WO2005113600A2 - Methodes de regulation, de traitement et de prise en charge de l'obesite - Google Patents

Methodes de regulation, de traitement et de prise en charge de l'obesite Download PDF

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WO2005113600A2
WO2005113600A2 PCT/GB2005/001916 GB2005001916W WO2005113600A2 WO 2005113600 A2 WO2005113600 A2 WO 2005113600A2 GB 2005001916 W GB2005001916 W GB 2005001916W WO 2005113600 A2 WO2005113600 A2 WO 2005113600A2
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peptide
general formula
monoclonal antibody
molecule
treatment
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PCT/GB2005/001916
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WO2005113600A3 (fr
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Keith Charlton
Andrew Porter
Gillian Strachan
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Haptogen Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)

Definitions

  • the present invention relates to conjugates comprising human (mammalian) cell signalling molecule(s) coupled to a carrier molecule, and methods for generating specific human binding molecules to cell signalling molecules.
  • the signalling molecules can be ligands of the endocannabinoid receptors of the brain, or peptide hormone molecules produced by the digestive tract and involved in the short-term regulation of food intake.
  • the conjugate where the carrier is an immunogenic molecule, and prepared in a pharmaceutical composition, is suitable for use as a vaccine for humans and other mammals to regulate a range of physiological processes controlled by the cell signalling molecules.
  • the conjugate is also used to isolate (human) antibodies, fragments thereof, or other specific binding molecules from protein display libraries, which are capable of binding to the cell signalling molecule.
  • the specific binding molecules (referred to hereafter as antibodies) are utilised in a method of disrupting and modulating cell signalling events in patients to therapeutic effect.
  • Obesity is linked to a number of serious co-morbidities including cardiovascular disease, type II diabetes, cancer and osteoarthritis, making the development of obesity much more than a cosmetic concern.
  • Current research has placed the hapten class known as endocannabinoids (Mol. Wt. approx 375 Da) (Mechoulam & Fride, 2001) and a signalling neuro-peptide called ghrelin (Bloom, 2003; Kojima et al, 1999; Tolle, et al, 2001), centre stage in obesity research, particularly as the classical appetite regulating hormone leptin suppresses both endocannabinoid and ghrelin activity.
  • Endocannabinoids are small non- immunogenic lipid-soluble molecules involved in the signalling process between the stomach and the hypothalamus region of the brain, and stimulate appetite following fasting.
  • the blocking of endocannabinoid activity in a related brain structure, the hippocampus may influence the cognitive abilities of an individual, changing the timeframe and length of information retention and so slowing degenerative brain disease (Caspari, 1999; Emrich et al, 1997; Fernandez-Ruiz et al, 2002).
  • the hypothalamus has been chosen as a target area for anti-obesity therapy because of its role in controlling food intake and body weight.
  • Chronic exposure to cannabinoids/endocannabinoids or ghrelins increases food intake and reduces energy balance with a net gain of body weight, hence the use of cannabis to prevent weight loss in AIDS patients (Williams & Kirkham, 2002).
  • down-regulation of cannabinoid and ghrelin activity in the hypothalamus, (particularly the arcuate nucleus) leads to a reduction in body weight through both appetite suppression and a slightly elevated (but within the normal range) basal metabolic rate (Harrold et al, 2002).
  • the arcuate nucleus is now seen as the central region of the brain controlling both short and long-term weight regulatory systems. It contains two principal types of neuron, one of which produces the neurotransmitters neuropeptide Y (NPY) and agouti-related peptide (AgRP) when activated, leading to increased appetite and reduced metabolism.
  • NPY neuropeptide Y
  • AgRP agouti-related peptide
  • POMC/CART neurons cause the release of ⁇ -MSH which inhibits eating.
  • the two neuron types therefore act in opposition to regulate weight.
  • increased leptin activates POMC/CART neurons and inhibits NPY/ AgRP release.
  • insulin has recently been shown to act on the arcuate nucleus to restrict NPY production.
  • PYY and ghrelin Two peptide hormones, PYY and ghrelin, produced by the large intestine and stomach respectively, also act upon the arcuate nucleus.
  • PYY suppresses NPY/ AgRP release and so suppresses food intake (Batterham et. al., 2002), whereas ghrelin stimulates their release, in addition to stimulating release of growth hormone by the pituitary gland.
  • Injection of ghrelin into human volunteers has been shown to significantly increase the amount of food subsequently eaten by the subjects. Analysis of the levels of endogenous ghrelin in human subjects has shown that it is present in increasing quantities in the hours leading up to regular meal times, and that levels then fall immediately after eating, adding to evidence that this is a major 'meal initiator'.
  • AEA N- arachidonylethanolamine
  • 2-arachidonyl glycerol (2-AG) Mechanismoulam et al, 1995
  • 2-arachidonyl glycerol ether noladin ether
  • Endocannabinoids play a significant role in the mediation of numerous cell functions, and are involved in processes as diverse as cardiovascular function, aggression, neuroprotection, reproduction, immune system function, and motor behaviour in addition to food intake and cognition.
  • Endocannabinoid activity arises from interaction with the well characterised cannabinoid receptors CB1 and CB2 through activation of G-proteins.
  • CB1 receptors are found on cells throughout the central nervous system, and are particularly abundant in the substantia nigra, the globus pallidus, the hippocampus and cerebral cortex.
  • the CB2 receptor in contrast is found in immune system tissues such as spleen, tonsil, lymph nodes, and many leucocytes including B-cells, ⁇ K-cells, macrophages and monocytes.
  • Both of the endocannabinoids AEA and 2-AG exhibit a characteristically high binding affinity for the CB1 receptor, and modest to low affinities for the CB2 receptor.
  • AEA has been shown to bind to other receptors such as the vanilloid receptor (VR1), and as a result can exert vasodilatory effects.
  • VR1 vanilloid receptor
  • AEA has further been shown to exert wide ranging physiological effects such as the inhibition of neurotransmitter release, nociception, hypotension, bradycardia, inhibition of inducible nitric oxide synthase and the growth of breast and prostate tumour cells, and the impairment of memory (Sugiura et al., 2002; Di Marza, V. 1998; Piomelli et al., 1998). Under certain circumstances it can also act as a partial agonist in some cases.
