EP1539232A1 - Immunization against autologous ghrelin - Google Patents

Immunization against autologous ghrelin

Info

Publication number
EP1539232A1
EP1539232A1 EP03794825A EP03794825A EP1539232A1 EP 1539232 A1 EP1539232 A1 EP 1539232A1 EP 03794825 A EP03794825 A EP 03794825A EP 03794825 A EP03794825 A EP 03794825A EP 1539232 A1 EP1539232 A1 EP 1539232A1
Authority
EP
European Patent Office
Prior art keywords
ghrelin
polypeptide
analogue
cell
nucleic acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03794825A
Other languages
German (de)
English (en)
French (fr)
Inventor
Tine Elisabeth Gottschalk Pharmexa A/S BOVING
Steen c/o Pharmexa A/S KLYSNER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Affitech AS
Original Assignee
Affitech AS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Affitech AS filed Critical Affitech AS
Publication of EP1539232A1 publication Critical patent/EP1539232A1/en
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • 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
    • A61K39/0005—Vertebrate antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to therapeutic vaccination ("active therapeutic immunothera- py").
  • therapeutic vaccination that targets autologous (“self”) ghrelin protein and therapy targeting obesity and other diseased characterized by excess body fat deposits or, alternatively, conditions where an increase in body weight are of interest.
  • the present invention thus relates to improvements in therapy and prevention of obesity characterized by excess fat deposition, but also to improvements in therapy and prevention of conditions characterized in loss of body weight. More specifically, the present invention pro- vides a method for down-regulating (undesired) deposits of fat by enabling the production of antibodies against ghrelin or components thereof in subjects suffering from or in danger of suffering from obesity involving excess fat deposition. The invention further provides a method for up-regulating desired deposits of body fat by enabling the production of antibodies against ghrelin or components thereof in subjects suffering from or in danger of suffering from emancipation. The invention also provides for methods of producing polypeptides useful in these methods as well as for the modified polypeptides as such.
  • nucleic acid fragments encoding the modified polypeptides as well as vectors incorporating these nucleic acid fragments and host cells and cell lines transformed therewith.
  • the invention also provides for a method for the identification of analogues of the deposit polypeptides which are useful in the methods of the invention as well as for compositions comprising modified polypeptides or comprising nucleic acids encoding modified polypeptides.
  • the present invention also provides for conjugate ghrelin peptide immunogens.
  • Overweight and obese individuals are at increased risk for physical ailments such as: High blood pressure, hypertension; High blood cholesterol, dyslipidemia; Type 2 (non-insulin dependent) Diabetes; Insulin resistance, glucose intolerance; Hyperinsu- linemia; Coronary heart disease; Angina pectoris; Congestive heart failure; Stroke; Gallstones; Cholescystitis and cholelithiasis; Gout; Osteoarthritis; Obstructive sleep apnoea and respiratory problems; Some types of cancer (such as endometrial, breast, prostate, and co- Ion); Complications of pregnancy; Poor female reproductive health (such as menstrual irregularities, infertility, irregular ovulation); Bladder control problems (such as stress incontinence); Uric acid nephrolithiasis; Psychological disorders (such as depression, eating disorders, distorted body image, and low self esteem).
  • physical ailments such as: High blood pressure, hypertension; High blood cholesterol, dyslipidemia; Type 2 (
  • Overweight and obesity result from an imbalance involving excessive calorie consumption and/or inadequate physical activity.
  • body weight is the result of a combination of genetic, metabolic, behavioural, environmental, cultural, and socio-economic influences. Behavioural and environmental factors are large contributors to overweight and obe- sity and provide the greatest opportunity for actions and interventions designed for prevention and treatment. Hence, many studies have demonstrated that reduction in obesity by diet and exercise reduces the risk factors mentioned above dramatically. Unfortunately, these treatments are largely unsuccessful with a failure rate reaching 95%. This failure may be due to a complex body mechanism, which in ancient times helped us survive when food supplies were unreliable. The mechanism may contribute to increased appetite, preference for highly caloric foods, reduced physical activity, and increased lipogenic metabolism as a response to dieting and exercising.
  • Leptin discovered in 1995, is a hormone which suppresses appetite. Produced primarily in fat tissue, leptin circulates generally in proportion to fat stores. It encourages people to stop eating when their fat cells are full. A newly discovered hormone (1999) called ghrelin seems to have the opposite effect.
  • the hormone is a gastric hormone that has been identified as an endogenous ligand for the growth hormone (GH) secretagouge receptor subtype la (GHS- Rla), which stimulates growth hormone secretion in rats and humans (Kojima M et al., Nature, 1999, 402: 656-60; Kojima M et al., Trends in Endocrinology and Metabolism, 2001, 12: 118-22; Takaya K et al., J Clin Endocrinol Metab, 2000, 85: 4908-11).
  • GH growth hormone
  • GSS- Rla growth hormone secretagouge receptor subtype la
  • ghrelin does play an important role in obesity and the need for a marked reduction in ghrelin production in obese patients undergoing dieting is essential for 1. loosing weight i.e. reducing excess body fat, and 2. subsequently retaining a diet-induced weight loss. Structure of ghrelin
  • Ghrelin shows a unique structure with an n-octanoyl ester in serine-3 residue. It has been shown that the first few residues of processed, mature ghrelin, Gly-Ser-Ser(n-octanoyl)-Phe segment constitute the active component of this peptide (Bednarek MA et al., 2000).
  • Ghrelin has been identified in the human stomach and is homologous to rat ghrelin apart from 2 amino acids.
  • Human pre-proghrelin isolated from a stomach cDNA library, consists of 117 amino acids. Rat and human pre-proghrelins are 82.9% identical. Ghrelin does not share high homology with any other non-ghrelin peptides identified so far.
  • Ghrelin is thought to alter feeding in part through the modulation of CNS control of gastric function (Date Y et al., 2001; Masuda Y et al., 2000). Specifically, ICV administration of ghrelin stimulated gastric acid secretion in a dose-dependent and atropine-sensitive manner. Immunohistochemistry demonstrated the induction of Fos expression in the nucleus of the soli- tary tract and dorsomotor nucleus of the rat vagus nerve. Asakawa A er al.
  • ghrelin exhibited gastroprokinetic activity with structural resemblance to motilin and potent orexigenic (feeding) activity through action on the hypothalamic neuropeptide Y and Y(l) receptor, which was lost after vagotomy. Ghrelin decreased gastric vagal afferent discharge in contrast to other anorexigenic peptides that increased the activity.
  • exogenous ghrelin also increased food intake, caused weight gain and reduced fat utilization in mice and rats (Wren AM et al, 2000; Tsch ⁇ p M et al, 2000).
  • intracerebroventricular (ICV) administration of ghrelin also generated a dose- dependent increase in food intake and body weight.
  • Rat serum ghrelin concentrations were increased by fasting and were reduced by re-feeding or oral glucose administration, but not by water ingestion.
  • ghrelin is involved in the hypothalamic regulation of energy homeostasis. Intracerebroventricular injections of ghrelin strongly stimulated feeding in rats and increased body weight gain. Ghrelin also increased feeding in rats that were genetically deficient in GH. Anti-ghrelin immunoglobulin G robustly suppressed feeding. After icv ghrelin administration, FOS protein, a marker of neuronal activation, was found in regions of primary importance in the regulation of feeding, including neuropeptide Y neurons and agouti-related protein neurons. Antibodies and antagonists of neuropeptide Y and agouti-related protein abolished ghrelin-induced feeding.
  • Ghrelin augmented neuropeptide Y gene ex- pression and blocked leptin-induced feeding reduction, implying that there is a competitive interaction between ghrelin and leptin in feeding regulation. It was therefore concluded that ghrelin is a physiologic mediator of feeding. In addition to animal studies ghrelin has also been investigated in a number of clinical studies. Levels of ghrelin demonstrated a two-fold increase 1 hour prior to eating and dropped to low levels within 1 hour of eating (see inset) suggesting that ghrelin plays an important role in the initiation of eating (Cummings DE er a/., 2001). This was confirmed by studies showing that ghrelin increased appetite and food intake in humans (Wren AM er a/., 2001).
  • Ghrelin represents one of the most promising breaking targets in the field of obesity. Although scientists only identified ghrelin in 1999, more than 200 papers on the substance have alrea- dy been published. Ghrelin acts to stimulate food intake but plasma levels are reduced in obese patients suggesting that this mediator represents a key regulator of food intake. Field- leaders currently believe that further reduction of ghrelin activity may offer a therapeutic target and hence antagonists of ghrelin receptor binding are emerging as a pharmacological option in the treatment of obesity. Correspondingly a number of tools are now available for the screening of ghrelin receptor antagonists. Despite the potential for drug discovery, ghrelin receptor antagonists have yet to appear although the publication of a number of patents suggests that such molecules may be on the way. Considering the proof of concept supporting the development of ghrelin antagonists, the potential size of the obesity market and the relative paucity of treatments available to the clinician, now is an ideal time to invest in the development of this exciting therapeutic class.
  • the object of the present invention is to provide novel therapies against conditions characterized by deposition of excess body fat resulting from energy intake exceeding energy expenditure as is characteristic for obesity. Another object is to provide therapies and treatments that induce an increase in body fat. A further object is to develop an autovaccine against ghrelin.
  • Described herein is the use of an autovaccination technology for generating strong immune responses against an otherwise non-immunogenic self-protein, ghrelin, involved in excess body fat deposition. Thereby, a strong immune response is generated against ghrelin. Described is also the preparation of such vaccines for the prevention, possible cure or alleviation of the symptoms of such diseases associated with excess body fat deposits, but also for inducing an increase in body fat.
  • the present invention relates to a method for inducing an immune response against autologous ghrelin in an animal, including a human being, the method comprising effecting presentation to the animal's immune system of an im- munogenically effective amount of an immunogen selected from the group consisting of
