NZ624875B2 - Anti-adrenomedullin (adm) antibody or anti-adm antibody fragment or an anti-adm non-ig scaffold for use in therapy - Google Patents

Abstract

Discloses use of an anti-adrenomedullin antibody or an anti-ADM antibody fragment binding to adrenomedullin or an anti-ADM non-Ig-protein scaffold binding to adrenomedullin in the manufacture of a medicament, wherein said antibody or fragment or scaffold binds to the N terminal part, amino acids 1 to 21, of adrenomedullin: YRQSMNNFQGLRSFGCRFGTC; SEQ ID No. 23. o 21, of adrenomedullin: YRQSMNNFQGLRSFGCRFGTC; SEQ ID No. 23.

Description

WO 72512 Anti-Adrenomedullin (ADM) antibody or anti-ADM antibody nt or an anti~ADM non-lg scaffold for use in therapy Field of the invention t matter of the present invention is an antiwadrenoniedullin antibody or an anti- adrenorneduliin antibody fragment or an antinADM non—1g scaffold, wherein said antibody or fragment or scaffold is a non-neutralizing antibody.

Subject matter of the present invention is an anti—adrenomedullin antibody or an anti- adrenomedullin antibody fragment or an anti-ADM non—lg scaffold, wherein said antibody or fragment or scaffold is 0 an ADM stabilizing dy or an ADM stabilizing antibody fragment or an ADM stabilizing non—1g scaffold that enhances the half life (tug half retention time) of adre- nomedullin in serum, blood, plasma at least it) %, preferably at least, 50 “/0, more preferably >50 %, most preferably 100 % and/or 0 wherein said ADM stabilizing antibody or an adrcnomedullin stabilizing antibody fragment or an ADM izing non—lg scaffold blocks the bioactivity ofADM to less than 80 ”/0, preferably to less than 50 %.

The stated above is in the sense of blocking the ADM bioactivity of not more than 80 “/0 or not more than 50 0/0, respectively, and thus is to be understood as limited blocking of ADM bioactivity or ion of ADM bioactivity by the respective ADM binder, being it an ADM stabiiizing antibody or antibody fragment or non—1g scaffold. By implication, this means that ADM bioactivity is remaining. For instance, in case of blocking the ADM bioactivity of not more than 80 %, is. approximately 20 % residual ADM ivity t. in case of block— ing the ADM bioactivity of not more than 50 %, tie. approximately 50 % residual ADM bio- activity present.

Subject matter of the present ion is an drenomedullin antibody or an anti— adrenomedullin antibody fragment or an anti—ADM non-1g scaffold, wherein said antibody or fragment or ld binds to the N-terminal part (aa 1—21) ofhuman adrenomedullin.

In a preferred embodiment the subject matter of the present invention is an anti— adrenomedullin antibody or an anti-adrenornedullin antibody fragment or an anti-ADM non- roADM). The peptide adrenomedullin (ADM) pp. 551—555 (1998). Abstract Only). There is also a g n (Pic, R., at 521., "Comple~ inent Factor H is a Serum—binding Protein for adrenomedullin, and the Resulting Complex Modulates the Bioactivities of Both Partners", The Journal of Biological Chemistry, Vol. 276(15), pp. 12300 (2001)) which is specific for ADM and ly likewise modu- lates the effect of ADM. Those physiological s of ADM as well as of PAMP which are of primary importance in the investigations to date were the effects influencing blood pres— sure .

Hence, ADM is an effective vasodilator, and thus it is possible to associate the hypotensive 3.0 effect with the particular peptide ts in the C-terminal part of ADM. It has furthermore been found that the above—mentioned further physiologically active peptide PAMP formed from pre—proADM likewise exhibits a nsive effect, even if it s to have an action mechanism differing from that of ADM (of. in addition to the abovementioned review articles (Etc, T., "A review of the biological properties and al implications of adrenomedullin and proadrenomedullin N—terrninal 20 peptide (PAMP), hypotensive and vasodilating pep- tides”, Peptides, Vol. 22, pp. 1693-1711 ) and (Hinson, er al, "Adrenomedullin, a Mul- tifiinctional tory Peptide", Endocrine Reviews, Vol. 21(2), pp. 138-167 (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. 105—110 (1997). Abstract only), (Kuwasaki, K., at at, "Increased plasma proadrenome- dullin N—tertninal 20 peptide in patients with ial hypertension", Ann. Clin. m, Vol. 36 (Pt. 5), pp. 622—628 (1999). Abstract only) or (Tsuruda, T., et al., "Secretion of proadrenornedullin N—terminalZO peptide from ed neonatal rat c cells", Life Sci, Vol. 69(2), pp. 239-245 (2001). Abstract only) and EP—A2 0 622 458). It has furthermore been found that the concentrations of ADM which can be measured in the circulation and other biological liquids are, in a number of pathological states, significantly above the concentra— tions to be found in healthy control persons. Thus, the ADM level in patients with congestive heart failure, myocardial tion, kidney diseases, hypertensive disorders, Diabetes melli- tus, in the acute phase of shock and in sepsis and septic shock are significantly increased, al~ though to different extents. The PAMP concentrations are also increased in some of said pathological states, but the plasma levels are lower relative to ADM ((Eto, T., "A review of the biological properties and clinical implications of adrenomedullin and proadrenomedullin N—terminal 20 peptide (PAMP), hypotensive and vasodilating peptides", Peptides, Vol. 22, pp. 1693-1711 (2001)); page 1702), It is furthermore known that unusually high concentrations of WO 72512 — 4 — ADM are to be observed in sepsis, and the t concentrations in septic shock (cf. (Etc, T., “A review of the biological properties and clinical implications of adrenomedullin and proadrenomedullin N—terminal 20 peptide (PAMP), hypotensive and vasodilating peptides", Peptides, Vol. 22, pp. 16934711 (2001)) and (Hirata, at at, "Increased Circulating Adreno— medullin, a Novel Vasodiiatory Peptide, in Sepsis", Journal of Clinical Endocrinology and Metabolism, Vol. 81(4), pp. 1449—1453 (1996)), (Ehlenz, K, et at, "High levels of circulating adrenomedullin in severe illness: Correlation with Cureactive n and evidence t the adrenal medulla as site of origin", Exp Clin Endocrinol Diabetes, Vol. 105, pp. 156-162 (1997)), (Tomoda, Y., e: at, ”Regulation of adrenomedullin secretion from cultured cells", es, Vol. 22, pp. 1783-1794 (2001)), (Ueda, 8., at at, ased Plasma Levels of Adrenomedullin in Patients with Systemic Inflammatory Response Syndrome", Am. .1. Res- pir. Crit. Care Med, Vol. 160, pp. 132-136 ) and (Wang, P., "Adrenomedullin and car- diovascular responses in sepsis", Peptides, Vol. 22, pp. 1835—1840 (2001)).

Known in the art is further a method for identifying adrenomedullin immunoreactivity in bio- l liquids for diagnostic purposes and, in particular Within the scope of sepsis diagnosis, cardiac diagnosis and cancer diagnosis. According to the invention, the midregional partial peptide of the proadrenomedullin, which ns amino acids (45-92) of the entire pre- proadrenomedullin, is measured, in particular, with an immunoassay which works with at ieast one labeled antibody that specifically recognizes a sequence of the oADM (W02004/090546).

WO—Al 97423 bes the use of an antibody against adrenomedullin for diagnosis, prognosis, and treatment of cardiovascular disorders. Treatment of es by blocking the ADM receptor are also described in the art, (eg. WO-Al 2006/027147, ) said diseases may be sepsis, septic shock, cardiovascular es, infections, dermatological diseases, endocrinological diseases, metabolic diseases, gastroenterological diseases, cancer, inflammation, hematological diseases, respiratory diseases, muscle skeleton diseases, neuro— logical 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 intestine. ADM—neutralizing antibodies neutralize the before mentioned effects during the early phase of sepsis (Wang, P., "Adrenomedullin and cardiovascular ses 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. (Schlitz et at, “Circu- lating Precursor levels of endothelin—1 and adrenomedullin, two endothelinrn—derived, coun~ teracting substances, in sepsis”, Endothelium, 14:345-351, ). Methods for the diagnosis and treatment of critically ill patients, 9. g. in the very late phases of sepsis, and the use of en- dothelin and endothelin agonists with vasoconstrictor ty for the ation of medica— ments for the treatment of ally ill patients have been described in WO—Al 2007/062676.

It is r described in WO—Al 20077062676 to use, in place of endothelin and/or endothelin agonists, or in combination therewith, medullin antagonists, Le. molecules which pre- vent or attenuate the vasodilating action of medulin, e.g. by blocking its relevant recep- tors, or substances preventing the binding of adrenomedullin to its receptor (ag. specific s as e. g. antibodies binding to adrenomedullin and blocking its receptor gs sites; “immunological lization“). Such use, or combined use, including a subsequent or pre— ceding separate use, has been described in certain cases to be desirable for example to im- prove the therapeutic success, or to avoid undesirable physiological 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. stration of ADM in combination with nding—Protein—l is described for treat- ment of sepsis and septic shock in the art. It is assumed that treatment of septic animals with ADM and ADM—binding—Protein—l prevents transition to the late phase of sepsis. It has to be noted that in a living organism ADM binding protein (complement factor H) is present in the circulation of said organism in high concentrations (Pio et al.: Identification, terization, 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 a1.; 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-protein—l may also be referred to as ADM-binding—protein—l {complement factor H).

Administration of ADM may thus be ial in an early stage of a disease like e.g. sepsis but may be detrimental in a later stage of sepsis. The opposite may be the case with the ad- - 6 – (Followed by page 6A) In one embodiment the t invention provides use of an anti-adrenomedullin antibody or an anti-ADM antibody fragment binding to adrenomedullin or an anti-ADM non-Ig-protein scaffold binding to adrenomedullin in the cture of a medicament, wherein said antibody or fragment or scaffold binds to the N-terminal part, amino acids 1 to 21, of adrenomedullin: YRQSMNNFQGLRSFGCRFGTC; SEQ ID No. 23.

In another embodiment the present invention provides the use of a vasopressor, enous fluid, a TNF-alpha-antibody, and/or antibiotics in the manufacture of a medicament, wherein the medicament is for use in combination with an anti-adrenomedullin antibody or an anti- ADM antibody fragment binding to adrenomedullin or an anti-ADM non-Ig-protein scaffold binding to adrenomedullin, wherein said antibody or fragment or scaffold binds to the N- terminal part, amino acids 1 to 21, of mature medullin: YRQSMNNFQGLRSFGCRFGTC; SEQ ID No. 23. - 6A – (Followed by page 7) In a still further embodiment the invention provides a pharmaceutical composition comprising an antibody, antibody nt or non-Ig-protein scaffold as defined herein. — 7 w scaffold would be more than 0%, preferably more than 5 %, preferably more than 10% more preferred more than 20 ”/0, more preferred more than 50 %. This is in the sense of blocking the ADM bioactivity of not more than 80 % or not more than 50 %, respectively, and thus is to be understood as limited blocking of ADM ivity by the respective ADM binder, being it an DM antibody or anti—ADM antibody nt or anti—ADM non—1g scaffold.

It has been understood that said limited blocking of the bioaetivity of ADM occurs even at excess concentration of the antibody, antibody fragment or non-lg scaffold, meaning an ex- cess of the antibody, fragment or scaffold in relation to ADM. Said limited blocking is an intrinsic ty of the ADM binder itself. This means that said antibody, fragment or scaf- fold has a maximal tion of 80% or 50%, respectively. In a preferred ment said anti—ADM antibody, anti—ADM antibody fragment or anti-ADM non-lg scaffold would block the bioactivity of ADM to at least 5 %. This means — by implication w approximately 95% residual ADM bioactivity is present.

Subject matter of the present invention is an medullin antibody or an adrenomeduliin antibody fragment for use in a treatment of a chronic or acute disease wherein said antibody or fragment is 9 an ADM stabilizing antibody or an adrenomeduilin antibody fragment that enhances the tug half retention time of adrenomedullin in serum, blood, plasma at least 10 %, preferably at least, 50 %, more preferably >50 %, most preferably 100 % and/or 0 wherein said antibody or said medullin antibody fragment blocks the bioactivity of ADM to less than 80 %, preferably to less than 50 %. t matter of the present invention is an adrenornedullin antibody or an adrenomedullin antibody fragment for use in a ent of a chronic or acute disease wherein said antibody or fragment binds to the N-terminal part (aa 1—21) of human adrenomedullin.

In a preferred embodiment the subject matter of the present invention is an adrenomedullin antibody or an adrenomedullin antibody fragment for use in a treatment of a chronic or acute disease wherein said antibody or nt binds to the Nwterminal end of human adrenome— dullin.

In this context (a) molecule(s), being it an antibody, or an antibody fragment or a non-lg scaf- fold with “nonmeutralizing anti—ADM activity”, tively termed here for simplicity as — 8 - “non—neutralizing” anti~ADM antibody, antibody nt, or non—lg scaffold, that eg. blocks the bioactivity ofADM to less than 80 0/0, 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 (calcitonin receptor like or) and RAMP3 toractivity modifying protein 3), reduces the amount of CAMP produced by the cell line through the action of parallel added human tic ADM peptide, wherein said added human tic ADM is added in an amount that in the absence of the non—neutralizing antibody to be analyzed, leads to half-maximal stimulation of CAMP synthesis, wherein the reduction of cAMP by said molecule(s) binding to ADM takes place to an 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 d more than the amount, which is needed to obtain the maximal re- duction of CAMP synthesis obtainable with the utralizing antibody to be an~ alyzed.

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

The bioactivity is defined as the effect that a substance takes on a living organism or tissue or organ or functional unit in vivo or in vitro (eg. in an assay) after its interaction. In case of ADM bioactivity this may be the effect of ADM in a human recombinant Adrenornedullin or CAMP functional assay. Thus, according to the t invention bioactivity is de— fined Via an Adrenomedullin receptor CAMP functional assay. The following steps may be performed in order to determine the bioactivity of ADM in such an assay: - Dose response curves are performed with ADM in said human recombinant Adreno— medullin receptor CAMP functional assay.

— The ADM—concentration of aximal CAMP stimulation may be calculated.

— At constant half~rnaximal cAMPmstimulating ADMtconcentrations dose response curves (up to 100 ng/ml final concentration) are performed by an ADM stabilizing an- tibody or an adrenomedullin stabilizing antibody fragment or an adrenomedullin stabi— lizing non—lg scaffold, respectively. - 9 — A maximai tion in said ADM bioassay of 50% means that said anti-ADM antibody or said anti-adrenomeduliin dy fragment or said anti—adrenomedullin non—1g scaffold, re»- spectively, blocks the bioactivity to 50% of baseline values. A l inhibition in said ADM bioassay of 80% means that said anti—ADM antibody or said anti—adrenomedullin anti— body fragment or said anti—adrenomedullin non—1g scaffold, respectively, blocks the bioactiv- ity of ADM to 80%. This is in the sense of ng the ADM ivity to not more than 80%. This means imately 20% residual ADM bioactivity remains present.

However, by the present specification and in the above context the expression “blocks the bioactivity of ADM” in relation to the herein disclosed DM antibodies, anti-ADM anti— body fragments, and anti—ADM non—1g scaffolds should be understood as mere decreasing the ivity of ADM, ably sing circulating ADM bioactivity from 100% to 20% ing ADM bioactivity at maximum, preferably decreasing the ADM bioactivity from 100% to 50% remaining ADM bioactivity; but in any case there is ADM bioactivity remain- ing that can be determined as detailed above.

The bioactivity of ADM may be determined in a human recombinant Adrenornedullin recep- tor CAMP functional assay (Adrenomedullin Bioassay) according to Example 2.

Anti—adrenomeduilin (ADM) antibody is an antibody that binds specifically to ADM, anti- adrenornedullin dy fragment is a fragment of an ADM antibody wherein said fragment binds Specifically to ADM. An antiwADM non—1g scaffold is a non—1g scaffold that binds spe— cifically to ADM. Specifically binding to ADM allows binding to other antigens as well. This means, this city would not exclude that the antibody may cross-react with other poly~ peptides than that against it has been raised. This also pertains the specificity of the anti—ADM antibody fragment, or anti-ADM non—lg scaffold in accordance with the invention.

Surprisingly it has turned out that non-neutralizing anti—ADM antibodies or non—neutralizing anti-ADM antibody fragments or a non—neutralizing anti—ADM non-lg scaffolds offer a sig— nificant therapeutic advantage over the neutralizing ones. in one specific embodiment it is preferred to use an anti—ADM antibody or an anti~ adrenomeduIIin antibody fragment or anti—ADM non—1g scaffold according to the present in— vention, wherein said anti—adrenomeduilin antibody or said anti—adrenomedullin antibody fragment or anti-ADM non—lg scaffold is an ADM stabilizing antibody or an adrenomedullin stabilizing antibody fragment or an adrenomedullin stabilizing non—lg scaffold that enhances the half life (t1[2; half ion time) of adrenomedullin in serum, blood, plasma at least 10 ”/0, preferably at least 50 0/9, more preferably >50 %, most preferably >100%.

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 adrenornedullin stabilizing antibody fragment or an adrenomedullin stabilizing non—lg scaffold, respectively, using an immunoas- say for the quantification of ADM.

The following steps may be conducted: — ADM may be diluted in human e plasma in absence and presence of an ADM stabilizing antibody or an adrenomedullin stabilizing antibody fragment or an adreno— medullin stabilizing non—lg scaffold, respectively, and may be incubated at 24 OC. — ts are taken at selected time points (eg. within 24 hours) and degradation of ADM may be stopped in said ts by fieezing at —20 °C.

- The quantity of ADM may be determined by an hADM immunoassay directly, if the selected assay is not influenced by the stabilizing antibody. Alternatively, the aliquot may be treated with denaturing agents (like HC1) and, alter clearing the sample (e.g. by centrifiigation) the pH can be neutralized and the ADM-quantified by an ADM immunoassay. Alternatively, non—immunoassay technologies (eg. rpHPLC) can be used for ADM~quantification — The half life of ADM is calculated for ADM incubated in absence and presence of an ADM stabilizing antibody or an adrenomedullin stabilizing antibody fragment or an mednllin izing 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 adre- llin stabilizing antibody fragment or an adrenomedullin stabilizing non—lg ld.

A two—fold increase of the half life of ADM is an enhancement ofhalf life of 100%. — ll — Half Life (half retention time) is defined as the period over which the concentration of a spec— ified chemical or drug takes to fall to half its baseline concentration in the specified fluid or blood.

