HK40006910B - Anti-adrenomedullin (adm) antibody or anti-adm antibody fragment or ant-adm non-ig scaffold for use in therapy - Google Patents

Description

Anti-adrenergic medullary (ADM) antibodies, anti-ADM antibody fragments, or anti-ADM non-Ig backbones for therapeutic use

Invention Field

The subject of this invention is an anti-adrenergic medullary antibody or an anti-adrenergic medullary antibody fragment or an anti-ADM non-Ig backbone, wherein the antibody or fragment or backbone is a non-neutralizing antibody.

The subject of this invention is an anti-adrenergic medullary antibody or an anti-adrenergic medullary antibody fragment or an anti-ADM non-Ig backbone, wherein said antibody, fragment, or backbone is

• An ADM-stabilized antibody or ADM-stabilized antibody fragment or ADM-stabilized non-Ig backbone that increases the half-life (t _1/2 half-retention time) of adrenomedullin in serum, blood, or plasma by at least 10%, preferably at least 50%, more preferably >50%, and most preferably 100%.

• The ADM stable antibody or adrenaline-medulloin stable antibody fragment or ADM stable non-Ig backbone blocker has less than 80%, preferably less than 50%, of ADM biological activity.

The above description means blocking no more than 80% or no more than 50% of the ADM biological activity, respectively, and should therefore be understood as limiting or reducing the ADM biological activity through the respective ADM binders (which are ADM-stabilized antibodies, antibody fragments, or non-Ig backbones). Implicitly, this means that some ADM biological activity remains. For example, assuming no more than 80% of the ADM biological activity is blocked, approximately 20% of the ADM biological activity remains. Assuming no more than 50% of the ADM biological activity is blocked, approximately 50% of the ADM biological activity remains.

The subject of this invention is an anti-adrenal medullary antibody or an anti-adrenal medullary antibody fragment or an anti-ADM non-Ig backbone, wherein the antibody or fragment or backbone binds to the N-terminal portion (aa 1-21) of human adrenal medullary.

In a preferred embodiment, the subject of the invention is an anti-adrenal medullary antibody or an anti-adrenal medullary antibody fragment or an anti-ADM non-Ig backbone, wherein the antibody or fragment or backbone binds to the N-terminus (aa1) of human adrenal medullary.

Background of the Invention

The peptide adrenomedullin (ADM) was first described in 1993 (Kitamura, K., et al., "Adrenomedullin: A Novel Hypotensive Peptide Isolated From Human Pheochromocytoma", Biochemical and Biophysical Research Communications, Vol. 192(2), pp. 553-560 (1993)) as a novel 52-amino acid antihypertensive peptide isolated from human pheochromocytoma, with the sequence SEQ ID No.: 21. In the same year, a cDNA encoding a 185-amino acid precursor peptide and its complete amino acid sequence were also described. The precursor peptide (which contains a 21-amino acid signal sequence at its N-terminus and other sequences) is referred to as "pre-proADM". In this specification, all amino acid positions specified generally refer to the 185-amino acid pre-proADM. Adrenomedullin (ADM) is a 52-amino acid peptide (SEQ ID NO: 21) containing amino acids 95 to 146 of the pre-proADM peptide, formed by proteolytic cleavage. To date, only a few fragments of the peptides formed from the cleavage of pre-proADM have been more precisely characterized, specifically the physiologically active peptides adrenomedullin (ADM) and "PAMP," which are 20-amino acid peptides (22-41) following the 21-amino acid signal peptide of the pre-proADM peptide. The discovery and characterization of ADM in 1993 sparked a concentrated research effort, the results of which were summarized in numerous review articles. In the context of this specification, specific references are made to articles appearing in “peptide” publications dedicated to ADM, particularly (Editorial, Takahashi, K., “Adrenomedullin: from a pheochromocytoma to the eyes”, Peptides, Vbl. 22, p. 1691 (2001)) and (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, Vbl. 22, pp. 1693-1711 (2001)). Other reviews include (Hinson, et al., "Adrenomedullin, a Multifunctional Regulatory Peptide", Endocrine Reviews, Vbl. 21(2), pp. 138-167 (2000)). In scientific research to date, among other things, ADM has been identified as a multifunctional regulatory peptide. It is released into circulation in an inactive form extended from glycine (Kitamura, K., et al., "The intermediate form of glycine-extended adrenomedullin is the major or circulating molecular form in human plasma", Biochem. Biophys. Res. Commun., Vol. 244(2), pp. 551-555 (1998). Abstract only). Another binding protein (Pio, R., et al., "Complement Factor His a Serum-binding Protein f or adrenomedullin, and the Resulting Complex Modulates the Bioactivities of Both Partners", The Journal of Biological Chemistry, Vol. 276(15), pp. 12292-12300(2001)) is specific for ADM and may similarly regulate the effects of ADM. The physiological effects of ADM and PAMP (which have been the main focus of research to date) are effects on blood pressure.

Therefore, ADM is an effective vasodilator, and thus, it is possible to associate its antihypertensive effect with a specific peptide segment in the C-terminal region of ADM. Furthermore, it has been found that another physiologically active peptide, PAMP, formed from pre-proADM, also exhibits an antihypertensive effect, even though it appears to have a different mechanism of action than ADM (see also Eto, T., "A review of the biological properties and clinical implications of adrenomedullin and prorenomedullin N-terminal 20 peptide (PAMP), hypotensive a..."). nd vasodilating peptides", Peptides, Vbl. 22, pp. 1693-1711 (2001)) and (Hinson, et al., "adrenomedullin, a Multifunctional Regulato ryPeptide", Endocrine Reviews, Vbl.21(2), pp.138-167(2000)), and (Kuwasako, K., et al., "Purification and characterization of PAM P-12(PAMP-20)in porcine adrenalmedulla as a major endogenous biologically active peptide″, FEBS Lett, Vol.414(1), pp.105-1 10 (1997). Abstract only), (Kuwasaki, K., et al., "Increased plasmaproadrenomedullin N-terminal 20 peptide in patients with essentialhy (See "pertension", Ann. Clin. Biochem., Vbl. 36(Pt. 5), pp. 622-628 (1999). Abstract only) or (Tsuruda, T., et al., "Secretion of proadrenomedullin N-terminal 20peptide from cultured neonatal rat cardiac cells", Life Sci., Vol. 69(2), pp. 239-245 (2001). Abstract only) and EP-A2 0 622 458). Furthermore, it has been found that, under various pathological conditions, the concentrations of ADM measurable in circulation and other biological fluids are significantly higher than those present in healthy controls. Therefore, ADM levels are significantly increased, albeit to varying degrees, in patients with congestive heart failure, myocardial infarction, nephropathy, hypertension, diabetes, acute shock, and sepsis and septic shock. PAMP concentrations also increase in some of the pathological conditions, but plasma levels are lower relative to ADM (Eto, T., "A review of the biological properties and clinical implementations of adrenomedullin and prorenomedullin N-terminal 20 peptide (PAMP), hypotensive and vasodilating peptides", Peptides, Vbl. 22, pp. 1693-1711 (2001); 1702). Furthermore, unusually high concentrations of ADM have been observed in sepsis, with the highest concentrations observed in septic shock (see (Eto, T., "A review of the biological properties and clinical implications of adrenomedullin and proadrenomedullin N-terminal 20peptide (PAMP), hypotensive and vasodilating peptides", Peptides, Vbl. 22, pp. 1693-1711 (2001)). (Hirata, et al., ‘Increased Circulating adrenomedullin, a NovelVasodilatory Peptide, in Sepsis’, Journal of Clinical Endocrinology andM etabolism, Vbl.81(4), pp.1449-1453(1996)), (Ehlenz, K., et al., "High levels of circulating adrenomedullin in severe illness: Correlation with C-reactiveprotein and evidence against the adrenal medulla as site of origin”, Exp ClinEndocrinol Diabetes, Vbl.105, pp.156-162( 1997)), (Tomoda, Y., et al., "Regulation of adrenomedullin secretion from cultured cells", Peptides, Vbl.22, pp.1783-1794(2001)), (Ueda, S., et al., "Increased Plasma Levels of adrenomedullin inPatients with Systemic Inflammatory Response Syndrome", Am.J.Respir.Crit.CareMe d., Vbl. 160, pp. 132-136 (1999)) and (Wang, P., "adrenomedullin and cardiovascular responses in sepsis", Peptides, Vbl. 22, pp. 1835-1840 (2001)).

Methods for identifying the immunoreactivity of adrenomedullin in biological fluids for diagnostic purposes, and specifically within the scope of sepsis diagnosis, cardiac diagnosis, and cancer diagnosis, are also known in the art. According to the invention, a central region peptide of the adrenomedullin precursor, comprising the amino acids (45-92) of the entire adrenomedullin precursor, is specifically measured using an immunoassay utilizing at least one labeled antibody that specifically recognizes the mid-proADM sequence (WO2004/090546).

WO-A1 2004/097423 describes the use of antibodies against adrenomedullin for the diagnosis, prognosis, and treatment of cardiovascular diseases. Treatment of diseases by blocking ADM receptors is also described in the art (e.g., WO-A1 2006/027147, PCT/EP2005/012844), and these diseases can include sepsis, septic shock, cardiovascular diseases, infections, skin diseases, endocrine disorders, metabolic diseases, gastrointestinal diseases, cancer, inflammation, hematological diseases, respiratory diseases, musculoskeletal diseases, neurological diseases, and urinary system diseases.

According to reports, ADM improves cardiac function and blood supply to the liver, spleen, kidneys, and small intestine in early sepsis. ADM neutralizing antibodies neutralize the previously mentioned effects in early sepsis (Wang, P., "Adrenomedullin and cardiovascular responses in sepsis", Peptides, Vol. 22, pp. 1835-1840 (2001).

In late-stage sepsis, i.e., the low-dynamic phase of sepsis, ADM constitutes a risk factor closely associated with mortality in patients with septic shock (Schütz et al., “Circulating Precurs or levels of endothelin-1 and adrenomedullin, two endothelium-derived, counteracting substances, insepsis”, Endothelium, 14:345-351, (2007)). The diagnosis and treatment of critically ill patients (e.g., in the very late stages of sepsis), and the use of vasoconstrictive endothelin and endothelin agonists in the preparation of medicines for the treatment of critically ill patients, are described in WO-A1 2007/062676. WO-A1 2007/062676 also describes the use of adremyelin antagonists in place of or in combination with endothelin and/or endothelin agonists, said adremyelin antagonists being molecules, for example, that block or attenuate the vasodilatory activity of adremyelin by blocking its associated receptors, or substances that prevent adremyelin from binding to its receptors (e.g., specific binders, such as antibodies that bind to adremyelin and block its receptor binding site, "immunologic neutralization"). Such use or combination, including subsequent or prior use alone, has been described in certain circumstances as suitable, for example, to improve treatment success rates, or to avoid adverse physiological stress or side effects. Thus, antibodies that neutralize ADM have been reported for use in the treatment of sepsis in advanced stages of sepsis.

Treatment of sepsis and septic shock with ADM and ADM-binding protein 1 is described in this art. It is presumed that treatment of sepsis-affected animals with ADM and ADM-binding protein 1 prevents the transition to advanced sepsis. It should be noted that in living organisms, ADM-binding protein (complement factor H) is present in high concentrations in the circulation of said organisms (Pio et al.: Identification, characterization, and physiological actions of factor H as an Adrenomedullin-binding Protein present in Human Plasma; Microscopy Res. and Technique, 55: 23-27 (2002) and Martinez et al.; Mapping of the Adrenomedullin-Binding domains in Human Complement factor H; Hypertens Res Vol. 26, Suppl (2003), pp. 56-59).

According to the present invention, ADM-binding protein 1 may also be referred to as ADM-binding protein 1 (complement factor H).

Therefore, administering ADM may be beneficial in early-stage diseases such as sepsis, but harmful in late-stage sepsis. The opposite could be administering anti-ADM antibodies. Furthermore, dosage can be crucial when administering ADM or anti-ADM antibodies.

For other diseases, partial blocking of ADM may be beneficial. However, complete neutralization of ADM may also be harmful, as a certain amount of ADM may be necessary for some physiological functions. Many reports emphasize that administration of ADM may be beneficial in certain diseases. Conversely, in other reports, ADM has been reported to be life-threatening when administered in certain disease states. The shortcomings of the prior art can be overcome by the anti-ADM antibody, anti-ADM antibody fragment, or anti-ADM non-Ig backbone provided by this invention.

Invention Overview

The subject of this invention is an anti-adrenergic medullary antibody or an anti-ADM antibody fragment or an anti-ADM non-Ig backbone that binds adrenergic medullary, used as a medicament, wherein the antibody or the fragment or the backbone is a non-neutralizing antibody or fragment or backbone.

Throughout this specification, the “antibody” or “antibody fragment” or “non-Ig backbone” of the present invention is capable of binding to ADM, and is therefore targeted at ADM, and may thus be referred to as an “anti-ADM antibody,” “anti-ADM antibody fragment,” or “anti-ADM non-Ig backbone.”

In another embodiment of the present invention, the anti-ADM antibody, anti-ADM antibody fragment, or anti-ADM non-Ig backbone provided by the present invention can bind to circulating ADM, and is therefore targeted at circulating ADM.

In a specific implementation, the anti-ADM antibody, anti-ADM antibody fragment, or anti-ADM non-Ig backbone is a non-neutralizing antibody, fragment, or non-Ig backbone. The neutralizing anti-ADM antibody, anti-ADM antibody fragment, or anti-ADM non-Ig backbone blocks approximately 100%, at least 90%, and preferably at least 95% of the ADM biological activity.

In contrast, non-neutralizing anti-ADM antibodies, anti-ADM antibody fragments, or anti-ADM non-Ig backbones block less than 100% of ADM biological activity, preferably less than 95%, more preferably less than 90%, more preferably less than 80%, and even more preferably less than 50% (baseline value). This means that the residual biological activity of ADM bound to the non-neutralizing anti-ADM antibody, anti-ADM antibody fragment, or anti-ADM non-Ig backbone is 0% or more, preferably more than 5%, more preferably more than 10%, more preferably more than 20%, and even more preferably more than 50%. This implies blocking no more than 80% or no more than 50% of ADM biological activity, and therefore should be understood as limited blocking of ADM biological activity by the respective ADM binders (which are anti-ADM antibodies, anti-ADM antibody fragments, or anti-ADM non-Ig backbones).

It should be understood that the limited blocking of ADM biological activity occurs even when the concentration of the antibody, antibody fragment, or non-Ig backbone is excessive (i.e., the antibody, fragment, or backbone is in excess relative to ADM). This limited blocking is an inherent property of the ADM binder itself. This means that the antibody, fragment, or backbone has a maximum inhibition of 80% or 50%, respectively. In a preferred embodiment, the anti-ADM antibody, anti-ADM antibody fragment, or anti-ADM non-Ig backbone blocks at least 5% of ADM biological activity. This means—implicitly—that approximately 95% of the residual ADM biological activity remains.

The subject of this invention is an adrenomedullin antibody or adrenomedullin antibody fragment for treating chronic or acute diseases, wherein said antibody or fragment is

• An ADM-stabilized antibody or adrenomedullin antibody fragment that increases the t1 _/2 half-retention time of adrenomedullin in serum, blood, or plasma by at least 10%, preferably at least 50%, more preferably >50%, and most preferably 100%.

• The antibody or the adrenomedullin antibody fragment therein blocks less than 80%, preferably less than 50%, of the ADM biological activity.

The subject of this invention is an adrenomedullin antibody or adrenomedullin antibody fragment for treating chronic or acute diseases, wherein the antibody or fragment binds to the N-terminal portion (aa 1-21) of human adrenomedullin.

In a preferred embodiment, the subject of the invention is an adrenomedullin antibody or adrenomedullin antibody fragment for treating chronic or acute diseases, wherein the antibody or fragment binds to the N-terminus of human adrenomedullin.

In this context, for simplification purposes, molecules with "non-neutralizing anti-ADM activity," collectively referred to as "non-neutralizing" anti-ADM antibodies, antibody fragments, or non-Ig backbones (which, for example, block less than 80% of ADM biological activity), are defined as having "non-neutralizing anti-ADM activity."

• One or more molecules of ADM, after being added to a eukaryotic cell line culture, reduce the amount of cAMP produced by the cell line by the action of parallel addition of synthetic human ADM peptides, wherein the cell line expresses a functional recombinant human ADM receptor consisting of CRLR (calcitonin receptor-like receptor) and RAMP3 (receptor activity modified protein 3), wherein the added synthetic human ADM is added in an amount that results in half-maximal stimulation of cAMP synthesis in the absence of the non-neutralizing antibody to be analyzed, wherein the degree to which the molecule binds to ADM results in a reduction of cAMP does not exceed 80%, even when the non-neutralizing molecule to be analyzed that binds to ADM is added in an amount more than 10 times (the amount required to obtain the maximum reduction of cAMP with the non-neutralizing antibody to be analyzed).

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

Bioactivity is defined as the effect of a substance in vivo or in vitro (e.g., in an assay) following its interaction, presenting itself as a living organism, tissue, organ, or functional unit. In the case of ADM bioactivity, this could be the effect of ADM in a human recombinant adrenomedullary receptor cAMP function assay. Therefore, according to the invention, bioactivity is defined by an adrenomedullary receptor cAMP function assay. To determine the bioactivity of ADM in such an assay, the following steps can be performed:

• A dose-response curves were generated using ADM in the human recombinant adrenal medullaris receptor cAMP function assay.

• It can calculate the ADM concentration for half-maximal cAMP stimulation.

• Under constant half-maximal cAMP stimulation, ADM concentration-dose response curves were obtained using ADM-stabilizing antibodies, adrenomedullin-stabilizing antibody fragments, or adrenomedullin-stabilized non-Ig backbones (up to a final concentration of 100 μg/ml).

The 50% maximum inhibition in the ADM bioassay indicates that the anti-ADM antibody, the anti-adrenergic medullary antibody fragment, or the anti-adrenergic medullary non-Ig backbone each blocks 50% of the baseline bioactivity. The 80% maximum inhibition in the ADM bioassay indicates that the anti-ADM antibody, the anti-adrenergic medullary antibody fragment, or the anti-adrenergic medullary non-Ig backbone each blocks 80% of the ADM bioactivity. This means that no more than 80% of the ADM bioactivity is blocked. This implies that approximately 20% of the residual ADM bioactivity remains.

However, in light of this specification and in the context described above, in conjunction with the anti-ADM antibody, anti-ADM antibody fragment, and anti-ADM non-Ig backbone disclosed herein, the expression "blocking the biological activity of ADM" should be understood as merely reducing the biological activity of ADM, preferably reducing the remaining ADM biological activity from 100% to 20% at most, and preferably reducing the remaining ADM biological activity from 100% to 50%, but in any case, there is still ADM biological activity that can be measured as described above.

ADM bioactivity can be determined in the human recombinant adrenal medullary receptor cAMP function assay (adrenal medullary bioassay) according to Example 2.

Anti-adrenergic medullary (ADM) antibodies are antibodies that specifically bind to ADM. Anti-adrenergic medullary antibody fragments are fragments of ADM antibodies, wherein said fragments specifically bind to ADM. Anti-ADM non-Ig backbones are non-Ig backbones that specifically bind to ADM. Specific binding to ADM also allows binding to other antigens. This means that this specificity does not preclude the antibody from cross-reacting with peptides other than those that stimulate the antibody. This also applies to the specificity of the anti-ADM antibody fragments or anti-ADM non-Ig backbones of the present invention.

Surprisingly, it has been demonstrated that non-neutralizing anti-ADM antibodies, or non-neutralizing anti-ADM antibody fragments, or non-neutralizing anti-ADM non-Ig backbones offer significant therapeutic advantages over neutralizing anti-ADM antibodies, or neutralizing anti-ADM antibody fragments, or neutralizing anti-ADM non-Ig backbones.

In a specific embodiment, the anti-ADM antibody or anti-adrenal medullary antibody fragment or anti-ADM non-Ig backbone of the present invention is preferably used, wherein the anti-adrenal medullary antibody or the anti-adrenal medullary antibody fragment or anti-ADM non-Ig backbone is an ADM-stabilized antibody or adrenal medullary-stabilized antibody fragment or adrenal medullary-stabilized non-Ig backbone that increases the half-life (half-retention time) of adrenal medullary in serum, blood, or plasma by at least 10%, preferably at least 50%, more preferably >50%, and most preferably >100%.