  • ghrelin polypeptide or subsequence thereof which has been formulated so that immunization of the animal with the ghrelin polypeptide or subsequence thereof induces production of antibodies against the animal's autologous ghrelin
  • At least one ghrelin analogue that incorporates into the same molecule at least one B- cell epitope of ghrelin and at least one chemical moiety not derived from ghrelin so that immunization of the animal with the analogue induces production of antibodies against ghrelin.
  • the method can be used to either effect in vivo down-regulation of ghrelin activity or to effect in vivo up-regulation of ghrelin activity.
  • the invention also relates to ghrelin analogues as well as to nucleic acid fragments encoding a subset of these. Also immunogenic compositions comprising the analogues or the nucleic acid fragments are part of the invention.
  • the invention also relates to a method of identifying analogues of ghrelin as well as a method for preparing a composition comprising the ghrelin analogues.
  • the invention also provides for passive immune therapy, where monoclonal anti-ghrelin antibodies are administered in order to obtain an effect similar to that of active vaccina- tion.
  • Fig. 2 Body weight of immunized rats.
  • the graph shows the cumulative food intake over the 10 weeks duration of the study. Hence, the demonstrated differences in body weight are not directly reflected in the cumulative food intake.
  • Fig. 4 Plasma ghrelin levels in vaccinated animals.
  • immunogen in the present context refers to an agent (a substance or a composition of matter) that induces an immune response. It will be understood that certain molecules (e.g. traditional small haptens or self-protein that are tolerated in the autologous host) are incapable of inducing an immune response. However, some self-proteins are, when formulated in very strong immunologic adjuvants, capable of inducing an immune response in spite of the normally tolerant state of the immunized animal. In such a context, the "immunogen” is therefore the composition of matter (self-protein with adjuvant) and not just a single molecule.
  • T-lymphocyte and "T-cell” will be used interchangeably for lymphocytes of thymic origin which are responsible for various cell mediated immune responses as well as for helper activity in the humeral immune response.
  • B-lymphocyte and “B-cell” will be used interchangeably for antibody-producing lymphocytes.
  • a "ghrelin polypeptide” is herein intended to denote polypeptides having the amino acid sequence of the above-discussed ghrelin proteins derived from humans and other mammals (or truncates thereof sharing a substantial amount of B-cell epitopes with intact ghrelin), but also polypeptides having the amino acid sequence identical to xeno-analogues of these proteins isolated from other species are embraced by the term; included in the term is both the mature ghrelin peptide as well as the ghrelin propeptide and the ghrelin pre-propeptide.
  • ghrelin polypeptide a self-protein or is a xeno-analogue of such a self-protein which will not normally give rise to an immune response against ghrelin of the animal in question.
  • a "ghrelin analogue” is a ghrelin polypeptide which has been subjected to changes in its primary structure. Such a change can e.g. be in the form of fusion of a ghrelin polypeptide to a suitable fusion partner ⁇ i.e. a change in primary structure exclusively involving C- and/or N- terminal additions of amino acid residues) and/or it can be in the form of insertions and/or deletions and/or substitutions in the ghrelin polypeptide's amino acid sequence. Also encom- passed by the term are derivatized ghrelin molecules, cf. the discussion below of modifications of ghrelin.
  • ghrelin analogue As defined above.
  • ghrelin this is intended as a reference to the amino acid sequence of wildtype ghrelin (also denoted “ghrelin” and “ghrelin-wt” herein). This term embraces both the propeptide and the mature peptide, so mature ghrelin is termed ghrelin- m.
  • Mature human ghrelin is denoted h-ghrelin, h-ghrelin-m, and murine mature ghrelin is de- noted m-ghrelin, m-ghrelin-m, or m-ghrelin-wt, etc.
  • a DNA construct includes information encoding a leader sequence or other material, this will normally be clear from the context.
  • polypeptide is in the present context intended to mean both short peptides of from 2 to 10 amino acid residues, oligopeptides of from 11 to 100 amino acid residues, and poly- peptides of more than 100 amino acid residues. Furthermore, the term is also intended to include proteins, i.e. functional biomolecules comprising at least one polypeptide; when comprising at least two polypeptides, these may form complexes, be covalently linked, or may be non-covalently linked.
  • the polypeptide(s) in a protein can be glycosylated and/or lipidated and/or comprise prosthetic groups. Also, the term "polyamino acid” is an equivalent to the term "polypeptide".
  • sequence means any consecutive stretch of at least 3 amino acids or, when relevant, of at least 3 nucleotides, derived directly from a naturally occurring ghrelin amino acid sequence or nucleic acid sequence, respectively.
  • animal is in the present context in general intended to denote an animal species (preferably mammalian), such as Homo sapiens, Canis domesticus, etc. and not just one single animal. However, the term also denotes a population of such an animal species, since it is important that the individuals immunized according to the method of the invention all harbour substantially the same ghrelin allowing for immunization of the animals with the same immu- nogen(s). If, for instance, genetic variants of ghrelin exist in different human population it may be necessary to use different immunogens in these different populations in order to be able to break the autotolerance towards ghrelin in each population. It will be clear to the skilled person that an animal in the present context is a living being which has an immune system. It is preferred that the animal is a vertebrate, such as a mammal.
  • ghrelin activity reduction in the li- ving organism of the number of interactions between ghrelin and its receptors (or between ghrelin and other possible biologically important binding partners for this molecule).
  • the down-regulation can be obtained by means of several mechanisms: Of these, simple interference with the active site in ghrelin by antibody binding is the most simple. However, it is also within the scope of the present invention that the antibody binding results in removal of ghre- lin by scavenger cells (such as macrophages and other phagocytic cells). Another possibility is binding of anti-ghrelin antibodies that are capable of interfering with the normal cleavage of proghrelin that result in mature ghrelin.
  • effecting presentation ... to the immune system is intended to denote that the animal's immune system is subjected to an immunogenic challenge in a controlled man- ner.
  • challenge of the immune system can be effected in a number of ways of which the most important are vaccination with polypeptide containing "pharmaccines” (i.e. a vaccine which is administered to treat or ameliorate ongoing disease) or nucleic acid "pharmaccine” vaccination.
  • pharmaceutical competent cells in the animal are confronted with the antigen in an immunologically effective manner, whereas the precise mode of achieving this result is of less importance to the inventive idea underlying the present invention.
  • immunogenically effective amount has its usual meaning in the art of immunology, i.e. an amount of an immunogen which is capable of inducing an immune response which significantly engages molecules which share immunological features with the immunogen.
  • ghrelin When using the expression that the ghrelin has been "modified" is herein meant a chemical modification of the polypeptide which constitutes the backbone of ghrelin.
  • a modification can e.g. be derivatization (e.g. alkylation, acylation, esterification etc.) of certain amino acid residues in the ghrelin sequence, but as will be appreciated from the disclosure below, the preferred modifications comprise changes of (or additions to) the primary structure of the ghrelin amino acid sequence.
  • One particular modification is the omission of the naturally occurring n-octanoyl group in ghrelin.
  • ghrelin is a self- protein in the population to be vaccinated, normal individuals in the population do not mount an immune response against ghrelin; it cannot be excluded, though, that occasional individu- als in an animal population might be able to produce antibodies against native ghrelin, e.g. as part of an autoimmune disorder.
  • an animal will normally only be autotolerant towards its own ghrelin, but it cannot be excluded that ghrelin analogues derived from other animal species or from a population having a different ghrelin phenotype would also be tolerated by said animal.
  • a “foreign T-cell epitope” is a peptide which is able to bind to an MHC molecule and which stimulates T-cells in an animal species.
  • Preferred foreign T-cell epitopes in the invention are "promiscuous" epitopes, i.e. epitopes which bind to a substantial fraction of a particular class of MHC molecules in an animal species or population. Only a very limited number of such promiscuous T-cell epitopes are known, and they will be discussed in detail below.
  • a "foreign T helper lymphocyte epitope" (a foreign T H epitope) is a foreign T cell epitope which binds an MHC Class II molecule and can be presented on the surface of an antigen presenting cell (APC) bound to the MHC Class II molecule.
  • APC antigen presenting cell
  • a "functional part" of a (bio)molecule is in the present context intended to mean the part of the molecule which is responsible for at least one of the biochemical or physiological effects exerted by the molecule. It is well-known in the art that many enzymes and other effector molecules have an active site which is responsible for the effects exerted by the molecule in question. Other parts of the molecule may serve a stabilizing or solubility enhancing purpose and can therefore be left out if these purposes are not of relevance in the context of a certain embodiment of the present invention. For instance it is possible to use certain cytokines as a modifying moiety in ghrelin (cf. the detailed discussion below), and in such a case, the issue of stability may be irrelevant since the coupling to ghrelin provides the stability necessary.
  • adjuvant has its usual meaning in the art of vaccine technology, i.e. a substance or a composition of matter which is 1) not in itself capable of mounting a specific immune response against the immunogen of the vaccine, but which is 2) nevertheless capable of enhancing the immune response against the immunogen. Or, in other words, vaccination with the adjuvant alone does not provide an immune response against the immunogen, vaccina- tion with the immunogen may or may not give rise to an immune response against the immunogen, but the combination of vaccination with immunogen and adjuvant induces an immune response against the immunogen which is stronger than that induced by the immunogen alone.
  • “Targeting" of a molecule is in the present context intended to denote the situation where a molecule upon introduction in the animal will appear preferentially in certain tissue(s) or will be preferentially associated with certain cells or cell types. The effect can be accomplished in a number of ways including formulation of the molecule in composition facilitating targeting or by introduction in the molecule of groups which facilitates targeting. These issues will be discussed in detail below.
