RU2790561C2 - Compound binding adrenomedullin (adm) for use for therapy or prevention of disease symptoms - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: pharmaceutical composition and use of an antibody against adrenomedullin (hereinafter – ADM), or a fragment of an antibody against ADM, or a non-Ig frame against ADM in therapy or prevention of disease symptoms or in therapy or prevention of a disease characterized by certain symptoms are presented. In this case, the specified antibody, or the fragment, or the frame binds to 1-52 amino acids of ADM or to fragments of this sequence.

EFFECT: inventions allow for use them in efficient therapy or prevention of inflammatory conditions, autoimmune diseases, metabolic diseases, brain diseases, and cardiovascular diseases, as well as in therapy or prevention of disease symptoms.

21 cl, 5 dwg, 3 tbl, 2 ex

Description

The present invention relates to an anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold for use in the treatment or prevention of symptoms of a disease and/or for use in the treatment or prevention of diseases characterized by these symptoms. Symptoms of the disease may be selected from the group consisting of nausea, headache, muscle pain, back pain, tremor, vomiting in an individual, wherein said antibody or fragment or scaffold can bind to amino acids 1-52 of ADM (SEQ ID NO: 1) or with fragments of this sequence.

State of the art

A variety of diseases or pathologies may share partially non-specific symptoms that may range from unpleasant to intolerable to the individual suffering from them. Quite often, individuals who have more than one symptom must take several drugs to alleviate these symptoms. There is a continuing need for new forms of therapy or prevention of symptoms associated with various underlying diseases or pathologies. In particular, it would be useful to provide medicaments or drugs that can be used to treat or prevent more than one symptom associated with the underlying disease or pathology. The present invention addresses this need.

The peptide adrenomedullin (ADM) was first described in 1993 ( Kitamura K. et al. 1993. Biochemical and Biophysical Research Communications Vol. 192 (2): 553-560 ) as a new antihypertensive peptide containing 52 amino acids, which was isolated from human pheochromocytoma. In the same year, a cDNA encoding a 185 amino acid precursor peptide and the complete amino acid sequence of this precursor peptide was also described. A precursor peptide that contains, inter alia, a 21 amino acid signal sequence at the N-terminus is also referred to as "preproadrenomedullin" (pre-proADM). As used herein, all amino acid positions indicated generally refer to pre-proADM, which contains 185 amino acids. Adrenomedullin (ADM) peptide is a 52 amino acid peptide (SEQ ID NO:1) and which contains amino acids 95-146 of pre-proADM from which it is formed by proteolytic cleavage. To date, only a few fragments of the peptide fragments resulting from cleavage of pre-proADM have been more accurately characterized, notably the physiologically active peptides adrenomedullin (ADM) and "PAMP", a 20 amino acid (22-41) peptide that follows 21 amino acid signal peptide in pre-proADM. The discovery and characterization of ADM in 1993 set off an intense research activity, the results of which are summarized in various review articles, for the purposes of this disclosure, reference is made in particular to the articles in the Peptides issue dealing in particular with ADM ( Editorial, Takahashi K. 2001. Peptides 22:1691) and (Eto T. 2001. Peptides 22: 1693-1711 ). Another review is ( Hinson et al. 2000. Endocrine Reviews 21(2):138-167 ). In scientific studies carried out to date, it has been found, inter alia, that ADM can be considered a polyfunctional regulatory peptide. It is released into the bloodstream in an inactive, glycine-extended form ( Kitamura K. et al. 1998. Biochem. Biophys. Res. Commun. 244(2):551-555 ). There is also a binding protein ( Pio R. et al. 2001. The Journal of Biological Chemistry 276(15):12292-12300 ) that is specific for ADM, and possibly similarly modulates the effect of ADM. These physiological effects of ADM, as well as PAMP, which are of major importance in research to date, were effects on blood pressure.

ADM is an effective vasodilator and it is possible to associate the hypotensive effect with specific peptide segments at the C-terminus of ADM. Moreover, the aforementioned physiologically active PAMP peptide derived from pre-proADM was found to similarly exhibit an antihypertensive effect, even though it appeared to have a different mechanism of action from ADM.

Moreover, it was found that the concentrations of ADM, which can be determined in the blood and other biological fluids in a number of pathological conditions, significantly exceed the concentration in healthy control individuals. Thus, the level of ADM in patients with congestive heart failure, myocardial infarction, kidney disease, hypertensive disorders, diabetes mellitus in the acute phase of shock and in sepsis and septic shock increases significantly, although to varying degrees. PAMP concentrations are also increased in some of these pathological conditions, however, plasma levels are lower relative to ADM ((Eto, T., supra). shock (see (Eto, T., supra) and ( Hirata et al. Journal of Clinical Endocrinology and Metabolism 1996. 81(4): 1449-1453; Ehlenz K. et al. 1997. Exp Clin Endocrinol Diabetes 105: 156- 162); Tomoda Y. et al. 2001. Peptides 22: 1783-1794; Ueda S. et al. 1999. Am. J. Respir. Crit. Care Med. 160: 132-136; and Wang P. Peptides 2001. 22: 1835-1840 ).

There is only limited information on the role of adrenomedullin in migraine. Since adrenomedullin has vasodilating effects in the cerebral circulation, it is presumed to be involved in migraine. On the one hand, administration of intravenous infusion of human ADM to patients suffering from migraine did not induce significantly more severe headache or migraine compared to placebo ( Petersen et al. 2008. Cephalalgia 29:23-30 ). Moreover, Akkali et al. reported low plasma adrenomedullin levels in the natural history of migraine in patients ( Akcali A. 2016. Medical Science and Discovery 3(4): 153-158 ). On the other hand, Kis et al. have argued that there is a need to develop new, effective, specific, and possibly non-peptide receptor antagonists as potential therapeutic tools to suppress ADM-mediated proliferative effects as anti-migraine drugs ( Kis et al. 2003. Hypertens Res 26 (Suppl): S61 -S70 ).

Definitions

Before a detailed description of the invention, it is advisable to provide definitions of certain technical terms used throughout the present description. Although the present invention has been described in relation to specific embodiments, the present description should not be construed as limiting. Before a detailed description of illustrative embodiments of the present invention, definitions are provided that are important for understanding the present invention.

As used herein and in the appended claims, the singular form also includes the corresponding plural form, unless the context clearly indicates otherwise.

In the context of the present invention, the terms "approximately" and "approximately" denote the range of accuracy, which, as understood by a person skilled in the art, still provides the technical effect of the feature in question. The term generally indicates a deviation from a numerical value of ±20%, preferably ±15%, more preferably ±10% and even more preferably ±5%.

It should be understood that the term "comprising" is not limiting. For the purposes of the present invention, the term "consisting of" is considered to be the preferred embodiment of the term "comprising". If a group is further defined to contain at least a certain number of embodiments, this implies that it also encompasses a group that preferably consists of only those embodiments.

It should also be understood that the terminology used in the present description is only intended to describe specific embodiments and is not intended to limit the scope of the present invention, which is limited only by the appended claims.

As used herein, the term "nausea" refers to a feeling of heaviness and discomfort in the upper stomach with an involuntary urge to vomit ( Metz A. 2017. Australian Family Physician Vol. 36 (9): 688-692 ). It may precede vomiting, however, the individual may have nausea without vomiting. When it is prolonged, it is a debilitating symptom. Nausea is a non-specific symptom, which means that it has many possible causes. Some common causes of nausea are motion sickness, dizziness, migraines, weakness, low blood sugar, gastroenteritis (stomach infection), or food poisoning. Nausea is a side effect of many medications, including chemotherapy, or early pregnancy toxemia. Nausea can also be caused by anxiety, disgust, and depression.

As used herein, the term "headache" is a symptom of pain anywhere in the head or neck. It occurs with migraine (sharp or throbbing pain), tension headache, and cluster headaches ( Waldman et al. 2014. J Yoga Phys Ther 2014, 4:1 ). There is also an increased risk of depression in individuals with severe headache. Headache can result from many conditions, both severe and non-severe. There are a number of different classification systems for headaches. It is generally accepted that causes of headaches can include fatigue, lack of sleep, stress, drug effects, psychoactive drug effects, viral infections, loud noise, colds, head trauma, rapid consumption of very cold food or drink, and dental or sinus problems.

Headaches are broadly classified into "primary" or "secondary" ( Oleson 2005. Functional Neurology 20(2): 61-68 ). Primary headaches are benign recurrent headaches that are not caused by an underlying disease or structural problem. For example, migraine is a type of primary headache. While primary headaches can cause significant daily pain, they are not dangerous. The four categories of primary headache are: migraine, tension headache (TTH), cluster headache and other autonomic trigeminal cephalgia, and other primary headaches.

Secondary headaches, which are of organic, metabolic or drug-induced origin, are caused by an underlying disease such as infection, brain injury, vascular disorders, cerebral hemorrhage, and tumors. Secondary headaches can be harmless or dangerous. Migraine is a primary headache disorder characterized by moderate to severe recurrent headache (for a review, see: Diener et al. 2012. Nat Rev Neurol. 8(3):162-71 ). As a rule, headaches affect one half of the head, have a throbbing character and last from two to 72 hours. Symptoms associated with it may include nausea, vomiting, and sensitivity to light, sound, or smell. The pain usually gets worse with physical activity. Up to one-third of people have an aura: usually a short period of blurred vision that signals that a headache is about to set in. Sometimes an aura may occur with little or no headache following it.

As used herein, the terms "muscle aches" or "muscle pain", also referred to as "myalgia", refer to a symptom of many diseases and disorders (for a review, see: Kyriakides et al. 2013. European Journal of Neurology 20: 997 -1005 ). The most common causes are overload or overextension of a muscle or muscle group. Myalgia without a history of trauma is often the result of viral infections. Prolonged myalgias may indicate metabolic myopathy, some nutrient deficiencies, or chronic fatigue syndrome.

As used herein, the term "back pain" refers to a painful sensation in any part of the back. Episodes of back pain can be acute, subacute, or chronic, depending on the duration. The pain may be characterized as aching, shooting or stabbing pain, or a burning sensation. The pain may radiate to the shoulders and arms, as well as the legs and feet, and may include paresthesia (tingling for no apparent reason), weakness, or numbness in the legs and arms. The anatomical classification of back pain is according to the segments of the back: neck pain (cervical), middle back pain (thoracic), lower back pain (lumbar) or coccygodynia (coccyx or sacral pain) with pain in the back being the most common. lumbar region of the spine. The pain may come from muscles, nerves, bones, joints, or other structures of the spinal column (spine), but internal structures such as the gallbladder and pancreas can also cause said back pain ( Cohen et al. 2008. BMJ. 337:a2718 ).

As used herein, the term shivering (also referred to as shivering) is a bodily function in response to prior hypothermia, or simply the sensation of cold in warm-blooded animals. As core body temperature drops, the shiver reflex is triggered to maintain homeostasis. Skeletal muscles begin to shake in small movements, generating heat through the expended energy. Shaking may also be a response to fever, as the individual may feel cold. During fever, the temperature set by the hypothalamus increases. An increased set temperature causes an increase in body temperature (pyrexia), but also causes the patient to feel cold until the new set temperature is reached. Severe shivering with violent shaking is called chills. Chills may be the result of the patient's body shaking in a physiological attempt to increase body temperature to a new set point. Trembling can also occur after surgery, which is known as postanesthetic trembling.

As used herein, the term "vomiting", also known as gagging and regurgitation, among other terms, is the involuntary, abrupt expulsion of an individual's stomach contents through the mouth and sometimes through the nose ( Metz A. 2017. Australian Family Physician Vol. 36 ( 9): 688-692 ). Vomiting can be caused by a wide variety of conditions; it may appear as a specific response to a disease such as gastritis or poisoning, or as a non-specific consequence of disorders ranging from brain tumors and increased intracranial pressure to excessive exposure to ionizing radiation.

As used within the scope of the invention, the symptoms of ailments or diseases in a patient in need of therapy and/or prevention of such symptoms are selected from the groups of indications including inflammatory conditions, autoimmune diseases, metabolic diseases, brain diseases, cardiovascular diseases and drug-induced diseases. diseases.

The types of symptoms and diseases associated with them are provided below in the form of a non-limiting list. It should be noted that the therapy or prophylaxis described herein may be directed to more than one type of symptom. In addition, it should be noted that a given medical condition, ailment, or disease may be associated with more than one of the symptoms.

Nausea may be associated with diseases of the abdomen, for example, obstructive disorders (eg, pyloric obstruction, small bowel obstruction, colonic obstruction, superior mesenteric artery syndrome), intestinal infections (eg, viral or bacterial infection), inflammatory diseases (such as cholecystitis, pancreatitis, appendicitis, hepatitis), sensorimotor dysfunction (eg, gastroparesis, intestinal pseudo-obstruction, gastroesophageal reflux, chronic idiopathic nausea, functional vomiting, cyclic vomiting syndrome, regurgitation syndrome) or biliary colic; diseases outside the abdomen, for example, cardiopulmonary disorders (such as cardiomyopathy, myocardial infarction), diseases of the inner ear (such as motion sickness, labyrinthitis, malignant tumors), intracerebral disorders (such as cerebral hemorrhage, abscess, hydrocephalus, malignant tumors) , psychiatric disorders (eg, anorexia and bulimia nervosa, depression), postoperative vomiting, nausea associated with therapies and drugs (eg, chemotherapy and biological therapy, antibiotics, antiarrhythmic drugs, digoxin, oral hypoglycemic agents, oral contraceptives), nausea associated with endocrine/metabolic diseases (such as pregnancy, uremia, ketoacidosis and parathyroid disease, adrenal insufficiency), nausea due to intoxication due to liver failure, alcohol abuse, etc.