The half-life (half-retention time) of ADM in human plasma can be determined using a quantitative ADM immunoassay, under conditions of absence and presence of ADM stabilizing antibodies, adrenomedullin stabilizing antibody fragments, or adrenomedullin stabilizing non-Ig backbones.

The following steps can be performed:

• ADM can be diluted in human citrate plasma in the absence of ADM stabilizing antibodies or adrenomedullin stabilizing antibody fragments or adrenomedullin stabilizing non-Ig backbones, and can be incubated at 24°C.

• Collect aliquots at selected time points (e.g., within 24 hours), and freeze at -20°C to terminate the degradation of ADM in the aliquots.

• If the selected assay is not affected by stable antibodies, the amount of ADM can be determined directly by hADM immunoassay. Alternatively, aliquots can be treated with a denaturing agent (such as HCl), and after sample clarification (e.g., by centrifugation), the pH can be neutralized and ADM can be quantified by ADM immunoassay. Alternatively, non-immunoassay techniques (e.g., rpHPLC) can be used for ADM quantification.

• Calculate the half-life of ADM for incubation under conditions of absence and presence of ADM stabilizing antibody or adrenomedullin stabilizing antibody fragment or adrenomedullin stabilizing non-Ig backbone, respectively.

• The calculated half-life of stable ADM is increased compared to ADM incubated under conditions where no ADM stabilizing antibody or adremyelin stabilizing antibody fragment or adremyelin stabilizing non-Ig backbone is present.

ADM's half-life is doubled, representing a 100% increase in half-life.

Half-life (half-retention time) is defined as the time it takes for the concentration of a specified chemical or drug in a specified liquid or blood to decrease to half of its baseline concentration.

The determination of the half-life (half-retention time) of adrenomedullin in serum, blood, and plasma is described in Example 3.

The antibodies of this invention are proteins, comprising one or more polypeptides specifically binding to antigens, substantially encoded by immunoglobulin genes. Recognized immunoglobulin genes include constant region genes for κ, λ, α (IgA), γ (IgG1, IgG2, IgG3, IgG4), δ (IgD), ε (IgE), and μ (IgM), as well as numerous variable region genes. The full-length immunoglobulin light chain is typically about 25 kDa or 214 amino acids long. The full-length immunoglobulin heavy chain is typically about 50 kDa or 446 amino acids long. The light chain is encoded by a variable region gene (about 110 amino acids long) located at the NH2-terminus and a κ or λ constant region gene located at the COOH-terminus. The heavy chain is also encoded by a variable region gene (about 116 amino acids long) and one of the other constant region genes.

The basic structural unit of an antibody is typically a tetramer composed of two pairs of identical immunoglobulin chains, each pair having one light chain and one heavy chain. In each pair, the variable regions of the light and heavy chains bind the antigen, while the constant regions mediate the effect or function. Immunoglobulins also exist in a variety of other forms, including, for example, Fv, Fab, and F(ab′)2, as well as bifunctional hybrid antibodies and single-chain antibodies (e.g., Lanzavecchia et al., Eur: J. Immunol. 17: 105, 1987; Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85: 5879-5883, 1988; Bird et al., Science 242: 423-426, 1988; Hood et al., Immunology, Benjamin, N.Y., 2nd ed., 1984; Hunkapiller and Hood, Nature 323: 15-16, 1986). The variable regions of the immunoglobulin light or heavy chains include framework regions interrupted by three hypervariable regions, also known as complementarity-determining regions (CDRs) (see Sequences of Proteins of Immunological Interest, E. Kabat et al., U.S. Department of Health and Human Services, 1983). As noted above, CDRs are primarily responsible for binding to epitopes of antigens. Immune complexes are antibodies, such as monoclonal antibodies, chimeric antibodies, humanized antibodies, or human antibodies, or functional antibody fragments, that specifically bind to antigens.

Chimeric antibodies are antibodies whose light and heavy chain genes are constructed by genetic engineering from variable and constant region genes of immunoglobulins belonging to different species. For example, a variable segment from a mouse monoclonal antibody gene can be linked to a human constant segment such as κ and γ1 or γ3. In one instance, a therapeutic chimeric antibody is thus a hybrid protein consisting of a variable domain or antigen-binding domain from a mouse antibody and a constant or effector domain from a human antibody, although the variable region can be generated using other mammalian species or through molecular techniques. Methods for preparing chimeric antibodies are well known in the art, see, for example, U.S. Patent No. 5,807,715. A “humanized” immunoglobulin is an immunoglobulin that includes one or more CDRs of a human framework region and a non-human (such as mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin that provides the CDR is called the “donor”, and the human immunoglobulin that provides the framework is called the “recipient”. In one embodiment, all CDRs of a humanized immunoglobulin are derived from the donor immunoglobulin. Constant regions are not required, but if they exist, they must be substantially identical to the constant regions of human immunoglobulins, i.e., at least about 85-90%, such as about 95% or more identical. Therefore, all parts of a humanized immunoglobulin, except for the CDR, are substantially identical to their corresponding parts of the natural human immunoglobulin sequence. A "humanized antibody" is an antibody containing humanized light and heavy chain immunoglobulins. Humanized antibodies can bind to the same antigen as the donor antibody that provides the CDR. The receptor framework of a humanized immunoglobulin or antibody may have a limited number of amino acid substitutions from the donor framework. Humanized or other monoclonal antibodies may have other conserved amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin function. Exemplary conserved substitutions are those such as gly, ala; val, ile, leu; asp, glu; asn, gln; ser, thr; lys, arg; and phe, tyr. Humanized immunoglobulins can be constructed using genetic engineering techniques (see, for example, U.S. Patent No. 5,585,089). Human antibodies are antibodies in which the light chain and heavy chain genes are derived from humans. Human antibodies can be produced using methods known in the art. Human antibodies can be produced by immortalizing human B cells that secrete the target antibody. Immortification can be achieved, for example, through EBV infection or by fusing human B cells with myeloma or hybridoma cells to produce tri-source hybridoma cells. Human antibodies can also be produced by phage display methods (see, for example, Dower et al., PCT Publication WO91/17271; McCafferty et al., PCT Publication WO92/001047; and Winter, PCT Publication WO92/20791), or selected from human combinatorial monoclonal antibody libraries (see Morphosys website). Human antibodies can also be prepared using transgenic animals carrying human immunoglobulin genes (e.g., see Lonberg et al., PCT Publication WO93/12227; and Kucherlapati, PCT Publication WO91/10741).

Therefore, anti-ADM antibodies can take the forms known in the art. Examples include human antibodies, monoclonal antibodies, humanized antibodies, chimeric antibodies, and CDR transplantation antibodies. In a preferred embodiment, the antibody of the present invention is a recombinant antibody such as IgG (a typical full-length immunoglobulin), or an antibody fragment containing at least a heavy chain and/or a light chain F-variable domain, such as a chemically conjugated antibody (fragment antigen binding), including but not limited to Fab fragments including Fab microantibodies; single-chain Fab antibodies; monovalent Fab antibodies with epitope tags such as Fab-V5Sx2; divalent Fab (microantibodies) dimerized with a CH3 domain; divalent or multivalent Fab, for example formed by the aided polymerization of heterologous domains, such as by dimerization of the dHLX domain, such as Fab-dHLX-FSx2; F(ab')2-fragments; scFv-fragments; multivalent and/or multispecific scFv-fragments; divalent and/or bispecific dimers; (bispecific T-cell engagers); trifunctional antibodies; multivalent antibodies, for example from classes other than G; and single-domain antibodies, such as nanobodies derived from camel or fish immunoglobulins.

Besides anti-ADM antibodies, other biopolymer backbones are known in the art to bind target molecules and be used to generate biopolymers with high target specificity. Examples include aptamers, specigelmers, anti-transporters, and cone snail toxins. See Figures 1a, 1b, and 1c for examples of antibody forms.

In a preferred embodiment, the anti-ADM antibody is selected from Fv fragments, scFv fragments, Fab fragments, scFab fragments, F(ab) ₂ fragments, and scFv-Fc fusion proteins. In another preferred embodiment, the antibody is selected from scFab fragments, Fab fragments, scFv fragments, and above conjugates with optimized bioavailability, such as PEGylated fragments. One of the most preferred forms is the scFab form.

Non-Ig backbones can be protein backbones and can be used as antibody mimics because they are capable of binding ligands or antigens. Non-Ig backbones can be selected from tetraligin-based non-Ig backbones (e.g., described in US 2010/0028995), fibronectin backbones (e.g., described in EP 1266025), lipid transport protein-based backbones (e.g., described in WO 2011/154420), ubiquitin backbones (e.g., described in WO 2011/073214), transfer backbones (e.g., described in US 2004/0023334), and protein A backbones (e.g., described in EP 2231860). The backbones include: ankyrin repeat-based backbones (e.g., those described in WO 2010/060748), microprotein (preferably microproteins that form cystine knots) backbones (e.g., those described in EP2314308), Fyn SH3 domain-based backbones (e.g., those described in WO 2011/023685), EGFR-A domain-based backbones (e.g., those described in WO 2005/040229), and Kunitz domain-based backbones (e.g., those described in EP 1941867).

In a preferred embodiment of the present invention, the antibody of the present invention can be generated as follows:

Balb/c mice were immunized with 100 μg of ADM-peptide-BSA-conjugate (emulsified in 100 μl Freund's complete adjuvant) on days 0 and 14, and with 50 μg of ADM-peptide-BSA-conjugate (in 100 μl Freund's incomplete adjuvant) on days 21 and 28. Three days prior to the fusion experiment, animals received 50 μg of the conjugate dissolved in 100 μl saline, administered once intraperitoneally and once intravenously.

Spleen cells from immunized mice and myeloma cell line SP2/0 were fused for 30 seconds at 37°C with 1 ml of 50% polyethylene glycol. After washing, the cells were seeded in 96-well cell culture plates. Hybrid clones were selected by culturing in HAT medium [RPMI 1640 medium supplemented with 20% fetal bovine serum and HAT supplement]. After two weeks, the HAT medium was replaced with HT medium for three passages, and then the cells were restored to normal cell culture medium.

Three weeks after fusion, the primary screening for antigen-specific IgG antibodies in the cell culture supernatant was conducted. Microcultures testing positive were transferred to 24-well plates for proliferation. After retesting, the selected cultures were cloned and subcloned using a limiting dilution technique, and the isotypes were determined (see also Lane, R.D. (1985). A short-duration polyethyleneglycol fusion technique for increasing production of monoclonal antibody-secreting hybridas. J. Immunol. Meth. 81: 223-228; Ziegler, B. et al. (1996) Glutamate decarboxylase (GAD) is not detectable on the surface of rat islet cells examined by cytofluorometry and complementary-dependent antibody-mediated cytotoxicity of monoclonal GAD antibodies, Horm. Metab. Res. 28: 11-15).

Antibodies can be generated via phage display using the following method:

The human natural antibody gene library HAL7/8 was used for the isolation of recombinant single-chain F-variable domains (scFv) targeting the adrenal medullarin peptide. The antibody gene library was screened using a panning strategy, including the use of biotinylate tags containing peptides linked to the adrenal medullarin peptide sequence via two distinct spacer regions. A mixed panning cycle of nonspecifically bound antigens and streptavidin-bound antigens was used to minimize the background of nonspecific binders. Phages eluted from the third round of panning were used to generate *E. coli* strains expressing monoclonal scFv. Supernatants from cultures of these cloned strains were used directly for antigen ELISA detection. (See also Hust, M., Meyer, T., Vbedisch, B., Rülker, T., Thie, H., El-Ghezal, A., Kirsch, M.I., Schütte, M., Helmsing, S., Meier, D., Schirrmann, T., Dübel, S., 201 1.A human scFv antibody generation pipeline for proteome research.Journal of Biotechnology 152, 159-170; Schütte, M., Thullier, P., Pelat, T., Wezler, tativeCrf splice variant and generation of recombinant antibodies for the specific detection of Aspergillus fumigatus.PLoS One4, e6625).

Humanization of mouse antibodies can be performed according to the following protocol:

For the humanization of murine antibodies, the structural interactions between the frame region (FR) and complementarity-determining region (CDR) of the antibody sequence and the antigen were analyzed. Based on structural modeling, a suitable human FR was selected, and the murine CDR sequence was transplanted into the human FR. Variations can be introduced into the amino acid sequences of the CDR or FR to restore structural interactions that are disrupted by species switching of the FR sequence. The restoration of these structural interactions can be achieved using a phage display library via a randomized method or via a specified method guided by molecular modeling (Almagro JC, Fransson J., 2008. Humanization of antibodies. Front Biosci. 2008 Jan 1; 13: 1619-33.).

In a preferred embodiment, the ADM antibody form is selected from Fv fragments, scFv fragments, Fab fragments, scFab fragments, F(ab) ₂ fragments, and scFv-Fc fusion proteins. In another preferred embodiment, the antibody form is selected from scFab fragments, Fab fragments, scFv fragments, and above conjugates with optimized bioavailability, such as PEGylated fragments. One of the most preferred forms is the scFab form.

In another preferred embodiment, the anti-ADM antibody, anti-ADM antibody fragment, or anti-ADM non-Ig backbone is a full-length antibody, antibody fragment, or non-Ig backbone.

In a preferred embodiment, the anti-adrenergic medullary antibody, or the anti-adrenergic medullary antibody fragment, or the anti-adrenergic medullary non-Ig backbone targets and binds to an epitope of at least 5 amino acids in length contained in the ADM.

In a specific implementation scheme, the anti-adrenal medullary antibody, or the anti-adrenal medullary antibody fragment, or the anti-adrenal medullary non-Ig backbone targets and binds to an epitope of at least four amino acids in length contained in ADM.

In a specific embodiment of the present invention, an anti-adrenergic medullaris (ADM) antibody or an anti-ADM antibody fragment or an anti-ADM non-Ig backbone that binds adrenergic medullaris is provided as a drug, wherein the antibody or fragment or backbone is not ADM-binding protein 1 (complement factor H).

In a specific embodiment of the invention, an anti-adrenergic medullaris (ADM) antibody, an anti-ADM antibody fragment binding to adrenergic medullaris, or an anti-ADM non-Ig backbone binding to adrenergic medullaris is provided as a drug, wherein the antibody, antibody fragment, or non-Ig backbone binds to a region preferably containing at least 4 or at least 5 amino acids within the aa 1-42 sequence of mature human ADM.

SEQ ID No.: 24

YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVA.

In a specific embodiment of the invention, an anti-adrenergic medullary (ADM) antibody, an anti-ADM antibody fragment binding to adrenergic medullary, or an anti-ADM non-Ig backbone binding to adrenergic medullary is provided as a drug, wherein the antibody, fragment, or backbone binds preferably to at least four or at least five amino acid regions within the aa 1-21 sequence of mature human ADM:

SEQ ID No.: 23

YRQSMNNFQGLRSFGCRFGTC.

Therefore, in a specific embodiment of the present invention, the anti-ADM antibody or anti-adrenal medullary antibody fragment or anti-ADM non-Ig backbone binds to ADM in the region of the N-terminal portion (aa 1-21) of adrenal medullary (see Figure 2).

In another preferred embodiment, the anti-antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig backbone recognizes and binds to the N-terminus (aa 1) of adrenomedullin. The N-terminus signifies amino acid 1, i.e., “Y” in SEQ ID No. 21 or 23; it is essential for antibody binding. The anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig backbone does not bind to N-terminally elongated adrenomedullin, nor to N-terminally modified adrenomedullin, nor to N-terminally degraded adrenomedullin.

The use of the ADM antibody or adrenomedullin antibody fragment of the present invention is also preferred, wherein the antibody or adrenomedullin antibody fragment is an ADM-stabilized antibody or ADM-stabilized adrenomedullin antibody fragment that increases the t1 _/2 half-retention time of adrenomedullin in serum, blood, or plasma by at least 10%, preferably at least 50%, more preferably >50%, and most preferably 100%. An analysis that can be used to determine the half-retention time of adrenomedullin in serum, blood, or plasma is described in Example 3.

It is also preferred to use the ADM antibody or adrenomedullin antibody fragment of the present invention, wherein the antibody or the adrenomedullin stable antibody fragment blocks less than 80%, preferably less than 50%, of the ADM biological activity.

In a preferred embodiment, the modulating antibody or modulating adrenomedullin antibody fragment is used in the treatment of sepsis. The modulating antibody or adrenomedullin antibody fragment increases the bioactivity of ADM in early sepsis but reduces the destructive effect of ADM in late sepsis. The “modulating” antibody or modulating adrenomedullin antibody fragment is an antibody or adrenomedullin antibody fragment that increases the t1 _/2 half-retention time of adrenomedullin in serum, blood, or plasma by at least 10%, preferably at least 50%, more preferably >50%, most preferably 100%, and blocks less than 80%, preferably less than 50%, of ADM bioactivity.

In another specific embodiment of the present invention, the anti-ADM antibody, anti-ADM antibody fragment, or anti-ADM non-Ig backbone provided herein does not bind to the C-terminal portion of ADM, i.e., ADM aa 43-52.

PRSKISPQGY-NH2

(SEQ ID NO: 25).

In specific implementations, the modulated anti-ADM antibody or modulated anti-adrenergic medullary antibody fragment or modulated anti-adrenergic medullary non-Ig backbone of the present invention is used to treat individuals to prevent or treat diseases or conditions, such as to prevent or treat chronic or acute diseases or conditions in individuals.

Such modulation of anti-ADM antibodies, anti-adrenergic medullary antibody fragments, or anti-adrenergic medullary non-Ig backbones is particularly useful for the treatment of sepsis. Modulation of anti-ADM antibodies, anti-adrenergic medullary antibody fragments, or anti-adrenergic medullary non-Ig backbones increases the bioactivity of ADM in early sepsis but reduces the role of ADM in late sepsis.

The "regulatory" antibody, or antibody fragment for regulating adrenomedullin, or non-Ig backbone for regulating adrenomedullin, is an antibody, antibody fragment, or non-Ig backbone that increases the half-life (t1 _/2 half-retention time) of adrenomedullin in serum, blood, or plasma by at least 10%, preferably at least 50%, more preferably >50%, and most preferably >100%, and blocks less than 80%, preferably less than 50%, of the ADM biological activity, wherein the ADM biological activity is blocked by at least 5%. These values related to half-life and biological activity blocking should be understood as relating to the up-analyses used to determine these values. It should be understood that the regulatory antibody, or antibody fragment for regulating adrenomedullin, or non-Ig backbone for regulating adrenomedullin, is an antibody, antibody fragment, or non-Ig backbone that binds to ADM.

It should be emphasized that blocking ADM bioactivity means blocking no more than 80%, thus leaving 20% residual ADM bioactivity. The same applies to blocking no more than 50% of ADM bioactivity, thus leaving 50% residual ADM bioactivity.

The subject of this invention is an anti-adrenergic medullary antibody or an anti-adrenergic medullary antibody fragment or an anti-ADM non-Ig backbone, wherein said antibody, fragment, or backbone is

• ADM-stabilizing antibody or adrenomedullin-stabilizing antibody fragment or ADM-stabilizing non-Ig backbone, which increases the t1 _/2 half-retention time of adrenomedullin in serum, blood, or plasma by at least 10%, preferably at least 50%, more preferably >50%, most preferably 100%, and/or

• The anti-adrenergic medullary antibody, the anti-adrenergic medullary antibody fragment, or the anti-ADM non-Ig backbone blocks less than 80%, preferably less than 50%, of ADM biological activity.

The above means that no more than 80% or no more than 50% of the ADM biological activity is blocked, and therefore should be understood as the limited blocking of ADM biological activity by the respective ADM binders (which are ADM-stabilized antibodies or antibody fragments or non-Ig backbones).