  • “Stimulation of the immune system” means that a substance or composition of matter exhibits a general, non-specific immunostimulatory effect. A number of adjuvants and putative adjuvants (such as certain cytokines) share the ability to stimulate the immune system. The result of using an immunostimulating agent is an increased "alertness" of the immune system meaning that simultaneous or subsequent immunization with an immunogen induces a significantly more effective immune response compared to isolated use of the immunogen.
  • Productive binding means binding of a peptide to the MHC molecule (Class I or II) so as to be able to stimulate T-cells that engage a cell that present the peptide bound to the MHC molecule.
  • MHC molecule Class I or II
  • T-cells that engage a cell that present the peptide bound to the MHC molecule.
  • a peptide bound to an MHC Class II molecule on the surface of an APC is said to be productively bound if this APC will stimulate a T H cell that binds to the presented peptide-MHC Class II complex.
  • the immunization against ghrelin can be active as well as passive. Even though the focus of the present invention is the administration of agents that in- prise an active immune response against ghrelin, it is also within the scope of the present invention to administer agents that bind ghrelin in vivo. For instance, it is possible to utilise the well-known technology of monoclonal antibody administration in the present invention. It is in this context preferred to use humanized or completely human monoclonal antibodies, e.g. by employing transgenic mice that express human immunoglobulin light and heavy chains. The skilled person will know how to dose and administer such antibody compositions. Alternatively, a soluble version of a ghrelin receptor can be injected, resulting in an effective binding in the bloodstream to ghrelin.
  • the preferred embodiment entails active immunization against ghrelin.
  • the ghrelin polypeptide used as an immunogen in the method of the invention is a modified molecule wherein at least one change is present in the ghrelin polypeptide amino acid sequence, since the chances of obtaining the all-important breaking of autotolerance towards ghrelin is greatly facilitated that way. It should be noted that this does not exclude the possibility of using such a modified ghrelin in formulations which further fa- cilitate the breaking of autotolerance against ghrelin, e.g. formulations containing certain adjuvants discussed in detail below.
  • T-cells recognizing the foreign element are activated upon recognizing the foreign epitope on an APC (such as, initially, a mononuclear cell).
  • APC such as, initially, a mononuclear cell.
  • Polyclonal B-lymphocytes capable of recognising self-epitopes on the modified self-protein also internalise the antigen and subsequently presents the foreign T-cell epitope(s) thereof, and the activated T- lymphocytes subsequently provide cytokine help to these self-reactive polyclonal B-lymphocytes.
  • the antibodies produced by these polyclonal B-lymphocytes are reactive with different epitopes on the modified polypeptide, including those which are also present in the native polypeptide, an antibody cross-reactive with the non-modified self-protein is induced.
  • the T-lymphocytes can be led to act as if the population of polyclonal B-lymphocytes have recognised an entirely foreign antigen, whereas in fact only the inserted epitope(s) is/are foreign to the host. In this way, antibodies capable of cross-reacting with non-modified self-antigens are induced.
  • the modification can include that
  • At least one third moiety is introduced which optimises presentation of the modified ghrelin polypeptide to the immune system.
  • One especially preferred version of this embodiment is the tech- nique described in WO 95/05849, which discloses a method for immunizing against self-proteins by immunising with analogues of the self-proteins wherein a number of amino acid sequence ⁇ ) has been substituted with a corresponding number of amino acid sequence(s), which each comprise a foreign immunodominant T-cell epitope, while at the same time maintaining the overall 3 dimensional structure of the self-protein in the analogue.
  • the number of amino acid insertions, deletions, substitutions or additions is at least 2, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 and 25 insertions, substitutions, additions or deletions. It is furthermore preferred that the number of amino acid insertions, substitutions, additions or deletions is not in excess of 150, such as at most 100, at most 90, at most 80, and at most 70. It is especially preferred that the number of substitutions, insertions, deletions, or additions does not exceed 60, and in particular the number should not exceed 50 or even 40. Most preferred is a number of not more than 30. With respect to amino acid additions, it should be noted that these, when the resulting construct is in the form of a fusion polypeptide, is often considerably higher than 150.
  • Preferred embodiments of the invention include modification by introducing at least one foreign immunodominant T-cell epitope.
  • T-cell epitope the question of immune dominance of a T-cell epitope depends on the animal species in question.
  • immunodominance simply refers to epitopes which in the vaccinated individual/population gives rise to a significant immune response, but it is a well-known fact that a T- cell epitope which is immunodominant in one individual/population is not necessarily immunodominant in another individual of the same species, even though it may be capable of binding MHC-II molecules in the latter individual.
  • the fraction of the population covered by a specific vaccine composition can be determined by means of the following formula
  • ⁇ ⁇ is the sum of frequencies in the population of allelic haplotypes encoding MHC molecules which bind any one of the T-cell epitopes in the vaccine and which belong to the / 11 of the 3 known HLA loci (DP, DR and DQ); in practice, it is first determined which MHC molecules will recognize each T-cell epitope in the vaccine and thereafter these are listed by type (DP, DR and DQ) - then, the individual frequencies of the different listed allelic haplotypes are summed for each type, thereby yielding ⁇ lt ⁇ 2 , and ⁇ 3 .
  • T-cell epitopes to be introduced in the analogue, it is important to include all knowledge of the epitopes which is available: 1) The frequency of responders in the population to each epitope, 2) MHC restriction data, and 3) frequency in the population of the relevant haplotypes.
  • T-cell epitopes which are active in a large proportion of individuals of an animal species or an animal population and these are preferably introduced in the vaccine thereby reducing the need for a very large number of different analogues in the same vaccine.
  • the modification of the ghrelin polypeptide can also include the introduction of a first moiety which targets the modified ghrelin polypeptide to an APC or a B-lymphocyte.
  • the first moiety can be a specific binding partner for a B-lymphocyte specific surface antigen or for an APC specific surface antigen. Many such specific surface an- 0 tigens are known in the art.
  • the moiety can be a carbohydrate for which there is a receptor on the B-lymphocyte or the APC (e.g. mannan or mannose).
  • the second moiety can be a hapten.
  • Suitable cytokines to be used according to the invention are those which will normally also function as adjuvants in a vaccine composition, i.e. for instance interferon ⁇ (IFN- ⁇ ), interle- ukin 1 (IL-1), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin 12 (IL- 12), interleukin 13 (IL-13), interleukin 15 (IL-15), and granulocyte-macrophage colony stimulating factor (GM-CSF); alternatively, the functional part of the cytokine molecule may suffice as the second moiety.
  • IFN- ⁇ interferon ⁇
  • IL-1 interle- ukin 1
  • IL-2 interleukin 2
  • IL-4 interleukin 4
  • IL-6 interleukin 6
  • IL-12 interleukin 12
  • IL-13 interleukin 13
  • IL-15 interleukin 15
  • GM-CSF granulocyte-
  • Integral membrane proteins from e.g. E. coli and other bacteria are also useful conju- gation partners.
  • the traditional carrier molecules such as keyhole limpet hemocyanin (KLH), tetanus toxoid, diphtheria toxoid, and bovine serum albumin (BSA) are also preferred and useful conjugation partners.
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • Preferred embodiments of covalent coupling of the ghrelin polypeptide to pharmaceutically acceptable polyhydroxypolymers such as carbohydrates involve the use of at least one ghrelin polypeptide and at least one foreign T-helper epitope which are coupled separately to the polyhydroxypolymer (i.e.
  • the foreign T-helper epitope and the ghrelin polypeptide are not fused to each other but rather bound to the polyhydroxypolymer which then serves as a carrier backbone).
  • the suitable B-cell epitope carrying regions of the ghrelin polypeptide are constituted by short peptide stretches - this is because this approach is one very convenient way to achieve multiple presentations of selected epitopes in the resulting immunogenic agent.
  • the coupling of the polyamino acids to the polyhydroxypolymer is by means of an amide bond which can be cleaved by a peptidase.
  • ghrelin peptides and T-cell epitopes prepared by means of conventional solid or liquid phase peptide synthesis techniques.
  • the resulting product consists of a polyhydroxypolymer backbone (e.g. a dextran backbone) that has, attached thereto by their N-termini or by other available nitrogen moieties, ghrelin polypeptides and foreign T-cell epitopes.
  • the human ghrelin polypeptide has been modified by substituting at least one amino acid sequence in SEQ ID NO: 11 with at least one amino acid sequence of equal or different length and containing a foreign T H epitope.
  • the foreign T H epitope may simply be inserted in SEQ ID NO: 15 11.
  • a T H containing (or completing) amino acid sequence which is introduced into SEQ ID NO: 11 may be introduced at any amino acid in SEQ ID NO: 11. That is, the introduction is possible after any one of amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,
  • the introduction will preferably include deletion of a substantial number of amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, and 51 in SEQ ID NO: 11. Consequently, it is pre- ferred that only non-destructive (i.e.
  • B-cell epitope conserving deletions/substitutions are made among amino acids 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, and 117 in SEQ ID NO: 11. In this embodiment it is most preferred that all of amino acids 1-51 are deleted.
  • the introduction of the foreign T H epitope should be accompanied by deletion of a substantial number of amino acids 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, and 117 in SEQ
  • non-destructive deletions/substitutions are made among amino acids 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, and 51 in SEQ ID NO: 11. It is in this embodiment most preferred that all of amino acids 52- 117 are deleted.
  • Another embodiment of the present invention is the presentation of the ghrelin analogues which do not include any subsequence of ghrelin that binds productively to MHC class II molecules initiating a T-cell response.
