All patent and non-patent references cited in the application, or in the present appli¬ cation, are also hereby incorporated by reference in their entirety.

Field of invention The present invention relates to a composition comprising a protective binding member capable of binding PYY for the production of a pharmaceutical composition for the treatment of metabolic disorders. The invention further relates to a protective binding member capable of increasing the Ti/2l plasma concentration and/or biologi¬ cal activity of PYY. There is further provided herein a method for treatment of an individual in need thereof, comprising administering to said individual an effective amount of the pharmaceutical compositions comprising said protective binding member of the present invention. The present invention further relates to isolated nucleic acid molecules encoding at least a part of the binding members disclosed herein and vectors comprising said nucleic acid molecules. Host cells comprising said nucleic acids are also disclosed, and the present invention further relates to a cell line engineered to express one or more of the binding members disclosed herein. In another aspect of the present invention, a method and a kit are provided for detecting a metabolic disorder in an individual.

Background of the invention

Metabolic disorders Metabolic disorders include overweight, obesity, syndrome X, bowel diseases, as well as eating disorders. The disease all result in an unbalanced energi-intake due to misregulation of appetite or nutrient uptake. All such diseases are associated with severe health risk as well as loss of the sense of well- being and quality of life for the individual.

Overweight and obesity The cause of overweight and obesity may be disease related, caused by unsuitable eating habits or lack of exercise or a combination of these.

Bodymass index Overweight and obesity may be defined by the body mass index (BMI) of the indi¬ vidual. BMI is calculated, by dividing the bodyweight in kilograms with the square of the height in meters. hodyweight{kg) Bodymass index: , . , 2 height1 (m)

According to guidelines from the US National institute of health three BMI categories have been defined as follows.

• Normal weight = 18.5-24.9

• Overweight = 25-29.9

• Obesity = BMI of 30 or greater

o Severe obesity = 35-39.9

o Morbid obesity = BMI of 40 or greater

Overweight and obesity increase the risk of heart diseases, type 2 diabetes, several cancer types, cardiovascular disease, results in breathing problems, and an overall risk of premature death.

Syndrome X Multiple metabolic disorders have been grouped under the name syndrome X as risk factors for cardiovascular diseases. These include; abdominal obesity, atherogenic dyslipedimia, raised blood pressure) insulin resistance, glucose intolerance, proinflammatory state, prothrombotic state. The criterias for diagnosis of syndrome X , include abdominal obesity (waist circumference/waisthip ratio), elevated plasma triglycerides, low HDL cholesterol, increased blood pressure, impaired fasting glucose, impaired glucose tolerance, type 2 diabes, anti-hypertensive medication and/or elevated blood pressures, urinary albymin excretion rate, hypertension. These symptoms are considered risk factors for the development of cardiovascular diseases, and are all related to the metabolism and thereby further indicate the need of treatment for metabolic disease (Grundy, SM et al, 2004).

Bowel diseases Problems related to the metabolism in the individual, may arise due to bowel diseases whereby absorption of nutrients and secretion of waste compounds are affected, such as irritable bowel syndrome, inflammatory bowel disease, ulcerative colitis, Crohn's disease, bowel atrophy, conditions characterized by loss of bowel mucosa or bowel mucosal function, and other conditions of the gastrointestinal tract.

Eating disorders A great number of people suffer from eating disorders, such as bulimia nervosa, binge eating disorder and/or night eating syndrome and thereby have an increased risk of acquiring additional health problems as well as a lack of quality of life. These types of syndromes have been getting more attention lately and are now being characterized as clinical diseases. A major problem is the diagnosis of these dis¬ eases, as the syndromes are merely characterised by a behavioural pattern of the patient. The characteristics of the three syndromes are summarised below.

Bulimia Nervosa Bulimia nervosa is a serious eating disorder marked by a destructive pattern of binge-eating and recurrent inappropriate behaviour to control one's weight. It can occur together with other psychiatric disorders such as depression, obsessive- compulsive disorder, substance dependence, or self-injurious behaviour. Binge eat¬ ing is defined as the consumption of excessively large amounts of food within a short period of time. The food is often sweet, high in calories, and has a texture that makes it easy to eat fast. "Inappropriate compensatory behaviour" to control one's weight may include purging behaviours (such as self-induced vomiting, abuse of laxatives, diuretics, or enemas) or non-purging behaviours (such as fasting or ex¬ cessive exercise). For those who binge eat, any amount of food, even a salad or half an apple, may be perceived as a binge and is vomited.

People with bulimia nervosa often feel a lack of control during their eating binges. Their food is usually eaten secretly and gobbled down rapidly with little chewing. A binge is usually ended by abdominal discomfort. When the binge is over, the person with bulimia feels guilty. Persons with bulimia, however, regularly purge, fast, or engage in strenuous exercise after an episode of binge eating. Purging means vomiting or using diuretics (water expelling compounds) or laxatives, sometimes in greater-than-recommended doses, to avoid gaining weight. Fasting is defined as not eating for at least 24 hours. Strenuous exercise, in this case, is defined as exer- cising for more than an hour solely to avoid gaining weight after binge eating. Purging, fasting, and strenuous exercise are dangerous ways to attempt weight control. To be diagnosed with bulimia, a person must have had, on average, a mini¬ mum of two binge-eating episodes a week for at least three months. The eating disorders are characterized by a constant concern with food and weight, to the extent that it overshadows other personal concerns.

Binge Eating Disorder Binge Eating Disorder (BED) is a type of eating disorder not otherwise specified and is characterized by recurrent binge eating without the regular use of compensatory measures to counter the binge eating. People with binge eating disorder frequently eat large amounts of food and feel a loss of control over their eating behaviour. This disorder is different from binge-purge syndrome (bulimia nervosa) because people with binge eating disorder usually do not purge afterward by vomiting or using laxa- tives. Most people with serious binge eating problems frequently experience epi¬ sodes of eating what others would consider an abnormally large amount of food and frequently feel unable to control what or how much to eat. A person suffering from BED often eats without being hungry, eats more rapidly than normal and eats until uncomfortably full. The patient may feel ashamed or disgusted by their behaviour. Eating alone or in secret out of embarrassment of the quantity of food being eaten are other characteristics of binge eating disorder. The patient may experience feelings of disgust, depression, or guilt after overeating.

Night eating syndrome Night-eating syndrome (NES) has not yet been formally defined as an eating disor¬ der. Underlying causes are being identified, and treatment plans are still being de¬ veloped. It seems likely that a combination of biological, genetic, and emotional fac¬ tors contribute to the problem.

A person suffering from NES shows little or no appetite for breakfast and delays first meal for several hours after waking up. This is due to lack of hunger or based on being upset about how much was eaten the night before. Often the patient eats more food after dinner than during the meal. This may mean that more than half of the daily food intake occurs after dinner but before breakfast. Most people who suf- fer from the syndrome are prone to stress, especially at night the person may be moody, tense, anxious, nervous, agitated, etc. and has trouble falling asleep or stay¬ ing asleep and thus have poor sleep, often waking up three to four times a night. Each time they wake up they walk to the kitchen to eat a "snack" of high carbohy¬ drate (sugar and starch) food such as biscuits, cakes or crisps. The person often feels tense, anxious, upset, or guilty while eating. This pattern should persist for at least two months before being characterised as a night eating syndrome. NES is thought to be stress related and is often accompanied by depression. The behaviour of an NES patient is different from the behaviour of an individual suffering from a binge eating disorder. During binge eating, relatively large amounts of food are con- sumed over a relatively short space of time. Night-eating syndrome involves con¬ tinuous eating throughout evening hours. The heavy preference for carbohydrates, which trigger the brain to produce so-called "feel-good" neuro-chemicals, suggests that night eating may be an unconscious attempt to self-medicate mood problems. NES may run in families. It appears to respond to treatment with the selective serotonin reuptake inhibitor (SSRI) sertraline (a prescription medication). NES is remarkable for characteristic disturbances in the circadian rhythm of food intake, while the circadian sleep rhythm remains normal.

Night-eating syndrome shows distinctive changes in hormones related to sleep, hunger and stress. The night time rise in the hormone that accompanies sleep, melatonin, is greatly decreased in night eaters, probably contributing to their sleep disturbances. Similarly, night-eaters fail to show a nighttime rise in the hormone leptin, which suppresses hunger, and the stress hormone Cortisol is elevated throughout a 24-hour period.

Regulation of energy uptake.

Energy uptake is regulated by hormones that regulate food intake, by modifing the sensing of appetite, and regulate absorbsion of nutrients and solutes from the gut.

Hormones can be separated into long and short term regulating hormones. Long term regulating hormones as insulin and leptin act slowly to promote the stability of body fat stores long whereas the short term regulating hormones, as ghrelin and cholecystokinin, act rapidly to influence the individual meal by sensing of satiety and "fullness". Insulin and leptin are released into the blood in proportion to the amount of body fat. When body fat stores are reduced, declining levels of these hormones are sensed by the brain and are transduced into increases in appetite and metabolic efficiency that persist until the lost weight is recovered. Ghrelin and cholecystokinin are factors that trigger the onset and termination of eating and thereby ghrelin and cholecystokini function in a meal-to-meal control system that itself is sensitive to changes in insulin and leptin levels. In this way, the size and frequency of individual meals can be adjusted so as to minimize changes in body fat content.

The uptake of nutrients from the gut is regulated by various factors including gut hormones. The gut hormones VIP, CCK, and motilin relates to the motility of the upper gastrointestinal tract whereas polypeptide YY (PYY) and Neuropeptide Y (NPY) affect the absorption in the intestine.

PYY The gut hormone peptide YY (PYY), and the neuropeptide, neuropeptide Y (NPY), are structurally related to pancreatic polypeptide (PP) (figure 1). PYY and NPY exert their action through NPY receptors (Y1 R, Y2R, Y4R and Y5R). The PP1 NPY and PYY peptides consist of 36 amino acids with an amidated C-terminal. Two forms of PYY, PYY1-36 and PYY3-36, the latter being a truncated form of the former, have been found in circulation. PYY3-36 is produced by the cleavage of PYY1-36 by the enzyme dipeptidyl peptidase IV (DPP-IV). PYY1-36 binds to and activates at least three NPY receptor subtypes (Y1 , Y2 and Y5) whereas PYY3-36 is more selective for the Y2 receptor (Y2R). Only the C-terminal part of the PYY3-36 peptide is required for the binding to the Y2 receptor. Throughout this document, the notation PYY covers both PYY1-36 and PYY3-36 (Berglund, M.M. et al., 2003).

PYY was initially isolated from porcine intestine (Tatemoto, K. and Mutt, C, 1980) and named Peptide YY due to the tyrosine residues present in the N- and C- terminal of the molecule.

PYY is expressed in endocrine cells lining the gastrointestinal tract and particularly in the distal portion. PYY is secreted in response to food ingestion. Within 15 minutes the plasma level of PYY rise and the level of PYY will reach a plateau after approximately 90 min. The maximum level of PYY reached is proportional to the calories ingested, suggesting that PYY may function as a sensor of food ingestion. In addition PYY is also expressed by neurones, such as in peripheral neurons, particularly enteric neurons. Furthermore, PYY is found in a restricted set of central neurons. The expression pattern of PYY in both endocrine cells and neurons suggest that PYY may be involved in regulation of multiple functions in the individual (Ekblad, E. and Sundler, F., 2002).

A suggested role of PYY may be to regulate the secretion and absorbance of fluid and electrolytes in the gastrointestinal tract and intestine and PYY have therefore been suggested as treatment of diarrhoea (US 6,588, 708) by prolonging of the residence time. Furthermore, PYY3-36 has been suggested to be involved in the system regulating feeding behaviour. It has been found that peripheral administration of PYY3-36 inhibited food intake in rodents. Moreover, direct intra- arcuate administration of PYY3-36 inhibited food intake. A linkage between the PYY effect on feeding behaviour and the NPY 2 receptor have been suggested by the demonstration that NPY receptor Y2 null mice are resistant to the anorectic effects of peripherally administered PYY3-36 (Batterham, R.L. and Bloom, S. R., 2003).

The hypothalamic arcuate nucleus, a key brain area regulating appetite, has access to nutrients and hormones within the peripheral circulation. NPY neurons within the arcuate nucleus express the Y2R. The arcuate nucleus contains two distinct sub- groups of neurons that control food intake. On group of neurons produces NPY, which acts in the brain to stimulate feeding (Stanley, B. G. et al, 1986), whereas an adjacent subgroup of neurones produces melanocortin peptides, which act in the same brain areas as NPY, but inhibit eating (Fan, W. et al, 1997). Typically, when one of these subsets is activated, the other one is inhibited.

References

Batterham, R.L. and Bloom, S. R. (2003). The gut hormone peptide YY regulates appetite. Ann. N. Y. Acad. Sci. Jun;994:162-8.

Berglund, M. M.; Hipskind, P.A. and Gehlert, D. R. (2003). Recent developments in our understanding of the physiological role of PP-fold peptide receptor subtypes. Exp Biol Med (Maywood). Mar;228(3):217-44 Ekblad, E. and Sundler, F. (2002). Distribution of pancreatic polypeptide and peptide YY. Peptides. Feb;23(2):251-61.

Fan, W.; Boston, B.A.; Kesterson, R.A.; Hruby, V.J. and Cone, R.D (1997). Role of melanocortinergic neurons in feeding and the agouti obesity syndrome. Nature. Jan 9;385(6612):165-168.

Grundy, S.M.; Brewer, H. B.; Cleeman, J.I.; Smith, S. C. and Lenfant, C.(2004). Definition of Metabolic Syndrome. NHLBI/AHA conference proceedings, 109: 433- 438.

Stanley, B. G.; Kyrkouli, S. E.; Lampert, S. and Leibowitz, S. F. (1986). Peptides 7, 1189-1192)

Tatemoto, K. and Mutt, V. (1980). Isolation of two novel candidate hormones using a chemical method for finding naturally occurring polypeptides. Nature, Jun 5;285(5764):417-8

Summary of invention The present invention relates to a protective binding member of PYY. The protective binding member may prolong the half-life of PYY or increase the binding of PYY to the NPY receptor. Thereby the binding member effectively potentiates the effects of PYY. The present invention relates to a composition comprising an isolated binding member capable of binding PYY for the production of a pharmaceutical composition for the treatment of metabolic disorders. The invention further relates to a method of treatment of metabolic disorders comprising administration of said binding member. Thus treatments including administration of said binding member may potentiate the action of PYY. The potentiating effect is preferably triggered by binding of the binding member to PYY molecules already present in the patient.

In an embodiment of the invention the composition is administered for the treatment of metabolic disorders. In an embodiment of the invention said composition comprising a binding member capable of binding PYY or a functional equivalent thereof is for the production of a pharmaceutical composition for a) the treatment of overweight and/or b) the treatment of obesity and/or c) the treatment of syndrome X and/or d) the treatment of eating disorders and/or e) the treatment of bowel diseases and/or f) inhibition of appetite and/or g) inhibition of weight gain and/or h) decreasing body weight and/or i) decreasing body fat mass j) treatment of bowel diseases or any combinations of the above.

In an embodiment said binding member is an antibody, a fragment of an antibody or an immunologically active molecule.

In an embodiment of the invention a binding member is conjugated to PYY or a functional equivalent thereof.

In a more preferred embodiment the binding member is capable of increasing the plasma concentration of PYY1-36, PYY3-36 or a functional equivalent thereof. In a further embodiment the binding member is capable of increasing the biological activity of PYY1 -36, PYY3-36 or a functional equivalent thereof.

The binding member may also be administrated in combination with a second active ingredient, such as PYY or a functional equivalent there of.

An aspect of the invention relates to a method of treatment comprising administration to an individual in need thereof an effective amount of the pharmaceutical composition defined by the invention.

Drawings Figure 1

Structure of the family of the PP-fold peptides NPY, PYY, and PP share a common hairpin-like three-dimensional structure called the PP-fold. All three peptides are 36 amino acids long with an amidated carboxy- terminus. The general structure of the PP-fold peptides has been established using x-ray crystallography of avian PP and confirmed in several studies using nuclear magnetic resonance. Amino acid residues 1-8 form a type Il proline helix followed by a loop. Residues 15-32 form an α-helix, and the four most carboxy-terminal resi- dues are in a flexible loop conformation. The amino acid sequence of mammalian NPY is highly conserved, PPY display 8 varible amino acids, whereas the amino acid sequence of PP is the least conserved PP-fold peptide. The general three- dimensional structure seems to be conserved in all PP-fold peptides (Berglund MM et al, 2003).

Figure 2

PYY plasma concentration in response to subcutaneous PYY1-36 administration.

PYY1-36 was administered to subjects as described in example 3. The plasma level of PYY was measured during a 4 hour time period following injection. The plasma level of PYY increases with in 15 minutes after administration. A plasma level of 80-100 pmol/l is obtained using a dosage of 200 pmol/kg FFM.

Figure 3

PYY plasma concentration in response to subcutaneous PYY3-36 administration.

PYY3-36 was administered to subjects as described in example 3. The plasma level of PYY was measured during a 4 hour time period following injection. The plasma level of PYY increases with in15 minutes after administration. A plasma level of 100-120 pmol/l is obtained using a dosage of 100 pmol/kg FFM.

Description of sequence listing Sequence ID 1 : amino acid sequence of Human PYY1-36

Definitions: Affinity: The term refers to the binding strength between receptors and their ligands, for example an antigen and an antibody or an antigen and an affibody. Af fi body: A recombinant immunologically active molecule, selected from a library constructed by combinatorial variegation of the Fc binding surface of of a protein that is not an antibody, preferably the 58 residue staphylococcal protein A (SPA). Agonist: A molecule that stimulates the action of the target. Amino acid: Entity comprising an amino terminal part (NH2) and a carboxy terminal part (COOH) separated by a central part comprising a carbon atom, or a chain of carbon atoms, comprising at least one side chain or functional group. NH2 refers to the amino group present at the amino terminal end of an amino acid or peptide, and COOH refers to the carboxy group present at the carboxy terminal end of an amino acid or peptide. The generic term amino acid comprises both natural and non- natural amino acids. Natural amino acids of standard nomenclature as listed in J. Biol. Chem., 243:3552-59 (1969) and adopted in 37 C.F.R., section 1.822(b)(2) be- long to the group of amino acids listed in Table 1 herein below. Non-natural amino acids are those not listed in Table 1. Examples of non-natural amino acids are those listed e.g. in 37 C.F.R. section 1.822(b)(4), all of which are incorporated herein by reference. Further examples of non-natural amino acids are listed herein below. Amino acid residues described herein can be in the "D" or or "L" isomeric form.

Symbols Amino acid 1 -Letter 3-Letter

Y Tyr tyrosine G GIy glycine F Phe phenylalanine M Met methionine A Ala alanine S Ser serine I lie isoleucine L Leu leucine T Thr threonine V VaI valine P Pro proline K Lys lysine H His histidine Q GIn glutamine E GIu glutamic acid W Trp tryptophan R Arg arginine D Asp aspartic acid N Asn asparagine C Cys cysteine

Table 1. Natural amino acids and their respective codes.

