom , or sepsis; other diseases as diabetes, cancer, acute and chronic vascular diseases as ag. heart failure, myocardial infarction, stroke, atherosclerosis; shock as eg. septic shock and organ dysfunction as e.g. kidney dysfunction, liver dysfunction, burnings, surgery, traumata, poisoning, damages induced by chemotherapy. Especially useful is the antibody or nt or scaffold according to the present invention for reducing the risk of mortality during sepsis and septic shock, Le. late phases of sepsis. The person skilled in the art is aware that said reducing the risk of mortality is associated with the stabilization of the circulation in accordance with the invention. Acute disease or acute condition may be a e or condition wherein the patient is characterized as being in need of stabilizing the ation. The need of stabilizing the circulation is characterized as outlined above, namely this may be a patient preferably with a heart rate of > 90 beats/min or even with a heart rate of > 100 beats/min. This may be a patient with mean al pressures < 65 1ang or even with < 60 mmHg. Mean arterial pressure is defined as MAP = R)+CVP where CO is cardiac output; SVR is systemic vascular resistance and CVP is central venous pressure and usually small enough to be neglected in this formula. A patient in need of stabilizing the ation may be also a patient having a respiratory rate > 20 /min.

The ation stabilizing effect of the anti—ADM antibody or the anti-ADM antibody fragment or anti—ADM non—lg scaffold is thus supporting the primary y of said chronic or acute disease or acute condition. This means in one embodiment that the anti—ADM antibody or the anti—ADM antibody fragment or anti-ADM non—lg scaffold is administered in addition to a first line treatment (primary therapy). In case of a chronic or acute e or acute condition like a severe infection, SIRS, sepsis or the like the primary y would be eg. the administration of antibiotics. The anti—ADM antibody or the DM antibody fragment or anti—ADM non-lg scaffold would stabilize the circulation and would help to prevent worsening of the critical condition of said patient until the cg. otic administration takes effect. As before mentioned the anti-ADM antibody or the anti-ADM antibody fragment or anti—ADM non—1g scaffold may be administered in a preventive way or in a therapeutic way, this means in order to prevent circulation problems or in order to stabilize the circulation when circulation problems are t in a said patient, It should be emphasized that the circulation problems comprised by the present invention may be acute circulation problems according to a specific embodiment of the invention.

In one embodiment of the invention an anti—Adrenomeduliin (ADM) antibody or an anti- ADM antibody fragment or anti—ADM non—1g scaffold is to be used in ation with vasopressors cg. catecholamine wherein said combination is for use in therapy of an acute disease or acute condition of a patient for stabilizing the circulation of said patient.

In one embodiment of the invention said patient having a c or acute disease or condition being in need for izing the circulation is characterized by the need of said patient to get administration of vasopressors cg. catecholamine stration.

Subject matter of the invention in one specific embodiment is, thus, an anti~adrenomeduliin (ADM) antibody or an anti-adrenomedullin antibody fragment g to ADM or anti-ADM non—lg scaffold binding to ADM for use in y of a patient in need of an administration of vasopressors, eg. a catecholamine administration.

Furthermore, in one embodiment of the ion an anti—Adrenomedullin (ADM) antibody or an anti-adrenomedullin dy fragment or an anti—ADM non-lg scaffold is to be used in combination with fluids administered intravenously, wherein said ation is for use in therapy of a patient having a chronic or acute disease or acute condition of a patient for stabilizing the circulation of said patient. In one embodiment of the invention said patient having a chronic or acute disease or ion being in need for stabilizing the circulation is characterized by the need of said t to get intravenous fluids. 2012/072932 In accordance with the invention the need of a patient to get intravenous fluids is also an acute need due to an acute disease or acute condition. This, however, does not exclude an underlying chronic or acute disease the patient is having and that is maybe associated with the acute need for fluids as well as acute need for stabilizing the circulation.

Subject matter of the invention in one specific ment is, thus, an drenomedullin (ADM) antibody or an drenomedullin antibody fragment or anti—ADM non-lg scaffold for use in therapy of a patient in need of intravenous fluids.

Furthermore, in one embodiment of the invention an anti~Adrenomedullin (ADM) antibody or an anti—adrenomedullin antibody fragment or an anti-ADM non—lg scaffold is monospecific.

Monospecific anti-Adrenomedullin (ADM) dy or monospecific anti—adrenomedullin antibody fragment or monospecific anti—ADM non-lg scaffold means that said antibody or antibody fragment or non—lg ld binds to one specific region encompassing at least 5 amino acids Within the target ADM. ecific anti—Adrenomedullin (ADM) antibody or monospecific anti—adrenomedullin antibody fragment or monospecific anti-ADM non-1g ld are antiuAdrenomedullin (ADM) antibodies or anti~adrenomedullin antibody fragments or anti—ADM non—lg scaffolds that ali have affinity for the same antigen.

In a c and preferred embodiment the present invention provides for a monospecific anti—Adrenomedullin (ADM) antibody or ecific anti~adrenomeduliin antibody fragment or monospecific anti—ADM non~Ig scaffold, characterized in that said antibody or antibody fragment or non-lg scaffold binds to one specific region encompassing at least 4 amino acids within the target ADM.

In another special embodiment the anti—ADM antibody or the antibody fragment binding to ADM is a monospecific antibody. Monospecific means that said antibody or antibody fragment binds to one specific region encompassing ably at least 4, or at. least 5 amino acids within the target ADM. Monospecific antibodies or fragments are antibodies or fragments that all have y for the same antigen. Monoclonal antibodies are monospecific, but monospecific antibodies may also be produced by other means than producing them from a common germ cell.

An antibody according to the present invention is a protein including one or more polypeptides substantially encoded by immunoglobulin genes that specifically binds an antigen. The recognized immunoglobulin genes include the kappa, lambda, alpha (IgA), gamma (IgGl, IgGg, IgGg, IgG4), delta (IgD), epsilon (IgE) and mu (IgM) constant region genes, as well as the myriad immunoglobulin variable region genes. Full-length immunoglobulin light chains are generally about 25 Kd or 214 amino acids in length. Full— length immunoglobulin heavy chains are generally about 50 Kd or 446 amino acid in length.

Light chains are encoded by a variable region gene at the NHE-terminus (about llO amino acids in length) and a kappa or lambda constant region gene at the COOI—Iw-terminns. Heavy chains are rly encoded by a variable region gene (about 116 amino acids in length) and one ofthe other constant region genes.

The basic structural unit of an antibody is generally a ner that consists of two identical pairs of immunoglobulin , each pair having one light and one heavy chain. In each pair, the light and heavy chain variable s bind to an antigen, and the constant regions mediate effector fimctions. immunoglobulins also exist in a y of other forms including, for example, Fv, Fab, and (Fab'b, as well as bifunctional hybrid antibodies and single chains (eg, Lanzavecchia et all, Eur. J. Immunol. l7:lOS,l987; Huston et al, Proc. Natl. Acad. Sci.

USA” 815879—5883, 1988; Bird er £11., Science 242:423—426, 1988; Hood et al, Immunology, Benjamin, N.Y., 2nd ed, 1984; Hunkapiller and Hood, Nature 323:15-16,1986).

An globulin light or heavy chain variable region includes a framework region interrupted by three hypervariable regions, also called complementarity ining regions (CDR's) (see, Sequences of Proteins of Immunological Interest, E. Kabat er £11., US.

Department of Health and Human Services, 1983). As noted above, the CDRs are primarily responsible for binding to an epitope of an antigen. An immune x is an antibody, such as a monoclonal antibody, chimeric antibody, humanized antibody or human antibody, or functional antibody fragment, specifically bound to the antigen.

Chimeric antibodies are antibodies Whose light and heavy chain genes have been constructed, typically by genetic engineering, from immunoglobulin le and constant region genes belonging to different species. For example, the variable segments of the genes from a mouse monoclonal antibody can be joined to human constant segments, such as kappa and gamma 1 or gamma 3. In one example, a therapeutic chimeric dy is thus a hybrid protein ed of the le or antigen—binding domain from a mouse antibody and the constant or effector domain from a human antibody, although other mammalian species can be used, or the variable region can be produced by molecular techniques. Methods of making chimeric antibodies are well known in the art, e.g., see U.S. Patent No. 5,807,715. A "humanized" globulin is an immunoglobulin including a human framework region and one or more CDRs from a nonshuman (such as a mouse, rat, or synthetic) noglobulin. The non- human immunoglobulin providing the CDRs is tenned a "donor" and the human immunoglobulin providing the framework is termed an "acceptor." In one embodiment, all the CDRs are from the donor iminunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85—90%, such as about 95% or more identical. Hence, all parts of a humanized globulin, except possibly the CDRS, are ntially identical to corresponding parts of natural human immunoglobulin sequences. A "humanized antibody" is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The or ork of a humanized immunoglobulin or antibody may have a limited number of substitutions by amino acids taken from the donor ork. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions. Exemplary conservative tutions are those such as gly, ala; val, ile, leu; asp, glu; asn, gln; ser, thr; lys, arg; and phe, tyr. Humanized immunoglobulins can be constructed by means of genetic engineering (eg, see US. Patent No. 5,585,089). A human antibody is an antibody n the light and heavy chain genes are of human origin.

Human antibodies can be generated using methods known in the art. Human antibodies can be produced by immortalizing a human B cell secreting the antibody of interest. Immortalization can be accomplished, 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 produced by phage display methods (see, ag, Dower e: (11., PCT ation No. W091/17271; McCafferty er al, PCT Publication No. WO92/001047; and Winter, PCT Publication No.

W092/2079l), or selected from a human atorial monoclonal antibody library (see the Morphosys website). Human dies can also be prepared by using transgenic s carrying a human immunoglobulin gene (for e, see Lonberg at all, PCT Publication No. 2227; and Kuoherlapati, PCT Publication No. W09l/1074]).

Thus, the anti—ADM antibody may have the formats known in the art. Examples are human antibodies, monoclonal antibodies, humanized antibodies, chimeric antibodies, CDR—grafted antibodies. In a preferred embodiment antibodies according to the present invention are WO 72513 recombinantly produced antibodies as ag. lgG, a typical full—length immunoglobulin, or antibody fragments containing at least the F—variable domain of heavy and/or light chain as eg. ally d antibodies (fragment antigen binding) including but not limited to Fab—fiagments including Fab minibodies, single chain Fab antibody, monovalent Fab antibody with epitope tags, e.g. Fab-VSSXZ; bivalent Fab (mini-antibody) dimerized with the CH3 domain, bivalent Fab or multivalent Fab, ag. formed via multirnerizaticn with the aid of a heterologous domain, ag. via zation of dHLX domains,e.g. Fab—dHLX-FSX2; F(ab‘)2—iiagments, scFvufragments, multimerized multivalent or/and multispecific SCFV~ fragments, bivalent and/or bispecific diabodies, BITE® (bispecific T-cell engager), trifunctional dies, polyvalent dies, egg. from a different class than G; single— domain antibodies, eg. nanobcdies derived from carnelid or fish immunoglobulinesand numerous others.

In addition to DM antibodies other biopolyrner scaffolds are well known in the art to complex a target molecule and have been used for the tion of highly target c biopelymers. Examples are aptamers, lmers, anticalins and conotoxins. For illustration of antibody formats please see Fig. la, lb and lo.

An antibody fragment according to the present ion is an antigen binding fragment of an antibody according to the present invention.

In a red embodiment the ADM antibody format is selected from the group comprising Fv fragment, scFv fragment, Fab fragment, scFab fragment, (Fab)? fragmentand scvaFc Fusion protein. in another preferred embodiment the antibody format is selected from the group sing scFab fragment, Fab nt, scFv fragment and bioavailability optimized conjugates thereof, such as PEGylated fragments. One of the most preferred formats is the scFab format.

Non—lg scaffolds may be protein scaffolds and may be used as antibody mimics as they are capable to bind to ligands or antigenes. Non—lg scaffolds may be selected from the group comprising tetranectin-based non-lg scaffolds (Lag. described in US 2010/0028995), fibronectin scaffolds (ag. described in EP 1266 025; lipocalin—based scaffolds ((e.g. described in ); ubiquitin lds (tag. described in ), transferring scaffolds (e.g. described in US 2004/0023334), protein A scaffolds (eg. described in EP WO 72513 2231860), ankyrin repeat based scaffolds (6g. described in ) , micr0proteins preferably microproteins forming a cystine knot) scaffolds (tag. described in EP 2314308), Fyn SH3 domain based scaffolds (cg. described in ) EGFR—A-domain based scaffolds (cg. described in ) and Kunitz domain based scaffolds (e. g. described in EP 1941867).

In one embodiment of the invention antibodies ing to the present invention may be produced as follows: A Balb/c mouse was zed with lOOug ADM-Peptide-BSA—Conjugate at day 0 and I4 (emulsified in lOOul complete Freund’s nt) and SOug at day 21 and 28 (in lOOul incomplete Freund’s adjuvant). Three days before the fusion experiment was performed, the animal received 50ug of the conjugate dissolved in lOOul saline, given as one intraperitoneal and one intravenous injection.

Spenocytes from the immunized mouse and cells of the myeloma cell line SPZ/O were fused with lml 50% polyethylene glycol for 303 at 37°C. After washing, the cells were seeded in 96~well cell culture plates. Hybrid clones were selected by growing in HAT medium [RPMI 1640 culture medium supplemented with 20% fetal calf serum and HATfiSupplement]. After two weeks the HAT medium is replaced with HT Medium for three passages ed by returning to the normal cell e medium.

The cell e supeinatants were primary screened for antigen specific IgG- antibodies three weeks after . The positive tested microcultures were transferred into 24-well plates for propagation. After retesting, the selected cultures were cloned and ed using the limiting—dilution technique and the isotypes were determined (see also Lane, RD. (1985). A short—duration polyethylene glycol fusion technique for increasing production of monoclonal antibody~secreting hybridomas. J. Immunol. Meth. 81: 223—228; Ziegler, B. at 611.0996) Glutamate oxylase (GAD) is not detectable on the surface of rat islet cells examined by cytofluorometry and ment—dependent dy—mediated cytotoxicity of monoclonal GAD antibodies, Horm. Metab. Res. 28: 11—15).

Antibodies may be produced by means of phage display according to the following procedure: 2012/072932 The human naive antibody gene libraries HAL7/8 were used for the isolation of recombinant single chain F—Variable domains (scFv) against adrenornedullin peptide. The dy gene libraries were screened with a panning strategy comprising the use of peptides containing a biotin tag linked via two different spacers to the adrenomedullin peptide sequence. A mix of panning rounds using ecifically bound antigen and streptavidin bound antigen were used to minimize background of non-specific binders. The eluted phages from the third round of panning have been used for the generation of monoclonal scFv sing E.coli s.

Supernatant from the cultivation of these clonal strains has been directly used for an n ELISA testing (Hust, M., Meyer, T., Voedisch, B., Riilker, T., Thie, 1-1., zal, A., Kirsch, Ml, Schiitte, M., Helmsing, 3., Meier, D., Schirrmann, TI, Dfibel, 8., 2011. A human scFyr antibody generation pipeline for proteorne research. Journal of Biotechnology 152, 159% 170; Schfitte, M., Thullier, P., Pelat, T., Wezler, X., Rosenstock, P., Hinz, D., Kirsch, M.I.,Hasenberg, M., Frank, R., Schirrmann, T., Gunzer, M., Hust, M., Dubel, S., 2009. fication of a putative Crf splice t and generation of recombinant antibodies for the specific detection of Aspergillus fumigatus. PLoS One 4, 66625). zation of murine antibodies may be conducted according to the following procedure: For humanization of an antibody of murine origin the antibody sequence is ed for the structural interaction of framework regions (FR) with the complementary ining regions (CDR) and the antigen. Based on structural modelling an appropriate FR of human origin is selected and the murine CDR sequences are transplanted into the human FR. ions in the amino acid sequence of the CDRS or FRs may be introduced to regain structural interactions, which were abolished by the species switch for the FR sequences. This recovery of structural interactions may be achieved by random approach using phage display libraries or via directed approach guided by molecular modeling (Almagro JC, Fransson 1., 2008.

Humanization of antibodies. Front Biosci. 2008 Jan l;l3:1619—33.).

In a preferred embodiment the ADM antibody format is selected from the group comprising Fv fragment, scFv fragment, Fab nt, scFa-b fragment, F(ab)2 fragment and scFquc Fusion protein. In another preferred embodiment the antibody format is selected from the group comprising scFab fragment, Fab fragment, scFv fragment and bioavailability optimized conjugates thereof, such as PEGylated fragments. One of the most red formats is scFab format.

In another preferred embodiment, the anti—ADM antibody, anti-ADM antibody fragment, or anti-ADM non—1g scaffold is a full length antibody, antibody fragment, or non—lg scaffold.

In a preferred embodiment the anti—adrenomedullin antibody or an drenomeduilin antibody nt or anti-ADM non—1g scaffold is directed to and can bind to an epitope of at least 5 amino acids in length contained in ADM.

In another preferred ment the drenomedullin antibody or an anti-adrenomedullin antibody fragment or DM non—lg scaffold is directed to and can bind to an epitope of at least 4 amino acids in length ned in ADM.

In one specific embodiment of the invention the anti—Adrenomedullin (ADM) antibody or anti—ADM antibody fragment binding to adrenomedullin or anti-ADM. non—1g scaffold binding to adrenomedullin is provided for use in therapy of an acute disease or acute 3.5 condition of a patient wherein said antibody or fragment or scaffold is not ADM—binding— Protein—l (compiement factor H).

In one specific ment of the invention the anti-Adrenomedullin (ADM) antibody or anti-ADM antibody nt binding to medullin or anti—ADM noang scaffold binding to adrenorneduilin is provided for use in therapy of an acute disease or acute condition of a patient wherein said antibody or antibody fragment or non—lg scaffold binds to a region of preferably at least 4, or at least 5 amino acids Within the sequence of aa 1-42 of mature human ADM: SEQ ID No.: 24 YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVA.

In one specific ment of the invention the anti-Adrenomeduliin (ADM) antibody or antimADM antibody fragment binding to medullin or anti-ADM non—1g scaffold binding to adrenomedullin is provided for use in therapy of an acute disease or acute condition of a patient wherein said antibody or fragment or scaffold binds to a region of preferably at least 4, or at least 5 amino acids within the sequence of aa l~21 ofmature human ADM: WO 72513 2012/072932 SEQ ID No.2 23 YRQSMNNFQGLRSFGCRFGTC.

In a preferred embodiment of the present invention said anti—ADM antibody or an anti— adrenomedullin antibody fragment or anti—ADM non—lg scaffold binds to a region of ADM that is located in the N-terminal part (aa 1—21) of adrenomedullin, (see Fig. 2).

In another preferred embodiment said anti-ADM antibody or an anti—adrenomedullin antibody fragment or anti—ADM non—lg scaffold recognizes and binds to the Naterminal end (aal) of adrenomedullin. N—terminal end means that the amino acid 1, that is “Y” of SEQ ID No. 21 or 23; is mandatory for antibody binding. Said antibody or fragment or non—1g scaffold would neither bind N—terminal ed nor N-terminal modified Adrenomedullin nor Nuterminal degraded adrenomedullin.

In a red embodiment the anti~adrenomedullin antibody or an anti—adrenomedullin antibody fragment or anti-adrenomedullin non—lg scaffold is directed to and can bind to an epitope of at least 5 amino acids in length contained in ADM, preferably in human ADM.

