NZ624877B2 - Anti-adrenomedullin (adm) antibody or anti-adm antibody fragment or anti-adm non-ig scaffold for regulating the fluid balance in a patient having a chronic or acute disease - 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 the regulation of fluid balance, wherein said antibody or antibody fragment or non-Ig scaffold binds to region of at least 4 amino acids within sequence of aa1-42 of mature human ADM (SEQ ID NO: 24), wherein the sequences are as defined in the complete specification. d balance, wherein said antibody or antibody fragment or non-Ig scaffold binds to region of at least 4 amino acids within sequence of aa1-42 of mature human ADM (SEQ ID NO: 24), wherein the sequences are as defined in the complete specification.

Description

Anti—Adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non- Ig scaffold for regulating the fluid balance in a patient having a chronic or acute disease Field of the invention Subject matter of the present invention is an anti-Adrenomcdullin (ADM) antibody or an anti- adrenomedullin antibody fragment or anti-adrenomedullin non-lg scaffold for regulating the fluid balance in a patient having a c or acute disease or acute condition.

Subject matter of the t invention is a method for regulating the fluid balance in a t having a chronic or acute disease or acute condition.

Background The peptide adrenomedullin (ADM) was described for the first time in 1993 (Kitamura, K., et at, "Adrenomedullin: A Novel Hypotensive Peptide Isolated From Human Pheochromocytoma", Biochemical and Biophysical Research Communications, Vol. 192 (2), pp. 0 (1993)) as a novel hypotensive peptide comprising 52 amino acids, which had been isolated from a human pheochromoeytome', SEQ ID No.2 21. In the same year, cDNA coding for a precursor e comprising 185 amino acids and the complete amino acid sequence of this precursor peptide were also described. The precursor peptide, which comprises, inter alia, a signal sequence of 2] amino acids at the inus, is referred to as "preproadrenomedullin" (pre-proADM). In the present description, all amino acid positions specified usually relate to the pre-proADM which comprises the 185 amino acids. The peptide 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 ge. To date, substantially only a few fragments of the peptide fragments formed in the cleavage of the pre-proADM have been more exactly terized, in particular the physiologically active es adrenomedullin (ADM) and , a peptide comprising 20 amino acids (22-41) which follows the 21 amino acids of the signal peptide in pre—proADM. The discovery and characterization ofADM 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 es to be found in an issue of "Peptides" d to ADM in particular (Editorial. Takahashi, K., "Adrenomedullin: from a pheochromocytoma to the eyes", Peptides, Vol. 22, p. 1691 (2001)) and (Eto, T., "A review of the ical properties and clinical implications of adrenomedullin and proadrenomedullin N—terminal 20 peptide (PAMP), hypotensive and vasodilating peptides", es, V01. 22, pp. 711 ). A further review is (Hinson, at at, "Adrenomedullin, a Multifunctional Regulatory Peptide", Endocrine Reviews, Vol. 21(2), pp. 7 (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 extended by glycine (Kitarnura, K., et all, "The intermediate form of glycine-extended adrenorncdullin is the major circulating molecular form in human plasma", Biochem. Biophys. Res. Commun, Vol. 244(2), pp. 551—555 (1998). Abstract Only). There is also a binding protein (Pio, R., at at, "Complement Factor H is a Serum—binding Protein for adrenomedullin, and the Resulting Complex Modulates the Bioactivities of Both Partners", The Journal of Biological try, Vol. 276(15), pp. 12292-12300 (2001)) which is specific for ADM and probably likewise modulates the effect of ADM. Those physiological effects of ADM as well as of PAM}J which are of primary ance in the investigations to date were the effects influencing blood pressure.

Hence, ADM is an effective vasodilator, and thus it is possible to associate the hypotensive effect with the particular peptide segments in the C-terminal part of ADM. It has furthermore been found that the above-mentioned further logically active peptide PAMP formed from pre—proADM likewise exhibits a hypotensive effect, even if it appears to have an action mechanism differing from that of ADM (cf. in addition to the entioned review articles (Eto, T., "A review of the biological ties and clinical implications of adrenomedullin and proadrenotnedullin inal 20 peptide (PAMP), hypotensive and vasodilating peptides", Peptides, Vol. 22, pp. 1693-1711 (2001)) and (Hinson, et at, omedullin, a Multifunctional Regulatory Peptide", ine Reviews, Vol. 21(2), pp. 138467 (2000)) also (Kuwasako, K., at al., "Purification and characterization of PAMP—12 (PAMP-ZO) in porcine adrenal medulla as a major endogenous biologically active peptide", FEBS Lett, Vol. 414(1), pp. l05—1 10 (1997). Abstract only), (Kuwasaki, K., at at, "Increased plasma proadrenornedullin N~ terminal 20 peptide in ts with essential hypertension", Ann. Clin, Biochem, Vol. 36 (Pt. ), pp. 622—628 (1999). Abstract only) or da, T., et at, "Secretion of proadrenornedullin N~termina120 peptide from cultured neonatal rat cardiac cells", Life Sci, Vol. 69(2), pp. 23 9—245 (2001). Abstract only) and EP—A2 0 622 458). It has furthermore been found that the trations of ADM which can be measured in the circulation and other biological liquids, 2012/072933 are in a number of pathological states, significantly above the concentrations to be found in healthy control persons. Thus, the ADM level in patients with congestive heart failure, myocardial infarction, kidney diseases, hypertensive ers, Diabetes mellitus, in the acute phase of shock and in sepsis and septic shock are significantly increased, although to different s. The PAMP concentrations are also increased in some of said pathological states, but the plasma levels are reduced relative to ADM ((Eto, T., ”A review of the biological properties and clinical implications of adrenomedullin and proadrenomedullin N~terrninal 20 peptide , hypotensive and vasodilating peptides“, Peptides, Vol. 22, pp. 16934711 (2001)); page 1702). It is firrthennore known that unusually high concentrations of ADM are to be observed in sepsis, and the t concentrations in septic shock (cf. (Etc, T., "A review of the biological ties and clinical implications of adrenomedullin and proadrenomedullin N-terminal 20 peptide (PAMP), hypotensive and vasodilating peptides", Peptides, Vol. 22, pp. 1693—1711 (2001)) and (Hirata, at at, “Increased Circulating Adrenomedullin, a Novel Vasodilatory e, in Sepsis", Journal of Clinical Endocrinology and lism, Vol. 81(4), pp. 1449—1453 (1996)), (Ehlenz, K., at 511., "High levels of circulating adrenomedullin in severe illness: Correlation with C— reactive protein and evidence against the l a as site of origin", Exp Clin Endocrinol Diabetes, Vol. 105, pp. 156—162 (1997)), (Tomoda, Y., at 611., "Regulation of adrenornedullin secretion from cultured cells", Peptides, Vol. 22, pp. 1783-1794 (2001)), (Ueda, S., et at, "Increased Plasma Levels of Adrenomedullin in Patients with Systemic Inflammatory se Syndrome", Am. J. Respir. Crit. Care Med, Vol. 160, pp. 132-136 (1999)) and (Wang, P., "Adrenomedullin and cardiovascular responses in sepsis", Peptides, Vol. 22, pp. 40 Known in the art is further a method for identifying medullin immunoreactivity in biological liquids for diagnostic purposes and, in ular within the scope of sepsis diagnosis, cardiac diagnosis and cancer sis. According to the invention, the midregional partial peptide of. the proadrenomedullin, which contains amino acids (4592) of the entire preproadrenornedullin, is ed, 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 receptor are also described in the art, (ag. WO—Al 2006/027147, ) said diseases may be sepsis, septic shock, cardiovascular diseases, infections, dermatological diseases, inological diseases, metabolic diseases, gastroenterological diseases, cancer, inflammation, hematological diseases, respiratory diseases, muscle skeleton diseases, neurological diseases, urological diseases.

It is reported for the early phase of sepsis that ADM improves heart function and the blood supply in liver, spleen, kidney and small ine. ADM-neutralizing antibodies lize the before mentioned effects during the early phase of sepsis (Wang, P., omedullin and cardiovascular responses in sepsis", Peptides, Vol. 22, pp. 1835—1840 (2001). in the later phase of sepsis, the hypodynamical phase of sepsis, ADM constitutes a risk factor that is strongly associated With the mortality of patients in septic shock. (Schiitz et at, “Circulating Precursor levels of endothelin—1 and adrenornedullin, two endothelium-derived, racting substances, in sepsis”, Endothelium, 14:345—351, (2007)). Methods for the diagnosis and treatment of critically ill patients, 6.g. in the very late phases of sepsis, and the use of endothelin and endothelin agonists with vasoconstrictor activity for the ation of medicaments for the treatment of critically ill patients have been described in WO—Al 62676. It is further described in WO~Al 2007/062676 to use, in place of endothelin and/or endothelin agonists, or in combination therewith, adrenornedullin nists, tie. molecules which prevent or ate the vasodilating action of adrenomedulin, e. g. by blocking its relevant receptors, or substances preventing the binding of adrenomedullin to its or (ag. specific binders as ag. antibodies g to adrenomedullin and blocking its receptor bindings sites; ological neutralization“). Such use, or combined use, including a subsequent or preceding separate use, has been described in certain cases to be desirable for e to improve the therapeutic success, or to avoid undesirable logical stress or side effects.

Thus, it is reported that neutralizing ADM antibodies may be used for the treatment of sepsis in the late stage of sepsis.

Administration of ADM in combination with ADM—binding-Protein—l is described for treatment of sepsis and septic shock in the art. It is assumed that ent 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 (Pio at £11.: Identification, characterization, and physiological actions of factor H as an Adrenomedullin binding Protein present in Human Plasma; Microscopy Res. and Technique, 55:23—27 (2002) and Martinez et (11.; Mapping of the Adrenomedullin- Binding domains in Human Complement factor H; Hypertens Res Vol. 26, Suppl (2003), 856- 59).

In accordance with the invention the ADM-binding—Proteintl may be also referred to as ADM— g—Protein-l (complement factor H). ts having a chronic or acute disease or acute condition, especially patients at the ICU (Intensive Care Unit), may suffer from fluid nce. This may cause severe adverse events such as kidney e and mortality.

It was the subject of the present invention to provide a medicament for regulating the fluid balance and/’or improving the fluid balance of such patients.

The expression “regulating fluid balance” with the context of the instant invention is directed to any correction of a manifested — imbalance w of a t’s fluid balance due to an underlying c or acute disease or acute condition. Said correction is in favour of re-establishing normotension in said patients. The person skilled in the art is fully aware that blood pressure in general, as well as hyper— and nsion is closely related to the fluid balance of a patient.

Fluid balance is the balance of the input and the output of fluids in the body to allow metabolic processes to function. ation is defined as a 1% or greater loss of body mass as a result of fluid loss. The three elements for assessing fluid balance and hydration status are: clinical assessment, body weight and urine output; review fluid balance charts and review of blood chemistry. All this is very well known to a man skilled in the art n rd, Nursing Tomes 19.07.11/Vol 107 No 28, pages 12 to 16).

Thus, in one embodiment a person in need of regulating the fluid balance and/or improving the fluid balance of such patients is a person that has a 1% or greater loss ofbody mass as a result of fluid loss. The fluid balance may be assessed according to Scales and Pilsworth (2008) Nursing Standard 22:47, 50—57. For instance, normal urine output is in the range of 0.5 to 2 ml/kg ofbody weight per hour. The minimum acceptable urine output for a patient with normal renal function is 0.5 nil/kg per hour. All these standards may be used to assess whether a patient is in need for regulating the fluid balance and/or improving the fluid balance.

(Followed by page 6A) In ular the present invention provides 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 ion of a patient for the regulation of fluid balance, wherein said antibody or antibody fragment or non-Ig scaffold binds to region of at least 4 amino acids within sequence of aa1-42 of mature human ADM (SEQ ID NO: 24).

(Followed by page 7) Other embodiments of the invention provide for the use of intravenous fluid, catecholamine or ADM binding protein in the manufacture of a medicament for use in therapy of an acute disease or acute condition of a patient for the regulation of fluid balance, n the therapy comprises use in combination with an drenomedullin (ADM) antibody or an anti-ADM antibody fragment binding to adrenomedullin or anti-ADM non-Ig scaffold binding to adrenomedullin, wherein said antibody or dy fragment or non-Ig scaffold binds to region of at least 4 amino acids within sequence of aa1-42 of mature human ADM (SEQ ID NO: 24).

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 that against it has been raised.

Patient in status of fluid imbalance may get fluid administered enously as a standard measure of care, especially in an ICU setting. It is, however, desirable to reduce or avoid the additional fluid administration e of complications that might occur as tag. the occurrence of edema (acroedema). Edema means swelling caused by fluid in the body’s tissues. It may occur in feet and legs, but can involve the entire body and can involve organs as eg. lung, heart, eye.

Thus, anti—ADM antibody or anti—ADM dy fragment or anti—ADM non-lg ld may be administered at a point of time when the patient is in need of fluid administration. According to the invention said patient is a patient in need of regulating the fluid balance.

Thus, subject matter of the present invention is also an anti-ADM antibody or anti-ADM dy fragment or anti—ADM non—lg scaffold for use in therapy of an acute disease or acute condition of a patient for the regulation of fluid balance which includes but is not d to the tion or ion of edema.

The anti-ADM antibody or the anti-ADM antibody fragment or anti—ADM non~lg scaffold may be also administered preventively before the patient exhibits any signs of fluid imbalance. This might be the case if the patient has a chronic or acute disease or acute condition where fluid imbalance problems may be expected, e.g. comprising severe infections as ag. meningitis, Systemic inflammatory Response-Syndrom (SIRS), ; other diseases as diabetes, cancer, acute and c vascular diseases as eg. heart failure, myocardial infarction, stroke, atherosclerosis; shock as eg. septic shock and organ dysfunction as ag. kidney dysfunction, liver dysfiinction, burnings, surgery, traumata, poisoning, damages by chemotherapy. Especially useful is the antibody or fragment or scaffold according to the present ion for reducing the risk of mortality during sepsis and septic shock, :22. late phases of sepsis.

In the following clinical criteria for SIRS, sepsis, severe sepsis, septic shock will be defined. 1) Systemic inflammatory host se (SIRS) characterized by at least two of the following ms 0 patients exhibit hypotension (mean al pressure is < 65 mm Hg) 0 elevated serum lactate level being > 4 mmol/L 2012/072933 a blood glucose > 7.7 mmol/L (in absence of diabetes) 0 central venous pressure is not Within the range 8—12 mm Hg o urine output is < 0.5 mL 2; kg1 x hr"I :- central venous (superior vena cava) oxygen tion is < 70% or mixed venous < 65% a heart rate is > 90 beats/min 0 temperature < 36°C or > 38°C 0 respiratory rate > ZO/min a white cell count < 4 or > 12 x log/L (leucocytes); > 10% immature neutrophils 2) Sepsis Following at least two of the symptoms mentioned under 1), and additionally a clinical suspicion of new infection, being it: o cough/sputum/chest pain a abdominal istension/diarrhoea 0 line infection 1 5 o endocarditis o dysuria o headache with neck stiffness o cellulitis/woundljoint infection 0 positive microbiology for any infection 3) Severe sepsis Provided that sepsis is manifested in patient, and additionally a clinical suspicion of any organ dysfunction, being it: 0 blood pressure systolic < 90/mean; < 65mmHG o lactate > 2 mmol/L o Bilirubine > /L «- urine output < 0.5 mL/kg/h for 2h 0 creatinine > 177 umol/L o platelets < 100x109/L o SpOz > 90% unless 02 given 4) Septic shock At least One sign of end—organ dysfunction as mentioned under 3) is manifested. Septic shock is indicated, if there is refractory hypotension that does not d to treatment and intravenous fluid administration alone is insufficient to in a patient's blood pressure from becoming nsive also provides for an administration of an antinADM antibody or an anti—ADM antibody fragment or an anti—ADM non—lg scaffold in accordance with the present invention.

Thus, acute e or acute conditions may be selected from the group but are not limited to the group comprising severe infections as eg. meningitis, Systemic inflammatory Response Syndrorn (SIRS), or sepsis; other diseases as diabetes, cancer, acute and chronic vascular diseases as eg. heart failure, myocardial infarction, stroke, atherosclerosis; shock as 9g. septic shock and organ dysfunction as cg. kidney dysfunction, liver dysfunction, burnings, surgery, traumata, ing, damages induced by chemotherapy. Especially useful is the dy or fragment or scaffold according to the present ion for reducing the risk of mortality during sepsis and septic shock, 119. late phases of sepsis.