Appetite: Appetite in an individual is assessed by measuring the amount of food ingested and by assessing the individual's desire to eat. Appetite (i.e., hunger) is typically assessed with a short questionnaire given to individuals on a random basis several times a week. Typically, subjects rate their hunger, preoccupation with food, and desire to eat greater quantities and different types of food by answering the questions using analogue scales ranging from 1 , not at all, to 5, extremely. Amino acid residue: the term "amino acid residue" is meant to encompass amino acids, either standard amino acids, non-standard amino acids or pseudo-amino ac¬ ids, which have been reacted with at least one other species, such as 2, for example 3, such as more than 3 other species. In particular amino acid residues may com¬ prise an acyl bond in place of a free carboxyl group and/or an amine-bond and/or amide bond in place of a free amine group. Furthermore, reacted amino acids resi- dues may comprise an ester or thioester bond in place of an amide bond Antagonist: A molecule that inhibits that action of its target. Antibody: Are immunoglobulin molecules and active portions of immunoglobulin- molecules. Antibodies are for example intact immunoglobulin molecules or frag¬ ments there of retaining the immunologic activity. Antibody fragment refers to a portion of a full-length antibody, generally the anti¬ gen binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab') 2 and Fv fragments. Papain digestion of antibodies produces two identical antigen binding fragments, called the Fab fragment, each with a single antigen bind¬ ing site, and a residual "Fc" fragment, so-called for its ability to crystallize readily. Pepsin treatment yields an F(ab') 2 fragment that has two antigen binding fragments which are capable of cross-linking antigen, and a residual other fragment (which is termed pFc'). Additional fragments can include diabodies, linear antibodies, single- chain antibody molecules, and multispecific antibodies formed from antibody frag¬ ments. Antigen: The molecule recognised by an antibody. Usually a peptide, polypeptide or a multimeric polypeptide. Antigens are preferably capable of eliciting an immune response. BED: Binge eating disorder. Binding member: A molecule that can bind to a specific molecule. Here for exam- pie a molecule that binds an epitope on PYY or functional equivalents thereof. Binding domain: The binding domain is a region of a molecule that comprises the binding site. Binding site: The binding site is the smallest region of a receptor molecule that me¬ diates the interaction with the ligand. BMI: Body mass index measures an individual's height/weight ratio. It is determined by calculating weight in kilograms divided by the square of height in meters. The BMI "normal" range is 18,5-24,9. BN: Bulimia nervosa. Body fat mass: Body fat mass can be measured e.g. by the fat fold technique: In this technique, a pincer-type caliper is used to measure subcutaneous fat by deter¬ mining skin fold thickness at representative sites on the body. These skin fold measurements are then used to compute body fat by either, adding the scores from the various measurements and using this value as an indication of the relative de¬ gree of fatness among individuals or by using the measurements in mathematical equations that have been developed to predict percent body fat. Chimera : A molecule consisting of at least two parts not found together in nature. A chimeric peptide or protein is a peptide or protein constructed by the fusion of two peptides or proteins. A chimeric DNA molecule is a DNA molecule that encodes a chimeric protein. Dissociation constant, Kd: a measure to describe the strength of binding (or affin¬ ity or avidity) between receptors and their ligands, for example an antibody and its antigen. The smaller the Kd, the stronger binding. Concentration equivalent: A concentration equivalent is an equivalent dosage be¬ ing defined as the dosage of binding member compound having in vitro or/and in vivo the same response (as evaluated e.g. from a dosage-response curve) as a known binding member. Eating disorder event: See "EDE". EDE: An eating disorder event refers to an inappropriate meal consumed by an individual suffering from an eating disorder, such as NES, BED or BN. Frequency: The number of occurrences of a certain event within a certain period of time (e.g. the number of occurrences per day or per week). Epitope: A peptide segment or segments of a polypeptide recognized by the binding site of a binding member. Fusion Polypeptide: A polypeptide comprised of at least two polypeptides and a linking sequence to operatively link the two polypeptides into one continuous poly¬ peptide. The two polypeptides linked in a fusion polypeptide are typically derived from two independent sources, and therefore a fusion polypeptide comprises two linked polypeptides not normally found linked in nature. Half-life: See T1 /2. Immunoglobulin: The serum antibodies, including, IgG, IgM, IgA, IgE and IgD Immunologically active: Description referring to the ability of a molecule to distin¬ guish between two of more polypeptide by preferentially binding to one of the poly¬ peptide. "Immunologically active" may also refer to functionality associated with an entity capable of eliciting an immunological response. Individual: A living animal or human. In preferred embodiments, the subject is a mammal, including humans and non-human mammals such as dogs, cats, pigs, cows, sheep, goats, horses, rats, and mice. In the most preferred embodiment, the subject is a human. Isolated: is used to describe any of the various secretagogues, polypeptides and nucleotides disclosed herein, that have been identified and separated and/or recov¬ ered from a component of its natural environment. Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified. Label: refers to single atoms or molecules that are either directly or indirectly involved in the production of a detectable signal to indicate the presence of a complex Ligand: A molecule, for example a peptide, capable of specific binding to one or more cognate receptors. An antigen is, for example, a ligand to its cognate antibodies. "Loss of body weight": Defined herein as a reduction of an individual's overall weight, which include both relative and absolute reductions of weight, as well as BMI reductions. "Loss of body fat": Defined herein as either a reduction of an individual's overall fat mass or a reduction in the percentage of an individual's body fat. Medical disorder: By "medical disorder" is meant any disease or syndrome having a detrimental effect on an individual's physical and/or mental health. Said medical disorder may have a genetic cause. Preferably, said medical disorder leads to one or more undesirable symptoms including increase of fat mass, increase of weight, increase of appetite, and/or causes a reduction in the individual's PYY levels. Multimeric: A polypeptide molecule comprising more than one polypeptide. A multimeric polypeptide may be dimeric and contain two polypeptides etc. Mulitmers may be homomeric and contain two or more identical polypeptides or a multimer may be heteromeric and contain two or more non-identical polypeptides. NES: Night eating syndrome. Neutralizing binding member: A neutralizing binding member is a binding member that when bound to its ligand decreases the effect of the ligand. Parenteral: For the purpose of this document "parenteral" is defined as being out¬ side the alimentary canal. Thus, the term "parenteral administration" of a compound encompasses e.g. subcutaneous, intramuscular, intravenous, intranasal, buccal, intradermal and transdermal administration, as well as inhalation of said compound. Furthermore, "parenteral" is also meant to encompass administration via rectal suppositories. Peptide: Plurality of covalently linked amino acid residues defining a sequence and linked by amide bonds. The term is used analogously with oligopeptide and polypeptide. The amino acids may be both natural amino acids and non-natural amino acids, including any combination thereof. The natural and/or non-natural amino acids may be linked by peptide bonds or by non-peptide bonds. The term peptide also embraces post-translational modifications introduced by chemical or enzyme-catalyzed reactions, as are known in the art. Such post-translational modifications can be introduced prior to partitioning, if desired. Amino acids as specified herein will preferentially be in the L-stereoisomeric form. Amino acid analogs can be employed instead of the 20 naturally-occurring amino acids. Several such analogs are known, including fluorophenylalanine, norleucine, azetidine-2- carboxylic acid, S-aminoethyl cysteine, 4-methyl tryptophan and the like. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino acid residues or a covalent bond to an amino-terminal group such as NH2 or acetyl or to a carboxy-terminal group such as COOH. Protective binding member: A protective binding member is a binding member that when bound to its ligand (here PYY) is capable of enhancing or prolonging the effect of the ligand. PYY: Peptide YY. PYY is used herein as a term to describe both PYY1-36 and PYY3-36. Receptor: A receptor is a molecule, such as a protein, glycoprotein and the like, that can specifically (non-randomly) bind to another molecule Recombinant DNA (rDNA) molecule: A DNA molecule produced by operatively linking two DNA segments. Thus, a recombinant DNA molecule is a hybrid DNA molecule comprising at least two nucleotide sequences not normally found together in nature. Ti/2: The half-life is the time for the concentration of a compound to decrease 50 %. Valency: The term valency refers to the number of potential antigen binding sites, i.e. binding domain, in a polypeptide. A polypeptide may be monovalent and contain one antigen binding site or a polypeptide may be bivalent and contain two antigen binding sites. Additionally, a polypeptide may be tetravalent and contain four antigen binding sites. Each antigen binding site specifically binds one antigen. When a poly- peptide comprises more than one antigen binding site, each antigen binding site may specifically bind the same or different antigens. Thus, a polypeptide may con¬ tain a plurality of antigen binding sites and therefore be multivalent and a polypep¬ tide may specifically bind the same or different antigens.

Detailed description of the invention

The present invention relates to a composition comprising a binding member as described below for the production of a pharmaceutical composition. In an embodiment of the invention the composition comprising a binding member is for the production of a pharmaceutical composition for the treatment of metabolic disorders and PYY deficiency. The binding member binds PYY or a functional equivalent as described in the following sections related to PYY.

Binding member A binding member according to the invention is any type of molecule as defined here below capable of binding to a ligand, here preferably PYY. The binding member may be a single moiety, e.g., a polypeptide or protein, or it may include two or more moieties, e.g., a pair of polypeptides such as a pair of single chain antibody domains. The binding member may be derived from a naturally occurring protein or polypeptide; it may be designed de novo, or it may be selected from a library. For example, the binding member may be or be derived from an antibody, a single chain antibody (scFv), a single domain antibody (VHH), a lipocalin, a single chain MHC molecule, an Anticalin™ (Pieris), an Affibody™ , a nanobody (Ablynx) or a Trinectin™ (Phylos).

Neutralizing binding member The binding member according to the invention may be a neutralizing binding member, which may serve as a carrier of PYY. A neutralizing binding member may inhibit the function of the ligand. It is although contemplated that such neutralising binding memers having an affinity for PYY with is less than the affinity of PYY for the NPY receptors (see tabel 1 ), may be usefull for the present invention.

Tabel 1 affinities for human PYY molecules for NPY Y1 and Y2

With out being bound by the theory a neutralising binding member may prolong the half-life of PYY, and due to the relative low affinity between PYY and the binding member, compared to the relatively high affinity between PYY and the NPY recep- tors, PYY will continously be released from the neutralising binding member and thereby free to active the appropriate receptors. In a preferred embodiment the neu¬ tralising binding member serves to protect PYY from degradation, in particular to protect PW1-36 from degradation bu DPPV-1. Such binding members preferably binds the N-terminal region of PYY1-36 as described below.

Accordingly, a neutralising binding members of the invention preferably have a dis¬ sociation constant Kd for PYY1-36, PYY3-36 or a functional equivalent thereof which is more than 1x 10"9 M. More preferably the dissociation constant Kd for PYY1 -36, or PYY3-36 or a functional equivalent thereof which is more than 5x 10"8 M, more preferably a dissociation constant Kd for PYY1-36, PYY3-36 or a func¬ tional equivalent thereof which is more than 1x 10'8 M, more preferably a dissocia¬ tion constant Kd for PYY1-36, PYY3-36 or a functional equivalent thereof which is more than 5x 10'7 M, more preferably a dissociation constant Kd for PYY1-36, PYY3-36 or a functional equivalent thereof which is more than 1x 10"7 M, more pref¬ erably a dissociation constant Kd for PYY1-36, or PYY3-36 or a functional equiva¬ lent thereof which is more than 5x 10~6 M, more preferably a dissociation constant Kd for PYY1-36, PYY3-36 or a functional equivalent thereof which is more than 1x 10"6 M.

In order to alow binding between the neutralizing binding member and PYY or the functional equivalent a binding member with a Kd for PYY1-36, PYY3-36 or a func¬ tional equivalent thereof which is less than 5 x 10"5 M, such as less than 1x 10"6 M. More preferably the dissociation constant Kd for PYY1-36, or PYY3-36 or a func- tional equivalent thereof which is less than 1x 10"7 M, more preferably a dissociation constant Kd for PYY1-36, PYY3-36 or a functional equivalent thereof which is less than 5x 10'7 M, more preferably a dissociation constant Kd for PYY1-36, PYY3-36 or a functional equivalent thereof which is less than 1x 10"8 M.

In a preferred embodiment the neutralizing binding member have an Kd for PYY1- 36, or PYY3-36 or a functional equivalent thereof of 1x 10"6 to 5x 10~9 M, such as 5x 1Q-7 to Ix IQ-8 M.

Protective binding member The binding member according to the invention is preferably a protective binding member. The protective binding member is capable of prolonging or enhancing the activity of the ligand. This is in contrast to an neutralizing binding member that may inhibit the function of its ligand. A protective binding member is expected not to interfere with binding of PYY or the functional equivalent to the receptor, or alternatively to facilitate transfer of PYY from the binding member to the receptor in case the respective affinities permit such tranfer.

In one embodiment of the invention the binding member is immunologically active, for example as an antibody or an affibody, such as being capable of binding to an antigen. In particular the invention relates to a binding member capable of recognis¬ ing and binding to a PYY1-36, PYY3-36 or a functional equivalent thereof and pref¬ erably able to bind said molecule specifically.

Accordingly, a protective binding members of the invention preferably have a disso¬ ciation constant Kd for PYY1-36, PYY3-36 or a functional equivalent thereof which is less than 1x 10"6 M. More preferably the dissociation constant Kd for PYY1-36, or PYY3-36 or a functional equivalent thereof which is less than 1x 10"7 M, more pref¬ erably a dissociation constant Kd for PYY1-36, PYY3-36 or a functional equivalent thereof which is less than 5x 10'7 M, more preferably a dissociation constant Kd for PYY1-36, PYY3-36 or a functional equivalent thereof which is less than 1x 10"8 M, more preferably a dissociation constant Kd for PYY1-36, PYY3-36 or a functional equivalent thereof which is less than 5x 10'8 M, more preferably a dissociation con¬ stant Kd for PYY1-36, or PYY3-36 or a functional equivalent thereof which is less than 1x 10"9 M, more preferably a dissociation constant Kd for PYY1-36, PYY3-36 or a functional equivalent thereof which is less than 5x 10"9 M, more preferably a disso¬ ciation constant Kd for PYY1-36, PYY3-36 or a functional equivalent thereof which is less than 1x 10-10 M.

Dissociation constants can be determined using methods well-known in the art, such as ELISA (e.g. as described in Orosz and Ovadi (2002) J. Immunol. Methods 270:155-162) or surface plasmon resonance analysis. Both, neutralizing and protective binding members may in an embodiment of the invention be an antibody, fragments of antibodies (including single chain antibodies) or other immunologically active molecule as further described here below.

In one embodiment of the invention the binding member is an antibody, such as any suitable antibody known in the art, in particular antibodies as defined herein, such as antibodies, other immunologically active fragments of antibodies or single chain an¬ tibodies. Antibody molecules are typically Y-shaped molecules whose basic unit consist of four polypeptides, two identical heavy chains and two identical light chains, which are covalently linked together by disulfide bonds. Each of these chains is folded in discrete domains. The C-terminal regions of both heavy and light chains are conserved in sequence and are called the constant regions, also known as C-dorήains. The N-terminal regions, also known as V-domains, are variable in sequence and are responsible for the antibody specificity. The antibody specifically recognizes and binds to an antigen mainly through six short complementarity- determining regions located in their V-domains.

In another embodiment of the invention the binding member is an affibody, such as any suitable affibody known in the art, in particular antibodies as defined herein, such as affibodies or immunologically fragments of affibodies. Affibodies are se¬ lected in vitro, from an affibody library constructed by combinatorial variation of the IgG binding domain of Protein A. Protein A is a surface protein from the bacterium Staphylococcus aureus. The biding domain consists of 58 residue, where of 13 are randomized to generate Affibody® libraries Thus, the size of an affibody is consid- erably less than of an antibody (www.affibody.com).

In yet another embodiment of the invention the binding member is a chimera of an antibody and an affibody. The chimera may be constructed by several methods, for example, but not exclusively the affibody may be fused with an Fc fragment, thus potentially allowing dimers to form by homodimerisation, alternatively even dimeric affibodies may be fused to the Fc fragment resulting in a tetravalent binding mem¬ ber. Multivalent In one preferred embodiment of the invention, the binding member is a multivalent binding member comprising at least two binding sites, thus in another embodiment the binding member comprises 4 binding sites, or more that 4 binding sites, such as 6, or such as 8 binding sites. The binding sites of the binding member may have identical or different binding sites

Multi-specificitv In one embodiment of the invention the binding member have multiple binding sites. The binding sites may have specificity for the same antigen or for different antigens, and thus an embodiment of the invention relates to a binding member with at least two binding sites with different specificity, thus a binding member capable of binding two different entities. The different entities may be two different antigens or two different epitopes of the same antigen.

Thus in another embodiment of the invention the binding member may be able to bind PYY1-36, PYY3-36 or a functional equivalent thereof, in addition a second, a third, and a fourth ligand. These ligands may for example be PYY1-36, PYY3-36 or functional equivalent molecules thereof.

Monoclonal/polvclonal antibodies In one embodiment of the invention the binding member is an antibody, wherein the antibody may be a polyclonal or a monoclonal antibody derived from a mammal or mixtures of monoclonal antibodies. In a preferred embodiment the binding member is a monoclonal antibody or a fragment thereof. The antibody may be any kind of antibody, however it is preferably a IgG antibody. More preferably the antibody is a IgGI antibody or a fragment thereof.

Monoclonal antibodies (Mab's) are antibodies, wherein every antibody molecule are similar and thus recognises the same epitope. Monoclonal antibodies are in general produced by a hybridoma cell line. Methods of making monoclonal antibodies and antibody-synthesizing hybridoma cells are well known to those skilled in the art. An¬ tibody producing hybridomas may for example be prepared by fusion of an antibody producing B lymphocyte with an immortalized cell line. A monoclonal antibody can be produced by the following steps. In all procedures, an animal is immunized with an antigen such as a protein (or peptide thereof) as de¬ scribed above for preparation of a polyclonal antibody. The immunization is typically accomplished by administering the immunogen to an immunologically competent mammal in an immunologically effective amount, i.e., an amount sufficient to pro¬ duce an immune response. Preferably, the mammal is a rodent such as a rabbit, rat or mouse. The mammal is then maintained on a booster schedule for a time period sufficient for the mammal to generate high affinity antibody molecules as described. A suspension of antibody-producing cells is removed from each immunized mammal secreting the desired antibody. After a sufficient time to generate high affinity anti¬ bodies, the animal (e.g., mouse) is sacrificed and antibody-producing lymphocytes are obtained from one or more of the lymph nodes, spleens and peripheral blood. Spleen cells are preferred, and can be mechanically separated into individual cells in a physiological medium using methods well known to one of skill in the art. The antibody-producing cells are immortalized by fusion to cells of a mouse myeloma line. Mouse lymphocytes give a high percentage of stable fusions with mouse ho¬ mologous myelomas, however rat, rabbit and frog somatic cells can also be used. Spleen cells of the desired antibody-producing animals are immortalized by fusing with myeloma cells, generally in the presence of a fusing agent such as polyethyl¬ ene glycol. Any of a number of myeloma cell lines suitable as a fusion partner are used with to standard techniques, for example, the P3-NS1/1-Ag4-1 , P3-x63- Ag8.653 or Sp2/O-Ag14 myeloma lines, available from the American Type Culture Collection (ATCC), Rockville, Md.

Monoclonal antibodies can also be generated by other methods well known to those skilled in the art of recombinant DNA technology. An alternative method, referred to as the "combinatorial antibody display" method, has been developed to identify and isolate antibody fragments having a particular antigen specificity, and can be utilized to produce monoclonal antibodies.

Polyclonal antibodies is a mixture of antibody molecules recognising a specific given antigen, hence polyclonal antibodies may recognise different epitopes within said antigen. In general polyclonal antibodies are purified from serum of a mammal, which previously has been immunized with the antigen. Polyclonal antibodies may for example be prepared by any of the methods described in Antibodies: A Labora¬ tory Manual, By Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, 1988. Polyclonal antibodies may be derived from any suitable mammalian species, for example from mice, rats, rabbits, donkeys, goats, and sheep.

An embodiment of the invention relates to a composition wherein the binding mem¬ ber is selected from monoclonal antibodies, polyclonal antibodies or a mixture of monoclonal antibodies.

The antibodies may be commercially available such as the antibodies produced by Phoenix Pharmaceuticals and Research Diagnostics Inc.

Antibody fragments In one embodiment of the invention the binding member is a fragment of an anti- body, preferably an antigen binding fragment or a variable region. Examples of anti¬ body fragments useful with the present invention include Fab, Fab', F(ab')2 and Fv fragments. Papain digestion of antibodies produces two identical antigen binding fragments, called the Fab fragment, each with a single antigen binding site, and a residual "Fc" fragment, so-called for its ability to crystallize readily. Pepsin treatment yields an F(ab') 2 fragment that has two antigen binding fragments which are capable of cross-linking antigen, and a residual other fragment (which is termed pFc').

Additional fragments can include diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

The antibody fragments Fab, Fv and scFv differ from whole antibodies in that the antibody fragments carry only a single antigen-binding site. Recombinant fragments with two binding sites have been made in several ways, for example, by chemical cross-linking of cysteine residues introduced at the C-terminus of the VH of an Fv (Cumber et al., 1992), or at the C-terminus of the VL of an scFv (Pack and Pluck- thun, 1992), or through the hinge cysteine residues of Fab's (Carter et al., 1992).

Preferred antibody fragments retain some or essential all the ability of an antibody to selectively binding with its antigen or receptor. Some preferred fragments are de- fined as follows: Fab is the fragment that contains a monovalent antigen-binding fragment of an anti¬ body molecule. A Fab fragment can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain.

Fab' is the fragment of an antibody molecule and can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a por¬ tion of the heavy chain. Two Fab' fragments are obtained per antibody molecule. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region.

(Fab')2 is the fragment of an antibody that can be obtained by treating whole anti¬ body with the enzyme pepsin without subsequent reduction. F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds.

Fv is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in a tight, non-covalent association (VH -V L dimer). It is in this con- figuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH -V L dimer. Collectively, the six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

In one embodiment of the present invention the antibody is a single chain antibody ("SCA"), defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable poly- peptide linker as a genetically fused single chain molecule. Such single chain anti¬ bodies are also refered to as "single-chain Fv" or "sFv" antibody fragments. Gener¬ ally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the sFv to form the desired structure for antigen binding. The antibody fragments according to the invention may be produced in any suitable manner known to the person skilled in the art. Several microbial expression systems have already been developed for producing active antibody fragments, e.g. the pro¬ duction of Fab in various hosts, such as E. coli (Better et al., 1988, Skerra and Pluckthun, 1988, Carter et al., 1992), yeast (Horwitz et al., 1988), and the filamen¬ tous fungus Trichoderma reesei (Nyyssonen et al., 1993) has been described. The recombinant protein yields in these alternative systems can be relatively high (1-2 g/l for Fab secreted to the periplasmic space of E. coli in high cell density fermentation, see Carter et al., 1992), or at a lower level, e.g. about 0.1 mg/l for Fab in yeast in fermenters (Horwitz et al., 1988), and 150 mg/l for a fusion protein CBHI-Fab and 1 mg/l for Fab in Trichoderma in fermenters (Nyyssonen et al., 1993) and such pro¬ duction is very cheap compared to whole antibody production in mammalian cells (hybridoma, myeloma, CHO).

The fragments can be produced as Fab's or as Fv's, but additionally it has been shown that a VH and a VL can be genetically linked in either order by a flexible polypeptide linker, which combination is known as an scFv.

Natural single domain antibodies Heavy-chain antibodies (HCAbs) are naturally produced by camelids (camels, dromedaries and llamas). HCAbs are homodimers of heavy chains only, devoid of light chains and the first constant domain (Hamers-Casterman et al., 1993). The possibility to immunise these animals allows for the cloning, selection and produc¬ tion of an antigen binding unit consisting of a single-domain only. Furthermore these minimal-sized antigen binding fragments are well expressed in bacteria, inter¬ act with the antigen with high affinity and are very stable.

New or Nurse Shark Antigen Receptor (NAR) protein exists as a dimer of two heavy chains with no associated light chains. Each chain is composed of one variable (V) and five constant domains. The NAR proteins constitute a single immunoglobulin variable-like domain (Greenberg, A. S., Avila, D., Hughes, M., Hughes, A., McKin- ney, E. C. & Flajnik, M. F. (1995) Nature (London) 374, 168-173.) which is much lighter than an antibody molecule. According to the invention natural single domain antibodies may be considered an antibody fragment. The proteins may be produced and purified by any suitable method know by a person skilled in the art as described above.

In a further embodiment the binding member is active fragments of antibodies se¬ lected from Fab, Fab', F(ab)2, Fv, HCAbs and NARs.

Humanised antibody It is not always desirable to use non-human antibodies for human therapy, since the non-human "foreign" epitopes may elicit immune response in the individual to be treated. To eliminate or minimize the problems associated with non-human antibod¬ ies, it is desirable to engineer chimeric antibody derivatives, i.e., "humanized" anti¬ body molecules that combine the non-human Fab variable region binding determi¬ nants with a human constant region (Fc). Such antibodies are characterized by equivalent antigen specificity and affinity of the monoclonal and polyclonal antibod¬ ies described above, and are less immunogenic when administered to humans, and therefore more likely to be tolerated by the individual to be treated.

Accordingly, in one embodiment the binding may be a humanised antibody.

Humanised antibodies are in general chimeric antibodies comprising regions derived from a human antibody and regions derived from a non-human antibody, such as a rodent antibody. Humanisation (also called Reshaping or CDR-grafting) is a well- established technique for reducing the immunogenicity of monoclonal antibodies (mAbs) from xenogeneic sources (commonly rodent), increasing the homology to a human immunoglobulin, and for improving their activation of the human immune system. Thus, humanized antibodies are typically human antibodies in which some CDR residues and possibly some framework residues are substituted by residues from analogous sites in rodent antibodies.