The symptom "back pain" can be associated with diseases due to inflammation, especially in the acute phase, which usually lasts from two weeks to three months, and it can be associated with lumbago, trauma, injury, infections, malignancy (especially malignant tumors known to spread to the spine, such as breast, lung and prostate cancer), etc.

The symptom "myalgia" or "muscle pain" may be associated with damage or injury, including sprain, hematoma or overuse, when the muscle is used excessively, too often, including when protecting from a separate injury, chronic tension, muscle pain due to rhabdomyolysis, associated with e.g. viral infections, compression injury, it may be drug related e.g. due to fibrates and statins, ACE inhibitors, cocaine, some antiretroviral drugs, severe potassium deficiency, fibromyalgia, Ehlers-Danlos syndrome, autoimmune disorders ( including, for example, mixed connective tissue disease, systemic lupus erythematosus, polymyalgia rheumatica, myositis such as polymyositis, dermatomyositis and inclusion body myositis, multiple sclerosis, myalgic encephalomyelitis (chronic fatigue syndrome), familial Mediterranean fever, polyarteritis nodosa, Devic's disease, annular scleroderma, sarcoidosis), metabolic diseases (such as carnitine palmitoyltransferase II deficiency, Conn's syndrome, adrenal insufficiency, hyperthyroidism, hypothyroidism, diabetes, hypogonadism), as well as canalopathy, Stickler's syndrome, hypokalemia, hypotension (low muscle tone), exercise intolerance, mastocytosis, peripheral neuropathy, eosinophilia-myalgia syndrome, Bark's fever, herpes, hemochromatosis also known as iron overload, delayed muscle soreness syndrome, AIDS, HIV infection, tumor-induced osteomalacia, hypovitaminosis D, myocardial infarction.

The symptom "headache" may be associated with primary headaches. 90% of all headaches are primary headaches. Primary headaches usually begin for the first time in a person between the ages of 20 and 40. The most common types of primary headaches are migraines and tension headaches. They have different characteristics. Migraines typically present as throbbing headaches, nausea, photophobia (sensitivity to light), and phonophobia (sensitivity to sounds). Tension headaches usually present as non-pulsating "girdle" pressure on both sides of the head without any other symptoms. Other very rare types of primary headaches include: cluster headaches: short episodes (15-180 minutes) of severe pain, usually around one eye, with autonomous symptoms (lacrimation, redness of the eyes, nasal congestion) that occur at the same time every day. Cluster headaches can be treated with triptans and prevented with prednisone, ergotamine, or lithium. In addition, they include trigeminal neuralgia or occipital neuralgia, characterized by shooting pain in the face, prolonged hemicrania, i.e. continuous unilateral pain with episodes of severe pain; primary shooting headaches, eg, recurrent episodes of shooting "ice breaker pain" or "bumps and thumps" for 1 second to several minutes without autonomic symptoms (lacrimation, red eyes, nasal congestion); a primary cough headache that starts suddenly and lasts a few minutes after coughing, sneezing, or straining (anything that can increase pressure in the head). Serious causes (see the important section on secondary headaches) must be ruled out before making a diagnosis of benign primary cough headache; primary exercise headache is characterized by a rhythmic, throbbing pain that begins during or after exercise, lasting from 5 minutes to 24 hours, the mechanism of these headaches is unclear, possibly due to the tension causing the veins in the head to dilate, causing pain; Primary intercourse headaches are characterized by a dull, bilateral headache that begins during sexual activity and worsens during orgasm. hypnagogic headache; secondary headaches, which can be caused by problems somewhere in the head or neck. Some of them are not harmful, such as cervicogenic headache (pain originating from the muscles of the neck), headache caused by overuse of drugs in individuals who use excessive amounts of headache painkillers, meningitis pain characterized by inflammation of the meningeal membranes, which is manifested by fever and meningeal symptoms, or stiffness of the neck; pain with bleeding in the brain (intracranial hemorrhage); subarachnoid hemorrhage (acute severe headache, neck stiffness without fever); pain during aneurysm rupture, arteriovenous malformation, intraparenchymal hemorrhage; temporal arteritis, i.e. inflammatory disease of the arteries, common in the elderly (mean age 70), polymyalgia rheumatica; pain in acute angle-closure glaucoma (increased pressure in the eyeball); post-seizure headaches that occur after a seizure or other type of seizure as part of the post-seizure period (post-seizure condition); gastrointestinal disorders can cause headaches, including Helicobacter pylori infection, celiac disease, non-celiac gluten sensitivity, irritable bowel syndrome, inflammatory bowel disease, gastroparesis, and hepatobiliary disorders. Treatment of gastrointestinal disorders may result in remission or reduction in headaches.

The term "headache" is defined as primary or secondary headache. Primary headache is defined as migraine, tension headache (TTH), cluster headache and other autonomic trigeminal cephalalgias, and other primary headaches.

The diagnosis or assessment of headache is well established in the art. The evaluation can be based on subjective indicators, such as characterization of symptoms by the patient. For example, migraine can be diagnosed based on the following criteria: 1) episodic attacks of headache lasting from 4 to 72 hours; 2) with two of the following symptoms: unilateral pain, throbbing, aggravated by movement, and pain of moderate or severe severity; and 3) with one of the following symptoms: nausea or vomiting, and photophobia or phonophobia ( Goadsby et al., N. Engl. J. Med. 346:257-270, 2002 ).

The shaking symptom may be associated with fever, cold sensitivity, menopause, panic attack, anxiety, bacterial infection, rickettsial infection, viral infection, drug withdrawal, etc.

Vomiting may be associated with gastritis (inflammation of the stomach wall), gastroenteritis, gastrointestinal reflux, celiac disease, non-celiac gluten sensitivity, pyloric stenosis, bowel obstruction, overheating, acute abdomen and/or peritonitis, ileus, food allergy (often associated with hives or edema) cholecystitis, pancreatitis, appendicitis, hepatitis; food poisoning, an allergic reaction to cow's milk proteins, such as a milk allergy or lactose intolerance; motion sickness, Meniere's disease, concussion, cerebral hemorrhage, migraine, brain tumors, benign intracranial hypertension and hydrocephalus, metabolic disorders such as hypercalcemia, uremia, adrenal insufficiency, hypoglycemia, hyperglycemia, drug reaction, alcohol intoxication, use of opioids, selective serotonin reuptake inhibitors; the use of chemotherapeutic drugs; inflammation of the stomach caused by a number of viruses and bacteria, for example, norovirus, influenza virus; or psychiatric/behavioral conditions such as bulimia nervosa and diarrhea.

Detailed description of embodiments of the invention

The following are embodiments of the invention. It should be noted that, as a rule, embodiments can be combined with any other embodiments of the same category (product, process, application, method).

One embodiment of the invention provides an anti-ADM antibody, an anti-ADM antibody or antibody fragment, or a non-Ig anti-ADM scaffold for use in the treatment or prevention of symptoms of a disease, or for use in the therapy or prevention of diseases characterized by such symptoms. The symptoms may be selected from the group consisting of nausea, headache, muscle pain, back pain, trembling, vomiting in the patient. Said antibody or fragment or framework binds to mature ADM, e.g. amino acids 1-52 ADM (SEQ ID NO:1) or fragments of mature ADM, e.g. mid-region ADM (MR-ADM) (SEQ ID NO:3 ), or with N-terminal ADM (SEQ ID NO:4), as detailed below.

Thus, another embodiment of the invention provides an anti-ADM antibody, or an anti-ADM antibody fragment, or an anti-ADM non-Ig scaffold, for use in the therapy or prevention of symptoms of a disease, or for use in the therapy or prevention of diseases characterized by such symptoms, wherein said antibody or fragment or scaffold binds to mature ADM, eg amino acids 1-52 of ADM (SEQ ID NO:1) or fragments thereof as mentioned above.

A further embodiment of the invention relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in the treatment or prevention of diseases characterized by symptoms such as nausea, headache, muscle pain, back pain, tremor, vomiting in a patient, and wherein said antibody or fragment or scaffold binds to a mature ADM, eg ADM amino acids 1-52 (SEQ ID NO:1), or fragments thereof as defined above.

In further embodiments, the invention relates to an anti-ADM antibody, or an anti-ADM antibody fragment, or a non-Ig anti-ADM scaffold for use in therapy or disease prevention in a patient, wherein said antibody, or fragment, or scaffold binds to a mature ADM. , for example, with amino acids 1-52 of ADM (SEQ ID NO:1), or fragments thereof, as defined above, and where the diseases are selected from indications where the amount of C-reactive protein (CRP) in the sample is ≥ 10 mg/l , and/or where the amount of TNF in the sample is ≥ 50 pg/ml, and/or where the amount of bio-ADM in the sample is ≥ 43 pg/ml, and/or where the mean arterial pressure is reduced.

In a further embodiment, the invention relates to an anti-ADM antibody or anti-ADM antibody fragment or non-Ig anti-ADM scaffold for use in therapy or disease prevention in a patient, wherein said antibody or fragment or scaffold binds to a mature ADM, for example, with amino acids 1-52 ADM (SEQ ID NO:1), or fragments thereof, as defined above, and where the disease is migraine.

In a further embodiment, the invention relates to an anti-ADM antibody or anti-ADM antibody fragment or non-Ig anti-ADM scaffold for use in therapy or disease prevention in a patient, wherein said antibody or fragment or scaffold binds to a mature ADM, for example, with amino acids 1-52 of ADM (SEQ ID NO:1), or fragments thereof, as defined above, and where the disease is migraine, where the individual in need of it has an amount of C-reactive protein (CRP) in the sample ≥ 10 mg/l, and/or where the amount of TNF in the sample is ≥ 50 pg/ml, and/or where the amount of bio-ADM in the sample is ≥ 43 pg/ml, and/or where the mean arterial pressure is reduced.

In a further embodiment, the invention relates to an anti-ADM antibody or anti-ADM antibody fragment or non-Ig anti-ADM scaffold for use in therapy or disease prevention in a patient, wherein said antibody or fragment or scaffold binds to a mature ADM, for example, with amino acids 1-52 of ADM (SEQ ID NO:1), or fragments thereof, as defined above, and where the disease is migraine, where the individual in need of it has an amount of C-reactive protein (CRP) in the sample ≥ 10 mg/l, and/or where the amount of TNF in the sample is ≥ 50 pg/ml, and/or where the amount of bio-ADM in the sample is ≥ 43 pg/ml, and/or where the mean arterial pressure is reduced, and where the individual in need thereof has at least one of the following disease symptoms selected from the group of nausea, vomiting, muscle pain, back pain, trembling, vomiting.

In one embodiment of the invention, the sample is selected from the group consisting of whole blood, plasma and serum.

Mean arterial pressure (MAP) is defined as the mean pressure in a patient's arteries over one cardiac cycle. It is considered an indicator of perfusion to vital organs. It is believed that a MAP greater than 70 mm. rt. Art., is sufficient to maintain the organs of the average individual. In the context of the present invention, the term "mean arterial pressure is reduced" means a MAP below 75 mm. rt. Art.

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

Another embodiment of the invention relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in the treatment or prevention of disease symptoms selected from the group of nausea, headache, muscle pain, back pain, tremor, vomiting, or for use in the treatment or prevention of diseases characterized by symptoms such as migraine in a patient according to the preceding embodiment, wherein said antibody or fragment or scaffold binds to an N-terminal portion (a.k. .1-21) ADM:

YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO:4).

Another embodiment of the invention provides an anti-ADM antibody, or an anti-ADM antibody fragment binding to ADM, or an anti-ADM antibody that binds to ADM, for use in the treatment or prevention of symptoms of a disease selected from the group of nausea, headache , muscle pain, back pain, tremor, vomiting, or for use in the treatment or prevention of diseases characterized by such symptoms, such as migraine, in a patient, in accordance with any of the preceding embodiments, as appropriate, characterized in that said antibody , antibody fragment or non-Ig scaffold binds to part of the middle region, a.k. 21-42, adrenomedullin:

CTVQKLAHQIYQFTDKDKDNVA (SEQ ID NO:3)

Another embodiment of the invention relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in the treatment or prevention of disease symptoms selected from the group of nausea, headache, muscle pain, back pain, tremor, vomiting, or for use in the treatment or prevention of diseases characterized by symptoms such as migraine in a patient, in accordance with any of the preceding embodiments, wherein said antibody, or antibody fragment, or non-Ig framework is monospecific, in particular, monoclonal.