  • this embodiment of the invention also serves to ensure that the immunogen does not include peptide sequences of the target ghrelin that could serve as "self-stimulating T H epitopes" including sequences that merely contain conservative substitutions in a sequence of the target protein that might otherwise function as a T H epitope.
  • Preferred embodiments of the immune system presentation of the analogues of ghrelin involve the use of a chimeric peptide comprising at least one ghrelin derived peptide, which does not bind productively to MHC class II molecules, and at least one foreign T-helper epitope. Moreover, it is preferred that the ghrelin derived peptide harbours a B-cell epitope. It is espe- daily advantageous if the immunogenic analogue is one, wherein the amino acid sequences comprising one or more B-cell epitopes are represented either as a continuous sequence or as a sequence including inserts, wherein the inserts comprise foreign T-helper epitopes.
  • the selected B-cell epitope or -epitopes of ghrelin should therefore comprise at most 9 consecutive amino acids of ghrelin of a relevant animal, that is, at least 9 consecutive amino acids in e.g. SEQ ID NO: 9, 10, 11, 12, 13, or 14. Shorter peptides are preferred, such as those having at most 8, 7, 6, 5, 4, or 3 consecutive amino acids from the ghrelin amino acid sequence.
  • the analogue comprises at least one subsequence of SEQ ID NO: 9, 10, 11, 12, 13, or 14 so that each such at least one subsequence independently consists of amino acid stretches from ghrelin selected from the group consisting of 9 consecutive amino acids, 8 consecutive amino acids, 7 consecutive amino acids, 6 consecutive amino acids, 5 consecutive amino acids, 4 consecutive amino acids, and 3 consecutive amino acids.
  • the consecutive amino acids begins at an amino acid residue selected from the group consisting of residue 1 and/or 2 and/or 3 and/or 4 and/or 5 and/or 6 and/or 7 and/or 8 and/or 9 and/or 10 and/or 11 and/or 12 and/or 13 and/or 14 and/or 15 and/or 16 and/or 17 and/or 18 and/or 19 and/or 20 and/or 21 and/or 22 and/or 23 and/or 24 and/or 25 and/or 26 and/or 27 and/or 28 and/or 29 and/or 30 and/or 31 and/or 32 and/or 33 and/or 34 and/or 35 and/or 36 and/or 37 and/or 38 and/or 39 and/or 40 and/or 41 and/or 42 and/or 43 and/or 44 and/or 45 and/or 46 and/or 47 and/or 48 and/or 49 and/or 50 and/or 51 and/or 52 and/or 53 and/or 54 and/or 55 and/or 56 and/or 57 and/or 58 and
  • the immunogenic construct in such a way that the n-octa- nyolation is absent (either by preparing the constructs by means of peptide synthesis or by using an expression system that will not introduce the n-octanyolation). In this way it is en- sured that the constructs are not physiologically active in the CNS.
  • the formulation of the polypeptide follows the principles generally acknowledged in the art.
  • vaccines which contain peptide sequences as active ingredients are generally well understood in the art, as exemplified by U.S. Patents 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792; and 4,578,770, all incorporated herein by reference.
  • such vaccines are prepared as injectables either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared.
  • the preparation may also be emulsified.
  • the active immunogenic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient.
  • Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof.
  • the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the vaccines; cf. the detailed discussion of adjuvants below.
  • Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandeiic, and the like. Salts formed with the free carboxyl groups may 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.
  • inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandeiic, and the like.
  • Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such
  • the vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic.
  • the quantity to be administered depends on the subject to be treated, including, e.g., the capacity of the individual's immune system to mount an immune response, and the degree of protection desired.
  • Suitable dosage ranges are of the order of several hundred micrograms active ingredient per vaccination with a preferred range from about 0.1 ⁇ g to 2,000 ⁇ g (even though higher amounts in the 1-10 mg range are contemplated), such as in the range from about 0.5 ⁇ g to 2,000 ⁇ g or 0.5 ⁇ g to 1,000 ⁇ g, preferably in the range from 1 ⁇ g to 500 ⁇ g and especially in the range from about 10 ⁇ g to 100 ⁇ g.
  • Suitable regimens for initial administration and booster shots are also variable but are typified by an initial administration followed by subsequent inoculations or other administrations.
  • polypeptides of the vaccine are sufficiently immunogenic in a vaccine, but for some of the others the immune response will be enhanced if the vaccine further comprises an adjuvant substance.
  • adjuvants include use of agents such as aluminium hydroxide or phosphate (alum), commonly used as 0.05 to 0.1 percent solution in buffered saline, admixture with synthetic polymers of sugars (e.g. Carbopol®) used as 0.25 percent solution, aggregation of the protein in the vaccine by heat treatment with temperatures ranging between 70° to 101°C for 30 second to 2 minute periods respectively and also aggregation by means of cross-linking agents are possible. Aggregation by reactivation with pepsin treated antibodies (Fab fragments) to albumin, mixture with bacterial cells such as C.
  • agents such as aluminium hydroxide or phosphate (alum), commonly used as 0.05 to 0.1 percent solution in buffered saline, admixture with synthetic polymers of sugars (e.g. Carbopol®) used as 0.25 percent solution, aggregation of the protein in the vaccine by heat treatment with temperatures ranging between 70° to 101°C for 30 second to 2 minute periods respectively and also
  • Liposome formulations are also known to confer adjuvant effects, and therefore liposome adjuvants are preferred according to the invention.
  • Suitable polymer adjuvants are selected from the group consisting of a carbohydrate such as dextran, PEG, starch, mannan, and mannose; a plastic polymer such as; and latex such as latex beads.
  • Particles consisting only of the antigen can be made by various spraying and freeze-drying techniques.
  • the super critical fluid technology that is used to make very uniform particles of controlled size (York P, 1999 & Shekunov B et al, 1999).
  • the vaccine should be administered at least once a year, such as at least 1, 2, 3, 4, 5, 6, and 12 times a year. More specifically, 1-12 times per year is expected, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 times a year to an individual in need thereof. It has previously been shown that the memory immunity induced by the use of the preferred auto- vaccines according to the invention is not permanent, and therefore the immune system needs to be periodically challenged with the analogues.
  • the vaccine may consequently comprise 3-20 different modified or unmodified polypeptides, such as 3-10 different polypeptides. However, normally the number of polypeptides will be sought kept to a minimum such as 1 or 2 polypeptides.
  • anti-idiotypic antibodies or even mimotopes.
  • the technologies for preparing anti-idio- typic antibodies that mimic a ghrelin epitope are known in the art, but one especially interesting version involves use of autologous anti-idiotypic antibodies, which are reactive with an anti-ghrelin antibody and which are modified by introduction of a foreign T helper epitope as generally described herein.
  • Mimotopes can be isolated from libraries of random peptides that are screened in phage display against antibodies that bind ghrelin specifically.
  • nu- cleic acid vaccination also known as “nucleic acid immunisation”, “genetic immunisation”, and “gene immunisation”
  • nu- cleic acid immunisation also known as “nucleic acid immunisation”, “genetic immunisation”, and “gene immunisation”
  • nucleic acid vaccination does not require resource consuming large-scale production of the immunogenic agent (e.g. in the form of industrial scale fermentation of microorganisms producing modified ghrelin). Furthermore, there is no need to device purification and refolding schemes for the immunogen.
  • a preferred embodiment of the invention comprises effecting presentation of modified ghrelin to the immune system by introducing nucleic acid(s) encoding the modified ghrelin into the animal's cells and thereby obtaining in vivo expression by the cells of the nucleic acid(s) introduced.
  • the introduced nucleic acid is preferably DNA which can be in the form of naked DNA, DNA formulated with charged or uncharged lipids, DNA formulated in liposomes, DNA included in a viral vector, DNA formulated with a transfection-facilitating protein or polypeptide, DNA formulated with a targeting protein or polypeptide, DNA formulated with Calcium precipitating agents, DNA coupled to an inert carrier molecule, DNA encapsulated in a polymer, e.g. in PLGA (cf. the microencapsulation technology described in WO 98/31398) or in chitin or chitosan, and DNA formulated with an adjuvant.
  • DNA which can be in the form of naked DNA, DNA formulated with charged or uncharged lipids, DNA formulated in liposomes, DNA included in a viral vector, DNA formulated with a transfection-facilitating protein or polypeptide, DNA formulated with a targeting protein or polypeptide, DNA formulated with Calcium precipitating agents, DNA coupled to an inert carrier molecule, DNA
  • nucleic acid vaccines can suitably be administered intraveneously and intraarterially.
  • nucleic acid vaccines can be administered by use of a so-called gene gun, and hence also this and equivalent modes of administration are regarded as part of the present invention.
  • VLN a VLN in the administration of nucleic acids has been reported to yield good results, and therefore this particular mode of administration is particularly preferred.
  • the nucleic acid(s) used as an immunization agent can contain regions encoding the 1 st , 2 nd and/or 3 rd moieties, e.g. in the form of the immunomodulating substances described above such as the cytokines discussed as useful adjuvants.
  • a preferred version of this embodiment encompasses having the coding region for the analogue and the coding region for the immunomodulator in different reading frames or at least under the control of different promoters. Thereby it is avoided that the analogue or epitope is produced as a fusion partner to the immunomodulator.
  • two distinct nucleotide fragments can be used, but this is less preferred because of the advantage of ensured co-expression when having both coding regions included in the same molecule.
  • the invention also relates to a composition for inducing production of antibodies against ghrelin, the composition comprising
  • the ghrelin variant-encoding nucleic acid is introduced in the form of a vector wherein expression is under control of a viral promoter.
  • vectors and DNA fragments according to the invention cf. the discussion below.
  • detailed disclosures relating to the formulation and use of nucleic acid vaccines are available, cf. Donnelly JJ et al, 1997, Annu. Rev. Immunol. 15: 617-648 and Donnelly JJ et al., 1997, Life Sciences 60: 163-172. Both of these references are incorporated by reference herein.
  • the non-pathogenic microorganism or virus is administered only once to the animal, but in certain cases it may be necessary to administer the microorganism more than once in a lifetime in order to maintain protective immunity. It is even contemplated that immunization schemes as those detailed above for polypeptide vaccination will be useful when using live or virus vaccines.