It is further important that humanized antibodies retain high affinity for the antigen and other favourable biological properties. To achieve this goal, according to a pre¬ ferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three- dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of certain residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is maximized, although it is the the CDR residues that directly and most substantially influence antigen binding.

One method for humanising MAbs related to production of chimeric antibodies in which an antigen binding site comprising the complete variable domains of one anti¬ body are fused to constant domains derived from a second antibody, preferably a human antibody. Methods for carrying out such chimerisation procedures are for example described in EP-A-O 120 694 (Celltech Limited), EP-A-O 125 023 (Genen- tech Inc.), EP-A-O 171 496 (Res. Dev. Corp. Japan), EP-A-0173494 (Stanford Uni¬ versity) and EP-A-O 194 276 (Celltech Limited). A more complex form of humanisa- tion of an antibody involves the re-design of the variable region domain so that the amino acids constituting the non-human antibody binding site are integrated into the framework of a human antibody variable region (Jones et al., 1986).

The humanized antibody of the present invention may be made by any method ca¬ pable of replacing at least a portion of a CDR of a human antibody with a CDR de- rived from a non-human antibody. Winter describes a method which may be used to prepare the humanized antibodies of the present invention (UK Patent Application GB 2188638A, filed on Mar. 26, 1987), the contents of which is expressly incorpo¬ rated by reference. The human CDRs may be replaced with non-human CDRs using oligonucleotide site-directed mutagenesis as described in the examples below.

As an example the humanized antibody of the present invention may be made as described in the brief explanation below. The humanized antibodies of the present invention may be produced by the following process: (a) constructing, by conventional techniques, an expression vector containing an operon with a DNA sequence encoding an antibody heavy chain in which the CDRs and such minimal portions of the variable domain framework region that are required to retain antibody binding specificity are derived from a non-human immunoglobulin, and the remaining parts of the antibody chain are derived from a human immunoglobulin, thereby producing the vector of the invention;

(b) constructing, by conventional techniques, an expression vector containing an operon with a DNA sequence encoding a complementary antibody light chain in which the CDRs and such minimal portions of the variable domain framework region that are required to retain donor antibody binding specificity are derived from a non-human immunoglobulin, and the remaining parts of the antibody chain are derived from a human immunoglobulin, thereby producing the vector of the invention;

(c) transfecting the expression vectors into a host cell by conventional techniques to produce the transfected host cell of the invention; and

(d) culturing the transfected cell by conventional techniques to produce the human- ised antibody of the invention.

The host cell may be cotransfected with the two vectors of the invention, the first vector containing an operon encoding a light chain derived polypeptide and the sec¬ ond vector containing an operon encoding a heavy chain derived polypeptide. The two vectors contain different selectable markers, but otherwise, apart from the anti¬ body heavy and light chain coding sequences, are preferably identical, to ensure, as far as possible, equal expression of the heavy and light chain polypeptides. Alterna¬ tively, a single vector may be used, the vector including the sequences encoding both the light and the heavy chain polypeptides. The coding sequences for the light and heavy chains may comprise cDNA or genomic DNA or both.

The host cell used to express the altered antibody of the invention may be either a bacterial cell such as Escherichia coli, or a eukaryotic cell. In particular a mammal¬ ian cell of a well defined type for this purpose, such as a myeloma cell or a Chinese hamster ovary cell may be used. The general methods by which the vectors of the invention may be constructed, transfection methods required to produce the host cell of the invention and culture methods required to produce the antibody of the invention from such host cells are all conventional techniques. Likewise, once produced, the humanized antibodies of the invention may be purified according to standard procedures as described below.

Human antibody framework In a more preferred embodiment the invention relates to a binding member, wherein the binding domain is carried by a human antibody framework, i.e. wherein the anti¬ bodies have a greater degree of human peptide sequences than do humanised an¬ tibodies.

Human mAb antibodies directed against human proteins can be generated using transgenic mice carrying the complete human immune system rather than the mouse system. Splenocytes from these transgenic mice immunized with the antigen of interest are used to produce hybridomas that secrete human mAbs with specific affinities for epitopes from a human protein (see, e.g., Wood et al. International Application WO 91/00906, Kucherlapati et al. PCT publication WO 91/10741 ; Lonberg et al. International Application WO 92/03918; Kay et al. International Application 92/03917; Lonberg, N. et al. 1994 Nature 368:856-859; Green, L. L. et al. 1994 Nature Genet. 7:13-21 ; Morrison, S. L. et al. 1994 Proc. Natl. Acad. Sci. USA 81 :6851-6855; Bruggeman et al. 1993 Year Immunol 7:33-40; Tuaillon et al. 1993 PNAS 90:3720-3724; Bruggeman et al. 1991 Eur J Immunol 21 :1323-1326).

Such transgenic mice are available from Abgenix, Inc., Fremont, Calif., and Me- darex, Inc., Annandale, N.J. It has been described that the homozygous deletion of the antibody heavy-chain joining region (IH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Ja- kobovits et al., Proc. Natl. Acad. Sci. USA 90:2551 (1993); Jakobovits et al., Nature 362:255-258 (1993); Bruggermann et al., Year in Immunol. 7:33 (1993); and Duchosal et al. Nature 355:258 (1992). Human antibodies can also be derived from phage-display libraries (Hoogenboom et al ., J. MoI. Biol. 227: 381 (1991 ); Marks et al., J. MoI. Biol. 222:581-597 (1991); Vaughan, et al., Nature Biotech 14:309 (1996)).

Non-immonoqlobulin binding members In one preferred embodiment, the present invention relates to binding members derived from a naturally occurring protein or polypeptide; said protein or polypeptide may for example be designed de novo, or may be selected from a library. The binding member may be a single moiety, e.g., a polypeptide or protein domain, or it may include two or more moieties, e.g., a pair of polypeptides such as a pair polypeptides. The binding member may for example, but exclusively, be a lipocalin, a single chain MHC molecule, an Anticalin™ (Pieris), an Affibody™, or a Trinectin™ (Phylos), Nanobodies (Ablynx). The binding member may be selected or designed by recombinant methods known by people well known in the art.

Affibodv In one embodiment of the present invention, said binding member is an affibody. Affibodies are produced recombinantly by methods well known to those skilled in the art of recombinant DNA technology. Phage display techniques may be used to identify affibodies capable of specifically recognising a particular antigen. Affibodies can be produced in any suitable host, as for example, but not exclusively £. coli or S. cerevisiae (se below) (Hansson M et al., "An in vitro selected binding protein (affibody) shows conformation-dependent recognition of the respiratory syncytial virus (RSV) G protein", Immunotechnology. 1999 Mar; 4(3-4): 237-52.)

An embodiment of the invention relates to a composition wherein the binding member is an affibody or a mixture of affibodies.

Affibody-antibodv chimeras In another embodiment of the present invention, said binding member is an affibody- antibody chimera (Ronnmark J et al, Construction and characterization of affibody- Fc chimeras produced in Escherichia coli. J Immunol Methods. 2002 Mar 1 ; 261(1- 2): 199-211). According to the invention affibody-antibody chimeras can be con¬ structed by several methods, for example by fusion of nucleotide sequences or fu¬ sion of polypeptide sequences. The nucleic acid sequence of an affibody maybe fused to a nucleic acid sequence of an antibody by DNA recombinant technology for the production of the binding member in a suitable host. The affibody nucleotide sequences may for example be fused to an antibody light chain nucleotide se¬ quence or an antibody heavy chain nucleic acid sequence. In an embodiment of the invention the affibody sequence may be fused with a fragment of an antibody se- quences. The affibody sequence may for example, but not exclusively, be fused with an Fc fragment of an antibody, thus potentially allowing dimers to form by homo- dimerisation. The affibody antibody chimeras may contain multiple affibody se¬ quences, such as at least two, three, four of at least six affibody sequences. In an embodiment of the invention a fusion of two affibodies may be fused with an Fc fragment resulting in a tetravalent binding member upon dimerisation.

Alternatively the chimeras may be obtained by linking of the two protein/polypeptide molecules together by methods known to people skilled in the art.

In a further embodiment of the invention the binding member may be an antibody- affibody chimera or a mixture of antibody-affibody chimeras.

In a preferredn embodiment the binding member binds to an epitope within amino acids corresponding to the 6 N-terminal amino acids of PYY1-36 as defined in SEQ ID NO: .1 (Tyr Pro lie Lys Pro GIu). The a further embodiment the binding member binds to an epitope within the 8 N-terminal amino acids of PYY1-36 as defined in SEQ ID (Tyr Pro lie Lys Pro GIu Ala Pro), or such as within the 10 N-terminal amino acids of PYY1-36 as defined in SEQ ID (Tyr Pro lie Lys Pro GIu Ala Pro GIy GIu) or such as within the 12 N-terminal amino acids of PYY1-36 as defined in SEQ ID (Tyr Pro Ne Lys Pro GIu Ala Pro GIy GIu Asp Ala), or such as within the 14 N-terminal amino acids of PYY1-36 as defined in SEQ ID (Tyr Pro lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro) ), or such as within the 16 N-terminal amino acids of PYY1-36 as defined in SEQ ID (Tyr Pro lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu), or such as within the 18 N-terminal amino acids of PYY1-36 as defined in SEQ ID (Tyr Pro lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn), or such as within the 20 N-terminal amino acids of PYY1-36 as defined in SEQ ID (Tyr Pro lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr), or such as within the 22 N-terminal amino acids of PYY1-36 as defined in SEQ ID (Tyr Pro lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala), or such as within the 24 N-terminal amino acids of PYY1-36 as defined in SEQ ID (Tyr Pro Ne Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu), or such as within the 26 N-terminal amino acids of PYY1-36 as defined in SEQ ID (Tyr Pro lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His), or such as within the 28 N-terminal amino acids of PYY1-36 as defined in SEQ ID (Tyr Pro lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu), or such as within the 30 N-terminal amino acids of PYY1-36 as defined in SEQ ID (Tyr Pro lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu), or such as within the 32 N-terminal amino acids of PYY1-36 as defined in SEQ ID (Tyr Pro lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu VaI Thr), or such as within the 34 N-terminal amino acids of PYY1-36 as defined in SEQ ID (Tyr Pro lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu VaI Thr Arg GIn).

In a further preferredn embodiment the binding member binds an epitope within the 6 N-terminal amino acids of PYY3-36 as defined in SEQ ID NO: .1 (He Lys Pro GIu Ala Pro), or such as within the 8 N-terminal amino acids of PYY3-36 as defined in SEQ ID (lie Lys Pro GIu Ala Pro GIy GIu) or such as within the 10 N-terminal amino acids of PYY3-36 as defined in SEQ ID (lie Lys Pro GIu Ala Pro GIy GIu Asp Ala), or such as within the 12 N-terminal amino acids of PYY3-36 as defined in SEQ ID (lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro) ), or such as 14 within the N-terminal amino acids of PYY3-36 as defined in SEQ ID (lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu), or such as 16 within the N-terminal amino acids of PYY3-36 as defined in SEQ ID (lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn), or such as 18 within the N-terminal amino acids of PYY3-36 as defined in SEQ ID (lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr), or such as 20 within the N-terminal amino acids of PYY3-36 as defined in SEQ ID (lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala), or such as within the 22 N-terminal amino acids of PYY3-36 as defined in SEQ ID (lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu), or such as within the 24 N-terminal amino acids of PYY3-36 as defined in SEQ ID (Tyr Pro lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His), or such as within the 26 N-terminal amino acids of PYY3-36 as defined in SEQ ID (lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu), or such as within the 28 N- terminal amino acids of PYY3-36 as defined in SEQ ID (lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu), or such as 30 within the N-terminal amino acids of PYY3-36 as defined in SEQ ID (lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu VaI Thr), or such as within the 32 N-terminal amino acids of PYY3-36 as defined in SEQ ID (lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu VaI Thr Arg GIn).

In an preferred embodiment the binding member binds an epitope within the amino acids corresponding to the 6 C- terminal amino acids of PYY1-36 as defined in SEQ ID NO: 1 (VaI Thr Arg GIn Arg Tyr), or such as within the 8 C-terminal amino acids of PYY1-36 as defined in SEQ ID NO 1 (Asn Leu VaI Thr Arg GIn Arg Tyr), or such as the 10 C-terminal amino acids of PYY1-36 as defined in SEQ ID NO 1 (Tyr Leu Asn Leu VaI Thr Arg GIn Arg Tyr), or such as within the 12 C-terminal amino acids of PYY1-36 as defined in SEQ ID NO 1 (Arg His Tyr Leu Asn Leu VaI Thr Arg GIn Arg Tyr), or such as within the 14 C- terminal amino acids of PYY1-36 as defined in SEQ ID NO 1 (Ser Leu Arg His Tyr Leu Asn Leu VaI Thr Arg GIn Arg Tyr), or such as within the 16 C- terminal amino acids of PYY1-36 as defined in SEQ ID NO 1 (Ser Leu Arg His Tyr Leu Asn Leu VaI Thr Arg GIn Arg Tyr Tyr Ala), or such as within the 18 C-terminal amino acids of PYY1-36 as defined in SEQ ID NO 1 (Arg Tyr Ser Leu Arg His Tyr Leu Asn Leu VaI Thr Arg GIn Arg Tyr Tyr Ala), or such as within the 20 C-terminal amino acids of PYY1-36 as defined in SEQ ID NO 1 (Leu Asn Arg Tyr Ser Leu Arg His Tyr Leu Asn Leu VaI Thr Arg GIn Arg Tyr Tyr Ala), or such as within the 22 C-terminal amino acids of PYY1-36 as defined in SEQ ID NO 1 (GIu GIu Leu Asn Arg Tyr Ser Leu Arg His Tyr Leu Asn Leu VaI Thr Arg GIn Arg Tyr Tyr Ala), or such as within the 24 C-terminal amino acids of PYY1-36 as defined in SEQ ID NO 1 (Ser Pro GIu GIu Leu Asn Arg Tyr Ser Leu Arg His Tyr Leu Asn Leu VaI Thr Arg GIn Arg Tyr Tyr Ala), or such as within the 26 C-terminal amino acids of PYY1-36 as defined in SEQ ID NO 1 (Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Ser Leu Arg His Tyr Leu Asn Leu VaI Thr Arg GIn Arg Tyr Tyr Ala), or such as within the 28 C-terminal amino acids of PYY1-36 as defined in SEQ ID NO 1 (GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Ser Leu Arg His Tyr Leu Asn Leu VaI Thr Arg GIn Arg Tyr Tyr Ala), or such as within the 30 C-terminal amino acids of PYY1-36 as defined in SEQ ID NO 1 (Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Ser Leu Arg His Tyr Leu Asn Leu VaI Thr Arg GIn Arg Tyr Tyr Ala), or such as within the 32 C-terminal amino acids of PYY1-36 as defined in SEQ ID NO 1 (Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Ser Leu Arg His Tyr Leu Asn Leu VaI Thr Arg GIn Arg Tyr Tyr Ala), or such as within the 34 C-terminal amino acids of PYY1-36 as defined in SEQ ID NO 1 (Tyr Ala lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Ser Leu Arg His Tyr Leu Asn Leu VaI Thr Arg GIn Arg Tyr).

In another preferred embodiment the binding member binds an epitope with in the internal amino acids of PYY1-36 such as amino acid 16-21 of PYY1-36 as defined in sequence ID NO: 1 (GIu Leu Asn Arg Tyr Tyr ), or such as within amino acid 15-22 of PYY1-36 as defined in sequence ID NO 1 (GIu GIu Leu Asn Arg Tyr Tyr Ala), or such as within amino acid 14-23 of PYY1-36 as defined in sequence ID NO 1 (Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser), or such as within amino acid 13-24 of PYY1 - 36 as defined in sequence ID NO 1 (Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu), or such as within amino acid 12-25 of PYY1-36 as defined in sequence ID NO 1 (Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg), or such as within amino acid 11-26 of PYY1-36 as defined in sequence ID NO 1 (Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His), or such as within amino acid 10-27 of PYY1-36 as defined in sequence ID NO 1 (GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr), or such as within amino acid 9-28 of PYY1-36 as defined in sequence ID NO 1 (GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu), or such as within amino acid 8-29 of PYY1-36 as defined in sequence ID NO 1 (Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn), or such as within amino acid 7-30 of PYY1-36 as defined in sequence ID NO 1 (Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu), or such as within amino acid 6- 31 of PYY1-36 as defined in sequence ID NO 1 (GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu VaI), or such as within amino acid 5-32 of PYY1-36 as defined in sequence ID NO 1 (Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu VaI Thr), or such as within amino acid 4-33 of PYY1-36 as defined in sequence ID NO 1 (Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu VaI Thr Arg), or such as within amino acid 3-34 of PYY1-36 as defined in sequence ID NO 1 (lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu VaI Thr Arg GIn), or such as within amino acid 2-35 of PYY1-36 as defined in sequence ID NO 1 (Pro lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu VaI Thr Arg GIn Arg).

In a preferred embodiment of the present invention the binding member binds to an epitope present in the N-terminal part of PYY1-36 defined as AA 1-18, or such as AA 1-10, or such as AA 1-7 of sequence ID NO:no. 1. In another preferred embodiment of the present invention the binding member binds to an epitope present in the N-terminal part of PYY3-36 defined as AA 3-20, such as AA 3-16, such as AA 3-12 such as AA 3-9 of sequence ID no. 1. In a preferred embodiment of the present invention the binding member binds to an epitope present in the C- terminal part of PYY1 -36 or PYY3-36 defined as AA 19-36, or such as AA 22-36, or such as AA 28-36 of sequence ID no. 1.

In a particularly preferred embodiment a neutralising binding member binds an epitope present in the N-terminal region of PYY1-36 defined as AA 1-15, or such as AA 1 -12, or such as AA 1 -8, or such as AA1 -6 of sequence ID NO: 1.

In a mostfurther preferred embodiment of the present invention the binding member, being a protective binding memeber binds to an epitope present in the middlean internal region of PYY1-36 or PYY3-36 defined as AA 64-30, or such as AA 96- 2722, or such as AA 126-2418, or such as AA 15-206-13 of sequence ID NO:no. 1.

In a preferred embodiment the binding member binds to a non-continous epitope on PYY1-36 or PYY3-36, such as an epitope that comprises sequences present in any two of the regions defined above.

For emproving the efficiency of treatment the composition may comprise a second active ingredient. This second active ingredient may be PYY or a functional equivalent thereof as described here below. In a further embodimeint the binding member binds to a functional equivalent know from the literature, such as the PYY agonists described in WO 03/057235 and references therein and futher functional equivalents as described herebelow.

Functional equivalents The isolated binding member of the pharmaceutical compositions of the present invention is capable of binding PYY or a functional equivalent thereof. The binding member is preferably an essentially pure isolated binding member.

Human peptide YY1-36 is identified by SEQ ID NO:1. PYY3-36 is a truncated form of PYY1-36 where the two most N-terminal residues are deleted. Functional equivalents of PYY1-36 and PYY3-36 may include molecules originating from different species, such as mouse, rat, monkey, swine, bovine or other mammalian species. A functional equivalent may be a homologue to PYY1-36 or PYY3-36. For simplicity the term PYY relates to both PYY1-36 and PYY3-36 in the current document.

In an embodiment of the invention the binding member is capable of binding PYY. In an embodiment of the invention the binding member is capable of binding PYY1-36 specifically. In a different embodiment of the invention the binding member is capable of binding PYY3-36 specifically.

Homologues A homologue shall be construed as a molecule which shares some identity to the molecule, here PYY1-36 and PYY3-36. The homology may be expressed as the percentage of amino acid residues in the candidate sequence that are identical with the residue of a corresponding sequence to which it is compared, after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent identity for the entire sequence, and not considering any conservative substitutions as part of the sequence identity. Neither N- or C-terminal extensions nor insertions shall be construed as reducing identity or homology. Methods and computer pro¬ grams for the alignment are well known in the art. Sequence identity may be meas¬ ured using sequence analysis software (e.g., Sequence Analysis Software Package, Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Ave., Madison, Wis. 53705). This software matches similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifi¬ cations.

A homologue of one or more of the sequences specified herein may vary in one or more amino acids as compared to the sequences defined, but is capable of perform¬ ing the same function, i.e. a homologue may be envisaged as a "functional equiva¬ lent" of a predetermined sequence.

As described above a, functional equivalent of any of the predetermined sequences herein may be defined as:

i) homologues comprising an amino acid sequence capable of being recog¬ nised by an antibody, said antibody also recognising PYY1-36 or PYY3-36, and/or

ii) homologues comprising an amino acid sequence capable of binding selec¬ tively to an NPY receptor, and/or

iii) homologues having a substantially similar or higher binding affinity to NPY receptors than PYY1-36 or PYY3-36, and/or

iv) .homologues with at least 60 % identity to human PYY identified by SEQ ID NO 1 , and/or

v) homologues consisting of fragments of human PYY identified by SEQ ID NO 1 , wherein the fragments comprise a strech of at least 6 continuous amino acids of SEQ ID NO L

In the above examples, PYY1-36 has the sequence shown in SEQ ID NO: 1. PYY3- 36 is 34 amino acids long and has the sequence shown in SEQ ID NO: 1 except for the deletion of the two N-terminal amino acids.

Examples of homologues may comprise one or more conservative amino acid sub¬ stitutions, including one or more conservative amino acid substitutions within the same group of predetermined amino acids, or a plurality of conservative amino acid substitutions, wherein each conservative substitution is generated by substitution within a different group of predetermined amino acids.