Another embodiment of the invention relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in the treatment or prevention of disease symptoms selected from the group of nausea, headache, muscle pain, back pain, tremor, vomiting, or for use in the treatment or prevention of diseases characterized by symptoms such as migraine in a patient, in accordance with any of the preceding embodiments, wherein said antibody, or fragment, or framework exhibits an ADM binding affinity of at least 10 ^-7 M by surface plasmon resonance without labeling using the Biacore 2000 system.

Another embodiment of the invention relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in the treatment or prevention of disease symptoms selected from the group of nausea, headache, muscle aches, back pain, tremors, emesis, or for use in the treatment or prevention of diseases characterized by symptoms such as migraine in a patient according to any of the preceding embodiments, wherein said antibody or fragment or scaffold is not an ADM binding protein 1 (complement factor H).

Another embodiment of the invention relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in the treatment or prevention of disease symptoms selected from the group of nausea, headache, muscle pain, back pain, tremor, vomiting, or for use in the treatment or prevention of diseases characterized by symptoms such as migraine in a patient, according to any of the preceding embodiments, wherein said antibody or fragment or scaffold recognizes and binds the N-terminus (a .k. 1) ADM.

Another embodiment of the invention relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in the treatment or prevention of disease symptoms selected from the group of nausea, headache, muscle pain, back pain, tremor, vomiting, or for use in the treatment or prevention of diseases characterized by symptoms such as migraine in a patient, according to any of the preceding embodiments, wherein said antibody or fragment or scaffold is an ADM stabilizing antibody, or a fragment or scaffold that increases the half-life (half residence time t _1/2 ) of ADM in serum, blood, plasma by at least 10%, preferably by at least 50%, more preferably by >50%, most preferably by > 100%.

Another embodiment of the invention relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in the treatment or prevention of disease symptoms selected from the group of nausea, headache, muscle pain, back pain, tremor, vomiting, or for use in the treatment or prevention of diseases characterized by symptoms such as migraine in a patient according to any of the preceding embodiments, wherein said antibody, or fragment, or scaffold blocks the biological activity of ADM by no more than 80 %, preferably not more than 50%, when using hADM 22-52 as a reference antagonist in CHO-K1 cells expressing the recombinant human ADM receptor.

Another embodiment of the invention relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in the treatment or prevention of disease symptoms selected from the group of nausea, headache, muscle pain, back pain, tremor, vomiting, or for use in the treatment or prevention of diseases characterized by such symptoms in an individual according to any of the preceding embodiments, wherein said individual in need thereof is selected from a group of individuals suffering from a disease selected from the group consisting of migraine , viral infections, drug-induced disease symptoms (eg, induced by chemotherapy or induced by treatment with biological agents, antibiotics, antiviral compounds, etc.). In a specific embodiment, the disease or pathology (these terms may be used interchangeably) is migraine.

Another embodiment of the invention relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in the treatment or prevention of disease symptoms selected from the group of nausea, headache, muscle pain, back pain, trembling, vomiting, in an individual according to any of the preceding embodiments, wherein said individuals are undergoing cancer therapy (chemotherapy).

Chemotherapy is a category of cancer therapy that uses one or more anti-cancer drugs (chemotherapeutic agents) as part of a standardized chemotherapy regimen. Chemotherapeutic drugs may include the following drug categories: alkylating agents, kinase inhibitors, vinca alkaloids, anthracyclines, antimetabolites, aromatase inhibitors, topoisomerase inhibitors, engineered anti-cancer agents such as monoclonal antibodies, cytokines, gene therapy vectors, antisense and peptide molecules .

Another embodiment of the invention relates to an anti-ADM antibody, or an anti-ADM antibody fragment, or a non-Ig anti-ADM scaffold, for use in the treatment or prevention of disease symptoms selected from the group of nausea, headache, muscle pain, back pain , tremors, vomiting, or for use in the treatment or prevention of diseases characterized by symptoms such as migraine in an individual according to any of the preceding embodiments, wherein said antibody or fragment is a human monoclonal antibody or fragment that binds to N- terminal region (a.k. 1-21) of ADM (SEQ ID NO:4), or a fragment of this antibody, where the heavy chain contains the sequences:

CDR1: SEQ ID NO:5

GYTFSRYW

CDR2: SEQ ID NO:6

ILPGSGST

CDR3: SEQ ID NO:7

TEGYEYDGFDY

and where the light chain contains the sequences:

CDR1: SEQ ID NO:8

QSIVYSNGNTY

CDR2:

RVS

CDR3: SEQ ID NO:9

FQGSHIPYT.

Another embodiment of the invention relates to a human monoclonal antibody or fragment that binds to ADM, or an antibody fragment thereof, for use in the treatment or prevention of disease symptoms selected from the group of nausea, headache, muscle pain, back pain, tremor, vomiting or for use in the treatment or prevention of diseases characterized by symptoms such as migraine in an individual according to any of the preceding embodiments, wherein said antibody or fragment contains a sequence selected from the group consisting of a VH region:

SEQ ID NO:10 (AM-VH-C)

QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYKK

SEQ ID NO:11 (AM-VH1)

QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYKK

SEQ ID NO:12 (AM-VH2-E40)

QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYKK

SEQ ID NO:13 (AM-VH3-T26-E55)

QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYKK

SEQ ID NO:14 (AM-VH4-T26-E40-E55)

QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYK

and contains the following sequence as a VL region:

SEQ ID NO:15 (AM-VL-C)

DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQRGDSKDSTYSLSSTLVSKADYEKHKVACE

SEQ ID NO:16 (AM-VL1)

DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESTKVTEQDSKDSPVSLSSTLVSSKAGLACEVEEKHKHK

SEQ ID NO:17 (AM-VL2-E40)

DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQRGSSKDSTYSLSSTLVSKADYEKHKVACE

Another embodiment of the invention relates to a human monoclonal antibody or fragment thereof that binds to ADM, or an antibody fragment thereof, for use in the treatment or prevention of disease symptoms selected from the group of nausea, headache, muscle pain, back pain, tremors, emesis or for use in the treatment or prevention of diseases characterized by symptoms such as migraine in an individual according to any of the preceding embodiments, wherein said antibody or fragment contains the following sequence as a heavy chain:

SEQ ID NO:22

QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGEILPGSGSTNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS c SVMHEALHNHYTQKSLSLSPGK

and contains the following sequence as a light chain:

SEQ ID NO:23

DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPRLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQNGSSKDSTYSLSSTLVSKADYEKHKVACE

In a specific embodiment of the invention, the antibody contains the following sequence as a heavy chain:

SEQ ID NO:22

QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGEILPGSGSTNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

or a sequence that is > 95% identical to it, preferably > 98%, preferably > 99% and contains the following sequence as a light chain:

SEQ ID NO:23

DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPRLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQNGSSKDSTYSLSSTLVSKADYEKHKVACE

or a sequence that is >95% identical to it, preferably >98%, preferably >99%.

Pairwise alignment is performed to assess identity between two amino acid sequences. Identity determines the percentage of amino acids with a direct match in an alignment.

Pharmaceutical composition containing an antibody as described above

Another embodiment of the invention relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in the treatment or prevention of disease symptoms selected from the group of nausea, headache, muscle pain, back pain, tremor, vomiting, or for use in the treatment or prevention of diseases characterized by symptoms such as migraine in a patient according to any of the preceding embodiments for use in combination with known anti-nausea drugs.

Another embodiment of the invention relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in the treatment or prevention of disease symptoms selected from the group of nausea, headache, muscle pain, back pain, tremors, vomiting, or for use in the treatment or prevention of diseases characterized by symptoms such as migraine in a patient, according to any of the preceding embodiments, for use in combination with known headache drugs.

Another embodiment of the invention relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in the treatment or prevention of disease symptoms selected from the group of nausea, headache, muscle pain, back pain, tremors, vomiting, or for use in the treatment or prevention of diseases characterized by symptoms such as migraine in a patient according to any of the preceding embodiments for use in combination with known anti-myalgia drugs.

Another embodiment of the invention relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in the treatment or prevention of disease symptoms selected from the group of nausea, headache, muscle pain, back pain, tremor, vomiting, or for use in the treatment or prevention of diseases characterized by such symptoms, such as migraine, in a patient, according to any of the preceding embodiments, for use in combination with known anti-tremor drugs.

Another embodiment of the invention relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in the treatment or prevention of disease symptoms selected from the group of nausea, headache, muscle pain, back pain, tremor, vomiting, or for use in the treatment or prevention of diseases characterized by symptoms such as migraine in a patient, according to any of the preceding embodiments, for use in combination with known antiemetic drugs.

Another embodiment of the invention relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in the treatment or prevention of disease symptoms selected from the group of nausea, headache, muscle pain, back pain, trembling, vomiting, or for use in the treatment or prevention of diseases characterized by symptoms such as migraine in a patient, according to any of the preceding embodiments, for use in combination with known drugs against back pain.

Another embodiment of the invention relates to a pharmaceutical composition for use in the treatment or prevention of symptoms of a disease selected from the group of nausea, headache, muscle pain, back pain, tremors, vomiting, or for use in the treatment or prevention of diseases characterized by such symptoms, such like migraine, in a patient containing an antibody or fragment or scaffold according to any of the preceding embodiments.

Another embodiment of the invention relates to a pharmaceutical composition for use in the treatment or prevention of symptoms of a disease selected from the group of nausea, headache, muscle pain, back pain, tremors, vomiting, or for use in the treatment or prevention of diseases characterized by such symptoms, such like migraine, in a patient according to the preceding embodiment, wherein said pharmaceutical composition is a solution, preferably a ready-to-use solution.

Another embodiment of the invention relates to a pharmaceutical composition for use in the treatment or prevention of symptoms of a disease selected from the group of nausea, headache, muscle pain, back pain, tremors, vomiting, or for use in the treatment or prevention of diseases characterized by such symptoms, such as migraine, in an individual according to the preceding embodiment, wherein said pharmaceutical composition is in a lyophilized state.

Another embodiment of the invention relates to a pharmaceutical composition for use in the treatment or prevention of symptoms of a disease selected from the group of nausea, headache, muscle pain, back pain, tremors, vomiting, or for use in the treatment or prevention of diseases characterized by such symptoms, such like migraine, in a patient, in accordance with any of the previous embodiments regarding such compositions, where the specified pharmaceutical composition is administered intramuscularly.

Another embodiment of the invention relates to a pharmaceutical composition for use in the treatment or prevention of symptoms of a disease selected from the group of nausea, headache, muscle pain, back pain, tremors, vomiting, or for use in the treatment or prevention of diseases characterized by such symptoms, such like migraine, in a patient, in accordance with any of the previous embodiments regarding such compositions, where the specified pharmaceutical composition is administered intravascularly.

Another embodiment of the invention relates to a pharmaceutical composition for use in the treatment or prevention of symptoms of a disease selected from the group of nausea, headache, muscle pain, back pain, tremors, vomiting, or for use in the treatment or prevention of diseases characterized by such symptoms, such as migraine, in an individual according to the preceding embodiment, wherein said pharmaceutical composition is administered by infusion.

Another embodiment of the invention relates to a pharmaceutical composition for use in the treatment or prevention of symptoms of a disease selected from the group of nausea, headache, muscle pain, back pain, tremors, vomiting, or for use in the treatment or prevention of diseases characterized by such symptoms, such as migraine, in an individual according to any of the preceding embodiments relating to such formulations, wherein said pharmaceutical formulation is to be administered systemically.

As used herein, the term "disease index" refers to LPS-induced clinical symptoms, including nausea, headache, muscle pain, back pain, tremors, and vomiting. These symptoms have been combined into one index (=disease index): as an example, each symptom is scored on a scale of 0 to 5, with the exception of vomiting (either 0 or 3, depending on the presence of vomiting in the last 30 minutes). The disease index is the sum of all symptoms (theoretical range is 0 to 28).

It should be emphasized that the embodiments described herein regarding an anti-ADM antibody, or an anti-ADM antibody fragment, or an anti-ADM non-Ig scaffold, are intended to be used to treat or prevent symptoms associated with a disease, and thus they are not necessarily intended for any primary or first line treatment of the most acute disease or acute condition considered to be the underlying disease(s). This means that the present invention does not relate to therapy for the cure/elimination of, for example, cancer, sepsis, septic shock, COPD, congestive heart disease (acute heart failure).

Moreover, in some embodiments, the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold is monospecific. A monospecific anti-ADM antibody, or a monospecific anti-ADM antibody fragment, or a monospecific anti-ADM non-Ig framework means that said antibody, or antibody fragment, or non-Ig framework binds to one specific region spanning at least 5 amino acids in the ADM- targets. An anti-ADM monospecific antibody, or a monospecific anti-ADM antibody fragment, or a monospecific anti-ADM non-Ig scaffold are anti-ADM antibodies, or anti-ADM antibody fragments, or anti-ADM non-Ig scaffolds, all of which have affinity for the same same antigen.