Such modulating antibodies, or antibody fragments that modulate adremyelin, or non-Ig backbones that modulate adremyelin, offer the advantage of facilitating quantitative dosing. The combination of partially blocking or partially reducing the biological activity of adremyelin, along with increasing its in vivo half-life (increasing adremyelin biological activity), advantageously simplifies the quantitative administration of anti-adremyelin antibodies, anti-adremyelin antibody fragments, or anti-adremyelin non-Ig backbones. In cases of endogenous adremyelin overdose (maximum stimulation, late sepsis, shock, asthenia), the reducing effect is the primary influence of the antibody, fragment, or backbone, limiting the (negative) effects of adremyelin. In cases of low or normal endogenous adremyelin concentrations, the biological effects of anti-adremyelin antibodies, anti-adremyelin antibody fragments, or anti-ADM non-Ig backbones are a combination of reducing (through partial blocking) and increasing (through increasing adremyelin half-life). If the half-life effect is stronger than the blocking effect, the net biological activity of endogenous adrenomedullin is favorably increased in early sepsis (low adrenomedullin, high-dynamic phase). Therefore, non-neutralizing and modulating antiadrenomedullin antibodies, antiadrenomedullin antibody fragments, or antiadrenomedullin non-Ig backbones act as buffers of ADM biological activity to maintain ADM biological activity within a certain physiological range.

Therefore, the quantitative administration of antibodies/fragments/backbone in, for example, sepsis can be selected from excessive concentrations, because, in terms of modulatory effects, both stages of sepsis (early and late) benefit from treatment with excessive amounts of anti-ADM antibodies or anti-adrenergic medullary antibody fragments or anti-ADM non-Ig backbones. Excess means that the concentration of anti-adrenergic medullary antibodies or anti-adrenergic medullary antibody fragments or anti-ADM non-Ig backbones is higher than, for example, adrenergic medullary in the late stage of sepsis (shock). This means that, in terms of modulatory antibodies or modulatory antibody fragments or modulatory non-Ig backbones, the quantitative administration in sepsis can be as follows:

The concentration of adrenomedullin in septic shock is 226 +/- 66 fmol/ml (Nishio et al., "Increased plasma concentrations of adrenomedullin correlate with relaxation of vascular tone in patients with septic shock.", Crit Care Med. 1997, 25(6): 953-7), and the equimolar concentration of antibody, fragment or backbone is 42.5 μg/L blood (based on 61 blood volume/80 kg body weight) 3.2 μg/kg body weight. Excessive dosage means at least twice the average concentration of adrenomedullin in septic shock, at least >3 μg anti-adrenomedullin antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig backbone/kg body weight, preferably at least 6.4 μg anti-adrenomedullin antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig backbone/kg body weight. Preferably >10 μg/kg, more preferably >20 μg/kg, most preferably >100 μg anti-adrenomedullin antibody or anti-adrenomedullin antibody fragment or anti-DM non-Ig backbone/kg body weight.

This can also be applied to other severe and acute conditions besides septic shock.

Furthermore, in one embodiment of the invention, the anti-adrenergic medullary (ADM) antibody, anti-adrenergic medullary antibody fragment, or anti-ADM non-Ig backbone is monospecific. Monospecific anti-adrenergic medullary (ADM) antibody, anti-adrenergic medullary antibody fragment, or monospecific anti-ADM non-Ig backbone indicates that the antibody, antibody fragment, or non-Ig backbone binds to a specific region, which preferably includes at least four or five amino acids within the target ADM. A monospecific anti-adrenergic medullary (ADM) antibody, anti-adrenergic medullary antibody fragment, or monospecific anti-ADM non-Ig backbone is an anti-adrenergic medullary (ADM) antibody, anti-adrenergic medullary antibody fragment, or anti-ADM non-Ig backbone having affinity for the same epitope. In another specific embodiment, the ADM-binding anti-ADM antibody or antibody fragment is a monospecific antibody, where monospecificity indicates that the antibody or antibody fragment binds to a specific region, which preferably includes at least four or five amino acids within the target ADM. A monospecific antibody or fragment is an antibody or fragment that has an affinity for the same epitope. Monoclonal antibodies are monospecific, but monospecific antibodies can also be produced by methods other than those that produce them from the same germ cells.

In a specific embodiment of the invention, the antibody is a monoclonal antibody or a fragment thereof. In one embodiment of the invention, the anti-ADM antibody or anti-ADM antibody fragment is a human antibody or a humanized antibody or an antibody derived therefrom; in a specific embodiment, one or more (mouse) CDRs are transplanted into a human antibody or antibody fragment.

In one aspect, the subject of this invention is a human CDR transplant antibody or antibody fragment thereof that binds to ADM, wherein the human CDR transplant antibody or antibody fragment thereof comprises an antibody heavy chain (H chain), which includes:

SEQ ID NO: 1

GYTFSRYW

SEQ ID NO: 2

ILPGSGST

and/or

SEQ ID NO: 3

TEGYEYDGFDY

And/or also contains an antibody light chain (L chain), which includes:

SEQ ID NO: 4

QSIVYSNGNTY

SEQ ID NO: 5

RVS

and/or

SEQ ID NO: 6

FQGSHIPYT.

In one specific embodiment of the invention, the subject matter is a human monoclonal antibody or antibody fragment thereof that binds to ADM, wherein the heavy chain comprises at least one CDR selected from:

SEQ ID NO: 1

GYTFSRYW

SEQ ID NO: 2

ILPGSGST

SEQ ID NO: 3

TEGYEYDGFDY

Furthermore, the light chain contains at least one CDR selected from the following:

SEQ ID No: 4

QSIVYSNGNTY

SEQ ID NO: 5

RVS

SEQ ID NO: 6

FQGSHIPYT.

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

SEQ ID NO: 1

GYTFSRYW

SEQ ID NO: 2

ILPGSGST

SEQ ID NO: 3

TEGYEYDGFDY

And the light chain contains the following sequence

SEQ ID NO: 4

QSIVYSNGNTY

SEQ ID NO: 5

RVS

SEQ ID NO: 6

FQGSHIPYT.

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

The anti-ADM antibody, anti-adrenergic medullary antibody fragment, or anti-ADM non-Ig backbone of the present invention exhibits an affinity for human ADM such that the affinity constant is greater than ^10⁻⁷ M, preferably ^10⁻⁸ M, more preferably greater than ^10⁻⁹ M, and most preferably greater than ^10⁻¹⁰ M. Those skilled in the art will understand that applying a higher dose of the compound can be considered to compensate for a lower affinity, and this measure will not exceed the scope of the present invention. The affinity constant can be determined according to the method described in Example 1.

The antibodies or fragments of the present invention can be used in combination with other reagents, such as with so-called ADM-binding proteins, for the purposes described herein.

It should be emphasized that the term "ADM-binding protein" includes ADM-binding protein 1 (complement factor H), but it does not manifest as the non-neutralizing and/or modulating anti-ADM antibody, antibody fragment, or non-Ig backbone of the present invention.

In a preferred embodiment, the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig backbone is used to reduce the risk of death in patients with chronic or acute disease or acute symptoms.

The chronic or acute diseases or conditions of this invention can be selected from severe infections, such as meningitis, systemic inflammatory response syndrome (SIRS), sepsis; other diseases such as diabetes, cancer, acute and chronic vascular diseases such as heart failure, myocardial infarction, stroke, and atherosclerosis; shock such as septic shock; and organ dysfunction such as renal dysfunction, liver dysfunction, burns, surgery, trauma, poisoning, and chemotherapy damage. Particularly useful are the antibodies, fragments, or backbones of this invention used to reduce the risk of death in sepsis and septic shock, i.e., late-stage sepsis.

In one embodiment, the anti-ADM antibody, anti-adrenergic medullary antibody fragment, or anti-ADM non-Ig backbone according to the invention is used for the treatment or prevention of a patient with a chronic or acute illness or condition, wherein the patient is an ICU patient. In another embodiment, the anti-ADM antibody, anti-adrenergic medullary antibody fragment, or anti-ADM non-Ig backbone according to the invention is used for the treatment or prevention of a patient with a chronic or acute illness, wherein the patient is critically ill. Critically ill means that the patient has a potentially fatal or imminent death condition or is in a potentially fatal or imminent death state.

The antibodies, antibody fragments, and non-Ig backbones of the present invention can be used to treat individuals to prevent or treat diseases or conditions, such as chronic or acute diseases or conditions. Such diseases can be selected from severe infections, such as meningitis, systemic inflammatory response syndrome (SIRS), and sepsis; other diseases such as diabetes, cancer, acute and chronic vascular diseases such as heart failure, myocardial infarction, stroke, and atherosclerosis; shock such as septic shock; and organ dysfunction such as renal dysfunction, hepatic dysfunction, burns, surgery, and trauma. Particularly useful are the antibodies, fragments, or backbones of the present invention for reducing the risk of death in sepsis and septic shock, i.e., late-stage sepsis.

The clinical criteria for SIRS, sepsis, severe sepsis, and septic shock will be defined below.

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

The patient presented with low blood pressure (mean arterial pressure <65 mmHg).

• Elevated serum lactate levels, >4 mmol/L

• Blood glucose > 7.7 mmol/L (in the absence of diabetes)

• Central venous pressure is not within the range of 8-12 mmHg

• Urine output < 0.5 mL × kg ^{- 1} × hr ^{- 1}

• Central vein (superior vena cava) oxygen saturation <70% or mixed vein oxygen saturation <65%

Heart rate > 90 beats/minute

• Temperature <36℃ or >38℃

• Respiratory rate > 20/minute

• White blood cell count <4× ^10⁹ /L or >12× ^10⁹ /L (white blood cells); >10% immature neutrophils

2) Sepsis

After having at least two of the symptoms mentioned in 1), other clinical suspicions of new infection include:

• Cough/phlegm/chest pain

• Abdominal pain/swelling/diarrhea

·Hematogenous infection

Endocarditis

Difficulty urinating

Headache accompanied by neck stiffness

Cellulitis/Injury/Joint Infection

• Any positive microorganisms that cause infection

3) Severe sepsis

If the patient presents with sepsis, other clinical suspicions of organ dysfunction include:

• Systolic blood pressure <90/average; <65 mmHg

Lactate > 2 mmol/L

• Bilirubin > 34 μmol/L

• 2-hour urine output < 0.5 mL/kg/h

Creatinine > 177 μmol/L

Platelet count < 100 × ^10⁹ /L

• _SpO2 > 90% unless _O2 is provided

4) Septic shock

The patient exhibits at least one of the signs of end-organ dysfunction mentioned in 3). If there is refractory hypotension that is unresponsive to treatment, and intravenous fluids alone are insufficient to maintain the patient's blood pressure, and administration of the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig backbone of the present invention is required, this indicates septic shock.

In one embodiment of the invention, the patient suffers from SIRS, severe infection, sepsis, or shock such as septic shock. The severe infection refers to, for example, meningitis, systemic inflammatory response syndrome (SIRS), sepsis, severe sepsis, and shock such as septic shock. Here, severe sepsis is characterized by the patient presenting with sepsis and other clinical suspicions of any organ dysfunction, namely:

• Systolic blood pressure <90/average; <65 mmHg

Lactate > 2 mmol/L

• Bilirubin > 34 μmol/L

• 2-hour urine output < 0.5 mL/kg/h

Creatinine > 177 μmol/L

Platelet count < 100 × _10⁹ /L

• _SpO2 > 90% unless _O2 is provided

In another specific implementation, the acute illness or acute condition is not sepsis, severe sepsis, SIRS, shock, or septic shock.

In another embodiment, the acute illness or acute condition is not sepsis.

The antibodies, fragments, or backbones of the present invention can be used in combination with other reagents, such as with so-called ADM-binding proteins, for the purposes described herein. ADM-binding proteins are also naturally present in the circulation of the patients.

It should be emphasized that the term "ADM-binding protein" includes ADM-binding protein 1 (complement factor H), but it does not manifest as the non-neutralizing and/or modulating anti-ADM antibody, antibody fragment, or non-Ig backbone of the present invention.

The subject matter of this invention also includes anti-ADM antibodies or anti-adrenergic medullary antibody fragments or anti-ADM non-Ig backbones for the treatment or prevention of chronic or acute diseases or acute symptoms in patients, wherein the antibodies or fragments or backbones are used in combination with other active ingredients.

The subject of this invention also includes anti-adrenergic medullary (ADM) antibodies or fragments of anti-adrenergic medullary antibodies or anti-ADM non-Ig backbones used in combination with another active drug (e.g., used as a principal drug), wherein the combination is used to stabilize the circulation of the patient, particularly the patient's systemic circulation, for the treatment or prevention of the patient's chronic or acute illness or condition.

The primary drug refers to a drug that acts against the primary cause of the disease or condition. In the case of infection, the primary drug may be an antibiotic.

In a specific implementation of the aforementioned combination, the combination is used in combination with a vasopressor such as catecholamine, wherein the combination is used to treat or prevent a patient’s chronic or acute illness or condition.

In one embodiment of the invention, the patient suffering from a chronic disease or acute disease or chronic condition is characterized in that the patient needs to be given a vasopressor, such as catecholamines.

Therefore, in a specific embodiment, the subject of the invention is to use in combination with ADM-binding proteins and/or other active ingredients to treat or prevent anti-adrenergic medullary (ADM) antibodies or fragments of anti-adrenergic medullary antibodies or anti-ADM non-Ig backbones in patients requiring vasopressor therapy such as catecholamine therapy.

In a specific implementation of the above combination, the combination is used in combination with intravenously administered fluids, wherein the combination is used to stabilize circulation, especially systemic circulation, for the treatment or prevention of chronic or acute diseases or conditions in patients.

In one embodiment of the present invention, the patient suffering from a chronic disease or an acute disease or acute condition who requires stable circulation is characterized in that the patient needs to receive intravenous fluids.

Therefore, in one specific embodiment, the subject of the invention is an anti-adrenergic medullary (ADM) antibody or anti-adrenergic medullary antibody fragment or anti-ADM non-Ig backbone, in combination with ADM-binding proteins and/or other active ingredients, for the treatment or prevention of patients requiring intravenous infusions. From the patient's perspective, this is where intravenous infusions are needed to regulate overall fluid balance.

It should be emphasized that the term ADM-binding protein also refers to ADM-binding protein 1 (complement factor H), but it is not the non-neutralizing and/or modulating anti-ADM antibody, anti-ADM antibody fragment, or anti-ADM non-Ig backbone of the present invention.

According to the present invention, ADM-binding protein 1 may also be referred to as ADM-binding protein 1 (complement factor H).

The anti-ADM antibody or anti-adrenergic medullary antibody fragment or anti-ADM non-Ig backbone or its combination with ADM-binding proteins and/or other active ingredients may be used in combination with vasopressors such as catecholamines and/or intravenously administered fluids to stabilize circulation, especially systemic circulation, for patients with chronic or acute illnesses or conditions.

The subject matter of this invention also includes anti-ADM antibodies or anti-adrenergic medullary antibody fragments or anti-ADM non-Ig used in combination with TNF-α antibodies. The TNF-α antibodies used to treat patients are commercially available.

In a preferred embodiment, the anti-ADM antibody, anti-ADM antibody fragment, or anti-ADM non-Ig backbone is used to reduce the risk of death in patients with the chronic or acute disease, wherein the disease is selected from sepsis, diabetes, cancer, acute and chronic vascular diseases such as heart failure, shock such as septic shock, and organ dysfunction such as renal dysfunction. Particularly useful are the antibodies, fragments, or backbones of the present invention for reducing the risk of death in sepsis and septic shock, i.e., late-stage sepsis.

The antibodies, antibody fragments, backbones, and combinations of the present invention can be used to treat or prevent the following chronic or acute diseases in patients:

• Used for the prevention of organ dysfunction or organ failure, especially renal dysfunction or renal failure, and/or

• Used to stabilize circulation, for example, to reduce the patient's need for vasopressors, such as catecholamines, and/or

• Used to regulate the fluid balance of the patient.

• Used to reduce the risk of death for the patient.

In one embodiment, the anti-ADM antibody, anti-adrenergic medullary antibody fragment, or anti-ADM non-Ig backbone according to the invention is used to treat or prevent a chronic or acute illness in a patient, wherein the patient is an ICU patient. In another embodiment, the anti-ADM antibody, anti-adrenergic medullary antibody fragment, or anti-ADM non-Ig backbone according to the invention is used to treat or prevent a chronic or acute illness or acute condition in a patient, wherein the patient is critically ill. Critically ill means that the patient has a potentially fatal or imminent death condition or is in a potentially fatal or imminent state.

The subject of this invention also includes anti-ADM antibodies or anti-adrenergic medullary antibody fragments or anti-ADM non-Ig backbones for the treatment or prevention of chronic or acute diseases or acute symptoms in patients, wherein the antibodies or fragments or backbones are used in combination with ADM binding proteins.

The subject of this invention also includes pharmaceutical formulations comprising the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig backbone of this invention.

The subject of this invention also includes pharmaceutical formulations of the invention, wherein the pharmaceutical formulation is a solution, preferably a ready-to-use solution.

The drug formulation can be administered intramuscularly. The drug formulation can be administered intravenously. The drug formulation can be administered by infusion.

In another embodiment, the subject matter of the invention also includes the pharmaceutical preparation of the invention, wherein the pharmaceutical preparation is in a dry state so as to be restored before use.

In another embodiment, the subject matter of the invention also includes the pharmaceutical formulation of the invention, wherein the pharmaceutical formulation is in a freeze-dried state.

In another embodiment of the invention, the pharmaceutical preparation of the invention can be administered intramuscularly, intravascularly, or preferably by infusion to the patient systemically.

Therefore, in another embodiment of the invention, the pharmaceutical preparation of the invention can be administered to the patient systemically.

Other embodiments within the scope of this invention are shown below:

1. Adrenomedullin ADM antibody or adrenomedullin antibody fragment, which is used to treat patients' chronic or acute diseases and to regulate fluid balance.

2. The ADM antibody or adrenomedullin antibody fragment according to claim 1, wherein the antibody form is selected from Fv fragment, scFv fragment, Fab fragment, scFab fragment, (Fab) ₂ fragment and scFv-Fc fusion protein.

3. The ADM antibody or adrenomedullin antibody fragment according to claim 1 or 2, wherein the antibody or fragment binds to the N-terminal portion (aa 1-21) of adrenomedullin.

4. The ADM antibody or adrenomedullin antibody fragment according to any one of claims 1 to 3, wherein the antibody or fragment is capable of recognizing and binding to the N-terminus (aal) of adrenomedullin.

5. The ADM antibody or adrenomedullin antibody fragment according to any one of claims 1 to 4, wherein the antibody or fragment is an ADM-stabilized antibody or ADM-stabilized antibody fragment that increases the t1 _/2 half-retention time of adrenomedullin in serum, blood, or plasma by at least 10%, preferably at least 50%, more preferably >50%, and most preferably >100%.

6. The ADM antibody or adrenomedullin antibody fragment according to any one of claims 1 to 5, wherein the antibody or fragment blocks less than 80%, preferably less than 50%, of the ADM biological activity.

7. An ADM antibody or adrenomedullin antibody fragment for treating a patient’s chronic or acute disease according to any one of claims 1 to 6, wherein the disease is selected from sepsis, diabetes, cancer, heart failure, shock, and renal dysfunction.

8. An ADM antibody or adrenomedullin antibody fragment for treating a patient with a chronic or acute disease according to any one of claims 1 to 7, wherein the patient is an ICU patient.

9. An ADM antibody or adrenomedullin antibody fragment for treating a patient’s chronic or acute disease according to any one of claims 1 to 7, wherein the antibody or fragment is a regulatory antibody or fragment that increases the t1 _/2 half-retention time of adrenomedullin in serum, blood, or plasma by at least 10%, preferably at least 50%, more preferably >50%, most preferably >100%, and blocks less than 80%, preferably less than 50%, of ADM biological activity.

10. A pharmaceutical preparation comprising the antibody or fragment according to any one of claims 1 to 9.

11. The pharmaceutical preparation according to claim 10, wherein the pharmaceutical preparation is a solution, preferably a ready-to-use solution.

12. The pharmaceutical preparation according to claim 10, wherein the pharmaceutical preparation is in a freeze-dried state.

13. The pharmaceutical preparation according to any one of claims 10 to 11, wherein the pharmaceutical preparation is administered intramuscularly.

14. The pharmaceutical preparation according to any one of claims 10 to 11, wherein the pharmaceutical preparation is administered intravenously.