In a preferred embodiment the anti—adrenomedullin antibody or an anti-adrenomeduilin antibody fragment or anti—adrenomedullin non-lg scaffold is directed to and can bind to an epitope of at least 4 amino acids in length contained in ADM, preferably in human ADM.

In another specific embodiment nt to the ion the herein provided DM antibody or anti—ADM antibody fragment or anti~ADM non—lg scaffold does not bind to the C-terminal portion ofADM, re. the aa 43 — 52 (SrADM PRSKISPQGY—NHZ (SEQ ID NO: 25).

In one specific embodiment it is red to use an anti—ADM antibody or an anti“ adrenornedullin antibody fragment or anti—ADM non-lg scaffold ing to the present invention, wherein said anti—adrenomeduilin antibody or said anti—adrenomedullin antibody fragment or DM non—lg scaffold is an ADM izing antibody or an adrenomeduilin stabilizing antibody fragment or an adrenomedullin stabilizing non—lg scaffold that enhances the half life 0112; half retention time) of adrenomedullin in serum, blood, plasma at least 10 %, preferably at least 50 %, more preferably >50 %, most ably >1 00%.

The half life (half retention time) of ADM may be determined in human plasma in absence and presence of an ADM stabilizing antibody or an adrenomedullin stabilizing antibody fragment or an adrenornedullin stabilizing non—lg scaffold, respectively, using an immunoassay for the quantification ofADM.

The following steps may be conducted: - ADM may be diluted in human citrate plasma in absence and presence of an ADM stabilizing antibody or an adrenomedullin stabilizing antibody fragment or an adrenomedullin stabilizing non~IG scaffold, respectively,and may be incubated at 24 — Aliquots are taken at selected time points (ag. within 24 hours) and degradation of ADM may be d in said aliquots by ng at —20 °C — The quantity of ADM may be determined by a hADM immunoassay directly, if the selected assay is not influenced by the stabilizing antibody. atively, the aliquot may be treated with denaturing agents (like HCl) and, after clearing the sample (eg. by centrifugation) the pH can be lized and the ADM-quantified by an ADM immunoassay. Alternatively, munoassay technologies (ag. rpHPLC) can be used for ADM—quantification. a The half life of ADM is calculated for ADM incubated in absence and presence of an ADM stabilizing antibody or an adrenomedullin stabilizing antibody nt or an medullin stabilizing non-1G scaffold, respectively, - The enhancement of half life is calculated for the stabilized ADM in comparison to ADM that has been ted in absence of an ADM stabilizing antibody or an adrenomedullin stabilizing antibody fragment or an adrenomedullin izing non—1G scaffold.

A two—fold increase ofthe half life ofADM is an enhancement ofhalf life of. 100%.

Half Life (half retention time) is defined as the period over which the concentration of a specified chemical or drug takes to fall to half baseline concentration in the specified fluid or blood. 2012/072932 An assay that may be used for the determination of the Half life (half retention time) of adrenornednllin in serum, blood, plasma is described in Example 3.

For other diseases blocking of ADM may be beneficial to a certain extent. However, it might also be detrimental if ADM is totally neutralized as a certain amount of ADM may be required for several physiological functions. In many reports it was emphasized that the administration of ADM may be beneficial in certain diseases. In contrast thereto in other reports ADM was reported as being life threatening when stered in certain conditions.

In a specific embodiment said DM antibody, anti—ADM antibody fragment or anti— ADM nonmlg scaffold is a non—neutralizing antibody, fragment or non—lg ld. A neutralizing anti—ADM antibody, DM antibody fragment or anti—ADM non—1g scaffold would block the bioactivity of ADM to nearly l00%, to at least more than 90%, preferably to at least more than 95%.

In contrast, a non—neutralizing anti—ADM antibody, or anti—ADM antibody fragment or anti— ADM non—lg scaffold blocks the bioactivity of ADM less than 100%, ably to less than 95%, preferably to less than 90%, more preferred to less than 80 % and even more preferred to tess than 50 %. This means that the residual bioactivity of ADM bound to the non- neutralizing anti—ADM antibody, or anti-ADM antibody fragment or anti-ADM non—lg scaffold would be more than 0%, preferably more than 5 %, preferably more than 10 % more preferred more than 20 %, more preferred more than 50 %.

In this context (a) molecule(s), being it an dy, or an antibody fragment or a non—lg scaffold with “non-neutralizing anti—ADM ty”, collectively termed here for city as “non—neutralizing” anti—ADM antibody, antibody fragment, or non—1g scaffold, that e.g. blocks the bioactivity of ADM to less than 80 %, is defined as - a molecule or molecules g to ADM, which upon addition to a culture of an eukaryotic cell line, which expresses functional human recombinant ADM receptor composed of CRLR tonin receptor like receptor) and RAMP3 (receptor-activity modifying protein 3), reduces the amount of CAMP produced by the cell line h the action of parallel added human synthetic ADM WO 72513 peptide, wherein said added human synthetic ADM is added in an amount that in the absence of the non-neutralizing antibody to be analyzed, leads to half— maximal stimulation of CAMP synthesis, wherein the reduction of CAMP by said molecule(s) binding to ADM takes place to an , which is not more than 80%, even when the utralizing molecule(s) binding to ADM to be analyzed is added in an amount, which is 10-fold more than the , which is needed to obtain the maximal reduction of cAMP synthesis obtainable with the non-neutralizing antibody to be analyzed.

The same ion applies to the other ranges; 95%, 90%, 50% etc.

In a specific embodiment according to the present invention an anti—ADM antibody or an anti— adrenomedullin antibody nt or anti—ADM non—Ig scaffold is used, wherein said antibody or antibody fragment or non—lg scaffold blocks the bioaetivity of ADM to less than 80 %, preferably less than 50% (of baseline values).

This is in the sense of blocking the circulating ADM of not more than 80% or not more than 50%, respectively.

It has been understood that said d blocking of the bioactivity of ADM occurs even at excess concentration of the antibody, antibody fragment or non—lg scaffold, meaning an excess of the antibody, antibody fragment or non—1g scaffold in relation to ADM. Said limited blocking is an intrinsic property of the ADM binder itself. This means that said antibody, antibody fragment or non—1g scaffold has a maximal inhibition of 80% or 50%, respectively.

By implication, this means that 20% or 50% al ADM bioactivity remains present gh appropriate amounts or excess amounts of antibody, antibody fragment or non~1g scaffold are administered, respectively. in a preferred embodiment said anti»ADM antibody, anti—ADM dy fiagment or anti- ADM non—1g scaffold would block the bioactivity of ADM at least 5 %. By implication, this means residual 95% circulating ADM bioactivity s present. This is the lower threshold of bioactivity remaining after administration of said DM antibody, anti~ADM antibody fragment or anti—ADM nonnig scaffold. The bioactivity is defined as the effect that a substance takes on a living organism or tissue or organ or functional unit in vivo or in vitro (eg. in an assay) after its ction. In case of ADM bioactivity this may be the effect of ADM in a human recombinant Adrenomeduliin receptor CAMP functional assay. Thus, according to the present invention bioactivity is defined via an Adrenomedullin receptor cAMP onal assay. The following steps may be med in order to determine the bioactivity ofADM in such an assay: - Dose response curves are performed with ADM in said human recombinant Adrenomedullin receptor CAMP functional assay.

— The ADM—concentration ofhalf—maximal CAMP stimulation may be calculated.

- At constant half—maximal cAMP~stimulating ADM-concentrations dose response curves (up to 100 ug/ml final concentration) are performed by an ADM stabilizing antibody or an adrenomedullin stabilizing dy fragment or an adrenorneduliin stabilizing non-lg scaffold, respectively.

A maximal (at maximal dose) inhibition by said ADM izing antibody of 50% means that said ADM antibody or said adrenomedullin antibody fragment or said adrenomedullin nOn—Ig scaffold, respectively, blocks the bioactivity to 50% of baseline values. A l inhibition in said ADM bioassay of 80% means that said anti—ADM antibody or said anti— adrenomedullin antibody fragment or said anti-adrenomedullin non—1g scaffold, respectively, blocks the bioactivity of ADM to 80%. This is in the sense of blocking the ADM bioactivity to not more than 80%.

The bioactivity of ADM may be determined in a human recombinant Adrenomedullin receptor CAMP functional assay omedullin Bioassay) according to Example 2. in a preferred embodiment a. modulating DM antibody or a modulating anti- adrenomedullin antibody fragment or a modulating anti—adrenomedullin non—lg scaffold is used in therapy of an acute disease or acute condition of a patient for stabilizing the circulation.

Such a ting anti—ADM antibody or a modulating DM adrenomedullin antibody fragment or a modulating antiuADM adrenomedullin non—lg scaffold may be especially useful in the treatment of . A ting anti-ADM antibody or a modulating anti- adrenomedullin antibody nt or a modulating anti—adrenomedullin non—1g scaffold enhances the bioactivity of ADM in the early phase of sepsis and reduces the damaging effects ofADM in the late phase of sepsis.

WO 72513 A “modulating” anti—ADM antibody or a modulating anti-adrenomedullin antibody fragment or a modulating anti—adrenomedullin non—lg scaffold is an antibody or an adrenornedullin antibody fragment or non—1g scaffold that enhances the half life (tug half retention time) of adrenornedullin in serum, blood, plasma at least 10 %, preferably at least, 50 %, more preferably >50 %, most preferably >100% and blocks the ivity of ADM to less than 80 %, preferably less than 50 % and n said anti-ADM antibody, anti-ADM antibody fragment or anti~ADM non-lg scaffold would block the ivity of ADM at least 5 %.

TheSe values related to half—life and blocking of ivity have to be understood in relation to the before—mentioned assays in order to determine these values. This is in the sense of blocking the circulating ADM ofnot more than 80% or not more than 50%, respectively. This means 20% residual ADM bioactivity remains present, or 50% residual ADM bioactivity remains present, respectively.

Such a modulating anti—ADM dy or a modulating anti~adrenomedullin antibody nt or a modulating anti—adrenornedullin non—lg scaffold offers the advantage that the dosing of the administration is facilitated. The combination of partially blocking or partially reducing Adrenomedullin bioactivity and increase of the in viva half life (increasing the Adrenomedullin bioactivity) leads to ial simplicity of anti—Adrenomedullin antibody or an anti—adrenornedullin antibody fragment or anti—adrenomedullin non—lg scaffold . In a situation of excess endogenous Adrenomedullin (maximal stimulation, late sepsis phase, shock, hypodynamic phase) the activity lowering effect is the major impact of the antibody or fragment or scaffold, limiting the (negative) effect of Adrenomedullin. In case of low or normal nous Adrenornedullin concentrations, the biological effect of anti- Adrenomedullin antibody or an anti~adrenomedullin antibody fi'agrnent or DM non—lg scaffold is a combination of lowering (by partially ng) and increase by increasing the Adrenornedullin half life. If the half life effect is stronger than the net blocking effect, the biological activity of endogenous Adrenomedullin is beneficially increased in early phases of sepsis (low medullin, hyperdynarnic phase). Thus, the non-neutralizing and ting Adrenomeduilin antibody or adrenomedullin antibody fragment or adrenomedullin non—lg scaffold acts like an ADM bioactivity buffer in order to keep the bioactivity of ADM within a certain physiological range.

Thus, the dosing of the anti-ADM antibody/fragment/scaffold in cg. sepsis may be selected from an excessive concentration, because both sepsis phases (early and late) benefit from excessive anti—ADM antibody or an anti—adrenomedullin antibody fragment or anti—ADM non-lg scaffold ent in case of a modulating effect. Excessive means: The anti Adrenomedullin antibody or an anti—adrenomednllin antibody fragment or anti~ADM non-lg ld concentration is higher than endogenous mednllin during late phase (shock) of ag. sepsis. This means, in case of a ting anti~ADM antibody or modulating anti— ADM antibody fragment or modulating DM non—1g scaffold dosing in sepsis may be as follows: The concentration of Adrenomednllin in septic shock is 226+/—66 frnol/ml (Nishio er al., ”Increased plasma trations of adrenomedullin ate with relaxation of vascular tone in patients with septic shock", Crit Care Med. 1997, 25(6):953—7), an equimoiar concentration of anti—ADM antibody or anti—ADM antibody fragment or anti—ADM non—1g scaffold is 42.5 ng/l blood, (based on 6 1 blood volume / 80kg body weight) 3.2 ng/kg body weight. Excess means at least double (mean) septic shock Adrenomedullin concentration, at least > Bug anti—Adrenomedullin antibody or an anti—adrenomedullin antibody fragment or anti—ADM non-1g scaffold / kg body weight, preferred at least 6.4ug anti—Adrenornedullin antibody or an anti—adrenomedullin antibody fragment or anti—ADM non—lg scaffold / kg body weight. Preferred > mug/kg, more preferred > mug/kg, most preferred > 100 pg anti— Adrenomedullin dy or an anti-adrenomedullin antibody fragment or anti-ADM non—1g scaffold / kg body weight.

This may apply to other severe and acute conditions than septic shock as well.

In a specific embodiment of the invention the anti—ADM antibody is a monodonal antibody or a fragment thereof. In one embodiment of the invention the anti-ADM antibody or the anti~ ADM antibody nt is a human or humanized antibody or derived therefrom. In one specific embodiment one or more e) CDR’S are grafted into a human antibody or antibody fragment.

Subject matter of the present invention in one aspect is a human CDR—grafted anti-ADM dy or DM antibody fragment thereof that binds to ADM, wherein the human CDR-grafied antibody or antibody nt thereof comprises an antibody heavy chain (H chain) comprising SEQ ID NO:1 GYTFSRYW SEQ ID NO: 2 ILPGSGST and/or SEQ ID NO: 3 DGFDY and /or further comprises an antibody light chain (L chain) sing: SEQ ID N0:4 QSIVYSNGNTY SEQ ID NO: 5 and/or SEQ ID NO: 6 FQGSI-IIPYT.

In one specific embodiment of the invention subject matter of the present invention is a human monoclonal antibody that binds to ADM or an antibody fragment thereof wherein the heavy chain comprises at least one CDR selected from the group comprising: SEQ ID NO: 1 GYTFSRYW SEQ ID NO: 2 ILPGSGST 2012/072932 SEQ ID NO: 3 TEGYEYDGFDY and wherein the light chain comprises at least one CDR selected from the group sing: SEQ ID No: 4 QSIVYSNGNTY SEQ ID NO: 5 RVS SEQ ID NO: 6 FQGSHIPYT.

In a more specific embodiment of the invention subject matter of the invention is a human monoclonal antibody that binds to ADM or an antibody fragment thereof wherein the heavy chain comprises the sequences SEQ ID NO: 1 GYTFSRYW SEQ ID NO: 2 ILPGSGST SEQ in NO: 3 TEGYEYDGFDY and wherein the light chain comprises the sequences SEQ ID NO: 4 QSIVYSNGNTY SEQ 11:) NO: 5 SEQ ID NO: 6 FQGSHIPYT.

In a very Specific embodiment the anti—ADM antibody has a sequence selected from the group comprising: SEQ ID NO 7, 8, 9,10,11, 12, 13 and 14.

The anti—ADM antibody or drenomedulhn antibody fragment or anti—ADM non—1g scaffold according to the present invention exhibits an affinity s human ADM in such that affinity constant is greater than 10'7 M, preferred i0‘8 M, preferred affinity is greater than '9 M, most preferred higher than 10'10 M. A person skilled in the art knows that it may be ered to compensate lower affinity by applying a higher dose of compounds and this e would not lead out—of—the-scope of the invention. The affinity constants may be determined according to the method as described in e 1.

In a preferred embodiment the antibody or the antibody fragment is used for reducing the risk of mortality during said chronic or acute disease of a patient wherein said disease is selected from the group comprising sepsis, diabetes, cancer, acute and chronic vascular diseases as eg. heart failure, shock as e.g. septic shock and organ dysfunction as eg. kidney dysfunction.

In a preferred ment the anti—ADM antibody or the anti-ADM dy fragment or anti—ADM non—lg scaffold is used for reducing the risk of mortality during said acute disease or acute condition of a patient.

It should be emphasized that the t having an acute disease or acute condition may be characterized by need for stabilizing the circulation g the systemic circulation or by the need for preventively stabilizing the circulation meaning the systemic circulation. c or acute disease or acute condition according to the present invention may be a e or condition seiected from the group comprising severe infections as eg. meningitis, Systemic inflammatory Response—Syndrome (SIRS) sepsis; other diseases as diabetes, cancer, acute and chronic vascular diseases as eg. heart failure, myocardial infarction, stroke, atherosclerosis; shock as ag. septic shock and organ dysfunction as eg. kidney dysfunction, WO 72513 liver dysfunction, burnings, y, traumata, poisoning, damages by chemotherapy.

Especially useful is the antibody or fragment or scaffold according to the present invention for reducing the risk of mortality during sepsis and septic shock, :26. late phases of sepsis. r, it should be emphasized that the medicaments provided by the present invention, being anti-ADM antibodies, anti-ADM antibody fragments, or anti—ADM non—lg scaffolds are only intended to be used for sake of stabilizing the systemic circulation in a patient in need for stabilizing the systemic circulation or by the need for preventively stabilizing the systemic circulation, and thus not for any methods of primary treatment to a chronic or acute disease or condition itself. This means the t invention does not provide for a therapy of healing/curing e. g. meningitis, ic inflammatory Response—Syndrorn (SIRS), or sepsis, or severe sepsis; other diseases as diabetes, cancer, acute and chronic vascular diseases as eg. heart failure, myocardial infarction, stroke, atherosclerosis; shock as eg. septic shock and organ dysfunction as ag. kidney dysfunction, liver ction, burnings, surgery, traumata, poisoning, or damages induced by herapy Within the scope of the invention.

In one ment the anti~ADM antibody or an anti-adrenomedullin antibody fragment or anti—ADM non—lg scaffold is used in therapy of acute disease or acute condition of a patient according to the present invention, wherein said patient is an ICU patient. In another ment the anti—ADM antibody or an anti-adrenomedullin antibody fragment or anti— ADM non—lg scaffold is used in therapy of acute disease of a patient according to the present invention, wherein said patient is critically ill. Critically ill means the patient is having a disease or state in which death is possible or imminent.

Subject of the present invention is further an anti—ADM antibody or an antiuadrenomedullin antibody fragment or anti-ADM non—lg ld for use in therapy of acute disease of a patient according to the present invention, wherein said dy or antibody fragment or non- Ig scaffold is to be used in combination of ADM binding protein. ADM binding protein is also lly present in the circulation of said t.

It should be emphasized that the term “ADM binding protein” ses ADM—binding- protein—l (complement factor H). However, said ADM binding protein by ion pursuant to the ion is neither a non—neutralizing anti—ADM antibody/antibody nt/non-Ig scaffold nor a modulating anti—ADM antibody/antibody fragment/nonulg scaffoid.

Subject of the present invention is further an anti—ADM dy or an anti-adrenomedullin antibody fragment or anti—ADM non-lg scaffold for use in therapy of acute disease or acute condition of a patient according to the present invention, wherein said antibody or antibody fragment or non—1g scaffold may be used in ation with further active ingredients.

Subject matter of the invention is also an anti-Adrenomedullin (ADM) antibody or an anti- adrenomedullin antibody fragment or an anti—ADM non-1g scaffold may be used in combination with a primary medicament, wherein said combination is for use in therapy of a acute disease or acute condition of a patient for stabilizing the circulation of said patient.