In a specific and preferred embodiment, the present invention relates to a monospecific anti-ADM antibody, or a monospecific fragment of an anti-ADM antibody, or a monospecific anti-ADM non-Ig framework, characterized in that said antibody, or antibody fragment, or non-Ig framework binds to one a specific region spanning at least 4 amino acids in the ADM target. In another specific embodiment, the anti-ADM antibody or ADM-binding antibody fragment is a monospecific antibody. "Monospecific" means that said antibody or antibody fragment binds to one specific region, preferably spanning at least 4 or at least 5 amino acids in the ADM target. All of the monospecific antibodies or fragments are antibodies or fragments all of which have affinity for the same antigen. Monoclonal antibodies are monospecific, however, monospecific antibodies can also be obtained by methods other than producing them from a common germ cell.

An antibody according to the present invention is a protein comprising one or more polypeptides substantially encoded by immunoglobulin genes that specifically binds an antigen. Recognized immunoglobulin genes include the kappa, lambda, alpha (IgA), gamma (IgG1, IgG2, IgG3, IgG4), delta (IgD), epsilon (IgE), and mu (IgM) constant region genes, as well as a variety of immunoglobulin variable region genes. Full-length immunoglobulin light chains typically have a mass of approximately 25 kDa or a length of approximately 214 amino acids. Full-length immunoglobulin heavy chains typically have a mass of approximately 50 kDa or a length of approximately 446 amino acids. Light chains are encoded by a variable region gene at the NH ₂ -terminus (approximately 110 amino acids in length) and a kappa or lambda constant region gene at the COOH-terminus. Heavy chains are similarly encoded by the variable region gene (approximately 116 amino acids in length) and one of the other constant region genes.

The basic structural unit of an antibody is usually a tetramer, which consists of two identical pairs of immunoglobulin chains, each pair having one light and one heavy chain. In each pair, the light and heavy chain variable regions bind to the antigen, and the constant regions mediate effector functions. Immunoglobulins also exist in various other forms, including, for example, Fv, Fab, and (Fab')2, as well as bifunctional hybrid antibodies and single chains (e.g., Lanzavecchia et al. 1987. Eur. J. Immunol. 17:105; Huston et al 1988 PNAS 85:5879-5883 Bird et al 1988 Science 242:423-426 Hood et al 1984 Immunology, Benjamin, NY, 2nd ed., Hunkapiller and Hood 1986 Nature 323:15 -16 ). The variable region of an immunoglobulin light or heavy chain comprises a framework region interrupted by three hypervariable regions, also called complementarity determining regions (CDRs) (see, Sequences of Proteins of Immunological Interest, E. Kabat et al, US Department of Health and Human Services, 1983 ). As noted above, CDRs are primarily responsible for binding to an antigen epitope. An immune complex is an antibody, such as a monoclonal antibody, a chimeric antibody, a humanized antibody, or a human antibody, or a functional fragment of an antibody that specifically binds to an antigen.

Chimeric antibodies are antibodies whose light and heavy chain genes are constructed, usually by genetic engineering, from immunoglobulin variable and constant region genes belonging to different species. For example, variable gene segments from a mouse monoclonal antibody can be linked to human constant segments such as kappa and gamma 1 or gamma 3. In one example, the therapeutic chimeric antibody is thus a fusion protein consisting of a variable or antigen-binding domain from a mouse antibody. and a constant or effector domain from a human antibody, although other mammalian species may be used, or the variable region may be generated by molecular methods. Methods for making chimeric antibodies are well known in the art, for example, see US Patent No. 5,807,715. ). A non-human immunoglobulin providing a CDR is referred to as a "donor", and a human immunoglobulin providing a framework region is referred to as an "acceptor". In one embodiment, in a humanized immunoglobulin, all CDRs are from a donor immunoglobulin. The constant regions need not be present, however, if present, they must be substantially identical to human immunoglobulin constant regions, ie. at least about 85-90%, for example, about 95% or more identical. Thus, all parts of a humanized immunoglobulin, with the possible exception of the CDR, are essentially identical to the corresponding parts of natural human immunoglobulin sequences. A "humanized antibody" is an antibody containing a humanized light chain and a humanized heavy chain. The humanized antibody binds to the same antigen as the donor antibody that provides the CDR. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions for amino acids taken from the donor framework. Humanized or other monoclonal antibodies may have additional conservative amino acid substitutions that have essentially no effect on antigen binding or other immunoglobulin functions. Illustrative conservative substitutions are substitutions such as gly, ala; val, ile, leu; asp, glu; asn, gin; ser, thr; lys, arg; and phe, tyr. Humanized immunoglobulins can be engineered by genetic engineering (eg, see US Pat. No. 5,585,089). A human antibody is an antibody where the light and heavy chain genes are derived from a human. Human antibodies can be obtained using methods known in the art. Human antibodies can be obtained by immortalizing a human B cell secreting the antibody of interest. Immortalization can be carried out, for example, by infection with EBV or by fusing human B cells with a myeloma or hybridoma cell to obtain a trioma cell. Human antibodies can also be generated by phage display methods (see, for example, Dower et al., PCT Publication No. WO 91/17271; McCafferty et al., PCT Publication No. WO92/001047; and Winter, PCT Publication No. WO 92/20791) , or choose from combinatorial human monoclonal antibody libraries (see the Morphosys website). Human antibodies can also be generated using transgenic animals containing the human immunoglobulin gene (eg, see PCT Publication No. WO 93/12227; and PCT Publication No. WO 91/10741).

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

In addition to anti-ADM antibodies, biopolymer scaffolds that complex with a target molecule are well known in the art and are used to produce highly target-specific biopolymers. Examples are aptamers, spiegelmers, anticalins and conotoxins. For an illustration of antibody formats, see figures la, lb and lc in WO 2013/072513.

An antibody fragment of the present invention is an antigen-binding fragment of an antibody of the present invention.

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

Non-Ig scaffolds can be protein scaffolds and can be used as antibody mimetics because they are able to bind to ligands or antigens. Non-Ig scaffolds can be selected from the group consisting of tetranectin-based non-Ig scaffolds (e.g., described in US 2010/0028995), fibronectin-based scaffolds (e.g., described in EP 1266 025; lipocalin-based scaffolds ((e.g., described in WO 201 1/154420); ubiquitin-based scaffolds (e.g., described in WO 2011/073214), transferrin-based scaffolds (e.g., described in US 2004/0023334), protein A-based scaffolds (e.g., described in EP 2231860), ankyrin repeat scaffolds (e.g. described in WO 2010/060748), scaffolds based on microproteins, preferably cystine knot-forming microproteins) (e.g. described in EP 2314308), Fyn SH3 domain scaffolds (e.g. described in WO 2011/023685), EGFR A-domain-based frameworks (eg as described in WO 2005/040229) and marten-domain-based frameworks (eg as described in EP 1941867).

In one embodiment, antibodies according to the present invention can be prepared as follows: a Balb/c mouse is immunized with 100 µg of ADM-peptide-BSA conjugate on days 0 and 14 (emulsified in 100 µl of Freund's complete adjuvant) and 50 µg on days 21 and 28 day (in 100 µl of incomplete Freund's adjuvant). Three days prior to the fusion experiment, the animal was given 50 μg of the conjugate dissolved in 100 μl of saline administered as one intraperitoneal and one intravenous injection. Splenocytes from an immunized mouse and SP2/0 myeloma cell line were fused with 1 ml of 50% polyethylene glycol for 30 s at 37°C. After washing, the cells were plated in 96-well cell culture plates. Hybrid clones were selected by growing in HAT medium (RPMI 1640 culture medium supplemented with 20% fetal calf serum and HAT supplement). Two weeks later, HAT medium was replaced with HT medium for three passages and then returned to normal cell culture medium. Cell structure supernatants were first screened for antigen-specific IgG antibodies three weeks after fusion. Positive microcultures were transferred to 24-well plates to increase in numbers. After retesting, selected cultures were cloned and recloned using the limiting dilution method and isotyped (also see Lane, RD 1985. J. Immunol. Meth. 81: 223-228; Ziegler B. et al. l996. Horm. Metab. Res. 28:11-15 ).

Antibodies can also be generated by phage display using the following procedure: HAL7/8 naive human antibody gene libraries were used to isolate recombinant single chain F variable domains (scFv) against the ADM peptide. The antibody gene libraries were screened using a panning strategy involving the use of peptides containing a biotin tag linked via two different spacers to an ADM peptide sequence. A combination of panning rounds using non-specific bound antigen and streptavidin-bound antigen was used to minimize the background level of non-specific binding molecules. The eluted passages after the third round of panning were used to generate E. coli strains expressing the monoclonal scFv. Supernatants from culture of these clonal strains were directly used for testing using antigen ELISA (see references cited in WO 2013/072513 incorporated herein in its entirety).

Humanization of mouse antibodies can be carried out according to the following procedure: to humanize an antibody derived from a mouse, the antibody sequence is analyzed for the structural interaction of framework regions (FRs) with complementarity determining regions (CDRs) and antigen. Based on structural modeling, a suitable human-derived FR is selected and mouse CDR sequences are transplanted into human FR. Changes can be made to the amino acid sequence of a CDR or FR to restore structural interactions that have been eliminated by species switching of FR sequences. This reconstruction of structural interactions can be done by a random approach using phage display libraries or by a directed approach using molecular modeling ( Almagro and Fransson 2008. Front Biosci. 13:1619-33 ) in a preferred embodiment, the anti-ADM antibody format is selected from the group , including Fv-fragment, scFv-fragment, Fab-fragment, scFab-fragment, F(ab)2-fragment and scFv-Fc fusion protein. In another preferred embodiment, the antibody format is selected from the group consisting of scFab fragment, Fab fragment, scFv fragment and its bioavailability optimized conjugates such as pegylated fragments. One of the most preferred formats is the scFab format. In another preferred embodiment, the anti-ADM antibody, anti-ADM antibody fragment, or anti-ADM non-Ig scaffold is a full-length antibody, antibody fragment, or non-Ig scaffold.

In a preferred embodiment, the anti-ADM antibody, or anti-ADM antibody fragment, or non-ADM Ig scaffold is directed to and can bind to an epitope of at least 5 amino acids in length located in the ADM.

In another preferred embodiment, the anti-ADM antibody, or anti-ADM antibody fragment, or non-Ig anti-ADM framework is directed to and can bind to an epitope of at least 4 amino acids in length located in the ADM.

In one particular embodiment, an anti-ADM antibody or anti-ADM antibody fragment that binds to ADM, or an anti-ADM antibody that binds to ADM, is provided for use in the treatment of an acute disease or acute condition of a patient, wherein said antibody or fragment, or the scaffold is not ADM-binding protein 1 (complement factor H). The acute disease or acute condition may be a migraine.

In one specific embodiment, an anti-ADM antibody or anti-ADM antibody fragment that binds to ADM, or an anti-ADM antibody that binds to ADM, is provided for use in the treatment of an acute disease or acute condition of a patient, wherein said antibody or antibody fragment , or non-Ig framework binds to a region of preferably at least 4 or at least 5 amino acids in the a.k.1-42 sequence of mature human ADM.

The acute disease or acute condition described above may be a headache, preferably a primary headache, most preferably a migraine.

In one specific embodiment, an anti-ADM antibody or anti-ADM antibody fragment that binds to ADM, or an anti-ADM antibody that binds to ADM, is provided for use in the treatment of an acute disease or acute condition of a patient, wherein said antibody or fragment, or the framework binds to a region preferably of at least 4 or at least 5 amino acids in the sequence a.k. 1-21 of mature human ADM:

YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO:: 4).

The acute disease or acute condition described above may be a migraine.

In a preferred embodiment of the present invention, said anti-ADM antibody, or anti-ADM antibody fragment, or anti-ADM non-Ig scaffold binds to an ADM region that is located at the N-terminus (amino acids 1-21) of the ADM.

In another preferred embodiment, said anti-ADM antibody, or anti-ADM antibody fragment, or anti-ADM non-Ig scaffold recognizes and binds the N-terminus (a.k. 1) of ADM.

The N-terminus means that amino acid 1, which is "Y" of SEQ ID NO:1 or 4, is required for antibody binding. Said antibody or fragment or non-Ig scaffold binds neither N-terminally extended, N-terminally modified ADM, nor N-terminal disrupted ADM.

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

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

In one particular embodiment, it is preferred to use an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold according to the present invention, wherein said anti-ADM antibody or said anti-ADM antibody fragment or non-ADM anti-Ig scaffold is is an ADM stabilizing antibody, or an ADM stabilizing antibody fragment, or an ADM stabilizing non-Ig scaffold that increases the half-life (t _1/2 ; half residence time) of ADM in serum, blood, plasma by at least 10%, preferably at least at least 50%, more preferably >50%, most preferably >100%. The half-life (half residence time) of ADM can be determined in human plasma in the absence and presence of an ADM stabilizing antibody, or an ADM stabilizing fragment, or an ADM stabilizing non-Ig scaffold, respectively, using an immunoassay to quantify ADM.