  • 2-AG The role of 2-AG is perhaps less well understood. It is thought to be the intrinsic natural receptor of both the CB1 and CB2 receptors for which it is a full agonist. Its interaction with the CB1 receptor is thought to be involved in attenuation of neurotransmission and the prevention of neuronal exhaustion (Sugiura et al., 2002; Sugiura and Waku, 2000; Sugiura et al., 1998).Apart from its role in the central nervous system, 2-AG induces hypotension and vascular relaxation, and therefore may play an important role during inflammatory and /or immune responses (Lee et al., 1995; Ouyang et al., 1998; Iwamura et al., 2001).
  • the present invention provides for methods for regulating food intake and weight gain/loss in mammals by selectively controlling extra-cellular concentrations of endogenous cell-signalling molecules involved in mediating food intake and fat metabolism.
  • the present invention targets the actual signalling molecules regulating short-term food intake, and provides methods for reducing fat deposition and increasing fat catabolism in a mammal.
  • the subject is a human.
  • compositions of the invention have particular applications in weight management as they target the body's short-term food intake regulatory mechanisms, and thus can be applied to reducing hunger cravings and increasing feelings of satiety in obese individuals. Further, the compositions of the invention can be used to stimulate increased food intake and have applications for individuals with weight-loss eating disorders and those with poor appetite resulting from other disease conditions.
  • a monoclonal antibody to an endocannabinoid molecule in which the endocannabinoid is of general formula (I):
  • X is NH or O
  • Y is H or CH 2 OH
  • Z is OH or NH 2 .
  • the endocannabinoids are the endogenous ligands of the G-protein-coupled cannabinoid receptors CBl and CB2, and particularly include anandamide, otherwise known as AEA (N- arachidonylethanolamine), and 2-AG (2-arachidonyl glycerol).
  • AEA N- arachidonylethanolamine
  • 2-AG 2-arachidonyl glycerol
  • the invention therefore provides for a receptor specific for endocannabinoid cell- signalling molecules that have a biological function in the regulation of food intake in mammals.
  • S* is S or an acylated serine residue -O-C(O)-(CH ) n -CH 3 , where n is from 6 to 14 X s R. or K X 2 is V, or A
  • the C-terminal residue of the peptide may have an ⁇ -amide (-NH 2 ).
  • Peptides of this aspect of the invention are based on the ghrelin cell signalling peptide hormones.
  • Amino acid designations are as follows:
  • A alanine
  • D aspartate
  • E glutamate
  • F phenylalanine
  • G glycine
  • H histidine
  • I isoleucine
  • K lysine
  • L leucine
  • N asparagine
  • P proline
  • Q glutamine
  • R arginine
  • S serine
  • T threonine
  • V valine
  • Y tyrosine
  • C cysteine
  • n may be 6, 8, 10, 12 or 14. Any four or more contiguous amino acid residues from within the structure of the formula given above may be used to generate the monoclonal antibody.
  • the peptide length may be from 4 to 28 residues in length from the N-terminus according to the structure given in general formula (II). Suitable chain lengths can be 6 to 28, 8 to 24, 10 to 26 residues. However, a chain length of 18, 19, 28 residues may be preferred.
  • the C-terminal residue may be amidated to include an ⁇ -amide.
  • a terminal cysteine residue (C) may also be added for ease of synthesis of the peptide or for ease of conjugating the peptide to a carrier molecule.
  • n may be 6 and the peptide length may be 28 residues and suitably a C-terminal ⁇ -amide may be included.
  • a receptor specific for the ghrelin peptide hormones that induce hunger cravings there is provided.
  • TTT HGP-18-C 0 ⁇ (CH 2 )n ⁇ / CH, O H N— G-S-S-F-L-S-P-E-H-Q-R-V-Q-Q-R-K-E-S-C — NH 5 Peptide (IV) RGP-18-C
  • N-octanoyl-(Ser3)-modified peptides can be synthesised according to the methods laid out in patent US 2003/0211967 Al. Each has a non-native cysteine residue added to the C-terminus.
  • Peptides (I) - (TV) include a C-terminal ⁇ -amine.
  • Peptides I, HI and V include the residues arginine (R) and valine (V) at positions 11 and 12 as are found in human ghrelins.
  • Peptides II, IV and VI include residues lysine (K) and alanine (A) at these positions, as are found in rat ghrelins
  • Z 1 is YP or is absent
  • the C-terminal residue of the peptide may have an ⁇ -amide (-NH 2 ).
  • a preferred molecule may be the truncated 3-36 form
  • the peptide may include a C-terminal ⁇ -amide and may comprise residues 3-36 only (PYY 3-36),
  • Any four or more contiguous amino acid residues from within the structure of the formula given above may be used to generate the monoclonal antibody.
  • a terminal cysteine residue (C) may also be added for ease of synthesis of the peptide or for ease of conjugating the peptide to a carrier molecule
  • the present invention provides for receptors that bind to cell signalling molecules involved in the regulation of food intake in mammals, and methods for the generation and isolation of such receptors from libraries of receptors.
  • the invention also provides for conjugates of said signalling molecules that can be applied to the methods of receptor isolation or can be used to immunise patients against the signal molecules.
  • the descriptions of the invention are divided into sections detailing the synthesis of reactive signal molecule analogues, the generation of conjugates, and the isolation of anti-signal molecule receptors.
  • the term 'antibody' is used to describe receptors, and does not limit the extent or scope of the invention.
  • three distinct classes of signal molecule are included that comprise the endogenous endocannabinoids of general formula (I), the acylated peptide hormones (ghrelins) of general formula (II) and the peptide hormones of general formula (III).
  • ghrelins acylated peptide hormones
  • III peptide hormones of general formula (III).
  • Endocannabinoid molecules of general formula (I) can be synthesised from a starting point of arachidonic acid (compound A, Scheme 1).
  • the acid-chloride (Compound B) can be produced by reacting arachidonic acid (compound A) with chloromethanoic acid in dry dichloromethane (DCM) and dry dimethylformamide (DMF) at ⁇ 0°C.
  • Anandamide (compound C) is then produced by reacting arachidonic acid chloride with ethanolamine at ⁇ 0°C in dry DMF, with Et ⁇ N included as a proton mop (base).
  • Method (a) (scheme 2) is as described for compound C. Briefly, 8-amino-octanoic acid (compound D) and arachidonic acid chloride (compound C) are reacted in dichloromethane at 0°C in the presence of Et ⁇ N.