In this regard, it should be emphasized that the anti—ADM dy/antibody fragment/non-Ig ld are not to be stered as primary medicament or as first—iine—treatment of any underlying disease or condition, irrespective of being acute or chronic, but administration of said anti—ADM antibody/antibody fragment/non-Ig scaffold pursuant to the invention is to be intended for patients with acute disease or acute condition associated with weak circulation or circulation problems, and thus who are in need for stabilizing the circulation.

Primary medicament means a medicament that acts against the primary cause of said disease or condition. Said primary ment may be antibiotics in case of infections.

In a specific embodiment of the before ned combinations said combinations are to be used in combination with vasopressors cg. catecholamine wherein said r combination is for use in therapy of a c or acute disease or condition of a patient for stabilizing the circulation.

In one embodiment of the invention said t having a chronic or acute disease or chronic condition being in need for stabilizing the circulation is characterized by the need of the t to get administration of vasopressors e.g. catecholamine administration.

Subject matter of the invention in one specific embodiment is, thus, an antiuAdrenornedullin (ADM) dy or an anti—adrenomeduilin antibody fragment or an anti-ADM nOn-Ig scaffold to be used in combination with ADM binding protein and/or further active ingredients for use in therapy of a patient in need of a treatment of vasopressors eg. catecholamine.

In a specific embodiment of the above ned combinations said combinations are to be used in combination with fluids administered intravenously, wherein said combination is for use in y of a chronic or acute disease or condition of a patient for stabilizing the circulation.

In one embodiment of the invention said patient having a c or acute disease or acute ion being in need for stabilizing the circulation is characterized by the need of the patient to get intravenous fluids.

Subject matter of the invention in one specific embodiment is, thus, an anti~Adrenomedullin (ADM) antibody or an anti—adrenomedullin antibody fragment or anti—ADM non~lg scaffold in combination with ADM g protein and/or further active ingredients for use in therapy of a patient in need of intravenous fluids.

Said DM antibody or an drenomedullin antibody fragment or anti—ADM non—lg scaffold or combinations f with ADM binding protein and/or further active ingredients may be used in combination with catecholamine and/or with fluids administered intravenously for use in a method of treating acute disease or acute condition of a patient for stabilizing the circulation. t matter of the invention is also an anti-ADM antibody or an anti-adrenomedullin antibody fragment or anti—ADM non—1g scaffold ing to the present invention to be used in combination with TNF—alpha—antibodies. TNF-alpha-antibodies are commercially available for the treatment of patients.

Subject matter of the invention is also an anti~ADM antibody or an anti-adrenomedullin antibody fragment or anti-ADM non-1g ld according to the t invention to be used in combination with antibiotics.

Subject of the present invention is further a pharmaceutical formulation comprising an anti~ ADM antibody or anti~ADM antibody fragment or anti—ADM non—1g scaffold according to the present invention. Subject of the present invention is further a pharmaceutical formulation according to the present invention n said pharmaceutical formulation is a solution, preferably a ready-to-use solution. In another ment subject of the present invention is further a ceutical formulation according to the present invention wherein said pharmaceutical formulation is in a dried state to be reconstituted before use.

Said pharmaceutical formulation may be administered intra—muscnlar. Said pharmaceutical formulation may be administered intra-vascular. Said pharmaceutical ation may be administered via infusion. In another embodiment subject of the present inventicn is further a pharmaceutical formulation according to the present invention wherein said pharmaceutical formulation is in a freeze—dried state.

It should be emphasized that the pharmaceutical formulation in accordance with the invention as may be administered intra-muscular, intra—vascular, or via infusion is preferably administered to a patient for stabilizing the systemic circulation with the o that said patient is in need for stabilizing the circulation. ore, in another embodiment of the t invention the pharmaceutical ation according to the present invention is to be administered to a patient for stabiiizing the systemic circulation with the proviso that said patient is in need for stabilizing the circulation.

In another more preferred embodiment the present invention provides for a pharmaceutical formulation comprising an anti—Adrenomedullin (ADM) dy or an anti—ADM antibody nt binding to adrenomeduilin or anti-ADM non—1g scaffold binding to adrenomeduilin for use in therapy of an acute disease or acute ion of a patient, wherein said pharmaceutical formulation is to be administered to a patient for izing the systemic circulation with the proviso that said patient is in need for stabilizing the systemic circulation. r embodiments Within the scope of the present invention are set out below: 1. Adrenomeduilin ADM antibody or an adrenomedullin antibedy nt for use in therapy of a chronic or acute disease of a patient for the regulation of liquid balance. 2. ADM antibody or an medullin dy fragment according to claim 1 wherein the antibody format is selected from the group comprising Fv fragment, scFv fragment, Fab fragment, scFab fragment, (Fab)2 fragment and scFv—Fc Fusion protein. 3. ADM dy or an adrenomeduilin antibody nt according claim 1 or 2 n said dy or fragment binds to the N—terminal part (aa 1—21) of adrenornedullin.

. ADM antibody or an adrenomedullin antibody fragment according to any of claims 1 to 3, wherein said antibody or fragment recognizes and binds to the N—terminal end (aal) of medullin.

. ADM antibody or an adrenomeduilin antibody fragment according to any of claims 1 to 4, wherein said antibody or fragment is an ADM stabilizing antibody or ADM stabilizing a antibody fragment that enhances the tug half retention time of adrenomedullin in serum, blood, plasma at least 10 %, ably at least 50 %, more preferably >50 ”/0, most preferably >100 %.

. ADM antibody or an adrenomedullin antibody fragment according to any of claims 1 to 5, wherein said antibody or fragment blocks the bioactivity ofADM to less than 80 %, preferably less than 50%.

. ADM antibody or an adrenomedullin antibody fragment for use in y of a chronic or acute disease of a patient according to any of claims 1 to 6 wherein said disease is selected from the group comprising sepsis, is, cancer, heart failure, shock and kidney dysfunction. an . ADM antibody or adrenomeduliin antibody fragment for use in y of a chronic or acute disease of a patient according to any of claims 1 to 7 wherein said t is an ICU patient.

. ADM antibody or an adrenomeduliin antibody fragment for use in therapy of a chronic or acute disease of a patient according to any of claims 1 to 7 wherein said antibody or fragment is a modulating antibody or fragment that enhances the tug half retention time of adrenomedullin in serum, blood, plasma at least. 10 %, preferably at least 50 %, more preferably >50 %, most preferably >100 % and that blocks the bioactivity of ADM to less than 80 %, preferably less than 50%.

. Pharmaceutical formulation sing an dy or fragment according to any of claims 1 to 9. ll. Pharmaceutical formulation according to claim 10 wherein said pharmaceutical formulation is a solution, preferably a ready—to—use solution. 12. Pharmaceutical formulation ing to claim 10 wherein said ceutical formulation is in a freeze—dried state. 13. Pharmaceutical formulation according to any of claims 10 to 1], wherein said pharmaceutical formulation is administered intra—rnuscular. 14. Pharmaceutical formulation according to any of claims 10 to 11, wherein said pharmaceutical formulation is administered intra~vascu1ar. . ceutical formulation according to claim 14, wherein said phannaceutical ation is administered via infusion.

Further embodiments Within the scope ofthe present invention are set out below: Adrenomedullin ADM antibody or an adrenomedullin antibody fragment an ADM non-1g scaffold for use in therapy of a c or acute disease or acute condition of a patient for the regulation of fluid balance.

ADM antibody or an adrenomedullin antibody fragment or ADM non-iG scaffold according to claim 1 wherein said ADM antibody or an adrenornedullin antibody fragment or ADM non—1G scaffold is a non~neutra1izing ADM antibody or a non— neutralizing adrenomedullin dy fragment or a non—neutralizing ADM non—1G medullin ADM antibody or an adrenomeduliin antibody fragment or an ADM non—lg scaffold for use in therapy of a chronic or acute disease or acute condition according to claim 1 or 2 for preventing or reducing edema in said patient. 2012/072932 ADM antibody or an adrenomedullin dy nt or ADM non—IG scaffold according to any of claims 1 to 3 n the antibody format is selected from the group comprising Fv fragment, scFV fragment, Fab fragment, scFab fragment, (Fab)2 fragment and scFv—Fc Fusion protein.

ADM antibody or an adrenomedullin antibody fragment or ADM non—1G scaffold according to any of claims 1 to 4, wherein said antibody or fragment or scaffold binds to the N—terminal part (aa L21) of adrenomedullin.

ADM antibody or an adrenornedullin antibody fragment or ADM non-1G scaffold according to any of claims 1 to 5, wherein said antibody or fragment scaffold recognizes and binds to the N—terminal end (aal) of medullin.

ADM antibody or an adrenomedullin antibody fragment or ADM non—1G scaffold according to any of claims 1 to 6, wherein said antibody or fragment or scaffold is an ADM stabilizing dy or ADM izing antibody fragment or ADM stabilizing non—IG scaffold that enhances the half life (tm half retention time) of adrenomedullin in serum, blood, plasma at least 10 %, preferably at least 50 “/0, more preferably >50 %, most preferably >100 %. or an adrenomedullin antibody nt or ADM non-1G . ADM antibody scaffold according to any of claims 1 to ’7, wherein said antibody or fragment blocks the bioactivity ofADM to less than 80 %, preferably less than 50%. or ADM non~IG scaffold for . ADM antibody or an adrenomedullin antibody fragment use in therapy of a chronic or acute disease of a patient ing to any of claims 1 to 8 wherein said disease is selected from the group comprising SIRS, sepsis, diabetis, , heart e, shock and kidney dysfunction 10. ADM antibody or an adrenomedullin antibody fragment according to any of claims 1 to 9, wherein said antibody or fragment is a human monoclonal antibody or fragment that binds to ADM or an antibody fragment thereof wherein the heavy chain comprises the sequences SEQ ID NO: 1 GYTFSRYW SEQ ID NO: 2 SEQ ID NO: 3 TEGYEYDGFDY and wherein the light chain comprises the sequences SEQ ID NO: 4 QSIVYSNGNTY SEQ ID NO: 5 SEQ ID NO: 6 FQGSHlPYT. 11. A human monoclonal antibody or fragment that binds to ADM or an antibody fragment thereof according to claim 10 wherein said antibody or fragment comprises a sequence selected from the group comprising : SEQ ID NO: 7 (AM-VH—C) QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEIL NYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGF DYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTK VDKRVEPKHHHHHH 2012/072932 SEQ ID NO: 8 (AM-VH1) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRI TNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDG FDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNT KVDKRVEPKHHHHHH SEQ ID NO: 9 (AM-VH2—E40) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRI LPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDG FDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKRVEPKHHHHHH SEQ ID NO: 10 (AM—VH3-T26-E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEI LPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDG FDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNWHKPSNT KVDKRVEPKHHHHHH SEQ ID NO: 11 (AM-VH4—T26—E40—E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEI LPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDG FDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKRVEPKI—IHHHHH SEQ ID NO: 12 (AM-VL—C) DVLLSQTPLSLPVSLGDQATESCRSSQSEVYSNGNTYLEWYLQKPGQSPKLLIY RVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTK LEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGBC SEQ ID NO: 13 (AM—VLi) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIY SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGT KLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC SEQ ID NO: 14 (AM—VLZ-E40) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIY RVSNRDSGVPDRPSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGT KLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC 12. ADM antibody or an adrenomedullin antibody fragment or ADM non—1G scaffold for use in therapy of a chronic or acute disease of a patient according to any of claims 1 to 9 wherein said patient is an ICU patient. 13‘ ADM antibody or an medullin antibody fragment or ADM non-1G scaffold for use in therapy of a chronic or acute disease of a patient according to any of claims I to 12 wherein said antibody or fragment or scaffold is a modulating dy or fragment or scaffold that enhances the half life (tug half retention time) of medullin in serum, blood, plasma at least 10 %, preferably at least 50 0/0, more preferably >50 0/o, most preferably >100 0/0 and that blocks the ivity of ADM to less than 80 0/0, ably less than 50%. 14. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG scaffold for use in therapy of a chronic or acute disease of a patient according to any of the claims 1 to 13 to be used in combination with catecholamine and/ or fluids administered intravenously. 15. ADM antibody or adrenomedullin antibody fragment or ADM noanG ld for use in therapy of a chronic or acute disease of a patient according to any of the claims 1 to WO 72513 13 or a combination according to claim 12 to be used in combination with ADM binding protein and/or further active ingredients. 16. Pharmaceutical formulation sing an antibody or fragment or scaffold according to any of claims I to 15. 17. Pharmaceutical ation according to claim 16 n said pharmaceutical formulation is a solution, preferably a to—use solution. 18. Pharmaceutical formulation according to claim 16 wherein said pharmaceutical formulation is in a freeze—dried state. 19. Pharmaceutical formulation according to any of claims 16 to 17, wherein said ceutical formulation is administered intramuscular. 20. Pharmaceutical fonnulation according to any of claims 16 to 17, wherein said pharmaceutical formulation is administered intra—vascular. 21. Pharmaceutical formulation according to claim 20, wherein said pharmaceutical formulation is administered Via infusion.

Further embodiments within the scope of the present invention are set out below: Adrenomedullin (ADM) antibody or an adrenomedullin antibody fragment for use in therapy of a chronic or acute disease of a patient for stabilizing the circulation.

ADM antibody or an adrenomedullin antibody nt according to claim 1 wherein said antibody or nt reduces the catechoiamine requirement of said patient.

ADM antibody or an adrenomedullin antibody fragment according to claim I or 2 wherein the antibody format is ed from the group comprising Fv fragment, scFv fragment, Fab fragment, scFab fragment, (Fab)2 fragment and scFV—Fc Fusion protein.

ADM antibody or an adrenomedullin antibody fragment according to any of claims 1 to 3 wherein said antibody or fragment binds to the N—terminal part (aa 1—21) of adrencmedullin.

ADM antibody or an adrenomedullin antibody fragment according to any of claims 1 to 4, wherein said antibody or fragment recognizes and binds to the N—terminal end (aal) of adrenomedullin.

ADM antibody or an adrenomedullin antibody fragment according to any of claims 1 to 5, wherein said antibody or fragment is an ADM stabilizing antibody that enhances the tl/2 half retention time of adrenomedullin in serum, blood, plasma at least 10 %, preferably at least, 50 %, more preferably > 50 %, most preferably >100 0/0.

ADM antibody or an adrenomedullin dy fragment according to any of claims 1 to 6, wherein said antibody or fragment blocks the bioactivity of ADM to less than 80 %, ably less than 50 %.

ADM antibody or an adrenomedullin antibody fragment according to any of claims 1 t0 '7, wherein said antibody or fragment is a modulating ADM antibody or a modulating adrenornedullin antibody fragment that enhances the HQ half retention time of adrenomedullin in serum, blood, plasma at least 10 %, preferably at least, 50 %, more preferably > 50 %, most preferably >100 % and that blocks the bioactivity of ADM to less than 80 0/0, preferably less than 50 %: ADM antibody or an adrenomedullin dy nt for use in therapy of a chronic or acute disease of a patient according to any of the claims 1 to 8 n said disease is ed from the group sing sepsis, diabetis, cancer, acute and chronic vascular diseases as cg. heart failure, shock as cg. septic shock and organ dysfunction as cg. kidney dysfunction. 10. Pharmaceutical formulation comprising an dy according to any of claims 1 to 9. 11. Pharmaceutical formulation according to claim 10 wherein said pharmaceutical formulation is a solution, preferably a to-use solution. 12. ceutical formulation according to claim 10 wherein said pharmaceutical formulation is in a fieeze—dried state. l3. Pharmaceutical formulation according to any of claims 10 to 11, n said phannaceutical formulation is administered intra—muscular. 14. Pharmaceutical formulation according to any of claims 10 to 11, wherein said pharmaceutical ation is administered intra—vascular. 15. Pharmaceutical formulation according to claim 14, wherein said pharmaceutical formulation is administered via infusion.

Further embodiments within the scope of the t invention are set out below: Adrenomedullin (ADM) antibody or an adrenomedullin antibody fragment or an ADM non—1G scaffold for use in therapy of a chronic or acute disease or condition of a patient for stabilizing the circulation.

ADM antibody or an adrenomedullin antibody fragment or ADM non—1G scaffold ing to claim 1 n said dy or fragment or scaffold reduces the essor requirement, tag, catecholamine requirement of said patient.

ADM antibody or an adrenomedullin antibody fragment or ADM non—IG scaffold according to claim 1 or 2 n said ADM antibody or an adrenomedullin antibody fragment or ADM non—1G scaffold is a non-neutralizing ADM antibody or a non— neutralizing adrenomedullin antibody fragment or a non—neutralizing ADM non—IG scaffold.

ADM antibody or an adrenomedullin antibody fragment according to any of claims 1 3O to 3 wherein the antibody format is selected from the group comprising Fv fragment, scFV fragment, Fab fragment, scFab nt, (Fab)2 fragment and scFV—Fc Fusion protein.

. ADM antibody or an adrenomedullin dy nt or ADM nonulG scaffold according to any of claims 1 to 4 wherein said antibody or fragment or scaffold binds to the inal part (aa 1-21) of adrenomedullin. 6. ADM antibody or an adrenomednllin antibody fragment or ADM non—1G scaffold according to any of claims 1 to 5, wherein said antibody or fragment or scaffold recognizes and binds to the N—terminal end (aal) of adrenomedullin. 7. ADM antibody or an adrenomedullin antibody fragment or ADM non-1G scaffold according to any of claims 1 to 6, wherein said antibody or fragment or scaffold is an ADM stabilizing antibody or fragment or scaffold that enhances the half life (ti/2 half retention time) of adrenomedullin in serum, blood, plasma at least 10 %, preferably at least, 50 %, more preferably > 50 %, most preferably >100 %. 8. ADM antibody or an adrenomedullin antibody fragment or ADM noanG ld according to any of claims 1 to 7, wherein said antibody or fragment or scaffold blocks the bioactivity ofADM to less than 80 ”/0, preferably less than 50 %. 9. ADM antibody or an adrenomedullin antibody fragment or ADM non-1G scaffold according to any of claims 1 to 8, wherein said dy or fragment or ld is a modulating ADM antibody or a modulating adrenomedullin antibody fragment or scaffold that enhances the half life (ti/2 half retention time) of adrenomedullin in serum, blood, plasma at least 10 %, preferably at least, 50 “/0, more ably > 50 %, most ably >100 % and that blocks the ivity of ADM to less than 80 0/0, preferably less than 50 %: . ADM dy or an adrenornedullin antibody fragment according to any of claims 1 to 9, wherein said antibody or fragment is a human monoclonal antibody or fragment that binds to ADM or an antibody fragment thereof wherein the heavy chain comprises the sequences SEQ ID NO: 1 GYTFSRYW 2012/072932 SEQ ID NO: 2 ILPGSGST SEQ ID NO: 3 TEGYEYDGFDY and wherein the light chain comprises the sequences SEQ ID NO:4 QSIVYSNGNTY SEQ ID NO: 5 SEQ ID NO: 6 FQGSHIPYT. 11. A human monoclonal dy or fragment that binds to ADM or an antibody fragment thereof according to claim 10 wherein said antibody or fragment comprises a sequence selected from the group comprising : SEQ ID NO: 7 (AM—VH—C) SGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEIL PGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGF DYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKRVEPKHHHHHH SEQ ID NO: 8 (AM—VH1) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRI LPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDG FDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKRVEPKHHHHHH SEQ ID NO: 9 (AM-VH2—E40) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRI LPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDG GTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKRVEPKHHHHHH SEQ ID NO: 10 (AM—VH3—T26—E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEI LPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDG FDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKRVEPKHHHHHH SEQ ID NO: 11 (AM—VH4—T26uE40-E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEI LPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDG FDYWGQGTTVTVSSASTKGPSVFPLA?SSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKRVEPKHHHHHH SEQ ID NO: 12 (AM—VL—C) DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIY RVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTK LEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC SEQ ID NO: 13 (AM—VLI) DVVMTQSPLSLPVTLGQPASiSCRSSQSiVYSNGNTYLNWFQQRPGQSPRRLIY SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGT KLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC SEQ ID NO: 14 (AM—VLZ-E40) DVVMTQSPLSLPVTLGQPASlSCRSSQSiVYSNGNTYLEWFQQRPGQSPRRLIY RVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGT KLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSL8STLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC 12. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG scaffold for use in therapy of a chronic or acute disease of a patient according to any of the claims 1 to 11 wherein said disease is selected from the group comprising SIRS, sepsis, diabetis, cancer, acute and chronic vascular diseases as e.g. heart failure, shock as eg septic shock and organ dysfunction as eg. kidney dysfunction. 13. ADM antibody or an adrenomedullin antibody fragment or ADM non-1G ld for use in therapy of a chronic or acute e of a patient according to any of the claims 1 to 12 to be used in combination with catecholamine and/ or fluids administered intravenously. 14. ADM antibody or adrenomedullin antibody fragment or ADM non—1G scaffold for use in therapy of a chronic or acute e of a patient according to any of the claims 1 to 13 or a combination according to claim 10 to be used in combination with ADM binding protein and/or fiirther active ingredients.