The following steps can be carried out:

- ADM can be diluted in human citrated plasma in the absence and presence of an ADM stabilizing antibody, or an ADM stabilizing antibody fragment, or an ADM stabilizing non-IG scaffold, respectively, and can be incubated at 24°C;

- Spend taking aliquots at selected time points (for example, within 24 hours), and the degradation of ADM can be stopped in these aliquots by freezing at -20°C;

- The amount of ADM can be determined using the hADM immunoassay directly if the stabilizing antibody does not interfere with the selected assay. Alternatively, an aliquot can be treated with denaturing agents (such as HCI) and after sample purification (eg, centrifugation), the pH can be neutralized and ADM quantified using an ADM immunoassay. Alternatively, non-immunoassay technologies (eg, RP-HPLC) can be used to quantify ADM.

- ADM half-life is calculated for ADM incubated in the absence and presence of an ADM stabilizing antibody, or an ADM stabilizing antibody fragment, or an ADM stabilizing non-IG scaffold, respectively.

- Calculate the increase in half-life for stabilized ADM compared to ADM that was incubated in the absence of an ADM stabilizing antibody, or an ADM stabilizing antibody fragment, or an ADM stabilizing non-Ig scaffold.

A two-fold increase in ADM half-life represents a 100% increase in half-life.

Half-life (half residence time) is defined as the period required to reduce the concentration of a particular chemical or drug to half its original concentration in said fluid or blood.

In a particular embodiment, said anti-ADM antibody, anti-ADM antibody fragment, or non-Ig anti-ADM framework is a non-neutralizing antibody, fragment, or non-Ig framework. An anti-ADM neutralizing antibody, an anti-ADM antibody fragment, or a non-ADM Ig scaffold can block the biological activity of ADM by up to 100%, at least more than 90%, preferably at least more than 95%.

In contrast, a non-neutralizing anti-ADM antibody or anti-ADM antibody fragment or non-ADM Ig scaffold blocks the biological activity of ADM by less than 100%, preferably less than 95%, preferably less than 90%, more preferably less than 80% and even more preferably less than 50%. This means that the residual biological activity of ADM bound to a non-neutralizing anti-ADM antibody or an anti-ADM antibody fragment or a non-ADM Ig scaffold can be greater than 0%, preferably greater than 5%, preferably greater than 10%, more preferably greater than 20 %, more preferably more than 50%. In this context, a molecule(s) that is an antibody, or antibody fragment, or non-Ig scaffold with "non-neutralizing" anti-ADM activity, collectively referred to herein for simplicity as a "non-neutralizing" anti-ADM antibody, antibody fragment, or non- Ig scaffolds that, for example, block the biological activity of ADM by less than 80%, are defined as an ADM-binding molecule or molecules that, when added to a culture of a eukaryotic cell line that expresses a functional recombinant human ADM receptor, consisting of CRLR (a receptor calcitonin receptor-like) and RAMP3 (receptor activity-modifying protein 3) reduce the amount of cAMP produced by the cell line by acting on the addition of a parallel added synthetic human ADM peptide, wherein said added synthetic human ADM is added in an amount that in the absence of a non-neutralizing antibody , to be analyzed, leads to half-maximal stimulation of cAMP synthesis, wherein the reduction of cAMP by said ADM-binding molecule(s) occurs to a degree that does not exceed 80%, even when the non-neutralizing ADM-binding molecule(s) to be analyzed is added in an amount that 10 times the amount required to achieve the maximum reduction in cAMP synthesis achieved using the non-neutralizing antibody to be analyzed. The same definition applies to other ranges; 95%, 90%, 50%, etc.

In a specific embodiment according to the present invention, an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold is used, wherein said antibody or antibody fragment or non-Ig scaffold blocks the biological activity of ADM by less than 80%, preferably less than 50% (from the initial values). This means blocking circulating ADM by no more than 80% or no more than 50%, respectively. It is understood that this limited blocking of the biological activity of ADM occurs even at an excess concentration of antibody, antibody fragment or non-Ig framework, which means an excess of antibody, antibody fragment or non-Ig framework relative to ADM. This limited blocking is a property inherent in the binding ADM connection itself. This means that said antibody, antibody fragment, or non-Ig scaffold has a maximum inhibition of 80% or 50%, respectively. As a consequence, this means that 20% or 50% residual biological activity of ADM remains, although proper amounts or excess amounts of antibody, antibody fragment, or non-Ig scaffold are administered, respectively.

In a preferred embodiment, said anti-ADM antibody, anti-ADM antibody fragment, or anti-ADM non-Ig scaffold can block the biological activity of ADM by at least 5%. As a consequence, this means that there remains a residual 95% biological activity of circulating ADM. This is the lower threshold of biological activity remaining after administration of said anti-ADM antibody, anti-ADM antibody fragment, or non-Ig anti-ADM framework. Biological activity is defined as the effect that a substance has on a living organism or tissue or organ or functional unit in vivo or in vitro (eg in an assay) following its interaction. In the case of biological activity of ADM, this may represent the effect of ADM in a functional assay with cAMP with the recombinant human ADM receptor. Thus, in accordance with the present invention, biological activity is determined by functional analysis of cAMP with the ADM receptor. The following steps can be carried out to determine the biological activity of ADM in such an assay:

- Dose-response curves are built using ADM in the specified cAMP functional assay with recombinant human ADM receptor.

- It is possible to calculate the ADM concentration at half-maximal cAMP stimulation.

- At constant half-maximal cAMP-stimulating concentrations of ADM, dose-response curves (up to a final concentration of 100 μl) are plotted with ADM stabilizing antibody or ADM stabilizing antibody fragment or ADM stabilizing non-Ig scaffold, respectively.

The maximum (at maximum dose) inhibition by said ADM stabilizing antibody of 50% means that said anti-ADM antibody, or said anti-ADM antibody fragment, or said anti-ADM non-Ig framework, respectively, blocks the biological activity by 50% of initial values. A maximum inhibition in said ADM bioassay of 80% means that said anti-ADM antibody, or said anti-ADM antibody fragment, or said anti-ADM non-Ig framework, respectively, blocks the biological activity of ADM by 80%. This means blocking the biological activity of ADM by no more than 80%.

In a preferred embodiment, a modulating anti-ADM antibody, or a modulating fragment of an anti-ADM antibody, or a modulating anti-ADM non-Ig scaffold is used. A "modulatory" anti-ADM antibody, or modulating anti-ADM antibody fragment, or modulating anti-ADM non-Ig scaffold is an antibody, or anti-ADM antibody fragment, or non-Ig scaffold that increases the half-life (half residence time t) of ADM in serum, blood, plasma by at least 10%, preferably at least 50%, more preferably >50%, most preferably >100%, and block the biological activity of ADM by less than 80%, preferably less than 50% and wherein said anti-ADM antibody, anti-ADM antibody fragment, or anti-ADM non-Ig scaffold can block the biological activity of ADM by at least 5%. These values associated with the half-life and blocking of biological activity must be taken in relation to the above methods of analysis to determine these values. This means blocking circulating ADM by no more than 80% or no more than 50%, respectively. This means that 20% residual biological activity of ADM remains, or 50% residual biological activity of ADM remains, respectively. Such a modulating anti-ADM antibody, or a modulating anti-ADM antibody fragment, or a modulating anti-ADM non-Ig scaffold provides the advantage of facilitating dosing upon administration. The combination of partially blocking or partially reducing ADM biological activity and increasing in vivo half-life (increasing ADM biological activity) results in useful ease of dosing of the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold. In the case of an excess of endogenous ADM, the activity-reducing effect is the main action of the antibody, or fragment, or scaffold, limiting the (negative) effect of ADM. In the case of low or normal concentrations of endogenous ADM, the biological effect of the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold is a combination of reduction (by partial blocking) and increase by increasing the half-life of ADM. Thus, a non-neutralizing and modulating anti-ADM antibody, or an anti-ADM antibody fragment, or a non-Ig anti-ADM scaffold acts like an ADM biological activity buffer to keep the ADM biological activity within a defined physiological range.

The anti-ADM antibody or anti-ADM antibody fragment or non-ADM Ig scaffold of the present invention exhibits an affinity for human ADM that exhibits an affinity constant greater than 10 ^-7 M, preferably 10 ^-8 M, preferably greater than 10 ^-9 M, most preferably above 10 ^-10 M. It is known to those skilled in the art that lower affinity can be compensated for by a higher dose of the compound, and that this measure is within the scope of the invention. Affinity constants can be determined in the manner described in Example 1 of WO 2013/072513.

It should be emphasized that the term "ADM binding protein" includes ADM binding protein 1 (complement factor H). However, said ADM binding protein of the invention is neither a non-neutralizing anti-ADM antibody/antibody fragment/non-Ig framework nor a modulating anti-ADM antibody/antibody fragment/non-Ig framework. In addition, the present invention provides an anti-ADM antibody or anti-ADM antibody fragment or non-Ig anti-ADM scaffold for use in the treatment of an acute disease or acute condition in a patient according to the present invention, wherein said antibody or antibody fragment, or the non-Ig scaffold can be used in combination with additional active ingredients.

In addition, the present invention relates to a pharmaceutical composition containing an anti-ADM antibody, or an anti-ADM antibody fragment, or a non-Ig anti-ADM scaffold according to the present invention. In addition, the present invention relates to a pharmaceutical composition according to the present invention, where the specified pharmaceutical composition is a solution, preferably a ready-to-use solution. In another embodiment, the present invention further relates to a pharmaceutical composition according to the present invention, wherein said pharmaceutical composition is in a dried state for reconstitution prior to use. Said pharmaceutical composition may be administered intramuscularly. Said pharmaceutical composition may be administered intravascularly. Said pharmaceutical formulation may be administered by infusion. In another embodiment, the present invention further relates to a pharmaceutical composition according to the present invention, wherein said pharmaceutical composition is in a lyophilized state.

In another more preferred embodiment, the present invention provides a pharmaceutical composition comprising an anti-ADM antibody or an anti-ADM antibody fragment that binds to ADM, or an anti-ADM antibody that binds to ADM, for use in the treatment or prevention of symptoms of a disease in a patient, wherein said pharmaceutical composition is administered to a patient in need thereof.

In another embodiment of the present invention, the pharmaceutical composition in accordance with the present invention is administered to a patient for the treatment and/or prevention of symptoms associated with a disease, as defined above, provided that said patient is in need of such treatment.

In one embodiment, the anti-ADM antibody or anti-ADM antibody fragment or non-IG anti-ADM framework of the invention is a non-neutralizing anti-ADM antibody or non-neutralizing anti-ADM antibody fragment or non-neutralizing non-Ig anti-ADM framework.

As used herein, the antibody format is selected from the group consisting of Fv fragment, scFv fragment, Fab fragment, scFab fragment, (Fab)2 fragment, and scFv-Fc fusion protein.

In some embodiments, an anti-ADM antibody, or an anti-ADM antibody fragment, or a non-Ig scaffold according to any of the preceding embodiments is used to treat a patient in need thereof, wherein said antibody or fragment can be administered at a dose of at least 0 .5 mg/kg of body weight, in particular, at least 1.0 mg/kg of body weight, more specifically, from 1.0 to 20.0 mg/kg of body weight, for example, from 2.0 to 10 mg/ kg body weight, 2.0 to 8.0 mg/kg body weight, or 2.0 to 5.0 mg/kg body weight.

In embodiments of the present invention, the symptoms of the disease referred to herein are not associated with any of the diseases selected from the group including sepsis, diabetes, malignancy, heart failure (acute heart failure) and septic shock.

In preferred embodiments of the present invention, the symptoms of the disease treated or prevented using any of the anti-ADM antibody or anti-ADM antibody fragment or non-Ig framework of any of the preceding embodiments are associated with migraine.

In preferred embodiments of the present invention, the symptoms of the disease treated or prevented using any of the anti-ADM antibody or anti-ADM antibody fragment or non-Ig scaffold of any of the preceding embodiments are associated with viral infections, wherein said viruses are selected from the group including hepadnaviridae, adenoviridae, herpesviridae, influenza viruses, arenaviridae, filoviridae, togaviridae, noroviruses, flaviviridae, retroviridae, measles virus, reoviridae, enteroviridae, picornaviridae, caliciviridae, etc.

In preferred embodiments of the present invention, the symptoms of the disease treated or prevented using any of the anti-ADM antibody or anti-ADM antibody fragment or non-Ig scaffold of any of the preceding embodiments are associated with medical treatment of the primary diseases, such as chemotherapy, therapy with biological agents (for example, antibodies or their fragments), antibiotics, or any medication that causes any of the above symptoms of the disease.

Further preferred embodiments of the present invention relate to methods of therapy (e.g., treatment, cure, amelioration, amelioration, amelioration, etc.) or prevention of symptoms as defined in any of the above embodiments, comprising administering to an individual in need thereof an antibody anti-ADM, or an anti-ADM antibody fragment, or a non-Ig scaffold according to any of the preceding embodiments. Preferably the subject is a human.

Administration of the anti-ADM antibody, or anti-ADM antibody fragment, or non-Ig scaffold can be by any of the methods known in the art, including: oral, intravenous, subcutaneous, intra-arterial, intramuscular, intracardiac, intravertebral, intrathoracic, intraperitoneal, intraventricular, sublingual , transdermal and / or inhalation. The administration may be systemic, eg intravenous or localized.