  • endocannabinoid derivatives can also be synthesised according to the methods of the invention, providing that a suitable 'signature' of the structure of the target signalling molecule is retained.
  • a suitable signature can be defined as a structure whereby an antibody (or other receptor) directed against the structure can also recognise and bind to the native endocannabinoid molecule.
  • a total of 6 peptide targets according to general formula IT were synthesised to the sequences given below:
  • Peptides I, LLT and V include the residues arginine (R) and valine (V) at positions 11 and 12 as are found in human ghrelins.
  • Peptides ⁇ , IV and VI include residues lysine (K) and alanine (A) at these positions, as are found in rat ghrelins.
  • the target molecule In order to generate anti-signal molecule antibodies, it is preferable to conjugate the target molecule, or a suitable derivative thereof, to two different carrier molecules, although a single conjugated species can be also used.
  • Cell signalling molecules such as endocannabinoids and small peptides are too small to stimulate an immune response in-vivo, or to be used directly as a sole source of antigen for the selection of high affinity receptors from antibody libraries. Larger peptides such as ghrelins and PYY can be adsorbed directly onto surfaces, and may be able to illicit an immune response in non-host mammals without conjugation.
  • Endocannabinoids and the carboxyl-containing derivatives are poorly soluble in aqueous solutions. Reactions are therefore carried out in organic solvents such as dimethylformamide (DMF). Briefly, an amount of the endocannabinoid is dissolved in DMF. To this, a molar equivalent of N-hydroxysuccinimide ( ⁇ HS) is added together with a 10% molar excess of a carbodiimide reagent, e.g. dicyclohexylcarbodiimide (DCC) and the reaction incubated. For signal molecules/analogues that are soluble in aqueous solutions, appropriate water-soluble carbodiimides and hydroxysuccinimide are used.
  • DMF dimethylformamide
  • the carrier molecule e.g. protein is dissolved in an appropriate aqueous buffer and organic solvent (DMF) added, typically ⁇ 20%.
  • DMF organic solvent
  • the amount of organic solvent added will vary with the tolerance of the carrier protein used.
  • the activated hapten- ester in DMF is added slowly to the carrier protein and the reaction incubated at 4°C for several hours. Conjugates are then purified to remove irrelevant and un-reacted material e.g. by dialysis or chromatography.
  • Peptides of the type described by general formula (II) and (LU) can also be conjugated to carrier proteins by a variety of chemistries familiar to those practised in the art, depending on the exact sequence of the peptide and the nature and location of the point of conjugation required.
  • peptides with a single cysteine residue incorporated into the C-terminus can be conjugated to carrier protein primary amine groups via the cysteine side chain, which does not limit the scope or extent of the present invention.
  • carrier protein is first activated with heterobifunctional cross-linker, such as succinimidyl-4-(N-maleimidomethyl)cyclohexane-l-carboxylate (SMCC), or a water soluble variant (sulfo-SMCC).
  • SMCC succinimidyl-4-(N-maleimidomethyl)cyclohexane-l-carboxylate
  • sulfo-SMCC water soluble variant
  • the cross-linker includes an ⁇ HS ester at one end that reacts with carrier protein primary amine groups to form a stable amide bond, and a maleimide group at the other end that reacts with sulphydryl groups.
  • the cross-linker is added to a suitable molar excess compared to the carrier molecule. The extent of the excess varies with carrier, and the number of available amine groups.
  • the reaction is preferably carried out in phosphate-buffered saline (PBS) at pH 7.2 for 1-2 h. Un-reacted or surplus cross-linker can be removed by techniques familiar to those practised in the art.
  • PBS phosphate-buffered saline
  • One appropriate method where the carrier is significantly larger than the cross-linker is by size exclusion chromatography, for example by passing the reaction mixture through a Sephadex G-25 column. The eluate is collected in a series of fractions, and those that contain the carrier retained and pooled. Larger molecules pass through sephadex G-25 than small ones, so late-eluted fractions containing un-reacted maleimide cross-linker are discarded.
  • the activated carrier is then combined with an appropriate molar excess of the peptide cysteine- containing peptide in PBS and reacted for 4-24 h.
  • the maleimide-activated carrier reacts with peptide cysteine sulphydryl groups to form a stable thioether bond. Un- conjugated peptide can be removed by size exclusion chromatography or dialysis.
  • Monoclonal antibodies can be produced from hybridomas. These can be formed by fusing myeloma cells and B-lymphocyte cells which produce the desired antibody in order to form an immortal cell line. This is the well known Kohler & Milstein technique (Nature 256 52-55 (1975)). The antibodies can then be purified by virtue of their binding to antigen or as described further below
  • the present invention includes derivatives thereof which are capable of binding to antigen.
  • the present invention includes antibody fragments and synthetic constructs. Examples of antibody fragments and synthetic constructs are given by Dougall et al in Tibtech 12 372-379 (September 1994).
  • Antibody fragments include, for example, Fab, F(ab') 2 and Fv fragments (see Roitt et al [supra]).
  • Fv fragments can be modified to produce a synthetic construct known as a single chain Fv (scFv) molecule.
  • scFv single chain Fv
  • the present invention therefore also extends to single chain antibodies or scAbs.
  • CDR peptides include CDR peptides. These are synthetic peptides comprising antigen binding determinants. Peptide mimetics may also be used. These molecules are usually conformationally restricted organic rings which mimic the structure of a CDR loop and which include antigen-interactive side chains. Synthetic constructs also include chimaeric molecules. Thus, for example, humanised (or primatised) antibodies or derivatives thereof are within the scope of the present invention. An example of a humanised antibody is an antibody having human framework regions, but rodent hyper-variable regions. Synthetic constructs also include molecules comprising a covalently linked moiety which provides the molecule with some desirable property in addition to antigen binding. For example the moiety may be a label (e.g. a detectable label, such as a fluorescent or radioactive label) or a pharmaceutically active agent.
  • a label e.g. a detectable label, such as a fluorescent or radioactive label
  • a pharmaceutically active agent e.g. a pharmaceutically active agent.
  • 'antigen' selection of antibodies specific for the cell signalling molecular (hereafter referred to as 'antigen') is carried out in the preferred embodiment using a repertoire (library) of first members of specific binding pairs (sbp), for example a library of antibodies displayed on the surface of filamentous bacteriophage.