. Pharmaceutical formulation comprising an antibody or nt or non-1G id 3O according to any of claims 1 to 14. 16. Pharmaceutical ation according to claim 15 wherein said pharmaceutical formulation is a solution, preferably a ready—to-use solution. 17. Pharmaceutical formulation ing to claim 15 wherein said pharmaceutical formulation is in a freeze—dried state. 18. Pharmaceutical formulation according to any of claims 15 to 16, wherein said ceutical formulation is administered intramuscular. 19. Pharmaceutical formulation according to any of claims 14 to 16, wherein said pharmaceutical ation is administered infra—vascular.

. Pharmaceutical formulation according to claim 16, n said ceutical formulation is administered Via infusion.

Further embodiments within the scope of the present invention are set out below: 1. Adrenomedullin antibody or an adrenomedullin antibody fragment for use in a treatment of a chronic or acute disease wherein said antibody or said nt is an ADM stabilizing antibody or fragment that enhances the t1]; half retention time of medullin in serum, blood, plasma at least 10 %, preferably at least, 50 %, more preferably >50 %, most preferably 100 % and/or n said antibody blocks the bioactivity ofADM to less than 80 %, preferably to less than 50 %.

Adrenomedullin antibody or an adrenomedullin antibody fragment for use in a treatment of a chronic or acute disease wherein said antibody or said fragment is a modulating ADM antibody or fragment that enhances the t1]; half retention time of adrenomedullin in serum, blood, plasma at least 10 %, preferably at least, 50 %, more preferably >50 %, most preferably 100 % and that blocks the bioactivity of ADM to less than 80 “/0, ably to less than 50 %.

Adrenomedullin antibody or an adrenomedullin antibody fragment for use in a treatment of a chronic or acute disease according to claim 1 or 2, wherein said antibody or fragment binds to the N-terminal part (aa 1—21) of adrenomedullin.

WO 72513 4. Adrenomedullin antibody or an adrenomedullin antibody fragment for use in a treatment of a chronic or acute disease wherein said antibody or said fragment according to claim 3 binds to the N—tenninal end of adrenomedullin.

. Adrenomedullin antibody or an adrenomedullin antibody fragment for use in use in a treatment of a chronic or acute disease ing to any of claims 1 to 4, wherein said dy or said nt is an ADM izing antibody or fragment that enhances the tug half retention time of medullin in serum, blood, plasma at least 10 %, preferably at least, 50 %, more preferably >50 %, most preferably 100 0/0. 6. Adrenomedullin antibody or an adrenomedullin antibody fragment for use in a treatment of a chronic or acute disease according to any of claims 1 to 5, n said antibody or said fragment blocks the bioactivity of ADM to less than 80 %, preferably to less than 50 %. '7. Adrenomedullin antibody or an adrencrnedullin antibody fragment according to any of the claims 1 to 6 for use in a treatment of a chronic or acute disease wherein said disease is selected from the group comprising SIRS, sepsis, septic shock, diabetis, cancer, heart failure, shock, organ failure, kidney dysfunction, acute liquid dysbalance, and low blood pressure. 8. Adrenomedullin antibody or an adrenornedullin antibody fragment according to any of the claims 1 to 7 for use in a treatment of a chronic or acute disease wherein said e is septic shock or sepsis. 9. Adrenomedullin antibody or an adrenomedullin antibody fragment for use in a treatment of a chronic or acute disease according to any of the claims 1 to 8 wherein said antibody or fragment regulates the liquid balance of said patient. . medullin antibody or an adrenornedullin antibody fragment for use in a treatment of a chronic or acute disease according to any of the claims 1 to 9 wherein said antibody or fragment used for prevention of organ ction or organ failure. ll. Adrenomeduliin antibody or an adrenomeduliin antibody fragment for use in a treatment of a chronic or acute disease according to claim 10 wherein said dy or fragment is used for tion of kidney dysfunction or kidney failure. 12. Adrenomedullin (ADM) antibody or an adrenomedullin antibody nt for use in a treatment of a chronic or acute disease in a patient according to claims 1 to ll wherein said antibody or fragment is used for stabilizing the circulation. 13. ADM antibody or an adrenomeduliin antibody fragment for use in a treatment of a chronic or acute disease in a patient according to claim 12 n said antibody or fragment reduces the catechoiamine requirement of said patient. 14. ADM antibody or an adrenomedullin antibody fragment for use in a treatment of a chronic or acute disease in a t according to any of claims 1 to 13 for the reduction of the mortality risk for said patient. 15. ADM antibody or an adrenomedullin antibody fragment for use in a treatment of a chronic or acute disease in a patient according to any of claims 1 to 14 wherein said antibody or fragment may be administered in a dose of at least 3 ug / Kg body weight. 16. Pharmaceutical composition comprising an antibody or fragment according to any of claims 1 to 15.

Further embodiments within the scope ofthe t invention are set out below: . Adrenomedullin antibody or an adrenomedullin antibody fragment or ADM non~1g scaffold wherein said antibody or said fragment or scaffold is a utralizing antibody. 2. Adrenomedullin antibody or an adrenomedullin antibody fragment or ADM nondg scaffold n said antibody or said fragment or scaffold is an ADM stabilizing antibody or fragment or scaffold that enhances the half life (111/2 half retention time) of adrenomedullin in serum, blood, plasma at least 10 0/0, preferably at least 50 %, more preferably >50 %, most preferably 100 % and/or wherein said antibody or fragment or scaffold blocks the bioactivity ofADM to less than 80 %, preferably to less than 50 %.

. Adrenomedullin antibody or an adrenomedullin antibody fragment or ADM non~lg scaffold wherein said dy or said nt is a modulating ADM antibody or fragment or scaffold that es the half life (121/2 half retention time) of adrenomedullin in serum, blood, plasma at least 10 %, preferably at least, 50 %, more preferably >50 %, most preferably 100 “/0 and that blocks the ivity of ADM to less than 80 ”/0, preferably to less than 50 %. 4. medullin antibody or an adrenomedullin antibody nt or ADM non—1g scaffold according to claim 1 or 2, wherein said antibody or fragment or scaffold binds to the N-terrninal part (aa 1-21) of adrenomedullin.

. Adrenomedullin antibody or an adrenomedullin antibody fragment or ADM non—1g scaffold wherein said antibody or said fragment or scaffold according to claim 3 binds to the N-terminal end of adrenomedullin. . medullin antibody or an adrenomedullin antibody fragment ADM non-lg scaffold according to any of claims 1 to 4, wherein said antibody or said fragment or said ld is an ADM stabilizing antibody or fragment that enhances the tyz half retention time of adrenomedullin in serum, blood, plasma at least 10 %, preferably at least, 50 %, more preferably >50 %, most preferably 100 %.

. Adrenomedullin antibody or an adrenomedullin antibody fragment or ADM non—lg scaffold according to any of the claims 1 to 6 for use as an active pharmaceutical substance.

. Adrenomedullin antibody or an adrenomedullin antibody nt ADM non—lg ld according to any of the claims 1 to 7 for use in a treatment of a chronic or acute disease or acute condition wherein said disease or condition is selected from the group comprising severe infections as ag. itis, systemic inflammatory Response—Syndrome ) sepsis; other diseases as diabetes, cancer, acute and chronic vascular diseases as eg. heart failure, myocardial infarction, stroke, atherosclerosis; shock as ag. septic shock and organ dysfunction as eg. kidney dysfunction, liver dysfunction, burnings, surgery, traumata.

. Adrenornednllin antibody or an adrenornedullin antibody fragment or ADM nonalg scaffold ing to any of the claims 1 to 8 for use in a ent of a chronic or acute disease or acute condition wherein said disease is septic shock or sepsis.

. ADM antibody or an adrenomedullin antibody fragment according to any of claims 1 to 9, wherein said antibody or fragment is a human monoclonal antibody or fragment that binds to ADM or an antibody fragment thereof wherein the heavy chain comprises at least one of the sequences 1 SEQ ID NO: 1 GYTFSRYW SEQ ID NO: 2 SEQ ID NO: 3 TEGYEYDGFDY And/or wherein the light chain comprises the at least one ofthe sequences SEQ ID NO:4 QSIVYSNGNTY SEQ ID NO: 5 SEQ ID NO: 6 FQGSHIPYT. 11. A human monoclonal antibody or fragment that binds to ADM or an antibody fragment thereof according to claim 10 wherein said antibody or fragment ses a sequence selected from the group sing: SEQ ID NO: 7 (AM-VH—C) QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEIL PGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAWYCTEGYEYDGF DYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKRVEPKHHHHHH SEQ ID NO: 8 (AM—VH1) WO 72513 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRI TNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDG FDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT S KVDKRVEPKHHHHHH SEQ ID NO: 9 (AM—VHZuEéiO) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRI LPGSGSTNYAQKFQGRVTITADBSTSTAYMELSSLRSEDTAVYYCTEGYEYDG FDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKRVEPKHHHHHH SEQ ID NO: 10 (AMwVH3—T26—E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEI LPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDG FDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKRVEPKHHHHHH SEQ ID NO: 11 (AM-VH4—T26-E40—E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWM6131 LPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDG FDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKRVEPKHHHHHH SEQ ID NO: 12 (AMfiVL—C) DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIY RVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTK VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC SEQ ID NO: 13 (AM-VLl) DWMTQSPLSLI’VTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIY RVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGT KLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC SEQ ID NO: 14 (AM—VLZ—E40) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIY RVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGT KLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKI)STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC 12. Adrenomedullin dy or an adrenomedullin antibody fragment or ADM non—lg scaffold according to any of the claims 1 to 11 for regulating the fluid balance in a patient having a c or acute disease or acute condition. . l3. Adrenornednllin antibody or an adrenomedullin antibody fragment or ADM nonalg scaffold according to any of the claims 1 to 11 for preventing or reducing organ dysfunction or organ failure in a patient having in a chronic or acute disease or acute condition. 14. Adrenomedullin antibody or an adrenomedullin antibody fragment or ADM non-1g scaffold according to claim 10 wherein organ is kidney or liver.

. Adrenomedullin (ADM) antibody or an medullin antibody nt or ADM non—lg Scaffold according to claims 1 to 14 for stabilizing the circulation in a patient having a chronic or acute disease or acute condition. 16. ADM antibody or an adrenomedullin antibody fragment or ADM non-1g scaffold for use in a ent of a chronic or acute disease in a patient according to claim 15 wherein said antibody or nt reduces the catechclamine ement of said patient. 17. Adrenomedullin dy or an adrenomedullin antibody fragment or ADM non-1g scaffold according to any of the claims 1 to 16 to be used in combination with vasopressors ag. catecholamine. 18. meduliin antibody or an adrenomedullin antibody fragment or ADM non—lg scaffold according to any of the claims 1 to 17 to be used in combination with intravenous fluid stration. 19. Adrenomedullin antibody or an rnedullin antibody fragment or ADM non—lg ld according to any of the claims 1 to 18 to be used in ation with an TNF—alpha—antibody.

. ADM antibody or an adrenomedullin antibody fragment or non—Ig-scaffold according to any of claims 1 to 19 for use in a treatment of a patient in need thereof wherein said antibody or fragment may be stered in a dose of at least 3 pg / Kg body weight. 21. Pharmaceutical composition comprising an antibody or fragment or scaffold according to any of claims 1 to 20. 22. ADM antibody or an adrenomeduliin antibody fragment or non—Ig-scaffold according to any of claims 1 to 20 for use in a treatment of a chronic or acute disease or chronic condition. 23. ADM antibody or an adrenornedullin antibody fragment or non—lg—scaffold according to claim 22 wherein said e is sepsis.

Further embodiments within the scope ofthe present invention are set out below: 1. Adrenomedullin ADM antibody or an adrenomedullin antibody t for use in therapy of a severe chronical or acute disease of a patient for the reduction of the mortality risk for said patient.

ADM antibody or an adrenomedullin antibody fragment according to claim 1 wherein the antibody format is ed from the group comprising FV fragment, seFV fragment, Fab fragment, scFab fragment, (Fab)2 fragment and scFv—Fc Fusion n.

ADM antibody or an adrenomeduilin antibody fragment according claim 1 or 2 wherein said antibody or fragment binds to the N—terminai part (aa 1—21) of adrenomeduilin. 2012/072932 4. ADM antibody or an adrenomedullin antibody fragment according to any of claims 1 to 3, wherein said dy or fragment recognizes and binds to the N-terminal end (aal) of adrenomedullin.

ADM antibody or an adrenomedullin dy fragment according to any of claims 1 to 4, wherein said antibody or fragment is an ADM stabilizing antibody or nt that enhances the t1/2 half retention time of adrenemedullin in serum, blood, plasma at least 10 0/0, preferably at least, 50 %, more preferably > 50 %, most preferably > 100 ADM antibody or an adrenomedullin antibody fragment according to any of claims 1 to 5, wherein said antibody or fragment blocks the bioactivity ofADM to less than 80 %, preferably less than 50%.

ADM antibody or an adrenomedullin antibody fragment for use in therapy of a chronicai or acute disease of a patient according to any of claims 1 to 6 wherein said disease is selected from the group sing sepsis, diabetis, cancer, heart failure, shock and kidney dysfunction.

ADM antibody or an adrenomedullin antibody fragment for use in therapy of a chronical or acute disease of a patient according to any of claims 1 to 7 wherein said patient is an ICU patient.

ADM antibody or an adrenemedullin antibody fragment for use in therapy of a chronical or acute e of a patient according to any of claims 1 to 8 n the mortality risk is reduced by preventing adverse event wherein the latter are ed from the group comprising SIRS, sepsis, septic shock, organ failure, kidney e, liquid dysbalance and low blood pressure. 10. ADM antibody or an adrenomedullin antibody nt for use in therapy of a chronicai or acute disease of a patient according to any of claims 1 to 8 wherein said antibody or fragment is to be used in combination ofADM binding protein. ll. Pharmaceutical formulation comprising an antibody or fragment ing to any of claims 1 to 10. 12. Pharmaceuticai formulation according to claim 11 n said ceutical formulation is a solution, preferably a ready-to-use solution. 13. Pharmaceutical formulation according to claim 11 wherein said pharmaceutical formulation is in a freeze—dried state. 14. Pharmaceutical formulation according to any of claims 11 to 12, wherein said pharmaceutical formulation is administered intra—muscular.

. Pharmaceutical formulation according to any of claims 11 to 12, wherein said pharmaceutical formulation is administered intra—vascular. 16. Pharmaceutical formulation according to claim 15, wherein said pharmaceutical formulation is administered Via on.

Further embodiments within the scope of the present invention are set out below: Adrenomedullin (ADM) antibody or an medullin antibody nt or ADM 2O non-1g scaffold for use in therapy of a severe chronic-a1 or acute disease or acute condition of a patient for the reduction of the mortality risk for said t n said antibody or fragment or scaffold is a non—neutralizing ADM antibody or a non- neutralizing adrenomedullin antibody fragment or a nonnneutralizing ADM non-lg scaffold.

ADM antibody or an adrenomedullin antibody fragment according to claim 1 n the antibody format is selected from the group comprising Fv fragment, scFv fragment, Fab nt, scFab fragment, (Fab)2 fragment and scFV-Fc Fusion protein.

ADM antibody or an adrenomedullin antibody fragment or an ADM non-lg scaffold according claim 1 or 2 wherein said antibody or fragment or scaffold binds to the N— terminal part (aa I~21) of adrenornedullin.

ADM dy or an adrenomedullin antibody fragment or an ADM non—lg scaffold according to any of claims 1 to 3, wherein said dy or fragment or scaffold recognizes and binds to the N—terrninal end (aal) of adrenomednllin.

ADM antibody or an adrenomeduilin antibody fragment or an ADM non—1g scaffold according to any of claims 1 to 4, wherein said antibody or fragment or scaffold is an ADM stabilizing antibody or fragment or scaffold that enhances the half life (tl/Z half retention time) of medullin in serum, blood, plasma at least 10 %, preferably at least, 50 ”/0, more preferably > 50 %, most preferably > 100 %.

ADM antibody or an adrenomedullin antibody fragment or an ADM non—1g scaffold according to any of claims 1 to 5, wherein said antibody or fragment or scaffold blocks the bioactivity ofADM to less than 80 0/0, Preferabiy less than 50%. adrenomedullin antibody fragment or an ADM non-1g ld . ADM antibody or an for use in y of a chronical or acute disease of a patient according to any of claims 1 to 6 n said disease is selected from the group comprising severe infections as ag. meningitis, Systemic inflammatory se-Syndrom ) sepsis; other diseases as diabetis, cancer, acute and chronic vascular diseases as e.g. heart failure, dial tion, stroke, atherosclerosis; shock as ag. septic shock and organ dysfunction as e.g. kidney dysfunction, liver dysfunction; burnings, surgery, traumata. or an ADM non—1g scaffold . ADM antibody or an adrenomeduilin antibody fragment for use in therapy of a chronical or acute disease of a patient according to any of claims 1 to 7 wherein said disease is selected from the group comprising SIRS, a severe infection, sepsis, shock agseptic shock . or an adrenornedullin antibody fragment or an ADM non-1g scaffold . ADM dy for use in therapy of a chronical or acute disease or acute condition of a t according to any of claims 1 to 8 wherein said patient is an ICU patient. ADM antibody or an adrenomedullin antibody fragment or an ADM non~lg scaffold for use in therapy of a chronical or acute disease or acute condition of a patient according to any of claims 1 to 9 wherein the mortality risk is reduced by preventing an adverse WO 72513 event wherein the latter are ed from the group comprising SIRS, sepsis, shock as e.g. septic shock, acute and chronic vascular diseases as ag. acute heart failure, myocardial infarction, stroke; organ failure as eg, kidney failure, liver failure, fluid dysbalance and low blood pressure.