Homologues may thus comprise conservative substitutions independently of one another, wherein at least one glycine (GIy) of said homologue is substituted with an amino acid selected from the group of amino acids consisting of Ala, VaI, Leu, and lie, and independently thereof, homologues, wherein at least one of said alanines (Ala) of said homologue thereof is substituted with an amino acid selected from the group of amino acids consisting of GIy, VaI, Leu, and lie, and independently thereof, homologues, wherein at least one valine (VaI) of said homologue thereof is substi¬ tuted with an amino acid selected from the group of amino acids consisting of GIy, Ala, Leu, and lie, and independently thereof, homologues thereof, wherein at least one of said leucines (Leu) of said homologue thereof is substituted with an amino acid selected from the group of amino acids consisting of GIy, Ala, VaI, and He, and independently thereof, homologues thereof, wherein at least one isoleucine (He) of said homologues thereof is substituted with an amino acid selected from the group of amino acids consisting of GIy, Ala, VaI and Leu, and independently thereof, homologues thereof wherein at least one of said aspartic acids (Asp) of said homo¬ logue thereof is substituted with an amino acid selected from the group of amino acids consisting of GIu, Asn, and GIn, and independently thereof, homologues thereof, wherein at least one of said phenylalanines (Phe) of said homologues thereof is substituted with an amino acid selected from the group of amino acids consisting of Tyr, Trp, His, Pro, and preferably selected from the group of amino acids consisting of Tyr and Trp, and independently thereof, homologues thereof, wherein at least one of said tyrosines (Tyr) of said homologues thereof is substituted with an amino acid selected from the group of amino acids consisting of Phe, Trp, His, Pro, preferably an amino acid selected from the group of amino acids consisting of Phe and Trp, and independently thereof, homologues thereof, wherein at least one of said arginines (Arg) of said fragment is substituted with an amino acid se- lected from the group of amino acids consisting of Lys and His, and independently thereof, homologues thereof, wherein at least one lysine (Lys) of said homologues thereof is substituted with an amino acid selected from the group of amino acids consisting of Arg and His, and independently thereof, homologues thereof, wherein at least one of said aspargines (Asn) of said homologues thereof is substituted with an amino acid selected from the group of amino acids consisting of Asp, GIu, and GIn, and independently thereof, homologues thereof, wherein at least one glutamine (GIn) of said homologues thereof is substituted with an amino acid selected from the group of amino acids consisting of Asp, GIu, and Asn, and independently thereof, homologues thereof, wherein at least one proline (Pro) of said homologues thereof is substituted with an amino acid selected from the group of amino acids consisting of Phe, Tyr, Trp, and His, and independently thereof, homologues thereof, wherein at least one of said cysteines (Cys) of said homologues thereof is substituted with an amino acid selected from the group of amino acids consisting of Asp, GIu, Lys, Arg, His, Asn, GIn, Ser, Thr, and Tyr.

Conservative substitutions may be introduced in any position of a preferred prede¬ termined sequence. It may however also be desirable to introduce non-conservative substitutions, particularly, but not limited to, a non-conservative substitution in any one or more positions.

A non-conservative substitution leading to the formation of a functionally equivalent homologue of the sequences herein would for example i) differ substantially in polar¬ ity, for example a residue with a non-polar side chain (Ala, Leu, Pro, Trp, VaI, lie, Leu, Phe or Met) substituted for a residue with a polar side chain such as GIy, Ser, Thr, Cys, Tyr, Asn, or GIn or a charged amino acid such as Asp, GIu, Arg, or Lys, or substituting a charged or a polar residue for a non-polar one; and/or ii) differ sub¬ stantially in its effect on polypeptide backbone orientation such as substitution of or for Pro or GIy by another residue; and/or iii) differ substantially in electric charge, for example substitution of a negatively charged residue such as GIu or Asp for a posi- tively charged residue such as Lys, His or Arg (and vice versa); and/or iv) differ sub¬ stantially in steric bulk, for example substitution of a bulky residue such as His, Trp, Phe or Tyr for one having a minor side chain, e.g. Ala, GIy or Ser (and vice versa).

Substitution of amino acids may in one embodiment be made based upon their hy- drophobicity and hydrophilicity values and the relative similarity of the amino acid side-chain substituents, including charge, size, and the like. Exemplary amino acid substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine. In a preferred embodiment the functional equivalent comprises a an amino acid se¬ quence of at least 60 % homologous to SEQ ID NO 1.

More preferably the homology identity is at least 65 %, such as at least 70 % ho- mologousidentical, such as at least 75 % homologousidentical, such as at least 80 % identicalhomologous, such as at least 85 % homologousidentical, such as at least 90 % homologousidentical, such as at least 95 % homologousidentical, such as at least 98 % homologous identical to SEQ ID NO 1.

In a more preferred embodiment the percentages mentioned above relates to the identity of the sequence of a homologue as compared to SEQ ID NO 1.

In a preferred embodiment the functional equivalent comprise the amino acids corresponding to the 6 N- terminal amino acids of PYY1-36 as defined in SEQ ID NO.1 (Tyr Pro lie Lys Pro GIu). The functional equivalent may comprise 8 N-terminal amino acids of PYY1-36 as defined in SEQ ID NO 1 (Tyr Pro lie Lys Pro GIu Ala Pro), or such as 10 N-terminal amino acids of PYY1-36 as defined in SEQ ID NO 1 (Tyr Pro lie Lys Pro GIu Ala Pro GIy GIu) or such as 12 N-terminal amino acids of PYY1-36 as defined in SEQ ID NO 1 (Tyr Pro lie Lys Pro GIu Ala Pro GIy GIu Asp Ala), or such as 14 N-terminal amino acids of PYY1-36 as defined in SEQ ID NO 1 (Tyr Pro lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro) ), or such as 16 N- terminal amino acids of PYY1-36 as defined in SEQ ID NO 1 (Tyr Pro lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu), or such as 18 N-terminal amino acids of PYY1-36 as defined in SEQ ID NO 1 (Tyr Pro lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn), or such as 20 N-terminal amino acids of PYY1- 36 as defined in SEQ ID NO 1 (Tyr Pro lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr), or such as 22 N-terminal amino acids of PYY1-36 as defined in SEQ ID NO 1 (Tyr Pro Ne Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala), or such as 24 N-terminal amino acids of PYY1-36 as defined in SEQ ID NO 1 (Tyr Pro lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu), or such as 26 N-terminal amino acids of PYY1-36 as defined in SEQ ID NO 1 (Tyr Pro He Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His), or such as 28 N- terminal amino acids of PYY1-36 as defined in SEQ ID NO 1 (Tyr Pro lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu), or such as 30 N-terminal amino acids of PYY1-36 as defined in SEQ ID NO 1 (Tyr Pro lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu), or such as 32 N-terminal amino acids of PYY1-36 as defined in SEQ ID NO 1 (Tyr Pro lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu VaI Thr), or such as 34N-terminal amino acids of PYY1-36 as defined in.SEQ ID NO 1 (Tyr Pro lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu VaI Thr Arg GIn).

In a further preferred embodiment the functional equivalent comprise the amino acids corresponding to the 6 N- terminal amino acids of PYY3-36 as defined in SEQ ID NO.1 (lie Lys Pro GIu Ala Pro), or such as 8 N-terminal amino acids of PYY3-36 as defined in SEQ ID NO 1 (lie Lys Pro GIu Ala Pro GIy GIu) or such as 10 N- terminal amino acids of PYY3-36 as defined in SEQ ID NO 1 (lie Lys Pro GIu Ala Pro GIy GIu Asp Ala), or such as 12 N-terminal amino acids of PYY3-36 as defined in SEQ ID NO 1 (lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro) ), or such as 14 N-terminal amino acids of PYY3-36 as defined in SEQ ID NO 1 (lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu), or such as 16 N-terminal amino acids of PYY3-36 as defined in SEQ ID NO 1 (He Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn), or such as 18 N-terminal amino acids of PYY3-36 as defined in SEQ ID NO 1 (lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr), or such as 20 N-terminal amino acids of PYY3-36 as defined in SEQ ID NO 1 (lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala), or such as 22 N-terminal amino acids of PYY3-36 as defined in SEQ ID NO 1 (lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu), or such as 24 N-terminal amino acids of PYY3-36 as defined in SEQ ID NO 1 (Tyr Pro lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His), or such as 26 N-terminal amino acids of PYY3-36 as defined in SEQ ID NO 1 (lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu), or such as 28 N-terminal amino acids of PYY3-36 as defined in SEQ ID NO 1 (lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu), or such as 30 N-terminal amino acids of PYY3-36 as defined in SEQ ID NO 1 (lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu VaI Thr), or such as 32 N-terminal amino acids of PYY3-36 as defined in SEQ ID NO 1 (lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu VaI Thr Arg GIn).

In a preferred embodiment the functional equivalent comprise the amino acids corresponding to the 6 C- terminal amino acids of PYY1-36 as defined in SEQ ID NO 1 (VaI Thr Arg GIn Arg Tyr), or such as the 8 C-terminal amino acids of PYY1-36 as defined in SEQ ID NO 1 (Asn Leu VaI Thr Arg GIn Arg Tyr), or such as the 10 C- terminal amino acids of PYY1-36 as defined in SEQ ID NO 1 (Tyr Leu Asn Leu VaI Thr Arg GIn Arg Tyr), or such as the 12 C-terminal amino acids of PYY1-36 as defined in SEQ ID NO 1 (Arg His Tyr Leu Asn Leu VaI Thr Arg GIn Arg Tyr), or such as the 14 C- terminal amino acids of PYY1-36 as defined in SEQ ID NO 1 (Ser Leu Arg His Tyr Leu Asn Leu VaI Thr Arg GIn Arg Tyr), or such as the 16 C- terminal amino acids of PYY1-36 as defined in SEQ ID NO 1 (Ser Leu Arg His Tyr Leu Asn Leu VaI Thr Arg GIn Arg Tyr Tyr Ala), or such as the 18 C-terminal amino acids of PYY1-36 as defined in SEQ ID NO 1 (Arg Tyr Ser Leu Arg His Tyr Leu Asn Leu VaI Thr Arg GIn Arg Tyr Tyr Ala), or such as the 20 C- terminal amino acids of PYY1-36 as defined in SEQ ID NO 1 (Leu Asn Arg Tyr Ser Leu Arg His Tyr Leu Asn Leu VaI Thr Arg GIn Arg Tyr Tyr Ala), or such as the 22 C-terminal amino acids of PYY1-36 as defined in SEQ ID NO 1 (GIu GIu Leu Asn Arg Tyr Ser Leu Arg His Tyr Leu Asn Leu VaI Thr Arg GIn Arg Tyr Tyr Ala), or such as the 24 C-terminal amino acids of PYY1-36 as defined in SEQ ID NO 1 (Ser Pro GIu GIu Leu Asn Arg Tyr Ser Leu Arg His Tyr Leu Asn Leu VaI Thr Arg GIn Arg Tyr Tyr Ala), or such as the 26 C-terminal amino acids of PYY1-36 as defined in SEQ ID NO 1 (Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Ser Leu Arg His Tyr Leu Asn Leu VaI Thr Arg GIn Arg Tyr Tyr Ala), or such as the 28 C-terminal amino acids of PYY1-36 as defined in SEQ ID NO 1 (GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Ser Leu Arg His Tyr Leu Asn Leu VaI Thr Arg GIn Arg Tyr Tyr Ala), or such as the 30 C-terminal amino acids of PYY1-36 as defined in SEQ ID NO 1 (Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Ser Leu Arg His Tyr Leu Asn Leu VaI Thr Arg GIn Arg Tyr Tyr Ala), or such as the 32 C-terminal amino acids of PYY1-36 as defined in SEQ ID NO 1 (Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Ser Leu Arg His Tyr Leu Asn Leu VaI Thr Arg GIn Arg Tyr Tyr Ala), or such as the 34 C-terminal amino acids of PYY1-36 as defined in SEQ ID NO 1 (Tyr Ala lie Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Ser Leu Arg His Tyr Leu Asn Leu VaI Thr Arg GIn Arg Tyr).

In another preferred embodiment the functional equivalent may comprises internal amino acids of PYY1-36 such as amino acid 16-21 of PYY 1-36 as defined in sequence ID NO 1 (GIu Leu Asn Arg Tyr Tyr ), or such as amino acid 15-22 of PYY 1-36 as defined in sequence ID NO 1 (GIu GIu Leu Asn Arg Tyr Tyr Ala), or such as amino acid 14-23 of PYY 1-36 as defined in sequence ID NO 1 (Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser), or such as amino acid 13-24 of PYY 1-36 as defined in sequence ID NO 1 (Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu), or such as amino acid 12-25 of PYY 1-36 as defined in sequence ID NO 1 (Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg), or such as amino acid 11-26 of PYY 1-36 as defined in sequence ID NO 1 (Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His), or such as amino acid 10-27 of PYY 1-36 as defined in sequence ID NO 1 (GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr), or such as amino acid 9-28 of PYY 1-36 as defined in sequence ID NO 1 (GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu), or such as amino acid 8-29 of PYY 1-36 as defined in sequence ID NO 1 (Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn), or such as amino acid 7-30 of PYY 1-36 as defined in sequence ID NO 1 (Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu), or such as amino acid 6-31 of PYY 1-36 as defined in sequence ID NO 1 (GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu VaI), or such as amino acid 5-32 of PYY 1-36 as defined in sequence ID NO 1 (Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu VaI Thr), or such as amino acid 4-33 of PYY 1-36 as defined in sequence ID NO 1 (Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu VaI Thr Arg), or such as amino acid 3-34 of PYY 1-36 as defined in sequence ID NO 1 (He Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu VaI Thr Arg GIn), or such as amino acid 2-35 of PYY 1-36 as defined in sequence ID NO 1 (Pro He Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu VaI Thr Arg GIn Arg) or such as amino acid 2-36 of PYY 1-36 as defined in sequence ID NO 1 (Pro He Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu VaI Thr Arg GIn Arg Tyr), or such as amino acid 4-36 of PYY 1-36 as defined in sequence ID NO 1 (Lys Pro GIu Ala Pro GIy GIu Asp Ala Ser Pro GIu GIu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu VaI Thr Arg GIn Arg Tyr).

In an embodiment the functional equivalent comprise any of the above sequences with conservative amino acid substitutions, such as one substitution, or such as two substitutions, or such as two substitutions, or such as more than two substitutions, or such as more than four substitutions.

Further included are functional equivalent know from the literature, such as the PYY agonists described in WO 03/057235 and references therein.

Covalent modifications The functional equivalent may comprise any type of modifications. Nearly 200 structurally distinct covalent modifications have been identified thus far, ranging in size and complexity from conversion of amides to carboxylic acids, to the attachment of multiple complex oligosaccharides. In an embodiment the PYY molecule or the functional equivalent may comprise modification(s) such as phosphorylation, acetylation, ubiquination, sumo attachment, lipidation (acetylation, prenylation, famesylation, geranylation, palmitoylation, myristoylation), methylation, carboxylation, sulfonation, O- and N-glycosylation. The PYY molecule of the functional equivalent may further comprise modification trough out the molecule.

A subset of modifications is dependent on vitamin C as a cofactor. This include proline and lysine hydroxylations and carboxy terminal amidation.

In an embodiment PYY or the functional equivalent comprise a C-terminal amida¬ tion. In a preferred embodiment the C-terminal tyrosine residue of PYY or a func- tional equivalent is amidated.

Protecting group

The functional equivalent may according to the invention comprise protecting group at the N-terminus or the C-terminus or at both. A protecting group covalently joined to the N-terminal amino group reduces the re¬ activity of the amino terminus under in vivo conditions. Amino protecting groups in¬ clude - C1-10 alkyl, -C1-10 substituted alkyl, -C2-10 alkenyl, -C2-10 substituted al- kenyl, aryl, -C1-6 alkyl aryl, -C(O)- (CH2) 1-6-COOH, -C(O)-CI -6 alkyl, -C(O)-aryl, -C (O)-O-CI -6 alkyl, or-C (O)-O-aryl. Preferably, the amino terminus protecting group is acetyl, propyl, succinyl, benzyl, benzyloxycarbonyl or tbutyloxycarbonyl.

A protecting group covalently joined to the C-terminal carboxy group reduces the reactivity of the carboxy terminus under in vivo conditions. The carboxy terminus protecting group is preferably attached to the a-carbonyl group of the last amino acid. Carboxy terminus protecting groups include amide, methylamide, and ethylamide.

In an embodiment the binding member is capable of binding a functional equivalent of PYY as described above.

Conjugates The binding member and PYY or the functional equivalent of PYY may conjugated to another entity, in order for example, to prolong its half-life. The binding member and PYY or the functional equivalent may further be conjugated with each other. Conjugation can improve the delivery of targeted doses, prevent breakdown, and increase bioavailablity in circulation The conjugate may be any molecule.

The preparation of conjugates is well known in the art se for example Hermanson GT. Bioconjugate Techniques. New York: Academic Press; 1996, Aslam M, Dent AH. Bioconjugation: protein coupling techniques for the biomedical sciences. Houndsmills, England: Macmillan Publishers; 1999, and Wong SS. Chemistry of protein conjugation and crosslinking. Boca Raton, FL: CRC Press; 1991.

Most methods use amine-reactive reagents or thiol-reactive reagent. In the prepara¬ tion of conjugates advantages may be achieved through the use of certain linkers. For example, linkers that contain a disulfide bond that is sterically "hindered" are often preferred, due to their greater stability in vivo, thus preventing release of the toxin moiety prior to binding at the site of action. It is generally desired to have a conjugate that will remain intact under conditions found everywhere in the body ex- cept the intended site of action, at which point it is desirable that the conjugate have good "release" characteristics.

Different conjugates have been described, for examplen, use of the A chain of ricin is described in US Pat. No. 4,340,535 incorporated herein by reference. Examples of peptide conjugates based on AC-RYY(RK)(WI)RK)-NH2 (where the brackets show allowable variation of amino acid residues) may be found in US patent application 2003040472.

A binding member and a ligand may be chemical linked with each other. This fusion may stabilize the complex and simplify the production and further minimize the amount of binding member necessary. In an embodiment of the invention the bind¬ ing member is chemically linked to the ligand. In an embodiment of the invention the binding member is conjugated to the ligand.

The molecules may be conjugated by peptide bonds, before or after synthesis and purification. The fusion may be obtained by any suitable methods, for example, but not exclusively, by recombinant DNA technology. In a preferred embodiment the fusion of is made by recombinant DNA technology, such as a fusion of the nucleo- tide sequence encoding the binding member and the nucleotide sequence encoding the ligand is made and the fusion is thereby encoded by a single nucleotide se¬ quence. The fusion polypeptide may be expressed and purified as a single polypep¬ tide molecule, using any suitable method, as described for the purification of a bind¬ ing member. The fusion polypeptide may include insertion of a linker, such as a pep- tide of at least 2 AA, such as at least 5 AA, such as at least 8 AA, such as at least 15 AA, such as at least 20 AA

In one embodiment the binding member is conjugated with PYY or a functional equivalent there of.

In a further embodiment of the invention the binding member is conjugated to PYY or a functional equivalent there of using a linker of at least 2AA. Purification of binding members After production, the binding members of the present invention are preferably puri¬ fied. The method of purification used is dependent upon several factors, including the purity required, the source of the binding member, the intended use for the bind- ing member, the species in which the binding member was produced. In addition the method of purification is dependent on the type of binding member.

Purification of antibodies is dependent of the class of the antibody, the subclass of the antibody and, whether the antibody is a monoclonal antibody. Purification of affi- bodies depends on there biochemical characteristics. Purification of antibody- affibodies chimeras are dependent on structure of the fusion, thus the molecules may be purified based on characteristics associated with the antibody part of the molecule or the affibody part of the molecule, or the total molecule.

Any suitable conventional methods of purifying polypeptides comprising antibodies and affibodies include precipitation and column chromatography are well known to one of skill in the purification arts, including cross-flow filtration, ammonium sulphate precipitation, affinity column chromatography, gel electrophoresis and the like may be used.

The method of purifying an antibody with an antiimmunoglobulin antibody can be either a single purification procedure or a sequential purification procedure. Methods of single and sequential purification are well known to those in the purification arts. In a single-step purification procedure, the antibody is specifically bound by a single antiimmunoglobulin antibody. Non-specifically bound molecules are removed in a wash step and the specifically bound molecules are specifically eluted. In a sequen¬ tial purification procedure, the antibody is specifically bound to a first anti¬ immunoglobulin antibody, non-specifically bound molecules are removed in a wash step, and the specifically bound molecules are specifically eluted. The elute from this first antiimmunoglobulin antibody purification step is then specifically bound to a second antiimmunoglobulin antibody. The non-specifically bound molecules are removed in a wash step, and the specifically bound molecules are specifically eluted. In a preferred embodiment, the antibody is sequentially purified by a first and second antiimmunoglobulin antibody selected from the group consisting of antibod- ies which specifically bind heavy and light chain constant regions. In a more pre- ferred embodiment, the antibody is sequentially purified by a first and second anti¬ immunoglobulin antibody selected from the group consisting of antibodies which specifically bind the heavy chain constant region of IgG and light chain constant regions of kappa and lambda. In an even more preferred embodiment, the anti- immunoglobulin antibody is selected from the group consisting of antibodies which specifically bind the light chain constant regions of kappa and lambda.

A commonly used method of purification is affinity chromatography in which the an¬ tibody to be purified is bound by protein A, protein G or by an antiimmunoglobulin antibody. Another method of affinity chromatography, which is well known to those of skill in the art, is the specific binding of the antibody to its respective antigen.

In particular for purifying a multispecific, including a bispecific antibody, a sequential purification procedure may be used, wherein the bispecific antibody comprising two or more variable domains is specifically bound to a first antigen and then to a sec¬ ond antigen.

In an alternative embodiment, a bispecific antibody comprising two or more variable regions is purified by sequential purification by specifically binding the antibody to a first antigen in a first purification step and to a second antigen in a second purification step.

The methods use for purification of antibodies may, when suitable, be applied for the purification of affibodies or antibody-affibody chimeras and other types of binding members.

Stabilisation of liqand In one embodiment the association of the protective binding member with the ligand, here for example PYY1-36, PYY3-36 or a functional equivalent should according to the invention result in stabilisation of the ligand. This may result from protection of the ligand from degradation by proteinases, for example by preventing access of proteinases to the ligand, by blocking the activity of a proteinase or by changing the three dimensional structure abolishing the association of the ligand with a proteinase. The stabilisation of the ligand may be beneficial in many situations, such as in the preparation of peptide drugs, that are normally very unstable and easily broken down after administration to the recipient. Thereby the half-life is increased and the bioavailability of said polypeptide is prolonged.

Half-life The stability of a compound may be described by the half-life of that compound which is the time for the concentration of the compound to decrease to the half. The T-i/2can be measured, using any suitable method known by a person skilled in the art. The TV2 of PYY may vary inbetween individuals, thus the T1/2 of PYY may initially be up to approximately 10 minutes or such as up to approximately 20 minutes or such as up to approximately 30 minutes or such as up to approximately 60 minutes. The concentration of a compound reflects the steady state level of a compound which is a result of synthesis and degradation, thus an increase of the T1/2of a compound will result in an overall increase in concentration of the compound if other parameters are constant.