15. The pharmaceutical preparation of claim 14, wherein the pharmaceutical preparation is administered by infusion.

Other embodiments within the scope of this invention are shown below:

1. Adrenomedullin ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone, which are used to treat patients' chronic or acute diseases and to regulate fluid balance.

2. The ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone according to claim 1, wherein the ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone is a non-neutralizing ADM antibody or a non-neutralizing adrenomedullin antibody fragment or a non-neutralizing ADM non-Ig backbone.

3. The adrenomedullin ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone for treating chronic or acute diseases or acute symptoms, as described in claim 1 or 2, for preventing or reducing edema in the patients.

4. The ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone according to any one of claims 1 to 3, wherein the antibody form is selected from Fv fragment, scFv fragment, Fab fragment, scFab fragment, (Fab) ₂ fragment and scFv-Fc fusion protein.

5. The ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone according to any one of claims 1 to 4, wherein the antibody or fragment or backbone binds to the N-terminal portion (aa 1-21) of adrenomedullin.

6. The ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone according to any one of claims 1 to 5, wherein the antibody or fragment backbone is capable of recognizing and binding to the N-terminal portion (aa1) of adrenomedullin.

7. The ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone according to any one of claims 1 to 6, wherein said antibody or fragment or backbone is an ADM stable antibody or ADM stable antibody fragment or ADM stable non-Ig backbone that increases the half-life (t _1/2 half-retention time) of adrenomedullin in serum, blood, or plasma by at least 10%, preferably at least 50%, more preferably >50%, and most preferably >100%.

8. The ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone according to any one of claims 1 to 7, wherein the antibody or fragment blocks less than 80%, preferably less than 50%, of the ADM biological activity.

9. An ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone for treating a patient’s chronic or acute disease according to any one of claims 1 to 8, wherein the disease is selected from SIRS, sepsis, diabetes, cancer, heart failure, shock, and renal dysfunction.

10. The ADM antibody or adrenomedullin antibody fragment according to any one of claims 1 to 9, wherein the antibody or fragment is a human monoclonal antibody or fragment binding to ADM or an antibody fragment thereof, wherein the heavy chain comprises the following sequence:

SEQ ID NO: 1

GYTFSRYW

SEQ ID NO: 2

ILPGSGST

SEQ ID NO: 3

TEGYEYDGFDY

Furthermore, the light chain contains the following sequences:

SEQ ID NO: 4

QSIVYSNGNTY

SEQ ID NO: 5

RVS

SEQ ID NO: 6

FQGSHIPYT.

11. The human monoclonal antibody or fragment binding to ADM according to claim 10, wherein the antibody or fragment comprises a sequence selected from:

12. An ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone for treating a patient with a chronic or acute disease according to any one of claims 1 to 9, wherein the patient is an ICU patient.

13. An ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone for treating a patient’s chronic or acute disease according to any one of claims 1 to 12, wherein the antibody or fragment or backbone is a regulatory antibody or fragment or backbone that increases the half-life (t _1/2 half-retention time) of adrenomedullin in serum, blood, or plasma by at least 10%, preferably at least 50%, more preferably >50%, most preferably >100%, and blocks less than 80%, preferably less than 50% of the ADM biological activity.

14. An ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone for treating a patient’s chronic or acute disease according to any one of claims 1 to 13, used in combination with catecholamines and/or intravenously administered fluids.

15. An ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone for treating a patient’s chronic or acute disease, as claimed in any one of claims 1 to 13, or a combination thereof as claimed in claim 12, used in combination with an ADM binding protein and/or other active ingredients.

16. A pharmaceutical preparation comprising an antibody, fragment, or backbone according to any one of claims 1 to 15.

17. The pharmaceutical preparation according to claim 16, wherein the pharmaceutical preparation is a solution, preferably a ready-to-use solution.

18. The pharmaceutical preparation according to claim 16, wherein the pharmaceutical preparation is in a freeze-dried state.

19. The pharmaceutical preparation according to any one of claims 16 to 17, wherein the pharmaceutical preparation is administered intramuscularly.

20. The pharmaceutical preparation according to any one of claims 16 to 17, wherein the pharmaceutical preparation is administered intravenously.

21. The pharmaceutical preparation of claim 20, wherein the pharmaceutical preparation is administered by infusion.

Other embodiments within the scope of this invention are shown below:

1. Adrenomedullin (ADM) antibody or adrenomedullin antibody fragment, used to treat patients with chronic or acute diseases and to stabilize circulation.

2. The ADM antibody or adrenomedullin antibody fragment of claim 1, wherein the antibody or fragment reduces the patient's catecholamine requirements.

3. The ADM antibody or adrenomedullin antibody fragment according to claim 1 or 2, wherein the antibody form is selected from Fv fragment, scFv fragment, Fab fragment, scFab fragment, (Fab) ₂ fragment and scFv-Fc fusion protein.

4. The ADM antibody or adrenomedullin antibody fragment according to any one of claims 1 to 3, wherein the antibody or fragment binds to the N-terminal portion (aa 1-21) of adrenomedullin.

5. The ADM antibody or adrenomedullin antibody fragment according to any one of claims 1 to 4, wherein the antibody or fragment is capable of recognizing and binding to the N-terminal portion (aa1) of adrenomedullin.

6. The ADM antibody or adrenomedullin antibody fragment according to any one of claims 1 to 5, wherein said antibody or fragment is an ADM-stabilizing antibody that increases the t1 _/2 half-retention time of adrenomedullin in serum, blood, or plasma by at least 10%, preferably at least 50%, more preferably >50%, and most preferably >100%.

7. The ADM antibody or adrenomedullin antibody fragment according to any one of claims 1 to 6, wherein the antibody or fragment blocks less than 80%, preferably less than 50%, of the ADM biological activity.

8. The ADM antibody or adrenomedullin antibody fragment according to any one of claims 1 to 7, wherein the antibody or fragment is an ADM-regulating antibody or adrenomedullin-regulating antibody fragment that increases the t1 _/2 half-retention time of adrenomedullin in serum, blood, or plasma by at least 10%, preferably at least 50%, more preferably >50%, most preferably >100%, and blocks less than 80%, preferably less than 50%, of ADM biological activity.

9. An ADM antibody or adrenomedullin antibody fragment for treating a patient’s chronic or acute disease according to any one of claims 1 to 8, wherein the disease is selected from sepsis, diabetes, cancer, acute and chronic vascular diseases such as heart failure, shock such as septic shock, and organ dysfunction such as renal dysfunction.

10. A pharmaceutical preparation comprising an antibody according to any one of claims 1 to 9.

11. The pharmaceutical preparation according to claim 10, wherein the pharmaceutical preparation is a solution, preferably a ready-to-use solution.

12. The pharmaceutical preparation according to claim 10, wherein the pharmaceutical preparation is in a freeze-dried state.

13. The pharmaceutical preparation according to any one of claims 10 to 11, wherein the pharmaceutical preparation is administered intramuscularly.

14. The pharmaceutical preparation according to any one of claims 10 to 11, wherein the pharmaceutical preparation is administered intravenously.

15. The pharmaceutical preparation of claim 14, wherein the pharmaceutical preparation is administered by infusion.

Other embodiments within the scope of this invention are shown below:

1. Adrenomedullin (ADM) antibody or adrenomedullin antibody fragment or ADM non-Ig backbone, used to treat patients with chronic or acute conditions and to stabilize circulation.

2. The ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone according to claim 1, wherein the antibody or fragment or backbone reduces the patient's vasopressor requirement, such as catecholamine requirement.

3. The ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone according to claim 1 or 2, wherein the ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone is a non-neutralizing ADM antibody or a non-neutralizing adrenomedullin antibody fragment or a non-neutralizing ADM non-Ig backbone.

4. The ADM antibody or adrenomedullin antibody fragment according to any one of claims 1 to 3, wherein the antibody form is selected from Fv fragments, scFv fragments, Fab fragments, scFab fragments, (Fab) ₂ fragments, and scFv-Fc fusion proteins.

5. The ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone according to any one of claims 1 to 4, wherein the antibody or fragment or backbone binds to the N-terminal portion (aa 1-21) of adrenomedullin.

6. The ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone according to any one of claims 1 to 5, wherein the antibody, fragment or backbone is capable of recognizing and binding to the N-terminal portion (aa1) of adrenomedullin.

7. The ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone according to any one of claims 1 to 6, wherein said antibody or fragment or backbone is an ADM stable antibody or fragment or backbone that increases the half-life (t _1/2 half-retention time) of adrenomedullin in serum, blood, or plasma by at least 10%, preferably at least 50%, more preferably >50%, and most preferably >100%.

8. The ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone according to any one of claims 1 to 7, wherein the antibody or fragment or backbone blocks less than 80%, preferably less than 50%, of ADM biological activity.

9. The ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone according to any one of claims 1 to 8, wherein the antibody or fragment or backbone is a regulatory ADM antibody or adrenomedullin antibody fragment or backbone that increases the half-life (t _1/2 half-retention time) of adrenomedullin in serum, blood, or plasma by at least 10%, preferably at least 50%, more preferably >50%, most preferably >100%, and blocks less than 80%, preferably less than 50% of the ADM biological activity.

10. The ADM antibody or adrenomedullin antibody fragment according to any one of claims 1 to 9, wherein the antibody or fragment is a human monoclonal antibody or fragment binding to ADM or an antibody fragment thereof, wherein the heavy chain comprises the following sequence:

SEQ ID NO: 1

GYTFSRYW

SEQ ID NO: 2

ILPGSGST

SEQ ID NO: 3

TEGYEYDGFDY

And the light chain contains the following sequence

SEQ ID NO: 4

QSIVYSNGNTY

SEQ ID NO: 5

RVS

SEQ ID NO: 6

FQGSHIPYT.

11. The human monoclonal antibody or fragment binding to ADM according to claim 10, wherein the antibody or fragment comprises a sequence selected from:

12. An ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone for treating a patient’s chronic or acute disease according to any one of claims 1 to 11, wherein the disease is selected from SIRS, sepsis, diabetes, cancer, acute and chronic vascular diseases such as heart failure, shock such as septic shock, and organ dysfunction such as renal dysfunction.

13. An ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone for treating a patient’s chronic or acute disease according to any one of claims 1 to 12, used in combination with catecholamines and/or intravenously administered fluids.

14. An ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone for treating a patient’s chronic or acute disease according to any one of claims 1 to 13, or a combination according to claim 13, used in combination with an ADM binding protein and/or other active ingredients.

15. A pharmaceutical formulation comprising the antibody or fragment or non-Ig backbone of any one of claims 1 to 14.

16. The pharmaceutical preparation according to claim 15, wherein the pharmaceutical preparation is a solution, preferably a ready-to-use solution.

17. The pharmaceutical preparation according to claim 15, wherein the pharmaceutical preparation is in a freeze-dried state.

18. The pharmaceutical preparation according to any one of claims 15 to 16, wherein the pharmaceutical preparation is administered intramuscularly.

19. The pharmaceutical preparation according to any one of claims 14 to 16, wherein the pharmaceutical preparation is administered intravenously.

20. The pharmaceutical preparation of claim 16, wherein the pharmaceutical preparation is administered by infusion.

Other embodiments within the scope of this invention are shown below:

1. An adrenomedullin antibody or adrenomedullin antibody fragment for treating chronic or acute diseases, wherein the antibody or fragment is an ADM-stabilized antibody or fragment that increases the t1 _/2 half-retention time of adrenomedullin in serum, blood, or plasma by at least 10%, preferably at least 50%, more preferably >50%, most preferably >100%, and/or wherein the antibody blocks less than 80%, preferably less than 50%, of ADM biological activity.

2. An adrenomedullin antibody or adrenomedullin antibody fragment for treating chronic or acute diseases, wherein the antibody or fragment is an ADM-regulating antibody or fragment that increases the t1 _/2 half-retention time of adrenomedullin in serum, blood, or plasma by at least 10%, preferably at least 50%, more preferably >50%, most preferably >100%, and blocks less than 80%, preferably less than 50%, of ADM biological activity.

3. An adrenomedullin antibody or adrenomedullin antibody fragment for treating chronic or acute diseases according to claim 1 or 2, wherein the antibody or fragment binds to the N-terminal portion (aa 1-21) of adrenomedullin.

4. An adrenomedullin antibody or adrenomedullin antibody fragment for the treatment of chronic or acute diseases, wherein the antibody or fragment according to claim 3 binds to the N-terminus of adrenomedullin.

5. An adrenomedullin antibody or adrenomedullin antibody fragment for treating chronic or acute diseases according to any one of claims 1 to 4, wherein the antibody or fragment is an ADM-stabilized antibody or fragment that increases the t1 _/2 half-retention time of adrenomedullin in serum, blood, or plasma by at least 10%, preferably at least 50%, more preferably >50%, and most preferably 100%.

6. An adrenomedullin antibody or adrenomedullin antibody fragment for treating chronic or acute diseases according to any one of claims 1 to 5, wherein the antibody or fragment blocks less than 80%, preferably less than 50%, of ADM biological activity.

7. An adrenomedullin antibody or adrenomedullin antibody fragment for treating chronic or acute diseases according to any one of claims 1 to 6, wherein the disease is selected from SIRS, sepsis, septic shock, diabetes, cancer, heart failure, shock, organ failure, renal dysfunction, acute fluid imbalance, and hypotension.

8. An adrenomedullin antibody or adrenomedullin antibody fragment for treating a chronic or acute disease according to any one of claims 1 to 7, wherein the disease is septic shock or sepsis.

9. An adrenomedullin antibody or adrenomedullin antibody fragment for treating a chronic or acute disease according to any one of claims 1 to 8, wherein the antibody or fragment regulates the fluid balance of the patient.

10. An adrenomedullin antibody or adrenomedullin antibody fragment for treating chronic or acute diseases according to any one of claims 1 to 9, wherein the antibody or tablet is used to prevent organ dysfunction or organ failure.

11. The adrenomedullin antibody or adrenomedullin antibody fragment for treating chronic or acute diseases according to claim 10, wherein the antibody or fragment is used to prevent renal dysfunction or renal failure.

12. An adrenomedullin (ADM) antibody or adrenomedullin antibody fragment for treating chronic or acute diseases according to any one of claims 1 to 11, wherein the antibody or fragment is used to stabilize circulation.

13. The ADM antibody or adrenomedullin antibody fragment for treating a patient’s chronic or acute disease according to claim 12, wherein the antibody or fragment reduces the patient’s catecholamine requirement.

14. An ADM antibody or adrenomedullin antibody fragment for treating a patient’s chronic or acute disease according to any one of claims 1 to 13, which is used to reduce the risk of said mitigating death.

15. An ADM antibody or adrenomedullin antibody fragment for treating a patient’s chronic or acute disease according to any one of claims 1 to 14, wherein the antibody or fragment may be administered at a dose of at least 3 μg/kg body weight.

16. A pharmaceutical composition comprising the antibody or fragment of any one of claims 1 to 15.

Other embodiments within the scope of this invention are shown below:

1. An adrenomedullin antibody or an adrenomedullin antibody fragment or an ADM non-Ig backbone, wherein the antibody or the fragment or backbone is a non-neutralizing antibody.

2. An adrenomedullin antibody or an adrenomedullin antibody fragment or an ADM non-Ig backbone, wherein the antibody or the fragment or backbone is an ADM-stabilized antibody or fragment or backbone that increases the half-life (t _1/2 half-retention time) of adrenomedullin in serum, blood, or plasma by at least 10%, preferably at least 50%, more preferably >50%, and most preferably 100%, and/or wherein the antibody or fragment or backbone blocks less than 80%, preferably less than 50%, of the ADM biological activity.

3. An adrenomedullin antibody or an adrenomedullin antibody fragment or an ADM non-Ig backbone, wherein the antibody or fragment is an ADM-regulating antibody or fragment or backbone that increases the half-life (t _1/2 half-retention time) of adrenomedullin in serum, blood, or plasma by at least 10%, preferably at least 50%, more preferably >50%, most preferably 100%, and blocks less than 80%, preferably less than 50%, of ADM biological activity.

4. The adrenomedullin antibody or adrenomedullin antibody fragment or ADM non-Ig backbone according to claim 1 or 2, wherein the antibody or fragment or backbone binds to the N-terminal portion (aa 1-21) of adrenomedullin.

5. An adrenomedullin antibody or an adrenomedullin antibody fragment or an ADM non-Ig backbone, wherein the antibody or the fragment or backbone according to claim 3 binds to the N-terminus of adrenomedullin.

6. The adrenomedullin antibody or adrenomedullin antibody fragment ADM non-Ig backbone according to any one of claims 1 to 4, wherein the antibody or the fragment or the backbone is an ADM-stabilized antibody or fragment that increases the t1 _/2 half-retention time of adrenomedullin in serum, blood, or plasma by at least 10%, preferably at least 50%, more preferably >50%, and most preferably 100%.

7. The adrenomedullin antibody or adrenomedullin antibody fragment or ADM non-Ig backbone according to any one of claims 1 to 6, which is used as an active pharmaceutical ingredient.

8. The adrenomedullin antibody or adrenomedullin antibody fragment ADM non-Ig backbone for treating chronic or acute diseases or conditions according to any one of claims 1 to 7, wherein the disease or condition is selected from severe infections such as meningitis, systemic inflammatory response syndrome (SIRS), sepsis; other diseases such as diabetes, cancer, acute and chronic vascular diseases such as heart failure, myocardial infarction, stroke, atherosclerosis; shock such as septic shock; and organ dysfunction such as renal dysfunction, liver dysfunction, burns, surgery, trauma.

9. An adrenomedullin antibody or adrenomedullin antibody fragment or ADM non-Ig backbone for treating a chronic disease or acute disease or acute condition, as claimed in any one of claims 1 to 8, wherein the disease is septic shock or sepsis.

10. The ADM antibody or adrenomedullin antibody fragment according to any one of claims 1 to 9, wherein said antibody or fragment is a human monoclonal antibody or fragment binding to ADM or an antibody fragment thereof, comprising one of the following sequences:

SEQ ID NO: 1

GYTFSRYW

SEQ ID NO: 2

ILPGSGST

SEQ ID NO: 3

TEGYEYDGFDY

and/or the light chain contains one of the following sequences:

SEQ ID NO: 4

QSIVYSNGNTY

SEQ ID NO: 5

RVS

SEQ ID NO: 6

FQGSHIPYT.

11. The human monoclonal antibody or fragment binding to ADM according to claim 10, wherein the antibody or fragment comprises a sequence selected from:

12. The adrenomedullin antibody or adrenomedullin antibody fragment or ADM non-Ig backbone according to any one of claims 1 to 11, for regulating fluid balance in patients with chronic or acute diseases or acute symptoms.

13. The adrenomedullin antibody or adrenomedullin antibody fragment or ADM non-Ig backbone according to any one of claims 1 to 11, for the prevention or relief of organ dysfunction or organ failure in patients with chronic or acute diseases or acute symptoms.

14. The adrenomedullin antibody or adrenomedullin antibody fragment or ADM non-Ig backbone according to claim 10, wherein the organ is the kidney or liver.

15. An adrenomedullin (ADM) antibody or adrenomedullin antibody fragment or ADM non-Ig backbone according to any one of claims 1 to 14, for stabilizing circulation in patients with chronic or acute diseases or acute conditions.

16. The ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone for treating a patient’s chronic or acute disease according to claim 15, wherein the antibody or fragment reduces the patient’s catecholamine requirement.

17. The adrenomedullin antibody or adrenomedullin antibody fragment or ADM non-Ig backbone according to any one of claims 1 to 16, used in combination with a vasopressor such as catecholamine.

18. The adrenomedullin antibody or adrenomedullin antibody fragment or ADM non-Ig backbone according to any one of claims 1 to 17, administered in combination with an intravenous injection solution.

19. The adrenomedullin antibody or adrenomedullin antibody fragment or ADM non-Ig backbone according to any one of claims 1 to 18, used in combination with a TNF-α antibody.

20. The ADM antibody or adrenomedullin antibody fragment or non-Ig-backbone according to any one of claims 1 to 19, for the treatment of patients in need, wherein said antibody or fragment may be administered at a dose of at least 3 μg/kg body weight.