  • a repertoire of first members of specific binding pairs (sbp)
  • sbp specific binding pairs
  • the library of receptors is of human origin.
  • signal molecule-specific clones can be selected from a panel of antibody secreting hybridoma cell lines generated from a mammal immunised with an antigen conjugate. In such instances and where applicable, it is preferred that isolated antibodies be subsequently rendered non-immunogenic to the subject where the subject is human.
  • the example of a library of antibody binding sites displayed on phage particles will be used.
  • a conjugate comprising an antigen coupled to a suitable carrier molecule which can be a protein, a peptide or any natural or synthetic compound or material (referred to hereafter as 'conjugate- 1') is immobilised onto a suitable solid support such as an 'immunotube' or microtitre plate, and the uncoated surface blocked with a nonspecific blocking agent such as dried milk powder.
  • suitable conjugate molecules can include, but are not limited to proteins such as bovine serum albumin (BSA), Keyhole Limpet Haemocyanin (KLH), Bovine Thyroglobulin (TG), Ovalbumin (Ova), or non- proteins such as biotin.
  • BSA bovine serum albumin
  • KLH Keyhole Limpet Haemocyanin
  • TG Bovine Thyroglobulin
  • Ova Ovalbumin
  • a library of first members of specific binding pairs (sbp's) ('the library') is applied to the immobilised conjugate and incubated for sufficient time for sbp members recognising conjugate-1 to bind.
  • Phage-antibodies ('phage') not recognising the conjugate are removed by stringent washing. Phage that remain bound are eluted, for example with tri-ethylamine or other suitable reagent, into a buffer solution to restore neutral pH.
  • bound phage-antibodies can be competitively eluted from the conjugate-1 by addition of an amount of the natural (non-conjugated) form of the signal molecule antigen.
  • Recovered phage particles are then infected into a suitable host organism, e.g. E. coli bacteria, and cultured to amplify numbers of each selected member and so generate a second 'enriched' library. The process is then repeated using the enriched library to select for phage recognising the antigen conjugated to a second carrier protein (conjugate-2).
  • a suitable host organism e.g. E. coli bacteria
  • Phage are selected against antigen conjugates as described previously, using initially conjugate-1, and alternating with conjugate-2 (where available) for each subsequent round.
  • Bound phage are eluted by incubating with a solution of free antigen, or antigen conjugated to small soluble selectable moieties, e.g. biotin, for sufficient time for sbp members with higher affinity for the bound form of the antigen to dissociate from the immobilised conjugate.
  • Those phage eluted with free antigen are infected into E. coli cells for amplification and re-selection, and those remaining bound to the immobilised antigen discarded.
  • all antibodies binding to conjugate may be eluted e.g. with low pH.
  • phage clones from each round of selection are screened for desired binding characteristics. This can be performed by a variety of methods that will be familiar to those with ordinary skill in the art, depending on requirements, including such techniques as SPR (Surface Plasmon Resonance) and ⁇ LISA (Enzyme Linked Immuno-Sorbant Assay). Selection criteria will include the ability to bind preferentially to the free soluble form of the antigen in the presence of conjugated derivatives.
  • the receptors of the invention are antibodies or fragments thereof that bind specifically to the signalling molecules involved in regulating food intake and metabolism.
  • antibodies will be generated from a na ⁇ ve human antibody phage display library (McCafferty et al., Nature 348: 552-554, 1990; and as described in WO 92/01047).
  • a library can be constructed from an animal pre-immunised with one or more conjugates of an endocannabinoid or peptide hormone signal molecule and a suitable carrier.
  • a further alternative is the generation of hybridoma cell lines from an animal immunised as described above.
  • the antibody can be engineered to include constant regions from different classes of human immunoglobulin (IgG, IgA, etc.) and produced as a whole antibody molecule in animal cells. In particular these approaches are desirable where the antibodies are to be used therapeutically
  • the antibody is a monoclonal antibody.
  • the antibodies are human.
  • Antibody fragments or derivatives such as Fab, F(ab').sup.2 (also written as F(ab') 2 ), Fv, or scFv, may be used, as may single-chain antibodies (scAb) such as described by Huston et al. (Int. Rev. Immunol. 10: 195-217, 1993), domain antibodies (dAbs), for example a single domain antibody, or antibody-like single domain antigen-binding receptors.
  • peptidomimetics or non-peptide mimetics can be designed to mimic the binding activity of antibodies, thus modulating the food intake of subjects to which they have been administered by inhibiting the binding of cell-signalling molecules to their natural receptors.
  • the receptors of the invention may have catalytic or enzymatic activity, such that administration of the receptors to subjects neutralises the signalling function of the signal molecules of the invention.
  • a suitable antibody After the preparation of a suitable antibody, it may be isolated or purified by one of several techniques commonly available (for example, as described in Antibodies: A Laboratory Manual, Harlow and Lane, eds. Cold Spring Harbor Laboratory Press (1988)). Generally suitable techniques include peptide or protein affinity columns, HPLC or RP-HPLC, purification on Protein A or Protein G columns, or combinations of these techniques. Recombinant antibodies can be prepared according to standard methods, and assayed for specificity using procedures generally available, including ELISA, dot-blot assays etc.
  • a method of screening a population of monoclonal antibodies for an anti-endocannabinoid monoclonal antibody comprising conjugating a endocannabinoid molecule of general formula (I) to a carrier molecule and using the conjugate so formed to identify a monoclonal antibody that specifically binds to the free soluble form of the endocannabinoid molecule of general formula (I) from the population of monoclonal antibodies in the presence of conjugated derivatives thereof.
  • a method of screening a population of monoclonal antibodies for an anti-peptide monoclonal antibody comprising conjugating a peptide of general formula (II) to a carrier molecule and using the conjugate so formed to identify a monoclonal antibody that specifically binds to the free soluble form of the peptide of general formula (II) from the population of monoclonal antibodies in the presence of conjugated derivatives thereof.
  • a method of screening a population of monoclonal antibodies for an anti-peptide monoclonal antibody comprising conjugating a peptide of general formula (TTT) to a carrier molecule and using the conjugate so formed to identify a monoclonal antibody that specifically binds to the free soluble form of the peptide of general formula (III) from the population of monoclonal antibodies in the presence of conjugated derivatives thereof.