  • live or virus vaccination is combined with previous or subsequent polypeptide and/or nucleic acid vaccination.
  • the microorganism or virus can be transformed with nucleic acid(s) containing regions encoding the 1 st , 2 nd and/or 3 rd moieties, e.g. in the form of the immunomodulating substances described above such as the cytokines discussed as useful adjuvants.
  • a preferred version of this embodiment encompasses having the coding region for the analogue and the coding region for the immunomodulator in different reading frames or at least under the control of different promoters. Thereby it is avoided that the analogue or epitopes are produced as fusion partners to the immunomodulator.
  • two distinct nucleotide fragments can be used as transforming agents.
  • having the 1 st and/or 2 nd and/or 3 rd moieties in the same reading frame can provide as an expression product, an analogue of the invention, and such an embodiment is especially preferred according to the present invention.
  • GSG-R ghrelin receptor
  • the methods of the invention may be practiced as a treatment of obesity and of ghrelin and ghrelin-receptor related cancers.
  • ghrelin is known to be a stimulater of the discharge of growth hormone, so in conditions or situations where the effects of an increase in growth hormone production is of interest, the present method affords an alternative, where repeated administrations of growth hormone can be avoided.
  • Such applications involve the use for in vitro fertilization (where GH treatment has been suggested as adjuvating therapy), the use in wound healing (e.g. after severe burns) and generally in treatment of severe trauma, as mentioned above for inducing growth in farm animals, for prolonging survival in patients with heart failure, and the use as an "anti- aging" drug.
  • the present invention is based on the concept of immunising individuals against the ghrelin antigen.
  • the preferred way of obtaining such an immunization is to use modified versions of ghrelin, thereby providing molecules which have not previously been disclosed in the art.
  • modified ghrelin molecules discussed herein are inventive in their own right, and therefore an important part of the invention pertains to a ghrelin analogue which is derived from an animal ghrelin wherein is introduced a modification which has as a result that immunization of the animal with the analogue induces production of antibodies cross-reacting with the unmodified ghrelin polypeptide.
  • the nature of the modification conforms with the types of modifications described above when discussing various embodiments of the method of the invention when using modified ghrelin.
  • any disclosure presented herein pertaining to modified ghrelin molecules are relevant for the purpose of describing the ghrelin analogues of the invention, and any such disclosures apply mutatis mutandis to the description of these analogues.
  • modified ghrelin molecules comprise modifications which results in a polypeptide having a sequence identity of at least 70% with ghrelin or with a subsequence thereof of at least 10 amino acids in length. Higher sequence identities are preferred, e.g. at least 75% or even at least 80% or 85%.
  • the sequence identity for proteins and nucleic acids can be calculated as (N ref - N dif )-100/N refl wherein N dif is the total number of non- identical residues in the two sequences when aligned and wherein N ref is the number of residues in one of the sequences.
  • the invention also pertains to compositions useful in exercising the method of the invention.
  • the invention also relates to an immunogenic composition comprising an immunogeni- cally effective amount of a ghrelin polypeptide which is a self-protein in an animal or a subsequence of such a ghrelin polypeptide, said ghrelin polypeptide or subsequence being formulated together with an immunologically acceptable adjuvant so as to break the animal's autotolerance towards the ghrelin polypeptide, the composition further comprising a pharma- ceutically and immunologically acceptable vehicle and/or carrier.
  • this part of the invention pertains to the formulations of naturally occurring ghrelin polypep- tides/subsequences which have been described in connection with embodiments of the method of the invention.
  • the invention also relates to an immunogenic composition
  • an immunogenic composition comprising an immunologically effective amount of a ghrelin analogue defined above, said composition further comprising a pharmaceutically and immunologically acceptable diluent and/or vehicle and/or carrier and/or excipient and optionally an adjuvant.
  • this part of the invention concerns formulations of modified ghrelin, essentially as described hereinabove.
  • the choice of adjuvants, carriers, and vehicles is accordingly in line with what has been discussed above when refer- ring to formulation of modified and unmodified ghrelin for use in the inventive method for the immunizing against autologous ghrelin.
  • polypeptides are prepared according to methods well-known in the art. Longer polypeptides are normally prepared by means of recombinant gene technology including introduction of a nucleic acid sequence encoding the ghrelin analogue into a suitable vector, transforma- tion of a suitable host cell with the vector, expression of the nucleic acid sequence, recovery of the expression product from the host cells or their culture supernatant, and subsequent purification and optional further modification, e.g. refolding or derivatization.
  • Shorter peptides are preferably prepared by means of the well-known techniques of solid- or liquid-phase peptide synthesis. However, recent advances in this technology has rendered possible the production of full-length polypeptides and proteins by these means, and therefore it is also within the scope of the present invention to prepare the long constructs by synthetic means. Nucleic acid fragments and vectors of the invention
  • modified ghrelin polypeptides can be prepared by means of recombinant gene technology but also by means of chemical synthesis or semisynthesis; the latter two options are especially relevant when the modification consists in coupling to protein carriers (such as KLH, diphtheria toxoid, tetanus toxoid, and BSA) and non-proteinaceous molecules such as carbohydrate polymers and of course also when the modification comprises addition of side chains or side groups to a ghrelin polypeptide-derived peptide chain.
  • protein carriers such as KLH, diphtheria toxoid, tetanus toxoid, and BSA
  • non-proteinaceous molecules such as carbohydrate polymers
  • nucleic acid fragments encoding modified ghrelin are important chemical products.
  • an important part of the invention pertains to a nucleic acid fragment which encodes a ghrelin analogue, i.e. a ghrelin derived polypeptide which either comprises the natural ghrelin sequence to which has been added or inserted a fusion partner or, preferably a ghrelin derived polypeptide wherein has been introduced a foreign T-cell epitope by means of insertion and/or addition, preferably by means of substitution and/or deletion.
  • the nucleic acid fragments of the invention are either DNA or RNA fragments.
  • the nucleic acid fragments of the invention will normally be inserted in suitable vectors to form cloning or expression vectors carrying the nucleic acid fragments of the invention; such novel vectors are also part of the invention. Details concerning the construction of these vec- tors of the invention will be discussed in context of transformed cells and microorganisms below.
  • the vectors can, depending on purpose and type of application, be in the form of plasmids, phages, cosmids, mini-chromosomes, or virus, but also naked DNA which is only expressed transiently in certain cells is an important vector.
  • Preferred cloning and expression vectors of the invention are capable of autonomous replication, thereby enabling high copy- numbers for the purposes of high-level expression or high-level replication for subsequent cloning.
  • the general outline of a vector of the invention comprises the following features in the 5' ⁇ 3' direction and in operable linkage: a promoter for driving expression of the nucleic acid fragment of the invention, optionally a nucleic acid sequence encoding a leader peptide enabling secretion (to the extracellular phase or, where applicable, into the periplasma) of or integration into the membrane of the polypeptide fragment, the nucleic acid fragment of the invention, and optionally a nucleic acid sequence encoding a terminator.
  • a promoter for driving expression of the nucleic acid fragment of the invention optionally a nucleic acid sequence encoding a leader peptide enabling secretion (to the extracellular phase or, where applicable, into the periplasma) of or integration into the membrane of the polypeptide fragment, the nucleic acid fragment of the invention, and optionally a nucleic acid sequence encoding a terminator.
  • vectors to be used for effecting in vivo expression in an animal i.e. when using the vector in DNA vaccination
  • the vectors of the invention are used to transform host cells to produce the modified ghrelin polypeptide of the invention.
  • Such transformed cells which are also part of the invention, can be cultured cells or cell lines used for propagation of the nucleic acid fragments and vectors of the invention, or used for recombinant production of the modified ghrelin polypeptides of the invention.
  • the transformed cells can be suitable live vaccine strains wherein the nucleic acid fragment (one single or multiple copies) have been inserted so as to effect secre- tion or integration into the bacterial membrane or cell-wall of the modified ghrelin.
  • Preferred transformed cells of the invention are microorganisms such as bacteria (such as the species Escherichia [e.g. E. coli], Bacillus [e.g. Bacillus subtilis], Salmonella, or Mycobacterium [preferably non-pathogenic, e.g. M. bovis BCG]), yeasts (such as Saccharomyces cere- visiae), and protozoans.
  • the transformed cells are derived from a multicellular organism such as a fungus, an insect cell, a plant cell, or a mammalian cell. Most preferred are cells derived from a human being, cf. the discussion of cell lines and vectors below.
  • the transformed cell is capable of replicating the nucleic acid fragment of the invention.
  • Cells expressing the nucleic fragment are preferred useful embodiments of the invention; they can be used for small-scale or large-scale preparation of the modified ghrelin or, in the case of non-pathogenic bacteria, as vaccine constituents in a live vaccine.
  • the modified ghrelin of the invention When producing the modified ghrelin of the invention by means of transformed cells, it is convenient, although far from essential, that the expression product is either exported out into the culture medium or carried on the surface of the transformed cell.
  • a stable cell line which carries the vector of the invention and which expresses the nucleic acid fragment encoding the modified ghrelin.
  • this stable cell line secretes or carries the ghrelin analogue of the invention, thereby facilitating purification thereof.
  • plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with the hosts.
  • the vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells.
  • E. coli is typically transformed using pBR322, a plasmid derived from an E.
  • the pBR322 plasmid contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells.