21. A pharmaceutical composition comprising an antibody, fragment, or backbone according to any one of claims 1 to 20.

22. The ADM antibody or adrenomedullin antibody fragment or non-Ig-backbone according to any one of claims 1 to 20, for the treatment of chronic or acute or chronic conditions.

23. The ADM antibody or adrenomedullin antibody fragment or non-Ig-backbone according to claim 22, wherein the disease is sepsis.

Other embodiments within the scope of this invention are shown below:

1. Adrenomedullin ADM antibody or adrenomedullin antibody fragment, used to treat patients with severe chronic or acute diseases and to reduce the risk of death in said patients.

2. The ADM antibody or adrenomedullin antibody fragment according to claim 1, wherein the antibody form is selected from Fv fragment, scFv fragment, Fab fragment, scFab fragment, (Fab) ₂ fragment and scFv-Fc fusion protein.

3. The ADM antibody or adrenomedullin antibody fragment according to claim 1 or 2, wherein the antibody or fragment binds to the N-terminal portion (aa 1-21) of adrenomedullin.

4. The ADM antibody or adrenomedullin antibody fragment according to any one of claims 1 to 3, wherein the antibody or fragment is capable of recognizing and binding to the N-terminal portion (aal) of adrenomedullin.

5. The ADM antibody or adrenomedullin antibody fragment according to any one of claims 1 to 4, wherein the antibody or fragment is an ADM-stabilizing antibody or fragment that increases the t1 _/2 half-retention time of adrenomedullin in serum, blood, or plasma by at least 10%, preferably at least 50%, more preferably >50%, and most preferably >100%.

6. The ADM antibody or adrenomedullin antibody fragment according to any one of claims 1 to 5, wherein the antibody or fragment blocks less than 80%, preferably less than 50%, of the ADM biological activity.

7. An ADM antibody or adrenomedullin antibody fragment for treating a patient’s chronic or acute illness according to any one of claims 1 to 6, wherein the acute illness is selected from sepsis, diabetes, cancer, heart failure, shock, and renal dysfunction.

8. An ADM antibody or adrenomedullin antibody fragment for treating a patient with a chronic or acute illness, as claimed in any one of claims 1 to 7, wherein the patient is an ICU patient.

9. An ADM antibody or adrenomedullin antibody fragment for treating a patient’s chronic or acute illness according to any one of claims 1 to 8, wherein the risk of death is reduced by preventing adverse events, wherein the adverse events are selected from SIRS, sepsis, septic shock, organ failure, renal failure, fluid imbalance, and hypotension.

10. An ADM antibody or adrenomedullin antibody fragment for treating a patient’s chronic or acute disease according to any one of claims 1 to 8, wherein the antibody or fragment is used in combination with an ADM-binding protein.

11. A pharmaceutical preparation comprising the antibody or fragment according to any one of claims 1 to 10.

12. The pharmaceutical preparation according to claim 11, wherein the pharmaceutical preparation is a solution, preferably a ready-to-use solution.

13. The pharmaceutical preparation according to claim 11, wherein the pharmaceutical preparation is in a freeze-dried state.

14. The pharmaceutical preparation according to any one of claims 11 to 12, wherein the pharmaceutical preparation is administered intramuscularly.

15. The pharmaceutical preparation according to any one of claims 11 to 12, wherein the pharmaceutical preparation is administered intravenously.

16. The pharmaceutical preparation of claim 15, wherein the pharmaceutical preparation is administered by infusion.

Other embodiments within the scope of this invention are shown below:

1. An adrenomedullin (ADM) antibody or an adrenomedullin antibody fragment or an ADM non-Ig backbone, used to treat patients with severe chronic or acute diseases or acute symptoms, for the purpose of reducing the risk of death of said patients, wherein said antibody or fragment or backbone is a non-neutralizing ADM antibody or a non-neutralizing adrenomedullin antibody fragment or a non-neutralizing ADM non-Ig backbone.

2. The ADM antibody or adrenomedullin antibody fragment according to claim 1, wherein the antibody form is selected from Fv fragment, scFv fragment, Fab fragment, scFab fragment, (Fab) ₂ fragment and scFv-Fc fusion protein.

3. The ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone according to claim 1 or 2, wherein the antibody or fragment or backbone binds to the N-terminal portion (aa 1-21) of adrenomedullin.

4. The ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone according to any one of claims 1 to 3, wherein the antibody, fragment or backbone is capable of recognizing and binding to the N-terminal portion (aa1) of adrenomedullin.

5. The ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone according to any one of claims 1 to 4, wherein the antibody or fragment or backbone is an ADM-stabilized antibody or fragment or backbone that increases the half-life (t _1/2 half-retention time) of adrenomedullin in serum, blood, or plasma by at least 10%, preferably at least 50%, more preferably >50%, most preferably >100%, and blocks less than 80%, preferably less than 50%, of ADM biological activity.

6. The ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone according to any one of claims 1 to 5, wherein the antibody or fragment or backbone blocks less than 80%, preferably less than 50% of the ADM biological activity.

7. An ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone for treating a patient’s chronic or acute disease according to any one of claims 1 to 6, wherein the disease is selected from severe infections such as meningitis, systemic inflammatory response syndrome (SIRS), sepsis; other diseases such as diabetes, cancer, acute and chronic vascular diseases such as heart failure, myocardial infarction, stroke, atherosclerosis; shock such as septic shock and organ dysfunction such as renal dysfunction, liver dysfunction; burns, surgery, trauma.

8. An ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone for treating a patient’s chronic or acute disease according to any one of claims 1 to 7, wherein the disease is selected from SIRS, severe infection, sepsis, shock such as septic shock.

9. An ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone for treating a patient with a chronic or acute disease according to any one of claims 1 to 8, wherein the patient is an ICU patient.

The ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone for treating a patient’s chronic or acute disease or acute condition according to any one of claims 1 to 9, wherein the risk of death is reduced by preventing adverse events, wherein the adverse events are selected from SIRS, sepsis, shock such as septic shock, acute and chronic vascular diseases such as acute heart failure, myocardial infarction, stroke, organ failure such as renal failure, liver failure, fluid imbalance and hypotension.

10. The ADM antibody or adrenomedullin antibody fragment according to any one of claims 1 to 9, wherein the antibody or fragment is a human monoclonal antibody or fragment binding to ADM or an antibody fragment thereof, wherein the heavy chain comprises the following sequence:

SEQ ID NO: 1

GYTFSRYW

SEQ ID NO: 2

ILPGSGST

SEQ ID NO: 3

TEGYEYDGFDY

Furthermore, the light chain contains the following sequences:

SEQ ID NO: 4

QSIVYSNGNTY

SEQ ID NO: 5

RVS

SEQ ID NO: 6

FQGSHIPYT.

12. The human monoclonal antibody or fragment thereof that binds to ADM according to claim 10, wherein the antibody or fragment comprises a sequence selected from:

13. An ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone for treating a patient’s chronic or acute illness according to any one of claims 1 to 12, used in combination with a vasopressor such as catecholamines and/or an intravenously administered fluid.

14. An ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone, or a combination thereof, according to any one of claims 1 to 13, for treating a patient's chronic or acute condition, used in combination with an ADM-binding protein and/or other active ingredients.

15. A pharmaceutical preparation comprising an antibody, fragment, or backbone according to any one of claims 1 to 14.

16. The pharmaceutical preparation according to claim 15, wherein the pharmaceutical preparation is a solution, preferably a ready-to-use solution.

17. The pharmaceutical preparation according to claim 15, wherein the pharmaceutical preparation is in a freeze-dried state.

18. The pharmaceutical preparation according to any one of claims 15 to 16, wherein the pharmaceutical preparation is administered intramuscularly.

19. The pharmaceutical preparation according to any one of claims 15 to 16, wherein the pharmaceutical preparation is administered intravenously.

20. The pharmaceutical preparation of claim 19, wherein the pharmaceutical preparation is administered by infusion.

21. An ADM antibody or an adrenomedullin antibody fragment or an AM non-Ig backbone, wherein the antibody or fragment or backbone binds to the N-terminal portion (aa 1-21) of adrenomedullin, preferably the N-terminal portion (aa 1-21) of adrenomedullin in human ADM.

22. The antibody, fragment, or backbone of claim 2, wherein the antibody, fragment, or backbone is capable of recognizing and binding to the N-terminus (aa 1) of adrenomedullin.

Other embodiments within the scope of this invention are shown below:

1. Adrenomedullin (ADM) antibody or adrenomedullin antibody fragment, used to treat patients with chronic or acute diseases, and to prevent organ dysfunction or organ failure.

2. The ADM antibody or adrenal medullary antibody fragment for treating chronic or acute diseases according to claim 1, wherein the organ is the kidney.

3. The ADM antibody or adrenomedullin antibody fragment according to claim 1, wherein the antibody form is selected from Fv fragment, scFv fragment, Fab fragment, scFab fragment, (Fab) ₂ fragment and scFv-Fc fusion protein.

4. The ADM antibody or adrenomedullin antibody fragment according to any one of claims 1 to 3, wherein the antibody or fragment binds to the N-terminal portion (aa 1-21) of adrenomedullin.

5. The ADM antibody or adrenomedullin antibody fragment according to any one of claims 1 to 4, wherein the antibody or fragment is capable of recognizing and binding to the N-terminal portion (aa1) of adrenomedullin.

6. The ADM antibody or adrenomedullin antibody fragment according to any one of claims 1 to 5, wherein the antibody or the fragment is an ADM-stabilizing antibody or fragment that increases the t1 _/2 half-retention time of adrenomedullin in serum, blood, or plasma by at least 10%, preferably at least 50%, more preferably >50%, and most preferably >100%.

7. The ADM antibody or adrenomedullin antibody fragment according to any one of claims 1 to 6, wherein the antibody blocks less than 80%, preferably less than 50%, of the ADM biological activity.

8. An ADM antibody or adrenomedullin antibody fragment for treating a patient’s chronic or acute disease according to any one of claims 1 to 7, wherein the disease is selected from sepsis, diabetes, cancer, heart failure, and shock.

9. An ADM antibody or adrenomedullin antibody fragment for treating a patient with a chronic or acute disease according to any one of claims 1 to 8, wherein the patient is an ICU patient.

10. An ADM antibody or adrenomedullin antibody fragment for treating a patient’s chronic or acute disease according to any one of claims 1 to 9, wherein the antibody or fragment is a regulatory antibody or fragment that increases the t1 _/2 half-retention time of adrenomedullin in serum, blood, or plasma by at least 10%, preferably at least 50%, more preferably >50%, most preferably >100%, and blocks less than 80%, preferably less than 50%, of ADM biological activity.

11. A pharmaceutical preparation comprising an antibody or fragment according to any one of claims 1 to 10.

12. The pharmaceutical preparation according to claim 11, wherein the pharmaceutical preparation is a solution, preferably a ready-to-use solution.

13. The pharmaceutical preparation according to claim 11, wherein the pharmaceutical preparation is in a freeze-dried state.

14. The pharmaceutical preparation according to any one of claims 11 to 12, wherein the pharmaceutical preparation is administered intramuscularly.

15. The pharmaceutical preparation according to any one of claims 11 to 12, wherein the pharmaceutical preparation is administered intravenously.

16. The pharmaceutical preparation of claim 15, wherein the pharmaceutical preparation is administered by infusion.

Other embodiments within the scope of this invention are shown below:

1. Adrenomedullin (ADM) antibody or adrenomedullin antibody fragment or ADM non-Ig backbone, used to treat a patient’s chronic or acute disease or acute condition, or to prevent or reduce organ dysfunction or prevent organ failure in said patient.

2. The ADM antibody or adrenal medullary antibody fragment or ADM non-Ig backbone for treating chronic or acute diseases or acute symptoms according to claim 1, wherein the organ is the kidney or liver.

3. The ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone according to claim 1 or 2, wherein the ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone is a non-neutralizing ADM antibody or a non-neutralizing adrenomedullin antibody fragment or a non-neutralizing ADM non-Ig backbone.

4. The ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone according to any one of claims 1 to 3, wherein the antibody form is selected from Fv fragment, scFv fragment, Fab fragment, scFab fragment, (Fab) ₂ fragment and scFv-Fc fusion protein.

5. The ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone according to any one of claims 1 to 4, wherein the antibody or fragment or backbone binds to the N-terminal portion (aa 1-21) of adrenomedullin.

6. The ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone according to any one of claims 1 to 5, wherein the antibody, fragment or backbone is capable of recognizing and binding to the N-terminal portion (aal) of adrenomedullin.

7. The ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone according to any one of claims 1 to 6, wherein the antibody or the fragment or backbone is an ADM stable antibody or fragment or backbone that increases the t _1/2 half-retention time of adrenomedullin in serum, blood, or plasma by at least 10%, preferably at least 50%, more preferably >50%, and most preferably >100%.

8. The ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone according to any one of claims 1 to 7, wherein the antibody or fragment or backbone blocks less than 80%, preferably less than 50%, of ADM biological activity.

9. An ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone for treating a patient’s chronic or acute disease or acute condition, according to any one of claims 1 to 8, wherein the disease is selected from sepsis, diabetes, cancer, heart failure, and shock.

10. The ADM antibody or adrenomedullin antibody fragment according to any one of claims 1 to 9, wherein the antibody or fragment is a human monoclonal antibody or fragment binding to ADM or an antibody fragment thereof, wherein the heavy chain comprises the following sequence:

SEQ ID NO: 1

GYTFSRYW

SEQ ID NO: 2

ILPGSGST

SEQ ID NO: 3

TEGYEYDGFDY

Furthermore, the light chain contains the following sequences:

SEQ ID NO: 4

QSIVYSNGNTY

SEQ ID NO: 5

RVS

SEQ ID NO: 6

FQGSHIPYT.

11. The human monoclonal antibody or fragment binding to ADM according to claim 10, wherein the antibody or fragment comprises a sequence selected from:

12. An ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone for treating a patient’s chronic or acute disease according to any one of claims 1 to 11, wherein the antibody or fragment or backbone is a regulatory antibody or fragment or backbone that increases the half-life (t _1/2 half-retention time) of adrenomedullin in serum, blood, or plasma by at least 10%, preferably at least 50%, more preferably >50%, most preferably >100%, and blocks less than 80%, preferably less than 50% of the ADM biological activity.

13. An ADM antibody or adrenomedullin antibody fragment or ADM non-Ig backbone for treating a patient’s chronic or acute disease or acute condition, as claimed in any one of claims 1 to 12, used in combination with a vasopressor such as catecholamines and/or an intravenously administered fluid.

14. An ADM antibody or adrenomedullin antibody fragment or AD non-Ig backbone for treating a patient’s chronic or acute disease or acute condition, as claimed in any one of claims 1 to 13, or a combination thereof as claimed in claim 13, used in combination with an ADM binding protein and/or other active ingredients.

15. A pharmaceutical preparation comprising the antibody or fragment according to any one of claims 1 to 13.

16. The pharmaceutical preparation according to claim 14, wherein the pharmaceutical preparation is a solution, preferably a ready-to-use solution.

17. The pharmaceutical preparation according to claim 14, wherein the pharmaceutical preparation is in a freeze-dried state.

18. The pharmaceutical preparation according to any one of claims 14 to 15, wherein the pharmaceutical preparation is administered intramuscularly.

19. The pharmaceutical preparation according to any one of claims 14 to 15, wherein the pharmaceutical preparation is administered intravenously.

20. The pharmaceutical preparation of claim 18, wherein the pharmaceutical preparation is administered by infusion.

Example

It should be emphasized that, according to the present invention, the antibodies, antibody fragments and non-Ig backbones in the embodiment section bind to ADM and should therefore be considered as anti-ADM antibodies/antibody fragments/non-Ig backbones.

Example 1

Antibody production and determination of its affinity constant

Several human and mouse antibodies were generated, and their affinity constants were determined (see Tables 1 and 2).

Peptides/conjugates used for immunity:

Peptides for immunization were synthesized (see Table 1, JPT Technologies, Berlin, Germany) and conjugated to bovine serum albumin (BSA) with an additional N-terminal cysteine residue (if no cysteine was present in the selected ADM sequence). The peptides were linked to BSA using a Sulfolink coupling gel (Perbio-science, Bonn, Germany). The coupling protocol was performed according to the Perbio manual.

Mouse antibodies were generated using the following method:

Balb/c mice were immunized with 100 μg of peptide-BSA conjugate (emulsified in 100 μl Freund's complete adjuvant) on days 0 and 14, and with 50 μg of peptide-BSA conjugate (in 100 μl Freund's incomplete adjuvant) on days 21 and 28. Three days prior to the fusion experiment, animals received 50 μg of the conjugate dissolved in 100 μl saline, administered once intraperitoneally and once intravenously.

Spleen cells from immunized mice and myeloma cell line SP2/0 were fused for 30 seconds at 37°C with 1 ml of 50% polyethylene glycol. After washing, the cells were seeded in 96-well cell culture plates. Hybrid clones were selected by culturing in HAT medium [RPMI 1640 medium supplemented with 20% fetal bovine serum and HAT supplement]. After two weeks, the HAT medium was replaced with HT medium for three passages, and then the cells were restored to normal cell culture medium.

Three weeks after fusion, the primary screening for antigen-specific IgG antibodies in the cell culture supernatant was conducted. Microcultures testing positive were transferred to 24-well plates for proliferation. After retesting, the selected cultures were cloned and subcloned using a limiting dilution technique, and the isotypes were determined (see also Lane, R.D. "A short-duration polyethylene glycol fusion technique for increasing production of monoclonal antibody-secreting hybridas", J. Immunol. Meth. 81: 223-228; (1985), Ziegler, B. et al. "Glutamate decarboxylase (GAD) is not detectable on the surface of rat islet cells examined by cytofluorometry and complementary-dependent antibody-mediated cytotoxicity of monoclonal GAD antibody", Horm. Metab. Res. 28: 11-15, (1996)).

Mouse monoclonal antibody production:

The antibody was generated using a standard antibody production method (Marx et al., Monoclonal Antibody Production, ATLA 25, 121, 1997) and purified using protein A. Based on SDS-PAGE analysis, the antibody purity was >95%.

Human antibodies

Human antibodies were generated using the phage display method according to the following protocol.

The human natural antibody gene library HAL7/8 was used for the isolation of recombinant single-chain F-variable domains (scFv) targeting the adrenal medullarin peptide. The antibody gene library was screened using a panning strategy, including the use of biotinylate tags containing peptides linked to the adrenal medullarin peptide sequence via two distinct spacer regions. A mixed panning cycle of nonspecifically bound antigens and streptavidin-bound antigens was used to minimize the background of nonspecific binders. Phages eluted from the third round of panning were used to generate *E. coli* strains expressing monoclonal scFv. Supernatants from cultures of these cloned strains were used directly for antigen ELISA detection (see also Hust, M., Meyer, T., Voedisch, B., Rülker, T., Thie, H., El-Ghezal, A., Kirsch, M.I., Schütte, M., Helmsing, S., Meier, D., Schirrmann, T., Dübel, S., 2011. A human scFv antibody generation pipeline for proteome research. Journal of Biotechnology 152, 159-170; Schütte, M., Thurlie). r, P., Pelat, T., Wezler, of a putative Crf splice variant and generation of recombinant antibodies for the specific detection of Aspergillus fumigatus.PLoS One 4, e6625).

Positive clones were selected based on a positive ELISA signal for the antigen and a negative result for streptavidin-coated micro-low-level plates. For further characterization, the scFv open reading frame was cloned into the expression plasmid pOFE107 (Hust et al., J. Biotechn. 2011), captured from the culture supernatant by immobilized metal ion affinity chromatography, and purified by size exclusion chromatography.

Affinity constant

To determine the affinity of the antibody for adrenomedullin, the kinetics of adrenomedullin and the fixed antibody were determined by label-free surface plasmon resonance using a Biacore 2000 system (GE Healthcare Europe GmbH, Freiburg, Germany). Anti-mouse Fc antibodies covalently coupled to the surface of the CM5 sensor at high density were used, and reversible fixation of the antibodies was performed according to the manufacturer's instructions (Mouse Antibody Capture Kit; GE Healthcare). (Lorenz et al., “Functional Antibodies Targeting IsaA of Staphylococcus aureus Augment Host Immunus Response and Open New Perspectives for Antibacterial Therapy”; Antimicrob Agents Chemother. Jan 2011; 55(1): 165-173.)