It is preferred that the binding member increase the T1/2of PYY1-36, PYY3-36 or a functional equivalent there of.

In an embodiment the binding meber increase the T1^Of PYY In an embodiment the binding meber increase the T^2 of PYY1-36 In an embodiment the binding meber increase the T-i/2of PYY3-36 In an embodiment the binding meber increase the T-|/2of a functional equivalent of PYY1-36 or 3-36.

In an embodiment the binding member increase the T1/2 of PYY or a functional equivalent there of, with approcimately 5 minutes, with approximately 10 minutes, or such as with approximately 15 minutes, or such as with approximately 20 minutes, or such as with approximately 25 minutes, or such as with approximately 30 minutes, or such as with approximately 40 minutes, or such as with approximately 50 minutes, or such as with approximately 60 minutes, or such as with approximately 80 minutes, or such as with approximately 100 minutes, or such as with approximately 120 minutes.

Plasma concentration The plasma concentration measures the steady state level of compound in vivo, thus alterations of the T1/2 of the compound will lead to an increase in plasma concentration. The endogenous plasma concentration of PYY is reported to be about 8.5 pM in a normal individual (www.phoenix.com). Other reports the plasma concentration of PYY to be 15-20 pM (WO 03/57235). The plasma concentration of PYY may be different depending on the method applied for mesaureing. Futhermore the plasma concentration may vary with in a group of people and in particular with in a group of people with metabolic disorders, so the numbers mentioned here should be used as a guide and not as absolute numbers.

An aspect of the invention relates to a method of treatment comprising administration of a pharmaceutical composition defined by the present invention.

In an embodiment of the present invention the treatment elevate the plasma concen- tration of PYY1-36 and/or PYY3-36 above 8.5 pM within an individual. In another embodiment the treatment elevates the plasma concentration of PYY1-36 and/ or PYY3-36 above 10 pM. In a further embodiment the treatment elevates the plasma concentration of PYY1-36 and/or PYY3-36 above 25 pM. In an additional embodi¬ ment said treatment elevates the plasma concentration of PYY1-36 and/or PYY3-36 above 50 pM. In a further embodiment said treatment elevates the plasma concentration of PYY1-36 and/or PYY3-36 above 75 pM. In a further embodiment said treatment elevates the plasma concentration of PYY1 -36 and/or PYY3-36 above 100 pM.

In an embodiment of the present invention the treatment increases the plasma con¬ centration of PYY1-36 and/or PYY3-36 1 to 2 times. In a further embodiment the treatment increases the plasma concentration of PYY1-36 and/or PYY3-36 2-3 times. In an embodiment of the present invention the treatment increases the plasma concentration of PYY1-36 and/or PYY3-36 3-4 times. In an embodiment of the present invention the treatment increases the plasma concentration of PYY1-36 and/or PYY3-36 more than 4 times, such as more than 5 times, or such as more than 6 times.

Biological activity The association of a compound with a protective binding member may affect the biological activity of the compound. The biological activity of PYY1-36, PYY3-36 or functional equivalents is reflected by the ability of the molecules to activate the NPY receptors. It is clear that the activity of both endogenous PYY molecules and exogenous supplied PYY molecules or functional equivalents of PYY may contribute to the total biological activity.

The activity of PYY1-36, PYY3-36 or a functional equivalent, in combination with a binding member can be measured by any suitable method known by a person skilled in the art (se example 1 ).

One simple measure of the ability of a PYY molecule or a functional equivlaent to activate a NPY receptor is to measure its EC50, i.e. the dose at which the com¬ pound is able to activate the signalling of the receptor to half of the maximal effect of the compound.

The NPY receptor can either be expressed endogenously on primary cells cultures, for example pituitary cells, or heterologously expressed on cells transfected with the NPY receptor. The NPY receptor activity can either be measured with endogenously expressed NPY receptors on primary cells cultures, for example pituitary cells, or with heterologously expressed NPY receptors on cells transfected with NPY recep¬ tor expression constructs. Whole cell assays or assays using membranes prepared form either of these cell types can be used depending on the type of assay.

The NPY receptor is generally believed to be primarily coupled to the G-protein sig¬ nalling pathways thus any suitable assay which monitor activity in this signalling pathway can be used. The Y2 receptor is coupled mainly to Gαi subpopulation of G- proteins that e.g. decreases the level of cAMP. However, through cotransfection chimeric G-proteins (Conklin B) between both Gαq and Gαi the Y2 receptor may be forced to activate Phospholipase C and increase (IP) turnover in the cell. Cells, for example NIH 3T3 or 293 cells, stably expressing the human Y2 receptor accumulate cAMP upon Forskolin stimulation. This is inhibited by simultaneous incubation with PYY. Thus simultaneous treatment with PYY and the PYY binding member can also be used as a measurement for the biological activity of PYY upon Forskolin stimula- tion. Several steps in the pathway may be used as markers of PYY activity, examples are:

1 ) The activity of phopholipase C (PLC), one of the first down-stream effector molecules in the pathway, for example by measuring the accumulation of inositol phosphate which is one of the products of PLC.

2) The inhibition of cAMP accumulation

3) The mobilization of calcium from the intracellular stores

4) The activity of different kinds of MAP kinases (p38, jun, eci), NF-K-B transloca¬ tion and CRE driven gene transcription.

5) Fluorescently tagged molecules able to detect the activated NPY Y2 receptor.

Binding assays can be performed using recombinantly produced NPY receptor polypeptides present in different environments. Such environments include, for ex- ample, cell extracts and purified cell extracts containing NPY receptor polypeptides expressed from recombinant nucleic acid or naturally occurring nucleic acid; and also include, for example, the use of a purified NPY receptor polypeptide produced by recombinant means or from naturally occurring nucleic acid which is introduced into a different environment.

The use of a recombinantly expressed NPY receptor offers several advantages such as the ability to express the receptor in a defined cell system so that a response to a compound at the NPY receptor can more readily be differentiated from responses at other receptors. For example, the NPY receptor can be expressed in a cell line such as HEK 293, COS 7, and CHO not normally expressing the receptor by an expression vector, wherein the same cell line without the expression vector can act as a control.

Complex formation between the binding member and PYY or the functional equivalent thereof may stimulate the biological activity of PYY or the functional equivalent thereof. The increase in T1/2of PYY or the functional equivalent thereof may result in an increase of PYY activity, likewise the raise of the plasma concentration of PYY may result in an increase of PYY activity. For PYY to retain or increase the activity the binding members must associate with PYY1-36 or PYY3-36 without interfering with the ability of the molecule to stimulate the NPY receptor. It is also possible that the association of PYY1-36 or PYY3-36 with the binding member enhances the ability of PYY1-36 or PYY3-36 to stimulate the NPY receptor, by stabilizing the interaction or by altering the three dimensional structure of PYY1-36 or PYY3-36 whereby the interaction with the receptor becomes more favourably.

In an embodiment the invention relates to a pharmaceutical composition comprising an isolated binding member capable of binding PYY1-36 or PYY3-36 specifically and thereby increases the biological activity of PYY1-36 or PYY3-36 within an individual.

It is preferred that the binding member is capable of prolonging the plasma half-life of PYY1-36 or PYY3-36 within an individual. For example, the binding of said binding member to PYY1-36 or PYY3-36 may prevent breakdown. It is further preferred that the binding of the binding member to PYY1-36 or PYY3-36 results in increased biological PYY activity, thus PYY1-36 or PYY3-36 retain the ability to active to the corresponding receptors. In another preferred embodiment, said binding member acts to increase the affinity of PYY1-36 or PYY3-36 for the receptor, for example by altering the 3D structure of PYY1-36 or PYY3-36 to make receptor binding more favourable and/or more frequent.

In a preferred embodiment of the present invention, said binding member is capable of elevating the plasma concentration of PYY1-36 or PYY3-36 within an individual, preferably by binding to the molecule.

In a further preferred embodiment of the present invention said binding member is capable of increasing the biological activity of PYY1-36 or PYY3-36 by elevation of the plasma concentration of PYY1-36 or PYY3-36. Individual in need According to the invention, any suitable individual who may draw benefit from the compositions of the present invention may be treated with said compositions.

Preferably, said individual is suffering from metabolic disorders (as described here below) or PYY deficiency. Thus an individual who would draw benefit of the treat¬ ment are considered an individual in need of treatment. An individual in need may be obese or overweight, and/or may suffer from a disease such as any of the above mentioned disorders. An individual in need may be an individual in risk of acquiring any of the above mentioned disorders.

In addition a person in need of losing weight for athletic performance or for cosmetic reasons may also be considered as an individual in need treatment.

Furthermore, the invention may relate to inhibition of appetite and decreasing the rate of weight gain in animals.

Metabolic disorders Metabolic disorders are, for example but not limited to, glucose intolerance, insulin resistance, glucose non-responsiveness, hyperglycemia, dyslipidemia, obesity (overweight), hyperlipedemia, hyperlipoproteinemia, hyperinsulinemia, hypertension, atherosclerosis, cardiovascular disorders, insulin-resistance syndrome (syndrome X), diabetes mellitus (incl.typel , type 2 (non-insulin dependent diabetes mellitus), gestational diabetes mellitus, maturity onset diabetes of the young (MODY), latent (or late-onset) autoimmune diabetes of adulthood (LADA) and secondary diabetes mellitus as a result of e.g. pancreatic disease, one example being chronic pancreati¬ tis, or drugs, one example being corticosteroids), disease of the gastrointestinal tract and eating disorders.

Overweight and obesity may be associated with developmental defects, e.g. Prader- Willi syndrome and potentially Turner's syndrome, Carpenter syndrome and mental retardation in general. Furthermore, obstructive sleep apnea syndrome/obesity- hypoventilation syndrome (pickwickian syndrome), arthrosis (especially of the knee and hip joints) may be associated with obesity. Diseases associated with single gene mutations may enhance the susceptibility and severity of obesity, such mutations include; MC4 receptor mutations (5% of all excessive obese children), Leptin protein and receptor mutations, Ghrelin peptide gene mutations, Adiponectin variation (Type Il diabetes), APOE and TGF-beta1 genes mutations, IL6 receptor gene variations (Pima Indians), Peroxisome proliferator-activated receptor gamma2 promoter mutations, Beta(3)-adrenergic receptor gene (BAR-3) allelic variant, complex haplotypes of IRS2 gene, functional polymorphism in the promoter of UCP2.

Bowel diseases Diseases of the gastrointestinal tract include, irritable bowel syndrome, inflammatory bowel disease, ulcerative colitis, Crohn's disease, Ileus or bowel obstruction, bowel obstruction, bowel atrophy, conditions characterized by loss of bowel mucosa or bowel mucosal function. Gastrointestinal disorders are further postgastrectomy syn¬ drome, dumping syndrome, AIDS-associated chronic diarrhea, diabetes-associated diarrhea, postvagotomy diarrhea, bariatric surgery-associated diarrhea (including obesity surgeries: gastric bypass, gastroplasties and intestinal bypass), short bowel syndrome (including resection of the small intestine after trauma, radiation induced complications, infarction of the intestine from vascular occlusion), tube-feeding re¬ lated diarrhea, chronic secretory diarrhea, carcinoid syndrome-associated diarrhea, gastrointestinal peptide tumors, endocrine tumors, chronic diarrhea associated with thyroid disorders, chronic diarrhea in bacterial overgrowth, chronic diarrhea in gas¬ trinoma, choleraic diarrhea, chronic diarrhea in giardiasis, antibiotic-associated chronic diarrhea, diarrhea-predominant irritable bowel syndrome, chronic diarrhea associated with maldigestion and malabsorption, chronic diarrhea in idiopathic pri- mary gastrointestinal motility disorders, chronic diarrhea associated with colla¬ genous colitis, surgery-associated acute diarrhea, antibiotic associated acute diar¬ rhea, infection-associated acute infectious diarrhea, and the like.

Eating disorders Some disorders of appetite regulation are caused by a direct malfunction of the ap¬ petite regulating system, others are psychological disorders. Such disorders include the binge eating disorders described (BED and NES), as well as anorexia nervosa (AN). Thus the invention relates to a pharmaceutical composition comprising a binding member to PYY or a functional equivalent thereof for the treatmen of metabolic dis¬ orders and PYY deficiency.

Pharmaceutical compositions Pharmaceutical compositions of the present invention may be prepared by conven¬ tional techniques, e.g. as described in Remington: The Science and Practice of Pharmacy 1995, edited by E. W. Martin, Mack Publishing Company, 19th edition, Easton, Pa. The compositions may appear in conventional forms, for example cap- sules, tablets, aerosols, solutions, suspensions or topical applications.

The pharmaceutical composition according to the present invention preferably fur¬ ther comprises pharmaceutically acceptable salts, a pharmaceutically acceptable carrier, diluent and/or stabilizer.

As used herein, the terms "pharmaceutically acceptable", "physiologically tolerable" and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capa¬ ble of administration to or upon an individual without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like.

In an embodiment of the present invention, an isolated binding member of the present invention is used for the production of pharmaceutical composition.

In a further embodiment the binding member is used for the production of a pharmaceutical composition for the treatment of a metabolic disorder or PYY deficiencies.

In an embodiment the composition according to the invention is for the production of a pharmaceutical composition for; a) the treatment of overweight and/or b) the treatment of obesity and/or c) the treatment of syndrome X and/or d) the treatment of eating disorders and/or e) the treatment of bowel diseases and/or f) inhibition of appetite and/or g) inhibition of weight gain and/or h) decreasing body weight and/or i) decreasing body fat mass and/or j) treatment of bowel diseases or any combinations of the above.

The composition comprises the binding member according to the invention may fur¬ ther comprises pharmaceutically acceptable salts and pharmaceutically acceptable carriers or diluents, vehicles and/or excipients and/or transport molecules.

Pharmaceutically acceptable salts Pharmaceutically acceptable salts of the instant compounds, where they can be prepared, are also intended to be covered by this invention. These salts will be ones which are acceptable in their application to a pharmaceutical use. By that it is meant that the salt will retain the biological activity of the parent compound and the salt will not have untoward or deleterious effects in its application and use in treating dis¬ eases.

Pharmaceutically acceptable salts are prepared in a standard manner. If the parent compound is a base it is treated with an excess of an organic or inorganic acid in a suitable solvent. If the parent compound is an acid, it is treated with an inorganic or organic base in a suitable solvent.

The pharmaceutical composition of the present invention can include pharmaceuti¬ cally acceptable salts of the compounds therein. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypep¬ tide).

Such salts include pharmaceutically acceptable acid addition salts, pharmaceutically acceptable metal salts, ammonium salts and alkylated ammonium salts. Acid addi¬ tion salts include salts of inorganic acids as well as organic acids. Representative examples of suitable inorganic acids include hydrochloric, hydrobromic, hydriodic, phosphoric, metaphosphoric, sulpfuric and nitric acids and the like. Representative examples of suitable organic acids include formic, acetic, trichloroacetic, trifluoroacetic, propionic, benzoic, cinnamic, citric, fumaric, glycolic, lactic, maleic, malic, malonic, mandelic, oxalic, picric, pyruvic, salicylic, arylsulphonic, succinic, p- toluenesulphonic methanesulfonic, ethanesulfonic, tartaric, ascorbic, pamoic, bis- methylene salicylic, ethanedisulfonic, gluconic, citraconic, aspartic, stearic, palmitic, ethylenediaminetetraacetic (EDTA), p-aminobenzoic, glutamic, benzenesulfonic and p-toluenesulfonic acids and the like. Further examples of pharmaceutically accept¬ able inorganic or organic acid addition salts include the pharmaceutical acceptable salts listed in J. Pharm. Sci. 1977,66,2, which is incorporated herein by reference. Examples of metal salts include lithium, sodium, potassium and magnesium salts and the like.

Examples of ammonium and alkylated ammonium salts include ammonium, methyl- ammonium, dimethylammonium, trimethylammonium, ethylammonium, hy- droxyethylammonium, diethylammonium, butylammonium and tetramethylammo- nium salts and the like.

Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

Also included within the scope of compounds or pharmaceutical acceptable acid addition salts thereof in the context of the present invention are any hydrates (hy- drated forms) thereof.

The compounds of the invention may be administered in the form of an alkali metal or earth alkali metal salt thereof, concurrently, simultaneously, or together with a pharmaceutically acceptable carrier or diluent, especially and preferably in the form of a pharmaceutical composition thereof, whether by oral, rectal, or parenteral (in- eluding subcutaneous) route, in an effective amount.

In an embodiment of the invention the composition comprise pharmaceutical ac¬ ceptable salts.

Stabilizers The active compound of the invention may be unstable, thus the composition pref¬ erably contain stabilizers, preservatives or conservatives to increase the stability of the compounds.

A pH-buffering agent may be used to stabilize the active compound of the composi¬ tion. The buffering agent may be acetate, carbonate, bicarbonate, phosphate, cit¬ rate, tris or hepes. In a preferred embodiment the buffering agent is acetate.

According to the invention the composition preferably has a pH between 2.0 and 9.0, or such as between 2.5 and 8.0, or such as 3.0 and 7.0, or such as between 3.5 and 6.0, or such as between 3.5 and 5.0 or such as between 4.0 and 5.5, or such as between 4.0 and 5.0, or such as between 4.0 and 4.5. Preferably the pH of the compositions is less than 6, preferably less than 5.5, preferably less than 5, prefera¬ bly less than 4.8, preferably less than 4.6, preferably less than 4.4, preferably less than 4.2.

Tween 20, Tween 60,Tween 80, Span 80, cetostearyl alcohol, benzyl alcohol, myristyl alcohol, glyceryl mono-stearate, mannitol, polysorbates and sodium lauryl sulphate are possible stabilizers.

In a preferred embodiment mannitol may be used as stabilizers

For the preparation of a lyophilised composition ad lyoprotectant may be used to stabilize the active ingredient (Townsend and DeLuca, "Use of lyoprotectants in the freeze-drying of a model protein, ribonuclease A" Journal of Parenteral Science & Technology 42 (6): 190-199 (Nov.-Dec. 1988)).

The lyoprotectant may preferably be a sugar such as sucrose or trehalose such as sucrose, dextran, or hydroxypropyl-/142-cyclodextrin.

Transport molecules Transport molecules act by having incorporated into or anchored to it the compound according to the invention. Any suitable transport molecules known to the skilled person may be used. Examples of transport molecules may be liposomes, micelles, and/or microspheres.

A variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos. 4, 235,871 , 4,501 ,728 and 4,837,028, all of which are incorporated herein by reference.

Micelles are formed by surfactants (molecules that contain a hydrophobic portion and one or more ionic or otherwise strongly hydrophilic groups) in aqueous solution. As the concentration of a solid surfactant increases, its monolayers adsorbed at the air/water or glass/water interfaces become so tightly packed that further occupancy requires excessive compression of the surfactant molecules already in the two monolayers. Further increments in the amount of dissolved surfactant beyond that concentration cause amounts equivalent to the new molecules to aggregate into micelles. This process begins at a characteristic concentration called "critical micelle concentration".

The shape of micelles formed in dilute surfactant solutions is approximately spherical. The polar head groups of the surfactant molecules are arranged in an outer spherical shell whereas their hydrocarbon chains are oriented toward the centre, forming a spherical core for the micelle. The hydrocarbon chains are randomly coiled and entangled and the micellar interior has a nonpolar, liquid-like character. In the micelles of polyoxyethylated nonionic detergents, the polyoxy- ethylene moieties are oriented outward and permeated by water. This arrangement is energetically favourable since the hydrophilic head groups are in contact with water and the hydrocarbon moieties are removed from the aqueous medium and partly shielded from contact with water by the polar head groups. The hydrocarbon tails of the surfactant molecules, located in the interior of the micelle, interact with one another by weak van der Waals forces.

The size of a micelle or its aggregation number is governed largely by geometric factors. The radius of the hydrocarbon core cannot exceed the length of the extended hydrocarbon chain of the surfactant molecule. Therefore, increasing the chain length or ascending homologous series increases the aggregation number of spherical micelles. If the surfactant concentration is increased beyond a few percent and if electrolytes are added (in the case of ionic surfactants) or the temperature is raised (in the case of nonionic surfactants), the micelles increase in size. Under these conditions, the micelles are too large to remain spherical and become ellipsoidal, cylindrical or finally lamellar in shape.

Common surfactants well known to one of skill in the art can be used in the micelles of the present invention. Suitable surfactants include sodium laureate, sodium oleate, sodium lauryl sulfate, octaoxyethylene glycol monododecyl ether, octoxynol 9 and PLURONIC F-127 (Wyandotte Chemicals Corp.). Preferred surfactants are nonionic polyoxyethylene and polyoxypropylene detergents compatible with IV injec¬ tion such as, TWEEN-80, PLURONIC F-68, n-octyl-.beta.-D-glucopyranoside, and the like. In addition, phospholipids, such as those described for use in the production of liposomes, may also be used for micelle formation.

Carriers and diluents The preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art. Typically such composi¬ tions are prepared as sterile injectables either as liquid solutions or suspensions, aqueous or non-aqueous, however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared. The preparation can also be emulsified.

The active ingredient can be mixed With excipients which are pharmaceutically ac¬ ceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein. Suitable excipients are, for example, wa- ter, saline, dextrose, glycerol, ethanol or the like and combinations thereof. In addi¬ tion, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which en¬ hance the effectiveness of the active ingredient.

Liquid compositions can also contain liquid phases in addition to and to the exclu¬ sion of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, organic esters such as ethyl oleate, and water-oil emulsions.

Suitable pharmaceutical carriers include inert solid diluents or fillers, sterile aqueous solution and various organic solvents. Examples of solid carriers are lactose, terra alba, sucrose, cyclodextrin, talc, gelatine, agar, pectin, acacia, magnesium stearate, stearic acid or lower alkyl ethers of cellulose. Examples of liquid carriers are syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene or water.

The pharmaceutical compositions formed by combining the compounds of the inven¬ tion and the pharmaceutical acceptable carriers are then readily administered in a variety of dosage forms suitable for the disclosed routes of administration. The com¬ positions may conveniently be presented in unit dosage form by methods known in the art of pharmacy.

In a preferred embodiment of the invention the composition comprises a binding member capable of binding PYY or a functional equivalent thereof or a salt thereof as a lyophilisate and the composition further comprises a solvent. In another em- bodiment the composition is a solution of a binding member according to the inven¬ tion or a salt thereof. Preferably, the solvent may be any suitable solvents, such as described herein, and preferably the solvent is saline or a physiological buffer like phosphate buffer.