  • the pBR plasmid, or other microbial plasmid or phage must also contain, or be modified to contain, promoters which can be used by the prokaryotic microorganism for expression.
  • promoters most commonly used in prokaryotic recombinant DNA construction include the B-lactamase (penicillinase) and lactose promoter systems (Chang et al., 1978; Itakura et a/., 1977; Goeddel et al, 1979) and a tryptophan (trp) promoter system (Goeddel et al, 1979; EP-A-0 036 776). While these are the most commonly used, other microbial promoters have been discovered and utilized, and details concerning their nucleotide sequences have been published, enabling a skilled worker to ligate them functionally with plasmid vectors (Siebwenlist et al., 1980). Certain genes from prokaryotes may be expressed efficiently in E. coli from their own promoter sequences, precluding the need for addition of another promoter by artificial means.
  • eukaryotic microbes such as yeast cultures may also be used, and here the promoter should be capable of driving expression.
  • Saccharomyces cerevisiae, or common baker's yeast is the most commonly used among eukaryotic microorganisms, although a number of other strains are commonly available.
  • the plasmid YRp7 for example, is commonly used (Stinchcomb et al., 1979; Kingsman et al., 1979; Tschemper et al., 1980).
  • This plasmid already contains the trpl gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan for example ATCC No. 44076 or PEP4-1 (Jones, 1977).
  • the presence of the trpl lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
  • Suitable promoting sequences in yeast vectors include the promoters for 3-phosphoglycerate kinase (Hitzman et a/., 1980) or other glycolytic enzymes (Hess et al., 1968; Holland et al, 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and gluco- kinase.
  • 3-phosphoglycerate kinase Hitzman et a/., 1980
  • other glycolytic enzymes Hess et al., 1968; Holland et al, 1978
  • enolase glyceraldehyde-3-phosphat
  • the termination sequences associated with these genes are also ligated into the expression vector 3' of the sequence desired to be expressed to provide polyadenylation of the mRNA and termination.
  • Other promoters which have the additional advantage of transcription controlled by growth conditions are the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phos ⁇ phatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and ga- lactose utilization.
  • Any plasmid vector containing a yeast-compatible promoter, origin of replication and termination sequences is suitable.
  • cultures of cells derived from multicellular organisms may also be used as hosts.
  • any such cell culture is workable, whether from vertebrate or invertebrate culture.
  • interest has been greatest in vertebrate cells, and propagation of vertebrate in culture (tissue culture) has become a routine procedure in recent years (Tissue Culture, 1973).
  • useful host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and W138, BHK, COS-7 293, Spodoptera frugiperda (SF) cells (commercially available as complete expression systems from a. Protein Sciences, 1000 Research Parkway, Meriden, CT 06450, U.S.A. and from Invitrogen), and MDCK cell lines.
  • an especially preferred cell line is S 2 available from Invitrogen, PO Box 2312, 9704 CH Groningen, The Netherlands.
  • Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located in front of the gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation site, and transcriptional terminator sequences.
  • control functions on the expression vectors are often provided by viral material.
  • promoters are derived from polyoma, Adenovirus 2, and most frequently Simian Virus 40 (SV40).
  • the early and late promoters of SV40 virus are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication (Fiers et al., 1978). Smaller or larger SV40 fragments may also be used, provided there is included the approximately 250 bp sequence extending from the Hind ⁇ l site toward the Bgll site located in the viral origin of replication.
  • promoter or control sequences normally associated with the desired gene sequence provided such control sequences are compatible with the host cell systems.
  • An origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV) or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient. Identification of useful ghrelin analogues
  • another part of the invention concerns a method for the identification of a modified ghrelin polypeptide which is capable of inducing antibodies against unmodified ghrelin in an animal species where the unmodified ghrelin polypeptide is a self-protein, the method comprising preparing, by means of peptide synthesis or by molecular biological means, a set of mu- tually distinct modified ghrelin polypeptides wherein amino acids have been added to, inserted in, deleted from, or substituted into the amino acid sequence of a ghrelin polypeptide of the animal species thereby giving rise to amino acid sequences in the set which comprise T-cell epitopes which are and foreign to the animal species, or preparing a set of nucleic acid fragments encoding the set of mutually distinct modified ghrelin polypeptides, testing members of the set for their ability to induce production of antibodies by the animal species against the unmodified ghrelin, and identifying and optionally isolating the member(
  • the "set of mutually distinct modified ghrelin polypeptides" is a collection of non-identical modified ghrelin polypeptides which have e.g. been selected on the basis of the criteria discussed above (e.g. in combination with studies of circular dichroism, NMR spectra, and/or ghrelin-ray diffraction patterns).
  • the set may consist of only a few members but it is contemplated that the set may contain several hundred members.
  • the set of nucleic acid fragments is a collection of non-identical nucleic acid fragments, each encoding a modified ghrelin polypeptide selected in the same manner.
  • test of members of the set can ultimately be performed in vivo, but a number of in vitro tests can be applied which narrow down the number of modified molecules which will serve the purpose of the invention.
  • T-cell proliferation can be tested by standardized proliferation assays in vitro.
  • a sample enriched for T-cells is obtained from a subject and subsequently kept in culture.
  • the cultured T-cells are contacted with APCs of the subject which have previously taken up the modified molecule and processed it to present its T-cell epitopes.
  • the proliferation of T-cells is moni- tored and compared to a suitable control (e.g. T-cells in culture contacted with APCs which have processed intact, native ghrelin).
  • proliferation can be measured by determining the concentration of relevant cytokines released by the T-cells in response to their recognition of foreign T-cells.
  • an immunogenic composition comprising at least one modified ghrelin polypeptide which is capable of inducing antibodies against unmodified ghrelin in an animal species where the unmodified ghrelin polypeptide is a self-protein, the method comprising admixing the member(s) of the set which significantly induces production of antibodies in the animal species which are reactive with ghrelin with a pharmaceutically and immunologically acceptable carrier and/or vehicle and/or diluent and/or excipient, optionally in combination with at least one pharmaceutically and immunologically acceptable adjuvant.
  • an immunogenic composition which as an immunogen contains a nucleic acid fragment encoding an immunogenic ghrelin analogue, cf. the discussion of nucleic acid vaccination above.
  • nucleic acid sequences and/or vectors are prepared by methods comprising exercise of a molecular amplification technique such as PCR or by means of nucleic acid synthesis.
  • Molecules comprising a T helper epitope and peptides representing or including B-cell epitopes linked covalently to a non-immunogenic polymer molecule acting as a vehicle, e.g. a multiva- lent activated poly-hydroxypolymer, will function as a vaccine molecule that only contains the immunologically relevant parts, can be obtained.
  • a non-immunogenic polymer molecule acting as a vehicle e.g. a multiva- lent activated poly-hydroxypolymer
  • a vaccine molecule that only contains the immunologically relevant parts, can be obtained.
  • Promiscuous or so-called universal T-helper epitopes can be used if e.g. the target for the vaccine is a self-antigen.
  • ele- ments that enhance the immunological response could be also co-coupled to the vehicle and thereby act as an adjuvant.
  • Such elements could be mannose, tuftsin, muramyl dipeptide, CpG motifs etc, e.g. immune stimulating or targeting peptides. In that case, subsequent adjuvant formulation of the vaccine product might be unnecessary and the product could be administered in pure water or saline.
  • CTL cytotoxic T cell
  • Elements that promote uptake of the product to the cytosol, such as mannose, of the APC, e.g. a macrophage could also be co-coupled to the vehicle together with the CTL- and the T helper epitope and enhance the CTL response.
  • the ratio of B-cell epitopes and T-helper epitopes (P2 and P30) in the final product can be varied by varying the concentration of these peptides in the synthesis step.
  • the immunogenic molecule can be tagged with e.g. mannose, tuftsin, CpG-motifs or other immune stimulating substances (described herein) by adding these, if necessary by using e.g. aminated derivatives of the substances, to the carbonate buffer in the synthesis step.
  • an insoluble activated polyhydroxy polymer is used to combine the peptides containing the B-cell epitope and the T-helper epitopes it can, as mentioned above be performed as a solid phase synthesis and the final product can be harvested and purified by wash and filtration.
  • the elements to be coupled to a tresyl activated polyhydroxypolymer (peptides, tags etc) can be added to the polyhydroxypolymer at low pH, e.g. pH 4-5, and allowed to be equally distributed in the "gel" by passive diffusion. Subsequently, the pH can be raised to pH 9-10 to start the reaction of the primary amino groups on the peptides and tags to the tresyl groups on the polyhydroxy polymer. After coupling of peptides and e.g. immune stimulating elements the gel is grinded to form particles of suitable size for immunization.
  • This particular part of the invention therefore generally relates to an immunogen that comprises at least one first amino acid sequence derived from a protein of interest, wherein the at least one first amino acid sequence contains at least one B-cell and/or at least one CTL epitope, and at least one second amino acid sequence that includes a foreign T helper cell epi- tope, wherein each of the at least first and at least second amino acid sequences are coupled to a pharmaceutically acceptable activated polyhydroxypolymer carrier.
  • polyhydroxypolymer is intended to have the same meaning as in WO 00/05316, i.e. the polyhydroxypolymer can have exactly the same characteristics as is specifically taught in that application. Hence, the polyhydroxypolymer can be water soluble or insoluble (thus requiring different synthesis steps during preparation of the immunogen).
  • the polyhydroxypolymer can be selected from naturally occurring polyhydroxy compounds and synthetic poly- hydroxy compounds.