Monoclonal antibodies were activated against the following ADM regions in both humans and mice. The table below shows the selection of antibodies used in other experiments. Selection was based on the following target regions:

Table 1:

The following is a list of other monoclonal antibodies obtained:

List of anti-ADM antibodies

Table 2:

Antibody fragments are produced through enzymatic digestion:

Fab and F(ab) ₂ fragments were generated by enzymatic digestion of the full-length mouse antibody NT-M. Antibody NT-M was digested using a) a pepsin-based F(ab) ₂ preparation kit (Pierce 44988) and b) a papain-based Fab preparation kit (Pierce 44985). Fragmentation protocols were performed according to the supplier's instructions. For F(ab) ₂ fragmentation, digestion was carried out at 37°C for 8 hours. Fab fragmentation digestion was carried out for 16 hours each.

Fab generation and purification scheme:

Equilibrate the immobilized papain by washing the resin with 0.5 ml of digestion buffer and centrifuging the column at 5000 × g for 1 min. Then discard the buffer. Prepare a desalted column by removing the storage buffer and washing with digestion buffer, then centrifuging at 1000 × g for 2 min each time. Add 0.5 ml of the prepared IgG sample to the centrifuge column containing the equilibrated immobilized papain. Incubate the digestion reaction at 37 °C on a benchtop shaker for 16 h. Centrifuge the column at 5000 × g for 1 min to separate the digestion fluid from the immobilized papain. Subsequently, wash the resin with 0.5 ml of PBS and centrifuge at 5000 × g for 1 min. Add the washed portion to the digested antibody to make a total sample volume of 1.0 ml. Equilibrate the NAb protein A column with PBS and IgG elution buffer at room temperature. Centrifuge the column for 1 min to remove the storage buffer (containing 0.02% sodium azide), and equilibrate by adding 2 ml of PBS, centrifuging again for 1 min, and discarding the pass. Apply the sample to the column and invert it to resuspend. Incubate at room temperature by tumbling for 10 minutes. Centrifuge the column for 1 minute, leaving the pass-through containing the Fab fragment.

(Reference: Coulter, A. and Harris, R. (1983). J. Immunol. Meth. 59, 199-203.; Lindner I. et al. (2010) {alpha}2-Macroglobulin inhibitors the malignantproperties of astrocytoma cells by impeding{beta}-catenin signaling.CancerRes.70, 277-87.;Kaufmann B.et al.(2010)Neutralization of West Nile virus bycross-linking of its surface proteins with Fab fragments of the humanmonoclonal antibody CR4354.PNAS.107, 18950-5.; Chen X.et al.(2010)Requirement of open headpiece conformation for activat ion of leukocyte integrinαxβ2.PNAS.107, 14727-32.; Uysal H.et al.(2009)Structure and pathogenicity ofantibodies specif ic for citrullinated collagen type II in experimentalarthitis.J.Exp.Med.206, 449-62.; Thomas G.M.et al.(2009)Cancer ce ll-derivedmicroparticles bearing P-selectin glycoprotein ligand 1 accelerate thrombusformation in vivo.J.Exp.Med.206 , 1913-27.; Kong F. et al. (2009) Demonstration ofcatch bonds between an integrin and its ligand.J.Cell Biol.185, 1275-84.)

F(ab′) ₂ fragment generation and purification protocol:

Equilibrate the immobilized pepsin by washing the resin with 0.5 ml of digestion buffer and centrifuging the column at 5000 × g for 1 min. Then discard the buffer. Prepare a desalted column by removing the storage buffer and washing with digestion buffer, then centrifuging at 1000 × g for 2 min each time. Add 0.5 ml of the prepared IgG sample to the centrifuge column containing the equilibrated immobilized pepsin. Incubate the digestion reaction at 37 °C on a benchtop shaker for 16 h. Centrifuge the column at 5000 × g for 1 min to separate the digestion fluid from the immobilized pepsin. Subsequently, wash the resin with 0.5 ml of PBS and centrifuge at 5000 × g for 1 min. Add the washed portion to the digested antibody to make a total sample volume of 1.0 ml. Equilibrate the NAb protein A column with PBS and IgG elution buffer at room temperature. Centrifuge the column for 1 min to remove the storage buffer (containing 0.02% sodium azide), and equilibrate by adding 2 ml of PBS, centrifuging again for 1 min, and discarding the pass. Apply the sample to the column and invert to resuspend. Incubate at room temperature by tumbling for 10 minutes. Centrifuge the column for 1 minute, leaving the pass-through containing Fab fragments.

(Reference: Mariani, M., et al. (1991). A new enzymatic method to obtain high-yield F(ab′)2suitable for clinical use from mouse IgG1. Mol. Immunol. 28: 69-77.; Beale, D. (1987). Molecular fragmentation: Some applications ons inimmunology.Exp Comp Immunol 11:287-96.; Ellerson, J.R., et al.(1972).A fragmentcorresponding to the CH2 region of immunoglobulin G(IgG)with complementfixing activity.FEBS Letters 24(3):318-22.;K erbel, R.S. and Elliot, B.E. (1983). Detection of Fc receptors. Meth Enzymol 93: 113-147.; Kulkami, P.N., et al. al.(1985).Conjμgation ofmethotrexate to IgG antibodies and their F(ab′)2fragments and the effect o f conjμgated methotrexate on tumor growth invivo.Cancer Immunol Immunotherapy 19: 211-4.; Lamoyi, E. (1986). Preparation of F(ab′)2Fragments from mouse IgG of various subclasses. Meth Enzymol 121: 652-663. ; Parham, P., et al. (1982). Monoclonal antibodies: purification, fragmentation and application to structural and functional studies of class I MHCantigens.J Immunol Meth 53: 133-73.; Raychaudhuri, G., et al.( 1985).Human IgG1and its Fc fragment bind with different affinities to the Fc receptors on thehuman U937, HL-60 and ML-1 cell lines. Mol Immunol 22(9): 1009-19.; Rousseaux, J., et al. (1980). The differentia l enzyme sensitivity of rat immunoglobulin Gsubclasses to papain an pepsin. Mol Immunoll7: 469-82.; Rousseaux, J., et al. (1983). Optimal condition fbr the preparation of Fab and F(ab′)2 fragments from mon oclonal IgG of different rat IgG subclasses.J Immunol Meth 64:141-6.; Wilson, K.M., et al.(1991).Rapid whole blood assay for HIV-1 seropositivityusing an Fab-peptide conjμgate.JImmunol Meth 138:111-9.)

Humanization of NT-H antibody fragments

Humanization of antibody fragments via CDR transplantation (Jones, P.T., Dear, P.H., Foote, J., Neuberger, M.S., and Winter, G. (1986) Replacing the complementarity-determining regions in a human antibody with those from a mouse. Nature 321, 522-525).

The following steps are performed to achieve humanized sequences:

Total RNA extraction: Total RNA was extracted from NT-H hybridomas using a Qiagen kit.

First round of RT-PCR: OneStep RT-PCR kit (Cat No. 210210) was used. RT-PCR was performed using heavy and light chain-specific primers. For each RNA sample, 12 separate heavy chain and 11 light chain RT-PCR reactions were established using a degenerate mixture of forward primers (covering the leader sequence of the variable region). Reverse primers were located in the constant regions of the heavy and light chains. No restriction sites were designed into the primers.

Reaction composition: 5.0 μl OneStep RT-PCR buffer, 0.8 μl dNTP Mix (containing 10 mM of each dNTP), 0.5 μl primer set, 0.8 μl OneStep RT-PCR Enzyme Mix, 2.0 μl template RNA, and 20.0 μl RNase-free water, for a total volume of 20.0 μl.

PCR conditions: Reverse transcription: 50℃, 30 min; Initial PCR activation: 95℃, 15 min

Cycles: 94℃, 25 sec; 54℃, 30 sec; 72℃, 30 sec; 20 cycles total; final extension: 72℃, 10 min

Second-round semi-nested PCR: The RT-PCR products from the first-round reaction were further amplified in the second-round PCR. Twelve separate heavy chain and eleven light chain RT-PCR reactions were established using antibody variable region-specific semi-nested primer sets.

Reaction composition: 10 μl of 2×PCR mix; 2 μl of primer set; 8 μl of first-round PCR product; total volume 20 μl; hybridoma antibody clone reporter.

PCR conditions: initial denaturation at 95℃ for 5 min; 25 cycles of 95℃ for 25 sec, 57℃ for 30 sec, and 68℃ for 30 sec; final extension at 68℃ for 10 min.

After PCR, the PCR reaction samples were run on an agarose gel to visualize the amplified DNA fragments. Following sequencing of DNA fragments from over 15 clones amplified by nested RT-PCR, several mouse antibody heavy and light chains were cloned and appeared suitable. Protein sequence alignment and CDR analysis identified one heavy chain and one light chain. After alignment with the homologous human frame sequence, the resulting humanized sequence of the variable heavy chain is as follows: See Figure 6 (Since the amino acids at positions 26, 40, and 55 of the variable heavy chain and position 40 of the variable light chain are crucial for binding properties, they can be reverted to the mouse source. The resulting candidates are described below) (Padlan, E.A. (1991) A possible procedure for reducing the immunogenicity of antibody variable domains while preserving their ligand-binding properties. Mol. Immunol. 28, 489-498.; Harris, L. and Bajorath, J. (1995) Profiles for the analysis of immunoglobulin sequences: Comparison of V gene subgroups. Protein Sci. 4, 306-310.).

Annotations for antibody fragment sequences (SEQ ID NO: 7-14): Bold and underlined CDRs 1, 2, and 3 are arranged in sequence; italics indicate constant regions; hinge regions are highlighted with bold letters; histidine tags are indicated with bold and italic letters; frame point mutations have a gray background.

Example 2

Effect of selected anti-ADM antibodies on the biological activity of anti-ADM

The effect of selected anti-ADM antibodies on ADM biological activity was detected in the human recombinant adrenal medulla receptor cAMP function assay (adrenal medulla bioassay).

Testing for antibodies targeting human or mouse adrenaline in the human recombinant adrenaline receptor cAMP function assay (adrenaline bioassay)

Material:

Cell line: CHO-K1

Receptor: Adrenal medullaris (CRLR+RAMP3)

Receptor accession cell lines: CRLR: U17473; RAMP3: AJ001016

Prior to the test, CHO-K1 cells (FAST-027C) expressing recombinant human adrenal medulla receptor were isolated from antibiotic-free culture medium by gently washing 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 KH2PO4} , 1.45 mM CaCl2, _0.5 g/L BSA).

Dose-response curves were performed in parallel with a reference agonist (hADM or mADM).

Antagonist test (96 wells):

For the antagonist assay, 6 μl of the reference agonist (human (5.63 nM) or mouse (0.67 nM) adrenomedullin) was mixed with 6 μl of the test sample diluted with different antagonists; or mixed with 6 μl of buffer. After incubating at room temperature for 60 min, 12 μl of cells (2,500 cells/well) were added. The plates were incubated at room temperature for 30 min. After adding lysis buffer, the percentage of DeltaF was estimated according to the manufacturer's instructions using the HTRF kit (cat n°62AM2 PEB) from Cis-Bio International. hADM 22-52 was used as the reference antagonist.

cAMP-HTRF assay for testing antibodies

In the human recombinant adrenal medullary receptor (FAST-027C) cAMP function assay, the antagonistic activity of anti-h-ADM antibodies (NT-H, MR-H, CT-H) was tested in the presence of 5.63 nM human ADM1-52 and the following final antibody concentrations: 100 μg/ml, 20 μg/ml, 4 μg/ml, 0.8 μg/ml, and 0.16 μg/ml.

In a human recombinant adrenal medullary receptor (FAST-027C) cAMP function assay, the antagonistic activity of anti-m-ADM antibodies (NT-M, MR-M, CT-M) was tested in the presence of 0.67 nM mouse ADM1-50 and the following final antibody concentrations: 100 μg/ml, 20 μg/ml, 4 μg/ml, 0.8 μg/ml, and 0.16 μg/ml. The data are plotted as relative inhibition against antagonist concentrations (see Figures 3a to 31). The maximum inhibition of individual antibodies is shown in Table 3.

Table 3:

Example 3

Data on stabilizing hADM with anti-ADM antibody

The stabilizing effect of human ADM antibodies on human ADM was tested using an hADM immunoassay.

Immunoassay for Quantitative Human Adrenal Medullary Insulin

The technique used is a luminescent immunoassay based on acridine ester-labeled sandwich-coated tubes.

Labeled compound (tracer): 100 μg (100 μL) of CT-H (1 mg/mL in PBS, pH 7.4, Adreno Med AG, Germany) was mixed with 10 μL of acridinium NHS-ester (1 mg/mL in acetonitrile, InVent GmbH, Germany) (EP 0353971) and incubated at room temperature for 20 min. Labeled CT-H was purified by gel filtration HPLC on a SEC 400-5 (Bio-Rad Laboratories, Inc., USA). The purified CT-H was diluted in (300 mmol/L potassium phosphate, 100 mmol/L NaCl, 10 mmol/L Na-EDTA, 5 g/L bovine serum albumin, pH 7.0). The final concentration was approximately 800,000 relative optical units (RLU) of the labeled compound (approximately 20 ng of labeled antibody) per 200 μL. The chemiluminescence of acridine ester was measured using an AutoLumat LB 953 (Berthold Technologies GmbH & Co. KG).

Solid phase: Polystyrene tubes (Greiner Bio-One International AG, Austria) were coated with MR-H (AdrenoMed AG, Germany) (1.5 μg MR-H/0.3 mL 100 mmol/L NaCl, 50 mmol/L TRIS/HCl, pH 7.8) at room temperature for 18 h. After blocking with 5% bovine serum albumin, the tubes were washed with PBS at pH 7.4 and vacuum dried.

calibration:

The assay was calibrated using dilutions of hADM (BACHEM AG, Switzerland) in 250 mmol/L NaCl, 2 g/L Triton X-100, 50 g/L bovine serum albumin, and 20 tabs/L Protease Inhibitor Cocktail (Roche Diagnostics AG, Switzerland).

hADM Immunoassay:

After adding 200 μl of labeled CT-H, 50 μl of sample (or calibrator) was pipetted into the coated tube and incubated at 4 °C for 4 h. Unbound tracers were removed by washing five times (1 ml each time) with washing solution (20 mM PBS, pH 7.4, 0.1% Triton X-100).

Chemiluminescence using LB 953 measuring tube

Figure 4 shows a typical hADM dose/signal curve and an hADM dose-signal curve in the presence of 100 μg/mL antibody NT-H.

NT-H does not affect the hADM immunoassay.

Stability of human adrenaline:

Human ADM was diluted in human citrate plasma (final concentration 10 nM) and incubated at 24 °C. Degradation of hADM was terminated at selected time points by freezing at -20 °C. Incubation was performed in the presence and absence of NT-H (100 μg/ml). Residual hADM was quantified using the hADM immunoassay described above.

Figure 5 shows the stability of hADM in human plasma (citric acid) in the absence and presence of NT-H antibody. The half-life of hADM alone is 7.8 h, while in the presence of NT-H, the half-life is 18.3 h (2.3-fold increase in stability).

Example 4

Sepsis mortality rate (early treatment)

animal models

Male C57Bl/6 mice (Charles River Laboratories, Germany), aged 12-15 weeks, were used for this study. Peritonitis was surgically induced under mild isoflurane anesthesia. An incision was made in the upper left abdomen (typically the location of the cecum). The cecum was exposed, and a tightly wrapped ligature was placed around it, distal to the small intestine entrance. A puncture wound was created inside the cecum using a 24-size needle, allowing a small amount of cecal contents to drain through the wound. The cecum was returned to the abdominal cavity, and the incision site was sutured. Finally, the animals were returned to their cages with free access to food and water. 500 μl of saline was administered subcutaneously as a fluid replacement.

Application and dosage of compounds (NT-M, MR-M, CT-M)

Mice were treated immediately after CLP (early treatment). CLP is an abbreviation for cecal ligation and puncture.

research group

Three compounds were used as the test compound, the medium, and the control compound. Five mice were in each group, and blood was drawn one day after BUN (blood urea nitrogen) measurement. Ten additional mice in each group were followed up for four days.

Group treatment (10 μl/g body weight) dose/follow-up:

1 NT-M, 0.2 mg/ml, survival within 4 days

2MR-M, 0.2 mg/ml, survival within 4 days

3CT-M, 0.2 mg/ml, survival within 4 days

4. Non-specific mouse IgG, 0.2 mg/ml, survival within 4 days.

5 controls - PBS 10 μl/g body weight, survival within 4 days

Clinical Chemistry

Renal function blood urea nitrogen (BUN) concentrations were measured at baseline and on day 1 after CLP. Blood samples were obtained from the cavernous sinus using a capillary tube under mild ether anesthesia. Measurements were performed using an AU 400 Olympus Multianalyser. 4-day mortality rates are provided in Table 4. Mean BUN concentrations are provided in Table 5.

Table 4:

4-day mortality rate Survival (%) PBS 0 Nonspecific mouse IgG 0 CT-M 10 MR-M 30 NT-M 70

Table 5:

The average number of 5 animals BUN (mM) before CLP Day 1 BUN (mM) PBS 8.0 23.2 Nonspecific mouse IgG 7.9 15.5 CT-M 7.8 13.5 MR-M 8.1 24.9 NT-M 8.8 8.2

As shown in Table 4, NT-M antibody significantly reduced mortality. After 4 days of treatment with NT-M antibody, 70% of the mice survived. After 4 days, 30% of the animals survived with MR-M antibody, and 10% survived with CT-M antibody. In contrast, all mice died after 4 days of treatment with nonspecific mouse IgG. Similar results were obtained in the control group receiving PBS (phosphate-buffered saline).

Blood urea nitrogen (BUN) tests are used to assess kidney function to aid in the diagnosis of kidney disease and to monitor patients with acute or chronic renal impairment or failure.

S-BUN test results revealed that NT-M antibody is the most effective in protecting the kidneys.

Sepsis mortality rate (late-stage treatment)

animal models

Male C57Bl/6 mice (Charles River Laboratories, Germany), aged 12-15 weeks, were used for this study. Peritonitis was surgically induced under mild isoflurane anesthesia. An incision was made in the upper left abdomen (typically the location of the cecum). The cecum was exposed, and a tightly wrapped ligature was placed around it, distal to the small intestine entrance. A puncture wound was created inside the cecum using a 24-size needle, allowing a small amount of cecal contents to drain through the wound. The cecum was returned to the abdominal cavity, and the incision site was sutured. Finally, the animals were returned to their cages with free access to food and water. 500 μl of saline was administered subcutaneously as a fluid replacement.

Application and dosage of compound (NT-M FAB2)

NT-M FAB2 was used to treat the test substance, media, and control compound. Treatment was initiated 6 hours after CLP, once sepsis had fully developed (late treatment). Each group consisted of 4 mice, and follow-up was conducted over 4 days.

Group treatment (10 μl/g body weight) dose/follow-up:

research group

1NT-M, FAB20.2mg/ml, survival within 4 days

2. Control: Non-specific mouse IgG, 0.2 mg/ml, surviving for 4 days.

3. Medium-PBS 10 μl/g body weight, survival within 4 days

Table 6:

4-day mortality rate Survival (%) PBS 0 Nonspecific mouse IgG 0 NT-M FAB2 75

As shown in Table 6, the NT-M FAB2 antibody significantly reduced mortality. After 4 days of treatment with the NT-M FAB2 antibody, 75% of the mice survived. In contrast, all mice died after 4 days of treatment with nonspecific mouse IgG. Similar results were obtained in the control group, which received mice PBS (phosphate-buffered saline).