  • TTT peptide of general formula
  • the carrier molecule may be a protein
  • the population of monoclonal antibodies may be a phage display library.
  • Such methods are therefore a means for identifying a specific binding molecule that can be used as a therapeutic agent, for example in the treatment of weight management and eating disorders.
  • Specific binding molecules identified by a method of the present invention may be used in medicine or a method of treatment as described above.
  • the specific binding molecules may further be used in the preparation of a medicament for the treatment of obesity or weight loss.
  • Such methods therefore extend to uses of an endogenous signal molecule involved in food intake and/or metabolism as defined by general formulae (I), (II) or (HI) to screen a population of specific binding molecules in order to identify a specific binding molecule that specifically binds to said signal molecule.
  • a method for the treatment of obesity comprising administering an effective amount of a monoclonal antibody as defined above to a subject in need thereof.
  • Such aspects of the invention therefore also extend to a monoclonal antibody as defined above for use in the treatment of obesity.
  • the antibodies of the invention may be administered to subjects in conjunction with other medicaments for the purpose of weight control.
  • the antibodies of the invention which will reduce the craving for food in subjects, made be applied together with signal molecules according to general formula (Hi) which induce a feeling of satiety.
  • a method for the treatment of anorexia comprising administering an effective amount of a monoclonal antibody as defined above to a subject in need thereof.
  • Such aspects of the invention therefore also extend to a monoclonal antibody as defined above for use in the treatment of anorexia.
  • a method for the treatment of loss of appetite comprising administering an effective amount of a monoclonal antibody as defined above to a subject in need thereof.
  • Such aspects of the invention therefore also extend to a monoclonal antibody as defined above for use in the treatment of loss of appetite
  • Loss of appetite may be the result of physical illness such as cancer or infection, or it may be the side-effect of a therapeutic course of treatment to treat cancer or another disease.
  • a pharmaceutical composition comprising a monoclonal antibody as defined above.
  • compositions may be prepared by any method known in the art of pharmacy, for example by admixing the active ingredient with a carrier(s), diluent (s) or excipient(s) under sterile conditions.
  • the pharmaceutical composition may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route.
  • Such compositions may be prepared by any method known in the art of pharmacy, for example by admixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions.
  • compositions adapted for oral administration may be presented as discrete units such as capsules or tablets; as powders or granules; as solutions, syrups or suspensions (in aqueous or non-aqueous liquids; or as edible foams or whips; or as emulsions)
  • Suitable excipients for tablets or hard gelatine capsules include lactose, maize starch or derivatives thereof, stearic acid or salts thereof.
  • Suitable excipients for use with soft gelatine capsules include for example vegetable oils, waxes, fats, semi-solid, or liquid polyols etc.
  • excipients which may be used include for example water, polyols and sugars.
  • suspensions oils e.g. vegetable oils
  • oil-in-water or water in oil suspensions may be used.
  • compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time.
  • the active ingredient may be delivered from the patch by iontophoresis as generally described in Pharmaceutical Research, 3 (6), page 318 (1986).
  • compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils.
  • the compositions are preferably applied as a topical ointment or cream.
  • the active ingredient may be employed with either a paraffinic or a water-miscible ointment base.
  • the active ingredient may be formulated in a cream with an oil-in-water cream base or a water-in-oil base.
  • compositions adapted for topical administration to the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent.
  • Pharmaceutical compositions adapted for topical administration in the mouth include lozenges, pastilles and mouth washes.
  • compositions adapted for rectal administration may be presented as suppositories or enemas.
  • compositions adapted for nasal administration wherein the carrier is a solid include a coarse powder having a particle size for example in the range 20 to 500 microns which is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.
  • suitable compositions wherein the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the active ingredient.
  • compositions adapted for administration by inhalation include fine particle dusts or mists which may be generated by means of various types of metered dose pressurised aerosols, nebulizers or insufflators.
  • compositions adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations.
  • Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solution which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation substantially isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • Excipients which may be used for injectable solutions include water, alcohols, polyols, glycerine and vegetable oils, for example.
  • compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carried, for example water for injections, immediately prior to use.
  • sterile liquid carried, for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.
  • compositions may contain preserving agents, solubilising agents, stabilising agents, wetting agents, emulsifiers, sweeteners, colourants, odourants, salts (substances of the present invention may themselves be provided in the form of a pharmaceutically acceptable salt), buffers, coating agents or antioxidants. They may also contain therapeutically active agents in addition to the substance of the present invention.
  • Dosages of the pharmaceutical compositions of the present invention can vary between wide limits, depending upon the disease or disorder to be treated, the age and condition of the individual to be treated, etc. and a physician will ultimately determine appropriate dosages to be used.
  • compositions may be formulated for human or for veterinary medicine.
  • the present application should be interpreted as applying equally to humans as well as to animals, unless the context clearly implies otherwise.
  • the receptor can be the natural receptor of the ligand (signal molecule), or a derivative of the natural receptor, altered for example to increase affinity for the ligand.
  • the receptor is administered to patients in order to sequester signal molecules, rendering them unavailable to the endogenous receptors of the patient.
  • the receptors are incorporated into the surface of a discrete 'compartment' such that signal molecules are bound and subsequently internalised into the compartment.
  • the compartment is a mono or bi-layer lipid vesicle or liposome. In other embodiments the compartment can be any package enclosing an internal space onto which one or more receptors can be embedded or displayed.
  • the vesicles can be produced to include internally an enzyme or other protein or reagent that digests, modifies or otherwise negates the signalling function of the signal molecule.
  • a monoclonal antibody as defined above in the preparation of a medicament for the treatment of obesity, anorexia or loss of appetite.
  • a pair of monoclonal antibodies as defined above for separate, simultaneous or subsequent administration in the treatment of obesity, anorexia or loss of appetite.
  • a monoclonal antibody identified by a method as defined above for use in the treatment of obesity, anorexia, or loss of appetite.
  • Such uses in accordance with this aspect of the invention therefore extend to methods of treatment of obesity, anorexia or loss of appetite, comprising administering an effective amount of such an antibody to a subject in need thereof.
  • a monoclonal antibody identified by a method as defined above in the preparation of a medicament for use in the treatment of obesity, anorexia or loss of appetite.