  • polyhydroxypolymers are polysaccharides selected from acetan, amy- lopectin, gum agar-agar, agarose, alginates, gum Arabic, carregeenan, cellulose, cyclodex- trins, dextran, furcellaran, galactomannan, gelatin, ghatti, glucan, glycogen, guar, karaya, konjac/A, locust bean gum, mannan, pectin, psyllium, pullulan, starch, tamarine, tragacanth, xanthan, xylan, and xyloglucan. Dextran is especially preferred.
  • the polyhydroxypolymer can also be selected from highly branched poly(ethyleneimine)(PEI), tetrathienylene vinylene, Kevlar (long chains of poly-paraphenyl terephtalamide), Poly(urethanes), Poly(siloxanes), polydimethylsiloxane, silicone, Poly(methyl methacrylate) (PMMA), Poly(vinyl alcohol), Poly(vinyl pyrrolidone), Poly(2-hydroxy ethyl methacrylate), Poly(N-vinyl pyrrolidone), Poly(vinyl alcohol), Poly(acrylic acid), Polytetra- fluoroethylene (PTFE), Polyacrylamide, Poly(ethylene-co-vinyl acetate), Poly(ethylene glycol) and derivatives, Poly(methacrylic acid), Polylactides (PLA), Polyglycolides (PGA), Poly(lactide- co-glycolides) (PLGA), Polyanhydrides, and Polyorthoesters.
  • the (weight) average molecular weight of the polyhydroxypolymer in question is typically at least 500, such as at least 1,000, preferably in the range of 2,500- 2,000,000, more preferably in the range of 3,000-1,000,000, in particular in the range of 5,000-500,000. It has been shown in the examples that polyhydroxypolymers having an average molecular weight in the range of 10,000-200,000 are particularly advantageous.
  • the polyhydroxypolymer is preferably water soluble to an extent of at least 10 mg/ml, pre- ferably at least 25 mg/ml, such as at least 50 mg/ml, in particular at least 100 mg/ml, such as at least 150 mg/ml at room temperature. It is known that dextran, even when activated as described herein, fulfils the requirements with respect to water solubility.
  • the ratio between C (carbon atoms) and OH groups (hydroxy groups) of the unactivated polyhydroxypolymers is in the range of 1.3 to 2.5, such as 1.5-2.3, preferably 1.6-2.1, in particular 1.85-2.05.
  • a C/OH ratio of the unactivated polyhydroxypolymer represents a highly advantageous level of hydrophilicity.
  • Polyvinylalcohol and polysaccharides are examples of polyhydroxypolymers which fulfil this requirement. It is believed that the above-mentioned ratio should be roughly the same for the activated polyhydroxypolymer as the activation ratio should be rather low.
  • polyhydroxypolymer carrier is intended to denote the part of the immunogen that carries the amino acid sequences.
  • the polyhydroxypolymer carrier has its outer limits where the amino acid sequences can be cleaved of by a peptidase, e.g. in an an- tigen presenting cell that is processing the immunogen.
  • the polyhydroxypolymer car- rier can be the polyhydroxypolymer with an activation group, where the bond between the activation group and the amino acid sequence is cleavable by a peptidase in an APC, or the polyhydroxypolymer carrier can be a polyhydroxypolymer with activation group and e.g. a linker such as a single L-amino acid or a number of D-amino acids, where the last part of the linker can bond to the amino acid sequences and be cleaved by a peptidase in an APC.
  • a linker such as a single L-amino acid or a number of
  • the polyhydroxypolymers carry functional groups (activation groups), which facilitates the anchoring of peptides to the carrier.
  • a wide range of applicable functional groups are known in the art, e.g. tresyl (trifluoroethylsulphonyl), maleimido, p-nitrophenyl cloroformate, cyanogenbromide, tosyl (p-toluenesulfonyl), triflyl (trifluoromethanesulfonyl), pentafluorobenzenesulfonyl, and vinyl sulphone groups.
  • Preferred examples of functional groups within the present invention are tresyl, maleimido, tosyl, triflyl, pentafluorobenzenesulfonyl, p-nitrophenyl cloroformate, and vinylsulphone groups, among which tresyl, maleimido, and tosyl groups are particularly relevant.
  • Tresyl activated polyhydroxypolymers can be prepared using tresyl chloride as described for activation of dextran in Example 1 in WO 00/05316 or as described in Gregorius et a/., J. Immunol. Meth. 181 (1995) 65-73.
  • Maleimido activated polyhydroxypolymers can be prepared using p-maleimidophenyl isocy- anate as described for activation of dextran in Example 3 of WO 00/05316.
  • maleimido groups could be introduced to a polyhydroxypolymer, such as dextran, by derivati- sation of a tresyl activated polyhydroxypolymer (such as tresyl activated dextran (TAD)) with a diamine compound (generally H 2 N-C n H 2n -NH 2 , where n is 1-20, preferably 1-8), e.g.
  • SMCC succinimidyl 4-(N-maleimidomethyl)cyclohexane-l-carboxylate
  • maleimide activated polyhydroxypolymers Although the different reagents and routes for activation formally results in slightly different maleimide activated products with respect to the linkage between the maleimide functionality and the remainder of the parent hydroxy group on which activa- tion is performed, all and every are considered as "maleimide activated polyhydroxypolymers".
  • Tosyl activated polyhydroxypolymers can be prepared using tosyl chloride as described for activation of dextran in Example 2 in WO 00/05316.
  • Triflyl and pentafluorobenzenesulfonyl activated polyhydroxypolymers are prepared as the tosyl or tresyl activated analogues, e.g. by using the corresponding acid chlorides.
  • Cyanogenbromide activated polyhydroxypolymer can be prepared by reacting the polyhydroxypolymer with cyanogenbromide using conventional methods. The resulting functional groups are normally cyanate esters with two hydroxy groups of the polyhydroxypolymer.
  • the degree of activation can be expressed as the ratio between the free hydroxy groups and the activation groups (i.e. functionalised hydroxy groups). It is believed that a ratio between the free hydroxy groups of the polyhydroxypolymer and the activation groups should be between 250: 1 and 4: 1 in order to obtain an advantageous balance between the hydrophilicity and the reactivity of the polyhydroxypolymer. Preferably the ratio is between 100: 1 and 6: 1, more preferably between 60: 1 and 8: 1, in particular between 40: 1 and 10: 1.
  • Especially interesting activated polyhydroxypolymers for use in the method for producing the generally applicable immunogen according to the invention are tresyl, tosyl and maleimido activated polysaccharides, especially tresyl activated dextran (TAD), tosyl activated dextran (TosAD), and maleimido activated dextran (MAD).
  • TAD tresyl activated dextran
  • TosAD tosyl activated dextran
  • MAD maleimido activated dextran
  • the bond between the polyhydroxypolymer carrier and the amino acid se- quences attached thereto are cleavable by a peptidase, e.g. as a peptidase active in the processing of antigens in an APC. It is therefore preferred that the at least first and at least second amino acid sequences are coupled to the activated polyhydroxypolymer carrier via an amide bond or a peptide bond. It is especially preferred that the at least first and at least second amino acid sequences each provide for the nitrogen moiety of their respective amide bond.
  • the polyhydroxypolymer carrier may be substantially free of amino acid residues, necessitating that the activation group provides for part of a peptidase cleavable bond, but as mentioned above, the carrier may also simply include a spacer including at least one L-amino acid. Nevertheless, the at least first and at least second amino acid sequences are normally bound to the activated version of the polyhydroxypolymer via the nitrogen at the N-terminus of the amino acid sequence.
  • 5 peptides were produced by standard methods on a peptide synthesis apparatus.
  • the pep- tides were: peptide 1, an irrelevant control peptide derived from IgE and also including the P2 and P30 epitopes (SEQ ID NO: 7 and 8, respectively), peptide 2, mature rat ghrelin, SEQ ID NO: 9, residues 24-53, and peptides 3-5, i.e. SEQ ID NOs: 15-17, respectively.
  • Blood samples were drawn following 18 hours of fasting at the study initiation and following one week after each injection.
  • Rat-Ghrelin Plasma Rat-Ghrelin using a commercial RIA kit from Phoenix (Ghrelin (Rat Mouse) - RIA Kit) according to the manufacturer's instructions, cf. Fig. 4.
  • Anti- Ghrelin titres were measured using an ELISA where the binding of anti-Ghrelin antibodies to wt Rat Ghrelin (Bachem) was detected with an HRP-conjugated anti-rat immunoglobulin secondary antibody (Dako), cf. Fig. 1.
  • Body weight as well as food and water intake was measured once a week, cf. Figs 2 and 3, respectively.
  • peptides 3-5 are capable of inducing antibodies reactive with rat ghrelin, whereas the irrelevant control (peptide 1) and the wildtype ghrelin (peptide 2) are not.
  • the antibody induction is correlated with increases in ghrelin levels in sera of the immunized rats and with an increase in body weight, whereas the food intake in the 5 groups of rats did not differ significantly.
  • the increase in body weight can therefore not be ascribed to an increase in food-intake but must probably be ascribed to a change in the metabolization in the animals demonstrating antibodies against ghrelin.
  • Example 1 The study in Example 1 can in principal be made with several other candidate vaccine molecules.
  • the exemplary candidates for a ghrelin autovaccine can e.g. be constructed according to the above described concepts (described i.a. in detail in WO 95/05849) by substitution or insertion with known promiscuous T cell epitopes into the ghrelin wild type protein (or into its propeptide variant).
  • the substitutions are peptide substitutions, where the inserted peptide may be of the same or different length than the deleted peptide in the wild- type sequence.
  • the constructs set forth in SEQ ID NOs: 1-5 may be used in mice.
  • Corresponding variants can be made with the P2 and P30 epitopes substituting the PADRE sequence.
  • constructs will be prepared synthetically by means of solid-phase peptide synthesis. This will La. ensure that the constructs will lack mature ghrelin's biological activity, since it appears that the n-octanoylation of serine-3 (in mature ghrelin) is essential for ghrelin's biological activity. Of course, this effect can also be attained by utilising a recombinant expression system that does not allow for this particular post-translational modification.
  • a population of experimental animals will be vaccinated according to a standard protocol (pri- ming with construct formulated in complete Freund's adjuvant and boosting with construct formulated in incomplete Freund's adjuvant) such as the one used in Example 1 with optimised amounts of these 5 constructs formulated according to standard procedures and animals will be compared to a control group with respect to weight gain/loss over time. Variants that produce a loss in weight (and also a decrease in ghrelin levels) will be suitable as candidates for indications where weight loss is of interest, whereas the variants that mimic the results in Example 1 will have the other herein described applications.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Obesity (AREA)
  • Hematology (AREA)
  • Diabetes (AREA)
  • Child & Adolescent Psychology (AREA)
  • Endocrinology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicinal Preparation (AREA)
EP03794825A 2002-09-12 2003-09-12 Immunization against autologous ghrelin Withdrawn EP1539232A1 (en)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US41016402P 2002-09-12 2002-09-12
DKPA200201345 2002-09-12
US410164P 2002-09-12
DK200201345 2002-09-12
PCT/DK2003/000592 WO2004024183A1 (en) 2002-09-12 2003-09-12 Immunization against autologous ghrelin