In one aspect, the present invention relates to a method for treating or preventing headache, preferably primary headache, more preferably migraine, in a subject, comprising administering to the subject an effective amount of an anti-ADM antibody, or an anti-ADM antibody fragment, or a non-Ig scaffold.

In a further embodiment, the invention relates to methods of alleviating, controlling, reducing the frequency or slowing the development or progression of headache, preferably primary headache, more preferably migraine, in an individual, comprising administering to the individual an effective amount of an anti-ADM antibody, or an anti-ADM antibody fragment, or not -Ig scaffold.

The anti-ADM antibody, or anti-ADM antibody fragment, or non-Ig scaffold can be administered before, during and/or after the headache. In some embodiments, the anti-ADM antibody, or anti-ADM antibody fragment, or non-Ig scaffold is administered prior to the headache attack.

In some embodiments, the anti-ADM antibody, or anti-ADM antibody fragment, or non-Ig scaffold can be co-administered with another agent, such as another headache treatment.

"Development" or "progression" of a headache means the initial manifestations and/or subsequent progression of a disorder. Headache progression can be determined and evaluated using standard clinical methods well known in the art. However, development also refers to progression, which may not be detectable. For the purpose of the present invention, development or progression refers to the biological course of symptoms. "Development" includes emergence, recurrence, and manifestation. As used herein, "manifestation" or "onset" of a headache includes initial manifestation and/or recurrence.

As used herein, an "effective dosage" or "effective amount" of a drug, compound, or pharmaceutical composition is an amount sufficient to achieve beneficial or desired results. For prophylactic use, useful or desirable results include results such as elimination or reduction of risk, reduction in severity or delay in the onset of a disease, including biochemical, histological and/or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes present during the course of the disease. For therapeutic use, useful or desirable results include clinical outcomes such as reduction in pain intensity, duration or frequency of headache attacks, and reduction in one or more headache symptoms (biochemical, histological and/or behavioral), including its complications and intermediate pathological phenotypes. , manifested during the development of the disease, improving the quality of life of those suffering from the disease, reducing the dose of other drugs required to treat the disease, enhancing the effect of another drug and / or slowing down the progression of the disease in patients. An effective dosage may be administered in one or more administrations. For the purposes of the present invention, an effective dosage of a drug, compound, or pharmaceutical composition is an amount sufficient to effect prophylactic or therapeutic treatment, either directly or indirectly. As understood in the clinical context, an effective dosage of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an "effective dosage" may be considered in the context of the administration of one or more therapeutic agents, and the administration of a single agent may be considered to be administered in an effective amount if, in conjunction with one or more other agents, the desired result can be or is achieved.

The present invention includes the following embodiments:

1. An anti-adrenomedullin (ADM) antibody, or anti-adrenomedullin antibody fragment, or anti-ADM non-Ig scaffold for use in the treatment or prevention of symptoms of a disease or for use in the treatment or prevention of a disease characterized by such symptoms in a patient, wherein said antibody, or a fragment or framework binds to ADM (1-52; SEQ ID NO:1) or a fragment thereof.

2. An anti-adrenomedullin (ADM) antibody, or an anti-adrenomedullin antibody fragment, or an anti-ADM non-Ig scaffold, for use in the treatment or prevention of symptoms of a disease, or for use in the treatment or prevention of a disease characterized by such symptoms in a patient, in accordance with regulation 1 wherein said antibody or fragment or scaffold binds to the N-terminal portion (a.k. 1-21) of ADM:

YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO:4).

3. An anti-adrenomedullin (ADM) antibody, or an anti-ADM antibody fragment that binds to ADM, or an anti-ADM non-Ig scaffold that binds to adrenomedullin, for use in the therapy or prevention of symptoms of a disease, or for use in the therapy or prevention of a disease characterized by: symptoms, in a patient, according to any of positions 1-2, characterized in that the specified antibody, antibody fragment, or non-Ig frame binds to a part of the middle region (MR) ADM, consisting of a.to. 21-42 ADM:

CTVQKLAHQIYQFTDKDKDNVA (SEQ ID NO:3).

4. An anti-adrenomedullin (ADM) antibody, or an anti-ADM antibody fragment that binds to ADM, or an anti-ADM non-Ig scaffold that binds to adrenomedullin, for use in the therapy or prevention of symptoms of a disease, or for use in the therapy or prevention of a disease characterized by: symptoms, in a patient, in accordance with any of provisions 1-3, where the symptoms of the disease are selected from the group of nausea, headache, muscle pain, back pain, trembling and/or vomiting.

5. An anti-adrenomedullin (ADM) antibody, or an anti-ADM antibody fragment that binds to ADM, or an anti-ADM non-Ig scaffold that binds to adrenomedullin, for use in the treatment or prevention of symptoms of a disease, or for use in the therapy or prevention of a disease characterized by: symptoms, in accordance with any of positions 1-4, where the disease is characterized by the amount of C-reactive protein (CRP) in the sample, which is ≥ 10 mg/l, and/or where the amount of TNF in the sample is ≥ 50 pg/ml, and /or where the amount of bio-ADM in the sample is ≥ 43 pg/ml, and/or where the mean arterial pressure in said individual in need thereof is reduced.

6. An anti-adrenomedullin (ADM) antibody, or an anti-ADM antibody fragment that binds to ADM, or an anti-ADM non-Ig scaffold that binds to adrenomedullin, for use in the treatment or prevention of symptoms of a disease, or for use in the therapy or prevention of a disease characterized by: symptoms, in a patient, in accordance with any one of provisions 1-5, where the disease in a patient in need of therapy and / or prevention of such symptoms or diseases is selected from the group of indications, including inflammatory conditions, autoimmune diseases, metabolic diseases, diseases of the brain , cardiovascular diseases and drug-induced diseases.

7. An anti-adrenomedullin (ADM) antibody, or an anti-ADM antibody fragment that binds to ADM, or an anti-ADM non-Ig scaffold that binds to adrenomedullin, for use in the treatment or prevention of symptoms of a disease, or for use in the therapy or prevention of a disease characterized by: symptoms, in a patient, in accordance with any of provisions 1-5, where the disease is a migraine.

8. Anti-adrenomedullin (ADM) antibody, or anti-adrenomedullin antibody fragment, or anti-ADM non-Ig scaffold for use in the treatment or prevention of symptoms of a disease or for use in the treatment or prevention of a disease characterized by such symptoms in a patient, according to any from positions 1-7, wherein said antibody or antibody fragment or non-Ig scaffold is monospecific.

9. Anti-adrenomedullin (ADM) antibody, or anti-adrenomedullin antibody fragment, or anti-ADM non-Ig scaffold, for use in the treatment or prevention of symptoms of a disease or for use in the treatment or prevention of a disease characterized by such symptoms in a patient, according to any from positions 1-8, wherein said antibody or fragment or framework exhibits an ADM binding affinity of at least 10 ^-7 M by unlabeled surface plasmon resonance using the Biacore 2000 system.

10. Anti-adrenomedullin (ADM) antibody, or anti-adrenomedullin antibody fragment, or anti-ADM non-Ig scaffold, for use in the treatment or prevention of symptoms of a disease or for use in the treatment or prevention of a disease characterized by such symptoms in a patient, according to any from positions 1-9, wherein said antibody or fragment or scaffold is not ADM-binding protein 1 (complement factor H).

11. Anti-adrenomedullin (ADM) antibody, or anti-adrenomedullin antibody fragment, or anti-ADM non-Ig scaffold for use in the treatment or prevention of symptoms of a disease or for use in the treatment or prevention of a disease characterized by such symptoms in a patient, according to any of the preceding points where the specified antibody, or fragment, or framework recognizes and binds the N-terminus (a.k. 1) ADM.

12. Anti-adrenomedullin (ADM) antibody, or anti-adrenomedullin antibody fragment, or anti-ADM non-Ig scaffold for use in the treatment or prevention of symptoms of a disease or for use in the treatment or prevention of a disease characterized by such symptoms in a patient, according to any of the preceding where said antibody or fragment or scaffold is an ADM stabilizing antibody or fragment or scaffold that increases the half-life (half residence time t1/2) of ADM in serum, blood, plasma by at least 10%, preferably at least 50%, more preferably > 50%, most preferably > 100%.

13. Anti-adrenomedullin (ADM) antibody, or anti-adrenomedullin antibody fragment, or anti-ADM non-Ig scaffold for use in the treatment or prevention of symptoms of a disease or for use in the treatment or prevention of a disease characterized by such symptoms in a patient, according to any from the preceding provisions, where the specified antibody, or fragment, or scaffold blocks the biological activity of ADM by no more than 80%, preferably no more than 50% using hADM 22-52 as a reference antagonist in CHO-K1 cells expressing the recombinant receptor human ADM.

14. Anti-adrenomedullin (ADM) antibody, or anti-adrenomedullin antibody fragment, or anti-ADM non-Ig scaffold for use in the treatment or prevention of symptoms of a disease or for use in the treatment or prevention of a disease characterized by such symptoms in a patient, according to any from the preceding provisions, where the specified individual is undergoing chemotherapy, treatment with biological agents, antibiotics or treatment with antiviral compounds.

15. Anti-adrenomedullin (ADM) antibody, or anti-adrenomedullin antibody fragment, or anti-ADM non-Ig scaffold for use in the treatment or prevention of symptoms of a disease or for use in the treatment or prevention of a disease characterized by such symptoms in a patient, according to any from the preceding provisions, where the specified antibody or fragment is a human monoclonal antibody or a fragment that binds to ADM, or a fragment thereof, where the heavy chain contains the sequences:

CDR1: SEQ ID NO:5

GYTFSRYW

CDR2: SEQ ID NO:6

ILPGSGST

CDR3: SEQ ID NO:7

TEGYEYDGFDY

and where the light chain contains the sequences:

CDR1: SEQ ID NO:8

QSIVYSNGNTY

CDR2:

RVS

CDR3: SEQ ID NO:9

FQGSHIPYT.

16. A human monoclonal antibody or fragment that binds to ADM, or an antibody fragment thereof, for use in the treatment or prevention of symptoms of a disease or for use in the therapy or prevention of a disease characterized by such symptoms in an individual according to position 15, wherein said antibody or fragment contains a sequence selected from the group including as a VH region:

SEQ ID NO:10 (AM-VH-C)

QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYKK

SEQ ID NO:11 (AM-VH1)

QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYKK

SEQ ID NO:12 (AM-VH2-E40)

QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYKK

SEQ ID NO:13 (AM-VH3-T26-E55)

QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYKK

SEQ ID NO:14 (AM-VH4-T26-E40-E55)

QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYK

and contains a sequence selected from the group containing the following sequence as the VL region:

SEQ ID NO:15 (AM-VL-C)

DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQRGDSKDSTYSLSSTLVSKADYEKHKVACE

SEQ ID NO:16 (AM-VL1)

DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESTKVTEQDSKDSPVSLSSTLVSSKAGLACEVEEKHKHK

SEQ ID NO:17 (AM-VL2-E40)

DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQRGSSKDSTYSLSSTLVNSKADYEKTHPVHKVACE

17. A human monoclonal antibody or fragment that binds to ADM, or an antibody fragment thereof, for use in the treatment or prevention of symptoms of a disease or for use in the therapy or prevention of a disease characterized by such symptoms in an individual according to position 15, wherein said antibody or fragment contains the following sequence as a heavy chain:

SEQ ID NO:22

QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGEILPGSGSTNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

or a sequence that is > 95% identical to it,

and contains the following sequence as a light chain:

SEQ ID NO:23

DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPRLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQNGSSKDSTYSLSSTLVSKADYEKHKVACE

or a sequence that is > 95% identical to it.

18. Pharmaceutical composition for use in therapy or prevention of symptoms of a disease, or for use in therapy or prevention of a disease characterized by such symptoms in a patient, containing an antibody, or fragment, or framework in accordance with any of provisions 1-17.

19. An antibody containing the following sequences as a heavy chain:

SEQ ID NO:22

QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGEILPGSGSTNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

or a sequence that is > 95% identical to it, preferably > 98%, and contains the following sequence as a light chain:

SEQ ID NO:23

DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPRLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQNGSSKDSTYSLSSTLVSKADYEKHKVACE

or a sequence that is > 95% identical to it, preferably > 98%.

20. Pharmaceutical composition containing an antibody according to position 19.

Examples

Obtaining antibodies and determining their affinity constants

Several human and mouse antibodies were prepared and their affinity constants were determined (see Table 1).

Peptides/conjugates for immunization:

Synthesized peptides for immunization, see table 1, (JPT Technologies, Berlin, Germany) with an additional N-terminal cysteine residue (if cysteine is not present in the selected ADM sequence) for conjugation of peptides with bovine serum albumin (BSA). The peptides were covalently linked to the BSA using a Sulfolink attachment tool (Perbio Science, Bonn, Germany). The attachment procedure was carried out according to the guide from Perbio.

Mouse antibodies were obtained according to the following method:

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

Splenocytes from an immunized mouse and SP2/0 myeloma cell line were fused with 1 ml of 50% polyethylene glycol for 30 s at 37°C. After washing, cells were plated in 96-well cell culture plates. Selection of hybrid clones was performed by growing in HAT medium (RPMI 1640 culture medium supplemented with 20% fetal calf serum and HAT supplement). Two weeks later, HAT medium was replaced with HT medium for three passages, and then returned to normal cell medium.