  • a kit of parts for the treatment of obesity, anorexia or loss of appetite comprising a monoclonal antibody as defined above which is provided in unit dosage form and where the kit contains instructions for use.
  • the antibody or immunogenic conjugate will usually be supplied as part of a sterile, pharmaceutical composition which will normally include a pharmaceutically acceptable carrier.
  • This pharmaceutical composition may be in any suitable form, (depending upon the desired method of administering it to a patient).
  • unit dosage form will generally be provided in a sealed container and may be provided as part of a kit.
  • kit of parts would normally (although not necessarily) include instructions for use. It may include a plurality of said unit dosage forms.
  • said method does not require the molecule of formula (I), or peptide of formula (II) or (in) to be conjugated to a carrier molecule.
  • Molecules of a certain size may be sufficiently immunogenic to provoke the formation of antibodies, or to bind to antibodies without the need to be conjugated to a carrier molecule.
  • a single chain antibody from E. coli clones 3AB12, 4AD8, 3BE10 or 3BH10 deposited as NCIMB 41282, NCIMB 41283, NC B 41284 and NCTMB 41285 respectively.
  • the invention further provides for conjugates comprising a cell signalling molecule according to general formulae (I), (H), or (HI), or a suitable derivative thereof, attached to a carrier.
  • immunogenic conjugates of cell signalling molecules according to general formulae (I), (H) or (III), wherein administration of said conjugates to patients results in an immune response that includes antibodies specific for the signalling molecule(s).
  • the present invention also relates to conjugates comprising a carrier molecule covalently linked or otherwise bound to a mammalian cell signalling molecule or suitable conjugatable derivative thereof of general formulae (I), (H), or (IH) above, and the use of said conjugates to generate receptors that bind specifically to the signal molecule and which can be applied to patients in order to help regulate food intake and fat metabolism.
  • the conjugates can also be applied to patients in order to induce an immune response leading to production of antibodies that bind to and neutralise the signal molecules of the invention.
  • a vaccine comprising a molecule according to general formulae (I), (H), or (HI), or a derivative thereof, attached to a carrier.
  • Suitable derivatives of the endocannabinoid and peptide signal molecules are prepared such that they can be conjugated to the carrier protein whilst retaining key structural characteristics that enable receptor binding.
  • the endocannabinoid molecules are synthesised to include a carboxylate moiety such that they can be conjugated to the amine group found on the side-chains of lysine residues of the carrier by methods familiar to those practised in the art.
  • peptide hormones are synthesised with an additional C-terminal cysteine residue, such that they can be conjugated to exposed lysine residues on the carrier.
  • any chemical group that allows conjugation to a suitable carrier can be incorporated into the signal molecule derivatives.
  • the carrier molecule is an immunogenic protein including one or more exposed lysine or acidic residues, including but not limited to bovine serum albumin, bovine thyroglobulin, chicken ovalbumin, keyhole limpet haemocyanin, tetanus toxoid and diphtheria toxoid.
  • Suitable carrier molecules can also include any molecule that is safe to administer to mammals, i.e. carries the minimal risk of allergic response and is not toxic to the subject at the administered dosage, and is immunogenic in the subject.
  • the administration of said conjugates to patients results in an immune response that includes neutralising antibodies specific for the signalling molecule(s).
  • an amount of an immunogenic conjugate comprising a carrier molecule covalently or otherwise bound to a signal molecule, or suitable derivative thereof, of general formula (I) and/or (II) or (HI) is administered to a subject.
  • the conjugates may be applied to subjects in the form of a vaccine such that the subjects are immunised against the signal molecule(s) of the invention.
  • the vaccine may be prepared with such pharmaceutically acceptable t diluents, adjuvants and/or carriers as required.
  • the subject is human.
  • a method for the treatment of obesity comprising the administration of an effective amount of a vaccine as defined above to a subject in need thereof.
  • the methods comprise administering to a patient an effective dose of a receptor specific for a signal molecule where the signal molecule is a hormone peptide or endogenous endocannabinoid.
  • the receptor is a human antibody or antigen-binding fragment thereof.
  • the invention also provides for diagnostic kits comprising the conjugates and receptors of the invention and directions for their use for detecting and quantifying levels of the aforementioned signalling molecules in patients' samples.
  • the methods comprise the steps of a) contacting the sample with receptor(s) specific for the signal molecule thereby forming a receptor-signal molecule complex, and applying to a conjugate of the signal molecule; (b) detecting the receptor complexed with the immobilised conjugate.
  • the signal molecule (or derivative) is conjugated to one or more carriers that need not be immunogenic, but that can be immobilised in some way.
  • the signal peptide hormones may alternatively be immobilised directly, without prior conjugation to a carrier.
  • compositions and methods disclosed herein may have broad ranging application in inhibiting, modulating, treating or diagnosing disease or conditions resulting in loss of control of body weight.
  • Figure 1 shows the response of rats immunised with EC-3-BSA conjugate.
  • ELISA data in graph (a) represents the binding of antibodies to EC-3-BSA (•), EC-3-KLH (O), EC-3-TG (T) conjugates and to BSA (V) and KLH ( ⁇ ) controls from pre- immune sera and following each of three immunisations/boosts.
  • Data in graph (b) shows the cross reactivity of EC-3-BSA immunised animals with EC-4-BSA (•), EC-4-KLH (O) andEC-4-TG (T).
  • FIG. 2 shows the response of rats immunised with EC-3-KLH conjugate.
  • ELISA data in graph (a) represents the binding of antibodies to EC-3-BSA (•), EC-3-KLH (O), EC-3-TG (T) conjugates and to BSA (V) and KLH ( ⁇ ) controls from pre- immune sera and following each of three immunisations/boosts.
  • Data in graph (b) shows the cross reactivity of EC-3-KLH immunised animals with EC-4-BSA (•), EC-4-KLH (O) andEC-4-TG (T).
  • FIG. 3 shows the response of rats immunised with EC-4-BSA conjugate.
  • ELISA data in graph (a) represents the binding of antibodies to EC-4-BSA (•), EC-4-KLH (O), EC-4-TG (V) conjugates and to BSA (V) and KLH ( ⁇ ) controls from pre- immune sera and following each of three immunisations/boosts.
  • Data in graph (b) shows the cross reactivity of EC-4-BSA immunised animals with EC-3-BSA (•), EC-3-KLH (O) and EC-3-TG (T ).