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

An antibody according to the t invention is a protein including one or more polypep- tides substantially encoded by irnmunoglobulin genes that specifically binds an antigen. The recognized immunoglobulin genes include the kappa, lambda, alpha (IgA), gamma (lgGi, Ing, lgG3, lgG4), delta (IgD), n (IgE) and mu (IgM) constant region genes, as well as the myriad immunoglobulin variable region genes. ength globulin light chains are generally about 25 Kd or 214 amino acids in length. Full—length immunoglobulin heavy chains are generally about 50 Kd or 446 amino acid in length. Light chains are encoded by a variable region gene at the rminus (about 110 amino acids in ) and a kappa or lambda constant region gene at the COOH——terminus. Heavy chains are similarly encoded by a variable region 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 n, and the constant regions mediate effector functions. lmmunoglobulins also exist in a variety of other forms including, for ex- ample, Fv, Fab, and F(ab')2, as well as bifunctional hybrid antibodies and single chains (e.g, Lanzavecchia et al., Eur. J. Immunol. ,1987; Huston et al, Proc. Natl. Acad. Sci.

[ZS/1., 85:5879—5883, 1988; Bird et al., Science 242:423—426, 1988; Hood er 511., Immunol- ogy, in, N.Y., 2nd ed., 1984; Hunkapiller and Hood, Nature 323:1 5,16,1986). An im— munoglobulin 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 oflmmunologieal Interest, E. Kabat et all, 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 antibody, chimeric dy, humanized antibody or human antibody, or func- tional antibody fragment, specifically bound to the antigen. — l2 — Chimeric antibodies are antibodies whose light and heavy chain genes have been constructed, typically by genetic engineering, from immunoglobulin variable and constant region genes belonging to different species. For example, the variable ts of the genes from a mouse monoclonal antibody can be joined to human nt segments, such as kappa and gamma 1 or gamma 3. In one example, a therapeutic chimeric antibody is thus a hybrid protein com- posed of the variable or antigenwbinding domain from a mouse antibody and the constant or effector domain from a human antibody, although other mammalian species can be used, or the variable region can be produced by molecular techniques. s of making ic antibodies are well known in the art, e.g., see [1.8. Patent No. 5,807,715. A "humanized" im- munoglobulin is an immunoglobulin including a human framework region and one or more CDRs from a non—human (such as a mouse, rat, or synthetic) irnmunoglobulin. The non— human immunoglobulin providing the CDRs is termed a "donor” and the human immu— noglobulin providing the framework is termed an "acceptor.” In one embodiment, all the CDRS are from the donor immunoglobulin in a humanized immuneglobulin. Constant regions need not be present, but if they are, they must be substantially identical to human immu« ulin constant regions, i.e., at least about , such as about 95% or more identical.

Hence, all parts of a zed immunoglobulin, except possibly the CDRS, are substantially identical to corresponding parts of natural human immunoglobulin ces. A "humanized antibody" is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The 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 sub~ stitutions 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, gin; ser, thr; lys, arg; and phe, tyr. Humanized immunoglobulins can be ucted by means of c engineering (eg, see US. Patent No. 5,585,089). A human antibody is an antibody wherein the light and heavy chain genes are of human . Human antibodies can be generated using methods known in the art. Human antibodies can be produced by im~ mortalizing a human B cell secreting the antibody of interest. lmmortalization can be accom» plished, for e, by EBV infection or by fusing a human B cell with a myeloma or hybri~ donia cell to produce a trioma cell. Human antibodies can also be produced by phage display methods (see, e.g., Dower et at, PCT Publication No. WOW/17271; McCafferty et at, PCT WO 72512 — l3 — Publication No. WO92/001047; and Winter, PCT Publication No. WO92/20791), or selected from a human combinatorial monoclonal antibody library (see the Morphosys website). Hu— man antibodies can also be prepared by using transgenic animals carrying a human immu- noglobulin gene (for e, see Lonberg et (11., PCT Publication No. WO93/12227; and Kucherlapati, PCT Publication No. WO91/10741).

Thus, the anti~ADM antibody may have the formats known in the art. Examples are human antibodies, onal antibodies, humanized antibodies, chimeric antibodies, CDR—grafted antibodies. In a preferred embodiment antibodies according to the present invention are re— combinantly produced antibodies as eg. igG, a typical full—length immunoglobnlin, or anti- body fragments containing at least the F-variable domain of heavy and/or light chain as eg. chemically coupled antibodies (fragment antigen g) including but not limited to Fab fragments including Fab minibodies, single chain Fab antibody, monovalent Fab antibody with epitope tags, 6.3, Fab—VSSXZ; nt Fab (mini—antibody) dimerized with the CH3 do- main; bivalent Fab or multivalent Fab, eg. formed Via multimerization with the aid of a het— erologous domain, tag. via dimerization of dHLX s,e.g. LX—FSXZ; F(ab‘)2~ fragments, scFv-fragments, multime‘rized multivalent or/and multispecific ‘agments, bivalent and/or biSpecific diabodies, BITE® (bispecific T~cell engager), ctional d- ies, polyvalent antibodies, 3.g. from a different class than G; single—domain antibodies, eg. nanobodies derived from camelid or fish immunoglobulines and numerous others.

In on to anti—ADM antibodies other biopolyrner scaffolds are well known in the art to complex a target molecule and have been used for the generation of highly target specific biopolyrners. es are rs, spiegelmers, anticalins and conotoxins. For illustration of antibody formats please see Fig. la, lb and 1c.

In a preferred embodiment the anti—ADM antibody format is selected from the group compris— ing Fv fragment, scFv fragment, Fab fragment, scFab fragment, Rab); 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 optimized conjugates thereof, such as PEGylated fragments. One of the most preferred s is the scFab format.

Non—lg lds may be protein scaffolds and may be used as antibody mimics as they are capable to bind to ligands or antigenes. Non—lg scaffolds may be selected from the group - 14 ~ comprising tetranectin—based non—lg scaffolds (eg. described in US 028995), f1- bronectin scaffolds (e. g. bed in EP 1266 025; lipocalin-based scaffolds ((e.g. described in WO 54420); ubiquitin scaffolds (cg. described in ), transferring scaffolds (e.g. described in US 2004/0023334), protein A scaffolds (eg. described in EP 2231860), ankyrin repeat based scaffolds (e. g. bed in ), microproteins preferably microproteins forming a cystine knot) scaffolds (eg. described in EP 2314308), Fyn SH3 domain based scaffolds (cg. described in WO 23685) EGFR-A—domain based scaffolds (cg. bed in WO 40229) and Kunitz domain based scaffolds (eg. described in EP 1941867).

In one preferred embodiment of the invention antibodies according to the present invention may be produced as follows: A Balb/c mouse was immunized with lOOug ADM-PeptidemBSA—Conjugate at day 0 and 14 ified in 100ul complete Freund’s adjuvant) and 50ug at day 21 and 28 (in 100ul in- complete Freund’s adjuvant). Three days before the fusion experiment was med, the animal received SOug of the ate dissolved in 100m , given as one eritoneal and one intra—venous injection.

Spenocytes from the zed mouse and cells of the myeloma cell line SP2/0 were fused with 11nl 50% polyethylene glycol for 30s; 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 mented with 20% fetal calf serum and HAT—Supplement]. After two weeks the HAT medium is replaced with HT Medium for three passages followed by returning to the normal cell culture 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 retesting, 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 anti" body—secreting hybridomas. J. Irnmunol. Meth. 81: 223—228; Ziegler, B. at 611.0996) Gluta— mate decarboxylase (GAD) is not detectable on the surface of rat islet cells examined by cyto- fluorometry and complement—dependent antibody-mediated cytotoxicity of monoclonal GAD antibodies, Horm. Metab. Res. 28: 11—15). ~ 15 w Antibodies may be produced by means of phage display according to the following proce- dure: The human naive antibody gene libraries HAL7/8 were used for the isolation of recombinant single chain F‘Variable domains (scFv) against medullin peptide. The antibody gene libraries were screened with a panning strategy sing the use of es containing a biotin tag linked Via two different 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 Ecoli strains.

Supernatant from the ation of these clonal strains has been directly used for an antigen ELISA testing. (see also Hust, M., Meyer, T., Voedisch, B., Riilker, T., Thie, H., ELGhezal, A., Kirsch, Ml, Schiitte, M., ng, 8., Meier, D., Schirrmann, T., Dfibel, 8., 2011. A human scFv antibody generation pipeline for proteome research. Journai of Biotechnology 152, 159—170; Schiitte, M., Thullier, P., Pelat, T., Wezler, X., Rosenstock, P., Hinz, D., Kirsch, asenberg, M., Frank, R., Schirrmann, T., Gunzer, M., Hust, M., , 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 ing to the following procedure: For humanization of an antibody of murine origin the antibody sequence is ed for the structural interaction of fiamework regions (FR) with the complementary determining regions (CUR) and the n. Based on ural modeling an appropriate FR of human origin is selected and the murine CDR ces are transplanted into the human FR. Variations in the amino acid sequence of the CDRs or FRs may be introduced to regain ural interactions, which were abolished by the species switch for the FR sequences. This recovery of structural interactions may be achieved by random approach using phage display libraries or via di- rected approach guided by molecular modeling. (Almagro JC, Fransson J., 2008. Humaniza— tion of antibodies. Front Biosci. 2008 Jan l;l321619~33.) In a preferred embodiment the ADM antibody format is selected from the group comprising FV fragment, scFv fragment, Fab fragment, scFab nt, F(ab)2 fragment and scFv—Fc Fu— sion protein. In another preferred embodiment the antibody format is selected from the group comprising scFab fragment, Fab fragment, scFv nt and bioavailability optimized con- jugates thereof, such as PEGylated fragments. One of the most preferred formats is scFab format.

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

In a preferred embodiment the anti—adrenornedullin antibody or an anti—adrenomedullin anti- body fragment or anti-adrenomeduilin non—lg scaffold is directed to and can bind to an epi- tope of at least 5 amino acids in length ned in ADM.

In a Specific ment the anti—adrenomedullin antibody or an anti—adrenornedullin anti— body nt or anti-adrenomedullin non—lg scaffold is directed to and can bind to an epi— tope of at least 4 amino acids in length contained in ADM.

In one specific embodiment of the invention the anti—Adrenomedullin (ADM) dy or anti—ADM antibody fragment binding to adrenornedullin or antiwADM non-1g scaffold bind— ing to adrenomedullin is provided for use as a medicament wherein said antibody or fragment or scaffold is not ADM-binding—Protein~i (complement factor H).

In one specific embodiment of the invention the anti-Adrenomedullin (ADM) antibody or DM antibody fragment binding to adrenomedullin or anti-ADM non-lg scaffold bind- ing to adrenomedullin is ed for use as a ment wherein said antibody or antibody fragment or non—1g ld binds to a region of preferably at least 4, or at least 5 amino acids within the sequence of aa 1—42 ofmature 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 medullin or anti—ADM non-lg scaffold bind— ing to adrenomedullin is provided for use as a medicament wherein said antibody or fragment or scaffold binds to a region of preferably at least 4, or at least 5 amino acids within the se— quence of aa 1-21 ofmature human ADM: — 17 — SEQ ID No.: 23 YRQSMNNFQGLRSFGCRFGTC.

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

In another red embodiment said anti-antibody or an antimadrenomedullin antibody frag- ment or DM non—1g scaffold recognizes and binds to the N-terminal end (aa 1) of adre— llin. N—terminal end means that the amino acid 1, that is “Y” of SEQ ID No. 21 or 23; is mandatory for antibody binding. Said anti-ADM dy or DM antibody fragment or anti—ADM non—lg ld would neither bind inal extended nor N—terminal modi- fied Adrenomedullin nor N—terminal degraded adrenomedullin, It also preferred to use an ADM antibody or an adrenomednllin antibody fragment according to the present invention, wherein said antibody or an adrenomednllin antibody fragment is an ADM stabilizing antibody or an ADM stabilizing adrenomedullin antibody fragment that en- hances the tug; half retention time of adrenomedullin in serum, blood, plasma at least 10 9/0, preferably at least 50 %, more preferably >50 %, most preferably >100. An assay that may be used for the determination of the half retention time of adrenomednllin in serum, blood, plasma is described in Example 3.

It is also preferred to use an ADM antibody or an adrenomedullin antibody fragment accord ing to the present ion, wherein said antibody or said adrenomedullin antibody fragment blocks the bioactivity ofADM to less than 80 %, preferably less than 50%.

In a preferred embodiment a modulating antibody or a modulating adrenomedullin dy fragment is used in the treatment of . A modulating antibody or an adrenomedullin anti- body fragment enhances the bioaetivity of ADM in the early phase of sepsis and reduces the damaging effects of ADM in the late phase of sepsis. A “modulating” antibody or a modulat— ing adrenomedullin antibody fragment is an antibody or an adrenomedullin antibody fragment that enhances the tm half retention time of adrenomednllin in serum, blood, plasma at least 10 %, preferably at least, 50 % more preferably >50 “/03 mOSt preferably >100% and blocks the bioactivity ofADM to less than 80 %, preferably less than 50 %. — 18 — In another specific embodiment pursuant to the invention the herein provided antinADM anti— body or anti~ADM antibody fragment or anti—ADM non—1g scaffold does not bind to the C— al portion ofADM, is. the aa 43 — 52 ofADM PRSKISPQGY-NHZ (SEQ ID NO: 25).

In a specific embodiment a modulating anti—ADM antibody or a modulating anti— medullin antibody fragment or a modulating anti—adrenomedullin non—lg scaffold ac— cording to the present invention is used for treatment of subjects for preventing or treating diseases or conditions, 6.g. for use in prevention or therapy or prevention of a chronic or acute disease or acute condition of a subject.

Such a modulating anti-ADM antibody or a modulating anti—adrenomedullin antibody frag- ment or a modulating anti—adrenomedullin non-lg scaffold may be especially useful in the treatment of sepsis. A modulating anti—ADM dy or a modulating anti—adrenomedullin antibody fragment or a modulating anti-adrenomedullin non-lg scaffold enhances the bioac— tivity of ADM in the early phase of sepsis and s the damaging effects of ADM in the late phase of sepsis.

A “modulating” antibody or a modulating adrenomedullin antibody fragment or a modulating adrenomedullin non—lg scaffold is an antibody or antibody fragment or nonnlg scaffold that enhances the half life (ll/2 half retention time) of adrenomedullin in serum, blood, plasma at least 10 %, preferably at least, 50 %, more preferably >50 %, most preferably >100% and blocks the bioactivity of ADM to less than 80 %, ably less than 50 % and wherein the bioactivity ofADM is blocked to at least 5%. These values related to half—life and blocking of bioactivity have to be understood in on to the before—mentioned assays in order to deters mine these values. It has to be understood that said modulating dy or a modulating adrenOmedullin antibody nt or a modulating medullin non-lg scaffold is an anti— body or dy fragment or non-lg scaffold that is binding to ADM.

It should be emphasized that blocking the ADM bioactivity is in the sense of no more than 80%, and thus 20% residual ADM ivity. The same applies to ng the ADM bioac— tivity to no more than 50%, and thus residual 50% ADM bioactivity. - 19 — Subject matter of the present invention is an anti—adrenomedullin dy or an anti- adrenornedullin antibody fragment or an anti—ADM non-lg scaffoid, wherein said antibody or fragment or scaffold is e an ADM stabilizing antibody or an adrenomedullin stabilizing antibody fragment or ADM izing non—1g scaffold 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 % and/or 0 wherein said anti—adrenomedullin antibody or said anti—adrenomedullin antibody fragment or said anti—ADM non~1g scaffold blocks the bioactivity of ADM to less than 80 %, preferably to less than 50 %.

The stated above is in the sense of ng the ADM bioactivity of not more than 80 % or not more than 50 0/0, respectively, and thus is to be understood as limited blocking of ADM bioactivity by the respective ADM binder, being it an ADM izing dy or antibody fragment or non—Kg scaffold, respectively.

Such a modulating antibody or a modulating adrenomedullin dy fragment or a modulat- ing adrenomedullin non—1g scaffold offers the advantage that the dosing of the administration is facilitated. The combination of partially blocking or partially reducing Adrenomedullin bioactivity and increase of the in vivo half life (increasing the Adrenomedullin bioactivity) leads to beneficial simplicity of drenomedullin antibody or an anti—adrenomednliin an— tibody fragment or anti~adrenomedullin non-1g scaffold dosing. In a situation of excess of endogenous Adrenomedullin (maximal stimulation, late sepsis phase, shock, hypodynamic phase) the activity lowering effect is the major impact of the antibody or fragment or scaffold, limiting the (negative) effect of Adrenomedullin. in case of low or normal nous Adre- nomedullin concentrations, the biological effect of anti—Adrenomedullin antibody or an anti~ adrenomedullin dy fragment or anti-ADM non-1g scaffold is a combination of lowering (by partially blocking) and increase by sing the Adrenomedullin half life. If the half life effect is stronger than the ng effect, the net biological activity of endogenous Adreno— medullin is beneficially increased in early phases of sepsis (low Adrenomedullin, hyperdy- namic phase). Thus, the non-neutralizing and ting anti—Adrenomedullin antibody or anti—adrenornedullin antibody fragment or anti-adrenomedullin non—1g scaffold acts like an ADM ivity buffer in order to keep the bioactivity of ADM within a certain physiologi— cal range. w 20 - Thus, the dosing of the antibody/fragment/scaffold in egg. sepsis may be selected from an ex— cessive concentration, because both sepsis phases (early and late phase) benefit from exces~ sive anti-ADM antibody or an anti~adrenomedullin antibody fragment or anti—ADM non—lg scaffold treatment in case of a modulating effect. Excessive means: The anti-Adrenomedullin antibody or an anti—adrenomedullin antibody fragment or anti—ADM non—lg scaffold concen- tration is higher than endogenous Adrenomedullin during late phase (shock) of eg. sepsis.

This means, in case of a modulating antibody or modulating antibody fragment or ting nonnlg scaffold dosing in sepsis may be as follows: The concentration of Adrenomedullin in septic shock is 226+/-66 fmol/rnl (Nishio et al., "In- creased plasma concentrations of adrenomedullin correlate with tion of vascular 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 1’ 80kg body weight) 3.2pg’kg body weight. Excess means at least double (mean) septic shock Adrenome- dullin concentration, at least > 3pg anti-Adrenomedullin antibody or an anti—adrenomedullin antibody fragment or DM non-lg scaffold / kg body , preferred at least 6.4ng anti~Adren0medullin antibody or an drenomedullin antibody fragment or DM non—lg scaffold / kg body . Preferred > IOng / kg, more preferred >20pg/kg, most pre- ferred >100ug anti-Adrenomedullin antibody or an anti-adrenomedullin antibody fragment or anti-DM non-lg Scaffold / kg body weight.