. ADM antibody or an adrenornedullin antibody fragment according to any of claims 1 to 9, wherein said antibody or fragment is a human monoclonal antibody or nt that binds to ADM or an antibody fragment thereof n the heavy chain comprises the sequences SEQ ID NO: 1 GYTFSRYW SEQ ID NO: 2 ILPGSGST SEQ ID NO: 3 TEGYEYDGFDY and wherein the light chain comprises the sequences SEQ ID NO:4 QSIVYSNGNTY SEQ ID NO: 5 SEQ ID NO: 6 FQGSHIPYT. 12. A human monoclonal antibody or fragment that binds to ADM or an antibody fragment thereof according to claim 10 wherein said antibody or fragment comprises a sequence ed from the group comprising : SEQ ID NO: 7 —C) QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEIL PGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGF DYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKRVEPKHHHHHH SEQ ID NO: 8 (AM—VH1) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRI LPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDG FDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKRVEPKHHHHHH SEQ ID NO: 9 (AM—VH2~E40) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRI LPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDG FDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYECNVNHKPSNT KVDKRVEPKHHHHHH SEQ ID NO: 10 (AM-VH3—T26—E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEI LPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDG FDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKRVEPKHHHHHH SEQ ID NO: 11 (AMmVH4—T26—E40—E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEE LPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDG FDYWGQGTTVTVSSASTKGPSVFPLA?SSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKRVEPKHHHHHH SEQ ID NO: 12 (AM—VLC) DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIY RVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTK LEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC SEQ ID NO: 13 (AM—VIA) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIY RVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGT KLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKI)STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC SEQ ID NO: 14 (AM—VLZ-E40) DVVMTQSPLSLPVTLGQPASISCRSSQSWYSNGNTYLEWFQQRPGQSPRRLIY RVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGT KLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC 13. ADM antibody or an adrenomedullin antibody fragment or ADM non-1G scaffold for use in therapy of a chronic or acute disease of a patient according to any of the claims 1 to 12 to be used in combination with vasopressors ag. catecholarnine and/ or fluids stered intravenously. 3O 14. ADM antibody or medullin antibody fragment or ADM non—1G ld for use in therapy of a chronic or acute disease of a patient according to any of the claims 1 to 2012/072932 13 or a combination according to claim 10 to be used in combination with ADM g protein and/or further active ingredients.

. Pharmaceutical formulation comprising an antibody or fragment or scaffold according to any of claims 1 to 14. 16. Pharmaceutical formulation according to claim 15 wherein said ceutical formulation is a solution, preferably a readymto—use solution. 17. Pharmaceutical formulation according to claim 15 n said pharmaceutical formulation is in a freeze—dried state. 1.8. Pharmaceutical formulation according to any of claims 15 to 16, wherein said pharmaceutical formulation is administered intra—muscular. 19. Pharmaceutical formulation according to any of claims 15 to 16, wherein said pharmaceutical formulation is administered intra-vascular. . ceutical formulation according to claim 19, wherein said pharmaceutical formulation is administered Via infusion. 21. ADM antibody or an Adrenomedullin dy fragment or AM non-lg scaffold, wherein said antibody or fragment or scaffold binds to the N-terminal part (aa 1—21) of Adrenomedullin in, ably human ADM. 22. Antibody or fragment or scaffold according to claim 2, wherein said antibody or fragment or ld recognizes and binds to the N-terminal end (aa 1) of Adrenomedullin.

Further embodiments within the sc0pe of the present invention are set out below: 1. Adrenomedullin (ADM) antibody or an adrenomedullin antibody fragment for use in therapy of a chronical or acute disease of a t for prevention of organ dysfunction or organ failure.

ADM antibody or an adrenornedullin dy fragment for use in therapy of a chronical or acute disease according to claim 1 wherein said organ is kidney.

ADM antibody or an adrenomedullin antibody fragment according to claim 1 wherein the antibody format is selected from the group comprising FV nt, scFv fragment, Fab fragment, scFab fragment, (Fab)2 fragment and scFv—Fc Fusion protein.

ADM antibody or an adrenomedullin antibody fragment ing any of claims 1 to 3 wherein said antibody or fragment binds to the N—terminal part (aa 1—21) of adrenomedullin .

ADM antibody or an adrenomedullin antibody fiagment according to any of claims 1 to 4, wherein said antibody or fragment recognizes and binds to the N—terminal end (aal) of medullin.

ADM antibody or an adrenomedullin antibody fragment according to any of claims 1 to 5, wherein said antibody or said fragment is an ADM stabilizing dy or fragment that enhances the tl/2 half retention time of adrenomedullin in serum, blood, plasma at least 10 %, preferably at least 50 %, more preferably >50 %, most preferably >100%.

ADM antibody or an adrenomedullin antibody fragment according to any of claims 1 to 6, wherein said antibody blocks the bioactivity of ADM to less than 80 %, preferably less than 50%.

ADM antibody or an adrenomedullin dy fragment for use in therapy of a cal or acute disease of a patient according to any of claims 1 to 7 wherein said disease is selected from the group comprising , diabetis, cancer, heart failure, and shock.

ADM antibody or an adrenomedullin antibody fragment for use in therapy of a chronical or acute e of a patient according to any of claims 1 to 8 wherein said t is an ICU patient. 2012/072932 . ADM antibody or an adrenomedullin antibody fragment for use in therapy of a cal or acute disease of a patient according to any of claims 1 to 9 wherein said antibody or fragment is a modulating antibody or fragment that enhances the tl/2 half retention time of medullin in serum, blood, plasma at least 10 %, preferably at least 50 0/0, more preferably >50 %, most ably >100% and that blocks the bioactivity ofADM to less than 80 %, preferably less than 50%. ll. Pharmaceutical formulation comprising an antibody or fiagment according to any of claims 1 to 10. 12. Pharmaceutical ation according to claim 11 wherein said pharmaceutical formulation is a solution, preferably a ready-to-use solution. 13. Pharmaceutical formulation ing to claim 11 wherein said pharmaceutical formulation is in a freeze-dried state. 14. Pharmaceutical formulation according to any of claims 11 to 12, wherein said pharmaceutical formulation is administered intra-muscular.

. Pharmaceutical formulation according to any of claims 11 to 12, wherein said pharmaceutical formulation is administered intra—vascular. 16. Pharmaceutical formulation according to claim 15, wherein said ceutical formulation is administered Via infusion.

Fuither embodiments Within the scope of the present invention are set out below: l. Adrenomedullin (ADM) antibody or an adrenomedullin antibody fragment or ADM non- Ig scaffold for use in therapy of a chronical or acute disease or acute condition of a patient for tion or reduction of organ dysfunction or prevention of organ failure in said patient. 2. ADM antibody or an medullin antibody fragment or ADM nonwlg ld for use in therapy of a chronical or acute disease or acute disease according to claim 1 wherein said organ is kidney or liver. 3. ADM antibody or an adrenomedullin antibody fragment or ADM non~iG scaffold according to claim 1 or 2 wherein said ADM antibody or an adrenomedullin dy nt or ADM nonulG ld is a non~neutralizing ADM antibody or a non neutralizing adrenomedullin antibody fragment or a non—neutralizing ADM non—1G scaffold ADM antibody or an adrenomeduliin antibody nt or ADM non-1G scaffold according to any of claims 1 or 3 wherein the antibody format is ed from the group comprising Fv fragment, scFV fragment, Fab fragment, scFab nt, (Fab)2 fragment and scFV—Fc Fusion protein.

ADM antibody or an adrenomedullin antibody fragment or ADM non—IG scaffold according any of claims 1 to 4 wherein said antibody or fragment or scaffold binds to the N—terminal part (aa 1—21) of adrenomedullin .

ADM antibody or an adrenomedullin antibody fragment or ADM non—1G scaffold according to any of claims 1 to 5, wherein said antibody or fragment or ld recognizes and binds to the N—terminal end (aal) of adrenomedullin.

ADM antibody or an adrenomedullin antibody fragment or ADM non—1G scaffold according to any of claims 1 to 6, wherein said antibody or said nt or scaffold is an ADM stabilizing antibody or fragment or scaffold that enhances the half life (ti/2 half ion time) of adrenomedullin in serum, blood, plasma at least 10 %, preferably at least 50 %, more ably >50 0/0, most preferably >100%.

ADM antibody or an adrenomeduliin antibody fragment or ADM non—1G scaffold according to any of claims 1 to 7, wherein said antibody or fragment or scaffold blocks the bioactivity ofADM to less than 80 0/0, preferably less than 50%.

ADM antibody or an adrenomedullin antibody fragment or ADM non-1G ld for use in therapy of a chronical or acute e or acute condition of a patient according to any of claims 1 to 8 wherein said disease is selected from the group comprising sepsis, diabetis, cancer, heart failure, and shock.

. ADM antibody or an adrenornedullin antibody fragment according to any of claims 1 to 9, wherein said antibody or fragment is a human monoclonal antibody or fragment that binds to ADM or an antibody fragment thereof wherein the heavy chain comprises the sequences SEQ ID NO: 1 GYTFSRYW SEQ ID NO: 2 ILPGSGST SEQ ID NO: 3 DGFDY and wherein the light chain comprises the sequences SEQ ID N014 QSIVYSNGNTY SEQ ID NO: 5 SEQ ID NO: 6 FQGSHIPYT. 11. A human monoclonal dy or fragment that binds to ADM or an antibody fragment thereof according to claim 10 wherein said antibody or fragment comprises a ce selected from the group comprising : SEQ ID NO: 7 (AM-VH-C) QVQLQQSGAELMKPGASVKISCKATGYT‘FSRYWIEWVKQRPGHGLEWIGEIL PGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGF DYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTK VDKRVEPKHHHHHH SEQ ID NO: 8 (AM-VH1) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRI LPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDG FDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKRVEPKHHHHHH SEQ ID NO: 9 (AMmVHZ—E40) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRI LPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDG FDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKRVEPKHHHHHH SEQ ID NO: 10 (AM—VH3—T26—E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEI LPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDG FDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT EPKHHHHHH SEQ ID NO: 11 (AM—VH4—T26-E40—E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEI LPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDG GTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT EPKHHHHHH SEQ ID NO: 12 (AM-VL—C) DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIY RVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTK VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC SEQ ID NO: 13 (AM-VLl) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIY RVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGT KLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC SEQ ID NO: 14 (AM-VLZ-E40) SPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIY SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGT KLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC 12. ADM antibody or an adrenomednllin antibody fragment or ADM non-1G scaffold for use in therapy of a chronical or acute disease of a patient according to any of claims 1 to 11 wherein said dy or fragment or scaffold is a modulating antibody or fragment or scaffold that es the half life ( tl/Z half retention time) of adrenomedullin in serum, blood, plasma at least 10 %, preferably at least 50 %, more preferably >50 0/0, most preferably >100% and that blocks the bioactivity of ADM to less than 80 %, preferably less than 50%. 13. ADM antibody or an adrenomedullin antibody fragment or ADM non—IG ld for use in therapy of a chronic or acute disease or acute condition of a patient ing to any of the claims 1 to 12 to be used in combination with vasopressors e.g.catecholamine and/ or fluids administered intravenously. 14. ADM antibody or adrenomedullin antibody fragment or ADM non—1G scaffold for use in therapy of a chronic or acute disease or acute condition ofa patient according to any of the claims 1 to 13 or a combination according to claim 13 to be used in combination with ADM binding protein and/or further active ingredients.

. Pharmaceutical formulation sing an antibody or fragment according to any of claims 1 to 13. 16. Pharmaceutical formulation according to claim 14 wherein said pharmaceutical formulation is a solution, preferably a rcady-to—use solution. 17. Phannaceutical formulation according to claim 14 wherein said pharmaceutical formulation is in a freeze—dried state. 18. Pharmaceutical formulation according to any of claims 14 to 15, wherein said pharmaceutical formulation is administered intra—muscular. 19. Pharmaceutical ation according to any of claims 14 to 15, wherein said pharmaceutical ation is administered intra—vascular.

. Pharmaceutical ation according to claim 18, wherein said pharmaceutical formulation is administered Via infusion.

EXAMPLES It should be emphasized that the dies, antibody fragments and non—lg scaffolds of the example portion in accordance with the invention are binding to ADM, and thus should be considered as anti—ADM antibodies/antibody fragments/non—Ig scaffolds.

Example 1 tion of Antibodies and determination of their affinity constants Several human and murine antibodies were produced and their affinity constants were determined (see tables 1 and 2).

Peptides/ conjugates for immunization: Peptides for immunization were synthesized, see Table l, (EPT Technologies, Berlin, Germany) with an additional N—terminal Cystein {if no Cystein is present within the selected ADM—sequence) residue for conjugation of the peptides to Bovine Serum Albumin (BSA).

The peptides were covalently linked to BSA by using Sulfolink—coupling gel o-science, Bonn, Germany). The ng procedure was performed according to the manual of Perbio.

The murine antibodies were ted according to the following method: A Balb/c mouse was immunized with lOOug Peptide—BSA—Conjugate at day 0 and 14 (emulsified in 100141 complete Freund’s adjuvant) and Song at day 21 and 28 (in 100m incomplete Freund’s adjuvant). Three days before the fusion experiment was performed, the animal received song of the conjugate dissolved in lOOul saline, given as one intraperitoneal and one intravenous injection.

Spenocytes from the zed mouse and cells of the myeloma cell line SP2/0 were fused with lml 50% hylene glycol for 303 at 370C. Alter washing, the cells were seeded in 96—well cell culture . Hybrid clones were selected by growing in HAT medium [RPMI 1640 culture medium supplemented with 20% fetal calf serum and HAT~Supplement]. After two weeks the HAT medium is ed with HT Medium for three passages followed by returning to the normal cell culture medium.

The cell culture supernatants were primary ed for antigen specific IgG antibodies three weeks after fusion. The positive tested inicrocultures were transferred into 24—well plates for propagation. After retesting, the selected es were cloned and recloned using the limiting—dilution technique and the isotypes were determined. (see also Lane, RD. “A short—duration hylene glycol fusion technique for increasing production of monoclonal antibody—secreting hybridoinas”, J. Immunol. Meth. 81: 223—228; (1985), Ziegler, B. et a1. “Glutamate decarboxylase (GAD) is not detectable on the surface of rat islet cells examined by cytofluorometry and complement—dependent antibody—mediated cytotoxicity of monoclonal GAD dies”, Horm. Metab. Res. 28: 1 1—15, (1996)).

Mouse monoclonal antibody tion: Antibodies were produced via standard antibody production methods (Marx et al, Monoclonal Antibody Production, ATLA 25, 121, 1997,) and purified via Protein A. The antibody purities were > 95% based on SDS gel electrophoresis analysis.

Human Antibodies Human Antibodies were produced by means ofphage y according to the following procedure: The human naive antibody gene libraries HAL7/8 were used for the isolation of recombinant single chain able domains (scFv) t adrenomedullin e. The antibody gene iibraries were screened with a panning strategy comprising the use of peptides containing a biotin tag linked via two different spacers to the adrenomedullin peptide ce. A mix of panning rounds using non—specifically bound antigen and streptavidin bound antigen were used to minimize ound of non~specific binders. The eluted phages from the third round of panning have been used for the generation of monoclonal scFV expressing E.coli strains. atant from the cultivation of these clonal strains has been directly used for an antigen ELISA testing (see also Hust, M., Meyer, T., Voedisch, B., Riilker, T., Thie, H., El—Ghezal, A., Kirsch, Ml, te, M., Helmsing, S., Meier, D., Schirrmann, T., Diibel, 8., 2011. A human 301% antibody generation pipeline for proteorne research. Journal of Biotechnology 152, 159—170; Schiitte, M., Thullier, P., Pelat, T., Wezler, X., Rosenstook, P., Hinz, D., Kirsch, M.I.,Hasenberg, M., Frank, R., Schirnnann, T., Gunzer, M., Hust, M., , S., 2009. Identification of a putative Crf splice variant and generation of recombinant antibodies for the specific detection of Aspergillus fumigatus. PLoS One 4, e6625).

Positive clones have been selected based on ve ELISA signal for antigen and negative for streptavidin coated micro titer plates. For further characterizations the scFv open reading frame has been cloned into the expression plasmid pOPE10’7 (Hust er al., J. hn. 2011), captured from the culture supernatant Via immobilised metal ion affinity chromatography and purified by a size ion chromatography.

Affinity Constants To determine the affinity of the antibodies to Adrenomedullin, the kinetics of binding of Adrenomedullin to immobilized antibody was determined by means of label-free surface plasmon resonance using a Biacore 2000 system (GE Healthcare Europe GmbH, Freiburg, Germany). Reversible immobilization of the dies was performed using an anti~mouse Fc antibody covalently coupled in high density to a CMS sensor surface according to the cturer‘s ctions (mouse dy capture kit; GE Healthcare). (Lorenz et LIL,“ Functional Antibodies Targeting lsaA of Staphylococcus aureus Augment Host Immune Response and Open New Perspectives for Antibacterial Therapy“; Antimicrob Agents Chemother. 2011 January; 55(1): 165—173.) The monoclonal antibodies were raised against the below depicted ADM regions of human and murine ADM, respectively. The following table represents a selection of obtained antibodies used in further experiments. ion was based on target region: Table 1: Sequence Antigen/lmmunogen Designation Affinity Number constants Kd (M) C-40—50 CT—M The following is a list of further obtained monoclonal antibodies: List of anti—ADM—antibodies Table 2 Target Source Klone number Affinity max inhibition (M) bioassay {0/0) (see example 2) NT—M Mouse ADM/63 5.8x10' 45 Mouse 4 2.2x10“ 48 Mouse ADM/3 65 3.0x10‘ Mouse ADM/3 66 1.7x10‘ Mouse ADM/3 67 1.3x10' Mouse ADM/3 68 1.9 x10" -Mouse AD1VL’369 2.0 x10' ADM/3 70 ADM/37 1 ADM/3 72 ADM/373 ADM/3 77 -Mouse ADM/378 =_ADM/380ADM/379 1.8 x10' Mouse ADM/381 2.4 X10" ADM/382 1.6 x10" Mouse 3 MR-M ADM/38 4.5x10" MR-M ADM/39 5.9 x10" '72 Mouse 100 NT-H Mouse ADM/34 1.6x10" MR—H Mouse ' Mouse ADM/43 2.0x10‘ —73 Mouse ADM/44 <1x10' CT-H Mouse ADM/15 CT~H Mouse ADM/16 CT—H Mouse CT—H Mouse Phage display <lx10” Phage display Phage display Phage display <1x10' - Phage display ADM/D8 <1x10’ - Phage display <lx10' — Phage y Generation of d fra entsb enz natic di estion: The generation of Fab and F(ab)2 fragments was done by enzymatic digestion of the murine fuii length antibody NT—M. Antibody NT—M was digested using a) the pepsin—based F(ab)2 Preparation Kit e 44988) and b) the papain—based Fab Preparation Kit (Pierce 44985).

The fragmentation procedures were performed according to the instructions provided by the supplier. Digestion was carried out in case of F(ab)2—frag1nentation for 8h at 37°C. The Fab- fragmentation digestion was carried out for 16h, respectively.

Procedure for Fab Generation and Purification: The immobilized papain was equilibrated by washing the resin with 0.5 ml of Digestion Buffer and centrifuging the column at 5000 x g for 1 minute. The buffer was discarded afterwards. The desalting column was prepared by removing the storage solution and washing it with digestion buffer, centrifuging it each time afterwards at 1000 X g for 2 minutes. 0.5m] of the prepared IgG sample where added to the spin coiumn tube containing the brated Immobilized Papain. Incubation time of the digestion reaction was done for 1611 on a tabletOp rocker at 37°C. The column was centrifuged at 5000 X g for 1 minute to separate digest from the Immobilized . Afterwards the resin was washed with 0.5ml PBS and centrifuged at 5000 X g for 1 minute. The wash fraction was added to the digested antibody that the total sample volume was l.0ml. The NAb n A Column was equilibrated with PBS and IgG Elution Buffer at room temperature. The column was centrifuged for 1 minute to remove storage on (contains 0.02% sodium azide) and equilibrated by adding 2ml of PBS, centrifuge again for 1 minute and the rough ded. The sample was applied to the column and resuspended by inversion. Incubation was done at room temperature with end— over—end mixing for 10 minutes. The column was centrifuged for 1 minute, saving the flow- through with the Fab fragments.