In one embodiment of the present invention the t is not suffering from SIRS, a severe infection, sepsis, shock as ag. septic shock. Said severe infection denotes eg. meningitis, Systemic inflammatory Response—Syndrome (SIRS), 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 t, being it: a blood pressure systolic < 90/mean; < 65mmHG o lactate > 2 mmol/L o bine > 34umol/L a urine output < 0.5 h for 2h 0 creatinine >177 umol/L :- platelets < lOOXlOg/L o SpOg > 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 ment said acute disease or acute condition is not sepsis.

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

Fluid balance! Fluid therapy In an acute hospital setting, being it ag. a setting in the ICU, commonly the fluid balance is monitored carefully by the clinical staff since this provides for particular information on a patient‘s actual state of hydration, and thus for renal and vascular function.

If, however, acute fluid loss is greater than fluid gain, the patient is referred to as being in negative fluid balance. In this case, logical fluid is often given intravenously by a ian to compensate for that loss.

In centrast, a positive fluid balance where fluid gain is greater than fluid loss may provides for information to a problem with either the renal or cardiovascular system.

This particularly means in context with sag. SIRS, sepsis, severe sepsis and septic shock, that also blood pressure is low nly referred to as hypotension), and the filtration rate in the kidneys will lessen, thus causing less fluid reabsorption and less urine output.

The term “fluid therapy” in l denotes the therapeutic administration of fluids (such as physiologic saline on or water for injection WVFD) to a patient as a treatment or preventative measure. It can be administered via intravenous, intraperitoneal, sseous, subcutaneous and oral routes.

Fluid therapy is indicated either when there is a loss of fluid or there is a risk of loss of fluid due to an underlying disease or ion. The severity of the fluid loss, and the compartment from which it has been lost, influences the choice of fluid and the speed at which it needs to be stered. If fluid therapy is performed as a treatment then it is necessary to diagnose and treat the underlying disease or condition. Fluid therapy is routinely indicated in case of nsion, hypovolemia, metabolic disorders, decreased oxygen delivery, SIRS, sepsis, severe sepsis, shock, and septic shock.

However, 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 regulating the fluid balance and thus not for any methods of y treatment to a chronic or acute disease or condition itself. This means the t invention does not provide for a therapy of healing/curing cg. meningitis, ic inflammatory Response—Syndrom (SIRS), or sepsis, or severe sepsis; other diseases as diabetes, cancer, acute and chronic vascular diseases as e. g. heart failure, myocardial tion, stroke, atherosclerosis; shock as cg. septic shock and organ dysfunction as cg. kidney dysfunction, liver dysfunction, burnings, surgery, traumata, poisoning, or damages induced by chemotherapywithin the scope of the ion.

The fluid regulating effect of the DM antibody or the anti—ADM antibody fragment or anti—ADM non—1g scaffold is thus supporting the primary therapy of said chronic or acute disease or acute condition. In case of a chronic or acute disease or acute condition like severe infections as cg. meningitis, Systemic atory Response-Syndrom (SIRS), sepsis or the like the primary therapy would be cg. the administration of antibiotics. The anti-ADM antibody or the anti-ADM antibody fragment or anti-ADM non-lg scaffold would te the fluid balance and would help to prevent worsening of the critical condition of said patient until the cg. antibiotic administration takes effect. As before mentioned the anti—ADM antibody or the anti—ADM dy nt or anti-ADM non—IG scaffold may be administered in a preventive way or in a therapeutic way, this means in order to prevent fluid imbalance problems or in order to reduce fluid imbalance when fluid imbalance problems are present in said patient. Edema is included in the term fluid imbalance ms.

It should be emphasized that in accordance with the invention the patients may have a chronic or acute e or acute condition as primary or underlying disease such as eg. cancer, or diabetes meilitus. However, those primary or underlying diseases are not prima facz‘e targeted by the therapeutic treatment according to the invention. By contrast, the therapeutic treatment pursuant to the invention is solely directed against acute symptoms that are diagnosed or indicated for fluid therapy.

Thus, the invention does not provide for a primary therapy for , es mellitus, meningitis, Systemic inflammatory Response-Syndrom (SIRS), sepsis or the like, but for a y of patients that suffer from fluid imbalance that is due to an acute disease or acute condition, and thus they are in need of fluid administration.

In one embodiment of the invention an antiADM antibody or an anti—ADM antibody fragment or an anti—ADM non-1g scaffold is to be used in combination with fluids administered intravenously, wherein said ation is for use in therapy of an acute disease or acute ion of a patient for the regulation of fluid balance of said patient.

In one embodiment of the invention said patient having a chronic or acute disease or ion being in need for regulation of fluid balance is characterized by the need of said patient to get intravenous fluids. In another ment of the invention said patient having a chronic or acute disease or condition being in need for regulation of fluid baiance is characterized by the risk of said patient of getting edema or by the presence of edema in said patient.

Subject matter of the invention in one specific embodiment is, thus, an drenomedullin (ADM) antibody or an drenornedullin antibody fragment or anti-ADM non-1g scaffold for use in therapy of a patient in need of intravenous fluids or for use in therapy of a patient having a risk of g edema or by the presence of edema in said patient.

In another embodiment of the ion an anti—Adrenomedullin (ADM) antibody or an anti* adrenomeduilin antibody nt or anti—ADM non—1g scaffold is to be used in combination with vasopressor agents, ag. catechoiamine, wherein said combination is for use in therapy of an acute disease or acute condition of a patient for regulation of fluid balance. 2012/072933 In one embodiment of the invention said patient having a chronic or acute disease or ion being in need for regulation of fluid balance is characterized by the need of said patient to get vasopressor agents, eg. catecholamine, stration.

Subject matter of the invention in one specific embodiment is, thus, an anti—Adrenomedullin (ADM) antibody or an anti—adrenomedullin antibody fragment or an anti»ADM nonrlg scaffold for use in therapy of a patient in need of a vasopressor agent, a.g. catecholamine treatment.

A patient in need of improvement of fluid balance may be characterized by a capillary leakage and may be a urine output </= 0.5 — l cc/kg per hour.

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

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

In a specific and preferred embodiment the present invention provides for a monospecific anti~ Adrenomedullin (ADM) antibody or monospecific anti—adrenomedullin antibody fragment or monospecific anti-ADM non-lg scaffold, characterized in that said antibody or dy fragment or non-1g scaffold binds to one specific region encompassing at ieast 4 amino acids within the target ADM.

In another special embodiment the anti-ADM antibody or the antibody nt 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 dies or fragments are antibodies or fragments that all have affinity 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 n including one or more polypeptides substantially encoded by immunoglobulin genes that specifically binds an antigen. The reengnized immunoglobulin genes include the kappa, , alpha (IgA), gamma (IgGl, IgGg, IgGg, lgG4), delta (IgD), epsilon (lgE) and mu (lgM) 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 . Full-length immunoglobulin heavy chains are generally about 50 Rd or 446 amino acid in . Light chains are encoded by a variable region gene at the NHB-tenninus (about 110 amino acids in length) and a kappa or lambda constant region gene at the COOH—wterminus. Heavy chains are similarly d by a variable regicn gene (about 116 amino acids in length) and one of the other constant region genes.

The basic structural unit of an antibody is generally a tetramer that consists of two identical pairs of immunoglobulin chains, each pair having one light and one heavy chain. In each pair, the light and heavy chain variable regions bind to an antigen, and the constant regions mediate effector functions. Immunoglobulins also exist in a variety of other forms including, for example, Fv, Fab, and (Fab');_, as well as tional hybrid antibodies and single chains (cg, Lanzavecchia er al., Eur. J. Immunol. l7:105,l987; Huston et (1]., Proc. Natl, Acad. Sci. U.S.A., 815879—5883, 1988; Bird et al, Science 242:423—426, 1988; Hood at £11., logy, Benjamin, N.Y., 2nd ed., 1984; Hunkapiller and Hood, Nature —16,1986). An immunoglobulin light or heavy chain le region includes a framework region interrupted by three hypervariable regions, also called complementarity determining regions (CDR's) (see, Sequences of Proteins of 2O Immunological Interest, E. Kabat er al, 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 complex is an antibody, such as a monoclonal dy, chimeric antibody, humanized antibody or human antibody, or onal antibody fragment, specifically bound to the antigen.

Chimeric antibodies are dies whose light and heavy chain genes have been constructed, typically by genetic engineering, from immunoglobnlin variable and constant region genes belonging to ent 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 antibody is thus a hybrid protein composed of the variable or n—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 le region can be produced by molecular techniques. Methods of making chimeric antibodies are well known in the art, rag, see U.S. Patent No. 5,807,715. A "humanized" immunoglobulin is immunoglobulin including a human framework region and one or more CDRs from a non human (such as a mouse, rat, or synthetic) immunoglobulin. The non—human globulin providing the CDRs is termed a "donor“ and the human immunoglobulin providing the framework is termed an "acceptor." In one embodiment, all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, 119., at least about 85—90%, such as about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences. A "humanized dy" is an antibody sing a humanized light chain and a zed heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that es the CDRs. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions by amino acids taken from the donor framework. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other globulin functions. Exemplary conservative substitutions are those such as gly, ala; val, ile, leu; asp, glu; asn, gln; ser, thr; Iys, 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 dies can be generated using methods known in the art.

Human antibodies can be produced by immortalizing a human B cell ing the dy of 2O 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, eg, Dower et all, PCT ation No.

W091/17271; McCafferty at 611., PCT Publication No. W092/001047; and Winter, PCT Publication No. WO92/20791), or selected from a human combinatorial monoclonal antibody library (see the Morphosys website). Human antibodies can also be prepared by using transgenic animals ng a human immunoglobulin gene (for example, see Lonberg e: 51]., PCT Publication No. W093/12227; and Kucherlapati, PCT Publication No. WO91/ l 0741).

Thus, the anti—ADM antibody may have the formats known in the art. Examples are human dies, monoclonal antibodies, humanized antibodies, chimeric antibodies, CUR—grafted antibodies. In a preferred ment antibodies according to the present invention are recombinantly produced antibodies as 8.81 IgG, a typical full—length globulin, or antibody fragments containing at least the F-variable domain of heavy and/or light chain as e.g. chemically coupled antibodies (fragment antigen binding) including but not limited to Fab- nts including Fab minibodies, single chain Fab antibody, monovalent Fab antibody with epitope tags, e.g. Fab-VSSX2; bivalent Fab (mini-antibody) dimerized with the CH3 ; bivalent Fab or multivalent Fab, ag. formed via multimerization with the aid of a heterolcgous domain, eg. via dimerization of dl-ILX domains,e.g. Fab—dHLX—FSX2; F(ab‘)2-fragments, scFv- fragments, multimerized alent or/and maltispecific scFV-fragments, bivalent and/0r bispecific diabodies, BITE® (bispecific T—cell engager), trifimctional antibodies, polyvalent antibodies, 9.g. from a different class than G; single—domain antibodies, e. g. nanobodies derived from camelid or fish immunoglobulines and numerous others.

IO In addition to anti—ADM dies other biopolymer scaffolds are well known in the art to complex a target molecule and have been used for the generation of highly target specific biopolymers, Examples are aptamers, spiegelmers, anticalins and conotoxins. For illustration of antibody formats please see Fig. la, lb and lo.

In a preferred embodiment the ADM antibody format is selected from the group comprising FV fragment, scFv fragment, Fab fragment, scFab fragment, (Fab)2 fragment and scFv-Fc Fusion protein. In another red embodiment the antibody format is selected from the group comprising scFab fragment, Fab fragment, scFV fragment and bioavailability zed conjugates thereof, such as PEGylated fragments. One of the most preferred formats is the scFab 2t) format.

Non—lg scaffolds may be protein scaffolds and may be used as antibody mimics as they are e to bind to ligands or antigenes. Non—lg scaffolds may be selected from the group comprising ectin—based non—lg scaffolds (tag. bed in US 2010/0028995), fibronectin scaffolds (ag. described in EP 1266 025; lipocalin-based scaffolds ((eg described in WO 54420); ubiquitin scaffolds (eg. described in ), transferring scaffolds (ag. described in US 023334), protein A scaffolds (6g. described in EP 0), ankyrin repeat based scaffolds (6g. described in ), microproteins, preferably microproteins forming a cystine knot) scaffolds (eg. described in EP 2314308), Fyn SH3 domain based lds (tag. described in ) EGFR-A—domain based scaffolds (e. g. described in ) and Kunitz domain based scaffolds (cg. described in EP 1941867) In one embodiment of the invention antibodies according to the present invention may be ed as follows: A Balb/c mouse was zed with ADM—lOOug I’eptide-BSA-Conjugate at day 0 and 14 (emulsified in lOOul complete Freund’s adjuvant) and SOug at day 21 and 28 (in IOOul incomplete Freund’s adjuvant). Three days before the fusion experiment was performed, the animal received SOug of the conjugate dissolved in 100p] saline, given as one intraperitoneal and one intravenous injection.

Spenocytes fi‘orn the immunized mouse and cells of the myeloma cell line SP2/O were fitsed with lml 50% polyethylene glycol for 303 at 37°C. After washing, the cells were seeded in 96—well cell e plates. 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 replaced with HT Medium for three es ed by returning to the normal cell e medium.

The cell culture supernatants were primary screened for antigen specific IgG antibodies three weeks after fusion. The positive tested microcultures were transferred into 24—well plates for propagation. After ing, the selected cultures were cloned and recloned 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 omas. J. Immunol. Meth. 81: 223—228; Ziegler, B. e: 651.0996) Glutamate oxylase (GAD) is not detectable on the surface of rat islet cells examined by cytofluorometry and ment—dependent antibody-mediated cytotoxicity of monoclonal GAD antibodies, Horm. Metab. Res. 28: 11-15).

Antibodies may be 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 F—Variable domains (scFv) against adrenomedullin e. The antibody 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 ce. A mix of panning rounds using non—specifically 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 tion of monoclonal scFv expressing E.coli strains.

Supernatant from the cultivation of these clonal strains has been directly used for an antigen ELISA testing (see Hust, M., Meyer, T., ch, 13., Riilker, T., Thie, H, El-Ghezal, A., Kirsch, M.I., Schiitte, M., Helmsing, S., Meier, D, Schirrmann, T., Diibel, 8., 2011. A human SCFV antibody generation pipeline for me research. Journal of hnology 152, 159— 170; Schfitte, M., Thullier, P., Pelat, T., Wezler, X., Rosenstock, P., Hinz, 1)., Kirsch, M.I.,Hasenberg, M., Frank, R., Schirrmann, T., Gunzer, M., Hust, M., Diibel, 8., 2009.

Identification of a putative Crf splice variant and generation of recombinant antibodies for the specific detection of Aspergillus fumigatus. PLoS One 4, e6625).

Humanization ofmurine antibodies may be conducted according to the following procedure: For zation of an antibody of murine origin the antibody sequence is analyzed for the structural interaction of framework s (FR) with the complementary determining regions (CDR) and the antigen. Based on structural modeling an appropriate FR of human origin is selected and the murine CDR sequences are lanted into the human FR. Variations in the amino acid sequence of the CDRs or FRs may be uced to regain structural interactions, which were abolished by the species switch for the FR ces. This ry of structural interactions may be achieved by random approach using phage y libraries or Via directed approach guided by molecular ng (see Almagro JC, Fransson LL, 2008. Humanization of antibodies. Front Biosci. 2008 Jan 619—33).

In a preferred embodiment the ADM antibody format is ed from the group comprising Fv fragment, scFv fragment, Fab fragment, scFab fragment, F(ab)2 fragment and scFV~Fc Fusion protein. In another preferred embodiment the antibody format is selected from the group comprising scFab fragment, Fab fragment, scFV fragment and bioavailability zed conjugates thereof, such as PEGylated fragments. One of the most preferred formats is scFab format.

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

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

In a more preferred embodiment the anti-ADM antibody or an anti—adrenomedullin antibody fragment or an DM non—1g 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 ld binding to adrenomedullin is provided for use in therapy of an acute e or acute condition of a patient wherein said antibody or fragment or scaffold is not ADM-binding—Protein—l (complement factor In one specific embodiment of the invention the anti—Adrenornedullin (ADM) antibody or anti- ADM antibody fragment binding to adrenomedullin or DM non—1g scaffold g to adrenornedullin is provided for use in therapy of an acute disease or acute condition of a patient wherein said antibody or antibody fragment or non—1g 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 embodiment of the invention the anti—Adrenomedullin (ADM) antibody or anti- ADM antibody fragment binding to adrenornedullin or anti—ADM non~Ig scaffold binding to adrenomedullin is provided for use in therapy of an acute disease or acute ion of a patient wherein said antibody or fragment or scaffold binds to a region of preferably at least 4, or at least amino acids within the sequence of aa 1—21 of mature human ADM: SEQ ID No 23 YRQSMNNFQGLRSFGCRFGTC.