The invention also relates to a method for preparing a pharmaceutical composition or pharmaceutical composition comprising a composition of the invention, compris¬ ing admixing at least one binding member as defined above with a physiologically acceptable carrier.

In a still further aspect, the invention relates to a pharmaceutical composition com¬ prising, as an active ingredient, a compound as defined above or a pharmaceutical acceptable salt thereof together with a pharmaceutical acceptable carrier.

Accordingly, the composition may further include the transport molecules as described above.

Compositions for oral administration The compounds of the present invention may be formulated in a wide variety of oral administration dosage forms. The pharmaceutical compositions and dosage forms may comprise the compounds of the invention or its pharmaceutically acceptable salt or a crystal form thereof as the active component. The pharmaceutically accept¬ able carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid car- rier can be one or more substances which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, wetting agents, tablet disintegrating agents, or an encapsulating material.

Preferably, the 0.5% to 75% by weight of a compound or compounds of the inven- tion, with the remainder consisting of suitable pharmaceutical excipients. For oral administration, such excipients include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, and the like.

In powders, the carrier is a finely divided solid which is a mixture with the finely di¬ vided active component. In tablets, the active component is mixed with the carrier having the necessary binding capacity in suitable proportions and compacted in the shape and size desired. The powders and tablets preferably containing from one to about seventy percent of the active compound. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. The term "preparation" is intended to include the compo¬ sition of the active compound with encapsulating material as carrier providing a cap¬ sule in which the active component, with or without carriers, is surrounded by a car- rier, which is in association with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be as solid forms suit¬ able for oral administration.

Drops according to the present invention may comprise sterile or non-sterile aque- ous or oil solutions or suspensions, and may be prepared by dissolving the active ingredient in a suitable aqueous solution, optionally including a bactericidal and/or fungicidal agent and/or any other suitable preservative, and optionally including a surface active agent. The resulting solution may then be clarified by filtration, transferred to a suitable container which is then sealed and sterilized by autoclaving or maintaining at 98-100 0C for half an hour. Alternatively, the solution may be steril- ized by filtration and transferred to the container aseptically. Examples of bacteri¬ cidal and fungicidal agents suitable for inclusion in the drops are phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride (0.01%) and chlorhexidine ace¬ tate (0.01%). Suitable solvents for the preparation of an oily solution include glyc- erol, diluted alcohol and propylene glycol.

Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for oral administration. Such liquid forms include solutions, suspensions, and emulsions. These preparations may con- tain, in addition to the active component, colorants, flavours, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.

Other forms suitable for oral administration include liquid form preparations including emulsions, syrups, elixirs, aqueous solutions, aqueous suspensions, toothpaste, gel dentrifrice, chewing gum, or solid form preparations which are intended to be con¬ verted shortly before use to liquid form preparations. Emulsions may be prepared in solutions in aqueous propylene glycol solutions or may contain emulsifying agents such as lecithin, sorbitan monooleate, or acacia. Aqueous solutions can be prepared by dissolving the active component in water and adding suitable colorants, flavours, stabilizing and thickening agents. Aqueous suspensions can be prepared by dis¬ persing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well known suspending agents. Solid form preparations include solutions, suspensions, and emulsions, and may contain, in addition to the active component, colorants, flavours, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.

Compositions for parenteral administration The compounds of the present invention may be formulated for parenteral admini¬ stration (e.g., by injection, for example bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, for example solutions in aqueous polyethylene glycol. Examples of oily or nonaqueous carriers, diluents, solvents or vehicles include propylene glycol, poly¬ ethylene glycol, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate), and may contain formulatory agents such as preserving, wetting, emulsifying or suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilisation from solution for constitution before use with a suitable vehicle, e.g., sterile, pyrogen-free water. Aqueous solutions should be suitably buffered if necessary, and the liquid diluents first rendered isotonic with sufficient saline or glu¬ cose. The aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. The sterile aqueous media em¬ ployed are all readily available by standard techniques known to those skilled in the art.

Solutions of a binding member or a pharmaceutically acceptable salt thereof, (and for example antigenic epitopes and protease inhibitors) can be prepared in water or saline, and optionally mixed with a nontoxic surfactant. Compositions for intravenous or intra-arterial administration may include sterile aqueous solutions that may also contain buffers, liposomes, diluents and other suitable additives.

Oils useful in parenteral compositions include petroleum, animal, vegetable, or syn¬ thetic oils. Specific examples of oils useful in such compositions include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral compositions include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

Suitable soaps for use in parenteral compositions include fatty alkali metal, ammo¬ nium, and triethanolamine salts, and suitable detergents include (a) cationic deter¬ gents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides; (b) anionic detergents such as, for example, alkyl, aryl, and olefin sul¬ fonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanola- mides, and polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-.beta.-aminopropionates, and 2-alkyl-imidazoline quater- nary ammonium salts, and (e) mixtures thereof. The parenteral compositions typically will contain from about 0.5 to about 25% by weight of the active ingredient in solution. Preservatives and buffers may be used. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such compositions will typically range from about 5 to about 15% by weight. Suitable surfactants in¬ clude polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol. The parenteral composi¬ tions can be presented in unit-dose or multi-dose sealed containers, such as am¬ pules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, im¬ mediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously de¬ scribed. As an alternative to standard injections the pharmaceutical composition may be administrated by infusions, such infusions are preferably short.

The pharmaceutical dosage forms suitable for injection or infusion can include ster- ile aqueous solutions or dispersions comprising the active ingredient that are adapted for administration by encapsulation in liposomes. In all cases, the ultimate dosage form must be sterile, fluid and stable under the conditions of manufacture and storage.

Sterile injectable solutions are prepared by incorporating PYY or an analogue or pharmaceutically acceptable salt thereof in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, fol¬ lowed by filter sterilization.

Compositions for topical administration The compounds of the invention can also be delivered topically. Regions for topical administration include the skin surface and also mucous membrane tissues of the rectum, nose, mouth, and throat. Compositions for topical administration via the skin and mucous membranes should not give rise to signs of irritation, such as swelling or redness. The topical composition may include a pharmaceutically acceptable carrier adapted for topical administration. Thus, the composition may take the form of a suspension, solution, ointment, lotion, cream, foam, aerosol, spray, suppository, implant, inha- lant, tablet, capsule, dry powder, syrup, balm or lozenge, for example. Methods for preparing such compositions are well known in the pharmaceutical industry.

The compounds of the present invention may be formulated for topical administra¬ tion to the epidermis as ointments, creams or lotions, or as a transdermal patch. Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will in general also containing one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or colouring agents. Compositions suitable for topical administra- tion in the mouth include lozenges comprising active agents in a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia; and mouth¬ washes comprising the active ingredient in a suitable liquid carrier.

Creams, ointments or pastes according to the present invention are semi-solid com¬ positions of the active ingredient for external application. They may be made by mix¬ ing the active ingredient in finely-divided or powdered form, alone or in solution or suspension in an aqueous or non-aqueous fluid, with the aid of suitable machinery, with a greasy or non-greasy base. The base may comprise hydrocarbons such as hard, soft or liquid paraffin, glycerol, beeswax, a metallic soap; a mucilage; an oil of natural origin such as almond, corn, arachis, castor or olive oil; wool fat or its deriva¬ tives or a fatty acid such as steric or oleic acid together with an alcohol such as pro¬ pylene glycol or a macrogel. The composition may incorporate any suitable surface active agent such as an anionic, cationic or non-ionic surfactant such as a sorbitan ester or a polyoxyethylene derivative thereof. Suspending agents such as natural gums, cellulose derivatives or inorganic materials such as silicaceous silicas, and other ingredients such as lanolin, may also be included.

Lotions according to the present invention include those suitable for application to the skin or eye. An eye lotion may comprise a sterile aqueous solution optionally containing a bactericide and may be prepared by methods similar to those for the preparation of drops. Lotions or liniments for application to the skin may also include an agent to hasten drying and to cool the skin, such as an alcohol or acetone, and/or a moisturizer such as glycerol or an oil such as castor oil or arachis oil.

The pharmaceutical agent-chemical modifier complexes described herein can be administered transdermally. Transdermal administration typically involves the deliv¬ ery of a pharmaceutical agent for percutaneous passage of the drug into the sys¬ temic circulation of the patient. The skin sites include anatomic regions for trans- dermally administering the drug and include the forearm, abdomen, chest, back, buttock, mastoidal area, and the like.

Transdermal delivery is accomplished by exposing a source of the complex to a patient's skin for an extended period of time. Transdermal patches have the added advantage of providing controlled delivery of a pharmaceutical agent-chemical modi¬ fier complex to the body. See Transdermal Drug Delivery: Developmental Issues and Research Initiatives, Hadgraft and Guy (eds.), Marcel Dekker, Inc., (1989); Con¬ trolled Drug Delivery: Fundamentals and Applications, Robinson and Lee (eds.), Marcel Dekker Inc., (1987); and Transdermal Delivery of Drugs, VoIs. 1-3, Kydo- nieus and Berner (eds.), CRC Press, (1987). Such dosage forms can be made by dissolving, dispersing, or otherwise incorporating the pharmaceutical agent-chemical modifier complex in a proper medium, such as an elastomeric matrix material. Ab¬ sorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate-controlling membrane or dispersing the compound in a polymer matrix or gel.

A variety of types of transdermal patches will find use in the methods described herein. For example, a simple adhesive patch can be prepared from a backing ma¬ terial and an acrylate adhesive. The pharmaceutical agent-chemical modifier com- plex and any enhancer are formulated into the adhesive casting solution and al¬ lowed to mix thoroughly. The solution is cast directly onto the backing material and the casting solvent is evaporated in an oven, leaving an adhesive film. The release liner can be attached to complete the system. Alternatively, a polyurethane matrix patch can be employed to deliver the pharma¬ ceutical agent-chemical modifier complex. The layers of this patch comprise a back¬ ing, a polyurethane drug/enhancer matrix, a membrane, an adhesive, and a release liner. The polyurethane matrix is prepared using a room temperature curing polyure- thane prepolymer. Addition of water, alcohol, and complex to the prepolymer results in the formation of a tacky firm elastomer that can be directly cast only the backing material.

A further embodiment of this invention will utilize a hydrogel matrix patch. Typically, the hydrogel matrix will comprise alcohol, water, drug, and several hydrophilic poly¬ mers. This hydrogel matrix can be incorporated into a transdermal patch between the backing and the adhesive layer.

The liquid reservoir patch will also find use in the methods described herein. This patch comprises an impermeable or semipermeable, heat sealable backing material, a heat sealable membrane, an acrylate based pressure sensitive skin adhesive, and a siliconized release liner. The backing is heat sealed to the membrane to form a reservoir which can then be filled with a solution of the complex, enhancers, gelling agent, and other excipients.

Foam matrix patches are similar in design and components to the liquid reservoir system, except that the gelled pharmaceutical agent-chemical modifier solution is constrained in a thin foam layer, typically a polyurethane. This foam layer is situated between the backing and the membrane which have been heat sealed at the pe- riphery of the patch.

For passive delivery systems, the rate of release is typically controlled by a mem¬ brane placed between the reservoir and the skin, by diffusion from a monolithic de¬ vice, or by the skin itself serving as a rate-controlling barrier in the delivery system. See U.S. Pat. Nos. 4,816,258; 4,927,408; 4,904,475; 4,588,580, 4,788,062; and the like. The rate of drug delivery will be dependent, in part, upon the nature of the membrane. For example, the rate of drug delivery across membranes within the body is generally higher than across dermal barriers. The rate at which the complex is delivered from the device to the membrane is most advantageously controlled by the use of rate-limiting membranes which are placed between the reservoir and the skin. Assuming that the skin is sufficiently permeable to the complex (i.e., absorption through the skin is greater than the rate of passage through the membrane), the membrane will serve to control the dosage rate experienced by the patient.

Suitable permeable membrane materials may be selected based on the desired degree of permeability, the nature of the complex, and the mechanical considera¬ tions related to constructing the device. Exemplary permeable membrane materials include a wide variety of natural and synthetic polymers, such as polydimethylsilox- anes (silicone rubbers), ethylenevinylacetate copolymer (EVA), polyurethanes, poly- urethane-polyether copolymers, polyethylenes, polyamides, polyvinylchlorides (PVC), polypropylenes, polycarbonates, polytetrafluoroethylenes (PTFE), cellulosic materials, e.g., cellulose triacetate and cellulose nitrate/acetate, and hydrogels, e.g., 2-hydroxyethylmethacrylate (HEMA).

Other items may be contained in the device, such as other conventional compo¬ nents of therapeutic products, depending upon the desired device characteristics. For example, the compositions according to this invention may also include one or more preservatives or bacteriostatic agents, e.g., methyl hydroxybenzoate, propyl hydroxybenzoate, chlorocresol, benzalkonium chlorides, and the like. These phar- maceutical compositions also can contain other active ingredients such as antim- , icrobial agents, particularly antibiotics, anesthetics, analgesics, and antipruritic agents.

Compositions for administration as suppositories The compounds of the present invention may be formulated for administration as suppositories. A low melting wax, such as a mixture of fatty acid glycerides or cocoa butter is first melted and the active component is dispersed homogeneously, for ex¬ ample, by stirring. The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool, and to solidify.

The active compound may be formulated into a suppository comprising, for exam¬ ple, about 0.5% to about 50% of a compound of the invention, disposed in a poly¬ ethylene glycol (PEG) carrier (e.g., PEG 1000 [96%] and PEG 4000 [4%]). Compounds for nasal administration The compounds of the present invention may be formulated for nasal administration. The solutions or suspensions are applied directly to the nasal cavity by conventional means, for example with a dropper, pipette or spray. The compositions may be pro- vided in a single or multidose form. In the latter case of a dropper or pipette this may be achieved by the patient administering an appropriate, predetermined volume of the solution or suspension. In the case of a spray this may be achieved for example by means of a metering atomizing spray pump.

Compounds for aerosol administration The compounds of the present invention may be formulated for aerosol administra¬ tion, particularly to the respiratory tract and including intranasal administration. The compound will generally have a small particle size for example of the order of 5 mi¬ crons or less. Such a particle size may be obtained by means known in the art, for example by micronization. The active ingredient is provided in a pressurized pack with a suitable propellant such as a chlorofluorocarbon (CFC) for example dichloro- difluoromethane, trichlorofluoromethane, or dichlorotetrafluoroethane, carbon diox¬ ide or other suitable gas. The aerosol may conveniently also contain a surfactant such as lecithin. The dose of drug may be controlled by a metered valve. Alterna- tively the active ingredients may be provided in a form of a dry powder, for example a powder mix of the compound in a suitable powder base such as lactose, starch, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidine (PVP). The powder carrier will form a gel in the nasal cavity. The powder composi¬ tion may be presented in unit dose form for example in capsules or cartridges of e.g., gelatin or blister packs from which the powder may be administered by means of an inhaler.

Compositions administered by aerosols may be prepared, for example, as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption pro- moters to enhance bioavailability, employing fluorocarbons, and/or employing other solubilizing or dispersing agents.

Combinations It is further envisaged that the active ingredient of the present invention can be ad- ministered in combination with a second active ingredient. By "in combination" is meant that said composition may be co-formulated with other compounds in the same composition, and/or that said other compound(s) and/or pharmaceutical com¬ positions) are administered before, during (including concurrently with) and/or after administration of the compositions of the present invention.

Thus an in embodiment of the present invention relates to a composition comprising a binding member as defined herein and a second active ingredient, said second active ingredient may be PYY or a functional equivalent there of.

Without being bound by theory, it is envisaged that administration of PYY or a functional equivalent thereof may be used to obtain loss of body weight and/or prevention of weight gain. It is furthermore believed, without being bound by theory, that a binding member that prolongs the half-life of PYY or a functional equivalent thereof may increases the action of PYY or a functional equivalent thereof, thus prolonging the actions (such as appetite inhibition) of said compound if administered to an individual. Thus, the binding member acts to enhance the effects of PYY or a functional equivalent there of. This enhancement is presumably increased when PYY or a functional equivalent there of is administered in combination with the binding member to the individual, which is particularly desirable in a situation of PYY deficiency.

A low plasma concentration of PYY1-36 or PYY3-36 in an individual may indicate that the individual suffer from PYY1-36 or PYY3-36 deficiency. A binding member capable of increasing T1Z2Of PYY or a functional equivalent may also be capable of increasing the plasma concentration of PYY or a functional equivalent if administered to an individual. In a person suffering from PYY deficiency this may normalize the PYY levels. In a normal individual administration of such binding members the plasma concentration of PYY may be raised above the normal level.

In a preferred embodiment, the composition according to the invention comprises a second active ingredient; said ingredient may be PYY1-36, PYY3-36 or a functional equivalent thereof. In an additional embodiment said composition is for the production of a pharmaceutical composition for the treatment of PYY1-36 or PYY3- 36 deficiency. Thus in an embodiment of the present invention relates to a composition comprising a binding member as defined herein and a second active ingredient. Said second active ingredient may be selected from the group of; sibutramine and other blockers of the presynaptic reuptake of serotonin and noradrenalin, orlistat and other inhibi- tors of Gl lipases, β3-adrenergic receptor agonists, uncoupling proteins agonists or inducers, (specific) antagonists of PPARY (Peroxisome Proliferator-Activated Recep¬ tor Y), GLP-1 agonists including (but not limited to) GLP-1 , exendin-4 (AC2993) and various formulations thereof, liraglutide (NN2211 ), CJC-1131 and ZP10/AVE-0010, insulin secretagogues, biguanides including (but not limited to) metformin, sulphony- lureas including (but not limited to) glibenclamide, glipizide and glimepiride, α- glucosidase inhibitors (acarbose and miglitol), thiazolidinediones, rimonabant and other CB1 endocannabinoid receptor antagonists, bupropion, topiramate, leptin agonists, ciliary neurotrophic factor, peptide analogues of the human growth hor¬ mone fragment 177-191 , cholecystokinin-A receptor agonists, melanocortin-3 ago- nists and noradrenergic drugs such as phentermine, diethylpropion, phendi- metrazine and benzphetamine.

Thus in an embodiment of the present invention relates to a composition comprising a binding member as defined herein and a second active ingredient. Said second active ingredient may be selected from the group of; anti-hypertensives including (but not limited to) beta-adrenoceptor antagonists, alpha-adrenoceptor antagonists, calcium antagonists, angiotensin converting enzyme (ACE) inhibitors, angiotensin Il receptor antagonists and diuretics (including, but not limited to, thiazide diuretics and loop diuretics) , insulin, DPP-IV inhibitors, anti-depressant medications including (but not limited to) selective serotonin reuptake inhibitors (SSRIs), mianserine and mirtazapine, classical as well as atypical antipsychotic drugs (neuroleptics), corticos¬ teroids as well as any other drug(s) that may increase body weight or body fat mass or that may be indicated for the treatment of hypertension, overweight, obesity, the • syndrome X and/or diabetes mellitus.

PYY composition According to the invention the PYY composition may comprimis any PYY molecule of functionel equivalent thereof described here in, including homologues of PYY and PYY analogs. PYY or functional equivalents thereof can be produced using techniques well known in the art. For example, a polypeptide region of a PYY can be chemically or bio¬ chemically synthesized and modified (se example 2). Techniques for chemical syn¬ thesis, such as solid phase synthesis of polypeptides are well known in the art. (See e.g., Vincent in Peptide and Protein Drug Delivery, New York, N. Y., Dekker, 1990.) Examples of techniques for biochemical synthesis involving the introduction of a nucleic acid into a cell and expression of nucleic acids are provided in Ausubel, Cur¬ rent Protocols in Molecular Biology, John Wiley, 1987-1998, and Sambrook et al., in Molecular Cloning, A Laboratory Manual, 2 d Edition, Cold Spring Harbor Laboratory Press, 1989.

PYY1-36, PYY3-36 or a functional equivalent thereof, according to the invention preferably has at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, functional activity relative to human PYY1-36 or human PPY3-36 as determined using the assay described herein above.

In one embodiment of the invention PYY1-36, PYY3-36 or a functional equivalent thereof has a potency (EC50) of less than 500 nmol. In another embodiment the compound has a potency (EC50) on the NPY receptor Y2 of less than 100 nmol, such as less than 80 nmol, for example less than 60 nmol, such as less than 40 nmol, for example less than 20 nmol, such as less than 10 nmol, for example less than 5 nmol, such as less than 1 nmol, for example less than 0.5 nmol, such as less than 0.1 nmol, for example less than 0.05 nmol, such as less than 0.01 nmol.

Administration The binding members according to the present invention are particular useful in therapeutic methods due to their high affinity and specificity.

Accordingly, the composition can be used towards disorders of appetite regulation such as overweight and obesity. Further the methods of include reducing food in¬ take, treating diabetes mellitus, and improving lipid profile (including reducing LDL cholesterol and triglyceride levels and/or changing HDL cholesterol levels). The composition may be used for the treatment of any disorder mentioned above. The scope of the present invention further includes methods of treatment of an indi¬ vidual in need thereof, comprising administering to said individual an effective amount of one or more of the pharmaceutical compositions described herein. Said individual is preferably suffering, or at risk of, one or more of the health problems described earlier herein such as a metabolic disorder. By "treatment" is also meant prophylaxis and aftercare, and/or lessening of disease symptoms and/or possible disease prevention and/or cure. Said method of treatment may comprise improving the sense of well-being and the quality of life in an individual. Said method may in¬ volve one or more of the combination treatments as disclosed herein.

The pharmaceutical compositions of the invention may be used both prophylactically as well as for therapeutic administration. Thus, the pharmaceutical composition comprising the binding members can be administered to patients in order to prevent the development of a metabolic disorder, in order to minimise the severity of a dis- order or to patients already suffering from a disorder. Furthermore the therapeutic method may prevent reoccurrence of disorders of metabolism.