Publications (1)

Publication Number Publication Date
EP1539232A1 true EP1539232A1 (en) 2005-06-15

Family

ID=31995413

Family Applications (1)

Application Number Title Priority Date Filing Date
EP03794825A Withdrawn EP1539232A1 (en) 2002-09-12 2003-09-12 Immunization against autologous ghrelin

Country Status (7)

Country Link
EP (1) EP1539232A1 (ja)
JP (1) JP2006504413A (ja)
AU (1) AU2003263150A1 (ja)
CA (1) CA2498739A1 (ja)
MX (1) MXPA05002699A (ja)
NO (1) NO20051779L (ja)
WO (1) WO2004024183A1 (ja)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050053664A1 (en) * 2003-09-08 2005-03-10 Eliezer Zomer Co-administration of a polysaccharide with a chemotherapeutic agent for the treatment of cancer
WO2005097830A2 (en) * 2004-04-07 2005-10-20 Aditech Pharma Ab Uses of isolated binding members capable of reducing the biological activity of secretagogue compounds
WO2005097831A2 (en) * 2004-04-07 2005-10-20 Gastrotech Pharma A/S Uses of isolated binding members capable of binding specifically to secretagogues
GB0411014D0 (en) * 2004-05-18 2004-06-23 Haptogen Ltd Methods for the control treatment and management of obesity
EP1856154A1 (en) * 2005-02-23 2007-11-21 Eli Lilly And Company Humanized anti-ghrelin antibodies
US8992928B2 (en) 2006-02-11 2015-03-31 Victor Raso Isolated monoclonal antibody or antigen-binding fragment that cleaves octanoylated native ghrelin
AU2010313282A1 (en) 2009-10-30 2012-05-24 Ocera Therapeutics, Inc. Macrocyclic ghrelin receptor antagonists and inverse agonists and methods of using the same

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001053312A1 (en) * 1999-12-23 2001-07-26 Hyseq, Inc. Novel nucleic acids and polypeptides
US20010020012A1 (en) * 2000-02-01 2001-09-06 Andersen Maibritt Bansholm Use of compounds for the regulation of food intake
EP1286697A2 (en) * 2000-05-17 2003-03-05 Eli Lilly And Company Method for selectively inhibiting ghrelin action
GB0027088D0 (en) * 2000-11-06 2000-12-20 Glaxo Group Ltd DNA expression vectors
WO2002066056A2 (en) * 2001-02-19 2002-08-29 Pharmexa A/S Synthetic vaccines comprising polyhydroxypolymer carriers
GB0105360D0 (en) * 2001-03-03 2001-04-18 Glaxo Group Ltd Chimaeric immunogens

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2004024183A1 *

Also Published As

Publication number Publication date
MXPA05002699A (es) 2005-09-20
WO2004024183B1 (en) 2004-05-13
WO2004024183A1 (en) 2004-03-25
NO20051779D0 (no) 2005-04-11
JP2006504413A (ja) 2006-02-09
NO20051779L (no) 2005-04-11
AU2003263150A1 (en) 2004-04-30
CA2498739A1 (en) 2004-03-25

Similar Documents

Publication Publication Date Title
US7070784B1 (en) Method for down-regulating GDF-8 activity using immunogenic GDF-8 analogues
EP1114166B1 (en) Method for down-regulating osteoprotegerin ligand activity
US7097837B2 (en) Synthetic vaccine agents
US20080253993A1 (en) Immunogenic Egfr Peptides Comprising Foreign T Cell Stimulating Epitope
US20020172673A1 (en) Method for down-regulating IgE
US20040037840A1 (en) Novel therapeutic vaccine formulations
WO2002034287A2 (en) Therapeutic vaccine formulations containing chitosan
US20050063952A1 (en) Immunogenic CEA
EP1539232A1 (en) Immunization against autologous ghrelin
AU2002233166B2 (en) Synthetic vaccines comprising polyhydroxypolymer carriers
ZA200502927B (en) Single chain recombinant T cell receptors.
AU2002233166A1 (en) Synthetic vaccines comprising polyhydroxypolymer carriers
BOEVING et al. Patent 2498739 Summary
KR20050052499A (ko) 자가 그렐린에 대한 면역화
AU2004235875A1 (en) Immunogenic human TNF alpha analogues with reduced cytotoxicity and methods of their preparation
EP1541587A2 (en) Method for down-regulating osteoprotegerin ligand activity

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20050412

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PT RO SE SI SK TR

AX Request for extension of the european patent

Extension state: AL LT LV MK

R17P Request for examination filed (corrected)

Effective date: 20050412

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20070402