(References: Coulter, A. and , R. (1983). J. Immunol. Meth. 59, 199—203.; Lindner 1. er a1. (2010) {alpha}2-Macroglobulin inhibits the malignant properties of astrocytoma cells by impeding {beta}—catenin signaling. Cancer Res. 70, 277—87.; Kaufmann B. et a1. (2010) Neutralization of West Nile virus by cross—linking of its surface proteins with Fab fragments of the human monoclonal antibody CR4354. PNAS. 107, 18950—5; Chen X. er a1. (2010) Requirement of open headpiece conformation for activation of leukocyte integrin (1)102.

PNAS. 107, 14727—32; Uysal H. at al. (2009) Structure and pathogenicity of antibodies specific for citrullinated collagen type II in experimental arthitis. If. Exp. Med. 206, 449—62.; Thomas G. M. at a]. (2009) Cancer cell—derived microparticles bearing P—selectin rotein ligand 1 rate thrombus formation in vivo. J. Exp. Med. 206, 7.; Kong F. er a1. (2009) Demonstration of catch bonds between an integrin and its iigand. J. Celi Biol. 185, 1275-84.) Procedure for tion and purification of Ft ab’ )3 nts: The immobilized Pepsin was equilibrated by washing the resin with 0.5 ml of Digestion Buffer and centrifuging the column at 5000 X g for 1 minute. The buffer was discarded afterwards. The desalting column was prepared by removing the storage on and washing it with digestion buffer, centrifuging it each time afterwards at 1000 x g for 2 minutes. 0.5mi of the ed lgG sample where added to the spin column tube containing the equilibrated Immobilized Pepsin. Incubation time of the digestion reaction was done for 1611 on a tabletop rocker at 37°C. The column was centrifuged at 5000 X g for 1 minute to separate digest from the lized Papain. Afterwards the resin was washed with 0.5mL PBS and centrifuged at 5000 X g for 1 . The wash on was added to the digested dy that the total sample volume was 1.0ml. The NAb Protein A Column was brated with PBS and IgG Elution Buffer at room temperature. The column was fuged for 1 minute to remove storage on (contains 0.02% sodium azide) and equilibrated by adding 2mL of PBS, centrifuge again for 1 minute and the flow—through discarded. The sample was applied to the column and resuspended by inversion. Incubation was done at room temperature with end- over—end mixing for 10 minutes. The column was centrifuged for 1 minute, saving the flow- through with the Fab fragments.

(References: Mariani, M., at a]. . A new enzymatic method to obtain high—yield F(ab')2 suitable for clinical use from mouse lgGl. Mollmmunol. 28: 69~77.;Beale, D. (1987).

Molecular fragmentation Some applications in immunology. Exp Comp Immune] 11:287- 96.; Ellerson, J.R., er al. (1972). A fragment corresponding to the CH2 region of ilnmunoglobulin G (IgG) with complement fixing activity. FEBS Letters 24(3):318~22.; , R.S. and Elliot, 13.13. (1983). Detection of Fe receptors. Meth Enzymol 93:113-147.; Kulkarni, P.N., er a1. (1985). Conjugation of methotrexate to lgG antibodies and their F(ab’)2 nts and the effect of conjugated methotrexate on tumor growth in vivo. Cancer l lmmunotherapy 1922114.; Lainoyi, E. (1986). Preparation of F(ab’)2 Fragments from mouse IgG of various subclasses. Meth Enzymol 1212652—663.; , P., et at. (1982). Monoclonal antibodies: purification, fragmentation and application to structural and functional studies of class I MHC antigens. J l Meth 531133—73; Raychaudhuri, 6., er al. (1985). Human IgG1 and its Fe fragment bind with different affinities to the Fc receptors on the human U937, HL—60 and ML—l cell lines. Mol Immunol 22(9):1009—l9.; Rousseaux, 1., er al. (1980). The differential enzyme sensitivity of rat immunoglobulin G subclasses to papain an . Mol Immunol 17:469-82.; Rousseaux, 1., er al. (1983). Optimal condition for the preparation of Fab and F(ab')2 fragments from monoclonal IgG of different rat IgG subclasses. J Immunol Meth 6411416.; Wilson, K.M., et a1. (1991). Rapid whole blood assay for HIV-1 seropositivity using an Fab-peptide conjugate. J Immunol Meth 138:111~9.) ntibody Fragment Humanization The antibody nt was humanized by the CDR-grafting method (Jones, P. T., Dear, P.

H., Foote, J., Neuherger, M. S., and Winter, G. (1986) Replacing the complementarity— deterrnining regions in a human antibody with those fiom a mouse. Nature 321, 522525).

The following steps where done to achieve the zed sequence: Total RNA extraction: Total RNA was extracted from NT—H hybridomas using the Qiagen kit.

First-round RT—PCR: QIAGEN® OneStep RTnPCR Kit (Cat No. 210210) was used. RT—PCR was performed with primer sets specific for the heavy and light chains. For each RNA sample, 12 individual heavy chain and 11 light chain RT—PCR reactions were set up using degenerate forward primer es covering the leader ces of variable s.

Reverse primers are located in the constant regions of heavy and light chains. No restriction sites were engineered into the primers.

Reaction Setup: 5); QIAGEN® OneStep RT-PCR Buffer 5.0 1.11, dNTP Mix (containing 10 mM of each dNTP) 0.8 ul, Primer set 0.5 ul, QIAGEN® OneStep RT—PCR Enzyme Mix 0.8 ul, Template RNA 2.0 ul, RNase-free water to 20.0 til, Total volume 20.0 [.11 PCR condition: Reverse transcription: 50°C, 30 min; Initial PCR activation: 95°C, 15 ruin Cycling: 20 cycles of 94°C, 25 see; 54°C, 30 sec; 72°C, 30 sec; Final extension: 72°C, 10 min Second—round semi—nested PCR: The RT—PCR products from the first-round reactions were further amplified in the second-round PCR. 12 individual heavy chain and I] light chain RT- PCR reactions were set up using semi—nested primer sets specific for dy variable regions.

Reaction Setup: 2x PCR mix 10 ul; Primer set 2 til; First-round PCR t 8 ul; Total volume 20 n1; Hybridorna dy Cloning Report PCR condition: Initial denaturing of 5 min at 95°C; 25 cycles of 95°C for 25 sec, 57°C for 30 sec, 68°C for 30 sec; Final extension is 10 min 68°C.

After PCR is finished, run PCR reaction samples onto agarose gel to visualize DNA fragments ampiifiedAfter cing more than 15 cloned DNA fragments amplified by nested RT—PCR, several mouse antibody heavy and light chains have been cloned and appear correct. Protein sequence alignment and CDR analysis identifies one heavy chain and one light chain. After aiignment with homologous human ork sequences the resulting humanized sequence for the variabie heavy chain is the following: see figure 6 (As the amino acids on positions 26, 40 and 55 in the variable heavy chain and amino acid on position 40 in the variable light are critical to the binding properties, they may be reverted to the murine al. The resulting candidates are depicted below) (Padlan, E. A. (1991) A possible ure for reducing the immunogenicity of antibody variable domains while preserving their —binding properties. Moi. Immunol. 28, 489498.; Harris, L. and Bajorath, J. (1995) Profiles for the analysis of immunoglobulin sequences: ison of V gene subgroups. Protein Sci. 4, 306—310.).

Annotation for the antibody nt ces (SEQ ID NO: 744): bold and underline are the CDR 1, 2, 3 in chronologically arranged; italic are constant regions; hinge regions are highlighted with bold letters and the histidine tag with bold and itaiic letters; framework point mutation have a grey 1etter~background SEQ ID NO: 7 (AM—VI—LC) QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGSG SINYNEKFKGKATITADTSSNTAYMQLSSLTSEDSA WYCTEGYEYDGFDYWGQGTTLT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSI/WSGALTSGWITFPA VLQS SGLYSLSSWTVPSSSLGTQW]CNVNHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 8 (AM«VH1) SGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILPGS WYAQKFQGRVTITADESTSTAYMELSSLRSEDTA GYEYDGFDYWGQGTTV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPWVSWNSGALTSGVHTFPA VLQ SSGLYSLSSWTVPSSSLGTQTYICNWHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 9 (AMwVH2-E40) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRILPGS _C_;S__’I_‘_NYAQKFQGRVTITADESTSTA YMELSSLRSEDTA VYYCTEGYEYDGFDYWGQGTTV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 10 (AMfiVH3—T26—E55) QVQLVQSGAEVKKPGSSVKVSCI :EjGYTFSRYWISWVRQAPGQGLEWMGfgILPGs GSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTA VYYCTEGYEYDGFDYWGQGTTV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSPWVSGALTSGVHTFPA VLQ SSGLYSLSSVVTVPSSSLGTQITICNVYVHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 11 (AM—VH4-T26—E4onE55) 325:: QVQLVQSGAEVKKPGSSVKVSCKA; izGYTFSRYWI EWVRQAPGQGLEWMGEJILPGS QKFQGRVTITADESTSTAYMELSSLRSEDTA VYYCTEGYEYDGFDYWGQGTTV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 12 -C) DVLLSQTPLSLPVSLGDQATISCRSSS QSIVYSNGNTYLEWYLQKPGQSPKLLIY_RV_SN RFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFEQGSHIPYTFGGGTKLEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 13 (AM—VLI) DWMTQSPLSLPVTLGQPASISCRSsg2SIVYSNGNTYLNWFQQRPGQSPRRLIYMN RDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCF!QGSHIPYTFGQGTKLEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYBKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 14 (AM-VLZ—E40) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLQZWFQQRPGQSPRRLIYMN RDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Example 2 Effect of ed anti-ADM-antibodies on anti-ADM—bioactivity The effect of selected ADM—antibodies on ADM~bioactivity was tested in an human recombinant Adrenomeduilin receptor CAMP functional assay (Adrenomedullin Bioassay).

Testing of antibodies targeting human or mouse adrenomedullin in human recombinant meduilin receptor cAMP functional assay (Adrenomedullin Bioassay) WO 72513 Materials: Cell line: CHO~K1 Receptor: Adrenornedullin (CRLR + RAMP3) Receptor Accession Number Cell line: CRLR: Ul 7473; RAMP3: AJ001016 CEO—Kl cells expressing human inant adrenornedullin receptor (FAST-027C) grown prior to the test in media without antibiotic were detached by gentle flushing with PBS—EDTA (5 mM EDTA), recovered by centrifugation and resuspended in assay buffer (KRH: 5 mM KC1, 1.25 mM MgSO4, 124 rnM NaCl, 25 mM HEPES, 13.3 mM Glucose, 1.25 mM KH2PO4, 1.45 mM CaClZ, 0.5 g/l BSA).

Dose response curves were performed in parallel with the reference agonists (hADM or mADM).

Antagonist test (96well): For antagonist testing, 6 ul of the reference t (human M) or mouse (0,67nM) adrenomedullin) was mixed with 6 ul of the test samples at ent antagonist dilutions; or with 6 ul buffer. After tion for 60 min at room temperature, 12 n] of cells (2.500 cells/well) were added. The plates were incubated for 30 min at room ature. After addition of the lysis buffer, percentage of DeltaF will be estimated, according to the manufacturer specification, with the HTRF kit from Cis—Bio International (cat n°62AM2 PEB). hADM 22-52 was used as reference antagonist.

Antibodies testing cAMP—I—ITRF assay The anti-h-ADM dies (NT—H, MR—l—i, CT—H) were tested for antagonist activity in human recombinant adrenoniedullin receptor (FAST-027C) CAMP functional assay in the presence of 5.63nM Human ADM 1—52, at the following final antibody concentrations: 100ug/rnl, 20ug/rnl, ding/ml, 0.8ug/rnl, 0.16ug/m1.

The anti-m-ADM antibodies (NT-M, MRMM, CT—M) were tested for antagonist activity in human recombinant adrenomedullin receptor (FAST-027C) CAMP functional assay in the presence of 0.67nM Mouse ADM 1—50, at the following final antibody concentrations: lOOug/ml, 20ug/ml, Ling/ml, 0.8ug/ml, 0.16ug/ml. Data were plotted relative inhibition vs. antagonist concentration (see figs. 3a to 31). The maximal inhibition by the individual antibody is given in table 3.

Table 3: Antibody Maximal inhibition ofADM bioactivity (ADM-Bioassay) (%) MR~H CT-H i 00 NT—M FAB 26 NT-M FAB2 Non specific mouse IgG Example 3 Data for stabilization of hADM by the DM antibody The stabilizing effect of human ADM by human ADM antibodies was tested using a hADM immunoassay.

Immunoassay for the quantification of human Adrenomedullin The technology used was a sandwich coated tube luminescence immunoassay, based on Acridinium ester labelling.

Labelled compound (tracer): iOOug (100111) CT-H (Img/ ml in PBS, pH 7.4, AdrenoMed AGGermany) was mixed with 10in nium NHS—ester (lmg/ ml in itriie, InVent GmbH, y) (EP 03 53971) and incubated for 20min at room ature. Labelled CT- H was d by Gel—filtration HPLC on Bio—Sil® SEC 400-5 (Bio~Rad Laboratories, Inc, USA) The purified CT—H was diluted in (300 mmol/L potassiumphosphate, 100 mmol/L NaCl, 10 mmoi/L Na—EDTA, 5 g/L Bovine Serum Albumin, pH 7.0). The final concentration was approx. 800.000 relative light units (RLU) of labelled compound (approx. 2011;; labeled antibody) per 200 pL. Acridiniumester chemiluminescence was measured by using an AutoLumat LB 953 (Berthold Technologies GmbH & Co. KG).

Solid phase: Polystyrene tubes (Greiner Bio-One ational AG, Austria) were coated (1811 at room ature) with MR—H (AdrenoMed AG, Germany) (1.5 ug MR—H/O.3 mL 100 mmol/L NaCl, 50 mmolfL TRIS/HCl, pH 7.8). After blocking with 5% bovine serum ne, the tubes were washed with PBS, pH 7.4 and vacuum dried.

Calibration: The assay was calibrated, using dilutions ofhADM (BACHEM AG, Switzerland) in 250 mmol/L NaCl, 2 g/L Triton X-100, 50 g/L Bovine Serum Albumin, 20 tabs/L Protease Inhibitor Cocktail (Roche Diagnostics AG, Switzerland» hADM Immunoassay: 50 pl of sample (or calibrator) was pipetted into coated tubes, after adding labeleld CT-H (200141), the tubes were incubated for 4h at 4°C. Unbound tracer was removed by washing 5 times (each lml) with washing solution (20mM PBS, pH 7.4, 0.1 “/0 Triton X400).

Tube—bound chemiluminescence was ed by using the LB 953 Figure 4 shows a typical hADM dose/ signal curve. And an hADM dose signal curve in the presence of 100 ug/mL antibody NT-H.

NT~H did not affect the described hADM immunoassay.

Stability of human Adrenomedullin: Human ADM was d in human Citrate plasma (final concentration 1011M) and incubated at 24 CC. At selected time points, the ation of hADM was stopped by freezing at ~20 °C. The tion was perfonned in absence and presence of NT—H (iOOug/ml). The remaining hADM was quantified by using the hADM immunoassay described above.

Figure 5 shows the stability of hADM in human plasma (citrate) in absence and in the presence of NT—H antibody. The half life ofhADM alone was 7,8h and in the presence ofNT- H, the half life was 18,3h. (2.3 times higher stability).

Example 4 Se sis Mortali earl treatment Animal model 12-15 week old male C57B1/6 mice (Charles River Laboratories, Germany) were used for the study. Peritonitis had been surgically induced under light isofluran anesthesia. ons were made into the left upper quadrant of the peritoneal cavity (normal location of the cecum). The cecum was exposed and a tight ligature was placed around the cecum with sutures distal to the insertion of the small bowel. One re wound was made with a 24—gauge needle into the cecum and small amounts of cecal contents were expressed through the wound. The cecum was replaced into the peritoneal cavity and the iaparotomy site was closed. Finally, animals were returned to their cages with free access to food and water. 500m saline were given so. as fluid ement.

Application and dosage of the compound (NT-M, MR—M, CT-M) Mice were treated immediately after CLP (early treatment). CLP is the iation for cecal ligation and puncture (CLP).

Study groups Three compounds were tested versus: vehicle and versus control compound treatment. Each group contained 5 mice for blood drawing after 1 day for BUN (serum blood urea nitrogen test) determination. Ten further mice per each group were followed over a period of 4 days.

Group Treatment (lOul/ g ight) dose! Follow-Up: 1 NT—M, 0.2 1 survival over 4 days 2 MR-M, 0.2 mg/ml survival over 4 days 3 CT-M, 0.2 nag/ml survival over 4 days 4 nonspecific mouse lgG, 0.2 mg/ml survival over 4 days l ~ PBS 10ul/g bodyweight survival over 4 days Clinical chemistry Blood urea nitrogen (BUN) concentrations for renal function were measured baseline and day 1 after CLP. Blood samples were obtained from the ous sinus with a capillary under light ether hesia. Measurements were performed by using an AU 400 Olympus Multianalyser. The 4—day mortality is given in table 4. The average BUN concentrations are given in table 5.

Table 4: 4 day mortality survival (0/0) PBS 0 non—specific mouse lgG CT—M Table 5: t Average from 5 animals BUN pre CLP (mM) * BUN day 1 (mM) PBS 8.0 23.2 non—specific mouse IgG 7.9 15.5 CT—M 7.8 13.5 24.9 It can be seen from Table 4 that the NT—M antibody reduced mortality considerably. After 4 days 70 % of the mice survived when treated with NTuM antibody. When treated with MR—M dy 30 % of the animals survived and when treated with CT—M antibody 10 % of the s ed after 4 days. In contrast thereto all mice were dead after 4 days when treated with unspecific mouse IgG. The same result was obtained in the control group where PBS (phosphate ed saline) was administered to mice.

The blood urea nitrogen or BUN test is used to evaluate kidney function, to help diagnose kidney disease, and to monitor patients with acute or chronic kidney dysfunction or failure.

The results of the S—BUN Test revealed that the NT-M antibody was the most effective to protect the kidney.

Sepsis Mortality (late treatment) Animal model 12—15 week old male CS7Bl/6 mice (Charles River Laboratories, Germany) were used for the study. Peritonitis had been surgically induced under light isotluran anesthesia. lncisions were made into the left upper quadrant of the peritoneal cavity (normal location of the cecum). The cecum was exposed and a tight ligature was placed around the cecum with sutures distal to the insertion of the small bowel. One re wound was made with a ge needle into the cecum and small amounts of cecal contents were expressed through the wound. The cecum was replaced into the peritoneal cavity and the laparotorny site was closed. Finally, animals were returned to their cages with free access to food and water. 5001;} saline were given so. as fluid replacement.

Application and dosage of the compound (NT-M FABZ) NT—M FABZ was tested versus: vehicle and versus control nd treatment. Treatment was med after full development of sepsis, 6 hours afier CLP (late treatment). Each group contained 4 mice and were followed over a period of 4 days.