In an embodiment of the invention the pharmaceutical composition is administered for the treatment of a metabolic disorder. In an embodiment of the invention the pharmaceutical composition is administered for the treatment of a bowel disease. In an embodiment of the invention the pharmaceutical composition is administered for the treatment of an eating disorder. In an embodiment of the invention the pharma¬ ceutical composition is administered for the treatment of a metabolic disorder. In an embodiment of the invention the pharmaceutical composition is administered for the treatment of syndrome X. In an embodiment of the invention the pharmaceutical composition is administered for the treatment of obesity. In a further embodiment the pharmaceutical composition is administered for inhibition of weight gain. In an em¬ bodiment the pharmaceutical composition is administered for inhibition of appetite. In an additional embodiment the pharmaceutical composition is administered for inhibition decreasing body weight. In a further embodiment the pharmaceutical com¬ position is administered for decreasing body fat. Another embodiment of the phar¬ maceutical composition is administration for the treatment of disorder of appetite regulation. The pharmaceutical compositions of the invention maybe administered for treatment of PYY deficiency, such as PYY1-36 or PYY3-36 deficiency, more specifically human PYY1-36 or human PYY3-36 deficiency. In an embodiment of the invention the method is for a) the treatment of overweight and/or b) the treatment of obesity and/or c) the treatment of syndrome X and/or d) the treatment of eating disorders and/or e) the treatment of bowel diseases and/or f) inhibition of appetite and/or g) inhibition of weight gain and/or h) decreasing body weight and/or i) decreasing body fat mass and/or j) treatment of bowel diseases or any combinations of the above.

or any combination of the above

The dosage ranges for the administration of the binding members of the invention are those large enough to produce the desired effect in which the symptoms of the disease are ameliorated. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any complication.

The pharmaceutical composition may be prepared so it is suitable for one or more particular administration methods. Furthermore, the method of treatment described herein may involve different administration methods.

The pharmaceutical composition comprising said binding member may be administered to an individual in need there of by any suitable method.

The compositions of the present invention may preferably be delivered to an individ¬ ual in any way so as to achieve a beneficial effect, preferably by inhibiting appetite and/or preventing malnutrition, and/or improving the individual's sense of well-being or quality of life. In one preferred embodiment, a composition according to the pre- sent invention is administered via an oral, nasal, pulmonary, transdermal or par- enteral route. More preferably, the composition is administered via the parenteral route. Other drug-administration methods, which are effective to deliver the drug to a target site or to introduce the drug into the bloodstream, are also contemplated.

The administration route must ensure that a non-degraded active form of the binding member will be the dominating form in the circulation, which will reach the ligands, for example the PYY or a functional equivalent thereof, and subsequently reach the NPY receptors, and allow PYY or a functional equivalent thereof to stimulate these.

The effect of PYY binding members and PYY or functional equivalents according to the invention is believed to be mediated via actions of PYY outside the central nervous system, with the exception of the NPY neurons located in the hypothalamus. Thereby PYY or functional equivalents do not affect NPY neurons located elsewhere in the central nervous system. Accordingly, PYY binding members, PYY and functional equivalents capable of binding to the NPY Y2 recep¬ tor may circulate in the bloodstream to the receptors in the hypothalamus; however, preferably neither of these molecules should be capable of crossing the blood-brain barrier, and thereby able to enter into other parts of the central nervous system.

The composition according to the invention may be administered parenterally, orally, nasally (inhalation or intranasal application), topically (to the skin or to the eye), rec- tally using suppositories or by intravaginal absorption.

The pharmaceutical composition according to the invention may be administered parenterally, e.g. by injection or by gradual infusion over time. The composition may be delivered by way of a pump, such as an implantable pump for constant and long term infusion or such as a peristaltic-type pump. Parenteral administration may be intravenously, intra-arterially, intraperitoneal^, intramuscularly, subcutaneously, intracavity, intranasally or transdermally.

The pharmaceutical compositions containing a binding member of this invention may be administered intravenously, as by injection of a unit dose, for example. The term "unit dose" when used in reference to a pharmaceutical composition of the pre¬ sent invention refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.

Suitable dosing regimens are preferably determined taking into account factors well known in the art including type of subject being dosed; age, weight, sex and medical condition of the subject; the route of administration; the renal and hepatic function of the subject; the desired effect; and the particular compound employed.

Optimal precision in achieving concentrations of drug within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the drug's availability to target sites. This involves a consideration of the distribution, equilibrium, and elimination of a drug.

The pharmaceutical compositions containing a binding member may be adminis¬ tered in unit dosis. The term "unit dose" when used in reference to a pharmaceutical composition of the present invention refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of ac¬ tive material calculated to produce the desired therapeutic effect in association with the required diluents; i.e., carrier, or vehicle.

A therapeutically effective amount of a binding member of this invention is typically an amount the binding member such that when administered in a physiologically tolerable composition is sufficient to achieve a plasma concentration of the binding member from about 0.1 mM to about 100 mM, preferably from about 1 mM to about 5 mM, and usually about 5 mM, in one or more dose administrations daily, for one or several days. The effective amount may also be measured as the amount of the binding member with is sufficient to increase the plasma concentration of PYY to the desired concentration. The dose may be measured in concentration equivalents, based on a dose response curve of a known compound.

In a preferred embodiment the pharmaceutical composition is administered in a concentration equivalent to from 0.1 unit to 1 unit per kg bodyweight, such as from 0.5 unit to 0.5 unit per kg bodyweight, such as from 1.0 unit to 0.1 unit per kg body- weight, such as from 1.0 unit to 50 unit per kg bodyweight, such as from 1.0 unit to 10 unit per kg bodyweight.

In a preferred embodiment the pharmaceutical composition is administered in a concentration equivalent to from 0.1 μg to1 g/per kg bodyweight, or such as from 0.5 μg to 0.5g/per kg bodyweight, or such as from 1 μg to100 mg/per kg bodyweight, or such as from 5 μg to 20 mg/per kg bodyweight, or such as from 20 μg to 1 mg per kg bodyweight, or such as from 50 μg to 1 mg per kg bodyweight, or such as from 100 μg to 1 mg per kg bodyweight, or such as from 100 μg to 500 μg per kg body- weight, or such as from 0.5 mg to 5 mg per kg bodyweight, such as from 1.0 mg to 10 mg per kg bodyweight, such as from 5 to 50, such as from 5 to 250 mg per kg bodyweight.

The pharmaceutical preparations described herein are preferably in unit dosage forms. In such form, the preparation is subdivided into unit doses containing appro¬ priate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form. When desired, compositions can be pre¬ pared with enteric coatings adapted for sustained or controlled release administra¬ tion of the active ingredient.

In one aspect of the present invention, a suitable dose of the compositions de- scribed herein is administered in pharmaceutically effective amounts to an individual in need of such treatment. Herein, "pharmaceutically effective amounts", is defined as an administration involving a total amount of each active component of the phar¬ maceutical composition or pharmaceutical composition or method that is sufficient to show a meaningful patient benefit. The term "unit dosage form" as used herein re- fers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of a compound, alone or in combination with other agents, calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier, or vehicle. The specifications for the unit dosage forms of the present invention depend on the particular compound or compounds employed and the effect to be achieved, as well as the pharmacodynamics associated with each compound in the host. The dose administered should be an "effective amount" or an amount necessary to achieve an "effective level" in the individual patient.

The dosage requirements will vary with the particular drug composition employed, the route of administration and the particular subject being treated. Ideally, a patient to be treated by the present method will receive a pharmaceutically effective amount of the compound in the maximum tolerated dose, generally no higher than that required before drug resistance develops. Suitable dosing regimens are preferably determined taking into account factors well known in the art including type of subject being dosed; age, weight, sex and medical condition of the subject; the route of administration; the renal and hepatic function of the subject; the desired effect; and the particular compound employed.

Optimal precision in achieving concentrations of drug within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the drug's availability to target sites. This involves a consideration of the distribution, equilibrium, and elimination of a drug.

The administration route must ensure that the non-degraded, bioactive form of the binding member will be the dominating form in the circulation, which will reach PYY and subsequently an NPY receptor. Thus, in order to obtain the maximum effect of the pharmaceutical composition it is preferably administered one time, two times, or three times daily, each administration being within 90 minutes of a meal, such as within 85 minutes of a meal, such as within 80 minutes of a meal, such as within 75 minutes of a meal, such as within 70 minutes of a meal, such as within 65 minutes of a meal, such as within 60 minutes of a meal, such as within 55 minutes of a meal, such as within 50 minutes of a meal, such as within 45 minutes of a meal, such as within 40 minutes of a meal, such as within 35 minutes of a meal, such as within 30 minutes of a meal, such as within 25 minutes of a meal, such as within 20 minutes of a meal, such as within 15 minutes of a meal, such as within 10 minutes of a meal, such as within 5 minutes of a meal. More preferred the pharmaceutical composition is administered prior to each main meal, such as administered three times daily. The pharmaceutical compositions may be administrated by intravenous infusion. The duration of an infusion may be less than 120 minutes, such as less than 100 minutes, such as less than 80 minutes, such as less than 60 minutes, such as less than 40 minutes, such as less than 20 minutes, such as less than 10 minutes or such as less than 5 minutes.]

For the present invention the dosage will vary depending on the compound em¬ ployed and the mode of administration.

Dosage levels of the binding member may vary between about 0.01 μg/kg body weight to 10 mg/kg body weight or such as between 0.05 μg/kg body weight to 2 mg/kg body weight, or such as between 0.1 μg/kg body weight to 1 mg/kg body weight, or such as between 0.1 μg/kg body weight to 500 μg/kg body weight, or such as between 1 μg/kg body weight to 500 μg/kg body weight, or such as between 1 μg/kg body weight to 100 μg/kg body weight, or such as between 100 μg/kg body weight to 500 μg/kg body weight, or such as between 200 μg/kg body weight to 1 mg/kg body weight, preferably between about 0.5 μg/kg body weight to 100 μg/kg body weight, more preferably between 0.7 to 50 μg /kg body weight.

In one preferred embodiment of the present invention, the compositions of the pre¬ sent invention are administered in doses of the binding member from about 0.1 μg/kg per day to about 1 mg/kg per day. In another preferred embodiment, the com¬ positions are administered in doses of from about 5 μg/kg per day to about 250 μg/kg per day of the binding member.

The dosages may be from 1 pmol to 5 nmol/kg, such as from 5 pmol to 1 nmol/kg, or such as from 100 pmol to 500 pmol/kg.

In a preferred embodiment the composition of the invention is administered in dos- age of a binding member from 5 nmol to 5 mmol, or such from 50 nmol to 1 mmol, 100 nmol to 500 μmole, or such from 500 nmol to 100 μmol, or such as from 100 nmol to 50 μmol, or such as from 50 nmol to 25 μmol, or such as from 1 μmol to 10 μmol, or such as from 5 μmol to 25 μmol, or such as from 200 nmol to 2.5 μmol. The pharmaceutical composition may be administered once a day, or such as two times, or such as three times a day, or such as four times a day, or such as five times a day. The composition of the present invention may be administered admixed with a pharmaceutically acceptable carrier or diluent.

For all methods of use disclosed herein for the compounds, the daily oral dosage regimen will preferably be from about 0.01 μg to about 10 mg/kg body weight. The daily parenteral dosage regimen will preferably be from about 0.01 μg to about 10 mg/kg body weight. The daily topical dosage regimen will preferably be from 0.01 μg to 150 mg, administered one to four, preferably two or three times daily. The daily inhalation dosage regimen will preferably be from about 0.01 μg /kg to about 10 mg/kg per day. It will also be recognized by one of skill in the art that the optimal quantity and spacing of individual dosages of a compound or a pharmaceutically acceptable salt thereof will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the particular patient being treated, and that such optimums can be determined by conventional tech¬ niques. It will also be appreciated by one of skill in the art that the optimal course of treatment, i.e., the number of doses of a compound or a pharmaceutically accept¬ able salt thereof given per day for a defined number of days, can be ascertained by those skilled in the art using conventional course of treatment determination tests.

Furthermore, since the "effective level" is used as the preferred endpoint for dosing, the actual dose and schedule can vary, depending on individual differences in pharmacokinetics, drug distribution, and metabolism. The "effective level" can be defined, for example, as the blood or tissue level desired in the patient that corre¬ sponds to a concentration of one or more compounds according to the invention.

Combination therapy The compounds according to the invention may be administered with at least one other compound. The compounds may be administered simultaneously, either as separate compositions or combined in a unit dosage form, or administered sequen¬ tially.

Administration of the pharmaceutical composition of the present invention may be used in combination with any suitable therapy. By treatment "in combination" is meant that another treatment regime may be carried out before, during (including concurrently with) and after treatment of an individual with a pharmaceutical compo¬ sition of the present invention.

In a preferred embodiment the pharmaceutical composition of the invention is ad¬ ministered in combination with a second pharmaceutical composition. Said second pharmaceutical composition may be administered together or separately from the pharmaceutical composition, of the present invention.

Said second pharmaceutical composition may comprise compound(s) selected from the group of; sibutramine and other blockers of the presynaptic reuptake of sero¬ tonin and noradrenalin, orlistat and other inhibitors of Gl lipases, β3-adrenergic re¬ ceptor agonists, uncoupling proteins agonists or inducers, (specific) antagonists of PPARy (Peroxisome Proliferator-Activated Receptor y), GLP-1 agonists including (but not limited to) GLP-1 , exendin-4 (AC2993) and various formulations thereof, liraglutide (NN2211 ), CJC-1131 and ZP10/AVE-0010, insulin secretagogues, bigua- nides including (but not limited to) metformin, sulphonylureas including (but not lim¬ ited to) glibenclamide, glipizide and glimepiride, α-glucosidase inhibitors (acarbose and miglitol), thiazolidinediones, rimonabant and other CB1 endocannabinoid recep- tor antagonists, bupropion, topiramate, leptin agonists, ciliary neurotrophic factor, peptide analogues of the human growth hormone fragment 177-191 , chole- cystokinin-A receptor agonists, melanocortin-3 agonists and noradrenergic drugs such as phentermine, diethylpropion, phendimetrazine and benzphetamine.

In a further embodiment said second pharmaceutical composition may comprise compound(s) selected from the group of; anti-hypertensives including (but not lim¬ ited to) beta-adrenoceptor antagonists, alpha-adrenoceptor antagonists, calcium antagonists, angiotensin converting enzyme (ACE) inhibitors, angiotensin Il receptor antagonists and diuretics (including, but not limited to, thiazide diuretics and loop diuretics) , insulin, DPP-IV inhibitors, anti-depressant medications including (but not limited to) selective serotonin reuptake inhibitors (SSRIs), mianserine and mirtazap- ine, classical as well as atypical antipsychotic drugs (neuroleptics), corticosteroids as well as any other drug(s) that may increase body weight or body fat mass or that may be indicated for the treatment of hypertension, overweight, obesity, syndrome X and/or diabetes mellitus. In an embodiment of the invention said second pharmaceutical composition com¬ prises PYY or a functional equivalent there of. In a further preferred embodiment the second pharmaceutical composition comprises PYY1-36 or a functional equivalent thereof. In a more preferred embodiment the second pharmaceutical composition comprises PYY3-36 or a functional equivalent thereof.

In an embodiment of the invention the pharmaceutical composition of the invention is administered in combination with a second pharmaceutical composition for the treatment of a metabolic disorder. In an embodiment of the invention the pharma¬ ceutical composition of the invention is administered in combination with a second pharmaceutical composition for the treatment of a bowel disease. In an embodiment of the invention the pharmaceutical composition of the invention is administered in combination with a second pharmaceutical composition for the treatment of an eat- ing disorder. In an embodiment of the invention the pharmaceutical composition of the invention is administered in combination with a second pharmaceutical composi¬ tion for the treatment of obesity. In a further embodiment the pharmaceutical com¬ position of the invention is administered in combination with a second pharmaceuti¬ cal composition for inhibition of weight gain. In an embodiment the pharmaceutical composition of the invention is administered in combination with a second pharma¬ ceutical composition for inhibition of appetite. In an additional embodiment the pharmaceutical composition of the invention is administered in combination with a second pharmaceutical composition for inhibition decreasing body weight. In a fur¬ ther embodiment the pharmaceutical composition of the invention is administered in combination with a second pharmaceutical composition for decreasing body fat. Another embodiment of the pharmaceutical composition of the invention is adminis¬ tered in combination with a second pharmaceutical composition for the treatment of disorder of appetite regulation. The pharmaceutical compositions of the invention maybe administered in combination with a second pharmaceutical composition for treatment of PYY deficiency, such as PYY1-36 or PYY3-36 deficiency, more spe¬ cifically human PYY1-36 or human PYY3-36 deficiency.

The doasage is generally calculated based on the bodyweight of the subject but in certain situations the dosages may be calculated base on the fat free mass (FFM) of the subject. Thus the dosage may be in concentration equivalent to PYY1-36 or PYY3-36.

For the present invention the dosage will vary depending on the compound em- ployed and the mode of administration. Dosage levels may vary between about 4 ng/kg body weight to 20 μg/kg body weight daily, preferably between about 10 ng/kg body weight to 1 μg/kg body weight, more preferably between 50 to 750 ng/kg body weight. Alternative dosages in relation to FFM may vary between about 5 ng/kg FFM to 25 μg/kg FFM daily, preferably between about 12.5 ng/kg FFM to 1.25 μg/kg FFM, more preferably between 62.5 to 875 ng/kg FFM. To obtain dosages in rela¬ tion to FFM the dosage/kg bodyweight should be multiplied by the factor 1.25.

The dosage may be administered when needed, such as up to ten times daily, such as one to five times daily, such as two or three times daily, or preferably such as once a day, thus the daily dosage maybe up to 2-5 times the dosages mentioned above. Alternatively, the dosage may be administered less frequently than once daily as described herein above.

A preferred dosage of a composition employed according to the invention is in a concentration equivalent to PYY or a functional equivalent there of from 4 ng to about 20 μg per kg bodyweight, or such as from 10 ng to 1 μg per kg bodyweight, more preferably from 50 to 750 ng per kg bodyweight. The dosage of PYY is pref¬ erably 20-200 ng/kg, 20-160 ng/kg, 40-160 ng/kg, 40-120 ng/kg, 40-80 ng/kg, 60- 120 ng/kg or such as approximately 60 ng/kg or 80 ng/kg.

The preferred dosages may be in a concentration equivalent to PYY or a functional equivalent there of from 1 pmol/kg to 5 nmol/ kg, such as from 5 pmol/kg to 1 nmol/kg, or such as from 20 pmol/kg to 500 pmol/kg altemativly such as from 40 to 160 pmol/kg or such as from 75 to 120 pmol/kg. PYY1-36 is preferably administered in dosages of, such as from 50-400 pmoi/kg, such as 80-320 pmol/kg, such as 150- 250 pmol/kg, such as about 200 pmol/kg. PYY3-36 is preferably administered in dosages of such as from 30-350 pmol/kg, such as 50-280 pmol/kg, such as 80- 200 pmol/kg, such as about 120 pmol/kg. Dosages in relation to FFM may be calculated by multiplying the indicated dosages with 1.25. In a second embodiment the dosage may be 5-50 pmol/kg, 5-40 pmol/kg, 5 to 30 pmol/kg, 10-40 pmol/kg, 10-30 pmol/kg such as 5 to 25 pmol/kg, such as 5 to 20 pmol/kg, and most preferably 10-20 pmol/kg, 15-30 pmol/kg or approximately 15 pmol/kg or 20 pmol/kg.

The dosages are preferably administrated once a day, or such as two times a day, or such as three times a day, or such as four times a day, or such as five times a day, or such as more than five times a day.

In one preferred embodiment of the present invention, the compositions are admin¬ istered in dosages of PYY or a functional equivalent from about 400 ng to about 2 mg, more preferably from about 10 μg to about 200 μg, or from about 5 μg to about 250 μg, more preferably from about 20 μg to about 200 μg, more preferably from about 20 μg to about 100 μg. Most preferably the dosage may be 1-20 μg, 2-16 μg, 4-16 μg, 4-12 μg, 4-8 μg, 6-12 μg, 6-10 μg or approximately 8 μg.

In a preferred embodiment the composition is administered in dosages of PYY or a functional equivalent from 100 pmol to 500 nmole, or such from 500 pmol to 100 nmol, or such as from 1 nmol to 50 nmol, or such as from 2 to 25 nmol, or such as from 4 to 20 nmol. Alternatively the preferred dosage may be 0,25-5 nmol, 0,5-4 nmol, 1-4 nmol, 1-3 nmol, 1-2 nmol, 1 ,5-3 nmol or more preferably 1 ,5-2,5 nmol or most preferably approximately 2 nmol. In a further preferred embodiment a PYY1-36 dosages includes such as from 5-40 nmol, such as 8-32 nmol, such as 15- 25 nmol, such as about 20 nmol, wheras a dosage of PYY3-36 includes such as from 3-35 nmol, such as 5-28 nmol, such as 8-200 nmol, such as about 12 nmol.

In another embodiment, the PYY or functional equivalent is administered subcuta- neously in a dosage of 5-30 pmol/kg, such as 5-25 pmol/kg, such as 5-20 pmol/kg or such as 10-20 pmol/kg bodyweight, in order to achieve an effective level in the indi- vidual treated. The presently preferred dosage is 10-20 pmol/kg bodyweight of PYY1-36 or PYY 3-36. In second preferred embodiment the dosages of PYY1-36 is 150-250 pmol/kg and/or the dosages of PYY3-36 is 80-150 pmol/kg. The dosages of PYY or the functional equivalent is preferably administered once a day, or such as two times a day, or such as three times a day, or such as four times a day, or such as five times a day.

The pharmaceutical preparations described herein may also be arranged in unit dosage forms. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as powders in compartments. In this embodiment the powders may be mixed with a solvent prior to or during use.

In a preferred embodiment the PYY composition is administrated in unit dosage form, from about 400 ng to about 2 mg of PYY or a functional equivalent thereof, more preferably from about 10 μg to about 200 μg, or from about 5 μg to about 250 μg, more preferably from about 20 μg to about 200 μg, more preferably from about 20 μg to about 100 μg. The unit dosage form may comprise from 100 pmol to 500 nmole, or such from 500 pmol to 100 nmol, or such as from 1 nmol to 50 nmol, or such as from 2 to 25, such as from 4 nmol to 20 nmol. of PYY or a functional equiva¬ lent thereof. The compositions are preferably administered once a day, or such as two times a day, or such as three times a day, or such as four times a day, or such as five times a day. In a further preferred embodiment a unit dosage of PYY1-36 includes such as from 5-40 nmol, such as 8-32 nmol, such as 15- 25 nmol, such as about 20 nmol, wheras a unit dosage of PYY3-36 includes such as from 3-35 nmol, such as 5-28 nmol, such as 8-200 nmol, such as about 12 nmol.