Example 5

The escalating effect of anti-ADM antibodies in CLP mice after antibiotic treatment and regulation of circulation and fluid homeostasis via catecholamines.

animal models

Male C57Bl/6 mice (8-12 weeks old, 22-30g) were used in this study. Multimicrobial sepsis induced by cecal ligation and puncture (CLP) was used as a model for studying septic shock (Albuszies G, et al: Effect of increased cardiac output on hepatic and intestinal microcirculatory bloodflow, oxygenation, and metabolism in hyperdynamic murine septic shock. Crit CareMed 2005;33:2332-8), (Albuszies G, et al: The effect of iNOS deletion on hepatic gluconeogenesis in hyperdynamic murine). e septic shock. Intensive Care Med 2007; 33: 1094-101), (Barth E, et al: Role of iNOS in the reduced responsiveness of themyocardium to catecholamines in a hyperdynamic, mu rine model of septicshock.Crit Care Med 2006;34:307-13), (Baumgart K, et al: Effect of SOD-1 over-expression on myocardial function during resuscitated murine septicsho ck. Intensive Care Med 2009; 35: 344-9), (Baumgart K, et al: Cardiac and metabolic effects of hypothermia and inhaled H2S in anesthetized and ventilated mice. Crit Care Med 2 010;38:588-95), (Simkova V, et al: The effect of SOD-1 over-expression on hepatic gluconeogenesis and whole-body gluconeoxidation during resuscitated, normotensive murine septic shock.Shock 2008;30:578-84), (Wagner F, et al.: Inflammatory effects of hypothermia and inhaled H2Sduring resuscitated, hyperdynamic murine septic shock.Shock, im Druck), (WagnerF, et al: Effects of intravenous H2S after murine blunt chest trauma: aprospective, randomized controlled trial. Crit Care 2011, submittes for publication)).

After weighing, mice were anesthetized by intraperitoneal injection of 120 μg/g ketamine, 1.25 μg/g midazolam, and 0.25 μg/g fentanyl. Body temperature was maintained at 37-38°C during the surgical procedure. A 1 cm abdominal incision was made along the centerline to approach the cecum. The cecum was then tied near the base with 3-0 silk suture and punctured once with an 18-size needle. The cecum was returned to its original position, and the incision was sutured again (with 4-0 suture). To compensate for perioperative fluid loss, 0.5 ml of lactated Ringer's solution containing 1 μg/g buprenorphine was subcutaneously injected into the dermis of the back as an analgesic. For antibiotics, mice were subcutaneously administered ceftriaxone 30 μg/g and clindamycin 30 μg/g via the hind limbs.

After CLP surgery, the animal is kept in an environment with sufficient heat, and water and food are provided freely.

Following short-term isoflurane anesthesia, fluid requirements were ensured by subcutaneous injection of 0.5 ml of lactated Ringer's solution containing 4 μg/g glucose and 1 μg/g buprenorphine in the back, administered in 8-hour cycles. Additionally, antibiotics were maintained by subcutaneous injection of ceftriaxone 30 μg/g and clindamycin 30 μg/g in the lower extremities.

Dosing of test substances

Early treatment

Immediately after CLP surgery and suturing the incision, the test substance antibody NT-M at a concentration of 500 μg/ml in phosphate-buffered saline (PBS) was administered via penile intravenous injection at a dose of 2 mg/kg body weight (dose volume 88-120 μl) (5 animals).

Late-stage treatment

After the development of complete sepsis, i.e. 15.5 hours after the CLP procedure, the animals were anesthetized as described above, and NT-M at a concentration of 500 μg/ml in phosphate-buffered saline (PBS) was administered via penile intravenous injection at a dose of 2 mg/kg body weight (dose volume 88-120 μl) (3 animals).

The control group (6 animals) received the corresponding amount of PBS solution immediately after CLP surgery, instead of antibodies (4 μl/g, 88-120 μl).

Research group and experimental setup

Septic shock in rats under intensive care monitoring:

Three groups of animals were monitored, with 3, 5, and 6 animals. Group 1 (5 animals) received antibody NT-M 15.5 hours after CLP, Group 2 received antibody NT-M immediately after CLP, and Group 3 received an equivalent amount of PBS (4 μl/g). After a 16-hour post-CLP incubation (to allow for the development of multimicrobial sepsis), the experiment continued under monitoring and interventions equivalent to intensive care protocols. Therefore, after weighing, animals were anesthetized as described in the CLP procedure section (except for animals receiving late treatment, which were anesthetized prior to treatment). For the remainder of the experiment, body temperature was maintained at 37–38°C. Respiration was monitored and maintained via experimental animal lung ventilator (Emka Technologies, FiO2 0.5, PEEP 10 H2O, VT 8 μl/g, I:E 1:1.5, AF 70–140, depending on body temperature) after tracheotomy and intubation.

Anesthesia was maintained throughout the experiment by continuous infusion of ketamine 30 μg/gxh and fentanyl 0.3 μg/gxh via the right external jugular vein through the cannula. Additionally, a cannula was inserted into the right carotid artery for continuous monitoring of heart rate and mean arterial pressure (MAP). A colloid (80 μL/gxh) was infused intravenously (jugular vein), and, if necessary, norepinephrine dissolved in the colloid was infused as a vasopressor to maintain MAP > 65 mmHg. Blood samples (120 μl) were collected via the cannulated carotid artery at 0 and 4 hours for creatinine determination. Urine was collected via bladder puncture and a bladder catheter. The experiment was terminated after 6 hours, or prematurely if MAP > 65 mmHg (jugular vein) could not be maintained with vasopressors.

Measured parameters

The following parameters were measured and analyzed: total norepinephrine consumption (μg NA/g), norepinephrine consumption rate (μg NA/g/h), total urine volume collected during the experiment, creatinine concentration at the end of the experiment (μg/mL), and average creatinine clearance rate (μL/min).

Table 7:

After administering nonspecific mouse IgG and CLP to 6 mice (control group), 5 mice were given NT-mouse antibody (early treatment) or CLP 15.5 h later, and 3 mice were given NT-mouse antibody (late treatment). Catecholamine requirements were then measured.

The reduction in catecholamine demand is a measure of circulatory stability. Therefore, the data indicate that ADM antibodies, particularly NT-M antibodies, lead to significant circulatory stability and a substantial reduction in catecholamine demand. This circulatory stabilizing effect was observed in both early treatment (immediately after CLP) and treatment following progression to complete sepsis (late treatment) (see Figure 7).

Fluid balance regulation

More absolute fluid balance accumulated in the early stages of resuscitation and within 4 days is associated with an increased risk of death in septic shock. Fluid balance control is extremely important in the course of sepsis (S. Boyd et al., 2011). Controlling fluid balance in critically ill patients remains a significant challenge in intensive care medicine. As shown in Table 8, treatment of mice with NT-M antibodies after CLP (see “Animal Models” for experimental protocol) resulted in an increase in total urine volume. NT-M-treated animals excreted approximately three times more urine than untreated animals. Positive therapeutic effects were observed in both early and late treatment. Fluid balance improved by approximately 20-30% in both early and late treatment. Therefore, the data suggest that the use of ADM antibodies, especially NT ADM antibodies, is beneficial for regulating fluid balance in patients (see Table 8 and Figures 8 and 9).

Table 8

Average urine volume/g body weight Mean volume of body fluid balance/g body weight Control (mouse IgG) (N=6) 0.042ml/g 0.23ml/g Early NT-M (N=5) 0.12ml 0.18ml/g Relative changes, early treatment +186% -21.7% Late NT-M stage (N=3) 0.125ml 0.16ml/g Relative changes, late-stage treatment +198% -30.5%

Improvement of kidney function

The combination of acute renal failure and sepsis is associated with a 70% mortality rate, compared to 45% for patients with acute renal failure alone (Schrier and Wang, “Mechanisms of Disease Acute Renal Failure and Sepsis”; The New England Journal of Medicine; 351:159-69; 2004). Creatinine concentration and creatinine clearance are standard laboratory parameters for monitoring renal function or renal dysfunction (Jacob, “Acute Renal Failure”, Indian J. Anaesth.; 47(5):367-372; 2003). Creatinine and creatinine clearance data from the aforementioned animal studies (early treatment) are provided in Table 9.

Table 9

Kidney function

Compared with control sepsis animals, creatinine concentration decreased by 42% and creatinine clearance increased by more than 100% due to NT-M treatment (Table 9). The data indicate that administration of ADM antibodies, especially NT-M, leads to improved renal function.

Improvement of liver inflammation

Liver tissues from control and early-treatment animals were homogenized and lysed in lysis buffer. To prepare cell extracts, cells were resuspended, lysed on ice, and centrifuged. The supernatant (protein extract) was stored at -80°C. Activation of the nuclear factor κ-light chain gene enhancer (NF-κB) in B cells was determined using electrophoretic mobility shift assay (EMSA)1,2 as described above. The cell extract (10 μg) was incubated on ice with poly(deoxy-inosinic-deoxy-cytidylic acid) (poly-dI-dC) and a 32P-labeled double-stranded oligonucleotide (Biomers, Ulm, Germany) containing NF-κB (HIVκB site) (5’-GGATCCTCAACAGAGGGGACTTTCCGAGGCCA-3’). The complex was separated on a non-denaturing polyacrylamide gel, dried, and exposed to X-ray film. Radiolabeled NF-κB was quantified by densitometry using a photosensitive imager and image analysis software (AIDA Image Analyzer; Raytest). For comparisons between individual gels, the intensity of each band was relative to the intensity of a simultaneously loaded control animal (which had not undergone surgical instruments and CLP). Therefore, EMSA data are expressed as a fold increase relative to the control value. Statistical analysis: All data are presented as median (range), and unless otherwise stated, differences between groups were analyzed using the Mann-Whitney rank-sum test for unpaired samples. Results: Animals treated with NT-M (2.27 (1.97–2.53)) showed significantly reduced liver tissue NF-κB activation (2.92 (2.50–3.81)) compared to the mediator animals (p < 0.001) (see Figure 10).

References

1.Wagner F, Wagner K, Weber S, Stahl B, 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 H2S during resuscitated, hyperdynamic murine septic shock. Shock 2011; 35(4): 396-402.

2.Wagner F, Scheuerle A, Weber S, Stahl B, McCook O, MW, Huber-Lang M, Seitz DH, Thomas J, Asfar P, Szabó C, P, Gebhard F, Georgieff M, Calzia E, Radermacher P, Wagner K: C ardiopulmonary, histologic, and inflammatory effects of intravenous Na2S after blunt chest trauma-induced lung contusion in mice. JTrauma2011;71(6):1659-67.

Example 6

In vivo side effects assay of antibody NT-M

Male C57Bl/6 mice (Charles River Laboratories, Germany) aged 12–15 weeks were used in this study. Six mice were treated with NT-M at a dose of 0.2 mg/ml (10 μl/g body weight). As a control, six mice were treated with PBS at a dose of 10 μl/g body weight. Survival and physical condition were monitored for 14 days. There were no deaths in either group, and there was no difference in physical condition between the NT-M and control groups.

Example 7

Gentamicin-induced nephrotoxicity

A non-septic acute kidney injury model has been established, utilizing gentamicin-induced nephrotoxicity (Chiu PJS. Models used to assess renal functions. Drμg Develop Res 32:247-255, 1994.). This model is used to evaluate whether treatment with anti-adrenal medullary antibodies can improve renal function.

The experiment will be conducted as follows.

Eight male Sprague-Dowley rats weighing 250 ± 20 g were used in the group. Animals were challenged with gentamicin 120 mg/kg intramuscularly for 7 consecutive days (groups 1 and 2). The test compound (anti-adrenergic medullary antibody NT-M) and the medium (phosphate-buffered saline) were administered intravenously 5 min before gentamicin on day 0, and subsequently on days 2, 4, and 6. Body weight and clinical signs were monitored daily. Twenty-four (24)-hour urine samples were collected on ice on days 0, 2, and 6. The concentrations of Na+, K+, and creatinine in the urine samples were determined. Blood samples were collected on day 1 (before gentamicin), day 3 (before gentamicin), and day 7 for clinical chemistry. Serum electrolytes (Na+ and K+), creatinine, and BUN were the primary analytes monitored for assessing renal function. Plasma samples were collected into EDTA tubes (100 μl on days 1 and 3; 120 μl on day 7). Creatinine clearance was calculated. Urine volume, urinary electrolytes, and creatinine were expressed as excretion per 100 g of animal body weight. All animals were sacrificed on day 7. Kidneys were weighed.

Urine collection involved placing animals in individual cages and collecting urine for 24 hours on days 0, 2, and 6. Urine volume, urinary Na+, K+, and creatinine were measured.

Endogenous creatinine clearance rate is calculated as follows:

CCr(ml/24h)=[UCr(mg/ml)×V(ml/24h)]/SCr(mg/ml)

Sodium (Na+) excreted in urine over 24 hours is calculated as follows:

UNaV(μEq/24h)=UNa(μEq/ml)×V(ml/24h)

Similarly, calculate NAG and NGAL for 24-hr urinary excretion.

The fractional excretion of Na+ (FENa), or the percentage of filtered sodium excreted into the final urine, is a measure of renal tubular Na+ reabsorption function. It is calculated as follows:

FENa(%)=100×[UNa(μEq/ml)×V(ml/24h)]/PNa(μEq/ml)×CCr(ml/24h)

Compared with the media, treatment with anti-adrenergic medullary antibody improved several measures of renal function on day 7: serum creatinine 1.01 mg/dL (NT-M) vs. 1.55 mg/dL (media) (Figure 11), BUN 32.08 mg/dL (NT-M) vs. 52.41 mg/dL (media) (Figure 12), endogenous creatinine clearance 934.43 mL/24h (NT-M) vs. 613.34 mL/24h (media) (Figure 13), and Na+ excretion fraction 0.98% (NT-M) vs. 1.75% (media) (Figure 14).

Example 8

In the mouse CLP model described above, the effects of treatment with the anti-adrenal medullary antibody NT-M on several parameters of renal function were investigated.

NT-M resulted in a two-fold and a three-fold increase in diuresis and creatinine clearance, respectively, ultimately leading to a decrease in serum creatinine, urea, and NGAL concentrations at the end of the experiment (see Table 10). Furthermore, NT-M treatment significantly reduced the concentration of keratinocyte-derived chemokines (KC) in the kidneys (Figure 15).

Table 10: Renal function parameters in media-treated (n=11) and NT-M-treated (n=9) animals. Blood concentrations were measured in samples collected at the end of the experiment. NGAL = neutrophil gelatinase-associated lipid transport protein. All data are median (interquartiles).

medium NT-M p-value <![CDATA[Urine output[μL·g^-1·h^-1]]]> 4.4 (3.5; 16.5) 15.2 (13.9; 22.5) 0.033 <![CDATA[Ccreatinine clearance rate[μL·min^-1]]]> 197(110; 301) 400(316; 509) 0.006 <![CDATA[creatine[μg·mL^-1]]]> 1.83 (1.52; 3.04) 1.28 (1.20; 1.52) 0.010 <![CDATA[Urea[μg·mL^-1]]]> 378 (268; 513) 175 (101; 184) 0.004 <![CDATA[NGAL[μg·mL^-1]]]> 16(15；20) 11(10；13) 0.008

Conduct the experiment as follows.

Creatinine, urea, and neutrophil gelatinase-associated lipotransferase (NGAL)

Blood NGAL concentrations were measured using a commercial ELISA (mouse NGAL, RUO 042, BioPorto Diagnostics A/S, Denmark, Gentotte). Urea and creatinine concentrations were measured using a capillary column (Optima-5MS, Macherey-Nagel, Düren, Germany) gas chromatography/mass spectrometry system (Agilent 5890/5970, Germany) with ^2H3 _- creatinine (CDN isotope, Pointe-Claire, QU, Canada) and methyl urea (Fluka Chemikalien, Buchs, Switzerland) as internal controls. After deproteinization with acetonitrile, the mixture was centrifuged and evaporated to dryness. The supernatant was reconstituted in formic acid and extracted on a weak anion exchange column (WCX, Phenomenex, Aschaffenburg, Germany). Acetonitrile with N,O-bis(trimethylsilyl)trifluoroacetamide and N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide allows the formation of urea tert-butyl-dimethylsilyl derivatives and creatinine trimethylsilyl derivatives, respectively. Ions were monitored at m/z 231 and 245 and m/z 329 and 332 for urea and creatinine analytes, and internal controls, respectively. Creatinine clearance was calculated from urine output and plasma and urine creatinine concentrations using a standard formula.

Sample preparation

Kidneys stored at -80°C were homogenized in PBS. Whole-cell lysates were lysed with a double-concentration buffer (100 mM Tris pH 7.6; 500 mM NaCl; 6 mM EDTA; 6 mM EGTA; 1% Triton-X-100; 0.5% NP40; 10% glycerol; protease inhibitors (β-glycerophosphate 2 mM; DTT 4 mM; Leupeptine 20 μM; Natrium orthovanadate 0.2 mM)) followed by centrifugation. Whole-cell lysates were obtained from the supernatant; the precipitate consisting of cellular residues was discarded. Protein levels were determined spectrophotometrically using a commercially available protein analyzer (Bio-Rad, Hercules, CA), and the sample was adjusted to a final protein concentration of 4 μg/μl. Samples used for multiplex and EMSA analysis were diluted 1:1 with EMSA buffer (10 mM Heps; 50 mM KCl; 10% glycerol; 0.1 mM EDTA; 1 mM DTT), and samples used for immunoblotting were diluted 1:1 with 2x sample buffer (2% SDS; 125 mM Tris-HCl (pH 6, 8, 25°C); 10% glycerol; 50 mM DTT; 0.01% bromophenol blue).

The concentration levels of keratinocyte-derived chemokines (KC) were determined using the Bio-Plex Pro Cytokine Assay (Bio-Rad, Hercules, CA) in mice, measured using the Bio-plex suspension array system according to the manufacturer's instructions (see also Wagner F, Wagner K, Weber S, Stahl B, 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 H2S during resuscitated, hyperdynamic murine s eptic shock. Shock 2011;35:396-402; and Wagner F, Scheuerle A, Weber S, Stahl B, McCook O, MW, Huber-Lang M, Seitz DH, Thomas J, Asfar P, Szabó C, P, Gebhard F, Georgieff M, Calzia E, Radermacher P, Wagner K. Cardiopulmonary, histologic, and inflammatory effects of intravenous Na2S after blunt chest trauma-induced lung contusion in mice. J Trauma 2011;71:1659-1667). In short, appropriate cytokine standards and samples are added to a filter plate. The samples are incubated with antibodies chemically linked to fluorescently labeled microbeads. Subsequently, premixed detection antibodies were added to each well, followed by streptavidin-phycoerythrin. The beads were then resuspended, and the cytokine reaction mixture was quantified using a Bio-Plex protein array reader. Data were automatically processed and analyzed using a standard curve generated from recombinant cytokine standards via Bio-Plex Manager Software 4.1. Levels below the detection limit were set to zero for statistical purposes.

Example 9

In the aforementioned mouse CLP model, the effects of treatment with the anti-adrenergic medullary antibody NT-M on the liver were investigated.

NT-M significantly reduced the concentration of keratinocyte-derived chemokines (KC) in the liver (Figure 16).

The keratinocyte-derived chemokine (KC) was measured in a manner similar to that in Example 8 (kidney).

Example 10

In the mouse CLP model described above, the effects of treatment with the anti-adrenergic medullary antibody NT-M on several cytokines and chemokines in the blood circulation (plasma) were investigated.

Cytokine and chemokine concentrations

Plasma levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, monocyte chemoattractant protein (MCP)-1, and keratinocyte-derived chemokine (KC) were measured using the Bio-Plex Pro Cytokine Assay (Bio-Rad, Hercules, CA) kit. Measurements were performed using the Bio-plex suspension array system according to the manufacturer's instructions (see also Wagner F, Wagner K, Weber S, Stahl B, 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 H2 during resuscitated , hyperdynamic murine septic shock. Shock 2011;35:396-402; and Wagner F, Scheuerle A, Weber S, Stahl B, McCook O, MW, Huber-Lang M, Seitz DH, Thomas J, Asfar P, Szabó C, P, Gebhard F, Georgieff M, Calzia E, Radermacher P, Wagner K. Cardiopulmonary, histologic, and inflammatory effects of intravenous Na2S after blunt chest trauma-induced lung contusion in mice. J Trauma 2011;71:1659-1667). In short, appropriate cytokine standards and samples are added to a filter plate. The samples are incubated with antibodies chemically linked to fluorescently labeled microbeads. Subsequently, premixed detection antibodies were added to each well, followed by streptavidin-phycoerythrin. The beads were then resuspended, and the cytokine reaction mixture was quantified using a Bio-Plex protein array reader. Data were automatically processed and analyzed using Bio-Plex Manager Software 4.1, employing a standard curve generated from recombinant cytokine standards. Levels below the detection limit were set to zero for statistical purposes.