Group Treatment (lOuL’ g bodyweight) dose/ Follow—Up: Study groups 1 NT—M, FABZ 0.2 mg/ml survival over 4 days 2 control : non-specific mouse lgG, 0.2 rug/ml survival over 4 days 3 vehicle: — PBS lOnl/g bodyweight survival over 4 days Table 6: 4 day mortality survival (%) NT-M FABZ 75 It can be seen from Table 6 that the NT—M FAB 2 dy d mortality erably.

After 4 days 75 % of the mice survived when treated with NT~M FAB 2 antibody. In contrast 2012/072932 o all mice were dead alter 4 days when treated with non—specific mouse IgG. The same result was obtained in the control group where PBS (phosphate buffered saline) was administered to mice.

Example 5 Incremental effect of anti-ADM antibody in CLP—animals on top of antibiotic treatment and circulation stabilization via cateeholamines as well as regulation of fluid balance.

Animal model In this study male 6 mice (8-12 weeks, 22-30g) were utilized. A polymicrobial sepsis induced by cecal on and puncture (CLP) was used as the model for studying septic shock ((Albuszies G, et al: Effect of increased cardiac output on hepatic and intestinal microcirculatory blood flow, oxygenation, and lism in hyperdynamic murine septic shock. Crit Care Med 2005;33:2332~8), (Albuszies G, er a]: The effect of iNOS deletion on hepatic gluconeogenesis in hyperdynamic murine septic shock. intensive Care Med 2007;33:1094-101), (Barth E, et al: Role of iNOS in the reduced responsiveness of the myocardium to catecholamines in a hyperdynamic, murine model of septic shock. Crit Care Med 2006;34:307—13), (Baumgart K, at al: Effect of SOD—l over-expression on myocardial function during resuscitated marine septic shock. Intensive Care Med 2009;35:344u9), (Baumgart K, et of: Cardiac and metabolic effects of hypothermia and inhaled H28 in etized and ventilated mice. Crit Care Med 2010;38:588—95), (Simkova V, et al: The effect of SOD—l over—expression on hepatic gluconeogenesis and whole-body glucose oxidation during itated, normotensive murine septic shock. Shock 2008;30:578—84), (Wagner F, er al.: Inflammatory effects of hypothermia and inhaled H28 during resuscitated, ynamic murine septic shock. Shock, irn Druck), (Wagner F, er al: Effects of intravenous H28 after murine blunt chest trauma: a prospective, ized lled trial.

Crit Care 201 l, submittes for publication)).

Afler weighing, mice were anesthetized by intraperitoneal injection of 120 ug/g Ketamin, 1.25 pig/g Midazolam and 0.25 ng/g Fentanyl. During the surgical procedure, body temperature was kept at 37—38°C. A lcm midline abdominal n was performed to get access to the cecum. The cecum then was ligated with 3—0 silk tie close to the basis and a single puncture with a lS-gauge needle was applied. The cecum was returned and the incision 2012/072932 was closed again (4-0 tie). For the compensation of perioperative loss of liquids, 0.5 m1 lacted Ringer’s solution with 1 ug/g Buprenorphin as analgetic was injected subcutaneously in dorsal dermis. For antibiosis the mice received Ceftriaxon BOug/g and Clindamycin 30ug/g subcutaneously via the lower extremities.

After CLP surgery the animal were kept in an adequately heated environment with water and food ad iibitum.

The covering of liquid requirements were ensured by a dorsal subcutaneous injections with 0.5 ml lactated ringer’s solution with 4 ug/g glucose and Buprenorphin lug/g, which were applied in an 8 hour cycle, after short term anesthesia by isofluran. In addition, antibiosis was maintained by subcutaneous ions of Ceftriaxon 30ug/g and mycin 30ug/g via the lower extremities.

Dosing of test substances Early treatment ately after the CLP surgery and closing of the incision, the test substance antibody NT—M was applied in a concentration of 500 ug/ml in phosphate buffered saline (PBS) via injection into the penis vein for a dose of 2mg per kg body weight (dose volume 88—120 iii) (5 animals).

Late treatment After full Sepsis development, 15.511 after CLP y, animals were anesthetized as bed above and NT—M was applied in a concentration of 500 pig/ml in ate buffered saline (PBS) via injection into the penis vein for a dose of 2mg per kg body weight (dose volume 88—120 ul) (3 animais).

The control group (6 animals) received a corresponding amount of the vehicle PBS solution without antibody (4ul/g, 88—120 [41) immediately after CLP surgery.

Study groups and experimental setting Murine septic shock model under intensive care monitoring: Three groups with 3, 5 and 6 animals were monitored. Group 1 (5 animals) received the antibody NT~M 15.5h after CLP, group 2 received the antibody NT—M immediately after CLP surgery and group 3 received a comparable amount of PBS (4ul/g). 16 hour incubation post CLP (to allow the polymicrobial sepsis to progress), the experiment was continued with monitoring and entions comparable to an intensive medical care regime. ore, after weighing the animals were anesthetized as described in the CLP surgery part (except the late treated animals, which were anesthized before treatment). Body temperature was maintained at 37—38%? for the rest of the experiment. After a tracheotomy and tion, respiration was monitored and supported by laboratory animal lung ator Flexivent®, (Emka Technologies, FiOZ 0,5, PEEP 10 H20, VT Sal/g, l:E 1:1,5, AF 70-140 ing on temperature).

Anesthesia was maintained throughout the experiment via the cannulated vena jugularis externa dextra with a continuous infusion of Ketamin 30 ug/gxh and Fentanyl 0.3 ug/gxh.

Furthermore, the right aorta s communis was ated for continuous monitoring of heart rate and the mean arterial pressure (MAP). The mean arterial pressure was maintained at MAP > 65 mmHg via enous (V. jugularis) infusion of colloids (80 uL/gxh, d®) and, if needed, Noradrenalin dissolved in ds as vasopressor. Blood samples (120 ul) were taken via the cannulated A. carotis at 0 and 4 hours for determination of creatinine. The bladder was punctured and urine was collected via a bladder catheter. The experiment was either terminated after 6 hours or prior to this, if the MAP > 65 mmHg (V. jugularis) could not be maintained with the vasorpressor .

Measured parameters The following parameters were measured and analyzed: Total consumption of noradrenalin (ug NA/g), consumption rate of enalin (ug NA/g/h), total volume of urine collected during the experiment, creatinine concentration (ug/mL) at the end of the experiment and mean creatinine clearance (uL/min).

Table 7: Total consumption of ption rate of Noradrenalin (pg NA/g) Noradrenalin (pg NA/g/h) (Average) (Average) Control (mouse IgG) (Nt6) NT—M (N=5) early treatment 0.07 pig/g 0.012ug/h/g Relative change (early treatment, 59% 62.5% amelioration) (59%) (62.5%) NT-M (N33) late treatment 0.04 Mfg/g 0.0075 ug/h/g Relative change (late ent, 76,5% 76,5% amelioration) (76.5%) (76.5%) The olamine requirement was measured alter administration of either non specific mouse IgG to a total of 6 mice as l group, NT—murine antibody to a group of 5 mice immediately after CLP (early treatment) or NT—murine antibody to a group of 3 mice 15.5h after CLP (late treatment).

The reduction of the catecholamine requirement is a measure for the stabilization of the circulation. Thus, the data show that the ADM antibody, especially the NT-M dy, leads to a considerable stabilization of the circulation and to a considerable reduction of the catecholamine requirement. The circulation—stabilizing effect was given in early treatment (immediately after CLP) and treatment after full sepsis development (late treatment) (see fig.

Regulation of Fluid Balance More positive fluid balance both early in resuscitation and cumulatively over 4 days is associated with an increased risk of mortality in septic shock. The control of the liquid e is of utmost importance for the course of disease of patients having sepsis. (s. Boyd er a1, 2011). Controlling the liquid balance of critical ill ts remains as a substantial challenge in ive care ne. As can be seen in table 8 treatment of mice after CLP (experimental procedures see “Animal Model”) with NT—M antibody lead to an enhancement of the total volume of urine excreted. The urine secreted was approx. three times higher in NT-M—treated animals compared to non—treated mice. The positive treatment effect was given in early- and in late ent. The fluid balance was improved by about 20-30%, also in both, early and late treatment. Thus, the data Show that the use ofADM antibody, especially the use ofNT ADM antibody, is favorable for regulating the fluid balance in patients. (see table 8 and figures 8 and 9).

Table 8 Urine average Fluid balance average volume/ g body volume/ gbody weight weight Control (mouse IgG) 0.042 ml/g 0,23 Inl/g (N=6) NT—M early (N=5) 0.12 ml 0,18 nil/g ve change early -21.7%% treatment NT—M late (N=3) 0.125 ml 0,16 rnl/g Relative change late + l98% «305% treatment Improvement of kidney function The ation of acute renal failure and sepsis is associated with a 70 percent mortality, as compared with a 45 percent ity among ts with acute renal failure alone. (Schrier and Wang, “Mechanisms of Disease Acute Renal Failure and Sepsis”; The New England Journal of Medicine; 351:159—69; 2004). Creatinine concentration and creatinine clearance are standard tory ters for monitoring kidney (dys)function (Jacob, “Acute Renal Failure”, Indian J. Anaesth; 47 (5): 367-372; 2003). Creatinine and creatinine clearance data from above described animal experiment (early treatment) are given in Table 9.

Table 9 Kidney function: creatinine mean creatinine concentration clearance (ML/min) (us/Hi) control mouse IgG (MW) 174 til/min NT—M (MW) 373 iii/min Relative change —42% +114% (amelioration) (42%) (114%) In comparision to control septic animals, the creatinine concentration was lowered by 42% and the nine clearance was improved by more than 100% as a result ofNT—M treatment (Table 9). The data show that the administration of ADM-antibody, especially NTnM, leads to an improvement ofkidney function.

Improvement of liver inflammatory status Liver tissue for control and early treated animals was homogenized and lysed in lysing buffer.

For cell extract preparation, cells were ended, lysed on ice, and centrifuged. The supernatant (protein extract) was stored at —80 0C. Activation of nuclear factor kappa-light chain gene er in B cells (NF-KB) was ined as previously described using an electrophoretic mobility shift assay (EMSA)1,2. Celi ts (IOng) were ted on ice with p0ly-doxyuinosinic—deoxy—cytidylic acid (poly-dI-dC) and beled double stranded oligonucleotide (Biomers, Ulm, y) containing the NF—KB (HIV KBsite) ( 5’— GGATCCTCAACAGAGGGGACTTTCCGAGGCCA—3’). Complexes were ted in native polyacrylamide gels, dried and exposed to X-ray tiirns. A phosphorimager and image analyzer software (AIDA Image Analyzer; Raytest) was used to quantify the radioactively labeled NF—KB by densitomctry. For comparison between individual gels, the intensity of each band was related to that of simultaneously loaded control s which had not undergone surgical instrumentation and CLP. Therefore, the EMSA data are expressed as fold increase over control values. Statistics: All data are presented as median (range) unless otherwise stated differences between the two groups were analyzed with the Mann—Whitney rank sum test for unpaired samples. Results: The s treated with NT—M presented with significantly attenuated liver tissue NF—KB activation (2.27 (1.97—2.53)) compared to vehicle animals (2.92 (2.50—3.81)) (p<0.001) (see figure 10).

References: l. Wagner F, Wagner K, Weber S, Stahl B, Knoferl MW, Huber-Lang M, Seitz DH, Asfar P, Calzia E, eben U, Gebhard F, GeorgieffM, Radermacher P, Hysa V: Inflammatory effects ofhypothermia and inhaled H28 during resuscitated, hyperdynamic murine septic shock. Shock 2011;35(4):396—402 2. Wagner F, Scheuerle A, Weber S, Stahl B, McCook 0, Knoferl MW, Huber—Lang M, Seitz DH, Thomas J, Asfar P, Szabo C, Moller P, d F, Georgieff M, Calzia E, Raderrnacher P, Wagner K: Cardiopulmonary, histologic, and inflammatory effects of intravenous N328 after blunt chest trauma—induced lung contusion in mice. J Trauma 2011;71(6):1659—67 Example 6 In vivo side effect determination of antibody NT—M 12~15 week old male 6 mice (Charles River Laboratories, Germany) were used for the study. 6 mice were treated with (lOul/ g bodyweight) dose ofNT—M, 0.2 mg/ml. As control, 6 mice were treated with (lOnl/g body weight) PBS. Survival and physical condition was red for 14 days. The mortality was 0 in both groups, there were no differences in physical condition between NT—M and control group. e 7 Gentamicin—induced toxieity A non—septic acute kidney injury model has been established, which makes use of the nephrotoxicity induced by Gentamicin (Chin P3S. Models used to assess renal functions. Drug Develop Res 32:247~255, 1994.). This model was used to assess Whether treatment with anti- Adrenomedullin antibody can improve kidney function.

The experiment was performed as s: Effect of a NT—M on Gentamicin-Induced Nephrotoxicity in Rats Study Design: --—- Route rug/kg NA x 4C aGentamicin at 120 mg/kg intramuscularly for 7 days (days 0-6). bVehicle; injected intravenously (i.v.) 5 min before icin on Day 0, followed by injections on Days 2, 4, and 6.

CNT—M at 4 mg/kg was injected intravenously (i.v.) 5 min before gentamicin on Day 0, followed by 2 mg/kg iv. on Days 2, 4, and 6. dPlasma samples were collected in EDTA tubes (Days 1 and 3 before Test and Control article: 100 pl; Day 7:120 ul. 24h urine tion on ice is initiated after gentamicin on Day 0, followed by Days 2 and 6; blood collection on days 1, 3, and 7.

Groups of 8 male Sprague~Dawley rats weighing 250 i 20 g were employed. Animals were challenged with gentamicin at 120 nag/kg i.m. for seven consecutive days (Groups 1 and 2).

Test compound (anti—adrenomedullin antibody NT-M) and vehicle (phosphate buffered saline) were injected intravenously 5 min before gentamicin on day 0, followed by injection on days 2, 4, and 6. Body s and clinical signs were monitored daily. Twenty—four (24) hour urine tions on ice were performed 011 Days 0, 2, and 6. Urine specimens were d for concentrations of Na+ and K+, and creatinine. Blood samples for clinical chemistry were collected on Days 1 (before gentamicin), 3 (before gentamicin), and 7. Serum olytes (Na+ and K+), nine, and BUN were the primary analytes that were monitored for assessing renal function. Plasma samples were collected in EDTA tubes (Days 1 and 3:100 n1; Day 7:120 til). Creatinine clearance was calculated. Urine volume, urinary electrolytes, and creatinine are expressed as amount excreted per 100 g of animal body weight. All animals were sacrificed on Day 7. s were weighed.

Urine collection. The animals were placed in individual cages where urine was collected for 24 h on Day 0, Day 2, and Day 6. Urine , urinary Na+, K+, and creatinine were measured.

Endogenous creatinine clearance was calculated as follows: CCr (ml/24 h) 2 [UCr (mg/ml) x V (ml/24 h)] / 80' (mg/ml) 24—hr urinary excretion of sodium (Na+) was calculated as follows: UNaV (qu124 h) = UNa (qu/ml) X V (ml/24 h) 24—hr urinary excretion ofNAG and NGALwas similarly calculated.

The fractional excretion of Na+ (FEM), or percentage of the filtered sodium that is excreted into the final urine, is a measure of tubular Na+reahsorptive function. It was computed as follows: FENa (%) =100 x [UNa (HEq/ml) x V (ml/24 h)] / PNa (HEq/ml) X Cc; (ml/24 11) Treatment with anti—Adrenomedullin antibody improved several es of kidney function on day 7 as ed to vehicle: serum nine 101 mg/dL (NT—M) vs 1.55 mg/dL le) (Fig. 11), BUN 32.08 mg/dL(NT—M) vs. 52.41 mg/dL (vehicle) (Fig. 12), endogenous nine clearance 934.43 mL/24 h (NT-M) vs. 68.34 mL/24 h (vehicle) (Fig. 13), fractional secretion of Nalr 0.98 % (NT—M) vs. 1.75 % (vehicle) (Fig. 14).

Example 8 In the mice CLP model described above, the effect of treatment with dren0medullin antibody NT—M on several parameters of kidney on was investigated.

NT—M caused a three— and two—fold higher diuresis and creatinine clearance, respectively, ultimately resulting in lower creatinine, urea, and NGAL blood concentrations at the end of the experiment (see Table 10). Moreover, keratinocyte—derived chemokine (KC) concentrations in the kidney were significantly lowered by treatment with NTnM (Fig. 15).

Table 10: Parameters of kidney function in the vehicle— (n=11) and NTFM—treated (n=9) animals. Blood concentrations were measured in samples taken at the end of the experiment.

NGAL = neutrophil gelatinase—associated lipocalin. All data are median (quartiles). e NT—M Urine output [pL-g‘I-hfi 4.4 (3.5;16.5) 15.2 (13.9;22.5) Creatinine clearance [uL-rnin‘l] 197 01) 400 (316509) 0-006 Creatinine [ug-mL’l} 1.83 3.04) 1.28 (1.20;1.52) 0.010 Urea [ug-mL'1] 378 (268;513) 175 (10l;184) 0.004 NGAL [pg-mm 16 0) 11 (1033) 0.008 The experiments were performed as follows: Creatinine, urea, and neurrophil gelatinase—associated lipocalin (NGAL) Blood NGAL concentrations were measured using a commercial ELISA (mouse NGAL, RUO 042, BioPorto Diagnostics A/S, Denmark, Gentofte). Urea and creatinine concentrations were measured with a ary column (OptimaéMS, Macherey—Nagel, Diiren, Germany) gas chromatography/mass spectrometry system (Agilent 5890/5970, Boblingen, Germany) using 2H3—creatinine (CDN isotopes, Pointe—Claire, QU, ) and methyl-urea (FlukaChemikalien, Buchs, Switzerland) as internal standards. After deproteinization with acetonitrile, centrifiigation and evaporation to dryness, the supernatant was reconstituted in formic acid, and extracted over a weak anion exchange column (WCX, Phenomenex, Aschaffenburg, Germany). Acetonitrile plus N,O—Bis(trimethylsiiyl)trifluoroacetamide and N» (tert-butyldimethylsilyl)~N—methyltrifluoroacetamide allowed ion of the urea tert—butyl- ylsilyl- and the creatininetrimethylsilyl-derivatives, respectively. Ions m/z 231 and 24S, and m/z 329 and 332 were monitored for urea and creatinine es and internal standards, tively. From the urine output and the plasma and urine creatinine concentrations creatinine clearance was calculated using the standard formula.

Sample preparation The kidney which was stored at -80°C was disrupted with a homogenizer in PBS and lysed with a 2—fold concentrated buffer for a whole cell lysate (100 mM Tris pH 7,6; 500 mM NaCl; 6 mM EDTA; 6 mM EGTA; 1 % Triton-X—IOO; 0,5 % NP 40; 10 % ol; Protease- lnhibitors (B-Glycerolphosphate 2 mM; DTT 4 111M; Leupeptine 20 nM; Natrimnorthovanadate 0,2 mM)) and subsequently centrifuged. The whole cell lysate was obtained out of the supernatant; the pellet consisting of cell remnants was discarded. The amount of n was determined photometrically with a commercially available protein assay (Bio—Rad, Hercules, CA) and the specimens were adjusted in the way that the final protein concentration was 4 ttg/ttl. The samples for the Multiplex— and EMSA analysis were diluted 1:1 with EMSA buffer (10 mM Hepes; 50 mM KCl; 10 % ol; 0,1 mM EDTA; 1 mM DTT), the samples for the immune blots 1:1 with 22—fold Sample Buffer (2 % SDS; 125 mM Tris—HCL (pH 6,8 at 25°C); 10 % Glycerol; 50 mM DTT; 0,01 % Bromophenol blue).