In certain embodiment the pharmaceutical composition may be administered by in¬ fusions. The dosages of PYY or a functional equivalent for infusion may be from 0.01 pmol/kg minute to 500 pmol/kg minute such as from 0.05 pmol/kg minute to 100 pmol/kg minute, or such as from 0.1 pmol/kg minute to 50 pmol/kg minute, or such as from 1 pmol/kg min. to 25 pmol/kg minute.

In a more preferred embodiment the method of treatment comprising administration of the pharmaceutical composition according to the invention in combination with a second composition comprising PYY or a functional equivalent thereof is for the treatment of PYY1 -36 or PYY3-36 deficiency. The kit-in-part may be used for simultaneous, sequential or separate administration.

In an embodiment the pharmaceutical composition according to the invention may be used for the treatment of an individual in need thereof. The composition may be used for the treatment of a metabolic disorder as described above, thus for the treatment of overweigt, obesity, syndrome X, disorder of appetite regulation, eating disorders and/or bowel diseases.

Isolated nucleic acid molecule/vector/host cell In one aspect the invention relates to an isolated nucleic acid molecule encoding at least a part of the binding member as defined above.

In one embodiment the nucleic acid molecule encodes a light chain and another nucleic acid encodes a heavy chain. The two nucleic acid molecules may be sepa¬ rate or they may be fused into one nucleic acid molecule, optionally spaced apart by a linker sequence. In particular in relation to antibody fragments the nucleic acid molecule may encode the whole binding member, however dependant on the design of the binding member this may also be relevant for some larger binding members.

I one embodiment the nucleic acid molecule encode an affibody sequence or and antibody-affibody chimera sequence. In a further aspect of the invention the nucleic acids encodes two nucleic acid molecules separate or fused. In particular more than one antibody-affibody chimera sequence may be included, for example two, three or four antibody-affibody chimera sequences may be included.

The nucleic acid molecule preferably is a DNA sequence, more preferably a DNA sequence comprising in its upstream end regulatory elements promoting the ex¬ pression of the binding member once the nucleic acid molecule is arranged in a host cell.

Accordingly, in one embodiment the invention relates to a polynucleotide selected from the group consisting of i) a polynucleotide encoding a fragment of a polypeptide, where in said fragment

a) is capable of recognising an antigen also being recognised by the binding member and/or.

b) is capable of binding selectively to an antigen, wherein said antigen is also bound selectively by the binding member and/or

c) has a substantially similar or higher binding affinity to an antigen bound selectively by the binding member

ii) a polynucleotide, the complementary strand of which hybridize under stringent conditions, with a polynucleotide as defined in i),

iii) a i polynucleotide comprising a nucleotide sequence which is degenerate to 3 the nucleotide sequence of a polynucleotide as defined in i)

and the complementary strand of such a polynucleotide.

The invention further relates to a vector comprising the nucleic acid molecule as defined above, either one vector per nucleic acid, or two or more nucleic acids in the same vector. The vector preferably comprises a nucleotide sequence which regu¬ lates the expression of the antibody encoded by the nucleic acid molecule.

In yet another aspect the invention relates to a host cell comprising the nucleic acid molecule as defined above. Such a host cell being a prokaryotic cell, for example E. coli, or a eukaryotic cell as for example a yeast strain or a mammalian cell line.

Also, the invention relates to a cell line engineered to express the binding member as defined above, this cell line for example being, Escherichia coli, Saccharomyces cerevisiea, Pichia pastoris, a hybridoma of a murine lymphocyte and an immortal¬ ised cell line. The cell line may be any suitable cell line, however the cell line P3 is preferred. In another embodiment a CHO cell line is preferred. Diagnostic methods The present invention also contemplates various assay methods for determining the presence, and preferably the amount of a PYY or functional equivalents thereof, typically in a biological sample.

Accordingly, the present invention relates to a method of detecting or diagnosing a metabolic disorder in an individual comprising

a) providing a biological sample from said individual, b) adding at least one binding member, capable of specifically binding PYY or functional equivalents thereof, to said biological sample c) detection of binding members bound to said biological sample, thereby detecting or diagnosing the disease or disorder.

The bound binding member may be detected either directly or indirectly and thereby the amount of PYY or functional equivalents thereof in the sample is measured.

Those skilled in the art will understand that there are numerous well known clinical diagnostic chemistry procedures in which a binding reagent of this invention can be used to form a binding reaction product whose amount relates to the amount of the ligand in a sample. Thus, while exemplary assay methods are described herein, the invention is not so limited.

Various heterogenous and homogeneous protocols, either competitive or noncompetitive, can be employed in performing an assay method of this invention.

Binding conditions are those that maintain the ligand-binding activity of the receptor. Those conditions include a temperature range of about 4 to 50 degrees Centigrade, a pH value range of about 5 to 9 and an ionic strength varying from about that of distilled water to that of about one molar sodium chloride.

The detecting step can be directed, as is well known in the immunological arts, to either the complex or the binding reagent (the receptor component of the complex). Thus, a secondary binding reagent such as an antibody specific for the receptor may be utilized.

Alternatively, the complex may be detectable by virtue of having used a labelled receptor molecule, thereby making the complex labelled. Detection in this case comprises detecting the label present in the complex.

A further diagnostic method may utilize the multivalency of a binding member composition of one embodiment of this invention to cross-link ligand, thereby forming an aggregation of multiple ligands and polypeptides, producing a precipitable aggregate. This embodiment is comparable to the well known methods of immune precipitation. This embodiment comprises the steps of admixing a sample with a binding member composition of this invention to form a binding admixture under binding conditions, followed by a separation step to isolate the formed binding complexes. Typically, isolation is accomplished by centrifugation or filtration to remove the aggregate from the admixture. The presence of binding complexes indicates the presence of the preselected ligand to be detected.

Thus, a further embodiment of the invention relates to a kit comprising at least one binding member capable of binding PYY specifically, said molecule being labelled.

The present invention also describes a diagnostic system, preferably in kit form (de¬ scribed below), for assaying for the presence of PYY, in a biological sample where it is desirable to detect the presence, and preferably the amount, of PYY in a sample according to the diagnostic methods described herein.

The diagnostic system includes, in an amount sufficient to perform at least one as¬ say, a binding member composition according to the present invention, preferably as a separately packaged reagent, and more preferably also instruction for use.

The biological sample can be a tissue, tissue extract, fluid sample or body fluid sample, such as blood, plasma or serum.

"Packaged" refers to the use of a solid matrix or material such as glass, plastic (e.g., polyethylene, polypropylene or polycarbonate), paper, foil and the like capable of holding within fixed limits a binding member of the present invention. Thus, for ex- ample, a package can be a glass vial used to contain milligram quantities of a con¬ templated labelled binding member preparation, or it can be a microtiter plate well to which microgram quantities of a contemplated binding member has been operatively affixed, i.e., linked so as to be capable of binding a ligand.

"Instructions for use" typically include a tangible expression describing the reagent concentration or at least one assay method parameter such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions and the like.

A diagnostic system of the present invention preferably also includes a label or indi¬ cating means capable of signalling the formation of a binding reaction complex con¬ taining a binding member complexed with the preselected ligand.

Any label or indicating means can be linked to or incorporated in an expressed poly¬ peptide, or phage particle that is used in a diagnostic method. Such labels are themselves well-known in clinical diagnostic chemistry.

The labelling means can be a fluorescent labelling agent that chemically binds to antibodies or antigens without denaturing them to form a fluorochrome (dye) that is a useful immunofluorescent tracer. Suitable fluorescent labelling agents are fluoro- chromes such as fluorescein isocyanate (FIC), fluorescein isothiocyante (FITC), 5- dimethylamine-1-naphthalenesulfonyl chloride (DANSC), tetramethylrhodamine isothiocyanate (TRITC), lissamine, rhodamine 8200 sulphonyl chloride (RB 200 SC) and the like. A description of immunofluorescence analysis techniques is found in DeLuca, "Immunofluorescence Analysis", in Antibody As a Tool, Marchalonis, et al., eds., John Wiley & Sons, Ltd., pp. 189-231 (1982), which is incorporated herein by reference.

In preferred embodiments, the indicating group is an enzyme, such as horseradish peroxidase (HRP), glucose oxidase, or the like. In such cases where the principal indicating group is an enzyme such as HRP or glucose oxidase, additional reagents are required to visualize the fact that a receptor-ligand complex (immunoreactant) has formed. Such additional reagents for HRP include hydrogen peroxide and an oxidation dye precursor such as diaminobenzidine. An additional reagent useful with glucose oxidase is 2,2'-amino-di-(3-ethyl-benzthiazoline-G-sulfonic acid) (ABTS).

Radioactive elements are also useful labelling agents and are used illustratively herein. An exemplary radiolabelling agent is a radioactive element that produces gamma ray emissions. Elements which themselves emit gamma rays, such as 124I, 1251, 1281, 132I and 51Cr, represent one class of gamma ray emission-producing radio¬ active element indicating groups. Particularly preferred is 125I. Another group of use¬ ful labelling means are those elements such as 11C, 18F, 15O and 13N which them¬ selves emit positrons. The positrons so emitted produce gamma rays upon encoun- ters with electrons present in the animal's body. Also useful is a beta emitter, such as 111indium Of 3H.

The linking of labels, i.e., labelling of, polypeptides and proteins or phage is well known in the art. For instance, proteins can be labelled by metabolic incorporation of radioisotope-containing amino acids provided as a component in the culture me¬ dium. See, for example, Galfre et al., Meth. Enzymol., 73:3-46 (1981). The tech¬ niques of protein conjugation or coupling through activated functional groups are particularly applicable. See, for example, Aurameas, et al., Scand. J. Immunol., Vol. 8 Suppl. 7:7-23 (1978), Rodwell et al., Biotech., 3:889-894 (1984), and U.S. Pat. No. 4,493,795.

The diagnostic systems can also include, preferably as a separate package, a spe¬ cific binding agent. A "specific binding agent" is a molecular entity capable of selec¬ tively binding a binding member species of the present invention or a complex con- taining such a species, but is not itself a binding member of the present invention. Exemplary specific binding agents are antibody molecules, complement proteins or fragments thereof, S. aureus protein A, and the like. Preferably the specific binding agent binds the binding member species when that species is present as part of a complex.

In preferred embodiments, the specific binding agent is labelled. However, when the diagnostic system includes a specific binding agent that is not labelled, the agent is typically used as an amplifying means or reagent. In these embodiments, the la¬ belled specific binding agent is capable of specifically binding the amplifying means when the amplifying means is bound to a reagent species-containing complex. The diagnostic kits of the present invention can be used in an "ELISA" format to detect the quantity of a preselected ligand in a fluid sample. "ELISA" refers to an Enzyme-Linked Immunosorbent Assay that employs an antibody or antigen bound to a solid phase and an enzyme-antigen or enzyme-antibody conjugate to detect and quantify the amount of an antigen present in a sample and is readily applicable to the present methods.

Thus, in some embodiments, a binding member of the present invention can be af- fixed to a solid matrix to form a solid support that comprises a package in the sub¬ ject diagnostic systems.

A reagent is typically affixed to a solid matrix by adsorption from an aqueous me¬ dium although other modes of affixation applicable to proteins and polypeptides can be used that are well known to those skilled in the art. Exemplary adsorption meth¬ ods are described herein.

Useful solid matrices are also well known in the art. Such materials are water in¬ soluble and include the cross-linked dextran available under the trademark SEPHADEX from Pharmacia Fine Chemicals (Piscataway, N. J.); agarose; beads of polystyrene beads about 1 micron to about 5 millimeters in diameter available from Abbott Laboratories of North Chicago, III.; polyvinyl chloride, polystyrene, cross- linked polyacrylamide, nitrocellulose- or nylon-based webs such as sheets, strips or paddles; or tubes, plates or the wells of a microliter plate such as those made from polystyrene or polyvinylchloride.

The binding member species, labelled specific binding agent or amplifying reagent of any diagnostic system described herein can be provided in solution, as a liquid dispersion or as a substantially dry power, e.g., in lyophilized form. Where the indi- eating means is an enzyme, the enzyme's substrate can also be provided in a sepa¬ rate package of a system. A solid support such as the before-described microtiter plate and one or more buffers can also be included as separately packaged ele¬ ments in this diagnostic assay system. Examples

The following examples illustrate the invention without limiting it thereto.

Example 1

Binding assay and functional assay Transfections and tissue culture: COS-7 cells can be grown Dulbecco's Modified Eagle'e Medium 1885 supplemented with 10% fetal calf serum, 2mM glutamine and 0.01 mg/ml gentamicin. The expression plasmids containing the cDNAs encoding the wild type or the mutated receptors can be transiently expressed after transfetion according to the calcium phosphate precipitation method and assay can be per¬ formed 48 hour after transfection. Binding assay: One day after transfection the cells will be transferred and seeded in multi-well plates for assay. The number of cells to be plated per well will be chosen so as to obtain 5 to 10% binding of the radioligand added. Two days after transfec¬ tion the cells will be assayed in competition binding assays using 125I- PYY(3-36) as a tracer. Radioligand will be bound in a buffer composed of 0.5 ml of 50 mM Hepes buffer, pH 7.4, supplemented with 1 mM CaCI2, 5 mM MgCI2, and 0.1% BSA, and displaced in a dose dependent manner by unlabelled ligands. The assay will be per¬ formed in duplicate for 3 hours at 40C, and stopped by washing twice in the buffer. Cell associated, receptor bound radioligand will be determined by the addition of lysis buffer (48% urea, 2% NP-40 in 3M acetic acid). The concentration of radioli- gand in the assay corresponds to a final concentration of approximately 20 pM. Functional assay. COS-7 can be cultured as described above and contransfections can be performed. The activation of Phospholipase C by chimeric G-proteins (Conklin B) formed be¬ tween both Gαq and Gαi the Y2 receptor can be measured through the inositol phosphate (IP) turnover in the cell. The IP turnover may be recorded by use of fol¬ lowing assay: One day after transfection COS-7 cells are incubated for 24 hours with 5 :Ci of [3H]- myo-inositol (Amersham, PT6-271 ) in 1 ml medium supplemented with 10% fetal calf serum, 2 mM glutamine and 0.01 mg/ml gentamicin per well. Cells are washed twice in buffer, 20 mM HEPES, pH 7.4, supplemented with 140 mM NaCI, 5 mM KCI, 1 imM MgSO4, 1 mM CaCI2, 10 mM glucose, 0.05 % (w/v) bovine serum; and are incubated in 0.5 ml buffer supplemented with 10 mM LiCI at 37EC for 30 min. The indicated curves are furthermore incubated with adenosine deaminase ADA (200U/mg, Boeringer Mannheim, Germany) for 30 min in a concentration of 1 U/ml . After stimulation with various concentrations of peptide for 45 min at 37 C, cells will be extracted with 10 % ice-cold perchloric acid followed by incubation on ice for 30 min. The resulting supernatants are neutralized with KOH in HEPES buffer, and the generated [3H]-inositol phosphate is purified on Bio-Rad AG 1-X8 anion-exchange resin. Determinations will be made in duplicates.

Example 2

Synthetic production of PYY and functional equivalents thereof The polypeptide of the present invention may be produced by a conventional peptide synthesis method. Amino acid derivatives and synthesis reagents, can be obtained from commercial sources. Peptide chain extension is performed by mainly using Applied Biosystem 433A synthesizer produced by Perkin Elmer, and a protected peptide derivative- resin is constructed by the Boc or Fmoc method. The protected peptide resin ob- tained by the Boc method is deprotected with anhydrous hydrogen fluoride (HF) in the presence of p-cresol thereby releasing the peptide, which is then purified. The protected peptide resin obtained by the Fmoc method is deprotected with trifluoroacetic acid (TFA) or dilute with TFA containing various scavengers, and the released peptide is purified. Purification is performed in reversed phase HPLC on a C4 or C18 column. The purity of the purified product is confirmed by reverse phase HPLC, and its structure is confirmed by amino acid composition analysis and mass spectrometry.

Example 3

Measurements of PYY plasma levels. The experiment is performed by subcutaneous injections of placebo and 4 escalat¬ ing doses of PYY1-36 or PYY3-36 as set out in table 2. The dosages of PYY is cal¬ culated base on the fat free mass (FFM) of the subject.

Table 2. Dosages of PYY and number of subjects (n)

The results are presented as mean±SE, paired t-test (SAS) and repeated measures (SAS).

The PYY injections is performed at time 0 minutes and the plasma concentrations of PYY upon PYY1-36 and PYY3-36 administration is measured at t=O, 15, 30, 45, 60, 75, 90, 120, 150, 180, 210 and 240 minutes.

PYY assay The plasma concentration of PYY is measured using radioimmunoassay of PYY. The assays are performed using PYY antiserum (code no. 8412-5) (Euro- Diagnostica, Malmoe, Sweden). The antiserum recognizes both human PYY 1-36 and PYY 3-36. Synthetic human PYY 1-36 (Peninsula, Merseyside, UK) and porcine 125I-PYY (code no. IM259) is purchased from Amersham Biosciences, Buckingham- shire UK) for use as standards. Detection limit of the assay is below 2 pmol/l and 50 % inhibition is obtained with 40 pmol/l PYY. Recovery of PYY added to plasma in concentrations between 5 and 50 pmol/l deviates less than 15 % from expected val- ues. Intra-assay coefficient of variation is below 5 %. The antiserum shows no cross reaction with human NPY or human PP in concentrations up to 500 pmol/l.

The results are shown in figure 2 and 3.

Measurements of PYY plasma levels upon administration of a binding member.

The PYY binding member may be administered peripherally as described herein, such orally or subcutaneously.

The plasma concentrations of PYY is measured as described above at diffemet time points after administration of the PYY binding member, or alternatively after admini¬ stration of the PYY binding member and PYY or a functional equivalent.

Example 4

Clinical protocol Binge Eating Disorder (BED)

45 subjects that meet the proposed diagnostic criteria/research criteria for BED ac¬ cording to the Diagnostic and Statistical Manual IV (DSM-IV) [see below] are in¬ cluded in the study. The discrete period of time in A1 is a 2-hour period, and the method of determining frequency under item D is counting the number of days on which binges occur.

The study is performed in a double-blinded, placebo-controlled fashion. Subjects are divided into three groups (n=15 in each), groups A, B and C. The subjects are given diaries where they note the type and amount of food ingested and at what time. This initial phase of the study is 4 weeks ("Run-in Phase"), after which the subjects start treatment with one of three regimens, as defined below. The subjects keep diaries where they note the type and amount of food ingested and at what time throughout the treatment phase. The treatment duration is 4 weeks ("Treatment Phase").

Dosing: The subjects of group A receive subcutaneous placebo injections (NaCI) three times daily (distributed evenly over the hours awake). The subjects of group B receive 60 pmol/kg body weight of PYY 3-36 binding member s.c. three times daily and the subjects of group C receive 100 pmol/kg body weight of PYY 3-36 binding member s.c. three times daily.

The subjects' diaries are reviewed by the investigators and the number of binge- eating episodes are determined and further the amount of food/calories ingested per binges/event (EDE) is recorded.

The reduction in the frequency of eating disorder events (binge-eating epi¬ sodes/events), as defined by calculating the number of binge-eating episodes during the 4-week Treatment Phase divided by the number of binge-eating episodes during the Run-in Phase for each subject, and then statistically comparing the result from group B with group A, and the result from group C with group A.

The change in the amount of food ingested during the events is calculated by calcu- lating the number of calories ingested during the binge-eating episodes during the 4- week Treatment Phase divided by the number of calories ingested during the binge- eating episodes during the Run-in Phase for each subject, and then statistically comparing the result from group B with group A, and the result from group C with group A.

Example 5

Measurement of anti-PYY binding member affinity

The following general description is applied.

Instrument and software:

a. BIAcore 3000, surface plasmon resonance instrument b. BiaEval v3.2, software for data analysis. c. All running buffers (HBS-EP/HBS-P) from Biacore.

Method to immobilize Protein G via amines on a CM5 chip (Amine coupling method)

a. Normalize the chip at least twice with appropriate buffer b. A 0.5 μg/mL dilution of Protein-G is made in 1OmM sodium acetate buffer of pH 2.9 c. Activate the CM5 chip for 7 minutes, by flowing freshly mixed EDC & NHS at a flow rate of 5 μL/min, according to the method mentioned in Biacore Handbook. d. Inject Protein-G sample for 22 minutes over this activated surface. e. Deactivate by flowing 1M ethonolamine-HCI for 10 minutes. f. This method couples about 10000 RUs of Protein-G on the activated sur¬ face. g. For the blank surface, the same activation and deactivation procedure is followed without the injection of Protein-G.

Method to immobilize PYY via amines on a CM5 chip (Amine coupling method)

a. A CM5 chip was normalized as above b. Human PYY dilutions are made in concentrations ranging from 50 to 150 μg/mL, in sodium 1OmM acetate buffer of pH 4.0 c. The chip is activated for 7 minutes by flowing freshly mixed EDC and NHS. d. Inject PsaA, made in acetate buffer, by manual injection until the amount captured on the chip reaches the desired level (in our case, 350 and 800 RUs) e. Deactivate the chip by injecting 1 M ethanolamine-HCI for 10 minutes f. Regenerate the chip with a mild acid or base to remove unbound/loosely bound molecules from the chip surface. g. The blank surface is generated in the same method, but without the step of injecting the protein.

Determination of the avidity of binding of anti-PYY binding member with human PYY

a. The general methodology followed to measure avidity of binding mem¬ bers is to flow binding memebers of at least five different concentrations over the antigen surface (described above) at high flow rates. The high flow rates are required to minimize the antibody re-binding to the antigen surface. The association and dissociation phases could vary between 5 to 10 minutes and 30 to 40 minutes respectively. This experimental de¬ sign will lead to the measurement of avidities of the binding members, we try to minimize the effect of this phenomenon in the estimation of avidity. The data analysis is carried out after carefully not including regions that exhibited biphasic behavior of association /dissociation, which was then fit to a 1 :1 Langmuir model. The method of selecting the data for analysis ensures that the estimates of avidity are closer to the true affinities.

b. Binding member concentration: 5, 4, 3, 2, 1 , 0.75 or 0.5 μg/mL All dilutions were made in the running buffer, HBS-EP, pH 7.2.

c. Flow rate of 30 μL/min. Association phase δmin, dissociation phase 30 to 40 min.

d. Regeneration of the surface: flow rate 100 μL/min, buffer: 10OmM HCI w/150mM NaCI, time: 1-2 minutes.