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

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

Monospecific anti—Adrenornedullin (ADM) antibody or monospecific anti~adrenomedullin antibody fragment or monospecific anti—ADM non-lg scaffold means that said antibody or dy nt or non—1g scaffold binds to one specific region encompassing preferably at least 4, or at least 5 amino acids within the target ADM. Monospecific anti—Adrenomedullin (ADM) antibody or monospecific anti-adrenornedullin antibody fragment or monospecific anti-ADM non-1g scaffold are anti-Adrenomedullin (ADM) antibodies or anti—adrenomedullin antibodiy fragments or anti-ADM non—1g scaffolds that all have affinity for the same n In another special embodiment the anti—ADM dy or the antibody fragment binding to ADM is a monospecific dy. Monospecific means that said dy or dy frag- ment binds to one specific region encompassing preferably at least 4, or preferably at least 5 — 21 — amino acids within the target ADM. Monospecific antibodies 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.

In a specific embodiment of the ion the antibody is a monoclonal antibody or a frag— ment thereof. In one embodiment of the invention the anti—ADM antibody or the anti~ADM antibody fragment is a human or humanized antibody or derived therefrom. In one specific embodiment one or more (marine) CDR’s are d into a human antibody or antibody fragment.

Subject matter of the present invention in one aspect is a human CDR-grafted antibody or dy fragment thereof that binds to ADM, n the human CDR-grafted antibody or antibody fragment thereof comprises an antibody heavy chain (H chain) comprising: SEQ ID NO: 1 GYTFSRYW SEQ ID NO: 2 lLPGSGST and/0r SEQ ID NO: 3 TEGYEYDGFDY and/or r comprises an antibody light chain (L chain) comprising: SEQ ID N014 QSIVYSNGNTY SEQ ID NO: 5 and/or — 22 — SEQ ID NO: 6 FQGSHIPYT.

In one specific ment of the invention subject matter of the present invention is a hu— man 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 SEQ ID NO: 2 ILPGSGST SEQ ID NO: 3 TEGYEYDGFDY and wherein the light chain comprises at least One CDR selected from the group comprising: SEQ ID No: 4 QSIVYSNGNTY SEQ ID NO: 5 SEQ ID NO: 6 FQGSHIPYT.

In a more specific embodiment of the ion 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 TEGYEYDGFDY and wherein the light chain comprises the ces SEQ ID NO: 4 QSIVYSNGNTY SEQ ID NO: 5 SEQ ID NO: 6 FQGSHIPYT.

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

The anti-ADM antibody or anti-adrenomedullin antibody fragment or anti-ADM non—lg scaf— fold ing to the present invention ts an affinity towards human ADM in such that affinity constant is greater than 10'7 M, preferred 10'8 M, red affinity is greater than 10‘9 M, most preferred higher than 10"0 M. A person skilled in the art knows that it may be corn sidered to compensate lower affinity by applying a higher dose of compounds and this meas- ure would not lead out—Of-themscope of the invention. The affinity constants may be deter— mined according to the method as described in Example 1.

The antibodies or fragments according to the present ion can be used in combinations with other agents, as e.g. with the so—called nding protein, for the uses as described therein. — 24 - It should be emphasized that the term “ADM g protein” comprises ADM—binding— protein—l (complement factor H), which however does not reflect a non—neutralizing and/or modulating anti—ADM antibody, antibody fragment, or non—1g scaffold in accordance with the invention.

In a preferred embodiment the anti-ADM antibody or the anti—ADM antibody fragment or DM non—1g scaffold is used for reducing the risk of mortality during a 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 dis- ease or condition selected from the group comprising severe infections as e.g. meningitis, Systemic inflammatory Response—Syndrom (SIRS) sepsis; other diseases as diabetes, cancer, acute and c vascular diseases as eg. heart failure, myocardial infarction, stroke, athero— sclerosis; shock as e.g. septic shock and organ dysfunction as e.g. kidney dysfiinction, liver dysfunction, burnings, surgery, trauma poisoning, by chemotherapy damages. Especially use— ful is the antibody or fragment or scaffold according to the present invention for reducing the risk of mortality during sepsis and septic shock, tie. late phases of .

In one embodiment the DM antibody or an anti—adrenomedullin antibody fragment or anti-ADM non—lg scaffold is used in y or prevention of a chronic or acute disease or acute condition of a patient according to the present invention wherein said patient is an ICU patient. In another embodiment the anti—ADM antibody or an anti—adrenornedullin antibody nt or anti—ADM non—lg scaffold is used in therapy or prevention of a chronic or acute disease of a patient according to the present invention wherein said patient is ally ill.

Critically ill means a patient having a disease or state in which death is possible or imminent.

The antibodies, antibody nts and nondg scaffolds ing to the present invention may be used for treatment of subjects for preventing or treating diseases or conditions, e.g. for use in prevention or y of a chronic or acute disease or acute condition of a subject. Such a disease may be selected from the group comprising severe infections as e. g. meningitis, Sys- temic inflammatory Response—Syndrome (SIRS,) sepsis; other es as diabetes, cancer, acute and chronic vascular diseases as e.g. heart failure, myocardial tion, stroke, athero- sclerosis; shock as e.g. septic Shock and organ dysfunction as cg. kidney dysfunction, liver dysfunction, burnings, surgery, ta. ally useful is the antibody or fragment or scaffold according to the present invention for reducing the risk of mortality during sepsis and septic shock, tie. late phases of .

In the following clinical criteria for SlRS, sepsis, severe sepsis, septic shock will be defined. 1) Systemic inflammatory host response (SIRS) characterized by at least two of the following patients exhibit hypotension (mean arterial pressure is < 65 mm Hg) elevated serum lactate level being > 4 mmol/L blood glucose > 7.7 mmol/L (in absence of diabetes) central venous re is not Within the range 842 mm Hg urine output is < 0.5 mL X kg'1 x hr"1 central venous (superior vena cava) oxygen saturation is < 70% or mixed venous < heart rate is > 90 beats/min temperature < 36°C or > 38°C respiratory rate > n White cell count < 4 or > lelOg/L (leucocytes); > l0% immature neutrophils 2) Sepsis 2O Following at least two of the ms mentioned under I), and additionally a clinical suspi— cion ofnew infection, being: cough/sputum/chest pain abdominal pain/distension/diarrhoea line infection endocarditis dysuria headache with neck stiffness o cellulitis/wound/joint 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: 0 blood pressure ic < 90/mean; < 651anG to lactate > 2 mmol/L - Bilirubine > 34umol/L - urine output < 0.5 mL/kg/h for 2h 0 creatinine > 177 umol/L - platelets < lOleOg/L 0 81302 > 90% unless 0; 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 respond to treatment and intrave- nous fluid administration alone is insufficient to maintain a patient's blood re from be- coming hypotensive also provides for an administration of an anti-ADM antibody or an anti» ADM antibody nt or an anti-ADM non—1g scaffold in ance with the present in- vention.

In one embodiment of the present invention the patient is not suffering from SIRS, a severe ion, sepsis, shock as cg. septic shock. Said severe infection denotes rag. meningitis, Systemic atory 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 ion of any organ dysfunction is present, being it: - 27 —— 0 blood pressure systolic < 90/1nean; < 65mml~lG o lactate > 2 mmol/L o Bilirubine > 34umol/L a urine output < 0.5 mL/kg/h for 2h 0 creatinine >177 umol/L o platelets < g/L a SpOg > 90% unless 02 given In another specific embodiment said acute disease or acute condition is not sepsis, severe sep— sis or is not SIRS or is not shock, or septic shock.

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

The antibodies or fragments or lds according to the present invention can be used in combinations with other agents, as e.g. with the so—called ADM binding protein, for the uses as bed therein. ADM g protein is also naturally present in the circulation of said patient.

It should be emphasized that the term “ADM binding protein” ses ADM—binding— protein—l (complement factor H), which however does not reflect a non—neutralizing and/or modulating anti—ADM antibody, antibody fragment, or non—lg scaffold in accordance with the invention.

Subject of the present invention is further an anti~ADM antibody or an anti—adrenomedullin antibody fragment or anti-ADM non-1g ld for use in therapy or prevention of a chronic or acute disease or acute condition of a patient according to the t invention, wherein said antibody or fragment or scaffold is to be used in combination with further active ingredi- ents .

Subject matter of the invention is also an anti-Adrenomedullin (ADM) antibody 01' an anti~ adrenomedullin antibody fragment or an anti—ADM non—lg scaffold to be used in combination with another active drug, e.g. used as primary medicament, n said combination is for — 28 - use in therapy or prevention of a chronic or acute disease or acute ion of a patient for stabilizing the circulation of said patient, in particuiar the systemic circulation of said patient. y medicament means a medicament that acts against the primary cause of said disease or condition. Said primary medicament may be antibiotics in case of infections.

In a specific embodiment of the before mentioned combinations said combinations are to be used in combination with vasopressors e. g. catecholatnine, wherein said further combination is for use in therapy or tion of a chronic or acute disease or condition of a patient.

In one ment of the invention said patient having a chronic or acute disease or chronic ion is characterized by the need of the patient to get administration of vasopressors e.g. catechoiamine administration.

Subject matter of the invention in one specific embodiment is, thus, an anti—Adrenomedullin (ADM) antibody or an anti-adrenomeduilin antibody fragment or an anti-ADM non~Ig scaf— fold to be used in combination with ADM binding protein and/or further active ingredients for use in therapy or prevention of a t in need of a treatment of vasopressors e.g. cate- cholamine treatment.

In a specific embodiment of the above~mentioned ations said combinations are to be used in combination with fluids administered enously, wherein said combination is for use in therapy or prevention of a chronic or acute disease or condition of a t for stabiliz- ing the ation, in particular the ic circulation.

In one ment of the invention said patient having a chronic or acute disease or acute condition being in need “for stabilizing the circulation is terized by the need of the pa— tient to get intravenous fluids.

Subject matter of the invention in one specific embodiment is, thus, an anti-Adrenornedullin (ADM) antibody or an anti-adrenomedullin antibody fragment or anti-ADM non-1g scaffold in combination with ADM binding protein and/or further active ingredients for use in therapy or prevention of a patient in need of intravenous fluids. This is in the sense of the patient is in need of enous fluids for regulating the systemic fluid balance.

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 .. 29 — anti—ADM antibody, anti—ADM antibody fragment, or anti—ADM non-lg scaffold as in accor- dance with the invention.

In accordance with the invention the ADMhinding—protein—l may also be ed to as ADM—binding—protein-l (complement factor H).

Said anti—ADM antibody or an anti—adrenornedullin dy fragment or DM non-1g ld or combinations thereof with ADM binding protein and/or further active ingredients may be used in combination With vasopressors eg. catecholamine and/or with fluids adminis- tered intravenously for use in a of a chronic or acute disease or acute condition of a patient for stabilizing the circulation, in particular for stabilizing the systemic circulation.

Subject matter of the invention is also an anti—ADM antibody or an anti—adrenomedullin anti- body fragment or DM non—lg according to the present invention to be used in combina- tion with TNF—alpha—antibodies. TNF-alpha—antibodies are commercially available for the treatment of patients. in a preferred embodiment the anti—ADM antibody or the DM antibody fragment or anti-ADM non—lg scaffold is used for reducing the risk of mortality during said chronic or acute disease of a patient wherein said disease is ed from the group comprising sepsis, diabetes, cancer, acute and chronic ar diseases as e.g. heart failure, shock as e.g. septic shock and organ dysfunction as eg. kidney dysfunction. Especially useful is the antibody or fragment or scaffold according to the t invention for reducing the risk of mortality dur~ ing sepsis and septic shock, 1'. 6. late phases of sepsis.

The antibodies, antibody fragments, lds and combinations of the present invention may be used in therapy or prevention of a chronic or acute disease of a patient: 0 for the prevention of organ ction or organ failure, especially kidney dysfunction or kidney failure and f or, o for stabilizing the circulation, e.g. for reducing the requirement of vasopressors eg. catecholamine requirement of said patient and / or, o for regulating the fluid balance in said t. a for reducing the mortality risk for said patient.

In one embodiment the anti-ADM antibody or an anti—adrenomedullin antibody fragment or anti—ADM non—1g scaffold is used in therapy or prevention of a chronic or acute disease of a patient according to the present invention n said patient is an ICU patient. In another embodiment the anti-ADM antibody or an anti—adrenomedullin antibody fragment or anti~ ADM non—1g scaffold is used in therapy or prevention of a chronic or acute disease or acute condition of a patient according to the present invention, wherein said patient is critically ill.

Critically ill means a t is having a disease or state in which death is possible or immi- nent.

Subject of the t invention is further an anti—ADM antibody or an anti-adrenomedullin dy fragment or anti—ADM non—lg scaffold for use in therapy or prevention of a chronic or acute disease or acute condition of a patient according to the t invention wherein said antibody or fragment or scaffold is to be used in combination ofADM binding protein.

Subject of the present ion is further a pharmaceutical formulation comprising an anti- ADM antibody or anti-ADM antibody fragment or anti-ADM non—1g scaffold according to the present invention.

Subject of the present invention is further a pharmaceutical formulation according to the pre— sent invention, wherein said pharmaceutical formulation is a solution, preferably a ready—to» use solution.

Said ceutical ation may be administered intramuscular. Said pharmaceutical formulation may be administered intra—vascular. Said pharmaceutical formulation may be administered via infusion.

In another embodiment subject of the present invention is further a ceutical formula- tion according 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 invention is r a pharmaceutical a- tion according to the present invention, wherein said pharmaceutical ation is in a freeze—dried state.

WO 72512 _ 31 _ In another embodiment of the ion the pharmaceutical formulation in accordance with the invention as may be administered intra—muscular, intra—vascular, or Via infusion is pref— erably administered to a t systemically.

Therefore, in another embodiment of the present ion the pharmaceutical ation according to the present invention is to be administered systemically to a patient.

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 therapy of a chronic or acute disease of a patient for the regulation of liquid balance. 2. ADM antibody or an adrenomedullin dy fragment according to claim 1 wherein the antibody format is ed from the group comprising FV fragment, scFv frag— ment, Fab fragment, scFab fragment, (Fab)2 fragment and scFv-Fc Fusion protein. 3. ADM antibody or an adrenomedullin antibody fragment according claim 1 or 2 wherein said antibody or fragment binds to the N—terminal part (aa 1—21) of adrenomedullin. 4. ADM antibody or an adrenomedullin antibody fragment acc0rding 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 adrenomeduilin antibody fragment ing to any of claims 1 to 4, wherein said antibody or fragment is an ADM stabilizing antibody or ADM sta~ bilizing a dy fragment that enhances the tm half retention time of adrenome- dullin in serum, blood, plasma at least 10 “/6, preferably at least 50 %, more preferably >50 %, most preferably >100 %. 6. ADM antibody or an adrenomedullin antibody fi'agment ing 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%. — 32 — ADM dy or an adrenomedullin antibody fragment for use in y of a chronic or acute disease of a patient according to any of claims 1 to 6 wherein said disease is selected from the group comprising sepsis, diabetis, cancer, heart failure, shock and kidney dysfixnction.

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

ADM antibody or an adrenomedullin antibody fragment for use in therapy of a chronic or acute disease of a patient according to any of claims 1 to 7 n said an— tibody or fragment is a modulating antibody or fragment that enhances the t1,2 half re~ tention time of adrenomeduilin in serum, blood, plasma at least 10 %, preferably at least 50 %, more preferably >50 %, most preferably >100 % and that blocks the bioac— tivity ofADM to less than 80 ”/0, preferably less than 50%. . ceutical formulation comprising an antibody or fragment according to any of claims 1 to 9. ll. Pharmaceutical formulation according to claim 10 n said pharmaceutical for- muiation is a solution, ably a ready-towuse solution 12. Pharmaceutical formulation according to claim 10 wherein said pharmaceutical for- mulation is in a freeze~dried state. 13. Pharmaceutical formulation according to any of claims 10 to 11, wherein said phar- maceutical formulation is administered muscular. 14. Pharmaceutical formulation according to any of claims 10 to 11, wherein said phar- maceutical formulation is administered intra‘vascular.

. Pharmaceutical formulation ing to claim 14, wherein said pharmaceutical for— mulation is administered via infusion.

Further embodiments within the scope of the present invention are set out below: - 33 u Adrenomedullin ADM dy or an adrenomedullin dy fragment an ADM non-lg scaffold for use in therapy of a chronic or acute disease or acute condition of a t for the regulation of fluid balance.

ADM antibody or an adrenomedullin antibody fragment or ADM non—1G scaffold ac cording to claim 1 wherein said ADM antibody or an adrenomedullin antibody frag— ment or ADM non—lG scaffold is a non—neutralizing ADM antibody or a non— neutralizing adrenomedullin antibody fragment or a non—neutralizing ADM non—1G scaffold.

DJ Adrenomedullin ADM antibody or an adrenomedullin antibody fragment or an ADM non—lg scaffold for use in therapy of a chronic or acute disease or acute condition ac— cording to claim 1 or 2 for preventing or reducing edema in said patient.

ADM antibody or an adi'enomedullin dy fragment or ADM non—IG ld ac— cording 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 frag— ment and scFV—Fc Fusion protein.

ADM antibody or an adrenomedullin antibody fragment or ADM non—1G ld ac- cording to any of claims 1 to 4, wherein said antibody or fragment or scaffold binds to the inal part (aa 1-21) of adrenomedullin.