Levels of keratinocyte—derived chemokine (KC) trations were determined using a mouse multiplex cytokine kit (Bio-Flex Pro Cytokine Assay, Bio—Rad, Hercules, CA), the assay was performed by using the Bio—plex suspension array system with the manufacturer’s instructions (see also Wagner F, Wagner K, Weber S, Stahl B, Knoferl MW, Huber-Lang M, Seitz DH, Asfar P, Calzia E, Senftleben U, Gebhard F, Georgieff M, Raderrnacher P, Hysa V.

Inflammatory effects of hypothermia and inhaled H28 during resuscitated, ynamic marine Septic shock. Shock 2011;35:396~402; and Wagner F, Scheuerle A, Weber S, Stahl B, McCook O, Knoferl MW, Huber—Lang M, Seitz DH, Thomas J, Asfar P, Szabo C, Moller P, Gebhard F, Georgieff M, Calzia E, Raderrnacher P, Wagner K. Cardiopulmonary, histologic, and inflammatory effects of intravenous NaZS after blunt chest traumadnduced lung contusion in mice. J Trauma 2011;71:1659—1667). In brief, the appropriate cytokine rds and samples were added to a filter plate. The s were incubated with antibodies chemically attached to fluorescent—labeled micro beads. Thereafter, premixed detection antibodies were added to each well, and subsequently, streptavidin—phycoerythrin was added.

Beads were then re-suspended, and the cytokines reaction e was quantified using the Bio-Flex protein array reader. Data were automatically sed and analyzed by Bio—Plex Manager Software 4.1 using the standard curve ed from recombinant cytokine standards. Levels below the detection limit of the assays were set to zero for statistical purposes.

Example 9 In the mice CLP model described above, the effect of treatment with anti-adrenomedullin antibody NT-M on the liver was igated.

NT—M caused a significant lowering of nocyte—derived chemokine (KC) concentrations in the liver (Fig. 16).

Measurement of nocyte—derived chemokine (KC) was done analogous to example 8 (kidney).

Example 10 in the mice CLP model described above, the effect of ent with anti—adrenomedullin antibody NT—M on several cytokines and chemokinesin the blood circulation (plasma) was investigated.

Cytokine and chemokine concentrations Plasma levels of tumor necrosis factor (TNF)-OL, eukin (lL)—6, monocyte chemoattractant protein l, and keratinocyte—derived chemokine (KC) trations were determined using a mouse multiplex. cytokine kit (Bio—Flex Pro Cytokine Assay, Bio—Rad, Hercules, CA), the assay was performed by using the Biouplex suspension array system with the manufacturer’s instructions (see also Wagner F, Wagner K, Weber 8, Stahl B, Knoferl MW, Huber—Lang M, Seitz DH, Asfar P, Calzia E, Senftleben U, Gebhard F, Georgieff M, Radermacher P, Hysa V. Inflammatory effects of hypothermia and inhaled H28 during resuscitated, hyperdynamic murine septic shock. Shock 5:396-402; and Wagner F, Scheuerle A, Weber S, Stabl B, McCook O, Knéferl MW, Huber—Lang M, Seitz DH, Thomas J, Asfar P, Szabo C, Mdller P, Gebhard F, Georgieff M, Calzia E, Raderrnacher P, Wagner K.

Cardiopulmonary, histologic, and inflammatory effects of intravenous NaZS after blunt chest trauma—induced lung contusion in mice. J Trauma 2011;71:1659-1667). In brief, the appropriate cytokine standards and samples were added to a filter plate. The samples were incubated with antibodies chemically attached to fluorescent—labeled micro beads. fter, premixed detection antibodies were added to each well, and subsequently, streptavidin- phycoerythrin was added. Beads were then re—suspended, and the cytokines reaction mixture was quantified using the Bio-Plex protein array reader. Data were automatically sed and analyzed by Bio-Flex Manager re 4.1 using the standard curve ed from recombinant cytokine standards. Levels below the detection limit of the assays were set to zero for statistical purposes.

Plasma levels and kidney tissue concentrations of tumor necrosis factor (TNF)—Gt, interleukin (IL)-6 and IL—10, monocyte chemoattractant protein (MCP)—l, and keratinocyte-dervived chemokine (KC) were determined using a commercially ble “Multiplex Cytokine Kit” (Bio-Plex Pro Precision Pro ne Assay, Bio—Rad, Hercules, CA), which allows to collect several parameters out of one single sample. The individual work steps of the assay were performed according to the cturer‘s instructions (see also Wagner F, Wagner K, Weber S, Stahl B, Knoferl MW, Huber-Lang M, Seitz DH, Asfar P, Calzia E, Senftleben U, Gebhard F, Georgieff M, Radermacher P, Hysa V. Inflammatory effects of hypothermia and d H28 during resuscitated, hyperdynamic marine septic shock. Shock 2011;35:396-402; and Wagner F, Scheuerle A, Weber S, Stahl B, McCook O, Knoferl MW, Huber-Lang M, Seitz DH, Thomas J, Asfar P, Szabo C, Moller P, Gebhard F, Georgieff M, Calzia E, Radermacher P, Wagner K. pulmonary, histologic, and inflammatory effects of enous NaZS after blunt chest trauma—induced lung contusion in mice. J Trauma 2011;71:1659-1667).

In brief, the fluorescence—labed microspheres (“beads”) were added to a 96—well plate, followed by two washing steps, the addition of internal standards and the addition of plasma- and kidney homogenate samples. During the subsequent incubation the single cytokines bind to the antibodies attached to polystyrene~beads After the addition of the cytokine-Specific biotin—labeled antibodies, which are for the detection of the single cytokines, and an additional incubation time, subsequently phycoerythrin—labeled streptavidine was added. After an additional incubation time, beads were then resuspended, and the plates could be measured with a specific flow cytometer iex suspension array system, Bio-Rad, Hercules, CA).

Data were tically processed and analyzed by Bio-Flex Manager Software 4.1 using the standard curve produced from inant cytokine standards. For the plasma levels the concentration was provided in pg * mL‘I, the concentration of the kidney homogenates were converted to the appropriate protein concentration and provided in pg * mg‘1 protein.

NT—M caused a significant ng of plasma concentrations of IL—6 (Fig. 17), IL—lO (Fig. 18), keratinocytenderived chemokine (KC) (Fig. 19), monocyte chemoattractant n—l (MCP—l) (Fig. 20), TNF~alpha (Fig. 21).

Example 11 Ischemia/Reperfusion-Induced Acute Kidney Injury 2012/072932 Another non—septic acute kidney injury model has been established, where acute kidney injury is induced by ischemia/reperfusion oto M, Shapiro 31, Shanley PF, Chan L, and Schrier RW. in Vitro and in vivo protective effect of atriopeptin III on ischemic acute renal failure. J ClinInvest 80:698-705, 1987., ia MS, dino V, and Chiu PJS. Cyclic GMP but not cyclic AMP prevents renal platelet accumulation following ischemia- reperfusion in anesthetized rats. J PharmacolExpTher 27121203-1208, 1994). This model was used to assess whether treatment with drenorneduilin antibody can improve kidney function.

The experiment was performed as s: Effect of a NT—M on Acute Kidney Injury Induced by Ischemia/Reperfusion in Rats Study Design: Test Cone Dosage Rats Group Article Route Ina/ml ml/kg 1112ka (Male) 1 LR + vehiclea IV 5 NA X 3 8 2 LR + NT—M 1v 5 x 3b 8 a vehicle; injected intravenously (iv) 5 min before reperfusion on day 0, followed by injections on days i and 2. bNT-M at 4 mg/kg was injected intravenously (iv) 5 min before reperfusion on day 0, followed by 2 mg/kg iv. each on days i and 2. cUrine collection on days -1, 0, l and 2, with blood chemistry and urine analysis on days 0, 1, 2 and 3, respectively. Plasma samples were collected in EDTA tubes (Days 0 (immediate before surgery), 1, 2: 100 gr], before vehicle or TA; Day 3 :120 ul.

Clinical obseiyations: daily before surgery, following surgery and throughout treatment.

Groups of 8 male Sprague—Dawiey rats ng 250 to 280 g were used. The animals were kept on a 12—hr light/dark cycle and receive a rd diet with distilled water ad libitum.

The s receive fluid supplements (0.9% NaCi and 5% dextrose/1:1, 10 ml/kg p.o.) 30 min prior to surgery (day 0). The rats were anaesthetized with pentobarbital (50 mg/kg, i.p.).

The abdominal cavity was exposed Via a midline incision, followed by intravenous administration of heparin (100 U/kg, iv) and both renal arteries were occluded for 45 min by using vascular clamps. Immediately after l of the renal clips, the kidneys were observed for additional 1 min to ensure color change indicating blood reperfusion. The test nd (NT-M) and vehicle (phosphate ed saline) were inj ectcd intravenously 5 min before reperfilsion, followed by daily injection on days 1 and 2.

Urine collection. The 24-h urine collection on ice was initiated at 24h before ischemia/reperfusion on day —1 (—24h to Oh), and day 0 (0-24h), day 1 (24-4811) and day 2 (48— 72h) afier reperfusion, Blood collection: 0.4 ml blood was collected through the tail vein into EDTA tubes at 011 (before I RI surgery), 2411 (before vehicle or TA), 48h (before vehicle or TA) and 72h for determination of plasma creatinine/Na+/K+, and BUN; 2 ml blood was collected through venal cava terminally.

The animals were placed in individual cages where urine was collected for 24 h day ~1 (—24h— 0h), day 0 ), day 1 (24—48h) and day 2 (48—7230) after reperfusion on day 0. Urine volume, urinary Na+, K+, and creatinine were measured.

The creatinine clearance (CCr) was calculated as s: CCr (ml/24 h) = [UCr (mg/m1) x V (ml/24 h)] / PCr (mg/ml) The 24-hr urinary excretion of sodium (N3+) was calculated as follows: UNaV (qu/24 h) = UNa (qu/ml) X V (ml/24 h) The fractional excretion of Na+ (FENa), or percentage of the filtered sodium that is excreted into the final urine, is a measure of tubular Na+ reabsorptive function. It was computed as follows: FENa (0/0) =100 x [UNa(qu/rn1) x v (ml/24 11)] / PNa l) X CCr (ml/24 11) Treatment with anti—Adrenomedullin dy improved several measures of kidney function: Blood urea nitrogen (BUN) showed a strong se in the vehicle group (0 h: 17.49 mg/dL, 24 h: 98.85 mg/dL, 48 11: 109.84 mg/dL, 72 h: 91.88 , which was less pronounced with NTnM treatment (0 h: 16.33 mg/dL, 24 h: 84.2 mg/dL, 48 h: 82.6l mg/dL, 72 11: 64.54 mg/dL) (Fig. 22).

Serum creatinine developed similarily: e group (0 h: 0.61 Ing/dL, 24 h: 3.3 mg/dL, 48 h: 3.16 lug/dL, 72 h: 2.31 rug/(1L), NT—M group: (O h: 0.59 mg/dL, 24 h: 2.96 ing/dL, 48 11: 2-31 mg/dL, 72 h: 1.8 mg/dL) (Fig. 23).

The endogenous creatinine clearance dropped massively on day one and thereafter improved better in the NT—M group than in the vehicle group. Vehicle group: (0 h: 65.171nL/h, 24 h: 3.5mL/h, 48 h: 12.61mL/h, 72 h: 20.88mL/h), NT—M group:(0 h: 70.11mL/h, 24 h: /h, 48 h: 21.23mL/h, 72 h: 26.611nL/h) (Fig. 24).

FIGURE DESCRIPTION Fig. la: Illustration of antibody s — FV and scFV-Variants Fig 1b: Illustration of antibody formats —u iogous fusions and bifunctional antibodies Fig 1c: Illustration of antibody formats m bivalentai antibodies and bispecific antibodies Fig. 2: hADM 1~52 (SEQ ID No.21) mADM 1-50 (SEQ ID No. 22) aa 1-21 ofhurnan ADM (SEQ ID No. 23) aa 1-42 n ADM (SEQ ID No. 24) aa 43-52 ofhuman ADM (SEQ ID No. 25) aa 144 ofhuman ADM (SEQ ID NO: 26) aa 1—10 of human ADM (SEQ ID NO: 27) aa 1—6 ofhuman ADM (SEQ ID NO: 28) aa 1-32 of human mature human ADM (SEQ ID NO: 29) aa 1-40 ofmature mun'ne ADM (SEQ ID NO: 30) aa 1—31 of mature murine ADM (SEQ ID NO: 31) Fig. 3: a: Dose response curve of human ADM. Maximal CAMP stimulation was adjusted to 100% activation b: DoseX inhibition curve of human ADM 22—52 (ADM—receptor nist) in the presence 0f5.63nM hADM. 0: Dose/ inhibition curve of CT—H in the presence of 5.63 nM hADM. (1: Dose/ inhibition curve ofMRHH in the presence of 5.63 nM hADM. e: Dose/ inhibition curve of NTmH in the presence of 5.63 nM hADM. f: Dose se curve of mouse ADM. Maximal CAMP stimulation was adjusted to 100% tion g: Dose/ inhibition curve of human ADM 22—52 eceptor antagonist) in the presence of 0,67 nM mADM. h: Dose/ inhibition curve of CT-M in the presence of 0,67 nM mADM. i: Dose/ inhibition curve of MR—M in the presence of 0,67 11M mADM. j: Dose/ inhibition curve ofNT-M in the presence of 0,67 nM mADM. k: shows the inhibition ofADM by F(ab)2 NT—M and by Fab NT-M 1: shows the inhibition ofADM by F(ab)2 NT-M and by Fab NT—M Fig. 4: This figure shows a typical hADM dose/ signal curve. And an hADM dose signal curve in the presence of 100 ug/mL antibody NT—H.

Fig. 5: This figure shows the stability of hADM in human plasma (citrate) in absence and in the presence of NT—H antibody.

Fig. 6: Alignment ofthe Fab with homologous human framework sequences Fig. 7: This figure shows the Noradrenalin requirements for early and late treatment with NT—M Fig. 8: This figure shows urine production after early and late treatment with NT-M Fig. 9: This figure shows the fluid balance after early and late ent with NT—M Fig. 10: Liver tissue activation of nuclear factor kappa—iight—chain gene enhancer in B cells (NF—KB) ed by elect0phoretic mobility shift assay (EMSA). # depicts p<0.001 vs. vehicle.

Fig. 11: Development of serum ereatinine over time. Mean +/- SEM are shown.

Fig. 12: Development ofblood urea en (BUN) over time. Mean +/— SEM are shown.

Fig. 13: Development of endogenous creatinine clearance over time. Mean +/— SEM are showu.

Fig. 14: Development of fractional secretion ofNa+ over time. Mean +/- SEM are shown.

Fig. 15: Keratinoeyte—derived chemokine (KC) levels determined in relation to the total kidney protein ted. The white box—plot shows results obtained with vehicle, the grey box-plot shows s obtained after treatment with NT—M.

Fig. 16: Keratinocyte—derived chemokine (KC) levels determined in relation to the total liver protein extracted. The White box—plot shows results obtained with vehicle, the grey box—plot shows results obtained after treatment with NT-M.

Fig. 17: Plasma IL-6 levels. The white box-plot shows results obtained with vehicle, the grey ot shows results obtained after treatment with NT—M.

Fig. 18: Plasma IL—IO levels. The white box—plot shows results obtained with vehicle, the grey box- plot shows results obtained after treatment with NT—M.

Fig. 19: Plasma keratinocyte-derived chemokine (KC) levels. The white box—plot shows results ed with e, the grey box-plot shows results obtained after ent with NT—M.

Fig. 20: Plasma monocyte chemoattractant protein—l (MCP—i) levels. The white box—plot shows results obtained with vehicle, the grey box—plot shows results obtained after treatment with NT-M.

Fig. 21: Plasma TNF—alpha . The white box—plot shows results obtained with vehicle, the grey box-plot shows results obtained after treatment with NT—M.

Fig. 22: Development ofblood urea nitrogen (BUN) over time. Mean +/~ SEM are shown.

Fig. 23: Development of serum creatinine over time. Mean +/- SEM are shown.

Fig. 24: Development of endogenous creatinine clearance over time. Mean +/- SEM are shown.

Claims (13)

1. Use of an anti-Adrenomedullin (ADM) antibody or an anti-ADM antibody fragment binding to adrenomedullin or anti-ADM non-Ig scaffold binding to adrenomedullin in the manufacture of a medicament for use in therapy of an acute disease or acute condition of a patient for stabilizing the systemic ation of said patient, and wherein said antibody or antibody fragment or non-Ig scaffold binds to a region of at least 4 amino acids within sequence of aa1-42 of mature human ADM (SEQ ID NO: 24).

2. Use according to claim 1, which is a. for use in therapy of an acute disease or acute condition of a t for stabilizing the systemic circulation of said patient wherein said patient is in need of izing the systemic circulation and exhibits a heart rate of > 100 beats /min and/ or < 65 mm Hg mean arterial pressure and wherein stabilizing the ic ation means increasing the mean arterial pressure over 65 mmHg or b. for preventive use in therapy of an acute disease or acute condition of a patient in order to prevent that the heart rate increases to > 100 beats /min and/ or mean arterial pressure decreases to < 65 mm Hg.

3. Use according to claim 1 or 2, wherein said antibody or fragment or scaffold reduces the vasopressor requirement of said patient.a

4. Use according to claim 3, wherein said antibody or fragment or scaffold reduces the olamine requirement of said patient.

5. Use according to any one of claims 1 to 4, n said antibody or antibody fragment or non-Ig scaffold is a non­neutralizing anti-ADM dy or a utralizing antiadrenomedullin antibody fragment or a non-neutralizing anti-ADM non-Ig scaffold.

6. Use according to any one of claims 1 to 5, n said antibody or antibody fragment or non-Ig scaffold is monospecific.

7. Use according to any one of claims 1 to 6, wherein said dy or fragment or scaffold ts a binding affinity to ADM of at least 10-7 M.

8. Use according to any one of claims 1 to 7, wherein said antibody or nt or scaffold is not ADM-binding-Protein-1 (complement factor H).

9. Use according to any one of claims 1 to 8, wherein said antibody or antibody fragment or non-lg scaffold binds to a region of at least 5 amino acids within the sequence of aa 1- 42 of mature human ADM: (SEQ ID NO: 24).

10. Use according to any one of claims 1 to 9, wherein said antibody or fragment or scaffold binds to a region of at least 4 amino acids within the sequence of aa 1-21 of mature human ADM: (SEQ ID NO: 23).

11. Use according to claim 10, wherein said antibody or nt or scaffold binds to a region of at least 5 amino acids within the sequence of aa 1-21 of mature human ADM: (SEQ ID NO: 23).

12. Use according to any one of claims 1 to 11, wherein said antibody or fragment or scaffold recognizes and binds to an epitope containing the N-terminal end amino acid 1 of mature human ADM and wherein said antibody or fragment or scaffold would neither bind N-terminal extended nor N­terminal modified adrenomedullin nor inal degraded adrenomedullin.

13. Use according to any one of claims 1 to 12, n said antibody or fragment or