Plasma and renal tissue concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-10, monocyte chemoattractant protein (MCP)-1 and keratinocyte-derived chemokine (KC) were measured using a commercially available "Multi-Cytokine Kit" (Bio-Plex Pro Precision Pro Cytokine Assay, Bio-Rad, Hercules, CA). This kit allows for the collection of several parameters from a single sample. The individual working steps for the assay were performed according to the manufacturer's instructions (see also Wagner F, Wagner K, Weber S, Stahl B, 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 H2S during resuscitated, hyperdynamic murine septic shock. Shock 2011; 35: 396-402; and Wagner F, Scheuerle A, Weber S, Stahl B, McCook O, MW, Huber-Lang M, Seitz DH, Thomas J, Asfar P, Szabó C, P, Gebhard F, Georgieff M, Calzia E, Radermacher P, Wagner K. Cardiop ulmonary, histologic, and inflammatory effects of intravenous Na2Safter blunt chest trauma-induced lung contusion in mice. J Trauma 2011;71:1659-1667).

In short, fluorescently labeled microspheres (“beads”) were added to 96-well plates, followed by two washing steps, the addition of internal controls, and the addition of plasma and kidney homogenate samples. During the subsequent incubation, an antibody linked to the polystyrene beads binds to a cytokine. After the addition of a cytokine-specific biotin-labeled antibody (used to detect a cytokine) and an additional incubation time, streptavidin labeled with phycoerythrin was subsequently added. After an additional incubation time, the beads were resuspended, and the plate was measured using a specific flow cytometer (Bio-Plex suspension array system, Bio-Rad, Hercules, CA). Data were automatically processed and analyzed using Bio-Plex Manager Software 4.1, utilizing a standard curve generated from recombinant cytokine standards. For plasma levels, concentrations were provided in pg* ^mL⁻¹ , and kidney homogenate concentrations were converted to appropriate protein concentrations and provided in pg* ^mg⁻¹ protein.

NT-M resulted in a significant decrease in plasma concentrations of IL-6 (Fig. 17), IL-10 (Fig. 18), keratinocyte-derived chemokine (KC) (Fig. 19), monocyte chemoattractant protein-1 (MCP-1) (Fig. 20), and TNF-α (Fig. 21).

Example 11

Acute kidney injury induced by local ischemia/reperfusion

Another non-septic acute kidney injury model was established in which acute kidney injury was induced by local ischemia/reperfusion (Nakamoto M, Shapiro JI, Shanley PF, Chan L, and Schrier RW. In vitro and invivo protective effect of atriopeptin III on ischemic acute renal failure. J Clin Invest 80: 698-705, 1987., Chintala MS, Bemardino V, and Chiu PJS. Cyclic GMP but not cyclic AMP prevents renal platelet accumulation followed by Wing ischemia-reperfusion in anesthetized rats. J Pharmacol Exp Ther 271: 1203-1208, 1994). This model is used to evaluate whether treatment with anti-adrenergic medullary antibodies can improve kidney function.

The experiment was conducted as follows:

Effects of NT-M on ischemia/reperfusion-induced acute kidney injury in rats

Research Design

^a. Medium: Intravenous (iv) injection 5 min before reperfusion on day 0, followed by injection on day 1 and day 2.

^b . NT-M was administered intravenously at a dose of 4 mg/kg 5 minutes before reperfusion on day 0, followed by intravenous injection of 2 mg/kg daily on days 1 and 2.

^c. Urine was collected on day -1, day 0, day 1, and day 2. Blood chemistry and urine analysis were performed on day 0, day 1, day 2, and day 3, respectively. Plasma samples were collected in EDTA tubes (day 0 (just before surgery), day 1 and day 2: 100 μl, before medium or TA; day 3: 120 μl).

Clinical observation: daily before surgery, then during surgery and throughout the treatment process.

Eight male Sprague-Dowley rats weighing 250–280 g were used in the group. Animals were maintained under a 12-hr light/dark cycle and received a standard diet with distilled water available at will. Animals received fluid supplementation (0.9% NaCl and 5% glucose/1:1, 10 ml/kg p.o.) 30 min prior to surgery (day 0). Rats were anesthetized with pentobarbital (50 mg/kg, i.p.). The peritoneum was exposed through a central incision, followed by intravenous administration of heparin (100 U/kg, i.v.), and both renal arteries were nasally occluded for 45 min using hemostatic forceps. Immediately after removal of the renal clamps, the kidneys were observed for 1 min to ensure color changes indicating reperfusion. A test compound (NT-M) and a mediator (phosphate-buffered saline) were administered intravenously 5 min prior to reperfusion, followed daily on days 1 and 2.

Urine collection: 24-hour urine collection on ice was initiated 24 hours before local ischemia/reperfusion, i.e., day -1 (-24h to 0h) and day 0 (0-24h), and day 1 (24-48h) and day 2 (48-72h) after reperfusion.

Blood collection: At 0h (before IRI surgery), 24h (before medium or TA), 48h (before medium or TA), and 72h, 0.4ml of blood was collected into an EDTA tube via the tail vein for the determination of plasma creatinine/Na+/K+ and BUN; 2ml of blood was collected via the vena cava.

Animals were placed in individual cages, and urine was collected at 24 hours: day -1 (-24h-0h), day 0 (0-24h), day 1 after reperfusion on day 0 (24-48h), and day 2 (48-72h). Urine volume, urinary Na+, K+, and creatinine were measured.

Creatinine clearance (CCr) is calculated as follows:

CCr(ml/24h)=[UCr(mg/ml)×V(ml/24h)]/PCr(mg/ml)

Sodium (Na+) excreted in urine over 24 hours is calculated as follows:

UNaV(μEq/24h)=UNa(μEq/ml)×V(ml/24h)

The fractional excretion of Na+ (FENa), or the percentage of filtered sodium excreted into the final urine, is a measure of renal tubular Na+ reabsorption function. It is calculated as follows:

FENa(%)=100×[UNa(μEq/ml)×V(ml/24h)]/PNa(μEq/ml)×CCr(ml/24h)

Treatment with anti-adrenergic medullin antibodies improved several measures of renal function:

Blood urea nitrogen (BUN) in the media group showed a strong increase (0h: 17.49 mg/dL, 24h: 98.85 mg/dL, 48h: 109.84 mg/dL, 72h: 91.88 mg/dL), which was less pronounced with NT-M treatment (0h: 16.33 mg/dL, 24h: 84.2 mg/dL, 48h: 82.61 mg/dL, 72h: 64.54 mg/dL) (Figure 22).

Serum creatinine levels were similar: mediator group (0h: 0.61 mg/dL, 24h: 3.3 mg/dL, 48h: 3.16 mg/dL, 72h: 2.31 mg/dL), NT-M group (0h: 0.59 mg/dL, 24h: 2.96 mg/dL, 48h: 2.31 mg/dL, 72h: 1.8 mg/dL) (Figure 23).

Endogenous creatinine clearance decreased significantly on day 1, and thereafter the improvement in the NT-M group was better than that in the mediator group. Mediator group: (0h: 65.17 mL/h, 24h: 3.5 mL/h, 48h: 12.61 mL/h, 72h: 20.88 mL/h), NT-M group: (0h: 70.11 mL/h, 24h: 5.84 mL/h, 48h: 21.23 mL/h, 72h: 26.61 mL/h) (Figure 24).

Attached Figure Description

Figure 1a: Illustration of antibody forms - Fv and scFv- variants

Figure 1b: Illustration of antibody forms - heterofusion and bifunctional antibodies

Figure 1c: Antibody Forms - Illustration of Bivalent Antibodies and Bispecific Antibodies

Figure 2:

hADM 1-52 (SEQ ID No. 21)

mADM 1-50 (SEQ ID No. 22)

Human ADM aa 1-21 (SEQ ID No. 23)

Human ADM aa 1-42 (SEQ ID No. 24)

Human ADM aa 43-52 (SEQ ID No. 25)

Human ADM aa 1-14 (SEQ ID NO: 26)

Human ADM aa 1-10 (SEQ ID NO: 27)

Human ADM aa 1-6 (SEQ ID NO: 28)

aa 1-32 of mature ADM (SEQ ID NO: 29)

aa 1-40 of mature mouse ADM (SEQ ID NO: 30)

aa 1-31 of mature mouse ADM (SEQ ID NO: 31)

Figure 3:

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

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

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

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

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

f: Dose-response curve of mouse ADM. Maximum 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 mADM.

i: Dose/inhibition curve of MR-M in the presence of 0.67 nM mADM.

j: Dose/inhibition curve of NT-M in the presence of 0.67 nM mADM.

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

l: This shows the inhibition of ADM by F(ab)2NT-M and Fab NT-M.

Figure 4: This figure shows the typical hADM dose/signal curve and the hADM dose-signal curve in the presence of 100 μg/mL antibody NT-H.

Figure 5: This figure shows the stability of hADM in human plasma (citric acid) in the absence and presence of NT-H antibodies.

Figure 6: Alignment of Fab and homologous human frame sequences

Figure 7: This figure shows the norepinephrine requirement for early and late treatment with NT-M.

Figure 8: This figure shows urine production after early and late treatment with NT-M.

Figure 9: This figure shows the fluid balance after early and late treatment with NT-M.

Figure 10: Liver tissue activation of nuclear factor κ-light chain gene enhancers in B cells (NF-κB) by electrophoretic mobility shift analysis (EMSA). # Depicts p < 0.001 relative to the medium.

Figure 11: Serum creatinine over time. Shows mean +/- standard error.

Figure 12: Blood urea nitrogen (BUN) over time. Shows mean plus/minus standard error.

Figure 13: Endogenous creatinine clearance over time. Showing mean +/- standard error.

Figure 14: Na+ excretion fraction over time. Shows mean plus/minus standard error.

Figure 15: Keratinocyte-derived chemokine (KC) levels measured relative to the extracted total renin. White boxes show results obtained with the medium, and gray boxes show results obtained after NT-M treatment.

Figure 16: Keratinocyte-derived chemokine (KC) levels measured relative to the extracted total liver protein. White boxes show results obtained with the medium, and gray boxes show results obtained after NT-M treatment.

Figure 17: Plasma IL-6 levels. The white box shows the results obtained using the medium, and the gray box shows the results obtained after NT-M treatment.

Figure 18: Plasma IL-10 levels. The white box shows the results obtained using the medium, and the gray box shows the results obtained after processing with NT-M.

Figure 19: Plasma keratinocyte-derived chemokine (KC) levels. White boxes show results obtained with the medium, and gray boxes show results obtained after NT-M treatment.

Figure 20: Plasma monocyte chemoattractant protein-1 (MCP-1) levels. White boxes show results obtained with the medium, and gray boxes show results obtained after NT-M treatment.

Figure 21: Plasma TNF-α levels. The white box shows the results obtained using the medium, and the gray box shows the results obtained after NT-M treatment.

Figure 22: Blood urea nitrogen (BUN) over time. Shows mean plus/minus standard error.

Figure 23: Serum creatinine over time. Shows mean +/- standard error.

Figure 24: Endogenous creatinine clearance over time. Showing mean +/- standard error.

Claims (31)

1. An anti-adrenergic medullary monoclonal antibody or a fragment thereof, wherein the anti-adrenergic medullary monoclonal antibody or fragment thereof specifically binds to at least four amino acids within the first 42 amino acid sequences of mature human adrenergic medullary (ADM), wherein the first 42 amino acid sequences of mature human ADM are: YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVA as shown in SEQ ID NO: 24, and wherein the anti-adrenergic medullary monoclonal antibody or fragment thereof exhibits an affinity of at least ^10⁻⁷ M for ADM. in: (a) The heavy chain variable region of the monoclonal antibody comprises: (i) CDR1 sequence: GYTFSRYW as shown in SEQ ID NO:1, (ii) CDR2 sequence: ILPGSGST shown in SEQ ID NO:2, (iii) CDR3 sequence: TEGYEYDGFDY as shown in SEQ ID NO:3, and (b) The light chain variable region of the monoclonal antibody comprises: (i) CDR1 sequence: QSIVYSNGNTY as shown in SEQ ID NO:4, (ii) CDR2 sequence: RVS shown in SEQ ID NO:5, and (iii) CDR3 sequence: FQGSHIPYT as shown in SEQ ID NO:6. 2. The anti-adrenergic monoclonal antibody or a fragment thereof of claim 1, wherein it is a human monoclonal antibody or a humanized antibody, wherein: (a) The heavy chain variable region of the monoclonal antibody comprises: (i) CDR1 sequence: GYTFSRYW as shown in SEQ ID NO:1, (ii) CDR2 sequence: ILPGSGST shown in SEQ ID NO:2, (iii) CDR3 sequence: TEGYEYDGFDY as shown in SEQ ID NO:3, and (b) The light chain variable region of the monoclonal antibody contains the following CDR sequence: (i) CDR1 sequence: QSIVYSNGNTY as shown in SEQ ID NO:4, (ii) CDR2 sequence: RVS shown in SEQ ID NO:5, and (iii) CDR3 sequence: FQGSHIPYT as shown in SEQ ID NO:6. 3. The anti-adrenergic medullary monoclonal antibody or a fragment thereof according to claim 1 or 2, wherein, (a) The heavy chain region comprises an amino acid sequence selected from SEQ ID NOs: 7-11, or a sequence comprising a non-CDR sequence of said heavy chain region that has at least 85% identity with a non-CDR sequence of one of the sequences in SEQ ID NOs: 7-11, and (b) The light chain region comprises an amino acid sequence selected from SEQ ID NOs: 12-14, or a non-CDR sequence comprising the light chain region having at least 85% identity with a non-CDR sequence of one of the sequences in SEQ ID NOs: 12-14. 4. The anti-adrenergic medullary monoclonal antibody or fragment thereof of claim 1 or 2, wherein the anti-adrenergic medullary monoclonal antibody or fragment thereof specifically binds to the N-terminal portion of mature human adrenergic medullary ADM as shown in SEQ ID NO: 23, amino acids 1-21: YRQSMNNFQGLRSFGCRFGTC. 5. The anti-adrenal medullary monoclonal antibody or fragment thereof of claim 1 or 2, wherein the anti-adrenal medullary monoclonal antibody or fragment thereof does not bind to either N-terminally elongated or N-terminally modified adrenal medullary or N-terminally degraded adrenal medullary. 6. The anti-adrenergic medullary monoclonal antibody or a fragment thereof of claim 1 or 2, wherein the anti-adrenergic medullary monoclonal antibody or the fragment thereof specifically binds to the N-terminal portion of mature human adrenergic medullary ADM, amino acids 1-19. 7. The anti-adrenergic medullary monoclonal antibody or fragment thereof of claim 1 or 2, wherein the anti-adrenergic medullary monoclonal antibody or fragment thereof binds to an epitope containing the first amino acid at the N-terminus of adrenergic medullary. 8. The anti-adrenergic medullary monoclonal antibody or a fragment thereof of claim 1 or 2, wherein the anti-adrenergic medullary monoclonal antibody or the fragment thereof does not bind to the C-terminal portion of ADM, wherein the C-terminal portion of ADM is amino acid 43-52 of ADM: PRSKISPQGY-NH2 as shown in SEQ ID NO:25. 9. The anti-adrenal medullary monoclonal antibody or fragment thereof of claim 1 or 2, wherein the anti-adrenal medullary monoclonal antibody or fragment thereof is an ADM-stable antibody or fragment that satisfies the following conditions: it increases the half-life of adrenal medullary in serum, blood, or plasma by at least 10%, or at least 50%, or >50%, or 100%, and/or wherein the antibody or fragment blocks no more than 80%, or no more than 50%, of the biological activity of circulating ADM. 10. The anti-adrenergic medullary monoclonal antibody of claim 9 or a fragment thereof, wherein the anti-adrenergic medullary monoclonal antibody or fragment thereof binds to ADM, wherein the anti-adrenergic medullary monoclonal antibody or fragment thereof comprises a sequence selected from: SEQ ID NO:7, AM-VH-C QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYWGQGTT LTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH; SEQ ID NO:8, AM-VH1 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTT VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH; SEQ ID NO:9, AM-VH2-E40 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTT VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH; SEQ ID NO:10，AM-VH3-T26-E55 QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTT VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH; SEQ ID NO:11，AM-VH4-T26-E40-E55 QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTT VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH; SEQ ID NO:12, AM-VL-C DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC; SEQ ID NO:13, AM-VL1 DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC; SEQ ID NO:14, AM-VL2-E40 DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. 11. The use of the anti-adrenergic medullary monoclonal antibody or a fragment thereof of any one of claims 1 to 10 in the preparation of a medicament for the treatment or prevention of septic shock. 12. The application of claim 11, wherein the drug is used to treat or prevent late-stage sepsis. 13. The use of the anti-adrenergic medullary monoclonal antibody or a fragment thereof of any one of claims 1 to 10 in the preparation of a medicament for reducing the risk of death in patients with chronic or acute diseases or acute conditions, wherein the chronic or acute diseases or acute conditions are selected from meningitis, systemic inflammatory response syndrome, sepsis, septic shock, renal dysfunction, and hepatic dysfunction. 14. The use of the anti-adrenergic medullary monoclonal antibody or a fragment thereof of any one of claims 1 to 10 in the preparation of a medicament for stabilizing circulation in a patient suffering from a chronic or acute disease or acute condition, wherein the chronic or acute disease or acute condition is selected from meningitis, systemic inflammatory response syndrome, sepsis, septic shock, renal dysfunction, and hepatic dysfunction. 15. The application of claim 14, wherein the circulation is systemic circulation. 16. The application of claim 14, wherein the drug is used to reduce the patient's need for vasopressors. 17. The application of claim 16, wherein the drug is used to reduce the patient's need for catecholamines. 18. The use of the anti-adrenergic medullary monoclonal antibody or a fragment thereof of any one of claims 1 to 10 in combination with another pharmaceutical agent in the preparation of a medicament for the treatment or prevention of septic shock. 19. The application of claim 18, wherein the drug is used to treat or prevent late-stage sepsis. 20. The application of claim 18, wherein the pharmaceutical agent is selected from vasopressors, intravenous solutions, TNF-α antibodies, and antibiotics. 21. The use of the anti-adrenergic medullary monoclonal antibody or a fragment thereof of any one of claims 1 to 10 in combination with another pharmaceutical agent in the preparation of a medicament for reducing the risk of death from a patient with a chronic or acute disease or acute condition, wherein the chronic or acute disease or acute condition is selected from meningitis, systemic inflammatory response syndrome, sepsis, septic shock, renal dysfunction, and hepatic dysfunction. 22. The application of claim 21, wherein the pharmaceutical agent is selected from vasopressors, intravenous solutions, TNF-α antibodies, and antibiotics. 23. The use of the anti-adrenergic medullary monoclonal antibody or a fragment thereof of any one of claims 1 to 10 in combination with another pharmaceutical agent in the preparation of a medicament for stabilizing circulation in a patient suffering from a chronic or acute disease or acute condition selected from meningitis, systemic inflammatory response syndrome, sepsis, septic shock, renal dysfunction, and hepatic dysfunction. 24. The application of claim 23, wherein the circulation is systemic circulation. 25. The application of claim 23, wherein the drug is used to reduce the patient's need for vasopressors. 26. The application of claim 25, wherein the drug is used to reduce the patient's need for catecholamines. 27. The application of any one of claims 23-26, wherein the pharmaceutical agent is selected from vasopressors, intravenous solutions, TNF-α antibodies, and antibiotics. 28. The application of any one of claims 11-26, wherein the drug can be formulated into a solution or a freeze-dried pharmaceutical preparation. 29. The application of claim 27, wherein the drug can be formulated into a solution or a freeze-dried pharmaceutical preparation. 30. The use of any one of claims 11-26, wherein the drug is formulated for intramuscular, intravascular, infusion administration or systemic administration to a patient. 31. The application of claim 27, wherein the drug can be formulated for intramuscular, intravascular, infusion administration or systemic administration to a patient.