In a preferred embodiment of the t invention said anti—ADM antibody or an anti- adrenomedullin antibody fragment or DM non~Ig scaffold binds to a region of ADM of preferably at least 4, or at least 5 amino acids that is located in the N-terminal part (aa 1~21) of adrenomedullin, (see Fig. 2).

In a preferred 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 centained in ADM, preferably in human ADM.

In a more preferred embodiment the anti-adrenomedullin antibody or an anti-adrenomedullin antibody fragment or anti—adrenoniedullin 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 preferred ment said anti-ADM antibody or an drenomedullin antibody fragment or anti—ADM non—lg scaffold recognizes and binds to the N—terminal end (aa 1) of adrenomedullin. N—teiminal end means that the amino acid 1, that is “Y” of SEQ 1D N0. 21 or 23; is mandatory for antibody binding. Said antibody or fragment or scaffold would r bind N—terminal extended nor N—terminal modified adrenomedullin nor N—terminal degraded adrenomedullin.

In another specific embodiment pursuant to the invention the herein provided anti-ADM antibody or anti—ADM antibody fragment or anti—ADM nonnlg scaffold does not bind to the C— terminal portion of ADM, 1'. e. the aa 43 — 52 ofADM (SEQ ID NO: 25): PRSKISPQGY-NHZ (SEQ ID N0225) In one specific embodiment it is preferred to use an anti-ADM antibody or an anti- adrenomedullin antibody nt or anti—ADM non—1g scaffold according to the present invention, wherein said adrenomedullin antibody or said adrenomedullin antibody nt or non—lg scaffold is an ADM stabilizing antibody or an adrenomednllin stabilizing antibody nt or an adrenomedullin stabilizing non—lg scaffold that enhances the half life (tug; half retention time) of adrenomedullin in serum, blood, plasma at least 10 %, preferably at least 50 ”/0, more preferably >50 ”/0, most ably >1000/o.

The half life (half ion time) of ADM may be determined in human plasma in absence and presence of an ADM stabilizing antibody or an adrenomedullin stabilizing dy fragment or an adrenomedullin stabilizing non—1g ld, 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 dy or an adrenomedullin stabilizing antibody fragment or an adrenornedullin stabilizing non- lg ld, respectively, and may be incubated at 24 °C Aliquots are taken at selected time points (8.g. within 24 hours) and degradation ofADM may be stopped in said aliquots by freezing at —20 CC The quantity of ADM may be determined by a liADM immunoassay directly, if the selected assay is not influenced by the izing dy. Alternatively, the aliquot may be treated with denaturing agents (like HCl) and, after clearing the sample (eg. by fugation) the pH can be lized and the ADM-quantified by an ADM immunoassay. Alternatively, non—immunoassay technologies (6.g. rpHPLC) can be used for ADM-quantification The half life of ADM is calculated for ADM incubated in absence and ce of an ADM stabilizing antibody or an adrenomedullin stabilizing antibody fragment or an adrenomedullin stabilizing non-lg scaffold, respectively, The enhancement of half life is calculated for the stabilized ADM in comparison to ADM that has been incubated in absence of an ADM stabilizing antibody or an adrenomedullin stabilizing antibody fragment or an adrenomedullin stabilizing non—1g scaffold.

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

Half Life (half retention time) is defined as the period over Which the tration of a specified al or drug takes to fall to half its baseline concentration in the specified fluid or blood.

An assay that may be used for the detennination of the Half life (half retention time) of adrenomedullin in serum, blood, plasma is described in Example 3.

For some diseases blocking of ADM may be beneficial to a n 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 es. In contrast thereto in other reports ADM was reported as being life threatening when administered in certain conditions.

In a specific embodiment said anti—ADM antibody, anti—ADM antibody fragment or anti~ADM nonmlg scaffold is a non—neutralizing antibody, nt or non—1g scaffold. A neutralizing anti« ADM dy, anti-ADM dy fragment or anti-ADM non—lg ld would block the bioactivity of ADM to nearly 100%, 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%, preferably to less than 95%, preferably to less than 90%, more preferred to less than 80 % and even more preferred to less than 50 %. This means that the residual bioactivity of ADM bound to the non—neutralizing anti—ADM antibody, or DM antibody fragment or anti-ADM non-lg scaffold would be more than 0%, ably more than 5 %, preferably more than 10 %, more preferred more than %, more preferred more than 50 %.

In this context (a) molecule(s), being it an antibody, or an antibody fragment or a nonalg scaffold with “non—neutralizing anti~ADM activity”, tively termed here for simplicity as “non- neutralizing” anti—ADM antibody, antibody fragment, or non—lg scaffold, that tag. blocks the bioactivity ofADM to less than 80 %, is defined as - a molecule or les binding to ADM, which upon addition to a culture of an eukaryotic cell line, which expresses functional human recombinant ADM receptor composed of CRLR (caicitonin or like receptor) and RAMP3 (receptor-activity modifying n 3), reduces the amount of CAMP produced by the cell line through the action of parallel added human synthetic ADM 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 ation of cAMP synthesis, wherein the reduction of CAMP by said molecule(s) binding to ADM takes place to an extent, which is not more than 80%, even when the non—neutralizing molecule(s) binding to ADM to be analyzed is added in an amount, which is 10—fold more than the amount, which is needed to obtain the maximal reduction of CAMP synthesis obtainable with the non- neutralizing antibody to be analyzed.

The same definition s to the other ranges; 95%, 90%, 50% etc.

In a specific ment according to the present ion an anti—ADM antibody or an anti- adrenornedullin antibody fragment or anti—ADM non—lg scaffold is used, wherein said antibody or an adrenomedullin antibody fragment blocks the bioactivity of ADM to less than 80 %, preferably less than 50% (of baseline values). This is in the sense of blocking the circulating ADM of no more than 80% or no 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, fragment or scaffold, meaning an excess of the antibody, fragment or scaffold in relation to ADM. Said limited blocking is an intrinsic property of the ADM binder itself This means that said antibody, fragment or scaffold has a maximal inhibition of 80% or 50% respectively. in a preferred ment said anti—ADM antibody, anti-ADM dy fragment or anti—ADM non—lg scaffold would block the bioactivity ofADM to at least 5 The stated above means that approximately 20% or 50% or even 95% residual ADM bioactivity remains present, respectively.

Thus, in ance with the present invention the provided anti—ADM antibodies, DM antibody fragments, and anti—ADM non—lg scaffolds do not neutralize the respective ating ADM bioactivity.

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 (ag. in an assay) after its interaction. In case ofADM bioactivity this may be the effect of ADM in a human recombinant Adrenornedullin receptor cAMP functional assay. Thus, according to the present invention bioactivity is defined via an Adrenomedullin receptor CAMP functional assay. The following steps may be performed in order to determine the bioactivity ofADM in such an assay: u 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 ated.

— At constant half-maximal CAMP—stimulating ADM—concentrations dose response curves (up to ml final concentration) are performed by an ADM stabilizing antibody or an adrenomedullin stabilizing antibody fragment or an adrenomedullin stabilizing non—lg scaffold, respectively, A maximal inhibition in said ADM bioassay of 50% means that said anti-ADM antibody or said anti—adrenomedullin antibody fragment or said anti~adrenomedullin non—lg scaffold, repectively, blocks the bioactivity to 50% of baseline values. A maximal inhibition in said ADM bioassay of 80% means that said antinADM antibody or said anti—adrenomedullin antibody nt or said drenomedullin non—lg scaffold, respectively, blocks the bioactivity ofADM to 80%. This is in the sense of blocking the ADM bioactivity to not more than 80%. This means approximately 20% residual ADM bioactivity remains present.

However, by the present specification and in the above context the expression “blocks the ivity of ADM” in relation to the herein disclosed anti—ADM antibodies, anti-ADM antibody fragments, and anti—ADM non—lg scaffolds should be understood as mere decreasing the bioactivity of ADM, preferably decreasing circulating ADM bioactivity from 100% to 20% remaining ADM bioactivity at maximum, preferably decreasing the ADM bioactivity from 100% to 50% remaining ADM bioactivity; but in any case there is ADM bioactivity remaining that can be determined as detailed above.

The bioactivity of ADM may be determined in a human recombinant Adrenomedullin receptor CAMP functional assay (Adrenomedullin Bioassay) according to e 2.

In a preferred embodiment a modulating anti—ADM antibody or a ting anti-ADM adrenomedullin antibody fragment or a modulating DM adrenomedullin non—lg scaffold is used in therapy of acute e or acute ion of a patient for tion of fluid balance.

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

A “modulating” antibody or a modulating adrenomeduilin antibody fragment or a modulating adrenomedullin non—1g scaffold is an antibody or an adrenomedullin antibody fragment or non—lg scaffold that enhances the half life (t1); half retention time) of adrenomedullin in serum, blood, plasma at least 10 %, preferably at least, 50 %, more preferably >50 %, most preferably >lOO% and blocks the bioaetivity of ADM to less than 80 %, preferably less than 50 %. These values related to half—life and ng of bioactivity have to be understood in on to the before— mentioned assays and definitions in order to determine these values.

It should be ized that blocking the ADM bioactivity is in the sense of no more than 80%, and thus 20% residual ADM bioactivity. The same applies to blocking the ADM bioactivity to no more than 50%, and thus residual 50% ADM bioactivity.

Such a modulating anti-ADM antibody or a modulating anti-ADM adrenomedullin antibody fragment or a modulating antiuadrenomedullin non—lg scaffold offers the age that the dosing of the administration is facilitated. The combination of partially blocking or partially ng Adrenomedullin bioactivity and increase of the in vivo half life (increasing the Adrenomedullin bioactivity) leads to beneficial simplificity of anti—Adrenomedullin antibody or an anti-adrenomedullin antibody fragment or anti—adrenomedullin non—lg scaffold . In a situation of excess of endogenous Adrenomedullin (maximal stimulation, late sepsis phase, shock, hypodynamic phase) the activity ng effect is the major impact of the antibody or fragment or scaffold, limiting the (negative) effect of Adrenomedullin. In case of low or normal endogenous Adrenomedullin concentrations, the biological effect of anti-Adrenomedullin dy or an drenomedullin antibody fragment or DM non—1g scaffold is a combination of lowering (by partially blocking) and increase by increasing the Adrenornedullin half life. if the half life effect is stronger than the blocking effect, the net ical activity of endogenous Adrenomednllin is beneficially sed in early phases of sepsis (low Adrenomedullin, hyperdynamic phase). Thus, the non—neutralizing and modulating anti— Adrenomedullin antibody or anti-adrenomednllin antibody fragment or anti~adrenomedullin non— Ig scaffold acts like an ADM ivity 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 eg. sepsis may be selected from an excessive concentration, because both sepsis phases (early and late) benefit from excessive anti—ADM dy or an anti—adrenomedullin antibody fragment or anti~ADM non—lg scaffold treatment in case of a modulating effect. Excessive means: The anti- Adrenomedullin dy or an anti—adrenornedullin antibody fragment or anti—ADM nonmlg scaffold concentration is higher than nous Adrenomedullin during late phase (shock) of e.g. sepsis. This means, in case of a modulating DM antibody or modulating anti—ADM antibody nt or modulating anti—ADM scaffold dosing in sepsis may be as follows: The concentration of Adrenomedullin in septic shock is 226+/-66 mi (Nishio at 61]., "Increased plasma concentrations of adrenomedullin correlate with tion of ar tone in patients with septic shock", Crit Care Med. 1997, 25(6):953—7), an equimolar concentration of antibody or fragment or scaffold is 42.5ug/l blood, (based on 6 1 blood volume / 80kg body weight) 3.2ngfkg body weight. Excess means at least double (mean) septic shock Adrenomedullin concentration, at least > 3ug anti-Adrenomedullin antibody or an anti— adrenomedullin antibody fragment or anti—ADM non—1g scaffold / kg body weight, preferred at least 6.4ug anti—Adrenomedullin antibody or an anti-adrenornedullin antibody fragment or anti— ADM non—lg scaffold /kg body weight. Preferred > lOug / kg, more preferred >20ng/kg, most preferred >100ug anti—Adrenomedullin antibody or an anti—adrenomedullin antibody fragment or anti—ADM non—lg ld / kg body . 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 monoclonal antibody or an anti~ADM antibody 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 (murine) CDR’s are grafted into a human antibody or antibody fragment. t matter of the present invention in one aspect is a human CDR—grafted antibody or antibody fragment thereof that binds to ADM, wherein the human CDRngrafted antibody or antibody fragment thereof comprises an antibody heavy chain (H chain) comprising SEQ ID NO:1 GYTFSRYW 2012/072933 SEQ ID NO: 2 ILPGSGST and/or SEQ ID NO: 3 TEGYEYDGFDY and/or further ses an antibody light chain (L chain) comprising: SEQ ID NO:4 QSIVYSNGNTY SEQ ID NO: 5 RVS and/01” SEQ ID NO: 6 FQGSHIPYT.

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 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 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 f wherein the heavy chain comprises the sequences SEQ ID NO: 1 GYTFSRYW SEQ ID NO: 2 ILPGSGST SEQ ID NO: 3 TEGYBYDGFDY and wherein the light chain comprises the sequences SEQ ID NO: 4 QSIVYSNGNTY SEQ ID NO: 5 RVS 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 DM antibody or anti—adrenomedullin antibody fragment or anti-ADM non—lg ld according to the present invention exhibits an affinity towards human ADM in such that affinity constant is greater than 10'7 M, preferred 10'8 M, preferred affinity is greater than 10'9 M, most preferred higher than 10’10 M” A person skilled in the art knows that it may be considered to compensate lower affinity by ng a higher dose of compounds and this measure would not lead out—of—the—scope of the invention. The affinity constants may be determined according to the method as described in Example 1.

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

Chronic or acute disease or acute condition according to the present invention may be a disease or ion selected from the group comprising severe infections as e. g. meningitis, ic inflammatory Response—'Syndrom (SIRS), ; other diseases as diabetes, , acute and chronic vascular diseases as ag. heart failure, myocardial infarction, stroke, artheriosclercsis; shock as e.g. septic shock and organ dysfunction as e.g. kidney dysfunction, liver dysfunction or capillary leakage, , poisoning, surgery. Especially useful is the anti—ADM antibody or anti-ADM antibody fragment or anti—ADM non—lg scaffold according to the present invention for reducing the risk of mortality during sepsis and septic shock, tie. late phases of sepsis.

Hereto it should be emphasized that the patient may be has a chronic or acute e or condition as primary and underlying e as outlined in the above paragraph; however, the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non—lg scaffold pursuant to the invention are not ed for y therapy of said diseases, but rather for regulating the fluid balance of a patient that is in need of administration of fluids, which can thus be considered as an acute disease or acute condition besides the primary disease. er, said need for fluid stration may be associated with a primary underlying disease but this is not mandatory Within the scope of the instant invention.

Thus, in one embodiment the anti—ADM antibody or an anti-adrenomedullin antibody fragment or anti-ADM nonulg scaffold is used in therapy of an acute disease or acute condition of a t according to the present invention wherein said patient is an ICU patient. In another embodiment the anti-ADM antibody or an anti—adrenomedullin antibody nt or anti-ADM non-1g scaffold is used in therapy of an acute disease of a patient ing to the present invention, wherein said patient is critically ill. Critically ill means that the patient is having a disease or state in which death is possible or imminent. t of the present invention is further an anti-ADM dy or an anti—adrenomedullin antibody nt or DM non-1g scaffold for use in therapy of an acute disease of a patient according to the present invention, wherein said antibody or fragment is to be used in combination of ADM binding protein. ADM binding protein is also naturally present in the circulation of said patient.

It should be emphasized that the term ADM binding protein also denotes ADM-binding—protein— l (complement factor H), which however is not a non-neutralizing and modulating anti-ADM antibody, anti-ADM antibody nt, or anti—ADM non-lg ld as in accordance with the ion.

Subject of the present invention is further an anti-ADM antibody or an anti—adrenomedullin antibody fragment or anti-ADM non—1g scaffold for use in therapy of an acute disease or acute condition of a patient according to the present invention n said antibody or fragment or scaffold is to be used in combination with further active ingredients. t matter of the invention is also an anti—Adrenomedullin (ADM) antibody or an anti— adrenornedullin antibody fragment or an antinADM non—lg scaffold to be used in combination with a primary medicament wherein said combination is for use in therapy of an acute disease or acute condition of a t for regulating the fluid balance of said patient.

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

It should be emphasized that said y cause is related to the primary and underlying disease or condition, and is not related to the acute disease or acute condition that is associated with fluid imbalance of a t, for which the herein provided therapy of regulating the fluid balance is intended.