Cell structure supernatants were first screened for antigen-specific IgG antibodies three weeks after fusion. Positive microcultures were transferred to 24-well plates to increase in numbers. After retesting, selected cultures were cloned and recloned using the limiting dilution method and isotyped (also see Lane, RD 1985. J. Immunol. Meth. 81: 223-228; Ziegler B. et al. l996. Horm. Metab. Res. 28:11-15 ).

Production of monoclonal antibodies:

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

Human antibodies:

Human antibodies by phage display using the following method:

naive human antibody gene libraries HAL7/8 were used to isolate recombinant single chain F variable domains (scFv) against the ADM peptide. Antibody gene libraries were screened using a panning strategy involving the use of peptides containing a biotin tag linked through two different spacers to an ADM peptide sequence. A combination of panning rounds using non-specific bound antigen and streptavidin-bound antigen was used to minimize the background level of non-specific binding molecules. The eluted passages after the third round of panning were used to generate E. coli strains expressing the monoclonal scFv. Culture supernatants from these clonal strains were directly used for antigen ELISA testing (see also Hust et al. 2011. Journal of Biotechnology 152, 159-170; Schütte et al. 2009. PLoS One 4, e6625 ).

Positive clones were selected based on a positive ELISA signal for antigen and negative for streptavidin-coated microtiter plates. For further characterizations, the scFv open reading frame was cloned into the expression plasmid pOPE107 ( Hust et al. 2011. Journal of Biotechnology 152, 159-170 ), recovered from the culture supernatant by immobilized metal ion affinity chromatography, and purified by size exclusion chromatography.

Affinity constants:

To determine the affinity of antibodies against ADM, the kinetics of ADM binding to the immobilized antibody was determined by unlabeled surface plasma resonance using the Biacore 2000 system (GE Healthcare Europe GmbH, Freiburg, Germany). Reversible antibody immobilization was performed using an anti-mouse Fc antibody covalently bound at high density to the CM5 sensor surface according to the manufacturer's instructions (Antibody Capture Kit; GE Healthcare) ( Lorenz et al. 2011. Antimicrob Agents Chemother. 55(1): 165-173 ).

Monoclonal antibodies were raised against the human and mouse ADM regions described below, respectively. The table below shows the set of obtained antibodies used for further experiments. The selection was based on the target area:

Table 1:

Sequence number Antigen/immunogen ADM area Designation Affinity constants Kd (M) SEQ ID: 4 YRQSMNNFQGLRSFGCRFGTC 1-21 NT-H 5.9×10 ^-9 SEQ ID: 21 CTVQKLAHQIYQ 21-32 MR-H 2×10 ^-9 Amidated SEQ ID: 2 (with an additional cysteine at the N-terminus C-APRSKISPQGY-NH ₂ C-42-52 CT-H 1.1×10 ^-9 SEQ ID: 18 YRQSMNQGSRSNGCRFGTC 1-19 NT-M 3.9×10 ^-9 SEQ ID: 19 CTFQKLAHQIYQ 19-31 MR-M 4.5×10 ^-10 Amidated SEQ ID: 20 (with an additional cysteine at the N-terminus C-APRNKISPQGY-NH ₂ C-40-50 CT-M 9×10 ^-9

Obtaining antibody fragments by enzymatic digestion:

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

Method for obtaining and purifying Fab:

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

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

The immobilized pepsin was equilibrated by washing the resin with 0.5 ml of digestion buffer and centrifuging the column at 5000×g for 1 minute. After that, the buffer was discarded. The desalting column was prepared by removing the storage buffer and washing it with the digestion buffer, centrifuging it each time at 1000×g for 2 minutes. 0.5 ml of the prepared IgG sample was added to the centrifuge tube containing the equilibrated immobilized pepsin. The incubation of the digestion reaction mixture was carried out for 16 hours on a table top shaker at 37°C. The column was centrifuged at 5000×g for 1 minute to separate the digested product from the immobilized pepsin. After that, the resin was washed with 0.5 ml of PBS and centrifuged at 5000×g for 1 minute. The wash fraction was added to the digested antibody to a total sample volume of 1.0 ml. The protein A NAb column was equilibrated with PBS and IgG elution buffer at room temperature. The column was centrifuged for 1 minute to remove the storage solution (containing 0.02% sodium azide) and equilibrated by adding 2 ml of PBS, centrifuged again for 1 minute and the eluate was discarded. The sample was applied to the column and resuspended by inversion. Incubation was carried out at room temperature with stirring by turning for 10 minutes. The column was centrifuged for 1 minute, leaving the eluate with Fab fragments. (refs: Mariani et al. 1991. Mol. Immunol. 28: 69-77; Beale 1987. Exp Comp Immunol 11:287-96; Ellerson et al. 1972. FEBS Letters 24(3):318-22; Kerbel and Elliot 1983 Meth Enzymol 93:113-147 Kulkarni et al 1985 Cancer Immunol Immunotherapy 19:211-4 Lamoyi 1986 Meth Enzymol 121:652-663 Parham et al 1982 J Immunol Meth 53:133- 73 Raychaudhuri et al 1985 Mol Immunol 22(9):1009-19 Rousseaux et al 1980 Mol Immunol 17:469-82 Rousseaux et al 1983 J Immunol Meth 64:141-6 Wilson et al 1991. J Immunol Meth 138:111-9 ).

Humanization of NT-H antibody fragments:

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

To obtain a humanized sequence, the following steps were performed:

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

- First round RT-PCR: QIAGEN® OneStep RT-PCR Kit (Cat No. 210210) was used. RT-PCR was performed with a set of primers specific for heavy and light chains. For each RNA sample, 12 individual heavy chain RT-PCR reactions and 11 individual light chain RT-PCR reactions were performed using degenerate forward primer mixtures spanning the leader sequences of the variable regions. The reverse primers are located in the constant regions of the heavy and light chains. The primers did not include any restriction sites.

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

- Reaction scheme: 2x PCR mix, 10 µl; primer set, 2 µl; PCR product of the first round 8 µl; total volume 20 μl; conditions from the report on the cloning of hybridoma antibodies by PCR: initial denaturation 5 min at 95°C; 25 cycles of 95°C for 25 s, 57°C for 30 s, 68°C for 30 s; the final elongation is 10 min at 68°C.

- After completion of PCR, separation of PCR samples on agarose gel to visualize amplified DNA fragments. After sequencing more than 15 cloned DNA fragments amplified by nested RT-PCR, several mouse antibody heavy and light chains were cloned and found to be correct. Protein sequence alignment and CDR analysis identified one heavy chain and one light chain. Because the amino acids at positions 26, 40, and 55 in the heavy chain variable region and the amino acid at position 40 of the variable region of the key chain are key to the binding property, they can be reverse-engineered into the original mouse sequence. The candidates received are listed below. ( Padlan 1991. Mol. Immunol. 28, 489-498; Harris and Bajorath. 1995. Protein Sci. 4, 306-310 ).

Abstract for the sequences of fragments of antibodies (SEQ ID NO:10-17): in bold and underlined CDR 1, 2, 3 in numerical order from N-terminus to C-terminus; constant regions are italicized; the hinge regions are shown in bold underlined letters and the C-terminal histidine tag is shown in bold and italic.

SEQ ID NO:10 (AM-VH-C)

QVQLQQSGAELMKPGASVKISCKAT GYTFSRYW IEWVKQRPGHGL EWIGE ILPGSGST NYNEKFKGKATITADTSS NTAYMQLSSLTSEDSAVYYC TE GYEYDGFDY WGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKRV EPK HHHHHH

SEQ ID NO:11 (AM-VH1)

QVQLVQSGAEVKKPGSSVKVSCKAS GYTFSRYW ISWVRQAPGQGL EWMGR ILPGSGST NYAQKFQGRVTITADE STSTAYMELSSLRSEDTAVYYC T EGYEYDGFDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKRV EPK HHHHHH

SEQ ID NO:12 (AM-VH2-E40)

QVQLVQSGAEVKKPGSSVKVSCKAS GYTFSRYW IEWVRQAPGQGL EWMGR ILPGSGST NYAQKFQGRVTITADE STSTAYMELSSLRSEDTAVYYC T EGYEYDGFDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKRV EPK HHHHHH

SEQ ID NO:13 (AM-VH3-T26-E55)

QVQLVQSGAEVKKPGSSVKVSCKAT GYTFSRYW ISWVRQAPGQGL EWMGE ILPGSGST NYAQKFQGRVTITADE STSTAYMELSSLRSEDTAVYYC T EGYEYDGFDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKRV EPK HHHHHH

SEQ ID NO:14 (AM-VH4-T26-E40-E55)

QVQLVQSGAEVKKPGSSVKVSCKAT GYTFSRYW IEWVRQAPGQGL EWMGE ILPGSGST NYAQKFQGRVTITADE STSTAYMELSSLRSEDTAVYYC T EGYEYDGFDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKRV EPK HHHHHH

SEQ ID NO:15 (AM-VL-C)

FQGSHIPYT FGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTKHPVACEVACE _

SEQ ID NO:16 (AM-VL1)

DVVMTQSPLSLPVTLGQPASISCRSS QSIVYSNGNTY LNWFQQRPGQSPRRLIY RVS NRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC

SEQ ID NO:17 (AM-VL2-E40)

DVVMTQSPLSLPVTLGQPASISCRSS QSIVYSNGNTY LEWFQQRPGQSPRRLIY RVS NRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC

SEQ ID NO:22 (adrecizumab heavy chain)

QVQLVQSGAEVKKPGSSVKVSCKAS GYTFSRYW IEWVRQAPGQGLEWIGE ILPGSGST NYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYC TEGYEYDGFDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:23 (adrecizumab light chain)

FQGSHIPYT FGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTKHPVACEVACE _

Example 2 - Randomized, double-blind, placebo-controlled, phase I trial of safety, tolerability, and pharmacokinetics/dynamics of increasing single intravenous doses of an anti-ADM antibody (ADRECIZUMAB) (HAM8101) in healthy males during experimental endotoxemia.

General test scheme

A randomized, double-blind, placebo-controlled phase I trial was conducted in healthy male volunteers during experimental endotoxemia with a single group-increasing dose of adrecizumab given by IV infusion over 1 hour. Used model with continuous administration of LPS (lipopolysaccharide) for the induction of experimental endotoxemia; administration of LPS as an initial bolus of 1 ng/kg followed by a continuous infusion at a dose of 1 ng/kg/h for 3 hours. The subject is administered one course of study drug (adrecizumab or placebo), 1 hour after the start of LPS administration. A systemic inflammation model has recently been described ( Kiers et al. 2017. Scientific Reports 7:40149 ).

Adrecizumab was administered as a single infusion over 1 hour at a dose of 0.5 mg/kg, and the dose was increased up to 2.0 and 8.0 mg/kg in subsequent administration groups (see Table 2).

Table 2: Study groups of 8 individuals were treated by infusion for 1 hour

Group Treatment Group 1 0.5 mg/kg HAM8101 (n=6) Placebo (n=2) Group 2 2.0 mg/kg HAM8101 (n=6) Placebo (n=2) Group 3 8.0 mg/kg HAM8101 (n=6) Placebo (n=2)

Sample selection for testing

The study sample consisted of healthy young male volunteers. To be included in the trial, individuals had to meet all of the inclusion criteria and not meet any of the exclusion criteria.

Inclusion Criteria

1. Written informed consent to participate in this trial prior to any study procedure.

2. Men aged 18 to 35 inclusive.

3. Subjects had to agree to a reliable method of contraception with their partners from the start of the study until 3 months after study drug administration.

4. BMI between 18 and 30 kg/m ² , with a lower body weight limit of 50 kg and an upper body weight limit of 100 kg.

5. Healthy as defined by medical history, physical examination, vital signs, 12-lead electrocardiogram, and clinical laboratory parameters.

Exclusion Criteria

1. Reluctance to abstain from any drug, psychoactive substance, or antioxidant vitamin supplement during the trial and within 7 days prior to the day of treatment.

2. Unwillingness to abstain from smoking or alcohol within 1 day before the day of administration.

3. Previous participation in a trial where LPS was administered.

4. Surgery or trauma with significant blood loss or blood donation within 3 months prior to the day of administration.

5. History, signs or symptoms of cardiovascular disease, in particular:

- Frequent vasovagal collapse or history of orthostatic hypotension

- Resting pulse ≤45 or ≥100 beats/min

- Hypertension (systolic RR >160 or diastolic RR >90 mmHg)

- Hypotension (systolic RR <100 or diastolic RR <50 mmHg)

- ECG conduction abnormalities consisting of 1st degree atriogastric block and complex bundle branch block

- Any chronic cardiac arrhythmias (with the exception of PAC, PVC)

6. Renal impairment: plasma creatinine >120 µmol/l

7. Liver function tests (alkaline phosphatase, AST, ALT and/or γ-GT) above 2x upper limit of normal.

8. History of asthma

9. Atopic constitution

10. CRP greater than 2x upper limit of normal, or clinically significant acute illness, including infections, within 2 weeks prior to the day of administration.