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

ADM dy or an adrenomedullin antibody fragment or ADM non-1G scaffold ac— cording to any of claims 1 to 6, wherein said antibody or fragment or scaffold is an ADM stabilizing antibody or ADM stabilizing antibody fragment or ADM stabilizing non-1G scaffold that enhances the half life (ll/2 half retention time) of adrenomedullin in serum, blood, plasma at least 10 “/0, ably at least 50 %, more preferably >50 %, most preferably >100 0/0. — 34 — 8. ADM antibody or an medullin dy fragment or ADM non—1G scaffold ac— cording to any of claims 1 to 7, wherein said antibody or fragment blocks the iv- ity ofADM to less than 80 0/6, preferably less than 50%. 9. 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 . ADM antibody or an adrenomedullin antibody fragment according to any of claims 1 to 9, wherein said antibody or fragment is a human onal antibody or fragment that binds to ADM or an dy fragment thereof wherein the heavy chain com— prises the sequences SEQ ID NO: 1 GYTFSRYW SEQ ID NO: 2 ILPGSGST SEQ ID NO: 3 TEGYEYDGFDY and wherein the light chain comprises the sequences SEQ ID NO:4 QSIVYSNGNTY SEQ ID NO: 5 3O RVS — 35 - SEQ ID N016 FQGSHIPYT. 11. A human monoclonal antibody or fragment that binds to ADM or an antibody frag— ment thereof according to claim 10 n said antibody or fragment comprises a se- quence selected from the group comprising: SEQ ID NO: 7 (AM-VH—C) SGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEIL PGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGF DYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTK VDKRVEPKHHHHHH SEQ ID NO: 8 {AM—VH1) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRI LPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDG FDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKRVEPKHHHHHH SEQ ID NO: 9 (AM—VH2—E40) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRI LPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSBDTAVYYCTEGYEYDG FDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKRVEPKHHHHHH SEQ ID NO: 10 (AM-VH3—T26—E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEI TNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDG FDYWGQGTTVTVSSASTKGPSVFPLA98SKSTSGGTAALGCLVKDYFPEPVTV — 36 - SWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNT KVDKRVEPKHHHHHH SEQ ID NO: 11 (AM—VH4-T26—E40—E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEI LPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDG FDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNT KVDKRVEPKHHHHHH SEQ ID NO: 12 (AM-VL—C) DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIY RVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTK LEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC SEQ ID NO: 13 (AM-NU) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIY RVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGT KLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC SEQ ID NO: 14 (AM—VLZ-E40) DWMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIY RVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGT KLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC 12. ADM dy or an adrenomeduflin antibody nt or ADM non—1G scaffold for use in therapy of a chronic or acute e of a patient according to any of claims 1 t0 9 wherein said patient is an ICU patient. - 37 _ 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 claims 1 to 12 wherein said antibody or fragment or scaffold is a modulating antibody or fragment or scaffold that enhances the half life (121/2 half retention time) of medullin in se~ rum, blood, plasma at least 10 "/0, preferably at least 50 %, more preferably >50 ”/0, most preferably >100 % and that blocks the bioactivity of ADM to less than 80 “/0, preferably less than 50%. i4. 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 13 to be used in combination with catecholamine and/ or fluids administered in— travenously. 15. ADM antibody or adrenomedullin antibody fragment or ADM non~IG scaffold for use in y of a chronic or acute disease 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 bind— ing protein and/or further active ingredients. 16. Pharmaceutical formulation comprising an antibody or fragment or scaffold according to any of claims 1 to 15. 17. Pharmaceutical formulation according to claim 16 wherein said pharmaceutical for- mulation is a solution, preferably a to—use solution. 18. Pharmaceutical formulation according to claim 16 wherein said pharmaceutical for— mulation is in a freeze—dried state. 19. Pharmaceutical ation ing to any of claims 16 to 17, wherein said phar— maceutical formulation is administered intramuscular.

. Pharmaceutical formulation according to any of claims 16 to 17, n said phar- maceutical formulation is stered intra-vascular. 21. Pharmaceutical formulation according to claim 20, wherein said pharmaceutical for— mulation is stered Via infusion.

Further embodiments within the scope of the present invention are set out below: I. Adrenomeduliin (ADM) antibody or an adrenomedullin antibody fragment for use in therapy of a chronic or acute disease of a patient for stabilizing the circulation. 2. ADM antibody or an medullin antibody fragment according to claim 1 wherein said antibody or fragment s the catecholarnine requirement of said patient. 3. ADM antibody or an adrenomednllin antibody fragment according to claim 1 or 2 wherein the dy format is selected from the group sing Fv fragment, SCFV fragment, Fab fragment, scFab fragment, (Fab)2 fragment and scFv—Fc Fusion protein. 4. ADM dy or an adrenomedullin antibody fragment according to any of claims 1 to 3 wherein said antibody or nt binds to the N~terminai part (aa 1—21) of adrenomedullin.

. ADM antibody or an adrenomednilin antibody fragment according to any of claims 1 to 4, wherein said antibody or fragment izes and binds to the N~terminal end (aal) of adrenomedullin. 6. ADM antibody or an adrenomedullin antibody fragment according to any of claims 1 to 5, wherein said antibody or fragment is an ADM izing antibody that es the tl/2 half retention time of adrenomedullin in serum, blood, plasma at least 10 ”/0, preferably at least, 50 %, more preferably > 50 %, most preferably >100 %. 7. ADM antibody or an adrenomeduliin antibody fragment according to any of claims 1 to 6, wherein said antibody or fragment blocks the ivity 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 antibody or a modulat- ing adrenomedullin antibody fragment that enhances the ti/Z half retention time of — 39 - adrenomedullin in serum, blood, plasma at least 10 %, preferably at least, 50 0/0, more preferably > 50 %, most preferably >100 % and that blocks the bioactivity of ADM to less than 80 %, ably less than 50 %: ADM antibody or an adrenomedullin antibody fragment for use in therapy of a chronic 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 e.g. septic shock and organ dysfunction as e.g. kidney dysfunction.

. Pharmaceutical formulation comprising an antibody according to any of claims 1 to 9. ll. Pharmaceutical formulation according to claim 10 wherein said pharmaceutical for- on is a solution, preferably a ready—to—use on. 12. Pharmaceutical formulation according to claim 10 wherein said pharmaceutical for- on is in a freeze—dried state. 13. Pharmaceutical formulation ing to any of claims l0 to 11, wherein said phar~ maceutical formulation is administered intra—muscular. 14. Pharmaceutical formulation according to any of claims 10 to ll, wherein said phar- maceutical ation is administered intra—vascular. 15. Pharmaceutical formulation according to claim 14, wherein said ceutical for— mulation is administered via infusion.

Further embodiments within the scope of the present invention are set out below: 1. medullin (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. — 40 — ADM antibody or an adrenomedullin antibody fragment or ADM non—1G scaffold ac- cording to claim 1 wherein said antibody or fragment or scaffold reduces the vasopres- sor requirement, e.g. catecholamine requirement of said patient.

ADM antibody or an adrenomedullin antibody fragment or ADM" non—IG scaffold ac— cording to claim 1 or 2 wherein said ADM antibody or an adrenomedullin antibody fragment or ADM non~lG scaffold is a utralizing ADM antibody or a nonm lizing adrenomedullin dy fragment or a non—neutralizing ADM non—IG scaf— fold.

ADM antibody or an adrenomedullin antibody fragment according to any of claims 1 t0 3 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 medullin antibody fragment or ADM non—IG scaffold ac- cording 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 nonulG scaffold ac— cording to any of claims 1 to 5, wherein said antibody or fragment or scaffold recog— nizes and binds to the N—terminal end (aal) of medullin.

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

ADM antibody or an adrenomcdullin antibody fragment or ADM nonmlG scaffold ac- cording to any of claims 1 to 7, wherein said antibody or fragment or ld blocks the bioactivity of ADM to less than 80 0/0, ably less than 50 %.

ADM antibody or an adrenomedullin antibody fragment or ADM non-IG scaffold ac— cording to any of claims 1 to 8, wherein said antibody or fragment or scaffold is a - 41 _ modulating ADM antibody or a modulating adrenomedullin antibody fragment or scaffold that enhances the half life (tl/2 half retention time) of adrenomedullin in se~ rum, blood, plasma at least 10 %, preferably at least, 50 %, more preferably > 50 %, most preferably >100 % and that blocks the bioactivity of ADM to less than 80 “/0, preferably less than 50 %: . ADM antibody or an adrenomedullin dy fragment according to any of claims 1 to 9, wherein said antibody or fragment is a human monoclonal antibody or fragment that binds to ADM or an antibody fragment thereof wherein the heavy chain comm prises the ces SEQ ID NO: 1 GYTFSRYW SEQ ID NO: 2 IL?GSGST SEQ ID NO: 3 DGFDY and wherein the light chain comprises the sequences SEQ ID N014 NGNTY SEQ ID NO: 5 SEQ ID NO: 6 FQGSHIPYT. 11. A human monoclonal antibody or fragment that binds to ADM or an antibody frag- ment thereof according to claim 10 wherein said antibody or fragment comprises a se- quence seiected from the group comprising : SEQ ID NO: 7 (AM-VH—C) QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEIL PGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAWYCTEGYEYDGF DYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEFVTVS TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKRVEPKHHHHHH SEQ ID NO: 8 (AM—VH1) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRI TNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDG FDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKRVEPKHHHHHH SEQ ID NO: 9 (AM—VH2-E40) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRI LPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDG GTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKRVEPKHHHHHH SEQ ID NO: 10 (AM—VH3-T26—E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEI LPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSBDTAVYYCTEGYEYDG FDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVV’I‘VPSSSLGTQTYICNVNHKPSNT KVDKRVEPKHHHHHH SEQ ID NO: 11 (AM—VH4~T26—E40-E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEI LPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDG FDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT EPKHHHHHH SEQ ID NO: 12 (AM—VL-C) DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIY RVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTK VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC SEQ ID No: 13 (AM—VLl) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIY RVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGT KLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC SEQ ID NO: 14 (AM~VL2—E40) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIY RVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGT KLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGBC 12. ADM antibody or an medullin antibody fragment or ADM non-1G scaffold for use in therapy of a chronic or acute disease of a patient according to any of the claims 1 to 11 wherein said disease is selected from the group comprising SIRS, sepsis, dia- betis, cancer, acute and chronic vascular es as e.g. heart failure, shock as e.g. septic shock and organ dysfunction as eg. kidney dysfunction. u 44 - 13. ADM dy or an adrenomedullin antibody fragment or ADM non—1G scaffold for use in therapy of a chronic or acute disease of a patient according to any of the claims 1 to 12 to be used in combination with catecholamine and/ or fluids stered in- travenously. 14. ADM antibody or 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 13 or a combination according to claim 10 to be used in combination with ADM bind- ing protein and/or further active ients.

. Pharmaceutical ation comprising an antibody or fragment or non-1G scaffold according to any of claims 1 to 14. 16. Pharmaceutical formulation according to claim 15 n said pharmaceutical for— mulation is a solution, preferably a ready—tomuse on. 17. Pharmaceutical formulation according to claim 15 wherein said pharmaceutical for— mulation is in a freeze—dried state. 18. Phannaceutical formulation according to any of claims 15 to 16, wherein said phar- maceutical fonnulation is administered muscular. 19. Pharmaceutical formulation according to any of claims 14 to l6, n said phar- maceutical formulation is administered intra—vascular.

. Pharmaceutical formulation according to claim 16, wherein said pharmaceutical for— mulation is administered via infusion.

Further embodiments within the scope of the present invention are set out beiow: Adrenomedullin antibody or an adrenornedullin antibody fragment for use in a treat— ment of a chronic or acute disease wherein said antibody or said fragment is an ADM izing antibody or fragment that enhances the iii/2 half retention time of adrcnome- dullin in serum, blood, plasma at least 10 %, preferably at least, 50 %, mere preferably - 45 a 2012/072931 >50 “/0, most preferably 100 % and/or wherein said antibody blocks the bioactivity of ADM to less than 80 0/0, preferably to less than 50 %. 2. Adrenomeduilin antibody or an adrenomedullin dy fragment for use in a treat- ment of a chronic or acute disease wherein said antibody or said fragment is a modu- lating ADM antibody or fragment that enhances the 131,12 half retention time of adrenomedullin in serum, blood, plasma at least 10 %, preferably at least, 50 %, more preferably >50 0/0, most preferably 100 % and that blocks the ivity of ADM to less than 80 %, preferably to less than 50 %. 3. Adrenornedullin antibody or an adrenomedullin antibody fragment for use in a treat- ment of a chronic or acute disease according to claim 1 or 2, wherein said antibody or fragment binds to the N—tenninal part (aa 1—21) of medullin. 4. Adrenomedullin antibody or an adrenomedullin antibody fragment for use in a treat— ment of a chronic or acute disease wherein said antibody or said fragment according to claim 3 binds to the N—terminal end of adrenomedullin.

. Adrenomednllin antibody or an adrenornedullin antibody fragment for use in use in a treatment of a c or acute disease ing to any of claims 1 to 4, wherein said antibody or said fragment is an ADM stabilizing antibody or fragment that enhances the the half retention time of adrenomedullin in serum, blood, plasma at least 10 %, preferably at least, 50 %, more ably >50 %, most preferably 100 %. 6. Adrenomedullin antibody or an adrenomedullin antibody fragment for use in a treat- ment of a chronic or acute disease according to any of claims 1 to 5, wherein said an— tibody or said fragment blocks the bioactivity of ADM to less than 80 %, preferably to less than 50 0/0. 7. Adrenomedullin antibody or an adrenomedullin dy fragment according to any of the claims 1 to 6 for use in a treatment of a chronic or acute disease wherein said dis- ease 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. n 46 _ 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 dis- ease is septic shock or .

Adrenomedullin antibody or an adrenomedullin antibody fi‘agment for use in a treat— ment of a c or acute disease according to any of the claims 1 to 8 wherein said antibody or fragment regulates the liquid balance of said patient.

. Adrenomedullin antibody or an adrenomedullin antibody fragment for use in a treat- ment of a chronic or acute disease according to any of the claims 1 to 9 wherein said antibody or fragment used for tion of organ dysfunction or organ failure. 11. Adrenomedullin antibody or an medullin antibody fragment for use in a treat- ment of a chronic or acute e according to claim 10 wherein said antibody or nt is used for prevention ofkidney dysfunction or kidney faiiure. 12. Adrenomedullin (ADM) antibody or an medullin antibody fragment for use in a treatment of a chronic or acute disease in a patient according to claims 1 to 11 wherein said antibody or fragment is used for stabilizing the circulation. 13. ADM antibody or an adrenomedullin antibody nt for use in a ent of a chronic or acute disease in a patient according to claim 12 n said antibody or fragment reduces the catecholalnine requirement of said patient. 14. ADM antibody or an adrenomedullin antibody fragment for use in a treatment of a c or acute disease in a patient according to any of claims 1 to 13 for the reduc- tion 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 wherein said antibody or fragment may be administered in a dose of at least 3 ng / Kg body weight. 16. Pharmaceutical composition comprising an antibody or fragment according to any of claims 1 to 15. - 47 _ Further embodiments within the scope of the present invention are set out below: Adrenomedullin antibody or an adrenomedullin antibody fragment or ADM non—lg scaffold wherein said antibody or said nt or scaffold is a utralizing anti— body.

Adrenornedullin antibody or an adrenomedullin antibody nt or ADM non—lg scaffold n said antibody or said fragment or scaffold is an ADM stabilizing an“ tibody or fragment or 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 100 % and/or wherein said dy or fragment or scaffold blocks the bioactivity ofADM to less than 80 %, preferably to less than 50 0/0.

Adrenomedullin dy or an adrenomedullin antibody fragment or ADM non—lg scaffold wherein said antibody or said fragment is a modulating ADM antibody or fragment or ld that enhances the half life (ll/2 half retention time) of adrenome- dullin in serum, blood, plasma at least 10 9/0, preferably at least, 50 %, more preferably >50 %, most preferably 100 % and that blocks the bioactivity of ADM to less than 80 “/0, preferably to less than 50 %.

Adrenomedullin antibody or an adrenomedullin antibody fragment or ADM non—lg scaffold according to claim 1 or 2, wherein said antibody or fragment or scaffold binds to the N—tenninal part (aa 1—21) of adrenomedullin. rnednllin antibody or an adrenomedullin dy fragment or ADM non-lg scaffold wherein said antibody or said nt or scaffold according to claim 3 binds to the N—tenninal end of adrenomedullin.

Adrenomedullin antibody or an adrenomedullin antibody fragment ADM non—lg scaf- fold according to any of claims 1 to 4, wherein said antibody or said fragment or said scaffold is an ADM stabilizing antibody or fragment that enhances the l1/2 half reten- tion time of adrenomcdullin in serum, blood, plasma at least 10 %, preferably at least, 50 %, more preferably >50 %, most preferably 100 %. '7. Adrenomedullin antibody or an adrenomedullin antibody fragment or ADM non—lg scaffold according to any of the claims 1 to 6 for use as an active pharmaceutical sub» stance. 8. Adrenomedullin antibody or an adrenornedullin antibody nt ADM non~Ig scaf— fold ing to any of the claims 1 to 7 for use in a treatment of a chronic or acute disease or acute condition wherein said e or condition is selected from the group comprising severe infections as e. g. meningitis, systemic inflammatory Response— Syndrome ) sepsis; other diseases as diabetes, cancer, acute and chronic vascum lar diseases as eg. heart failure, myocardial infarction, stroke, atherosclerosis; shock as e.g. septic shock and organ dysfunction as e.g. kidney dysfunction, liver dysfunc- tion, burnings, surgery, traumata. 9. Adrenomedullin antibody or an adrenomedullin antibody fragment or ADM non—1g ld according 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. l0. ADM antibody or an adrenomedullin antibody fragment according to any of claims 1 to 9, wherein said antibody or fragment is a human monoclonal dy or fragment that binds to ADM or an antibody fragment thereof wherein the heavy chain com— prises at least one of the sequences : SEQ ID NO: 1 SEQ ID NO: 2 ILPGSGST SEQ ID NO: 3 TEGYEYDGFDY And/or wherein the light chain comprises the at least one of the sequences WO 72512 _ 49 ..

SEQ ID NO:4 QSIVYSNGNTY SEQ ID NO: 5 RVS SEQ ID NO: 6 FQGSHIPYT. 11. A human monoclonal antibody or nt that binds to ADM or an antibody frag ment thereof ing to claim 10 wherein said antibody or fragment comprises a se- quence selected from the group composing: SEQ ID NO: 7 (AM—VH—C) QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEIL PGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGF DYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKRVEPKHHHHHH SEQ ID NO: 8 (AM-VH1) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRI LPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDG FDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKRVEPKHHHHHH SEQ ID NO: 9 (AM—VH2—E40) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRI LPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDG ~ 50 - GTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKRVEPKHHHHHH SEQ ID NO: 10 (AM—VH3—T26-E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEI LPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDG FDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNT KVDKRVEPKHHHHHH SEQ ID NO: 11 (AM—VH4—T26-E40—ESS) SGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEI LPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDG FDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKRVEPKHHHHHH SEQ ID NO: 12 (AM—VL-C) DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIY RVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTK LEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS VTEQDSKDSTYSLSSTLTLSKADYBKHKVYACEVTHQGLSSPVTKSF NRGEC SEQ ID NO: 13 (AM—VLI) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIY RVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGT KLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC SEQ ID NO: 14 (AM-VLZ—E40) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIY RVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGT - 5] _ KLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC 12. Adrenomedullin antibody or an adrenomedullin antibody fragment or ADM nondg scaffold according to any of the claims 1 to 11 for regulating the fluid balance in a pa- tient having a c or acute disease or acute condition. 13. Adrenomedullin antibody or an adrenomedullin antibody fragment or ADM non-1g scaffold according to any of the claims 1 to 11 for preventing or reducing organ dys— function or organ e in a patient having in a chronic or acute disease or acute con— dition. 14. Adrenomedullin antibody or an adrenomedullin antibody fragment or ADM non-1g scaffold according to claim 10 wherein organ is kidney or liver.