In a specific embodiment of the before mentioned combinations said combinations are to be used in ation with vasopressors e.g. catecholamine wherein said further combination is for use in therapy of an acute disease or condition of a patient for regulating the fluid balance.

In one ment of the invention said patient having a chronic or acute disease or chronic condition being in need for regulating the fluid balance is terized by the need of the patient to get administration of vasopressors e.g. of catecholainine.

It should be emphasized that said patient is having a chronic or acute disease or chronic condition such as , or diabetes, and thus this can be considered as primary, underlying disease, but in addition said patient is in acute need for regulating the fluid balance that is may be due to another acute disease or acute condition such as ag. SIRS, sepsis, severe sepsis, or shock, or septic shock.

Subject matter of the invention in one specific embodiment is, thus, an anti—Adrenomedullin (ADM) antibody or an anti—adrenomeduliin dy fiagment or an anti-ADM non—1g scaffold to be used in combination with ADM binding protein and/or further active ingredients for use in therapy of a t in need of a ent of vasopressors ag. catecholamine treatment.

In a specific embodiment of the above mentioned combinations said combinations are to be used in combination with fluids administered intravenously, wherein said combination is for use in therapy of an acute disease or ion of a patient for regulating the fluid balance.

In one embodiment of the invention said patient having a chronic or acute disease or acute condition being in need for regulating the fluid balance is characterized by the need of the patient to get intravenous fluids. t matter of the invention in one specific embodiment is, thus, an anti~Adrenomedullin (ADM) antibody or an drenomedullin antibody fragment or anti—ADM non-1g scaffold in combination with ADM binding protein and/or further active ients for use in therapy of a patient in need of intravenous fluids.

Said anti—ADM antibody or an drenomedullin antibody fragment or anti-ADM non—1g scaffold or combinations thereof with ADM binding protein and/or further active ients may be used in combination with vasopressors ag. catecholamine and/or with fluids administered intravenously for use in therapy of an acute disease or acute condition of a patient for regulating the fluid balance.

Subject matter of the invention is also an anti-ADM antibody or an anti—adrenomedullin antibody fragment or anti—ADM non-lg scaffold according to the present invention to be used in combination with TNF—alpha~antibodies. TNF-alpha-antibodies are commercially available for the ent of patients. t of the present invention is r a pharmaceutical formulation comprising an anti~ IO ADM antibody or anti—ADM antibody fragment or anti—ADM antibody ld according to the present invention.

Subject of the present invention is further a pharmaceutical formulation according to the present invention wherein said phannaceutical formulation is a solution, preferably a ready-to—use on.

Said pharmaceutical formulation may be stered intramuscular. Said pharmaceutical formulation may be administered intra~vascular. Said ceutical formulation may be administered via infusion.

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 regulating the ic fluid balance with the proviso that said patient is in need of regulating the fluid balance.

Therefore, in another embodiment of the present invention the pharmaceutical ation according to the present invention is to be administered to a patient for ting the systemic fluid balance with the proviso that said patient is in need of ting the fluid balance.

The expression “regulating fluid e” with the context of the instant invention is directed to any correction of a manifested ~ imbalance — of a patient’s fluid balance due to an underlying chronic or acute disease or acute condition. Said correction is in favour of re—establishing normotension in said patients. The person skilled in the art is fully aware that blood pressure in l, as well as hyper— and hypotension is closely related to the fluid balance of a patient.

Fluid balance is the balance of the input and the output of fluids in the body to allow lic ses to function. Dehydration is defined as a 1% or greater loss of body mass as a result of fluid loss. The three ts for assessing fluid balance and hydration status are: clinical assessment, body weight and urine output; review fluid balance charts and review of blood chemistry. All this is very well known to a man d in the art (Alison Shepherd, Nursing Tomes 19.07.11N01107 No 28, pages 12 to 16).

Thus, in one embodiment a person in need of regulating the fluid balance and/or improving the fluid balance of such patients is a person that has a 1% or r loss of body mass as a result of fluid loss. The fluid balance may be assessed according to Scales and Pilsworth (2008) Nursing Standard 22:47, 5067. For instance, normal urine output is in the range of 0.5 to 2 ml/kg of body weight per hour. The minimum able urine output for a patient with normal renal function is 0.5 rill/kg per hour. All these standards may be used to assess whether a patient is in need for regulating the fluid balance and/or improving the fluid balance.

In another embodiment subject of the present invention is further a pharmaceutical formulation ing to the present ion wherein said pharmaceutical formulation is in a dried state to be reconstituted before use.

In another embodiment subject of the present ion is further a pharmaceutical formulation according to the present invention wherein said pharmaceutical formulation is in a freeze—dried state.

Further embodiments Within the scope of the present invention are set out below: 1. Adrenomedullin ADM antibody or an adrenomeduilin antibody fragment for use in therapy of a chronic or acute disease of a patient for the regulation of liquid balance. 2. ADM antibody or an adrenomedullin antibody 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.

WO 72514 3. ADM antibody or an adrenornedullin antibody fragment ing claim 1 or 2 wherein said antibody or fragment binds to the N—terminal part (aa 1—521) of adrenomedullin. 4. 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 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 ADM stabilizing a antibody fragment that enhances the t1 /2 half retention time of adrenornedullin in serum, blood, plasma at least 10 %, preferably at least 50 %, more preferably >50 %, most preferably >100 %. 6. ADM antibody or an adrenomedullin antibody fragment according to any of claims 1 to 5, wherein said antibody or fragment blocks the bioactivity of ADM to less than 80 %, preferably less than 50%. 7. ADM antibody or an medullin antibody nt for use in therapy of a chronic or acute disease of a patient ing to any of claims 1 to 6 wherein said disease is ed from the group comprising sepsis, diabetis, cancer, heart failure, shock and kidney dysfunction. 8. ADM antibody or an adrenomedullin antibody fragment for use in therapy of a chronic or acute e of a patient according to any of claims 1 to 7 wherein said patient is an lCU patient. 9. ADM antibody or an adrenomedullin antibody fragment for use in therapy of a chronic or acute e of a patient according to any of claims 1 to 7 n said antibody or fragment is a modulating antibody or fragment that enhances the t; /2 half retention time of adrenomedullin in serum, blood, plasma at least 10 %, preferably at ieast 50 %, more preferably >50 %, most preferably >100 “/0 and that blocks the bioactivity ofADM to less than 80 “/0, preferably less than 50%.

. Pharmaceutical ation comprising an antibody or fragment according to any of claims 1 to 9. ll. Pharmaceutical formulation according to claim 10 wherein said ceutical formulation is a solution, preferably a ready—to—use solution. 12. Pharmaceutical formulation according to claim 10 wherein said pharmaceutical formulation is in a —dried state. 13. ceuticai formulation according to any of claims 10 to 11, wherein said pharmaceutical formulation is administered uscular. 14. Pharmaceutical formulation according to any of claims 10 to 11, wherein said pharmaceutical formulation is administered intranvascular.

. Pharmaceutical formulation according to claim 14, 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 an ADM non- Ig scaffold for use in therapy of a chronic or acute disease or acute ion 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 dy or an adrenomedullin dy fragment or ADM non—1G ld is a non-neutralizing ADM antibody or a non- neutralizing adrenomeduilin antibody fragment or a non—neutralizing ADM non—1G scaffold.

Adrenomedullin ADM antibody or an adrenomedullin antibody nt 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.

WO 72514 4. ADM dy or an adrenomedullin antibody fragment or ADM non—1G scaffold according to any of claims 1 to 3 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 dy or an adrenomedullin dy fragment or ADM nonan scaffold according to 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 scaffold recognizes and binds to the N—tenninal end (aal) of adrenomeduilin.

ADM antibody or an adrenoinedullin antibody fragment or ADM non-1G scaffold according to any of claims 1 to 6, wherein said dy or fragment or scaffold is an ADM izing antibody or ADM stabilizing antibody fragment or ADM stabilizing non-JG scaffold that enhances the half life 2 half retention time) of adrenomedullin in serum, blood, plasma at least 10 %, preferably at least 50 %, more preferably >50 “/0, most preferably >100 %.

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

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 8 wherein said disease is selected from the group comprising SIRS, sepsis, diabetis, cancer, heart failure, shock and kidney dysfunction 10. ADM antibody or an adrenomedullin antibody nt 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 TEGYEYDGFDY and wherein the light chain comprises the ces SEQ ID N024 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 nt comprises a sequence selected from the group comprising : SEQ ID NO: 7 (AM—VH—C) QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPG SGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYW GQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEP KHHHHHH SEQ ID NO: 8 1) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILP GSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS SALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKHHHHHH SEQ ID NO: 9 2—E40) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRILP GSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS'WNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKHHHHHH SEQ 1D NO: 10 (AM-VH3wT26-E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEILP GSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKHHHHHH SEQ ID NO: 11 (AM—VH4—T26—E40-E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEILP GSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKHHHHHH SEQ ID NO: 12 (AM~VL-C) DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYRV SNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 13 (AM—VLI) DVVMTQSPLSLPVTLGQPASISCRSSQSlVYSNGNTYLNWFQQRPGQSPRRLIYRV SNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLE1K RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 14 (AM—VLZ-E40) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRV SNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 12. ADM antibody or an medullin dy 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 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 12 wherein said antibody or fragment or scaffold is a modulating antibody or fragment or scaffold that enhances the half life (“:1 ,2 half retention time) of adrenornedullin in serum, blood, plasma at least 10 “/0, ably at least 50 ”/0, more preferably >50 %, most ably >100 % and that blocks the bioactivity of ADM to less than 80 %, preferably less than 50%. 14. ADM antibody or an adrenornedullin antibody fragment or ADM non-1G scaffold for use in therapy of a chronic or acute disease of a t 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 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 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 ofclaims l to 15. 17. Pharmaceutical formulation according to claim 16 wherein said ceutical formulation is a solution, preferably a ready—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, n said pharmaceutical formulation is administered muscular.

. Pharmaceutical formulation according to any of claims 16 to 17, wherein said pharmaceutical formulation is administered intra—vascular. 21. ceutical 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 ation.

ADM antibody or an adrenomedullin antibody fragment according to claim l wherein said antibody or fragment reduces the catecholamine requirement of said patient.

ADM antibody or an medullin dy fragment according to claim 1 or 2 n 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. 4, ADM antibody or an adrenomedullin antibody fragment ing to any of claims 1 to 3 wherein said antibody or fragment binds to the N-tenninal part (aa 1-21) of adrenomedullin. 5. ADM antibody or an medullin 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. 6. ADM antibody or an medullin 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 %, ably at least, 50 %, more preferably > 50 %, most preferably >100 %. 7. ADM antibody or an adrenomedullin antibody fragment according to any of claims 1 to 6, wherein said antibody or fragment blocks the bioactivity of ADM to less than 80 %, preferably less than 50 %. 8. ADM antibody or an adrenomedullin antibody fragment according to any of claims 1 to 7, wherein said antibody or fragment is a modulating ADM dy or a modulating adrenornedullin antibody nt 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 ably >100 % and that blocks the bioactivity of ADM to less than 80 %, preferably less than 50 %: 9. ADM antibody or an medullin dy fragment for use in therapy of a c or acute disease of a patient according to any of the claims 1 to 8 wherein said disease is selected from the group comprising sepsis, diabetis, cancer, acute and chronic vascular diseases as eg. heart failure, shock as eg. septic shock and organ dysfunction as eg. kidney dysfunction.

. Pharmaceutical formulation comprising an antibody according to any of claims I to 9. ll. Pharmaceutical formulation according to claim 10 wherein said pharmaceutical formulation is a solution, preferably a ready-to~use solution. 12. Pharmaceutical ation according to claim 10 wherein said pharmaceutical formulation is in a freeze—dried state. 13. Pharmaceutical formulation according to any of claims 10 to 11, wherein said pharmaceutical formulation is administered intra-muscular. l4. Pharmaceutical formulation according to any of claims 10 to 11, wherein said pharmaceutical formulation is administered intramvascnlar.

. Pharmaceutical formulation according to claim 14, wherein said pharmaceutical forrnuiation is stered Via infusion. r embodiments within the SCOpe of the present invention are set out below: I. 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 izing the circulation. 2. ADM antibody or an adrenornedullin antibody fragment or ADM non—1G scaffold according to claim 1 wherein said dy or fragment or scaffold reduces the vasopressor requirement, 9g. catecholamine requirement of said patient. 3. ADM antibody or an adrenomedullin antibody fragment or ADM non—1G scaffold according to claim 1 or 2 wherein said ADM antibody or an adrenomedullin antibody fragment or ADM non—1G ld is a non—neutralizing ADM antibody or a non- neutralizing adrenomedullin antibody fragment or a utralizing ADM non-1G scaffold. 4. ADM antibody or an adrenornedullin antibody nt 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 Nmterrninal part (aa l~21) of adrenomedullin. 6. 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 scaffold recognizes and binds to the N—terminal end (aal) of adrenomednllin- 7. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG 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 adrenorneduliin in serum, blood, plasma at ieast 10 %, preferably at least, 50 %, more preferably > 50 %, most preferably >100 %. 8. ADM antibody or an medullin antibody fragment or ADM non—1G scaffold ing to any of claims 1 to 7, wherein said antibody or fragment or scaffold blocks the bioactivity ofADM to less than 80 %, preferably less than 50 %. 9. ADM antibody or an adrenomedullin antibody fragment or ADM non—1G scaffold ing to any of claims 1 to 8, wherein said antibody or nt or ld is a modulating ADM antibody or a modulating adrenomedullin antibody fragment or scaffold that enhances the half life (t1/2 half retention time) of adrenomedullin in serum, blood, plasma at least 10 %, preferably at least, 50 %, more preferably > 50 %, most ably >100 % and that blocks the bioactivity of ADM to less than 80 ”/0, preferably less than 50 %: . ADM dy or an adrenomedullin antibody fragment according to any of claims 1 to 9, n said antibody or fragment is a human monoclonal dy or fragment that binds to ADM or an antibody nt thereof wherein the heavy chain comprises the sequences SEQ ID NO: 1 GYTFSRYW 2012/072933 SEQ 11:) 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 N016 FQGSHIPYT. 11. A human monoclonal antibody or nt that binds to ADM or an dy 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) QVQLQQSGAELMKPGA§VKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPG SGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYW GQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEP KHHHHHH SEQ ID NO: 8 (AM—VH1) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILP GSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKHHHHHH SEQ ID NO: 9 (AM-VH2-E40) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRILP GSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDY VTVSSASTKGPSVFPLA?SSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKHHHHHH SEQ ID NO: 10 (AM~VH3-T26-E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEILP GSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKHHHHHH SEQ ID NO: 11 (AM—VH4-T26-E40nE55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGBILP YAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKHHHHHH SEQ ID NO: 12 -C) DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYRV SNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 13 (AM—VLI) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYRV SNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 14 (AM-VL2-E40) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRV SNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 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 the claims 1 to 11 wherein said disease is selected from the group comprising SIRS, sepsis, diabetis, cancer, acute and chronic vascular diseases as eg. heart failure, shock as eg. septic shock and organ dysfunction as ag. kidney dysfunction. 13. ADM antibody or an adrenomedullin antibody fragment or ADM non—IG scaffold 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 stered intravenously. 14. ADM antibody or adrenomeduliin antibody fragment or ADM non-IG 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 n andfor further active ingredients. 15. Pharmaceutical formuiation comprising an antibody or fragment or non-1G scaffold according to any of claims 1 to 14. 16. Pharmaceutical formulation according to claim 15 wherein said pharmaceutical ation is a solution, ably a ready—to—use solution. 17. Pharmaceutical formulation according to claim 15 wherein said pharmaceutical formulation is in a freeze-dried state. 18. ceutical formulation according to any of claims 15 to 16, wherein said pharmaceutical formulation is stered intra—mnscular. 19. Pharmaceutical formulation ing to any of claims 14 to 16, n said pharmaceutical formulation is administered intra-vascular. 20. Pharmaceutical formulation according to claim 16, wherein said pharmaceutical formulation is administered Via infusion.