11. Treatment with trial drugs or participation in any other clinical trial within 30 days prior to the day of administration.

12. Known or suspected failure to follow the test protocol.

13. Known hypersensitivity to any excipients of the medicinal formulations used.

14. Failure to personally provide written informed consent (eg, for linguistic or psychiatric reasons) and/or to participate in a trial.

Lipopolysaccharide (LPS) from Escherichia coli type O113

Lipopolysaccharide (LPS, US reference endotoxin [Escherichia coli O:113, List Biological Laboratories Inc., Campbell, California, US]) was administered intravenously to achieve a controlled inflammatory state of the subject.

Test structure and time windows

Pre-dose sampling carried out at T=0 (baseline). The main vital signs were determined after 5 min at rest in the supine or semi-supinated position. Scheme of sampling and follow-up visits (see Table 2 for more details):

- On the day of administration (Day 0), blood samples should have been taken at the scheduled time ± 5 min.

- After 24 hours (1 day), an observation visit and blood draw should have been scheduled ± 1 hour after IMP administration.

- On Day 7, an observation visit could be scheduled at any time ± 1 day.

- On days 14 and 28, an observation visit could be scheduled at any time ±2 days.

- On days 60 and 90, an observation visit could be scheduled at any time ± 3 days.

Laboratory monitoring

The following hematology parameters were analyzed: hemoglobin, hematocrit, RBC, MCV, MCH, MCHC, WBC, platelets, differential leukocyte count.

Blood samples were drawn into serum separation tubes and the following biochemical parameters were analyzed: CRP, AF, ALT, AST, γ-GT, creatine kinase, LDH, urea-N, creatinine, sodium, potassium, PT and APTT.

Primary end variables are: adverse events; vital signs during the first 10 hours after administration of adrecizumab and during the observation period (T=24 hours, T=7 days, T=14 days, T=28 days, T=60 days, T=90 days), for example, frequency heart rate, blood pressure, oxygen saturation, temperature; local tolerability in the field of intravenous infusion; safety laboratory parameters (Hb, Ht, leukocytes, platelets, differential leukocyte count, sodium, potassium, creatinine, urea, alkaline phosphatase, ALT, AST, GGT, CK, CRP, PT, APTT); 12-lead electrocardiogram (ECG), 2 and 8 hours after administration of adrecizumab.

Secondary outcomes are: pharmacokinetics of adrecizumab during experimental endotoxemia (including AUC, Cmax, terminal T1 / 2, Cl, V); plasma levels of inflammatory mediators on the day of endotoxemia (including, but not limited to, TNFα, IL-6, IL-8, IL-10); disease symptom index; optional: markers of kidney damage (including but not limited to creatinine clearance, pro-enkephalin and KIM-1).

Table 3: Baseline characteristics per group (mean ± SD)

placebo 0.5 mg/kg 2.0 mg/kg 8.0 mg/kg P* value Age 22.0±1.1 22.0±2.8 23.3 ± 2.5 22.5 ± 2.7 0.745 BMI 23.7 ± 1.3 23.3±1.5 24.7 ± 1.9 25.6 ± 3.0 0.263

*Tested using one-way ANOVA

No serious adverse events (SAE) or suspected unexpected serious adverse reactions (SUSAR) were observed during the entire duration of the trial, including the 90-day follow-up period.

results

The disease index declines faster with adrecizumab compared with placebo.

Both 2 and 8 mg/kg had a significantly lower disease index than placebo between time points of 210 min (T210) to 450 min (T450) (p=0.011 and 0.005, respectively; repeated GLM scores) (Fig. 1).

2 shows the distribution of disease scores by administration group at time T270 (time 270 min) and T300 (time 300 min), respectively (FIG. 2A) and B)), confirming the surprisingly beneficial effect of the antibody of the present invention.

The contribution of single disease index components to the effect is illustrated in Figure 3: A) nausea, B) headache, C) muscle pain, D) back pain, and E) trembling. All of the symptoms presented contribute significantly to the effect. Vomiting occurred only in two patients, once in each case, and could not be analyzed.

Figure 4 presents an alternative assessment. Here, another measure, the Nausea Index, was assessed using a single question regarding nausea in general on a scale of 0 to 10. Again, the Nausea Index decreased faster in patients treated with the anti-ADM antibody (adrecizumab).

Administration of NT-H to healthy people

The trial was performed on healthy male individuals as a randomized, double-blind, placebo-controlled trial with single incremental doses of NT-H antibody administered as an intravenous (IV) infusion in 3 consecutive groups of 8 healthy male individuals each (1- I group 0.5 mg/kg, 2nd group 2 mg/kg, 3rd group mg/kg) of healthy male individuals (n=6 active substance, n=2 placebo for each group).

The main inclusion criteria were written informed consent, age 18-35 years, consent to use a reliable method of contraception and a BMI between 18 and 30 kg/m².

Subjects received a single intravenous dose of NT-H antibody (0.5 mg/kg; 2 mg/kg; 8 mg/kg) or placebo by slow infusion over a 1 hour period in the research unit.

Baseline ADM levels in the 4 groups did not differ. Median ADM levels were 7.1 pg/mL in the placebo group, 6.8 pg/mL in the first treatment group (0.5 mg/kg), 5.5 pg/mL in the second treatment group (2 mg/kg), and 7.1 pg/ml in the third treatment group (8 mg/ml).

The results demonstrate that ADM levels rapidly increased during the first 1.5 hours after administration of the NT-H antibody in healthy individuals and then plateaued and slowly decreased (FIG. 5).

Two individuals in the placebo group and 3 individuals in total from the administration groups reported migraine symptoms prior to administration of a single dose of placebo and NT-H antibody, respectively. After each time point, questions about symptoms were asked again. Individuals injected with the NT-H antibody did not report any migraine symptoms 4 hours after administration, while two individuals administered placebo still suffered from migraine symptoms as before the infusion.

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SEQUENCE LIST

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<120> ADRENOMEDULIN (ADM) BINDING COMPOUND FOR USE

FOR THERAPY OR PREVENTION OF SYMPTOMS OF THE DISEASE

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys His His His His His

210 215 220

His

225

<210> 13

<211> 225

<212> PROTEIN

<213> Artificial sequence

<400> 13

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys His His His His His

210 215 220

His

225

<210> 14

<211> 225

<212> PROTEIN

<213> Artificial sequence

<400> 14

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys His His His His His

210 215 220

His

225

<210> 15

<211> 219

<212> PROTEIN

<213> Artificial sequence

<400> 15

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 16

<211> 219

<212> PROTEIN

<213> Artificial sequence

<400> 16

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 17

<211> 219

<212> PROTEIN

<213> Artificial sequence

<400> 17

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 18

<211> 19

<212> PROTEIN

<213> Mus musculus

<400> 18

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

1 5 10 15

Gly Thr Cys

<210> 19

<211> 12

<212> PROTEIN

<213> Mus musculus

<400> 19

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

1 5 10

<210> 20

<211> 11

<212> PROTEIN

<213> Mus musculus

<400> 20

Ala Pro Arg Asn Lys Ile Ser Pro Gln Gly Tyr

1 5 10

<210> 21

<211> 12

<212> PROTEIN

<213> Homo sapiens

<400> 21

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

1 5 10

<210> 22

<211> 448

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Human antibody heavy chain transplanted with mouse CDRs

<400> 22

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

<210> 23

<211> 219

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Human antibody light chain transplanted with mouse CDRs

<400> 23

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<---

Claims (47)

1. The use of an anti-adrenomedullin (ADM) antibody, or anti-adrenomedullin antibody fragment, or anti-ADM non-Ig scaffold in the treatment or prevention of symptoms of a disease or in the treatment or prevention of a disease characterized by such symptoms in a patient, where the specified antibody, or fragment, or frame binds to ADM (1-52; SEQ ID NO:1) or a fragment thereof; where the specified antibody, or fragment, contains the following sequence as a heavy chain: SEQ ID NO: 22 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGEILPGSGSTNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK or a sequence that is > 95% identical to it; and contains the following sequence as a light chain: SEQ ID NO: 23 DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPRLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQNGSSKDSTYSLSSTLVSKADYEKHKVACE or a sequence that is > 95% identical to it; where the disease is selected from the group consisting of inflammatory conditions, autoimmune diseases, metabolic diseases, brain diseases and cardiovascular diseases; And where the symptoms of the disease are selected from the group consisting of nausea, headache, muscle pain, back pain and vomiting. 2. Use according to claim 1, wherein said antibody or fragment or scaffold binds to the N-terminal portion (a.k. 1-21) of ADM: YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO:4). 3. Use according to any one of claims 1, 2, where the specified antibody, antibody fragment or non-Ig frame binds to a part of the middle region (MR-) ADM, consisting of a.to. 21-42 ADM: CTVQKLAHQIYQFTDKDKDNVA (SEQ ID NO:3). 4. Use according to any one of claims 1 to 3, wherein the disease is characterized by an amount of C-reactive protein (CRP) in the sample that is ≥ 10 mg/l and/or where the amount of TNF in the sample is ≥ 50 pg/ml, and /or where the amount of bio-ADM in the sample is ≥ 43 pg/ml, and/or where the mean arterial pressure of said patient is reduced. 5. Use according to any one of claims 1 to 3, wherein the disease is migraine. 6. Use according to any one of claims 1 to 5, wherein said antibody or antibody fragment or non-Ig scaffold is monospecific. 7. Use according to any one of claims 1 to 6, wherein said antibody or fragment or framework exhibits an ADM binding affinity of at least 10 ^-7 M by unlabeled surface plasmon resonance using the Biacore 2000 system. 8. Use according to any one of claims 1 to 7, wherein said antibody or fragment or scaffold is not ADM binding protein 1 (complement factor H). 9. Use according to any of the preceding claims, wherein said antibody or fragment or scaffold recognizes and binds to the N-terminus (a.k. 1) of ADM. 10. Use according to any one of the preceding claims, wherein said antibody or fragment or scaffold is an ADM stabilizing antibody or fragment or scaffold that increases the serum, blood, plasma half-life of ADM by at least 10%, preferably by at least 50%, more preferably > 50%, most preferably > 100%. 11. The use according to any one of the preceding claims, wherein said antibody or fragment or scaffold blocks the biological activity of ADM by no more than 80%, preferably no more than 50% using hADM 22-52 as a reference antagonist in CHO- K1 expressing the recombinant human ADM receptor. 12. Use according to any one of the preceding claims, wherein said individuals are undergoing chemotherapy, treatment with biological agents, antibiotics, or treatment with antiviral compounds. 13. Pharmaceutical composition for use in the treatment or prevention of symptoms of a disease, or for the treatment or prevention of a disease characterized by such symptoms in a patient, characterized in that the composition contains an antibody, or fragment, or scaffold, where the specified antibody, or fragment, or frame binds to ADM (1-52; SEQ ID NO:1) or a fragment thereof; where the specified antibody, or fragment, contains the following sequence as a heavy chain: SEQ ID NO: 22 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGEILPGSGSTNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK or a sequence that is > 95% identical to it; and contains the following sequence as a light chain: SEQ ID NO: 23 DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPRLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQNGSSKDSTYSLSSTLVSKADYEKHKVACE or a sequence that is > 95% identical to it; where the disease is selected from the group consisting of inflammatory conditions, autoimmune diseases, metabolic diseases, brain diseases and cardiovascular diseases; And where the symptoms of the disease are selected from the group consisting of nausea, headache, muscle pain, back pain and vomiting. 14. The composition of claim 13, wherein said antibody, or fragment, or scaffold binds to the N-terminal portion (a.k. 1-21) of ADM: YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO:4). 15. The composition according to any one of claims 13, 14, where the specified antibody, antibody fragment or non-Ig frame binds to a part of the middle region (MR-) ADM, consisting of a.to. 21-42 ADM: CTVQKLAHQIYQFTDKDKDNVA (SEQ ID NO:3). 16. A composition according to any one of claims 13-15, wherein said antibody or antibody fragment or non-Ig scaffold is monospecific. 17. A composition according to any one of claims 13-16, wherein said antibody or fragment or framework exhibits an ADM binding affinity of at least 10 ^-7 M by unlabeled surface plasmon resonance using the Biacore 2000 system. 18. The composition of any one of claims 13-17, wherein said antibody or fragment or scaffold is not ADM binding protein 1 (complement factor H). 19. A composition according to any one of claims 13-18, wherein said antibody or fragment or scaffold recognizes and binds to the N-terminus (a.k. 1) of ADM. 20. Composition according to any one of claims 13-19, wherein said antibody, or fragment, or framework is an ADM stabilizing antibody, or fragment, or framework, which increases the half-life of ADM in serum, blood, plasma by at least 10% , preferably at least 50%, more preferably > 50%, most preferably > 100%. 21. The composition according to any one of claims 13-20, where the specified antibody, or fragment, or scaffold blocks the biological activity of ADM by no more than 80%, preferably no more than 50% when using hADM 22-52 as a reference antagonist in CHO-K1 cells expressing the recombinant human ADM receptor.

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