. Adrenomedullin (ADM) antibody or an adrenomedullin dy fragment or ADM non—1g scaffold according to claims 1 to 14 for izing the ation in a patient having a chronic or acute disease or acute condition. 16. ADM antibody or an adrenomedullin dy fragment or ADM non-lg 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 catecholamine requirement of said pa- ti ent. 17. Adrenomedullin antibody or an adrenornedullin antibody fragment or ADM non-1g scaffold according to any of the claims 1 to 16 to be used in combination with vaso- pressors e. g. olamine. 18. Adrenomedullin dy or an medullin antibody fragment or ADM non—1g scaffold according to any of the claims 1 to 1’? to be used in combination with intrave— nous fluid administration. 19. Adrenomedullin antibody or an adrenomedullin antibody fragment or ADM non—1g scaffold according to any of the claims 1 to 18 to be used in combination with an TNF—alpha—antibody. - 52 _ 2012/072931 . ADM antibody or an adrenomedullin antibody nt or non—Ig—scaffold according to any of claims 1 to 19 for use in a treatment of a patient in need thereof wherein said dy or fragment may be administered in a dose of at least 3 ug / Kg body weight. 21. Pharmaceutical composition comprising an antibody or fragment or id according to any of claims 1 to 20. 22. ADM antibody or an adrenornedullin antibody fragment or —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 adrenomedullin antibody fragment or non—lg-scaffold according to claim 22 wherein said disease is .

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 severe chronicai or acute disease of a patient for the reduction of the mor- tality risk for said t.

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

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

ADM antibody or an adrenomedullin antibody fragment according to any of claims 1 to 33 wherein said antibody or nt recognizes and binds to the Nfiterminai end (aal) of adrenomedullin.

WO 72512 - 53 _ ADM antibody or an adrenomedullin antibody fragment according to any of claims 1 to 4, wherein said antibody or nt is an ADM stabilizing 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 ADM antibody or an medullin antibody fragment ing to any of claims 1 to 5, wherein said antibody or fragment blocks the ivity of ADM to less than 80 %, preferably less than 50%.

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

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

ADM antibody or an adrenomedullin antibody fragment for use in y of a chroni- cal or acute disease of a patient according to any of claims 1 to 8 wherein the ity risk is reduced by preventing adverse event wherein the latter are ed from the group comprising SIRS, sepsis, septic shock, organ failure, kidney failure, liquid dys~ balance and low blood pressure.

. ADM antibody or an adrenomedullin antibody fragment for use in therapy of a chroni— cal or acute disease of a patient according to any of claims 1 to 8 wherein said anti- body or fragment is to be used in combination ofADM g protein. ll. Pharmaceutical formulation comprising an dy or fragment according to any of claims 1 to 10. 12. Pharmaceutical formulation according to claim 11 wherein said pharmaceutical formu- lation is a solution, preferably a ready—to-use solution. _ 54 .. 13. Pharmaceutical formulation according to claim 11 wherein said pharmaceutical formu— lation is in a freeze—dried state. 14. ceutical formulation according to any of claims 11 to 12, n said - ceutical formulation is administered ultra—muscular.

. Pharmaceutical formulation according to any of claims 11 to 12, wherein said ~ ceutical formulation is administered intra—vascular. 16. Pharmaceutical formulation according to claim 15, wherein said pharmaceutical for- mulation is stered Via infiision. r embodiments within the scope of the present invention are set out below: Adrenomedullin (ADM) antibody or an adrenomedullin antibody fragment or ADM non-1g scaffold for use in therapy of a severe chronical or acute disease or acute condi— tion of a patient for the reduction of the mortality risk for said patient wherein said an— tibody or fragment or scaffold is a non-neutralizing ADM antibody or a non- neutralizing adrenomedullin antibody fragment or a non—neutralizing ADM non-lg scaffold.

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

ADM antibody or an medullin antibody fragment or an ADM non-lg scaffold according claim 1 or 2 wherein said antibody or fragment or scaffold binds to the N- al part (aa 1—21) of adrenorneduilin.

ADM antibody or an adrenomedullin antibody fragment or an ADM non—1g scaffold according to any of claims 1 to 3, wherein said antibody or fragment or scaffold rec- ognizes and binds to the N—terminal end (aal) of adrenomedullin. - 55 _ ADM antibody or an adrcnomedullin antibody fragment or an ADM non—lg scaffold ing 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 (“cl/2 half retention time) of adrenomedullin in serum, blood, plasma at least 10 %, preferably at least, 50 ”/0, more preferably > 50 %, most preferably > 100 %.

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

ADM antibody or an adrenoniedullin antibody nt 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 t0 6 wherein said disease is selected from the group comprising severe infec— tions as e.g. meningitis, Systemic atory Response—Syndrom (SIRS,) sepsis; other diseases as diabetis, cancer, acute and chronic vascular diseases as eg. heart failure, myocardial infarction, , atherosclerosis; shock as e.g. septic shock and organ dysfunction as e.g. kidney dysfunction, liver dysfiinction; burnings, surgery, traumata.

ADM antibody or an medullin antibody fragment or an ADM non~1g ld for use in therapy of a chronical or acute disease of a patient according to any of claims 1 to 7 wherein said disease is selected from the group comprising SIRS, a se- vere infection, sepsis, shock e.g.septic shock .

ADM antibody or an adrenomedullin antibody nt or an ADM non-lg scaffold for use in therapy of a chronicai or acute disease or acute condition of a patient ac- cording to any of claims 1 to 8 wherein said patient is an ICU patient. ADM antibody or an adrenomedullin antibody fragment or an ADM non-lg scaffold for use in therapy of a chronical or acute disease or acute condition of a patient according to any of claims 1 to 9 n the mortality risk is reduced by preventing an adverse event wherein the latter are ed from the group comprising SIRS, sepsis, shock as cg. septic shock, acute and c vascular diseases as e.g. acute heart failure, myocar- dial infarction, stroke; organ failure as eg, kidney failure, liver failure, fluid dysbal- ance and low blood pressure.

. ADM antibody or an adrenomedullin antibody fragment according to any of claims 1 to 9, wherein said dy or fragment is a human onal antibody or fragment that binds to ADM or an antibody fragment thereof wherein the heavy chain com— prises the ces SEQ ID NO: 1 GYTFSRYW SEQ ID NO: 2 ILPGSGST SEQ ID NO: 3 TBGYEYDGFDY and wherein the light chain comprises the sequences SEQ ID NO:4 NGNTY SEQ ID NO: 5 SEQ ID NO: 6 FQGSHIPYT. 12. A human monoclonal antibody or fragment that binds to ADM or an antibody frag- ment thereof according to claim 10 wherein said antibody or fragment comprises a se- quence selected from the group comprising : - 57 _ SEQ ID NO: 7 (AM—VH-C) SGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEIL PGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGF DYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTK VDKRVEPKHHHHHI—I SEQ ID NO: 8 (AM-VH1) SGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRI LPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDG FDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKRVEPKHHHHHH SEQ ID NO: 9 (AM-VH2~E40) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRI LPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDG FDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKRVEPKHHHHHH SEQ ID NO: 10 B—TZG—ESS) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLBWMGEI LPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDG FDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKRVEPKHHHHHH SEQ ID NO: 11 (AM-VH4—T26-E40-E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEI LPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDG FDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKRVEPKHHHHHH _ 58 « SEQ ID NO: 12 (AM—VL-C) DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLlY RVSNRFSGVPDRFSGSGSGTEFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTK LEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTI—IQGLSSPVTKSF NRGEC SEQ ID NO: 13 (AM—VLl) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIY RVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGT KLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC SEQ ID NO: 14 (AM-VLZ—E40) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIY i5 RVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGT KLEIKRTVAA?SVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC 13. ADM antibody or an adrenomedullin antibody nt or ADM non—1G scaffold for use in therapy of a c or acute disease of a patient ing to any of the claims 1 to 12 to be used in combination with vasopressors e.g. olamine and/ or fluids administered intravenously. 14. ADM antibody or adrenornedullin 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 13 or a combination according to claim 10 to be used in combination with ADM bind— ing protein and/or further active ingredients. 15. Pharmaceutical formulation comprising an dy or fragment or scaffold according to any of claims 1 to 14.

WO 72512 _ 59 — 16. Pharmaceutical ation according to claim 15 wherein said pharmaceutical for— muiation is a solution, preferably a ready—to-use solution. 17. ceutical formuiation according to claim 15 wherein said pharmaceuticai for— rnuiation is in a freeze—dried state. 18. Pharmaceutical formulation according to any of claims 15 to 16, wherein said phar- ical formulation is administered intra—muscular. 19. Pharmaceutical formulation according to any of claims 15 to 16, wherein said phar— ical ation is administered intrawvascular.

. Pharmaceutical formulation according to claim 19, n said pharmaceutical for— mulation is administered via infusion. 21. ADM antibody or an Adrenomeduiiin antibody nt or AM non—1g scaffold, wherein said antibody or fragment or scaffold binds to the N-terminal part (aa 1-21) of Adrencmeduliin in, preferably human ADM. 22. Antibody or fragment or scaffold according to claim 2, wherein said antibody or frag- ment or scaffold recognizes and binds to the inal end (aa 1) of Adrenomeduliin.

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

ADM antibody or an adrenomeduliin antibody fragment for use in therapy of a chronical or acute disease according to claim 1 wherein said organ is kidney. - 6U - ADM antibody or an adrenornedullin dy fragment according to claim 1 wherein the antibody format is selected from the group comprising FV nt, scFV fragment, Fab fragment, scFab fragment, (Fab)2 fragment and scFV—Fc Fusion protein.

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

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

ADM antibody or an adrenomedullin dy fragment according to any of claims 1 to , wherein said antibody or said fragment is an ADM stabilizing 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%.

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

ADM antibody or an adrenomedullin antibody fragment for use in therapy of a chronical or acute disease of a patient ing to any of claims 1 to 7 wherein said disease is se- lected from the group comprising sepsis, diabetis, cancer, heart failure, and shock.

ADM antibody or an adrenomeduilin antibody nt for use in therapy of a chronieal or acute disease 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 for use in therapy of a chronical or acute disease of a patient according to any of claims 1 to 9 wherein said dy or fragment is a modulating antibody or fragment that enhances the t1/2 half retention time of meduliin in serum, blood, plasma at least 10 %, preferably at least 50 %, more preferably >50 0/0, most preferably >100% and that blocks the bioactivity of ADM to less than 80 %, preferably less than 50%. _ 61 _ 11. Pharmaceutical ation comprising an antibody or fragment according to any of claims I to 10. 12. Pharmaceutical formulaticn according to claim 11 wherein said pharmaceutical formula- tion is a solution, preferably a ready—to—use solution. 13. Pharmaceutical formulation according to claim 11 wherein said pharmaceutical formula— tion is in a -dried state. 14. ceutical formulation according to any of claims 11 to 12, wherein said phanna» ceutical formulation is administered intra—rnuscular.

. Pharmaceutical formulation according to any of claims ll to 12, wherein said - ceutical formulation is administered intra—Vascular. 16. Pharmaceutical formulation according to claim 15, wherein said pharmaceutical formu— lation is administered via infusion.

Further embodiments within the scope of the present invention are set out below: 1. Adrenornedullin (ADM) antibody or an adrenomedullin antibody fragment or ADM non—lg ld for use in therapy of a chronical or acute disease or acute condition of a patient for tion or reduction of organ dysfunction or tion of organ fail— ure in said patient. 2. ADM antibody or an adrenomedullin antibody fragment or ADM non—1g scaffold for use in therapy of a chronical or acute disease or acute e according to claim 1 wherein said organ is kidney or liver. 3. ADM antibody or an adrenomedullin antibody fragment or ADM non—1G scaffold ac- cording to claim 1 or 2 wherein said ADM dy or an adrenomedullin antibody fragment or ADM non—IG scaffold is a non—neutralizing ADM antibody or a non— neutralizing adrenomedullin dy fiagment or a non—neutralizing ADM non—IG scaffold _ 62 _ 4. ADM antibody or an adrenomedullin antibody fragment or ADM non~lG scaffold ac- cording to any of claims 1 or 3 wherein the antibody format is selected from the group comprising FV fragment, scFV fragment, Fab fragment, scFab nt, (Fab)2 frag- ment and scFv—Fc Fusion protein.

. ADM antibody or an adrenomedullin antibody fragment or ADM non-1G scaffold ac— cording any of claims 1 to 4 n said antibody or fragment or ld binds to the N—terminal part (aa 1—21) of adrenomedullin. 6. ADM antibody or an adrenomedullin antibody fragment or ADM non—1G scaffold ac~ cording to any of claims 1 to 5, wherein said antibody or fragment or scaffold recog- nizes and binds to the N—terminal end (aal) of adrenomedullin. 7. ADM dy or an adrenomedullin antibody fragment or ADM non—1G scaffold ac~ cording to any of claims 1 to 6, wherein said antibody or said fragment or scaffold is an ADM stabilizing antibody or fragment or ld that enhances the half life (tl/2 half retention time) of adrenomeduilin in serum, blood, plasma at least 10 %, prefera- bly at least 50 %, more preferably >50 %, most preferably >100%. 8. ADM antibody or an adrenomedullin antibody fragment or ADM non~lG scaffold ac— cording to any of claims 1 to 7, wherein said antibody or fragment or scaffold blocks the bioactivity of ADM to less than 80 0/0, preferably less than 50%. 9. ADM antibody or an adrenomedullin antibody nt or ADM non-IG ld for use in y of a chronical or acute disease or acute condition of a t according to any of claims 1 to 8 wherein said disease is selected from the group comprising sep— sis, is, cancer, heart failure, and shock.

. ADM antibody or an adrenorneduilin antibody fragment according to any of claims 1 3O 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 com- prises the sequences SEQ ID N0: 1 _ 63 — GYTFSRYW SEQ ID NO: 2 ILPGSGST SEQ ID NO: 3 TEGYEYDGFDY and n the light chain comprises the sequences SEQ ID N024 QSIVYSNGNTY SEQ ID NO: 5 SEQ ID NO: 6 FQGSHIPYT. 11. A human monocloual antibody or fragment that binds to ADM or an antibody frag— ment thereof according to claim 10 wherein said antibody or fragment ses a se- quence selected from the group comprising: SEQ ID NO: 7 (AMMH-C) QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEIL PGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGF DYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKRVEPKHHHHHH .. 64 _ SEQ ID NO: 8 (AM—VH1) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRI LPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDG FDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKRVEPKHHHHHH SEQ ID NO: 9 (AM-VH2—E40) QVQLVQSG‘AEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRI LPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDG FDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEI’VTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKRVBPKHHHHHH SEQ ID NO: 10 (AM—VH3-T26-E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEI LFGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDG FDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKRVEPKHHHHHH SEQ ID NO: 11 (AM—VH4~T26-E40~E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEI LPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSBDTAVYYCTEGYEYDG FDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT EPKHHHHHH SEQ ID NO: 12 (AM—VL-C) DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIY SGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTK LEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC WO 72512 _ 65 _ SEQ ID No: 13 (AM-VLI) DWMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIY RVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGT KLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC SEQ ID NO: 14 (AM—VLz—E40) DVVMTQSPLSLPVTLGQPASISCRSSQSWYSNGNTYLEWFQQRPGQSPRRLIY RVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGT KLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SG-NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC 12. ADM antibody or an adrenomedullin antibody fragment or ADM non—1G ld for use in therapy of a cal or acute disease of a patient according to any of claims 1 to 11 wherein said antibody or fragment or scaffold is a modulating antibody or frag— ment or scaffold that enhances the half life ( tl/2 half ion time) of adrenome- dullin in serum, blood, plasma at least 10 “/0, preferably at least 50 ”/0, more preferably >50 %, most preferably >100% and that blocks the ivity ofADM to less than 80 %, preferably less than 50%. 13. ADM antibody or an adrenornedullin antibody nt or ADM non—IG scaffold for use in therapy of a c 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 adrenornedullin 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 13 or a combination according to claim 13 to be used in combination with ADM binding protein and/or further active ingredients.

. Pharmaceutical formulation comprising an antibody or fragment according to any of claims 1 to 13.

WO 72512 .. 66 _ 16. ceutical formulation according to claim 14 wherein said ceutical formu- lation is a solution, preferably a ready—to-use solution. 17. Phannaceutical formulation according to claim 14 wherein said phannaceutical formu- lation is in a freeze-dried state. 18. Phannacautical formulation according to any of claims 14 to 15, wherein said - ceutical formulation is administered intra—muscular. 19. Pharmaceutical formulation according to any of claims 14 to 15, wherein said pharma— ceutical formulation is administered intra—vascular.

. Pharmaceutical formulation according to claim 18, wherein said pharmaceutical for- mulation is administered Via infusion. _ 67 _ 2012/072931 EXAMPLES 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 I Generation of Antibodies and determination of their affinity constants Several human and murine antibodies were ed and their affinity constants were deter- mined (see tables l and 2).

Peptides/ conjugates for Immunization: Peptides for immunization were synthesized, see Table l, (JPT Technologies, Berlin, Ger- many) with an additional inal Cystein (if no n 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 coupling procedure was performed ing to the manual of Perbio.

The murine antibodies were generated according to the following method: A Balb/c mouse was immunized with lOOug Peptide—BSAuConjugate at day 0 and 14 i— fied in lOOnl complete Freund’s nt) and SOng at day 21 and 28 (in 100m incomplete Freund’s adjuvant). Three days before the fusion experiment was performed, the animal re- ceived SOug of the conjugate dissolved in lOOul saline, given as one intraperitoneal and one intra-venous ion.