Further embodiments within the scope of the present invention are set out below: 1) Adrenorneduilin antibody or an adrenomedullin antibody fragment for use in a treatment of a chronic or acute disease wherein said antibody or said fragment is an ADM stabilizing antibody or fragment that enhances the 111/2 half ion time of adrenomedullin in serum, blood, plasma at least 10 %, preferably at least, 50 %, more preferably >50 %, most preferably 100 % and/or wherein said antibody blocks the bioactivity of ADM to less than 80 ”/0, preferably to less than 50 “/0. 2) Adrenornedullin antibody or an adrenomedullin dy nt for use in a treatment of a chronic or acute disease wherein said antibody or said fragment is a modulating ADM antibody or nt that enhances the fig half retention time of adrenomedullin in serum, blood, plasma at least 10 %, preferably at least, 50 0/0, more preferably >50 %, most ably 100 % and that blocks the bioactivity of ADM to less than 80 %, preferably to less than 50 %. 3) Adrenomedullin antibody or an adrenomedullin antibody nt for use in a treatment of a chronic or acute disease according to claim 1 or 2, n said antibody or fragment binds to the N~terminal part (aa 1—21) of adrenomedullin. 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—terrninal end of adrenomedullin.

WO 72514 ) Adrenomeduilin antibody or an adrenomedullin antibody fragment for use in use in a treatment of a chronic or acute disease according to any of claims 1 to 4, wherein said antibody or said fragment is an ADM izing antibody or fragment that enhances the t1 ,2 half retention time of adrenomedullin in serum, blood, plasma at least 10 %, preferably at least, 50 %, more preferably >50 %, most preferably 100 ”/0. 6) medullin 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, wherein said antibody or said fragment blocks the bioactivity ofADM to less than 80 %, preferably to less than 50 %. 7) Adrenomedullin antibody or an adrenomedullin antibody fragment ing 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 adrenomedullin antibody fragment according to any of the claims 1 to 7 for use in a treatment of a chronic or acute disease wherein said disease is septic shock or sepsis. 9) Adrenomedullin antibody or an adrenomedullin antibody fragment for use in a treatment of a chronic or acute e according to any of the claims 1 to 8 wherein said antibody or fragment regulates the liquid balance of said patient. 10) Adrenomeduliin antibody or an adrenomedullin antibody fragment for use in a treatment of a chronic or acute e according to any of the claims 1 to 9 n said antibody or fragment used for prevention of organ dysfunction or organ failure. ii) Adrenomedullin antibody or an adrenomedullin dy fragment for use in a treatment of a chronic or acute disease according to claim 10 wherein said antibody or nt is used for prevention ofkidney ction or kidney failure. 12) Adrenomedullin (ADM) antibody or an adrenomedullin antibody fragment for use in a treatment of a chronic or acute disease in a patient ing to claims 1 to 11 wherein said dy or fragment is used for stabilizing the circulation. 13) ADM dy or an adrenomedullin antibody fragment for use in a treatment of a chronic or acute disease in a patient according to claim 12 n said dy or fragment reduces the catecholarnine requirement of said patient. 14) ADM antibody or an adrenomeduliin antibody fragment for use in a treatment of a chronic or acute disease in a patient according to any of claims I to 13 for the reduction of the mortality risk for said patient.

) 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 n said antibody or fragment may be administered in a dose of at least 3 ng / Kg body weight. 16) Pharmaceutical composition sing an antibody or fragment according to any of claims 1 to 15.

Further embodiments within the scope of the present invention are set out below: . Adrenomedullin antibody or an adrenomedullin antibody fragment or ADM non~lg ld wherein said antibody or said fragment or scaffold is a non—neutralizing antibody.

. Adrenomednllin antibody or an adrenomedullin antibody fragment or ADM non—lg scaffold wherein said antibody or said nt or scaffold is an ADM stabilizing antibody or fragment or ld that enhances the half life 01/2 half retention time) of medullin in serum, blood, plasma at least 10 %, 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 nt or ADM non-lg scaffold wherein said dy or said fragment is a modulating ADM antibody or fragment or scaffold that enhances the half life (t1 ,2 half retention time) of adrenomedullin in serum, blood, plasma at least 10 0/6, preferably at least, 50 0/0, more preferably >50 %, most preferably 100 % and that blocks the bioactivity of ADM to less than 80 %, preferably to less than 50 %. 4. Adrenomedullin antibody or an adrenomeduilin antibody fragment or ADM non—1g scaffold according to claim 1 or 2, wherein said antibody or fragment or scaffold binds to the N-terminai part (aa 1—21) of medullin.

. Adrenornedullin 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—terrninal end of adrenomedullin. 6. Adrenomedullin 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 scaffold is an ADM stabilizing antibody or nt that enhances the t“; half retention time of adrenomedullin in serum, blood, plasma at least 10 %, preferably at least, 50 ”/0, more preferably >50 %, most preferably 100 0/0. 7. Adrenomedullin dy or an medullin antibody fragment or ADM non-lg Scaffold according to any of the claims 1 to 6 for use as an active pharmaceutical substance. 8. Adrenomedullin antibody or an adrenomedullin antibody fragment ADM non-lg scaffold according to any of the claims 1 to 7 for use in a treatment of a chronic or acute e or acute condition wherein said disease or condition is ed from the group comprising severe infections as ag. meningitis, systemic inflammatory Response- Syndrome (SlRS,) sepsis; other diseases as diabetes, , acute and chronic ar diseases as cg. heart e, myocardial infarction, stroke, atherosclerosis; shock as e. g. septic shock and organ dysfunction as ag. kidney dysfunction, liver dysfunction, burnings, surgery, traumata. 9. Adrenomedullin antibody or an adrenomedullin antibody fragment or ADM non-lg ld ing to any of the claims 1 to 8 for use in a treatment of a chronic or acute disease or acute condition wherein said disease is septic shock or sepsis.

. ADM antibody or an adrenomedullin antibody nt 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 : SEQ ID NO: I GYTFSRYW SEQ ID NO: 2 SEQ ID NO: 3 TEGYEYDGFDY And/or wherein the light chain comprises the at least one of the sequences SEQ ID NO:4 QSIVYSNGNTY SEQ ID NO: 5 SEQ ID NO: 6 PYT. 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) QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPG SGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYW GQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEP KHHHHHH SEQ ID NO: 8 (AM—VH1) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILP GSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKHHHHHH SEQ ID NO: 9 (AM—VH2—E40) SGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRILP GSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDY VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKHHHHHH SEQ ID NO: 10 (AM-VH3—T26—E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEILP GSGSTNYAQKPQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKHHHHHH SEQ ID NO: 11 (AM—VH4-T26~E40~E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEILP GSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKHHHHHH SEQ ID NO: 12 -C) DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYRV SNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 13 (AM—VLi) DWMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYRV SNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 14 (AM-VLZ—E40) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRV SNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 12. Adrenomedullin antibody or an adrenomedullin antibody fragment or ADM non—1g scaffold according to any of the claims 1 to 11 for regulating the fluid balance in a t having a chronic or acute disease or acute condition. . 13. Adrenomedullin antibody or an adrenoniedullin dy fiagment or ADM nondg scaffold ing 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 e 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. 1.5. Adrenoniedullin (ADM) antibody or an adrenomedullin antibody fragment or ADM non— Ig 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 rnedullin antibody fragment or ADM non—1g scaffold for use in a treatment of a chronic or acute disease in a patient according to claim 15 wherein said antibody or fragment reduces the olamine requirement of said patient. 17. Adrenornedullin antibody or an adrenomedullin antibody fragment or ADM non-1g scaffold ing to any of the claims 1 to 16 to be used in combination with vasopressors e.g. catecholamine. 18. Adrenomeduliin 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 dy or an adrenomedullin antibody fragment or ADM non-lg scaffold according to any of the claims 1 to 18 to be used in combination 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 n said dy or fragment may be administered 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 adrenomedullin 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 adrenomeduliin antibody fragment or non-Ig-scaffold according to claim 22 n said disease is sepsis.

Further embodiments within the scepe of the present invention are set out below: Adrenomedullin ADM antibody or an adrenomedullin antibody fragment for use in 2O therapy of a severe chronical or acute disease of a patient for the reduction of the mortality risk for said patient.

ADM dy or an medullin antibody fragment ing to claim 1 wherein the antibody format is selected from the group sing Fv fragment, scFv fragment, Fab fragment, scFab fragment, (Fab)2 fragment and ScFv—Fc Fusion protein.

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

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 adrenomedullin.

ADM antibody or an adrenornedullin antibody fragment according to any of claims 1 to 4, wherein said antibody or fragment is an ADM izing antibody or fragment that enhances the ti /2 half retention time of adrenornednllin in serum, blood, plasma at least %, preferably at least, 50 %, more ably > 50 %, most preferably > 100 %.

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

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 6 wherein said disease is Selected from the group comprising sepsis, diabetis, , heart e, 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 t.

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 8 n the mortality risk is reduced by preventing adverse event wherein the latter are selected from the group comprising SIRS, sepsis, septic shock, organ failure, kidney failure, liquid dysbalance and low blood pressure.

. ADM antibody or an adrenomedullin antibody fragment for use in therapy of a chronical or acute disease of a t according to any of ciairns l to 8 wherein said antibody or fragment is to be used in combination ofADM binding protein. 11. ceutical formulation comprising an dy or fragment according to any of claims 1 to 10. 12. Pharmaceutical formulation according to claim 11 wherein said pharmaceutical formulation is a solution, preferably a ready-to-use solution. l3. ceutical formulation ing to claim 11 wherein said pharmaceutical formulation is in a freeze-dried state. 14. Pharmaceutical ation 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 stered Via infusion.

Further embodiments within the scope of the present invention are set out below: . Adrenomedullin (ADM) dy or an adrenornedullin antibody nt or ADM non— 2O Ig ld for use in therapy of a severe chronical or acute disease or acute condition of a patient for the reduction of the mortality risk for said patient wherein said antibody or fragment or scaffold is a non-neutralizing ADM antibody or a non—neutralizing adrenomedullin antibody nt or a non-neutralizing ADM non—1g scaffold.

. ADM antibody or an adrenomedullin antibody 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.

ADM antibody or an adrenomedullin antibody fragment or an ADM non—lg ld ing claim 1 or 2 wherein said antibody or fragment or scaffold binds to the N- terminal part (aa 1—21) of adrenomedullin. 4. ADM antibody or an medullin antibody fragment or an ADM non~Ig scaffold ing to any of claims 1 to 3, wherein said antibody or fragment or scaffold recognizes and binds to the N-terminal end (aal) of adrenomedullin. 5. ADM antibody or an adrenomedullin 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 adrenomedullin in serum, blood, plasma at least 10 %, preferably at least, 50 %, more preferably > 50 %, most preferably > 100 0/0. 6. ADM antibody or an adrenomedullin antibody fragment or an ADM non-lg scaffold according to any of claims 1 to 5, wherein said antibody or nt or scaffold blocks the bioactivity ofADM to less than 80 %, preferably less than 50%. 7. ADM dy or an adrenomedullin antibody fragment or an ADM non—lg ld for use in therapy of a chronical or acute disease of a patient according to any of claims 1 to 6 wherein said disease is selected from the group comprising severe infections as eg. meningitis, Systemic inflammatory Response—Syndrom (SIRS,) sepsis; other es as diabetis, cancer, acute and chronic ar diseases as rag. heart failure, myocardial infarction, stroke, atherosclerosis; shock as eg. septic shock and organ dysfunction as e. g. kidney dysfunction, liver dysfunction; gs, surgery, traumata. 8. ADM antibody or an adrenomedullin antibody fragment or an ADM nonmlg scaffold for use in y of a chronical or acute disease of a patient ing to any of claims 1 to 7 wherein said disease is selected from the group comprising SIRS, a severe infection, sepsis, shock e.gseptic shock . 9. 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 ion of a patient 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 t according to any of claims 1 to 9 wherein the mortality risk is reduced by preventing an adverse event wherein the latter 2012/072933 are selected from the group comprising SIRS, sepsis, Shock as eg. septic shock, acute and chronic vascular diseases as e.g. acute heart failure, myocardial infarction, stroke; organ failure as e. g, kidney failure, liver failure, fluid dysbalance and low blood pressure.

. ADM antibody or an adrenornedullin antibody nt 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 dy fragment thereof wherein 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 ces SEQ ID NO:4 2O QSIVYSNGNTY SEQ ID NO: 5 SEQ ID NO: 6 FQGSHIPYT.

WO 72514 12. A human monoclonal antibody or fitagment that binds to ADM or an antibody fragment thereof according to claim 10 wherein said antibody or fragment comprises a sequence selected fiom the group comprising : SEQ ID NO: 7 (AM—VH—C) QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPG SGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYW TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEP KHHHHHH SEQ ID NO: 8 (AM—VH1) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRIL? GSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKHHHHHH SEQ ID NO: 9 (AM—VH2-E40) SGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRILP GSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV SEQ ID NO: 10 (AM-VH3~T26—E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEILP GSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKHHHHHH SEQ ID NO: 11 (AM-VH4—T26—E40-E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEILP GSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDY WGQGTTV’I‘VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS VHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKHHHHHH SEQ ID NO: 12 (AM—VL-C) DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYRV SNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 13 (AM—VLl) DVVMTQSPLSLPVTLGQPASiSCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYRV SNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ in NO: 14 (AM—VLZ-E40) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRV SNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIK RTVAAPSVFIFPPSDBQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 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 ing to any of the claims 1 to 12 to be used in combination with vasopressors ag. catecholamine and/ or fluids administered intravenously. 14. ADM antibody or adrencmedullin antibody fragment or ADM non—IG scaffold for use in y of a chronic or acute disease of a patient ing to any of the claims 1 to 13 or a combination according to claim 10 to be used in combination with ADM g 3O protein and/or further active ingredients. 2012/072933 . Pharmaceutical formulation comprising an antibody or fragment or scaffold ing to any of claims 1 to 14. 16. Pharmaceutical formulation according to claim 15 wherein said pharmaceutical ation is a solution, preferably a to-use solution. 17. Pharmaceutical formulation according 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 pharmaceutical formulation is administered ultra-muscular. 19. Pharmaceutical formulation according to any of claims 15 to 16, wherein said pharmaceutical formulation is administered intra—Vascular.

. Pharmaceutical 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 ld, 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 scaffold recognizes and binds to the N-terminal end (aa 1) of Adrenomedullin.

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

ADM dy or an adrenomedullin antibody 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 ing to claim I 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.

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

ADM antibody or an medullin 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 adrenornedullin.

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 antibody or fragment that enhances the t1/2 half retention time of adrenornedullin in serum, blood, plasma at least 10 “/0, preferably at least 50 %, more preferably >50 %, most ably >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 0/0, preferably less than 50%.

ADM antibody or an medullin antibody 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 sepsis, diabetis, cancer, heart failure, and shock.

ADM antibody or an adrenoniedullin dy fragment for use in y of a chronical or acute disease of a patient according to any of claims 1 to 8 wherein said patient is an ICU patient. 10. ADM antibody or an medullin antibody nt for use in therapy of a chronical 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 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%. 11. Pharmaceutical formulation comprising an dy or fragment according to any of claims lto 10. 12. Pharmaceutical ation according to claim 11 wherein said ceutical formulation is a solution, preferably a ready—to~use solution. 13. Pharmaceutical formulation according to claim ll n said pharmaceutical formulation is in a freeze-dried state.

I4. Pharmaceutical formulation according to any of claims 11 to 12, wherein said pharmaceutical formulation is stered intramuscular.

. 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 infusion.

Further embodiments within the scope of the present invention are set out below: 1. Adrenomedullin (ADM) dy or an adrenomedullin antibody fragment or ADM non—lg scaffold for use in therapy of a chronical or acute disease or acute condition of a patient for prevention or reduction of organ dysfunction or prevention of organ e in said patient.

ADM antibody or an adrenomedullin antibody fragment or ADM non—lg scaffold for use in therapy of a chronical or acute disease or acute disease according to claim 1 wherein said organ is kidney or liver.

ADM antibody or an adrenomedullin dy fragment or ADM non—1G scaffold according to claim 1 or 2 wherein said ADM antibody or an adrenomedullin antibody fragment or ADM non—1G scaffold is a nonnneutralizing ADM antibody or a non~neutra1izing adrenomedullin dy fragment or a non-neutralizing ADM nonulG scaffold ADM antibody or an adrenomedullin dy fragment or ADM non-1G ld according to any of claims 1 or 3 wherein the antibody format is selected from the group comprising Fv fragment, scFv fragment, Fab fragment, scFab fragment, (Fab)2 nt and scFV~Fc Fusion protein.

ADM dy or an adrenornedullin antibody fragment or ADM non—1G scaffold according any of claims 1 to 4 n said dy or fragment or scaffold binds to the N-terminal part (aa 1—21) of adrenomeduliin .

ADM antibody or an adrenornedullin antibody fragment or ADM non—IG ld 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 adrenornedullin.

ADM antibody or an adrenomedullin antibody fragment or ADM nonmIG scaffold according to any of claims 1 to 6, wherein said antibody or said fragment or scaffold is an ADM stabilizing antibody or fragment or scaffold that enhances the half life (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 nt or ADM non-IG scaffold according to any of claims 1 to 7, wherein said antibody or fragment or scaffold blocks the bioactivity ofADM to less than 80 %, preferably less than 50%.