  • FIG. 4 shows the response of rats immunised with EC-4-KLH conjugate.
  • ELISA data in graph (a) represents the binding of antibodies to EC-4-BSA (•), EC-4-KLH (O), EC-4-TG (V) conjugates and to BSA (V) and KLH ( ⁇ ) controls from pre- immune sera and following each of three immunisations/boosts.
  • Data in graph (b) shows the cross reactivity of EC-4-KLH immunised animals with EC-3-BSA (•), EC-3-KLH (O) and EC-3-TG ( ⁇ ).
  • Figure 5 shows polyclonal sera from a rat immunised with EC-3-KLH binding to EC- 3-TG in the presence of free soluble EC-3 at 50 ⁇ M (O), at 5 ⁇ M ( ⁇ ) or the absence of free EC-3 (•).
  • Figure 6 shows the binding of polyclonal phage-antibodies from panning a na ⁇ ve human antibody library against EC-3 conjugates for 3 rounds of selection to the antigens EC-3-BSA (•), EC-3-KLH (O), EC-3-TG (T), BSA (V), TG ( ⁇ ), EC-4- BSA (D), EC-4-KLH ( ⁇ ) and EC-4-TG (O).
  • Figure 7 shows the binding profile and cross-reactivity of monoclonal phage antibody 3BE10 to EC-3 conjugates, EC-4 conjugates and to carrier proteins alone.
  • Figure 8 shows the binding profile and cross-reactivity of monoclonal phage antibody 4AD8 to EC-3 conjugates, EC-4 conjugates and to carrier proteins alone.
  • Figure 9 shows the binding profile and cross-reactivity of monoclonal phage antibody 3AH1 to EC-3 conjugates, EC-4 conjugates and to carrier proteins alone.
  • Figure 10 shows the binding of monoclonal phage-antibodies binding to either EC-3- TG (clones 3AH1 and 3BE10) or EC-4-TG (clone 4AD8) in competition with no free antigen ( ⁇ ), 100 ⁇ M free EC-3 ([TJ ) or 100 ⁇ M free EC-4 (H ).
  • Figure 11 shows the binding of pre-selection and post-panning polyclonal phage- antibodies to short ghrelin-BSA (•), short ghrelin-TG (O), long ghrelin-BSA (T) and to the carrier proteins BSA ( ⁇ ) and TG (D) alone.
  • Figure 12 shows the binding of scAb clones 3AB12 (O) and 3BH10 (•) to short ghrelin-BSA conjugate in the presence of free full-length ghrelin peptide
  • the examples described herein relates to endocannabinoid cell signalling molecules. This given only as an example, the scope of the invention not being limited to the example but including cell signalling molecules described by the present invention that directly or indirectly regulate food intake and metabolism.
  • 2-arachidonoyl glycerol (2-AG) was purchased from EMD Biosciences Inc., San Diego CA.
  • Arachidonic acid-chloride was produced by treating arachidonic acid with chloromethanoic acid in dry dichloromethane (DCM) and dry dimethylformamide
  • a conjugatable derivative of anandamide (compound D), hereafter referred to as EC- 3, was synthesised by reacting arachidonic acid chloride with 8-amino-octanoic acid in dichloromethane at 0°C as described above. However a greater yield of product was obtained when the base (Et 3 N) was omitted.
  • a conjugatable derivative of 2-AG hereafter referred to as EC- 3
  • EC-3 and EC-4 (0.2 mmol) were each dissolved separately in 0.5 ml aliquots of dry dimethylformamide (DMF).
  • DMF dry dimethylformamide
  • ⁇ HS N-hydroxysuccinimide
  • DCC dicyclohexylcarbodiimide
  • O.5 ml DCC/ ⁇ HS solution was added to each of the compound D/E solutions, and the reactions stirred at room temperature for 2 h to produce activated ester intermediates.
  • a total of 14 rats were immunised with either EC-3-BSA (3 rats), EC-3-KLH (3 rats), EC-4-BSA (3 rats), EC-4-KLH (3 rats), BSA (1 rat), or KLH (1 rat).
  • Each animal was given 200 ⁇ g antigen admixed with 200 ⁇ l Freunds' complete adjuvant by subcutaneous injection.
  • the rats were subsequently given boost injections comprising the same amount of antigen in Freunds' incomplete adjuvant at two weekly intervals for a total of three boosts.
  • Test bleeds were taken pre-immunisation, and 3 days post-boost. Samples were allowed to clot then centrifuged for 10 min at 13,000 g. The supernatant (serum) was pipetted off and analysed for anti-signal molecule binding activity (Figs. 1 to 4). Rats immunised with EC-3-BSA showed strong binding to antigen and to BSA alone as expected, and also had good responses to EC-3-KLH and EC-3-TG (Fig. la). In addition there was significant cross reactivity with all three EC-4 conjugates (Fig. lb).
  • Rats immunised with EC-3 -KLH showed a similar response in binding to all EC-3 conjugates and to the immunising carrier protein, though binding to EC-3-TG was weak (Fig. 2a). Binding to EC-4-KLH was strong as expected, but binding to EC-4- BSA was significantly lower than that seen to EC-3-BSA, indicating higher specificity than serum from EC-3-BSA immunised animals (Fig. 2b).
  • a human na ⁇ ve antibody library displayed on the DBDx replicable package system (WO 99/11785) was screened for antibodies with binding activity to endocannabinoid signal molecules.
  • EC-3-TG and EC-4-TG conjugates were immobilised onto separate immunotubes (Nunc) at 20 ⁇ g/ml in PBS for 2 h at 37°C. Unbound material was removed by washing with PBS, and the immunotubes blocked with dried milk powder in PBS (MPBS) overnight at 4 °C. The tubes were washed with PBS and incubated with the library of phage-antibodies (referred to hereafter as 'phage') for sufficient time for phage recognising the conjugate to bind. Approximately 1.1 x 10 14 phage were applied to each tube in PBS.
  • the resulting amplified libraries of enriched clones were then used for the following round of panning against each target antigen.
  • the immobilised conjugates were alternated with EC-3- BSA and EC-4-BSA respectively for successive rounds of selection.
  • pan 2 antigen-conjugates were coated at 1 ⁇ g/ml to drive selection towards antibodies with higher affinity for antigen. This was reduced further to 10 ng/ml for pan 3.