Spenccytes from the immunized mouse and cells of the myeloma cell line SPZ/O were fused with lml 50% polyethylene glycol for 303 at 37°C. After g, 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 passages followed by returning to the normal cell culture 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 limit- ing~dilution technique and the isotypes were determined. (see also Lane, RD. “A short-duration polyethylene glycol fusion technique for sing production of onal antibody—secreting hybridomas”, J. l. Meth. 81: 223—228; (1985), Ziegler, B. er al. “Glutamate decarboxylase (GAD) is not detectable on the surface of rat islet cells examined by cytofluorometry and complement-dependent antibody-mediated cytotoxicity of monoclonal GAD antibodies”, Horm. Metab. Res. 28: 11-15, (1996)).

Mouse monoclonal antibody production: Antibodies were produced Via standard antibody production methods (Marx et a1, Monoclonal Antibody Production, ATLA 25, 121, 1997,) and d Via Protein A. The antibody purities were > 95% based on SDS gel electrophoresis analysis.

Human Antibodies Human Antibodies were produced by means of phage display according to the following pro— cedure: 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 g strategy comprising the use of peptides ning a biotin tag linked Via two different spacers to the medullin e sequence. A mix of panning rounds using non-specifically bound antigen and streptavidin bound antigen were used to ze background of non—specific binders. The eluted phages from the third round of panning have been used for the generation of monoclonal scFv expressing Ecoli strains.

Supernatant from the cultivation of these clonal strains has been directly used for an antigen ELISA testing (see also Host, M., Meyer, T., Voedisch, 13., Riilker, T., Thie, H., El—Ghezal, A., Kirsch, Ml, Schiitte, M., Helmsing, 8., Meier, D., Schirrmann, T., Diibel, S., 2011. A human SCFV antibody tion pipeline for proteorne research. Journal of Biotechnology 152, 159—170; Schiitte, M., Thullier, P., Pelat, T., WBZICI‘, X., Rosenstock, P., Hinz, D, Kirsch, M.1.,Hasenberg, M., Frank, R., Schirrmann, T., Gunzer, M., Hust, M., Dfibel, 8., 2009. fication of a putative Crf splice variant and generation of recombinant antibodies for the specific ion of Aspergiilus fumigatus. PLoS One 4, e6625). _ 69 ..

Positive clones have been selected based on positive ELISA Signal for antigen and ve for streptavidin coated micro titer plates. For further characterizations the scFv open reading frame has been cloned into the expression plasmid pOPElO7 (Hust et al, J. Biotechn. 2011), captured from the culture supernatant Via immobilised metal ion affinity chromatography and purified by a size ion chromatography.

Affinity Constants To determine the affinity of the antibodies to Adrenomedullin, the kinetics ofbinding of Adrenomedullin to lized antibody was determined by means of label—free surface plasmon resonance using a Biacore 2000 system (GE Healthcare Europe GmbH, Freiburg, Germany). Reversible immobilization of the antibodies was performed using an anti—mouse Fc dy covalently coupled in high density to a CMS sensor surface according to the manufacturer‘s instructions (mouse dy capture kit; GE Healthcare). (Lorenz et a .,“ Functional Antibodies Targeting IsaA of Staphylococcus aureus Augment Host Immune Re— sponse and Open New Perspectives for Antibacterial Therapy“; Antimicrob Agents Chemo~ ther. 2011 January; 55(1): {65—173.} The monoclonal antibodies were raised against the below depicted ADM regions of human and murine ADM, respectively. The following table ents a ion of obtained anti~ bodies used in further experiments. Selection was based on target region: Table 1: Sequence Antigen/Immunegen vTADM ation [ Affinity Number Region constants L Kd (M) SEQ ID: 15 "WRQSMNNFQGLRSFGCRFGEC—l 1—21 NT-H T9 xml—OT L .l SEQ ID: 16 EVQKLAHQIYQ 21—32 MR-H 2 x 10‘9— . “T SEQ ID: 17 CAPRSKISPQGY—NHZ | C—42—52 CT—H 1.1 x 10 } SEQ ID: 18 YRQSMNQGSRSNGCRFGTC 1—1 9 NT~M 3.9 x 10' SEQ ID: 19 CTFQKLAHQIYQ 19—31 MR—M Es x 10'” j SEQ ID: 20 ISP GY-NHZ (3—40-50 CT-M 9 x 167g” i_ _J Q l , 7O - The following is a list of fin'ther obtained oual antibodies: List of anti-ADM-antibodies Table 2: Target Source Klone number y max inhibition (M) bioassay (%) (see example 2) NT-M Mouse ADM/63 -Mouse ADM/365 Mouse ADM/366 Mouse ADM/367 -Mouse ADM/368 Mouse ADM/369 Mouse 0 JiM—ouse fluke/371 Mouse ADM/372 -Mouse $342373 Mouse ADM/3 77 WWI/379Mouse ADM/3 78 Mouse fiDMBSO 1.8 211633— Mouse ADM/382 1.6 1110*" Mouse WWI/383 1.8 1110' Mouse EDM/BSA 1.7 xlO'g— ‘_‘ Mouse ADM/3 85 1.7 x10'8_w Mouse ADM/403 1.2 1110's it Mouse ‘1 ADM/396ADM/395 1.2 x1113— -Mouse 3.0 KW Mouse ADM/397 1 .5x10'3_ T —l MR—M Mouse ADM/38 4.5X10T 68 MR—M Mouse ADM/39 5.9 KW 72 L_u WO 72512 T ADM/65 ADM/66 Mouse ADM/33 Mouse ADM/34 MR-H Mouse ADM/41 MR-H Mouse ADM/42 MR—H Mouse ADM/43 Mouse ADM/44 CT—H Mouse ADM/15 CT—H Mouse ADM/16 100 ADM/17 100 CT-H Mouse ADM/1 8 hADM Phage display i ADM/A7 Phage displayfiFADM/B7 Phage display ADM/C7 Phage display ADM/G3 Phage display ADM/B6 Phage display ADM/B11 Phage display ADM/D8 i Phage displayPhage display ADM/D11 who“? ‘ i ADM/G12 <1x15‘T i Generation of d fra nents‘o enz atic di estion: The generation of Fab and F(ab)2 fiagments was done by enzymatic ion of the murine full length antibody NT-M. dy NT—M was digested using a) the pepsin—based F(ab)2 Preparation Kit (Pierce 44988) and b) the ~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- fragmentation digestion was carried out for 16h, respectively.

Procedure for Fab Generation and Purification: .. 72 _ The immobilized papain was brated by washing the resin with 0.5 ml of Digestion Buffer and centrifilging the column at 5000 x g for 1 minute. The buffer was discarded after— wards. The desalting column was prepared by removing the storage solution and g 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 equilibrated Immobilized Papain. Incubation time of the digestion reaction was done for 16h on a tabletop rocker at 37°C. The column was centrifuged at 5000 X g for 1 minute to separate digest from the Immobilized Papain. ards the resin was washed with 0.5ml PBS and centrifuged at 5000 X g for 1. minute. The wash fraction was added to the digested antibody that the total sample volume was 1.0ml. The NAb Protein A Column was equilibrated with PBS and IgG Elation Buffer at room temperature. The column was centrifuged for 1 minute to remove stor- age solution (contains 0.02% sodium azide) and brated by adding 2ml of PBS, centri— fuge again for 1 minute and the hrough discarded. The sample was d to the col— umn and resuSpended by inversion. tion was done at room temperature with end-over— end mixing for 10 minutes. The column was centrifuged for 1. minute, saving the flow— h with the Fab fragments.

(References: Coulter, A. and Harris, R. (1983). J. Immunol. Meth. 59, 199-203.; Lindner I. et 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 al. (2010) Neu- tralization of West Nile virus by cross-linking of its surface proteins with Fab fragments of the human monoclonal antibody CR43 54. PNAS. 107, 18950—53, Chen X. et a1. (2010) Re— ent of open headpiece conformation for activation of leukocyte integrin 00432. PNAS. 107, 14727—32; Uysal H. et a1. (2009) Structure and pathogenicity of antibodies specific for citrullinated collagen type II in experimental arthitis. J. Exp. Med. 206, 449-62.; Thomas G.

M. et al. (2009) Cancer cell—derived microparticles bearing P—selectin glycoprotein ligand 1 accelerate thrombus ion in vivo. J. Exp. Med. 206, 1913-27.; Kong F. et a1. (2009) Demonstration of catch bonds n an integrin and its ligand. J. Cell Biol. 185, 1275—84.) Procedure for generation and purification of F1 ab' L2 Framents: The immobilized Pepsin was equilibrated by washing the resin with 0.5 ml of Digestion Buffer and centrifuging the column at 5000 x g for 1 minute. The buffer was discarded after- wards. The desalting column was prepared by ng the storage solution and washing it with ion buffer, centrifuging it each time afterwards at 1000 x g for 2 minutes. 0.5ml of _ 73 _ the prepared IgG sample where added to the spin column tube containing the equilibrated Immobilized Pepsin. Incubation time of the digestion reaction was done for 1611 on a tabletop rocker at 37°C. The column was centrifuged at 5000 X g for 1 minute to separate digest from the Immobilized Papain. Afterwards the resin was washed with 0.51nL PBS and centrifuged at 5000 X g for 1 minute. The wash traction was added to the ed antibody that the total sample volume was 1.0ml. The NAb Protein A Column was equilibrated with PBS and lgG- Elution Buffer at room temperature. The column was centrifuged for 1 minute to remove stor— age solution (contains 0.02% sodium azide) and equilibrated by adding 2mL of PBS, centri— fuge again for 1 minute and the flow—through discarded. The sample was applied to the col- umn 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 flown through with the Fab fragments.

(References: Mariam, M., et al. (1991). A new enzymatic method to obtain high—yield F(ab’)2 suitable for clinical use from mouse IgGl. unol. 28: 69—77.;Beale, D. (1987). Mo- r fragmentation: Some applications in immunology. Exp Comp lmmunol 1128196.; Ellerson, J.R., et al. (1972). A fragment corresponding to the CH2 region of immunoglobulin G (IgG) with complement fixing activity. FEBS Letters 24(3):318-22.; , R.S. and El- liot, 13.151. (1983). Detection of Fc receptors. Moth Enzymol 93: 1 13—147.; Kulkarni, P.N., et a1. (1985). Conjugation of methotrexate to lgG antibodies and their 2 nts and the effect of conjugated methotrexate on tumor growth in Vivo. Cancer Immunol Immunotherapy 19:211—4.; Lamoyi, E. (1986). Preparation of 2 Fragments from mouse igG of various sses. Meth Enzyme] 121:652—663.; Parham, P., et a1. (1982). Monoclonal antibodies: purification, fragmentation and application to structural and functional studies of class I MHC antigens. J Immunol Meth 53:133-733.; Raychaudhuri, (3., et a1. . Human igGl and its Fc fragment bind with different affinities to the Fc receptors on the human U937, HL-60 and ML-l cell lines. Mol l 22(9):1009—19.; Rousseaux, J a1. (1980). The differential ., et enzyme ivity of rat immunoglohulin G subclasses to papain an pepsin. Mol immunol 173469—82; Rousseaux, 1., et al. (1983). Optimal ion for the preparation of Fab and F(ab')2 fragments from onal lgG of different rat IgG subclasses. J Immunol Meth 64:141-6.; Wilson, K.M., et a1. (1991). Rapid whole blood assay for HIV—1 seropositivity us- ing an ptide conjugate. J Immunol Meth 138:1 I 1~9.) _ 74 _ NT-H—Antibody Fragrn ent Humanizaticn The antibody fragment was humanized by the CDR-grafiing method (Jones, P. T., Dear, P.

H., Foote, 3., Neuberger, M. S., and Winter, G. (1986) Replacing the complementarity- determining s in a human antibody with those from a mouse. Nature 321, 522—525).

The ing steps where done to achieve the humanized ce: Total RNA extraction: Total RNA was extracted from NT—H hybridomas using the Qiagen kit.

First—round RT—PCR: QIAGEN® OneStep 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 sam— ple, 12 individual heavy chain and 11 light chain RT—PCR reactions were set up using degenu crate 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 engineered into the primers.

Reaction Setup: 5x QIAGEN® OneStep RT—PCR Buffer 5.0 nl, dNTP Mix (containing 10 mM of each dNTP) 0.8 pl, Primer set 0.5 n1, ® OneStep RT—PCR Enzyme Mix 0.8 gal, Template RNA 2.0 n.1, RNase—free water to 20.0 n1, Total volume 20.0 n1 PCR condition: Reverse ription: 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 Second~round semi—nested PCR: The RT—PCR products from the first-round reactions were further amplified in the second-round PCR. 12 individual heavy chain and ll light chain RT— PCR reactions were set up using semi-nested primer sets specific for dy variable re— gions.

Reaction Setup: 2x PCR mix 10 n1; Primer set 2 n1; round PCR product 8 141; Total vol— ume 20 n1; Hybridoma dy Cloning Report PCR condition: Initial denaturing of 5 min at 95°C; 25 cycles of 95°C for 25 sec, 57°C for 30 sec, 68°C for 30 sec; Final extension is 10 min 68°C. _ 75 - After PCR is finished, run PCR reaction samples onto agarose gel to visualize DNA frag- ments amplifiedAfter sequencing more than 15 cloned DNA nts amplified by nested RT-PCR, several mouse dy 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 alignment with homologous human framework sequences the resulting humanized se— quence for the variable heavy chain is the following: see figure 6 (As the amino acids on posi- tions 26, 40 and 55 in the variable heavy chain and amino acid on position 40 in the variable light are critical to the binding ties, they may be reverted to the murine original. The resulting candidates are depicted below) (Padlan, E. A. (1991) A possible procedure for re— ducing the immunogenicity of antibody variable domains while ving their ligand— g properties. Mol. Immunol. 28, 4894198.; Harris, L. and Bajorath, J. (1995) Profiles for the analysis of immunoglobulin sequences: Comparison ofV gene subgroups. Protein Sci. 4, 306—310.).

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

SEQ ID NO: 7 (AM—VH~C) SGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEiLPGSG ~SLENYNEKFKGKATI'I‘AI)TSSNTAYMQLSSLTSEDSAWYCTEGYEYDGFDYWGQGTTLT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQS SGLYSLSSVVTVPSSSLGTQQ’YICNIWHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 8 (AM-VH1) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILPGS MNYAQKFQGRVTITADESTSTAYMELSSLRSEDTA VYYCTEGYEYDGFDYWGQGTTV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQ SSGLYSLSSVVTVPSSSLGTQTY]CNVNHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 9 2-E40) QVQLVQSGAEVKKPGssvxvsCKASGvrrsRYWIEWVRQAPGQGLEWMGRILPGS vaAQKrQGerirADESTSTAYMELSSLRSEDTA VYYCTEGYEYDGFDYWGQGTTV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPA VLQ LSSVVTVPSSSLGTQTYICNWHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 10 (AM-VH3-T26—E55) _ 76 _ QVQLVQSGAEVKKPGSSVKVSCKAQGYTFSRYMSWVRQAPGQGLEWMGEILifiGS QENYAQKFQGRVTITADESTSTAYMELSSLRSEDTA VYYCTEGYEYDGFDYWGQGTTV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 11 (AM~VH4~T26—E40—E55) QVQLVQSGAEVKKPGSSVKVSCKAgEGYTFSRYW ZWVRQAPGQGLEWMG‘ES GSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTA VYYCTEGYEYDGFDYWGQGTTV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQ SSGLYSLSSVVTVPSSSLGTQYTICNWHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 12 (AM-VL—C) TPLSLPVSLGDQATISCRSSg3SIVYSNGNTYLEWYLQKPGQSPKLLIYMN RFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKLEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 13 (AM-VLi) DVVMTQSPLSLPVTLGQPASISCRSS!3SIVYSNGNTYLNWFQQRPGQSPRRLIYBILSN RDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFSEGSHIPYTFGQGTKLEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 14 (AM-VLZ-E40) DVVMTQSPLSLPVTLGQPASISCRSS!ESIVYSNGNTY PGQSPRRLIYMN RDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFSQGSHIPYTFGQGTKLEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Example 2 Effect of selected anti-ADM-antibodies on anti—ADM-bioactivity The effect of selected ADM-antibodies on ADM—bioactivity was tested in an human recombi- nant medullin receptor CAMP functional assay (Adrenomedullin Bioassay).

Testing of antibodies targeting human or mouse adrenomeduflin in human inant Adrenomedullin receptor CAMP functional assay (Adrenomedullin Bioassay) _ 77 _ Materials: Cell line: CHO—Kl Receptor: Adrenomedullin (CRLR + RAMPB) Receptor ion Number Cell line: CRLR: U17473; RAMP3: AJ001016 CHO-Kl cells expressing human recombinant adrenomedullin receptor (FAST—027C) grown prior to the test in media without antibiotic were detached by gentle g with PBS-EDTA (5 mM EDTA), recovered by centrifugation and resuspended in assay buffer (KRH: 5 mM KCl, 1.25 mM MgSO4, 124 mM NaCl, 25 mM HEPES, 13.3 mM Glucose, 1.25 mM KHZPO4, 1.45 mM CaClZ, 0.5 gfl BSA).

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

Antagonist test (96well): For antagonist testing, 6 pl of the reference agonist (human (5,63nM) or mouse (0,67nM) adrenornedullin) was mixed with 6 al of the test samples at different antagonist dilutions; or with 6 n1 buffer. After incubation for 60 min at room temperature, 12 til of cells (2,500 cells/well) were added. The plates were ted for 30 min at room ature. After addi— tion of the lysis buffer, percentage of DeltaF will be estimated, ing to the manufacturer cation, with the HTRF kit from Cis—Bio International (cat n°62AM2 PEB). hADM 22- 52 was used as reference antagonist.

Antibodies testing cAMP-HTRF assay The anti~h~ADM antibodies (NT-H, MR—H, CT—H) were tested for antagonist activity in hu— man recombinant adrenomedullin receptor (FAST-027C) CAMP functional assay in the pres- ence of 5.63nM Human ADM 1-52, at the following final antibody concentrations: 100ng/m1, 20pgi’ml, Ling/ml, 0.8ng/ml, 0.16ng/m1.

The anti-m-ADM antibodies (NT-M, MR—M, CT—M) were tested for antagonist activity in human inant adrenomedullin receptor (FAST—027C) CAMP functional assay in the presence of 0.67nM Mouse ADM 1-50, at the following final antibody concentrations: ml, 20ng/ml, ting/m1, 0.8pg/ml, 0.16ng/ml. Data were plotted relative inhibition vs. antagonist concentration (see figs. 3a to 31). The maximal inhibition by the individual anti- body is given in table 3. _ 78 _.