ADM antibody or an adrenomedullin antibody fragment or ADM non—1G scaffold 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 e is selected from the group comprising sepsis, diabetis, cancer, heart failure, and shock.

. ADM antibody or an adrenomedullin antibody fragment according to any of claims 1 to 9, n said antibody or fragment is a human monoclonal antibody or fragment that binds to ADM or an antibody fragment fwherein the heavy chain comprises the sequences SEQ ID NO: l GYTFSRYW SEQ in NO: 2 ILPGSGST SEQ ID NO: 3 TEGYEYDGFDY and wherein the light chain comprises the sequences SEQ ID N014 QSIVYSNGNTY SEQ ID NO: 5 SEQ ID NO: 6 FQGSHIPYT.

A human monoclonal antibody or fragment that binds to ADM or an antibody fragment f according to claim 10 wherein said dy or fragment comprises a sequence selected from the group comprising: SEQ ID NO: 7 (AM—VH—C) QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPG SGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYW GQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEP KHHHHHH SEQ ID NO: 8 (AM-VH1) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILP YAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS W0 2013/072514 VHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKHHHHHH SEQ ID NO: 9 (AM-VH2-E40) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRILP GSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKHHHHHH SEQ ID NO: 10 (AMmVH3—T26—E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEILP GSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSBDTAVYYCTEGYEYDGFDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKHHHHHH SEQ ID NO: 11 (AM—VH4—T26—E40-E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEILP GSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKHHHHHH SEQ ID NO: 12 (AMA/LC) TPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYRV SNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKLEIK SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 13 (AM—VLI) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYRV SNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIK WO 72514 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 14 (AM-VLZ-E40) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRV VPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 12. ADM antibody or an adrenomedullin 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 ll wherein said antibody or fragment or scaffold is a modulating antibody or fragment or scaffold that enhances the half life ( tl/Z half retention time) of adrencmedullin 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%. 13. ADM dy or an adrencmedullin antibody fragment or ADM non—1G scaffold for use in therapy of a chronic or acute disease or acute condition of a patient according 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 nt or ADM non—1G scaffold for use in therapy of a chronic or acute disease or acute condition of a patient according to any of the claims 1 to 13 or a combination ing to claim 13 to be used in combination with ADM binding protein and/or r active ingredients.

. Pharmaceutical formulation comprising an antibody or fragment according to any of claims 1 to 13. 16. Pharmaceutical formulation according to claim 14 wherein said ceutical formulation is a solution, preferably a ready—to-use solution. 17. ceutical formulation according to claim 14 wherein said ceutical fonnulation is in a freeze—dried state. 18. Pharmaceutical formulation according to any of claims 14 to 15, wherein said pharmaceutical formulation is stered intramuscular. 19. Pharmaceutical formulation according to any of claims 14 to 15, wherein said ceutical formulation is administered intra—vascular.

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

WO 72514 It should be emphasized that the antibodies, 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-lg scaffolds.

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

Peptides:l conjugates for Immunization: es for immunization were synthesized, see Table 1, (JPT Technologies, Berlin, y) 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 (Perbio—science, Bonn, Germany). The ng procedure was performed according to the manual of Perbio.

The murine antibodies were generated according to the following method: A Balb/c mouse was zed with lOOug Peptide-BSA—Conjugate at day 0 and 14 (emulsified in lOOul complete Freund’s adjuvant) and Seug at day 21 and 28 (in lOOul incomplete Freund’s adjuvant). Three days before the fusion experiment was performed, the animal received SOng of the ate dissolved in lOOul saline, given as one intraperitoneal and one intravenous inj ection.

Spenocytes from the immunized mouse and cells of the myeloma cell line SPZ/O were qued with lml 50% polyethylene glycol for 305 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 HAT-Supplement]. After two weeks the HAT medium is replaced with HT Medium for three es followed by returning to the normal cell e medium. 2012/072933 The cell culture supernatants were primary screened for antigen specific IgG antibodies three weeks after . The positive tested microcultures were transferred into 24~weli plates for propagation. Afier retesting, the selected cultures were cloned and recloned using the limiting- dilution technique and the isotypes were determined. (see also Lane, RD. “A short—duration polyethylene glycol fusion technique for sing production of monoclonal antibody—secreting hybridomas”, J. Immunol. Meth. 81: 223228; (1985), Ziegler, B. at at. “Glutamate decarboxylase (GAD) is not detectable on the surface of rat islet cells examined by cytofluorometry and complementwdependent antibody—mediated cytotoxicity of monoclonal GAD antibodies”, Horm. Metab. Res- 28: 11—15, (1996)).

Mouse monoclonal antibody production: Antibodies were ed via standard antibody production s (Marx er a1, Monoclonal dy Production, ATLA 25, 121, 1997,) and d via Protein A. The antibody purities were > 95% based on SDS gel ophoresis analysis.

Human Antibodies Human Antibodies were produced by means of phage y according to the following procedure: The human naive antibody gene libraries HAL7/8 were used for the isolation of recombinant single chain F—Variable domains (scFv) against adrenomedullin peptide. The antibody gene libraries were screened with a panning strategy comprising the use of peptides containing a biotin tag linked via two ent spacers to the adrenomedullin peptide sequence, A mix of panning rounds using non-specifically 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 expressing E.coli strains.

Supeinatant 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, 1—1., zai, A., Kirsch, Ml, Schfitte, M., Helmsing, 8., Meier, D., Schirrrnann, T., Dfibel, S., 2011. A human scFv antibody generation pipeline for proteome research. Journal of Biotechnology 152, 159— 170; Schiitte, M., Thullier, P., Pelat, T., Wezler, X., Rosenstock, P., Hinz, D., Kirsch, M.i.,Hasenberg, M., Frank, R., Schirnnann, T., Gunzer, M., Hust, M., Diibel, 8., 2009.

Identification of a ve Crf splice variant and generation of recombinant antibodies for the specific detection of Aspergillus tus. PLoS One 4, e6625).

WO 72514 Positive clones have been selected based on positive ELISA signal for antigen and negative for streptavidin coated micro titer . For further characterizations the scFv open reading frame has been cloned into the expression plasmid pOPE107 (Hust er al., J. Biotechn. 2011), captured from the culture supernatant via lised metal ion affinity chromatography and purified by a size ion chromatography.

Affinity nts To determine the affinity of the antibodies to Adrencmedullin, the kinetics ofbinding of Adrenomedullin to immobilized antibody was determined by means of free surface plasmon nce using a Biacore 2000 system (GE Healthcare Europe GmbH, Freiburg, Germany). Reversible immobilization of the antibodies was med using an antinmouse Fe antibody covalently coupled in high density to a CMS sensor surface according to the manufacturer's instructions (mouse antibody capture kit; GE Healthcare). (Lorenz er al.,“ Functional Antibodies Targeting IsaA of Staphylococcus aurens 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 ofhuman 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/Immunegen ADM Designation Affinity Number Region nts Kd (M) SEQ ID: 15 YRQSMNNFQGLRSFGCRFGTC 1-21 NT—H SEQ ID: 16 CTVQKLAHQIYQ MR—H 2 x10" SEQ ID: 17 CAPRSKISPQGY—NH2 042—52 CT—H 1.1 x10- SEQ ID: is YRQSMNQGSRSNGCRFGTC NT-M 3.9 x10" SEQ 1D:19 CTFQKLAHQIYQ 19—31 MR—M 4.5x10" 'SEQIDr20 CAPRNKISPQGY—NH2 (3—40—50 cr—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 (%) (see example 2) .82110 45 Mouse ADM/364 2.2x10'8 48 Mouse 5 3.0x10’ - --—-— Mouse ADM/367 1.3x10‘ - Mouse 9 2.0 x10' ADM/370 Mouse ADM/371 2.0 x10' - ADM/372 - ADM/373 -Mouse ADM/377 1.5 x10" -Mouse ADM/378 2.2 x10' -Mouse ADM/379 1.6 x10" ——-— -—-_ Mouse ADM/397 1.5X10' E7:: 68 E7.“E Mouse ADM/39 5.9 x10" 72 W0 2013/072514 2012/072933 CT—M Mouse ADM/65 CT-M Mouse ADM/66 -—--— -——-—— ADM/15 <1x10' CT-H Mouse ADM/18 hA Phage display ADM/A7 -Phage display ADM/B7 <1x10' -Phage y <1X10" Phage display ADM/D1]; Phage display 2 Generation of antibod fra entsb enz atic di : The generation of Fab and F(ab)2 fragments was done by enzymatic digestion of the murine full length antibody NT—M. Antibody NT-M was digested using a) the pepsin—based F(ab)2 Preparation Kit (Pierce 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—fragrnentation for 8h at 37°C. The Fab-fiagmentation digestion was carried out for 16h, respectively.

Procedure for Fab Generation and Purification: 2012/072933 The lized 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 e solution and washing it with digestion , centrifiiging it each time afterwards at 1000 x g for 2 minutes. 0.5ml of the prepared lgG sample where added to the spin column tube containing the equilibrated Immobilized Papain.

Incubation time of the digestion reaction was done for 16h on a tabletop rocker at 37°C. The column was fuged at 5000 X g for 1 minute to separate digest from the Immobilized . Afterwards the resin was washed with 0.5m1 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 1.0ml. The NAb Protein A Column was equilibrated with PBS and IgG Elation Buffer at room temperature. The column was fuged for 1 minute to remove storage solution (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: Coulter, A. and Harris, R. (1983). J. Immunol. Meth. 59, 199—203.; Lindner I. 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 at. (2010) Neutralization of West Nile Virus by cross-linking of its surface proteins with Fab fragments of the human monoclonal antibody CR4354. PNAS. 107, 189506.; Chen X. at of. (2010) Requirement of open headpiece conformation for activation of yte integrin (1)432. PNAS. 107, 32; Uysal H. at al. (2009) Structure and pathogenicity of antibodies Specific for citruilinated collagen type II in experimental arthitis. J. Exp. Med. 206, 449—62.; Thomas G. M. er a2. (2009) Cancer cell—derived microparticles bearing i3~selectin glycoprotein ligand 1 rate thrombus formation in Vivo. J. Exp. Med. 206, 1913-27.; Kong F. at a]. (2009) tration of catch bonds between an integrin and its ligand. J. Cell Biol. 185, 4.) Procedure for generation and purification of Ffab'); Fragments: The immobilized Pepsin was equilibrated by washing the resin with 0.5 ml of Digestion Buffer and fuging 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. 0.5ml of the prepared IgG sample where added to the spin column tube containing the cquilibrated Immobilized Pepsin.

Incubation time of the digestion reaction was done for 1611 on a tabletop rocker at 37°C. The column was fuged 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 fraction was added to the digested dy that the total sample volume was 1.0ml. The NAb Protein A Column was equilibrated with PBS and IgG Elution Buffer at room temperature. The column was centrifuged for 1 minute to remove e solution (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: i, M., et al. (1991). A new enzymatic method to obtain high-yield F(ab’)2 suitable for clinical use from mouse IgGl. Moliinniunol. 28: 69~77.;Beale, D. (1987). Molecular ntation: Some applications in immunology. Exp Comp Immunol 11:287—96.; Ellerson, J.R., et a1. (1972). A fragment ponding to the CH2 region of immunoglobulin G (IgG) with complement fixing activity. FEBS Letters 24(3):318—22.; Kerbel, RS. and Elliot, BB. (1983).

Detection of Fc receptors. Meth Enzymol 93:113-147.; Kulkami, P.N., et al. (1985). Conjugation of methotrexate to IgG dies and their F(ab')2 fragments and the effect of conjugated methotrexate on tumor growth in vivo. Cancer Immunol Immunotherapy -4.; Lamoyi, E. (1986). Preparation of F(ab')2 Fragments from mouse IgG of various subclasses. Meth Enzymol 121:652—663.; Parharn, P., at al. (1982). Monoclonal antibodies: purification, fragmentation and application to structural and functional studies of class I MHC ns. J Immunol Meth 531133—73; Raychaudhuri, G., er a1. (1985). Human lgGl and its Fe fragment bind with different affinities to the Fc receptors on the human U937, HL-60 and ML—l cell lines. M01 lmmunol 1009—19.; Rousseaux, J., et at. (1980). The differential enzyme sensitivity of rat globulin G subclasses to papain an . Mol Immunol 17:469-82.; Rousseaux, J., et 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 64:141-6.; Wilson, K.M., er al. (1991).

Rapid whole blood assay for HIV—1 seropositivity using an Fab—peptide conjugate. J Immunol Meth 138:111-9.) WO 72514 NT—H~Antihody Fragment Humanization The antibody fragment was humanized by the afting method (Jones, P. T., Dear, P. H., Foote, 1., Neuberger, M. S., and Winter, G. (1986) Replacing the complementaritywdetermining regions in a human antibody with those from a mouse. Nature 321, 522—525).

The following steps where done to e the humanized seguence: Total RNA tion: Total RNA was extracted from NT—H hybridomas using the Qiagen kit.

First-round RT—PCR: QIAGEN® p RT—PCR 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 ll light chain RT-PCR reactions were set up using degenerate forward primer mixtures covering the leader sequences of variable regions. Reverse primers are located in the constant regions of heavy and light chains. No restriction sites were ered into the s.

Reaction Setup: 5): QIAGEN® OneStep RT—PCR Buffer 5.0 ul, dNTP Mix (containing 10 mM of each dNTP) 0.8 01, Primer set 0.5 in, QIAGEN® OneStep RT—PCR Enzyme Mix 0.8 01, Template RNA 2.0 01, RNase—free water to 20.0 pl, Total volume 20.0 pi PCR condition: Reverse transcription: 50°C, 30 min; Initial PCR activation: 95°C, 15 min Cycling: 20 cycles of 94°C, 25 sec; 54°C, 30 sec; 72°C, 30 sec; Final extension: 72°C, 10 min —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 1] light chain RT- PCR reactions were set up using semi-nested primer sets specific for antibody variable s.

Reaction Setup: 2}: PCR mix 10 n1; Primer set 2 pl; First—round PCR product 8 at; Total volume pl; Hybridoma Antibody 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. 2012/072933 After PCR is finished, run PCR reaction samples onto agarose gel to ize DNA fragments amplifiedAfier sequencing more than 15 cloned DNA fragments amplified by nested , 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 ent with homologous human framework sequences the resulting humanized sequence for the variable 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 ed to the murine al. The resulting candidates are depicted below) (Padlan, E. A. (1991) A possible procedure for reducing the immunogenicity of antibody variable domains while preserving their —binding properties. Mol. Immunol. 28, 489—498.; Harris, L. and Bajorath, J. (1995) Profiles for the analysis of immunoglobulin sequences: COmparison ofV gene subgroups. Protein Sci. 4, 306% 10.).

Annotation for the antibody fragment sequences (SEQ ID NO: 7—14): bold and underline are the CDR 1, 2, 3 in chronologically arranged; italic are constant s; hinge regions are highlighted with bold letters and the histidine tag with bold and italic letters; framework point mutation have a grey letter-background.

SEQ 11) NO: 7 (AM—VH—C) QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGSGST NYNEKFKGKATITADTSSNTAYMQLSSLTSEDSA VYYCTEGYEYDGFDYWGQGTTLTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPA LYSLS SVVTVPSSSLGTQTYICNWHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 8 (AM—VH1) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILPGSGS INYAQKFQGRVTITADESTSTAYMELSSLRSEDTAWYCTEGYEYDGFDYWGQGTTVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWSGALTSGVHTFPA VLQSSGLYSL SSVVTVPSSSLGTQWICNVNHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 9 (AMNHZ-EélO) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEZWVRQAPGQGLEWMGRILPGSGS INYAQKFQGRVTITADESTSTAYMELSSLRSEDTA VYYCTEGYEYDGFDYWGQGTTVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSL SSVVTVPSSSLGTQTY1CNWHKPSNTKVDKRVEPKHHHHHH SEQ ED NO: 10 (AM-VH3~T26—ESS) QVQLVQSGAEVKKPGSSVKVSC gisvrrsnymSWVRQAPGQGLEWMG INYAQKFQGRVTITADESTSTAYMELSSLRSEDTA VYYCTEGYEYDGFDYWGQGTTVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA LYSL SSVVTVPSSSLGTQTYICNWHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 11 (AMMVH4—T26~E4O—E55) QVQLVQSGAEVKKPGSSVKVSCKAEéGYTFSRYW 3WVRQAPGQGLEWMG§§1LPoses INYAQKFQGRVTITADESTSTA YMELSSLRSEDTA WCTEGYEYDGFDYWGQGTTVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 12 (AM—VL—C) DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYBLSNRF I0 SGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQBSVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 13 (AM—VLI) SPLSLPVTLGQPASISCRSSgQSIVYSNGNTYLNWFQQRPGQSPRRLIYMNRD SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCF!QGSHIPYTFGQGTKLEIKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 14 (AM-VLZ-E40) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYlgngRD SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Example 2 Effect of selected anfi-ADM-anfibodies on anti-ADM-bioactivity The effect of selected ADM—antibodies on ADM—bioactivity was tested in an human inant Adrenomedullin receptor CAMP functional assay (Adrenomedullin Bioassay).