  • the chosen antigen (anandamide or 2-AG) was used to competitively elute phage-antibodies during rounds 2 (at 10 nM final concentration) and 3 (at 100 pM final concentration), rather than low pH.
  • Phage eluted from round (pan) 2 were infected into E. coli as before and re-amplified for use in pan 3. Phage eluted from pan 3 were similarly amplified.
  • Rescued polyclonal phage from each of the three pans and from the un-selected library were subsequently analysed by ⁇ LISA for antigen binding (Fig. 5). Briefly, antigen was coated onto 96 well ⁇ LISA plates at 1 ⁇ g/ml, and the plates blocked with MPBS. Fifty microlitres 4% MPBS was applied to each well, and 50 ⁇ l rescued phage added. The plates were incubated for 1 h at room temperature, then washed with PBST and PBS. Bound phage were detected with horse-raddish peroxidase labelled anti-M13 antibody (Sigma).
  • each was assayed for binding to the TG-conjugate of the target it was raised against (EC-3-TG or EC-4-TG) with or without 100 ⁇ M free EC-3 or EC-4. Binding to free antigen was found to correlate with binding to conjugate with respect to specificity (Fig. 10).
  • a human na ⁇ ve antibody library displayed on the DBDx replicable package system was screened for antibodies with binding activity to ghrelin signal molecules.
  • the peptide HGP-18-C (peptide in) was chemically conjugated to the carrier proteins Bovine serum albumin (BSA) and Bovine thyroglobulin (TG) via the sulphidryl side chain of the terminal cysteine residue and carrier protein surface primary amines (lysine residues) using standard methods.
  • BSA Bovine serum albumin
  • TG Bovine thyroglobulin
  • HGP-18-C-BSA was immobilised onto an immunotube (Nunc) at 10 ⁇ g/ml in PBS for 2 h at 37°C. Unbound material was removed by washing with PBS, and the immunotubes blocked with dried milk powder in PBS (MPBS) overnight at 4 °C. The tube was washed with PBS and incubated with a suspension of the library of phage in PBS for sufficient time for phage recognising the conjugate to bind. Approximately 1.0 x 10 13 phage were applied to the tube in PBS. An excess of free BSA (1 ml at 1 mg/ml) was also included in order to minimise the number of phage recognising the carrier protein being retained.
  • Phage not bound to the immobilised conjugate were removed by stringent washing with PBS-Tween (x 20) followed by PBS (x 20), and the remaining bound phage eluted by incubation at low pH with 1 ml 100 mM triethylamine for 10 min. Eluted phage were immediately neutralised with 0.5 ml buffered Tris-HCl at pH 7.4. Half of the eluted phage (0.75 ml) were then infected into log-phase E. coli bacteria (strain TGI) and amplified ('rescued') as described previously. The resulting amplified library of enriched clones were then used for the following round of panning.
  • the immobilised conjugate was alternated between HGP-18-C-BSA and HGP-18-C-TG for successive rounds of selection.
  • antigen-conjugate was coated at 1 ⁇ g/ml to drive selection towards antibodies with higher affinity for antigen. This was reduced further to 100 ng/ml for pan 3.
  • Pans 2 and 3 were performed in the presence of free BSA and TG each at 0.25 mg/ml final concentration.
  • Phage eluted from round (pan) 2 were infected into E. coli as before and re-amplified for use in pan 3. Phage eluted from pan 3 were similarly amplified.
  • Rescued polyclonal phage from each of the three pans and from the un-selected library were subsequently analysed by ELISA for antigen binding (Fig. 11).
  • binding was also determined to carrier protein alone and to HGP-28-C-BSA (the long 28 residue peptide represents the full-length biologically active ghrelin molecule). Briefly, antigen was coated onto 96 well ELISA plates at 10 ⁇ g/ml, and the plates blocked with MPBS.
  • HRP horse-raddish peroxidase
  • the scAb was then further assayed by competitive binding ELISA to establish that were able to recognise the free form of the native full-length ghrelin peptide (Fig. 12).
  • a sub-saturating concentration of scAb (with respect to binding to HGP-18-C-BSA conjugate at 1 ⁇ g/ml) was applied to replicate wells of an ELISA plate pre-coated with HGP-18-C- BSA.
  • a series of concentrations of HGP-28-C in PBS, or PBS alone, were added to the wells, and the plates incubated for 1 h at room temperature.
  • Antibody not bound to the immobilised conjugate was removed by washing and the residual bound material detected using a HRP labelled anti-human kappa antibody.
  • the concentration-dependant reduction in signal in the presence of free peptide indicates that both clones are able to bind to native full-length ghrelin.
  • microorganism identified under I above was accompanied by: I j a scientific description a proposed taxonomic designation X (Mark with a cross where applicable) m. RECEIPT AND ACCEPTANCE
  • microorganism identified under I above was accompanied by: I a scientific description I a proposed taxonomic designation (Mark with a cross where applicable) IH. RECEIPT AND ACCEPTANCE

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Abstract

La présente invention concerne des méthodes de régulation des troubles du système nerveux central par le biais de la modulation de la concentration extracellulaire de molécules de signalisation de cellules hormonales de peptide et de cannabinoïdes endogènes. Des dérivés de molécules de signalisation cellulaire sont conjugués à des protéines porteurs appropriées et utilisés pour isoler des récepteurs à haute affinité reconnaissant les molécules signal endogènes parmi les bibliothèques de récepteurs appropriés. Du fait de leur liaison aux molécules de signalisation, les récepteurs réduisent les concentrations disponibles de molécules signal et modulent les réponses physiologiques commandées par ces dernières. De plus, les conjugués selon l'invention peuvent être utilisés en tant que vaccins pour stimuler chez des patients les réponses immunes de molécule anti-signal.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8420602B2 (en) * 2004-09-14 2013-04-16 Landon C. G. Miller Endocannabinoid conjugate and a pharmaceutical composition for treatment of neuronal disorders
KR101481036B1 (ko) 2008-05-15 2015-01-09 (주)아모레퍼시픽 아라키도닐 도파민을 함유하는 지방세포 분화 억제 및지방세포 내의 지질체 수준 감소용 약학 조성물
WO2013014269A1 (fr) 2011-07-28 2013-01-31 Intervet International B.V. Procédé pour la promotion de la croissance d'un animal nouveau-né

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