Table 3: Antibody Maximal inhibition ofADM bioactivity (ADM—Bioassay) (you NT-M FABZ NT-M MR—M CT—M 100 l Non specific mousa IgG Example 3 Data for ization of hADM by the anti-ADM antibody The stabilizing effect of human ADM by human ADM antibodies was tested using a hADM assay.

Immunoassay for the quantification of human Adrenomedullin The technology used was a sandwich coated tube scence immunoassay, based on Ac- ridinium ester labelling.

Labelled compound (tracer): lOOug (100111) CT-H (lmg/ ml in PBS, pH 7.4, AdrenoMed AGGermany) was mixed with 10m Acridinium NHS—ester (1mg/ ml in acetonitrile, InVent GmbH, Germany) (EP 0353971) and incubated for 20min at room temperature. Labelled CT— H was purified by Gel—filtration HPLC on Bio-Sil® SEC 400—5 ad Laboratories, Inc, USA) The purified CT—H was diluted in (300 mmol/L potassiumphosphate, 100 mmol/L NaCl, 10 mmol/L Na—EDTA, 5 gyL Bovine Serum Albumin, pH 7.0). The final concentration was approx. 800.000 relative light units (RLU) of labelled compound (approx. 20mg labeled dy) per 200 uL. Acridiniuniester chemiluminescence was measured by using an Aut0~ Lumat LB 953 (Berthold Technologies GmbH & Co. KG). _. 79 _ Solid phase: Polystyrene tubes er Bio—One International AG, Austria) were coated (18h at room temperature) with MR—I—i oMed AG, Germany) (1.5 pg MR—H/03 mL 100 mmol/L NaCl, 50 mmol/L TRIS/HCl, 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 (BACHEM AG, Switzerland) in 250 mmol/L NaCl, 2 g/L Triton X—100, 50 g/L Bovine Sen rum Albumin, 20 tabs/L Protease Inhibitor Cocktail (Roche Diagnostics AG, Switzerland)) hADM Immunoassay: 50 ul of sample (or calibrator) was pipetted into coated tubes, after adding labeleld CT—H (200M), the tubes were incubated for 411 at 4°C. Unbound tracer was removed by washing 5 times (each lml) with washing solution (20mM PBS, pH 7.4, 0.1 “/0 Triton X—l 00).

Tube-bound uminescence was measured by using the LB 953 Figure 4 shows atypical 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 0C. At selected time points, the degradation of hADM was stopped by freezing at —20 °C. The tion was performed in absence and ce ofNT—H (lOOug/ml). The remain- ing hADM was quantified by using the hADM immunoassay described above.

Figure 5 shows the stability of hADM in human plasma (citrate) in absence and in the pres- ence of NT~H dy. 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). _ 80 - Example 4 Se sis Mortali 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. lncisions were made into the left upper quadrant of the peritoneal cavity (normal on of the cecum). The cecum was exposed and a tight re was placed around the cecum with sutures distal to the insertion of the small bowel. One re wound was made with a 24—gauge needle into the cecum and small amounts of cecal contents were expressed through the wound. The cecum was replaced into the peritoneal cavity and the laparotomy site was closed. Finally, animals were returned to their cages with free access to food and water. 500m saline were given 3.0. 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 en test) determination. Ten further mice per each group were ed over a period of4 days.

Group Treatment (l Oul/ g bodyweight) dose! Folloqup: 1 NT—M, 0.2 trig/ml survival over 4 days 2 MR-M, 0.2 rug/ml survival over 4 days 3 CT—M, 0.2 mg/ml survival over 4 days 4 non-specific mouse lgG, 0.2 mg/ml survival over 4 days control - PBS lOul/g bodyweight survival over 4 days al chemistry _ 81 “ Blood urea en (BUN) concentrations for renal on were measured baseline and day 1 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 s 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 (%) PBS 0 j non—specific mouse IgG 0 CT—M 10 fl MR~M 3o 1 NT—M 70 mm...._J Table 5: Average fiom 5 animals BUN pre CLP (mM) 1 BUN day 1 (mM) PBS 8.0 23.2 non—specific mouse IgG 7.9 15.5 7.8 13.5 8.1 24.9 8.8 8.2 It can be seen from Table 4 that the NT-M antibody reduced mortality considerably. Alter 4 days 70 % of the mice survived when treated with NT—M antibody. When treated with MR-M antibody 30 % of the animals ed and when treated with CT-M antibody 10 0/0 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 s of. the S-BUN Test revealed that the NT—M antibody was the most effective to protect the kidney. , 32 _ Sepsis Mortality (late 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 cecnm 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 contents were expressed through the wound. The cecurn was replaced into the peritoneal cavity and the laparotomy site was closed. y, animals were ed to their cages with free access to food and water. 500141 saline were given so. as fluid replacement.

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

Group Treatment (1 Oul/ g bodyweight) dose/ Follow—Up: Study groups 1 NT-M, FABZ 0.2 mg/ml survival over 4 days 2 control: non—specific mouse IgG, 0.2 111ng survival over 4 days 3 vehicle: - PBS lOul/g bodyweight al over 4 days Table 6: 4 day mortality j survival (“/o) PBS 0 Non—specific mouse IgG 0 iNT~M FAB2 ’75 It can be seen from Table 6 that the NT—M FAB 2 antibody reduced ity considerably.

After 4 days 75 % of the mice survived when treated with NT—M FAB 2 dy. In contrast WO 72512 _ 83 _ thereto all mice were dead alter 4 days when treated with non—specific mouse IgG. The same result was obtained in the control group where PBS hate buffered saline) was s- tered 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, ,) were utilized. A polymicrobial sepsis induced by cecal ligation and puncture (CLP) was used as the model for studying septic shock ((Albuszies G, et al: Effect of increased cardiac output on hepatic and intestinal ircula— tory blood flow, oxygenation, and metabolism in hyperdynamic murine septic shock. Crit Care Med 2005;33:2332—8), (Albuszies G, et al: The effect of iNOS deletion on hepatic glu~ coneogenesis in hyperdynamic marine septic shock. Intensive Care Med 2007;33:1094—101), (Barth E, et al: Role of iNOS in the reduced responsiveness of the myocardium to catechola- mines in a hyperdynamic, murine model of septic shock. Crit Care Med 2006;34:307-13), art K, et al: Effect of SOD~1 over-expression on myocardial function during resusci— tated murine septic shock. Intensive Care Med 2009;35:344-9), (Baumgart K, et al'. Cardiac and metabolic effects of hypothermia and inhaled HZS in anes~ thetized and ventilated mice. Crit Care Med 2010;38:588-95), (Simkova V, et al: The effect of 8013—1 over-expression on hepatic gluconeogenesis and whole—body glucose oxidation during resuscitated, ensive murine septic shock. Shock 2008;30:578-84), (Wagner F, et al.: Inflammatory effects of hypothermia and inhaled H28 doling resuscitated, hyperdynarnic murine septic shock. Shock, im Druck), (Wagner F, et al: Effects of intravenous H28 after murine blunt chest : a prospective, randomized controlled trial. Crit Care 2011, submit“ tes for publication)).

After weighing, mice were etized by intraperitoneal injection of 120 ug/g Ketamin, 1.25 ugr’g Midazolam and 0.25 ug/g Fenianyl. During the surgical procedure, body tempera ture was kept at 376800 A lcm midline nal section was performed to get access to the cecum. The cecum then was ligated with 3—0 silk tie close to the basis and a single punc— ture with a lit-gauge needie was applied. The cecum was returned and the incision was closed _ 84 _ again (4—0 tie). For the compensation of perioperative loss of liquids, 0.5 m1 lacted Ringer’s solution with 1 pg/g orphin as analgetic was injected subcutaneously in dorsal dermis.

For antibiosis the mice received Ceftriaxon 30ng/g and Clindamycin 30pg/g subcutaneously via the lower extremities.

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

The covering of liquid requirements were d by a dorsal subcutaneous injections with 0.5 m1 lactated ringer’s solution with 4 ng/g glucose and Buprenorphin lug/g, which were applied in an 8 hour cycle, after short term anesthesia by isofluran. In on, antibiosis was maintained by subcutaneous injections of Ceftriaxon 30ag/g and Clindamycin 3014ng 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 pig/m1 in phosphate ed saline (PBS) via injection into the penis vein for a dose of 2mg per kg body weight (dose volume 88-120 91) (5 animals).

Late treatment Afier full Sepsis development, 15.511 after CLP y, animals were anesthetized as de— scribed above and NT—M was applied in a concentration of 500 ngfml in ate buffered saline (PBS) via injection into the penis vein for a dose of 2mg per kg body weight (dose vol“ ume 88-120 n1) (3 animals).

The control group (6 animals) received a corresponding amount of the vehicle I’BS solution without antibody (4pl/g, 88—120 {41) immediately after CLP surgery.

Study groups and mental setting Murine septic shock model under intensive care monitoring: .. 85 _ 2012/072931 Three groups with 3, 5 and 6 animals were monitored. Group 1 (5 animals) received the anti- body NT—M 15.5h after CLP, group 2 received the antibody NT-M immediately after CLP surgery and group 3 received a comparable amount of PBS ). 16 hour incubation post CLP (to allow the polymicrobial sepsis to progress), the experiment was continued with moni~ toring and interventions comparable to an intensive medical care regime. Therefore, after weighing the animals were anesthetized as described in the CLP surgery part (except the late treated animals, which were anesthized before treatment). Body temperature was maintained at 37~3 8°C for the rest of the experiment. After a tracheotomy and intubation, ation was red and ted by laboratory animal lung ventilator F1exivent®, (Ernka Technolo~ gies, Fi02 0,5, PEEP 10 H20, VT 8 pi/g, l:E 121,5, AF 70-140 depending on temperature).

Anesthesia was maintained throughout the experiment Via the cannulated vena jugularis ex— terna dextra with a continuous infusion of Ketamin 30 ug/gxh and Fentanyl 0.3 ug/gxh. Fur- thermore, the right aorta carotis is was cannulated for continuous monitoring of heart rate and the mean arterial pressure (MAP). The mean arterial pressure was maintained at MAP > 65 mmHg via intravenous (V. jugularis) on of colloids (80 uL/gxh, Hextend®) and, if needed, Noradrenalin dissolved in colloids as vasopressor. Blood samples (120 pl) were taken via the cannuiated 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 1111an (V. ris) could not be maintained with the vasorpressor dosing. ed parameters The following parameters were measured and analyzed: Total consumption of noradrenalin (ug NAfg), ption rate of noradrenalin (pg NAfg/h), total volume of urine collected during the experiment, creatinine concentration (ug/mL) at the end of the experiment and mean creatinine clearance (uL/rnin).

Table 7: Total consumption of consumption rate of Noradrenalin (ug NA/g) Noradrenalin (ug NA/g/h) (Average) (Average) - 86 a Control (mouse IgG) (N=6) T017 rag/g $032 ng/h/g NTuM (N35) early treatment r“07)7 pg/g m—l 0.012 ng/h/g Relative change (early treatmem 59% 62.5% amelioration) (59%) (62.5%) NT—M (Nms) rate treatment 0.04 rig/g 0.0075 lig/h/g Relative change (late ent, 76,5% 76,5% amelioration) (76.5%) (76.5%) The catecholamine requirement was measured after administration of either non specific mouse IgG to a total of 6 mice as l group, NT—murine antibody to a group of 5 mice immediately after CLP (early treatment) or NT—murine dy to a group of 3 mice 15.5h after CLP (late treatment).

The reduction of the catecholarnine requirement is a measure for the ization of the circu— lation. Thus, the data show that the ADM antibody, especially the NT—M antibody, leads to a erable stabilization of the circulation and to a considerable reduction of the catechola- mine requirement. The circulation-stabilizing effect was given in early treatment (immedi- ately after CLP) and treatment after full sepsis development (late treatment) (see fig. 7). tion of Fluid Balance More positive fluid balance both early in resuscitation and cumulatively over 4 days is associ- ated with an increased risk of mortality in septic shock. The control of the liquid balance is of utmost ance for the course of disease of patients having sepsis. (s. Boyd et a1, 2011).

Controlling the liquid balance of critical ill patients remains as a substantial challenge in in- tensive care medicine. As can be seen in table 8 ent of mice after CLP (experimental procedures see “Animal Model”) with NT-M antibody lead to an enhancement of the total volume of urine ed. The urine secreted was approx. three times higher in NT-M-treated animals compared to nonntreated mice. The positive treatment effect was given in early— and in late treatment. 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 dy, is favorable for regulating the fluid balance in patients. (see table 8 and fig- ures 8 and 9). _ 87 _ Table 8 Urine average vol— Fluid balance average ume/ g body weight volume/ g body weight Control (mouse IgG) 0.042 ml/g 0,23 ml/g (N26) NT—M early (N=5) 0.12 ml 0,18 ml/g Relative change early + 186% -2l.7%% fl treatment NT—M late (N23) 0.125 ml 0,16 ml/g Relative change late + 198% -30,5% Improvement of kidney function The combination 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 e alone. (Schrier and Wang, nisms of Disease Acute Renal Failure and Sepsis”; The New England Journal of Medicine; 351:159-69; 2004). Creatinine — tration and creatinine clearance are standard laboratory parameters for monitoring kidney mcti0n (Jacob, “Acute Renal Failure”, Indian J. Anaesth; 47 (5): 367—372; 2003).

Creatinine and nine clearance data from above described animal experiment (early treatment) are given in Table 9.

Table 9 Kidney fimction: creatinine concentra— mean creatiniwnej tion (ng/mL) ' clearance (nL/min) control mouse IgG (MW) 2.6 rig/ml 174 til/111m 1.5 ttg/ml 373 ul/min Relative change —42% +1 14% l (amelioration) (42%) (114%) _ 88 _ In comparision to control septic animals, the creatinine tration was lowered by 42% and the creatinine clearance was improved by more than 100% as a result ofNT-M treatment (Table 9). The data Show that the administration of ADM-antibody, especially NT—M, leads to an improvement of kidney 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 resuspcnded, lysed on ice, and centrifuged. The super— natant (protein extract) was stored at —80 °C. Activation of nuclear factor kappa—light—chain gene enhancer in B cells (NF-KB) was determined as previously described using an electro— phoretic mobility shift assay (EMSA)1,2. Cell extracts (lOug) were incubated on ice with poly—doxyuinosinic—deoxy—cytidylic acid (polyudI—dC) and beled double stranded oli- gonucleotide (Biomers, Ulm, y) containing the NF-KB (HIV KBsite) ( 5’~ GGATCCTCAACAGAGGGGACTTTCCGAGGCCA—3‘). xes were separated in native polyacrylamide gels, dried and exposed to X—ray films. A phosphorimager and image analyzer software (AIDA image Analyzer; t) was used to quantify the radioactively d NF-KB by densitometry. For comparison between individual gels, the intensity of each band was related to that of aneously loaded control animals which had not undergone surgical instrumentation and CLP. ore, the EMSA data are expressed as fold increase over control values. Statistics: All data are presented as median (range) unless otherwise stated differences between the two groups were analyzed with the Mann—Whitney rank sum test for unpaired samples. Results: The animals treated with NT—M presented with signifi- cantly attenuated liver tissue NF-KB activation (2.27 2.53)) compared to vehicle animals (2.92 (2.50—3.81)) (p<0.001) (see figure 10).

References: l. Wagner F, Wagner K, Weber S, Stab] B, Knoferl MW, Huber—Lang M, Seitz DH, Asfar P, Calzia E, eben U, Gebhard F, Georgieff M, nacher P, Hysa V: Inflammatory ef- fects ofhypothermia and inhaled H23 during resuscitated, hyperdynamic murine septic shock.

Shock 2011;35(4):396—402 _ 89 _ 2. Wagner F, Scheuerle A, Weber S, Stahl B, McCook O, Knoferl MW, Lang M, Seitz DH, Thomas J, Asfar P, Szabd C, Mdller P, Gebhard F, Georgieff M, Calzia E, acher P, Wagner K: Cardiopulmonary, histologic, and inflammatory effects of intravenous NaQS after blunt chest trauma—induced lung confusion in mice. J Trauma 201 l ;71 (6): 1659—67 nannies 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 d with (lOul/ g bodyweight) dose ofNT-M, 0.2 mg/rnl. As control, 6 mice were treated with (10 allg body weight) PBS. Survival and physical condition was moni— tored for 14 days. The mortality was 0 in both groups, there were no differences in physical condition between NT—M and control group.

Examnle 7 icin-induced nephrotoxicigy A non—septic acute kidney injury model has been established, which makes use of the nephro- toxicity d by Gentamicin (Chin P]S. Models used to assess renal functions. Drug De— velop 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 icimlnduced Nephrotoxicity in Rats Study Design: Article ml/kg mg/kg (Male) 1 Gentamicina + IV vehicleb NA x 4° 2 IV NT—M X 4° aGentamicin at 120 mg/kg intramuscularly for 7 days (days 0-6). _ 90 _ Vehicle; injected intravenously (i.v.) 5 min before gentarnicin 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 i.v. on Days 2, 4, and 6.

ClPlasma samples were collected in EDTA tubes (Days 1 and 3 before Test and Control article: 100 ul; Day 7:120 ul. 24h urine collection on ice is initiated after gentamicin on Day 0, followed by Days 2 and 6; blood collection on days 1, 3, and 7.

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

Test nd (anti—adrenomedullin antibody NT-M) and e (phosphate buffered sa— line) were ed intravenously 5 min before icin on day 0, ed by injection on days 2, 4, and 6. Body weights and clinical signs were monitored daily. Twenty-four (24) hour urine collections on ice were performed on Days 0, 2, and 6. Urine specimens were as— sayed for concentrations ofNa+ and K+, and creatinine. Blood samples for clinical chemistry were collected on Days 1 (before gentamicin), 3 (before icin), and 7. Serum o- lytes (Na+ and K+), creatinine, and BUN were the primary analytes that were monitored for assessing renal function. Plasma samples were collected in EDTA tubes (Days I and 3:100 ul; Day 7:120 ul). nine clearance was calculated. Urine volume, urinary electrolytes, and nine are expressed as amount excreted per 100 g of animal body weight. All animals were sacrificed on Day 7. Kidneys were weighed.

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

Endogenous creatinine clearance was calculated as follows: CCr (ml/24 h) = {UCr (mg/ml) K V (ml/24 h)] / SCr (mg/m1) 24-hr urinary excretion of sodium (N51+) was calculated as follows: UNaV (qu/24 h) m UNa ) x V (ml/24 h) 24~hr urinary excretion ofNAG and NGALwas similarly calculated.

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 ed as follows: FENa (%) =100 x {UNa(qu/m1) x V (ml/24 h)] / lE’Na (qu/ml) X Cc,— (ml/24 h) ent with anti—Adrenomedullin antibody improved several measures of kidney function on day 7 as compared to vehicle: serum creatinine 1.01 mg/dL mT—M) vs 1.55 mg/dL (vehicle) (Fig. 11), BUN 32.08 mg/dLmT—M) vs. 52.41 mg/dL (vehicle) (Fig. 12), endogenous creatinine nce 934.43 mL/24 h (NT—M) vs. 613.34 mL/24 h (vehicle) (Fig. 13), fractional secretion ofNa+ 0.98 % (NT-M) vs. 1.75 % (vehicle) (Fig. 14).

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

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

Table 10: Parameters of kidney on in the e-(11:11) and NT~threated (n=9) ani- mals. Blood concentrations were measured in samples taken at the end of the experiment.

NGAL z neutrophil gelatinase—associated lipocalin. All data are median (quartiles).

Vehicle ¥NT-M p-Value Urine output [uL-g'l-h‘l] 4.4 65) 15.2 (13.9225) 0.033 Creatinine clearance [uL-min'l] 0;301)_l 400 (316;509) 0.006 f._.. J Creatinine [pg-mm 1.83 (1.52;3.04) 1.28 (1.20;1.52) J—0.010 gee [ug-mijl] 0.004 L 378(268;513)4175(101;184) NGAL [ug-mL‘l] 16 (15;20) 11 (1033) —J 0.008 WO 72512 _ 92 - The experiments were performed as follows: Creatim'ne, urea, and neutrophil gelatinase-associared lipocali'n (NGAL) Blood NGAL concentrations were measured using a commercial ELISA (mouse NGAL, RUO 042, to 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, Boblingen, Germany) using 2Hg-creatinine (CDN isotopes, -Claire, QU, Canada) and methyl—urea (Flu- kaChemikalien, Buchs, Switzerland) as internal standards. After deproteinization with ace— tonitrile, centrifugation and evaporation to dryness, the supernatant was reconstituted in for— mic acid, and extracted over a weak anion exchange column (WCX, Phenomenex, Aschaffen— burg, y). Aeetonitrile plus s(trimethylsilyl)trifluoroacetamide and N—(tert~ butyldimethylsilyl)—N-rnethyltrifluoroacetamide 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 internal stan- dards, respectively. From the urine output and the plasma and urine creatinine concentrations creatinine nce was calculated using the standard formula.

Sample preparation The kidney which was stored at —80°C was disrupted with a nizer in PBS and lysed with a 2—fold concentrated buffer for a whole cell lysate (100 111M Tris pH 7,6; 500 mM NaCl; 6 mM EDTA; 6 mM EGTA; 1 % Triton—X—IOO; 0,5 % NP 40; 10 % Glycerol; Protease- tors (B—Glycerolphosphate 2 111M; DTT 4 mM; Leupeptine 20 uM; Natriumofihova— nadate 0,2 mM)) and subsequently centrifuged. The whole cell lysate was obtained out of the supernatant; the pellet ting 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 pg/pl. The samples for the Multiplex~ and EMSA is were diluted 1:1 with EMSA buffer (10 mM Hepes; 50 mM KCl; 10 ”/0 Glycerol; 0,1 mM EDTA; 1 mM DTT), the samples for the immuno blots 1:1 with 2~fold Sample Buffer (2 % SDS; 125 mM Tris—HCL (pl-l 6,8 at °C); 10 % Glycerol; 50 mM DTT; 0,01 % Bromophenol blue). _ 93 _ Levels of keratinocytevderived chemokine (KC) concentrations were determined using a mouse multiplex cytokine kit (BionPlex Pro Cytokine Assay, Bio—Rad, es, CA), the assay was performed by using the Bio-pies suspension array system with the manufacturer’s instructions (see also Wagner F, Wagner K, Weber S, Stahl B, Knc'iferl 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, Scheuerle A, Weber S, Stahl B, McCook O, Knoferl MW, Huber~Lang M, Seitz DH, Thomas J, Asfar P, Szabo C, Moller P, Gebhard F, Georgieff M, Calzia E, Radermacher P, Wagner K. Cardiopulmonary, histologic, and inflammatory effects of intravenous NaZS after blunt chest -induced lung contu— sion in mice. J Trauma 2011;71:1659—1667). In brief, the appropriate cytokine standards and samples were added to a filter plate. The samples were incubated with dies ally attached to fluorescent—labeled micro beads. Thereafter, premixed detection antibodies were added to each well, and uently, streptavidin—phycoerythrin was added. Beads were then re—suspended, and the cytokines reaction mixture was fied using the ex protein array reader. Data were tically processed and analyzed by Bio—Flex Manager Software 4.1 using the standard curve produced from recombinant cytokine rds. Levels below the detection limit of the assays were set to zero for statistical purposes. flannel).

In the mice CLP model described above, the effect of treatment with anti-adrenornedullin antibody NT—M en the liver was investigated.

NTmM caused a significant lowering of nocyteuderived chemokine (KC) concentrations in the liver (Fig. 16).

Measurement of keratinocyte—deiived chemokine (KC) was done analogous to example 8 (kidney).

Example 10 In the mice CLP model described above, the effect of treatment with anti-adrenomedullin antibody NT—M on several cytokines and ehemokinesin the blood circulation (plasma) was investigated. - 94 _ Cytokine and chemokine concentrations Plasma levels of tumor necrosis factor o, eukin (lL)-6, monocyte chemoattractant protein (MCP)-l, and keratinocyte~derived chemokine (KC) concentrations were determined using a mouse multiplex cytokine kit (Bio—Plex Pro Cytokine Assay, Bio-Rad, Hercules, CA), the assay was performed by using the ex suspension array system with the manufac- turer’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, y- namic murine septic shock. Shock 2011;35:396—402; and Wagner F, Scheuerle A, Weber S, Stahl B, McCook O, Knoferl MW, Huber-Lang M, Seitz DH, Thomas J, Asfar P, Szabo C, Moller P, Gebhard F, Georgieff M, Calzia E, Raderrnacher P, Wagner K. Cardiopulmonary, ogic, and inflammatory effects of intravenous NaZS after blunt chest trauma—induced lung contusion in mice. J Trauma 2011;71:1659~l667). In brief, the appropriate cytokine standards and samples were added to a filter plate. The s were incubated with antibod- ies 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 fied using the Bio—Flex protein array reader. Data were automatically processed and analyzed by Bio’Plex Manager Software 4.1 using the standard curve produced from recombinant cytokine stan~ dards. Levels below the detection limit of the assays were set to zero for statistical purposes.

Plasma levels and kidney tissue concentrations of tumor necrosis factor (TNF)—o., interleukin (IL)—6 and IL— 10, monocyte ttractant protein (MCP)—l, and keratinocyte—dervived chemokine (KC) were determined using a commercially available plex ne Kit” (Bio—Plex Pro ion Pro ne Assay, Bio—Rad, Hercules, CA), which allows to collect several parameters out of one single sample. The individual work steps of the assay were per— formed according to the manufacturer‘s instructions (see also Wagner F, Wagner K, Weber S, Stahl B, Kniiferl MW, Huber-Lang M, Seitz DH, Asfar P, Calzia E, Senitleben U, Gebhard F, Georgieff M, acher P, Hysa V. Inflammatory effects of hypothermia and d H28 during resuscitated, hyperdynamic murine septic shock. Shock 2011;35:396-402; and Wagner F, Scheuerle A, Weber S, Stahl B, McCook O, Knéiferl MW, Huber—Lang M, Seitz DH, Tho- mas J, Asfar P, Szabo C, Mdller P, Gebhard F, Georgieff M, Calzia E, Raderrnacher P, Wag— ner K. Cardiopulmonary, histologic, and inflammatory effects of intravenous NaZS after blunt chest trauma—induced lung contusion in mice. J Trauma 2011;71:1659—1667). _ 95 _ In brief, the fluorescence—labed microspheres s”) were added to a l plate, fol- lowed by two washing steps, the addition of internal standards and the addition of plasrna— and kidney homogenate samples. During the subsequent incubation the single cytokines bind to the antibodies attached to polystyrene—beads. After the addition of the cytokine~specific biotin—labeled antibodies, which are for the detection of the single cytokines, and an addi- tional tion time, subsequently phycoerythrin—labeled streptavidine was added. Alter an additional incubation time, beads were then resuspended, and the plates could be measured with a specific flow cytometer (Bio-Flex sion array system, Bio—Rad, es, CA).

Data were automatically processed and analyzed by ex Manager Software 4.1 using the standard curve produced from recombinant cytokine standards. For the plasma levels the con- centration was provided in pg * ELL-E, the concentration of the kidney homogenates were con— verted to the appropriate protein concentration and provided in pg * mg-1 protein.

NTwM caused a significant lowering of plasma concentrations of lL—6 (Fig. 17), lL—lO (Fig. 18), keratinocyte—derived chemokine (KC) (Fig. 19), monocyte chemoattractant protein—1 ) (Fig. 20), TNF—alpha (Fig. 21).

Exam le 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 ll, y PF, Chan L, and Schrier RW. In vitro and in Vivo protective effect of atriopeptin III on ischemic acute renal failure. I Clinlnvest 80:698—705, 1987., Chintala MS, dino V, and Chiu PJS. Cyclic GMP but not cyclic AMP prevents renal platelet accumulation foliowing ischernia- reperfusion in anesthetized rats. J PharmacolExpTher 271:1203—1208, 1994). This model was used to assess r treatment with anti—adrenomedullin antibody can improve kidney func- tion.

The experiment was performed as follows: _ 96 _ Effect of a NT-M on Acute Kidney Injury Induced by lschemia/Reperfusion in Rats Study Design: Test Cone Dosage Rats Groun Article Route Inn/ml mllkg rug/kg (Male) 1 LR + vehiclea IV 5 NA X 3 8 2 LR + NT—M 1v 5 x 3b 8 a vehicle; injected intravenously (iv) 5 min before usion on day 0, followed by injections on days 1 and 2. bNT—M at 4 mg/kg was injected enously (i.v.) 5 min before reperfusion on day 0, followed by 2 rag/kg i.v. each on days 1 and 2. cUrine collection on days ~1, 0, 1 and 2, with blood try and urine analysis on days 0, l, 2 and 3, respectively. Plasma samples were collected in EDTA tubes (Days 0 (im— mediate before surgery), 1, 2: 100 pl, before vehicle or TA; Day 3:120 ul.

Clinical observations: 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 e a rd diet with led water ad libitum.

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

The abdominal cavity was eXposed via a midline incision, followed by intravenous adminiu stration of heparin (100 U/kg, i.v.) and both renal arteries were occluded for 45 min by using vascular clamps. Immediately after removal of the renal clips, the s were observed for additional 1 min to ensure color change ting blood reperfusion. The test nd (NT-M) and vehicle (phosphate buffered saline) were injected intravenously 5 min before reperfusion, followed by daily injection on days i and 2.

Urine collection. The 24~h urine collection on ice was initiated at 2411 before ische~ mia/reperfusion on day —l ("2411 to Oh), and day 0 (0-2411), day l (24—48h) and day 2 (48—7211) after reperfusion, Blood collection: 0.4 ml blood was collected through the tail vein into EDTA tubes at 011 (be— fore 1 RI surgery), 24h (before vehicle or TA), 48h (before vehicle or TA) and 72b for deter— mination of plasma creatinine/Na+fK+, and BUN; 2 ml blood was collected through venal cava terminally. - 97 _ The animals were placed in individual cages where urine was collected for 24 h day —1 (—24h— 0h), day 0 ), day 1 (24«48h) and day 2 (48—72h) after reperfusion on day 0. Urine vol- ume, urinary Na+, K+, and creatinine were measured.

The creatinine clearance (CCr) was calculated as follows: CCr (ml/24 h) 2 [UCr (mg/ml) X V (ml/24 h)] / PCr (mg/ml) The 24-hr urinary excretion of sodium (N21+) was calculated as follows: UNaV (qu/24 h) : UNa (qu/Inl) x V (inlf24 h) The fractional excretion of Na+ , or percentage of the filtered sodium that is excreted into the final urine, is a measure of tubular Na+ reabsorptive function. It was ed as follows: FENa (%) =100 x [UNa (qu/rnl) x v (ml/24 11)} /PNa (nEq/rnl) X CCr (ml/24 11) Treatment with anti—Adrenomedullin antibody improved several measures of kidney function: Blood urea nitrogen (BUN) showed a strong increase in the vehicle group (0 h: 17.49 ing/dL, 24 h: 98.85 mg/dL, 48 h: 109.84 mg/dL, 72 h: 91.88 ing/dL), which was less pronounced with NT—M ent (0 h: 16.33 mg/dL, 24 h: 84.2 mg/dL, 48 h: 82.61 mg/dL, 72 h: 64.54 mg/dL) (Fig. 22).

Serum creatinine ped similarily: Vehicle group (0 h: 0.61 ing/dL, 24 h: 3.3 mg/dL, 48 h: 3.16 mg/dL, 72 h: 2.31 mg/dL), NT—M group: (0 h: 0.59 rng/dL, 24 h: 2.96 mg/dL, 48 h: 2.31 Ing/dL, 72 h: i .8 mg/dL) (Fig. 23).

The endogenous creatinine clearance dropped massively on day one and thereafter ed better in the NT-M group than in the vehicle group. Vehicle group: (O h: 65.17mth, 24 h: 3.5mL/h, 48 h: 12.61mL/h, 72 h: n), NT—M group: (O h: 70.11mL/h, 24 h: 5.84mL/h, 48 h: 21.23mL/h, 72 h: 26.61rnL/h) (Fig. 24). _ 98 ..

FIGURE DESCRIPTION Fig. 1a: Illustration of antibody formats — FV and scFV—Variants Fig 1b: Illustration of antibody formats — heteroiogous fusions and bifunctional antibodies Fig 1c: illustration of antibody formats _. bivalental antibodies and bispecific antibodies Fig. 2: hADM 1—52 (SEQ ID No. 21) mADM 1—50 (SEQ ID No. 22) aa 1—21 ofhuman ADM (SEQ ID No. 23) aa 1-42 ofhuman ADM (SEQ ID No. 24) aa 43—52 ofhuman ADM (SEQ D No. 25) aa 1-14 of human 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 ofhuman mature human ADM (SEQ 11) NO: 29) aa 1—40 of mature mutine ADM (SEQ ID NO: 30) aa 1~31 ofmature marine ADM (SEQ ID NO: 31) Fig. 3: 3: Dose response curve of human ADM. l CAMP stimuiation was ed to 100% activation Dose/ inhibition curve of human ADM 22~52 (ADM-receptor antagonist) in the presence of 5.63nM hADM.

Dose/ inhibition curve of CT—H in the presence of 5.63 nM hADM.

Dose/ tion curve of MR—H in the presence of 5.63 nM hADM.

Dose/ tion curve ofNT-H in the presence of 5.63 nM hADM. a 99 _ f: Dose response curve of mouse ADM. Maximal CAMP stimulation 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 niADM. i: Dose/ inhibition curve of MRuM in the presence of 0,67 nM mADM. j: Dose/ inhibition curve ofNT—M in the presence of 0,67 nM mADM. k: shows the inhibition ofADM by F(ab)2 NT—M and by Fab NT—M 1: shows the tion 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 ofhADM in human plasma (citrate) in e and in the pres ence ofNT—H antibody.

Fig. 6: Alignment of the Fab with homologous human framework sequences Fig. 7: This figure shows the Noradrenalin requirements for early and late treatment with NT-M Fig. 8: This figure shows urine production after early and late 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 er in B cells (NF—KB) analyzed by electophoretic mobility shift assay (EMSA)- # depicts p<0.001 vs. vehicle.

Fig. 11: — 100 — 2012/072931 Development of serum creatinine over time. Mean +/— SEM are shown.

Fig. 12: Development of blood urea nitrogen (BUN) overtime. Mean +/— SEM are shown.

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

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

Fig. 15: Keratinocyte-deiived chemokine (KC) levels determined in relation to the total kidney protein ted. The white box—plot shows results obtained with e, the grey box—plot shows results obtained after ent with NT~M.

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

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

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

Fig. 19: Plasma keratinocyte—derived chemokine (KC) levels. The white box—plot shows results ob- tained with vehicle, the grey box—plot shows results obtained after treatment with NT—M. - 101 ~ Fig. 20: Plasma monocyte Chemoattractan’: protein—1 (MCP-l) levels. The white ot shows re- sults obtained with vehicle, the grey box~plot shows results obtained after treatment with NT- Fig. 21: Plasma TNF—alpha - The white box—plot shows results obtained with vehicle, the grey ot shows results obtained after treatment with NT—M.

Fig. 22: Development of blood urea nitrogen (BUN) overtime. Mean +/~ SEM are shown.

Fig. 23: Development of serum creatinine overtime. Mean +/— SEM are Shown.

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

Claims (7)

Claims

1. Use of an anti-adrenomedullin antibody or an anti-ADM antibody fragment binding to adrenomedullin or an anti-ADM -protein scaffold binding to adrenomedullin in the manufacture of a medicament, wherein said antibody or fragment or scaffold binds to the N- al part, amino acids 1 to 21, of adrenomedullin: YRQSMNNFQGLRSFGCRFGTC; SEQ ID No. 23.

2. Use according to claim 1, wherein said antibody or fragment or scaffold is ecific.

3. Use according to claim 1 or 2, wherein said antibody or fragment or ld exhibits a g affinity to ADM of at least 10-7 M.

4. Use according to any one of claims 1 to 3, wherein said antibody or fragment or scaffold is not ADM-binding-Protein-1, complement factor H.

5. Use of an according to any one of claims 1 to 4 binds to an epitope ning the N- al end; amino acid 1; of mature adrenomedullin.

6. Use according to any one of the claims 1 to 5, characterized in that said antibody, antibody fragment or non-Ig-protein scaffold does not bind to the C-terminal portion of mature ADM, being amino acids 43-52 of mature ADM PRSKISPQGY-NH2 ; SEQ ID NO: 25.

7. Use according to any one of claims 1 to 6, wherein said dy or said fragment or said scaffold is an ADM izing antibody or fragment or scaffold that enhances the half life, t