Testing of dies targeting human or mouse adrcnomedullin in human recombinant Adrenomedullin receptor cAMP functional assay omedullin Bioassay) Materials: Cell line: CHO—Kl 2012/072933 Receptor: Adrenomedullin (CRLR + RAMP3) Receptor Accession Number Cell line: CRLR: U17473; RAMP3: AJ001016 CHO-Kl cells expressing human recombinant 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 KC], 1.25 mM MgSO4, 124 mM NaCl, 25 mM HEPES, 13.3 mM Glucose, 1.25 111M KH2PO4, 1.45 mM CaCl2, 0.5 g/l BSA).

Dose response curves were performed in parallel with the reference agonists (hADM or mADM). nist test (96well): For antagonist testing, 6 ul of the reference agonist (human (5,63nM) or mouse (0,67nM) adrenomedullin) was mixed with 6 pl of the test samples at different nist dilutions; or with 6 u] buffer. After incubation for 60 min at room temperature, 12 ul of cells (2,500 well) were added. The plates were incubated for 30 min at room temperature. 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 nce antagonist.

Antibodies testing cAMP-HTRF assay The anti—h—ADM antibodies (NT—H, MR—H, CT-H) were tested for antagonist activity in human recombinant adrenornedullin or (FAST—027C) CAMP functional assay in the presence of .63nM Human ADM 1—52, at the following final antibody concentrations: lOOug/ml, 20ug/ml, drug/n11, m1,0.16ng/ml.

The anti—ni—ADM antibodies (NT-M, MR-M, CT-M) were tested for antagonist activity in human recombinant adrenomedullin receptor 027C) CAMP functional assay in the presence of 0.67nM Mouse ADM 1—50, at the following final dy concentrations: lOOug/ml, 20pg/rnl, ding/ml, 0.8ug/rnl, 0.16pg/1nl. 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 l inhibition ofADM bioactivity (ADM—Bioassay) (%) NT—H 3 8 Non specific mouse IgG 0 Example 3 Data for stabilization of hADM by the anti-ADM antibody The stabilizing effect of human ADM by human ADM dies 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): IOOng (100ul) CT-H (lmg/ ml in PBS, pH 7.4, AdrenoMed AGGermany) was mixed with 10u1 Acridinium NHS~ester (1mg! ml in acetonitrile, InVent Gmbl-l, y) (EP 0353971) and incubated for 20min at room temperature. Labelled CT—l—l was purified by tration HPLC on Bio—Sil® SEC 400—5 (BionRad tories, 1110., USA) The purified CTmH was diluted in (300 mmol/L potassiumphosphate, 100 mmcl/L NaCl, 10 mmol/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. 20mg labeled antibody) per 200 nL. Acridiniumester chemiluminescence was measured by using an AutoLurnat LB 953 (Berthold Technologies GmbI-l & Co. KG).

Solid phase: ?oiystyrene tubes er Bio—One International AG, Austria) were coated (18h at room ature) with MR-H (AdrenoMed AG, Germany) (15 pg MR—H/0.3 mL 100 mmol/L NaCl, 50 mmol/L TRIS/HG}, pH 7.8). After blocking with 5% bovine serum albumine, the tubes were washed with PBS, pH 7.4 and vacuum dried.

Calibration: The assay was calibrated, using dilutions ofhADM M AG, Switzerland) in 250 mmol/L NaCl, 2 g/L Triton X400, 50 g/L Bovine Serum Albumin, 20 tabs/L Protease Inhibitor Cocktail (Roche Diagnostics AG, Switzerland)) hADM Immunoassay: 50 p1 of sample (or calibrator) was pipetted into coated tubes, after adding labeleid CT—H (200M), 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 % Triton X-lOO).

Tube-bound chemiluminescence was measured 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 diluted in human Citrate plasma (final concentration 1011M) and incubated at 24 °C. At selected time points, the degradation of hADM was d by freezing at —20 0C. The incubation was performed in absence and presence of NT—H (100ug/ml). The remaining hADM was quantified by using the hADM immunoassay bed above.

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

Example 4 Se sis i earl treatment Animal model 12—15 week old male C57Bl/6 mice (Charles River Laboratories, Germany) were used for the study. Peritonitis had been surgically induced under light isofluran anesthesia. Incisions 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 ion of the small bowel. One puncture wound was made with a 24-gauge needle into the cecum and small amounts of cecal contents were sed through the wound. The cecum was replaced into the peritoneal cavity and the laparotomy site was closed. Finally, animals were returned to their cages with free access to food and water. 500a} saline were given so. as fluid replacement.

Application and dosage of the compound (NT—M, MR—M, CT—M) Mice were treated immediately after CLP (early treatment). CLP is the abbreviation for cecal on 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 (10 ul/ g bodyweight) dose/ FollownUp: l NT—M, 0.2 mg/ml survival over 4 days 2 MR-M, 0.2 mg/ml al over 4 days 3 CT-M, 0.2 mg/ml survival over 4 days 4 nonmspecific mouse IgG, 0.2 mg/ml survival over 4 days control — PBS lOul/g bodyweight survival over 4 days Clinical chemistry Blood urea nitrogen (BUN) trations for renal function were measured baseline and day I after CLP. Blood samples were obtained from the cavernous sinus with a capillary under light ether anaesthesia. 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 (%) non—specific mouse IgG 0 ‘ CT—M MR—M NT—M Table 5: Average from 5 animals BUN pre CL? (mM) BUN day 1 (mM) It can be seen from Table 4 that the NT—M antibody d mortality considerably. After 4 days 70 % of the mice survived when treated with NT—M antibody. When treated with MR-M dy 30 “/0 of the animals ed and when treated with CTnM antibody 10 % of the animals survived after 4 days. In contrast thereto all mice were dead after 4 days when treated with unspecific mouse lgG. The same result was obtained in the control group where PBS (phosphate buffered saline) was administered to mice.

The blood urea nitrogen or BUN test is used to te 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.

WO 72514 Sepsis Mortality (late ent) Animal model 12—15 week old male C57Bl/6 mice (Charles River Laboratories, Germany) were used for the study. Peritonitis had been surgically induced under light isofluran esia. Incisions 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 puncture wound was made with a 24—gauge needle into the cecum and small amounts of cecal ts were expressed through the wound. The cecum was replaced into the peritoneal cavity and the tomy site was closed. Finally, animals were returned to their cages with free access to food and water. SOOul 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 compound treatment. Treatment was performed after full development of , 6 hours after 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 : nonspecific mouse IgG, 0.2 mg/ml survival over 4 days 3 vehicle: ~ PBS lOul/g bodyweight survival over 4 days Table 6: 4 day mortality survival (%) Non—specific mouse IgG NT—M FAB2 It can be seen from Table 6 that the NT—M FAB 2 antibody reduced mortality considerably. After 4 days 75 % of the mice survived when treated with NT—M FAB 2 antibody. In contrast o all mice were dead after 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 catecholamines as well as regulation of fluid balance.

Animal model In this study male C57Bl/6 mice (8—12 weeks, 22n30g) were utilized. A polymicrobial sepsis induced by cecal ligation and puncture (CLP) was used as the model for studying septic shock ((Albuszies G, at al: Effect of increased cardiac output on c and intestinal microcirculatory blood flow, oxygenation, and metabolism in hyperdynamic marine septic shock. Crit Care Med 2005;33:2332—8), (Albuszies G, et al'. The effect of iNOS on on hepatic gluconeogenesis in hyperdynamic marine septic shock. Intensive Care Med 2007;33:1094—101), (Barth E, at a]: Role of iNOS in the reduced responsiveness of the myocardium to catecholamines in a ynamic, murine model of septic shock. Crit Care Med 2006;34:307—13), art K, et al: Effect of SOD—l over—expression on dial function during resuscitated murine septic shock.

Intensive Care Med 2009;333:3449), (Baumgart K, et (.11: Cardiac and metabolic effects of ermia and inhaled H28 in anesthetized and ventilated mice. Crit Care Med 2010;38:588-95), (Simkova V, et al: The effect of SOD—l overnexpression on hepatic gluconeogenesis and whole—body glucose oxidation during resuscitated, ensive murine septic shock. Shock 2008;30:578—84), (Wagner F, et all: Inflammatory effects of hypothermia and inhaled H28 during resuscitated, hyperdynamic marine septic shock. Shock, im Druck), (Wagner F, er al: Effects of enous H28 after marine blunt chest trauma: a prospective, ized lled trial. Crit Care 201i, submittes for publication)).

After weighing, mice were anesthetized by intraperitoneal injection of 120 gig/g Ketamin, 1.25 pg/g Midazolam and 0.25 ug/g Fentanyl. During the surgical procedure, body temperature was kept at 37-38°C. A lcm midline abdominal section was med 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 18~ and the on was closed again (4~O tie). gauge needle was applied. The cecum was returned For the compensation of perioperative loss of liquids, 0.5 ml lacted Ringer’s solution with lug/g Buprenorphin as analgetic was injected subcutaneously in dorsal dermis. For antibiosis the mice received Ceftriaxon 30ug/g and mycin 30ug/g subcutaneously via the lower extremities.

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

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 orphin lug/g, which were applied in an 8 hour cycle, after shert term esia by isofluran. In addition, antibiosis was maintained by subcutaneous injections of xon 30ug/g and Clindamycin 30ug/g via the lower extremities.

Dosing of test substances Early treatment Immediately after the CLP surgery and closing of the incision, the test substance antibody NT—M was applied in a concentration of 500 rig/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 pl) (5 animals).

Late treatment After full Sepsis development, 15.511 after CLP surgery, animals were anesthetized as described above and NT—M was d 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 pl) (3 s).

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

Study groups and experimental g 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 alter 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 interventions comparable to an intensive medical care regime. ore, after weighing the s 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°C for the rest of the experiment. After a tracheotomy and intubation, respiration was monitored and supported by laboratory animal lung ventilator Flexivent®, (Emka Technologies, FiOZ 0,5, PEEP lO H20, VT Sal/g, IzE 121,5, AF 70—140 depending 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 carotis communis was cannulated for continuous monitoring of heart rate and the mean arterial re (MAP). The mean arterial re was ined at MAP > 65 mmHg via intravenous (V. jugularis) infusion of colloids (80 uL/gxh, Hextend®) and, if needed, Noradrenalin dissolved in colloids as vasopressor, Blood samples (120 pl) were taken via the cannulated A. carotis at 0 and 4 hours for determination of creatinine. The r was punctured and urine was collected via a r 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 dosing.

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

Table 7: Total consumption of consumption rate of Noradrenalin (ug NA/g) Noradrenalin (pg NA/g/h) (Average) (Average) Control (mouse igG) (N=6) 0.032 ug/h/g NT-M (N25) early treatment 0.07 lug/g 0.012ug/h/g Relative change (early treatment, 59% 62.5% amelioration) (59%) (62.5%) NT—M (N33) late treatment 0.04 ug/g 0.0075 ug/h/g Relative change (late ent, 76,5% 76,5% amelioration) ) (76.5%) The catecholamine requirement was measured after administration of either non specific mouse IgG to a total of 6 mice as control group, NT—murine antibody to a group of 5 mice immediately afier CLP (early treatment) or NT—murine antibody to a group of 3 mice 15.5h after CLP (late treatment).

The ion 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 antibody, 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. 7).

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 balance is of utmost importance for the course of disease of patients having sepsis. (s. Boyd 3: a], 201 t). Controlling the liquid balance of critical ill ts remains as a substantial challenge in intensive care medicine. As can be seen in table 8 treatment of mice after CLP (experimental procedures see “Animal Model”) with NT-M antibody lead to an ement of the total volume of urine excreted. The urine secreted was . three times higher in NT—M~treated animals ed 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 of ADM antibody, especially the use of NT ADM antibody, is favorable for regulating the fluid balance in patients. (see table 8 and figures 8 and 9).

WO 72514 Table 8 Urine average Fluid balance average volume/ g body volume/ gbody weight weight Control (mouse IgG) 0.042 ml/g (N=6) NT~M early (N=5) 0,18 ml/g Relative change early -21.7%% treatment NT-M late (N=3) 0.125 ml 0,16 ml/g Relative change late + 198% ~30,5% 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 mortality among patients with acute renal failure alone. (Schrier and Wang, “Mechanisms of e Acute Renal Failure and Sepsis”; The New England Journal of Medicine; 351:159-69; 2004). Creatinine concentration and creatinine nce are standard laboratory parameters for monitoring kidney (dys)function (Jacob, “Acute Renal Failure”, Indian J. Anaesth.; 47 (5): 361372; 2003). Creatinine and creatinine clearance data from above described animal ment (early treatment) are given in Table 9.

Table 9 Kidney function: nine mean creatinine concentration clearance (uL/min) (Hg/HID control mouse IgG (MW) 174 til/min NT-M (MW) 373 til/min Relative change —42% +1 14% (amelioration) (42%) (114%) In comparision to control septic animals, the creatinine concentration was lowered by 42% and the creatinine clearance was improved by more than 100% as a result of NT—M treatment (Table 9). The data show that the administration of ADM—antibody, especially NT—M, leads to an ement 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 oC. Activation of nuclear factor kappa-light—chain gene enhancer in B cells (NF—KB) was ined as previously described using an electrophoretic mobility shift assay (EMSA)1,2. Cell extracts (lOug) were incubated on ice with poly-doxy—inosinic—deoxy~cytidylic acid (poly—dI—dC) and 32P-1abeled double ed oligonucleotide (Biomers, Ulm, Germany) ning the NF—KB (HIV KBsite) ( 5’— GGATCCTCAACAGAGGGGACTTTCCGAGGCCAJ’). Complexes were ted in native polyacrylamide gels, dried and exposed to X—ray films. A phosphorirnager and image analyzer software (AIDA Image Analyzer; Raytest) was used to quantify the radioactively labeled NF—KB by densitometry. For comparison between individual gels, the intensity of each band was related to that of simultaneously loaded control animals 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 ise stated differences between the two groups were analyzed with the Mann—Whitney rank sum test for unpaired samples. Results: The animals d 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)) 01) (see figure 10).

Between: 1. Wagner F, Wagner K, Weber S, Stahl B, Kntiferl MW, Huber—Lang M, Seitz DH, Asfar P, Calzia E, Senftleben U, Gebhard F, Georgieff M, Radermacher P, Hysa V: Inflammatory s of hypothermia and inhaled H28 during itated, hyperdynamic rnurine septic shock. Shock 2011;35(4):396-402 WO 72514 2. Wagner F, Scheuerle A, Weber S, Stahl B, McCook O, Knoferl MW, Huber—Lang M, Seitz DH, Thomas J, Asfar P, Szabé C, Moller P, Gebhard F, GeorgieffM, Calzia E, Radermacher P, Wagner K: Cardiopulmonary, histologic, and inflammatory effects ofintravenous NaZS after blunt chest trauma-induced lung contusion in mice. J Trauma 2011;71(6)11659~67 Example 6 In vivo side effect determination of antibody NT—M 12—15 week old male C57Bl/6 mice (Charles River Laboratories, Germany) were used for the study. 6 mice were treated with (lOui/ g bodyweight) dose of NT—M, 0.2 rug/m1. As l, 6 mice were treated with (lOul/g body ) PBS. Survival and physical condition was red for 14 days. The mortality was 0 in both groups, there were no differences in physical ion between NT-M and control group.

Examgle 7 Gentamicin-induced nephrctoxicity A non-septic acute kidney injury model has been established, which makes use of the nephrotoxicity induced by Gentamicin (Chin PJS. 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 follows: Effect of a NT-M on Gentamicin-Induced Nephrotoxicity in Rats Study Design: Route mg/ml -1/kg_glkg (Male) ‘iicina - b vehlcle . . a ::X.-4 micin at 120 mg/kg intramuscularly for 7 days (days 06). bVehicle; injected intravenously (iv) 5 min before gentamicin 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. dl-"lasrna samples were collected in EDTA tubes (Days 1 and 3 before Test and Control article: 100 111; Day 7:120 n1. 24h urine collection on ice is initiated after gentamicin on Day 0, followed by Days 2 and 6; blood tion on days 1, 3, and 7.

Groups of 8 male SpragueuDawley rats weighing 250 i: 20 g were employed. Animals were challenged with gentamicin at 120 mg/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 weights and clinical signs were monitored daily. Twentynfour (24) hour urine collections on ice were performed on Days 0, 2, and 6. Urine specimens were assayed for concentrations of Na+ and K+, and nine. Blood samples for clinical chemistry were collected on Days 1 (before gentamicin), 3 (before gentamicin), and 7. Serum olytes (Na+ and Kt), creatinine, and BUN were the y analytes that were monitored for assessing renal function. Plasma samples were collected in EDTA tubes (Days I and 3:100 1.11; Day 7:120 n1). Creatinine clearance was calculated. Urine volume, urinary electrolytes, and creatinine are sed as amount excreted per 100 g of animal body weight. All animals were sacrificed on Day 7.

Kidneys were weighed.

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

Endogenous creatinine nce was calculated as follows: CCr (ml/24 h) I {UCr (mg/m1) X V (ml/24 h)] / SCr (mg/ml) 24—hr y excretion of sodium (Na+) was ated as follows: UNaV (”liq/24 h) = UNa (nEq/ml) x V (ml/24 h) 24-hr urinary excretion ofNAG and s similarly calculated. 2012/072933 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: PENa (%) E100 x [UNa (qu/ml) x V (ml/24 h)] / PNa (qu/ml) X Cc, (ml/24 h) Treatment with anti—Adrenomedullin antibody improved l measures of kidney function on day 7 as compared to vehicle: serum creatinine 1.01 nig/dL (NTuM) vs 1.55 Ing/dL (vehicle) (Fig. 11), BUN 32.08 Ing/dL(NT-M) vs. 52.41 mg/dL (vehicle) (Fig. 12), endogenous nine clearance 934,43 mL/24 h (NT-M) vs. 613.34 mL/24 h (vehicle) (Fig. 13), fractional secretion of Nat 0.98 % (NT—M) vs. 1.75 % (vehicle) (Fig. 14).

Examgle 8 In the mice CLP model described above, the effect of treatment with antiuadrenomedullin antibody NT-M on several parameters of kidney on was investigated.

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

Table 10: Parameters of kidney on in the vehicle— (11:1 1) and NTnM—treated (n29) animals.

Blood trations were measured in s taken at the end of the experiment. NGAL : neutrophil gelatinase~associated lipocalin. All data are median (quartiles).

Urineoutput[nng‘1-h"1] 4.4(3.5;16.5) 15.2(13.9;22.5) 0.033 Creatinine clearance [nL-min'l] 197 (110;301) 400 (316;509) Creatinine[ng-mL'1] 1.83 (1523.04) 1.28 (1.20;1.52) Urea [ng'mL't] 378 (268;513) 175 (101,184) 0.004 NGAL [ng-rnL'E] 16 (15;:30) 11 (10;13) 0.008 The experiments were performed as follows: Creatinine, urea, and neutrophil gelatinase—associated lipocalin WGAL) Blood NGAL concentrations were measured using a commercial ELISA (mouse NGAL, RUO 042, BioPorto Diagnostics A/S, Denmark, Gentofie). Urea and creatinine concentrations were measured with a capillary column (Optima—5M8, Macherey—Nagel, Diiren, Germany) gas chromatography/mass spectrometry system (Agilent 5890/5970, Bo‘blingen, Germany) using 2lalg—creatiriine (CDN es, Pointe-Claire, QU, Canada) and methyl—urea (FlukaChernikalien, Buchs, Switzerland) as internal rds. After deproteinization with itrile, fugation 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(trirnethylsilyl)trifluoroacetamide and N~(tert—butyldimethy1silyl)~N~ methyltrifluoroacetamide allowed formation of the urea tert—butyl-dimethylsilyl— and the creatininetrimethylsilyl-derivatives, respectively. Ions m/z 231 and 245, and m/z 329 and 332 were monitored for urea and creatinine analytes and al rds, 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 Tn's pH 7,6; 500 mM NaCl; 6 mM EDTA; 6 mM EGTA; 1 “/0 Triton~X—100; 0,5 % NP 40; 10 “/0 Glycerol; Protease—Inhibitors (f3— Glycerolphosphate 2 mM; DTT 4 mM; Leupeptine 20 MM; Natriurnorthovanadate 0,2 mM)) and subsequently centrifuged. The whole cell lysate was obtained out of the supernatant; the pellet consisting of cell ts was discarded. The amount of protein 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 ug/ul. The samples for the Multiplex— and EMSA analysis were diluted 1:1 with EMSA buffer (10 mM Hepes; 50 mM KCl; 10 % Glycerol; 0,1 mM EDTA; 1 mM DTT), the s for the immune blots 1:1 with 2~fold Sample Buffer (2 “/0 SDS; 125 mM Tris-HCL (pH 6,8 at 25°C); 10 “/0 Glycerol; 50 mM DTT; 0,01 % Bromophenol blue).

Levels of keratinocyte—derived ine (KC) concentrations were determined using a mouse multiplex ne 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, Radermacher P, Hysa V. Inflammatory effects of hypothermia and inhaled H28 during resuscitated, hyperdynamic marine septic shock.

Shock 2011;35:396—402; and Wagner F, rle A, Weber S, Stahl B, McCook O, Knoferl MW, Huber-Lang M, Seitz DH, Thomas J, Asfar P, Szabo C, Meller P, d F, GeorgieffM, Calzia E, Radermacher P, Wagner K. Cardiopulmonary, histologic, and inflammatory effects of intravenous Na2S after blunt chest trauma—induced lung contusion in mice. J Trauma 1:1659—1667). In brief, the appropriate cytokine standards and samples were added to a filter plate. The samples were incubated with antibodies ally attached to fluorescent— labeled micro beads. Thereafier, premixed detection dies were added to each well, and subsequently, streptavidin—phycoerytlnin was added. Beads were then re—suspended, and the nes reaction e was quantified using the Bio—Flex protein array reader. Data were automatically processed and analyzed by Bio-Flex Manager Software 4.1 using the standard curve produced 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 drenomedullin dy NT—M on the liver was investigated.

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

Measurement of keratinocyte-derived chemokine (KC) was done analogous to example 8 (kidney) .

Example 10 In the mice CLP model bed above, the effect of treatment with anti—adrenomedullin dy NT-M on several nes and chemokinesin the blood circulation (plasma) was investigated. ne and chemokine concentrations Plasma levels of tumor is factor u, interleukin (IL)—6, monocyte chemoattractant protein (MCPH, and keratinocyte-derived chemokine (KC) concentrations were determined using a mouse multiplex cytokine kit (Bio—Plea Pro Cytokine Assay, Bio-Rad, Hercules, CA), the assay was performed by using the Bio—pie}: suspension array system with the manufacturer’s instructions (see also Wagner F, Wagner K, Weber 8, Stahl B, l MW, Huber—Lang M, Seitz DH, Asfar P, Caizia E, Senftleben U, Gebhard F, Georgieff M, acher P, Hysa V.

Inflammatory effects of hypothermia and inhaled HZS during itated, hyperdynamic murine septic shock. Shock 2011;35:396-402; and Wagner F, Scheuerle A, Weber S, Stahl B, McCook O, Knéferl MW, Huber-Lang M, Seitz DH, Thomas J, Asfar P, Szabo C, Moller P, Gebhard F, Georgieff M, Calzia E, Radermacher P, Wagner K. Cardiopulmonary, histologic, and inflammatory effects of intravenous Na2S after blunt chest trauma-induced lung contusion in mice. J Trauma 1:1659-1667). 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. Thereafter, 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—Flex protein array reader. Data were automatically processed and analyzed by Bio—Pie): Manager Software 4.1 using the standard curve produced from recombinant cytokine standards. Levels below the detection limit of the assays were set to zero for statistical es.

Plasma levels and kidney tissue concentrations of tumor necrosis factor (TNF)—oc, interleukin (IL)—6 and IL~10, monocyte chemoattractant protein (MCP)-l, and keratinocyte—dervived chemokine (KC) were determined using a commercially available “Multiplex Cytokine Kit” (Bio-Flex Pro Precision Pro Cytokine Assay, Bio—Rad, Hercules, CA), which allows to collect l parameters out of one single sample. The individual work steps of the assay were performed according to 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, Radermacher P, Hysa V. Inflammatory effects of ermia and d H28 during resuscitated, hyperdynamic murine septic shock. Shock 201l;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, Szabé C, Mdller P, Gebhard F, Georgieff M, Calzia E, Radennacher 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 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 s. During the subsequent tion the single cytokines bind to the antibodies attached to polystyrenewbeads. After the addition of the ne-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 (Bio-Flex suspension array system, Bio-Rad, Hercules, CA). Data were automatically processed and analyzed by Bio~Plex Manager Software 4.1 using the standard curve produced from recombinant cytokine standards. For the plasma levels the concentration * mL'l, the concentration of the kidney homogenates were converted to the was provided in pg appropriate protein concentration and provided in pg *_ rng”i protein.

NT-M caused a significant lowering of plasma concentrations of IL—6 (Fig. 17), IL-lO (Fig. 18), keratinocyte—derived chemokine (KC) (Fig. 19), te chemoattractant protein-1 (MCP—l) (Fig. 20), TNF—alpha (Fig. 21). e 11 Ischemia/Reperfusion-Induced Acute Kidney Injury Another non—septic acute kidney injury model has been established, where acute kidney injury is induced by ischemia/reperfusion (Nakamoto M, Shapiro JI, Shanley PF, Chan L, and Schrier RW. In vitro and in vivo protective effect of eptin Ill on ischemic acute renal failure. J Clinlnvest 80:698~705, 1987., Chintala MS, Bernardino V, and Chiu PJS. Cyclic GM]? but not cyclic AMP ts renal et lation following ischernia—reperfusion in anesthetized rats. J colEXpTher 27112034208, 1994). This model was used to assess whether ent with anti—adrenomedullin antibody can improve kidney function.

The experiment was performed as follows: Effect of a NT—M on Acute Kidney Injury Induced by Ischemia/Reperfusion in Rats Study Design: Test Cone Dosage Rats Groun Article Route HIE/[Ill milks mg/kg (Male) 1 I—R + vehiclea IV 5 NA x 3 8 2 LR + NT-M 1v 5 x 3" 8 a vehicle; injected intravenously (iv) 5 min before reperfusion on day 0, followed by injections on days 1 and 2. bNT-M at 4 mg/kg was injected intravenously (iv) 5 min before reperfusion on day 0, followed by 2 nag/kg i.v. each on days 1 and 2.

”Urine 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 it], before e or TA; Day 3:120 ul.

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

Groups of 8 male Sprague—Dawley rats weighing 250 to 280 g were used. The animals were kept on a 12—hr light/dark cycle and receive a standard diet with distilled water ad libiturn. The animals receive fluid supplements (0.9% NaCl and 5% dextrose/1:1, 10 ml/kg 13.0.) 30 min prior to surgery (day 0). The rats were anaesthetized with pentobarbital (50 mg/kg, i.p.). The nal 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 removal of the renal clips, the kidneys were observed for additional 1 min to ensure color change indicating blood usion. The test compound (NT—M) and vehicle (phosphate buffered ) were ed intravenously 5 min before reperfusion, followed by daily injection on days 1 and 2.

Urine coilection. The 24wh urine tion on ice was initiated at 24h before ia/reperfusion on day —l (-24h to Oh), and day 0 (0—24h), day 1 h) and day 2 (48— 72b) after reperfusion, Blood collection: 0.4 ml blood was collected through the tail vein into EDTA tubes at 011 (before I RI surgery), 24h (before vehicle or TA), 48h (before vehicle or TA) and 72h for determination of plasma creatinine/NaflKfi 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—Oh), day 0 ), day 1 (24—48h) and day 2 11) after reperfusion on day 0. Urine volume, urinary Na+, 81+, and creatinine were measured.

The creatinine clearance (CCr) was calculated as follows: CCr (ml/24 h) z [UCr (mg/ml) X V (ml/24 h)} / PCI (mg/ml) The 24~hr urinary excretion of sodium (Na+) 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+ rptive n. It was computed as follows: FENa (96) $100 :9; [UNa (qu/rnl) x V (ml/2411)] / PNa ) X CCr (ml/24 h) Treatment with anti—Adrenomedullin antibody improved l measures ofkidney function: Blood urea nitrogen (BUN) showed a strong increase 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 mg/dL), which was less pronounced with NT— M treatment (0 h: 16.33 mg/dL, 24 h: 84.2 mg/dL, 48 h: 82.61 mg/dL, 72 h: 64.54 rug/d1.) (Fig. 22).

Serum creatinine developed similarily: Vehicle group (0 h: 061 mg/dL, 24 h: 3.3 lug/(1L, 48 h: 3.16 mg/dL, 72 h: 2.31 , NT—M group: (0 h: 0.59 mg/dL, 24 h: 2.96 mg/dL, 48 h: 2.31 mg/dL, 72 h: 1.8 mg/dL) (Fig. 23).

The endogenous creatinine clearance dropped ely on day one and thereafter improved better in the NT—M group than in the vehicle group. Vehicle group: (0 h: 65.17mL/h, 24 h: 3.5mL/h, 48 h: 12.6lmL/h, 72 h: 20.88mL/h), NT—M group:(0 h: 70.11mL/h, 24 h: 5.84mL/h, 48 h: 21.23mth, 72 h: 26.61mL/h) (Fig. 24).

FIGURE DESCRIPTION Fig. 1a: Illustration of antibody formats w FV and scFv~Variants Fig 1b: Illustration of antibody formats — heterologous fusions and bifunctional antibodies Fig 1c: Illustration of dy formats — bivalentai antibodies and bispecific dies Fig. 2: hADM l~52 (SEQ ID No. 21) mADM 1-50 (SEQ ID NO. 22) aa 1-21 ofhuman ADM (SEQ ID No. 23) aa 1—42 of human ADM (SEQ ID No. 24) aa 43-52 n ADM (SEQ ID No. 25) aa 1-14 ofhuman ADM (SEQ ID NO: 26) aa 1-10 ofhuman 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 of mature murine ADM (SEQ ID NO: 30) aa 1—31 ofmature murine ADM (SEQ ID NO: 31) Fig. 3: a: Dose response curve of human ADM. Maximal CAMP stimulation was adjusted to 100% activation b: Dose/ inhibition curve of human ADM 22—52 (ADM—receptor antagonist) in the ce of .63nM hADM. 0: Dose/ inhibition curve of CT-H in the presence of 5.63 nM hADM. d: Dose/ inhibition curve ofMR—H in the presence of 5.63 nM hADM. e: Dose/ inhibition curve ofNT-H in the presence of 5-63 nM hADM. f: Dose response curve of mouse ADM. Maximal CAMP ation was adjusted to 100% activation g: Dose/ inhibition curve of human ADM 22—52 (ADM—receptor 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/ tion curve ofMR—M in the presence of 0,67 nM mADM. j: Dosef inhibition curve ofNT-M in the presence of 0,67 11M 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 ofNT—H antibody.

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

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

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

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

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

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

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

Fig. 18: Plasma IL—EO 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 obtained with vehicle, the grey box—plot shows results obtained after ent with NT—M.

Fig. 20: Plasma monocyte chemoattractant protein—1 (MCP—l) levels. The white box—plot shows results ed with vehicle, the grey box—plot shows s obtained after treatment with NT—M.

Fig. 21: Plasma TNF—a1pha levels. The white box~plot shows results obtained with vehicle, the grey box— plot shows results obtained after treatment with NT—M.

Fig. 22: Development of blood 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 nous creatinine clearance over time. Mean +/- SEM are shown.

Claims (7)

1. Use of an anti-Adrenomedullin (ADM) antibody or an anti-ADM antibody nt 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 t for the regulation of fluid balance, wherein said antibody or antibody fragment or non-Ig scaffold binds to 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 n said patient is a patient in need of regulating the fluid balance.

3. Use according to claim 1 or 2, wherein the medicament is for preventing or ng edema in said t.

4. Use according to any one of claims 1 to 3, wherein said antibody or antibody fragment or non-Ig scaffold is monospecific.

5. Use according to any one of claims 1 to 4, characterized in that said antibody or fragment or scaffold exhibits a binding affinity to ADM of at least 10-7 M.

6. Use according to any one of claims 1 to 5, wherein said antibody or fragment or scaffold is not ADM-binding-Protein-1, complement factor H.

7. Use according to any one of claims 1 to 6, wherein said dy or antibody fragment or non-Ig scaffold binds to a region of at least 5 amino acids within the sequence of aa 1-42 of mature human ADM: