EP1012171A1 - Modulation de l'activite d'hormones ou de leurs recepteurs - peptides, anticorps, vaccins et utilisations correspondantes - Google Patents

Modulation de l'activite d'hormones ou de leurs recepteurs - peptides, anticorps, vaccins et utilisations correspondantes

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Publication number
EP1012171A1
EP1012171A1 EP97921529A EP97921529A EP1012171A1 EP 1012171 A1 EP1012171 A1 EP 1012171A1 EP 97921529 A EP97921529 A EP 97921529A EP 97921529 A EP97921529 A EP 97921529A EP 1012171 A1 EP1012171 A1 EP 1012171A1
Authority
EP
European Patent Office
Prior art keywords
peptide
animal
immunised
sstr
seq
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Withdrawn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
EP97921529A
Other languages
German (de)
English (en)
Inventor
Norman L. Gerraty
Simon L. Westbrook
David J. Kingston
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.)
Northstar Biologicals Pty Ltd
Original Assignee
Northstar Biologicals Pty Ltd
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 Northstar Biologicals Pty Ltd filed Critical Northstar Biologicals Pty Ltd
Publication of EP1012171A1 publication Critical patent/EP1012171A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • 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
    • 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
    • A61P3/02—Nutrients, e.g. vitamins, minerals
    • 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
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • 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
    • A61P3/12—Drugs for disorders of the metabolism for electrolyte homeostasis
    • A61P3/14—Drugs for disorders of the metabolism for electrolyte homeostasis for calcium homeostasis
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4743—Insulin-like growth factor binding protein
    • 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
    • C07K14/655—Somatostatins
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • 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

Definitions

  • the present invention relates to peptides, immunologically reactive molecules (IRM) specific for the peptides, pharmaceutical compositions including vaccines incorporating the peptides or IRM and to uses of these in animals.
  • IRM immunologically reactive molecules
  • LH luteinizing hormone
  • TSH thyroid-stimulating hormone
  • inhibin Scanlon et al . , 1993
  • growth hormone Pell and James, 1995
  • insulin like growth factor Pell and Aston, 1995
  • prolactin Lindstedt, 1994
  • growth hormone releasing factor Moore et al . , 1992
  • vasopressin Kamoi et al . , 1977
  • somatostatin Westbrook et al .
  • Fecundin® an androstenedione antigen, which results in an improved fecundity (Scaramuzzi et al , 1977)
  • Vaxtrate® a LHRH antigen, which induces immuno-neutralisation of LHRH with resulting inhibition of steroidogenesis in the ovaries and testes of cattle (Hoskinson et al . , 1990) .
  • the inventors produced a number of peptides based on native hormones, or receptors of native hormones and administered these to animals.
  • Administration of the peptides together with an adjuvant resulted in a number of economically significant effects in the animals such as improved live weight gain, improved milk production, improved wool production, meat quality, efficiency of food utilisation and the like.
  • the invention provides a series of peptides, of various amino acid sequences which are capable of eliciting specific antibodies that alter an array of physiological functions in animals. These physiological functions include digestion, nutrient uptake and metabolism of absorbed substrates, which result in improved productive capacities of immunised animals. Particularly notable are the modifications of the gastrointestinal tract resulting in improved digestion and associated benefits.
  • the present invention provides a non-naturally occurring peptide with an amino acid sequence which is derived from, or is similar to a native animal hormone, carrier protein, binding protein or receptor for said hormone, wherein said peptide is capable of eliciting one or more antibodies which are able to modulate the activity of said hormone or receptor in vivo.
  • non-naturally occurring means that the peptide is not the same as that produced by protein synthesis in nature but is produced by the human hand.
  • Peptides may be produced by standard peptide synthesis techniques, or by recombinant DNA techniques or the like.
  • peptide refers to any molecule formed at least partly of amino acids wherein the amino acids are joined by peptide bonds.
  • the peptides may be made up of only a few amino acids or may be polypeptides. Therefore, proteins and microproteins are also encompassed by this term.
  • the amino acids comprising - A - the peptides may be naturally occurring or synthetic amino acids.
  • the peptides may be essentially amino acid sequences, and may also comprise non-amino acid components such as carbohydrates or fatty acids, or comprise non-natural amino acid-like structures.
  • amino acid sequence derived from, or is similar to refers to the fact that the amino acid sequence may be based on a native sequence or be similar to a native sequence.
  • the term “derived from” does not indicate the actual origin of the peptide (as it may be synthetic or recombinant in origin) but indicates a peptide at least partly homologous to the native hormone or receptor.
  • the peptide is of course different to the native hormone or receptor in that it may comprise a fragment thereof or two or more non ⁇ contiguous fragments thereof. It may also comprise other amino acids not present in the native hormone or receptor.
  • the term "native animal hormone, carrier protein, binding protein or receptor of the hormone” refers to hormones, carrier proteins, binding proteins or receptors which occur in animals. This may be any type of animal but is preferably a vertebrate, more preferably a mammal.
  • the term “capable of eliciting one or more antibodies” refers to the ability of the peptide to elicit or stimulate what appears to be primarily an antibody mediated immune response. There may be some cell mediated immunity involved also. The peptide may not be able to stimulate an immune response by itself especially in the case of small peptides which may need to be present on a carrier molecule. Nonetheless such a peptide still falls within the definition of "capable of eliciting one or more antibodies”.
  • the term "able to modulate the activity of said hormone or receptor in vivo" refers to the ability of the antibody to alter, adjust or vary the activity of the hormone or its receptor in a live animal.
  • the alteration, adjustment or variation will generally be in the form of a down regulation or inhibition of the hormone or receptor although other effects such as an increase in hormone or receptor activity are also contemplated.
  • the peptide is not biologically active. Although it is based on a native hormone or receptor, preferably the peptide does not have the biological activity of that hormone or receptor.
  • the peptide may be based on any hormone or receptor involved in regulating physiological functions.
  • the peptides are able to elicit antibodies to the following hormones or hormone receptors: somatostatin, glucagon, gastrin, cholecystokinin, somatostatin receptors, insulin-like growth factor binding proteins (IGFBP) .
  • the peptides elicit antibodies to hormones of the reproductive tract, in particular luteinising hormone releasing hormone (LHRH) , hormones of the adrenal gland, such as adrenal corticotropic hormone (ACTH) , or of the stomach such as gastrin and cholecystokinin.
  • the inventors have produced a number of peptides based on portions of somatostatin (Patel 1992) , somatostatin receptors (SSTR) (Resine & Bell, 1995) and insulin-like growth factor binding protein 1 to 4 (IGFBP) (Cohick & Clemmons, 1993) .
  • the invention provides a non-naturally occurring peptide with an amino acid sequence based on that of somatostatin (SRIF) .
  • SRIF somatostatin
  • the preferred sequence produces antibodies which preferentially target SSTR2, SSTR3 and SSTR5, in contrast to somatostatin which produces antibodies that bind to the receptor comprising SSTR1 to 6.
  • the peptide of the invention is able to increase levels of circulating insulin, IGF-I and IGF-II and, increase levels of anabolism, decrease gastric activity and/or improve digestion in an animal.
  • F-W-K-T is the key to blocking of SSTR2, SSTR3 and SSTR5.
  • the invention also relates to a derivative or variant of SEQ ID NO:l in which the size and the shape of the exposed rings of the antigen are the smallest diameter possible.
  • SEQ ID NO:l is a relatively soluble peptide in sterile physiological saline.
  • the peptide is presented as- a cyclised sequence, e.g. :
  • the peptide comprising the core sequence FWKT may also be cyclised by means other than inclusion of cysteine residues which form disulphide bonds.
  • a particulary preferred form of the cyclic peptide of the invention is one wherein the linking group or sequence is as short as possible, and the structural configuration is one which allows the portion of the molecule comprising FWKT to bind to the somatostatin receptor.
  • the spatial configuration corresponding to the sequence FWKT in the cyclic peptide is that it occupies minimal space.
  • the arc in the cyclic peptide that is formed by the sequence FWKT is as small as possible and complements its target receptor.
  • a cyclic peptide which stimulates production of antibodies with an affinity for SSTR2, 3 and 5 is particularly preferred.
  • the peptide may also be presented as a linear peptide singly, e.g: F-W-K-T-S-G-G (SEQ ID NO:4) or as a dimer, e.g:
  • the invention provides an immunogenic protein or molecule which comprises a sequence which produces antibodies which have a particular affinity for SSTR2, 3 and 5.
  • the protein or molecule may be of any type as long as the binding of antibodies to the receptors is stimulated.
  • the invention provides a non-naturally occurring peptide with an amino acid sequence which is at least partly homologous to a native animal hormone receptor, wherein said peptide is capable of eliciting one of more antibodies which are able to modulate the activity of said receptor in vivo
  • the invention provides a non-naturally occurring peptide with an amino acid sequence at least partly homologous to that of a somatostatin receptor (SSTR) .
  • SSTR somatostatin receptor
  • Such peptides have the ability to increase at least one biological activity selected from the group consisting of weight gain, birth weight, growth rates, milk production, levels of circulating insulin, IGF-I and IGF-II, fibre production, milk production, and muscle weight. More particularly preferred is a peptide having an amino acid sequence homologous to amino acid residues 1 to 11, 30-57 and/or between the sixth and seventh transmembrane domain of SSTR. The transmembrane domain in question has been reported to occur at residues 274 to 305.
  • the amino acid positions for SSTR are relative to the NH 3 + terminal of the SSTR for all vertebrate species.
  • the peptide is based on SSTR from humans, pigs, cattle, mice or rats.
  • the peptides may be based on SSTRs from other species such as sheep, goats, camels, llamas, alpacas, chickens, ducks, turkeys, ostriches, emus and fish.
  • the invention provides a peptide having the following sequence as represented by the single letter amino acid code, and derivatives and variants thereof:
  • derivatives and variants refers to peptides with different amino acid sequences having substantially the same or similar antigenic activity. Such derivatives or variants may have amino acid substitutions, insertions or deletions compared to the preferred sequences listed above. Typical substitutions are those made in accordance with Table 1 below.
  • the amino acids are generally replaced by other amino acids having like properties such as hydrophobicity, hydrophilicity, electronegativity, bulky side chains and the like.
  • Amino acid substitutions are typically of single residues.
  • Amino acid insertions will usually be in the order of about 1-10 amino acid residues and deletions will range from about 1-20 residues.
  • deletions or insertions are made in adjacent pairs, i.e. a deletion of two residues or insertion of two residues.
  • the peptide of the invention in the cyclised or cyclic form may be varied by substitution of some of the key amino acids with similar amino acids, or amino acid-like structures.
  • a particularly preferred sequence is :
  • the peptides and immunologically active molecules of the invention may be administered as the sole active agent, or be co-administered with one or more other agents.
  • SEQ ID NO:l can be co- administered with gastrin and/or cholecystokinin, together with a suitable carrier.
  • sequences such as SEQ ID NO: 2 to 5 may be co-presented as separate peptides or as one peptide molecule comprising sequences specifically targeting other hormones such as gastric hormones.
  • An example of such a sequence is :
  • anti-SRIF and anti-cholecystokinin antibodies were detectable at 84 days of age. By this time, the immunised lambs had grown an average of 20% more than non-immunised lambs.
  • Reproductive hormones may also be targetted and an example of a sequence for co-administration with the peptide preferred for this invention is:
  • the invention provides a non-naturally occurring peptide with an amino acid sequence at least partly homologous to insulin-like growth factor binding protein (IGFBP) .
  • IGFBP insulin-like growth factor binding protein
  • Such peptides are able to modulate carbohydrate metabolism and thereby improve growth of animals.
  • the peptides may also be useful in the prevention or treatment of diabetes. More particularly preferred is a peptide which includes in its sequence a portion of a native IGFBP which binds insulin-like growth factor, preferably at least some of the region of residues 1 to 10 or 1 to 13 of a native IGFBP.
  • the amino acid residues are relative to the NH 3 + terminal of IGFBP.
  • the peptide is based on IGFBP from humans, pigs, cattle, mice or rats.
  • the peptide may also be based on IGFBPs from other species such as sheep, goats, camels, llamas, alpacas, chickens, ducks, geese, turkeys, ostriches, emus and fish.
  • the invention provides a peptide having one of the following sequences as represented by the single letter amino acid code, or a derivative or variant thereof.
  • peptides of the invention may be referred to herein as "peptides based on SSTR or peptides based on IGFBP", for example.
  • Peptides may be based on one or more hormones, carrier proteins or hormone receptors.
  • the peptides contemplated herein may be chemically synthesised, for example by solid phase peptide synthesis or may be prepared by subjecting the native peptides to hydrolysis or other chemically disruptive processes to produce fragments of the molecule.
  • the peptides may be made by in vitro or in vivo recombinant DNA synthesis.
  • the peptides may need to be synthesised in combination with other proteins and then subsequently isolated by chemical or enzymic cleavage, or the peptides or polyvalent peptides may be synthesised in multiple repeat units.
  • the selection of a particular method of producing the subject peptides will depend on factors such as the required type, quantity and purity of the peptides as well as ease of production and convenience.
  • the peptides of the invention are at least partially purified. More preferably the peptides are in a substantially purified form.
  • the invention provides an immunologically reactive molecule (IRM) which is specific for the peptide of the first aspect of the invention.
  • immunologically reactive molecule refers to a molecule which is able to bind to another molecule, such as an antigen or a peptide capable of functioning as an antigen when present on a carrier.
  • Immunologically reactive molecules are typically antibodies including naturally occurring antibodies, recombinant antibodies, scantibodies, synthetic antibodies including fusions or chimeras of antibodies, and functional fragments of any of the foregoing, such as Fab and F(ab') 2 .
  • the molecule may be encoded by a naturally occurring or synthetic nucleotide sequence and expressed in any convenient expression system. Where the molecule is synthetic, it is conveniently prepared by the step-wise addition of single amino acid groups or amino acid fragments of antibodies. With regard to the latter, the synthetic antibody may be a fusion or chimeric antibody comprising light or heavy chains derived from other antibodies.
  • Antibodies and other IRMs of the present invention may be of any animal origin, including from mammals such as humans, livestock animals, companion animals, wild animals and laboratory test animals (eg. mice, rats, rabbits and guinea pigs) .
  • An "animal" antibody also extends to an antibody from non-mammalian species such as birds, eg. chickens and other poultry, emus and ostriches.
  • Binding of the peptide or IRM may occur in the target hormone receptor site (usually by the use of biologically active antigenic sequences) , or to areas of the cell membrane adjacent to the targeted receptor site, in which case the antigenic fragment has no biological activity.
  • the present invention provides a peptide of the first aspect of the invention coupled to a suitable carrier such that a peptide/carrier complex is formed.
  • the peptide/carrier complex is particularly advantageous where the peptide is relatively small (MW less than 10,000) and not particularly immunogenic when administered alone.
  • Suitable carriers are generally large molecules which are capable of coupling with the peptide.
  • the peptides In order to elicit an immune response, the peptides generally need to be coupled to carrier proteins, to make the antigen significantly "foreign” and to increase the molecular weight of the antigen.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an immunogenically effective amount of the peptide of the first aspect of the invention, or an amount of the IRM of the second aspect of the invention sufficient to confer passive immunity, together with a pharmaceutically or veterinarily acceptable carrier or excipient, and optionally comprising an adjuvant.
  • the peptide of the present invention is referred to as the "active ingredient" .
  • the active ingredient of the pharmaceutical composition is contemplated to exhibit excellent activity in stimulating, enhancing or otherwise facilitating a humoral immune response in animals when administered in an amount which depends on the particular case. For example, for about 0.5 ⁇ g to about 20 mg of protein which may be considered per kilogram of body weight per day may be administered in one or more of daily, weekly or monthly or in other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the situation.
  • the active compound may be administered by injection in a convenient manner or via a genetic sequence in a viral or bacterial vector.
  • the active ingredient may also be administered in dispersions prepared in sterile physiological saline, glycerol, liquid polyethylene glycols, and/or mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for in vivo administration include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases the form must be sterile and must be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like) , suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thormerosal and the like.
  • isotonic agents for example, sugars or sodium chloride
  • gelling agents such as cyclodextrins, gelatin, alginate, and the like. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example.
  • Sterile injectable solutions are prepared by incorporating the active ingredient in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilisation.
  • dispersions are prepared by incorporating the various sterilised active ingredient (s) into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.
  • pharmaceutically or veterinarily acceptable carriers and/or diluents include any and all solvents, dispersion media, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.
  • the use of such media and agents for pharmaceutically or veterinarily active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the composition is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • the pharmaceutical composition comprises the protein coupled to a carrier. More preferably the pharmaceutical composition is in the form of a vaccine preparation.
  • the invention provides a novel delivery vehicle for the peptide of the invention, comprising an oil derived from a deep sea shark.
  • a veterinarily or pharmaceutically acceptable carrier comprising shark oil which has immune adjuvant activity.
  • the oil is desirably of a type which stimulates antibody production in epithelial surfaces of the lung, the respiratory, gastrointestinal or urogenital tract, or the like.
  • the oil enhances antibody secretion in the mucosa of the mammary gland, thereby producing immuno-active colostrum or milk.
  • the oil typically comprises the following components: Hydrocarbons nil - 2%
  • the oil is preferably alkoxyglycerol-rich in that it comprises a triacylglycerol with the general formula CH 2 OH.CHOH.CH 2 OR, where R is a long chain radical, primarily and preferably C ⁇ 6 and C ⁇ 8 .
  • glycerol ethers are especially preferred:- 20-70% octadec - 9 - enylglyceryl ether
  • non-mineral oil typically having a triacylglycerol structure
  • the invention provides a method of producing an immunogenic composition, comprising the steps of contacting a peptide capable of eliciting an immune response with an oil and bringing said peptide and oil into a form suitable for administration, wherein said oil comprises 30-50% diacylglycerol-ethers.
  • the ethers comprise:
  • the immunogenic composition comprises peptide No. 1 emulsified with the oil (“FEEDMIZA”) .
  • FEEDMIZA oil
  • the term "immunogenic composition” refers to a pharmaceutical or veterinary composition which is able to elicit an immune response. This includes vaccines and the like.
  • the composition may be formulated for various forms of administration such as injection (intraperitoneal, subcutaneous, intramuscular or intramammary) , orally, nasal spray, skin patch or the like.
  • a peptide capable of eliciting an immune response refers to any peptide which is immunogenic per se or is capable of inducing an immune response once administered in the composition.
  • the peptide of the first aspect of the invention is used in the composition, but other peptides, such as bacterial and viral antigens, are also contemplated.
  • the composition may include other components such as other active ingredients, drugs or adjuvant as desired.
  • the present invention provides a method of delivering to an animal a peptide capable of eliciting an immune response, comprising the step of administering said peptide together with an effective amount of an oil comprising: 0-70% octadec - 9 - enylglyceryl ether
  • the peptide is preferably emulsified with an effective amount of the oil.
  • an effective amount of oil refers to an amount of oil which will be effective in enabling the peptide to elicit an immune response, particularly where the peptide is not immunogenic by itself or only induces low levels of immunity. Such an amount will generally be about 50 to 80% of the total volume of the composition, preferably 60-70% of the total volume, more preferably about 66 to 67% of the total volume of the composition.
  • the present invention provides a method of modulating one or more hormonal responses in an animal, comprising the step of administering to said animal a hormone-modulating effective amount of the peptide of the first aspect of the invention or the IRM of the second aspect of the invention.
  • the hormonal responses include endocrine and/or paracrine responses.
  • the term "modulating one or more hormonal responses” refers to altering, adjusting or varying the hormonal responses in the animal concerned.
  • the animal may be any animal, preferably a vertebrate, more preferably a mammal.
  • the term animal includes humans, ruminants, birds and reptiles.
  • the animal is a domestic or production animal such as a pig, goat, camel, sheep, alpaca, llama, chicken, goose, duck, turkey, ostrich, emu, fish or other economically important animal.
  • the peptide administered is the peptide complex discussed above, preferably having more than one antigen. More preferably the peptide is administered to the animal in the form of a pharmaceutical preparation, still more preferably, a vaccine formulation.
  • the invention relates to a method of modulating the hormonal responses to one or more of somatostatin, gastrin, insulin, glucagon, prolactin, molitin, cholecystokinin, secretin, prostaglandins, IGF-I, IGF-II, growth hormone and thyroid hormones by administration of the peptide or IRM of the invention.
  • the invention in another preferred aspect relates to a method of enhancing gastrointestinal function in an animal, comprising the step of administering an effective amount of a peptide based on SSTR and/or IGFBP to said animal.
  • enhancing gastrointestinal function refers to promoting digestion of and absorption of key metabolic substrates.
  • the invention provides a method of increasing anabolism and/or body weight in an animal, comprising the step of administering an effective amount of a peptide based on SSTR and/or IGFBP to said animal.
  • the peptide is that of the invention described earlier.
  • the invention provides a method of increasing circulating insulin, IGF-I and/or IGF-III in an animal, comprising the step of administering an effective amount of a peptide based on SSTR and/or IGFBP to said animal.
  • the invention provides a method of suppressing gastric enzymes in an animal, comprising the step of administering an effective amount of a peptide based on SSTR to said animal.
  • the peptide is that of the invention described earlier. More preferably the peptide is that based on SSTR.
  • the invention provides a method of increasing fibre production and optionally further altering the proportion of secondary to primary follicles in a fibre-producing animal, comprising the step of administering an effective amount of a peptide based on SSTR and/or IGFBP to said animal.
  • a peptide is that of the invention described earlier.
  • a fibre producing animal refers to any animal which produces a useful fibre such as wool or the like and includes sheep, goats, llamas and alpacas.
  • the invention provides a method of increasing milk production in a milk-producing animal, comprising the step of administering an effective amount of a peptide based on SSTR and/or IGFBP to said animal.
  • a peptide is that of the invention described earlier.
  • milk producing animal refers to any animal which produces milk either for human consumption or for suckling its young, and includes cows, goats, sheep, camels and the like.
  • the invention provides a method of decreasing the activity of the c- fos gene and/or increasing the activity of the c-jun gene in an animal, comprising the step of administering an effective amount of a peptide based on SSTR to said animal.
  • the peptide is that of the invention described earlier.
  • the invention provides a method of altering calcium metabolism in an animal, comprising the step of administering an effective amount of a peptide based on SSTR to said animal.
  • the peptide is that of the invention described earlier.
  • the invention provides a method of stimulating an immune response to a hormone, carrier protein, binding protein or a hormone receptor in an animal wherein said immune response modulates hormone activity, said method comprising the step of administering an immune response-inducing effective amount of the peptide of the first aspect of the invention to said animal.
  • the invention provides a method of stimulating an antibody response to a hormone, carrier protein, binding protein or hormone receptor wherein said response modulates hormone or receptor activity in an animal and wherein said antibody response results in antibodies being secreted on the mucosa of the animal and/or in the milk of said animal, said method comprising the step of administering to said animal an antibody stimulating effective amount of the peptide of the invention.
  • the mucosa onto which antibodies are secreted is the lung, mammary, gastrointestinal and/or urogenital mucosa.
  • the invention provides a kit comprising the novel peptides or molecule of the invention, optionally further comprising the immuno- adjuvant oil of the invention.
  • Figure 1 is a diagrammatic representation of multiple antigen peptide (MAP) showing 7 branching lysine with peptide antigens.
  • MAP multiple antigen peptide
  • Figure 2 is a diagrammatic representation of MAP showing 16 branching lysine with 36 peptide antigens.
  • Figure 3 is a graphic representation of live weights of pigs during pregnancy and lactation in Example 6.
  • Figure 4 is a graphic representation of live weights of piglets in Example 6.
  • Figure 5 is a graphic representation of milk produced by cows in Example 9.
  • Figure 6 is graphic representation of live weight of chickens in Example 10.
  • Figure 7 is a graphic representation of live weight of pigs in Example 11.
  • Figure 8 is a graphic representation of live weight of chickens in Example 14.
  • Figure 9 is a graphic representation of live weight of male pigs in Example 15.
  • Figure 10 is a graphic representation of live weight of female pigs in Example 15.
  • Figure 11 is a graphic representation of the live weight of pigs in Example 16.
  • Figure 12 is a graphic representation of the net, overall live weight gain of sows in Example 17.
  • Figure 13 is a graphic representation of the live weight of the sows in Example 17.
  • Figure 14 is a graphic representation of the live weight of the piglets in Example 18.
  • Figure 15 is a graphic representation of the live weight of the twin lambs in Example 19.
  • Figure 16 is a graphic representation of the yield of milk from the ewes in Example 19.
  • Figure 17 is a graphic representation of the live weight of the cows in Example 20.
  • Figure 18 is a graphic representation of the live weight of the calves in Example 20.
  • ChemTech ACT Models 396, 348 or 90 ChemTech ACT Models 396, 348 or 90.
  • ELISA enzyme-linked immunosorbent assay
  • the remaining absorbent sites were blocked by dispensing 100 ⁇ L of PBS plus 5% skim milk solution per well for 2 hours at room temperature.
  • the plates were washed three times with a solution of PBS containing 0.1% (v/v) Tween-20 (PBST) .
  • PBST 0.1% Tween-20
  • To each well was added an aliquot of 100 ⁇ L of diluted sample (1/400 v/v; plasma, fat-free colostrum or gastric mucosae in PBST) and the plates were incubated for a further 2 hours at room temperature.
  • the final wash consisted of two washes with PBST then one wash with distilled water.
  • the plates were developed using 100 ⁇ L of substrate [1 mM 2,2' azino-di (3-ethylbenzthiazoline sulphonate) crystallised diammonium salt (ABTS; Sigma Chemical Co., St Louis, U.S.A.) and 2.5 mM H 2 0 2 in 10 mL citrate phosphate buffer (0.1 M citric acid, pH was adjusted to 4.2 with 0.5 M Na 2 HP0 4 ) ] per well and absorbances were read after 30 minutes and 60 minutes with a Titertek MC plate reader at 450 nm. Each sample was tested in duplicate and both positive and negative controls were included for each ELISA plate.
  • substrate 1 mM 2,2' azino-di (3-ethylbenzthiazoline sulphonate) crystallised diammonium salt (ABTS; Sigma Chemical Co., St Louis, U.S.A.
  • Titres of anti-SSTR or IGFBP antibodies in the plasma, colostrum, milk and gut mucosa were expressed as the ratio of the optical density (OD) reading for positive control sample relative to the OD reading for the test sera (Steward and Lew, 1985; Reynolds et al . , 1990) . This ratio was multiplied by the dilution factor of each sample to establish relative quantities of each isotype produced.
  • Antibody titre (OD 450 test sera) x (OD 450 standard) -1 x sample dilution "1
  • the avidities of anti-SSTR or IGFBP antibodies were determined using scatchard plot analyses as outlined by Hoist et al . , (1992a) .
  • a multiple antigen peptide (MAP) system is employed to present/carry the antigens to the immune system.
  • This approach to prepare peptide immunogens of relatively small molecular weight overcomes the ambiguity of conventional carrier systems (i.e KLH, BSA thyriodglobulin etc.) .
  • the MAP approach produces chemically defined peptide antigens with a high degree of homogeneity.
  • immunogens prepared by the MAP approach elicit high and uniform antibody response to the immunogen in immunised animals.
  • the MAP approach to presenting the antigens described in the present application is particularly suitable for eliciting a uniform and site-specific antibody response to the desired antigens.
  • the MAP system consists of an oligomeric branching lysine core, usually composed of three or seven lysines, and four or eight copies of dendritic arms of peptide antigens (see Fig 1) .
  • the MAP system employed in the present application consists preferably of 18 to 20 branching lysine core giving rise to 36 to 40 copies of dedritic arms of the peptide antigens (see Fig 2) . Since each peptide arm may consist of 5-20 amino acids, the overall appearance of the MAP system is of a macromolecule with a high density of surface peptide antigens and a molecule weight exceeding 40,000.
  • the MAP system can be used to present a single antigen alone or any combination of antigens thereof. Furthermore, the antigen/carrier system displays no apparent biological activity, even if injected actively.
  • the MAP system described in the present application was synthesised using a solid phase automated peptide synthesiser (for example Advanced ChemTech ACT Models 396, 348 or 90) .
  • the MAP/antigen is then suspended (ideally emulsified) into a non-inflammatory delivery vehicle or immunostimulator (coded as NSB-050) .
  • a non-inflammatory delivery vehicle or immunostimulator coded as NSB-050
  • the product is administered usually by intraperitoneal or subcutaneous injection.
  • the product may also be injected intramammarily, intramuscularly, or delivered orally.
  • subcutaneous or intraperitoneal injection the emulsion rapidly is taken up by the lymphatic system, and presented to the immune system. At this point there is attachment of the various antigens to receptor sites on the processing cell of the immune system, resulting in the production of the specific antibodies of high titre and affinity.
  • the delivery system (NSB-050) is a particularly effective immunostimulator when attempting to elicit an immune response to a number of different antigens concurrently.
  • the oil has the following composition: diacylglycerol ethers 30-50% triacylglycerols 40-70% polar lipids 10-15% free fatty acids >2% hydrocarbons >2% wax esters >2%
  • the oil is of the following typical analysis: octadec-9-enylglyceryl ether 45% (20-70) 1-hexadecylglyceryl ether 11% (3-25) hexadec-7-enylglyceryl ether 5% (1-15) octadecyl glyceryl ether 7% (1.5-20) eicosa-9-enylglyceryl ether 5% (1-15) lecithin 13% (1-25)
  • the delivery vehicle except for lecithin and non-mineral oil (vegetable or animal origin) are oils extracted from the livers of deep sea sharks, specifically the livers of the Pacific sleeper shark ( Somniosus pacificus) and the Plunket shark ( Centroscymnus pl unketi ) .
  • the oils are recovered from the livers of these sharks at less than or equivalent to 125°C and at a pressure less than or equivalent to 666.6 Pa.
  • the livers are collected fresh from these sharks which are preferred because the oils are devoid of or contain minimum levels of hydrocarbons and wax esters.
  • the excised livers are washed in fresh sea or tap water, and then macerated.
  • the macerated mass is allowed to stand at room temperature (ideally 25°C) for 3 hours, after which the oil is decanted. Clarification of the oil occurs firstly by centrifugation, then by washing with water, followed by a deproteinisation step with bentonite, or similar material. Following a further washing of the oil, it is then stored at 4°C storage for a few weeks to sediment out any winterable material.
  • the clear supernatant is then decanted, mixed with the lecithin emulsifier, and with any of the oil soluble vitamins desired to form the oily delivery vehicle. It is stored in appropriate containers and treated with nitrogen gas to prevent oxidation.
  • Example 5 Preparation of the Vaccine
  • the constructed protein molecule was dispersed in phosphate buffered saline (pH 7.4) by use of ultrasonic agitation. (It is helpful to the performance of the immunogen if the antigen is not soluble in the aqueous phase, and if the protein is dispersed into single molecules in the liquid) .
  • the antigen was mixed preferably in saline (aqueous phase) and emulsified with the oil phase at the rate of 2 parts of oil to 1 aqueous part to produce a stable water-in-oil emulsion.
  • the concentration of antigen in the aqueous phase is such that a 3 ml dose of emulsion contains 100 ⁇ g of antigen macromolecule.
  • the amount of oil in the dose is 2ml (about 66.6% of the total volume of the vaccine) , the remaining 1ml accounts for the aqueous phase containing phosphate buffered saline and antigen.
  • the vaccine may be injected by the intraperitoneal, or subcutaneous routes (for preference), but may also be administered via intramammary, intramuscular, or oral routes.
  • Booster injections preferable two were generally administered within given 2 weeks or longer after the primary vaccination.
  • Liveweights of the gilts/sows were measured each week from mating to parturition, thence at weaning which occurred at three weeks post partum. Immediately after parturition, birthweights of piglets were recorded and thereafter liveweights were measured at weekly intervals until weaning, thence at five weeks of age.
  • Titres of anti-SSTR IgG antibodies were significantly greater (P ⁇ 0.01) than levels measured for anti-SSTR IgA antibodies in the colostrum collected from immunised sows at, or near, the time of parturition. Similar differences in the levels of IgG relative to IgA anti-SSTR antibodies were recorded in the mucosa scrapings of piglets sucking immunised dams; this was more apparent for piglets at 21 than at three days of age. In contrast, no significant differences were measured for titres of IgG and IgA anti-SSTR antibodies in the plasma of immunised piglets on days three and 21.
  • levels of IgG antibodies measured in the stomach mucosa did not differ significantly (P > 0.10) from levels detected in plasma of immunised piglets, at both three and 21 days post partum .
  • titres of anti-SSTR IgA antibodies in the plasma were significantly (P ⁇ 0.05) greater than levels detected in the gastric mucosa for those piglets from immunised sows.
  • levels of circulating concentrations of insulin and IGF I and II were significantly greater for immunised piglets relative to corresponding control piglets.
  • the level or amount of gastric acid secretion following pentagastrin stimulation was observed to be retarded significantly for those piglets sucking immunised than control dams.
  • the activity of key gastric enzymes were suppressed in these piglets. The results from these studies suggest that antibodies which block the SSTR 1 through to 5 alter gut gut function and thereby improve digestion.
  • the antigens used for immunisation comprised of the SSTR 2 (peptide #18 and 20), SSTR 3 (peptide #36 and 38) and SSTR 5 (peptide #48 and 50) coupled individually to the MAP system and were administered concomitantly.
  • the yields of wool were approximately 10% greater for immunised than control ewes during pregnancy and subsequent lactation. Furthermore, the yields of wool for lambs consuming colostrum/milk from immunised dams was improved by some 20% than corresponding control lambs. In addition the wool harvested from immunised lambs was significantly finer than that collected from control lambs. The changes in wool characteristic has been postulated to be attributed to improved nutritional status of those lambs sucking immunised dams leading to greater populations of secondary and primary follicles (see Table 4) .
  • Example 9 Administration of peptides SEQ ID NO: 18 20, 36, 38, 48 & 50 to cattle. Twenty pregnant beef heifers were selected from a grazing herd and allocated to two groups of ten; immunised and control.
  • Immunised cows were injected subcutaneously in the neck with a complex of SSTR 2 (peptide #18 and 20), SSTR 3 (peptide #32 and 34) and SSTR 5 (peptide #48 and 50) antigens coupled to the MAP system emulsified in NSB-050 at c. 5, 7 and 8 months of pregnancy.
  • the remaining ten cows were injected with placebo injections administered subcutaneously in the neck at the corresponding times.
  • Calves from immunised cows were significantly heavier at birth (38 v 46 kg; P ⁇ 0.05) than corresponding calves from non-immunised cows.
  • Example 10 Administration of peptides SEQ ID NO: 18, 20, 36, 38, 48 & 50 to sheep.
  • Immunised ewes were injected subcutaneously in the neck with a complex of SSTR 2 (peptide #18 and 20), SSTR 3 (peptide #36 and 38) and SSTR 5 (peptide #48 and 50) peptides and MAP emulsified in NB 050, the remaining ewes received placebo injections.
  • Ewes were administered corresponding injections at approximately 90, 110 and 130 days of pregnancy.
  • Example 11 Administration of peptides SEQ ID NO: 27, 29, 36, 38, 48 & 50 to chickens. Forty day old chickens were allocated randomly two treatment groups; immunised and control. Immunised birds were injected intraperitoneally at one day age and subsequent booster vaccinations were administered orally at 7 and 14 days of age with 20 ⁇ g of a complex of SSTR 2 (peptide #27 and 29), SSTR 3 (peptide #36 and 38) and SSTR 5 (peptide #48 and 50) coupled to the MAP system emulsified in NSB 050. Control birds received placebo injections at the corresponding site and time.
  • SSTR 2 peptide #27 and 29
  • SSTR 3 peptide #36 and 38
  • SSTR 5 peptide #48 and 50
  • the patagialis muscle was dissected from the each of the birds.
  • the yield of wet muscle collected from immunised birds was recorded to be some 23% and 30% heavier than control birds at 21 and 42 days of age respectively.
  • the mass of the patagialis muscle for immunised birds was observed to be heavier than corresponding control birds, the total yield of RNA per gram of muscle tissue was significantly less for immunised relative to control birds.
  • Example 12 Administration of peptides SEQ ID NO: 21, 23, 36 & 38 to pigs
  • Twenty six week old cross-bred male pigs were allocated randomly to two treatment groups; immunised and control. Pigs were injected subcutaneously in the neck with either a mixture of SSTR 2 (peptide #21 and 23), SSTR 3 (peptide #36 and 38) and SSTR 5 (peptide #48 and 50) antigens coupled to the MAP system emulsified in NSB 050 or placebo injections at 42, 63 and 84 days of age.
  • SSTR 2 peptide #21 and 23
  • SSTR 3 peptide #36 and 38
  • SSTR 5 peptide #48 and 50
  • Example 13 Administration of peptides SEQ ID NO: 54 to 57 to rats
  • Twenty laboratory rats were allocated randomly to two treatment groups immunised and control. Immunised rats were injected subcutaneously in the medial thigh with a 3 ml emulsion containing 100 ⁇ g of a mixture of IGFBP 1 (peptide #47), IGFBP 2 (peptide #55), IGFBP 3 (peptide #56) and IGFBP 4 (peptide #57) antigens described in the present patent coupled to the MAP system suspended in NSB 050.
  • Control rats received placebo injections administered subcutaneously in the medial thigh. The rats were administered three booster injections at intervals of 21 days with the corresponding vaccines.
  • FEEDMIZA The peptide and delivery vehicle is hereinafter referred to as FEEDMIZA
  • the FEEDMIZA dosage was 80 ⁇ g of peptide in 0.1ml of emulsion.
  • the heaviest mean liveweights of the chickens at the end of the experiments were recorded.
  • the weights were 2.19 ⁇ 0.057 kg.
  • Mean liveweight was 2.07 ⁇ 0.049 kg and 1.95 ⁇ 0.047 kg for Group B and A respectively.
  • the differences between each group were stastistically significant (P ⁇ 0.05) .
  • the chickens which were immunised by the intraperitoneal route were approximately 12.3% heavier than the non-immunised birds. Those immunised by the subcutaneous route were an average of 7.2% heavier than the non-immunised birds.
  • Antibodies to the FEEDMIZA antigen were detected in 100% and 60% of the samples of bile obtained from Group C and Group B respectively. No antibodies to the antigen could be detected in the bile of the birds in Group A.
  • Antibodies were detected in gut scrapings of 100% of the Group C birds, 50% of the Group B birds, and in none of the Group A birds.
  • Antibodies were detected in the blood of 100% of the birds in Group B, in 40% of the birds in Group C, and in none of the birds in Group A.
  • Each piglet was individually eartagged and weighed.
  • the treatment groups were:-
  • Group C Immunised by the intraperitoneal route
  • Group D Immunised by the intramuscular route
  • the antigen used in the following experiments was the identical antigen to that used in Example 14.
  • the immunised groups received FEEDMIZA containing 80 ⁇ g of antigen.
  • the placebo group received 3ml of emulsion devoid of the antigen. Pigs were injected at 5, 8 and 12 weeks of age.
  • the pigs were reared in conventional weaning, growing and finishing pens. They were offered weaner, grower and finisher feeds ad libi tum .
  • the liveweights at 21 weeks of age, and the average daily liveweight gains of the various treatment groups are presented in Table 5 (the males) and Table 6 (the females) .
  • Figs.9 and 10 show the weights of the male and female pigs respectively at different ages during the experiment.
  • Group A non-immunised controls Group B immunised with FEEDMIZA Group C immunised with a SRIF-conjugate
  • the gilts were immunised by subcutaneous injection of 3ml of the relevant antigen at the neck on the day of mating.
  • the animals were held in conventional single dry sow stalls.
  • the treatment groups were reduced to 5 confirmed pregnant animals per treatment group.
  • Subsequent vaccinations were performed at weeks 6, 9 and 12 of pregnancy.
  • Each gilt was fed 3kg of feed (Breedmore sow ration, Barastoc, Melbourne) each day from mating until farrowing.
  • Each animal was weighed at mating, and at intervals of 3 weeks until farrowing.
  • the non-immunised sows had gained an average of 60 kg per head, had eaten 115 x 3kg of feed (345 Kg) and had converted that feed into liveweight gain at a ratio of 5.75:1.
  • the FEEDMIZA- immunised sows had likewise consumed 345 Kg of feed, but had gained an average of 138 kg each during pregnancy, at an efficiency ratio of 2.5:1.
  • the SRIF-immunised group had gained an average of 75kg per head during the test period at an efficiency of converion of feed to liveweight gain ratio of 4.6:1.
  • SRIF-conjugate vaccine was not successful in modulating growth stastistically, but the FEEDMIZA preparation significantly improved liveweight gain. It is clear that the FEEDMIZA antigen and the SRIF- conjugate antigens are very different in the effect which they have on the modulation of growth of gestating sows.
  • Sows immunised with FEEDMIZA preparation were substantially better converters of feed to liveweight gain than were the SRIF immunised and the non-immunised sows.
  • the four immunisations provided to these sows are probably more than is necessary to achieve successful immunomodulation.
  • the regime was selected to ensure high levels of antibody were present in the animals at all times since the objective was to measure the efficacy of antibodies in modulating the performance.
  • a more practical regime might be vaccination at selection, at confirmation of pregnancy and at transfer to the farrowing house. Further testing may indicate that less than 3 vaccinations per pregnancy produces acceptable results.
  • Example 16 This experiment was a continuation of the trial of Example 16. It sought to observe the rate of liveweight gain of piglets during the lactation period up to weaning for piglets whose dams were immunised during gestation with FEEDMIZA, or with a SRIF-conjugate vaccine, using piglets being reared by non-immunised dams as controls.
  • Protocol Sows from Example 16 continued to be reared without any further immunisations or manipulations, with the exception that; a) daily feed offered was increased to 6kg per head from farrowing to weaning 21 days later. b) 3 days after farrowing the number of piglets on each sow was reduced to 8 to ensure that each litter of pigs in the experiment was benefiting from the combined effects of the quantity of milk produced, and ingested any antibodies that might be in them, without confounding influences of varying litter sizes, social interactions, and competition for teats. Sows were weighed at weaning.
  • Piglets were individually identified at birth, and were weighed at birth, and each 7 days until weaning at 21 days of age. All piglets received a routine oral dose of broad spectrum antibiotic (Tribrissen Piglet Suspension, Intervet, Melbourne) at three days of age, as well as an intramuscular supplemental iron injection Pignaemia, Intervet, Melbourne)
  • weight loss The weights of the sows at farrowing and at weaning were: Weaning weight (kg) Farrowing weight (kg) Weight loss
  • Sows which were immunised with the FEEDMIZA vaccine were noted to have prolific milk production as evidenced by teats frequently leaking copious quantities of milk, especially during the first week of lactation.
  • Sows lost weight during the lactation phase irrespective of whether they had been vaccinated during pregnancy, or not. This is obviously a response to the demands of lactation which are not adequately met from the increased daily ration offered.
  • the magnitude of the loss of weight of sows during lactation which had been immunised with FEEDMIZA during gestation, and which had so dramatically increased in liveweight during pregnancy as reported in the previous trial was double that which was observed for either the non-immunised or SRIF-conjugate immunised sows. This is a dramatically different pattern from that observed with either of the two other treatment regimes as shown in Figure 13.
  • FEEDMIZA antibodies are associated with improving the protential productive (liveweight gain or milk production) characteristics of pregnant/lactating pigs. Since both of these functions involve the metabolism of calcium, carbohydrate and protein in particular, these results indicate that antibodies produced to the SRIF-conjugate were less potent in the terms of promoting liveweight gain than were the anti-FEEDMIZA antibodies.lt is hypothesised that immunisation with FEEDMIZA into gestating pigs results in increased efficiency of the normal metabolic pathways involving these key metabolites. Whether the effect of the antibodies is stimulatory, or whether it occurs by removal of normal inhibitory mechanisms is not clear.
  • the piglets (8 per litter) on each of the 5 sows in the treatment groups were individually eartagged for identification, and were weighed at birth, 7, 14 ,21 days (the day of weaning) and 35 days of age (2 weeks post-weaning) .
  • each litter of piglets was placed in a flatdeck weaning cage with 50% of the floor being bedding area, and 50% woven wire mesh.
  • the cage was provided with a small silo feeder containing the same creep/weaner feed which had been offered to the piglets for the 3 days prior to weaning. Feed and fresh drinking water were offered ad libi tum.
  • the mean birthweights of the piglets were:
  • the piglets born to the FEEDMIZA-immunised sows were stastistically heavier (P ⁇ 0.05) than those born to the other groups (which were not stastistically different from each other) .
  • the liveweights of the piglets at weaning were:
  • Immunisation of pregnant sows with SRIF- conjugate antigen, or with FEEDMIZA resulted in increased rates of liveweight gain of piglets sucking those dams compared with non-immunised sows. This suggests that immunomanipulation of animals for the purpose of increasing milk production is possible.
  • Immunisation of pregnant sows with FEEDMIZA vaccine produced stastistically significanly heavier weights of piglets which sucked them for 3 weeks than was observed for piglets sucking either non-immunised , or SRIF- immunised sows at the observation points of birth, weaning and 2 weeks post-weaning. This observation suggests that the process of immunomanipulation had been more effectively achieved with FEEDMIZA than with the SRIF-conjugate.
  • Sheep have been the subject of numerous published attempts at immunomodulation with a variety of hormones. Sheep have many production characteristics that are similar to the pig production cycle, viz a relatively short period of gestation, rapid rate of gain of the sucking young, and a relatively short period of lactation. They are relatively easy animals for measuring milk production, and they lend themselves readily to blood collection for measurement of antibodies, circulating hormones or metabolites.
  • the pregnant ewes were maintained on pasture during pregnancy as a single mob of sheep. Free choice was provided to lucerne hay during the last four weeks of pregnancy. Immediately after the sheep lambed they were moved indoors and offered 2.5 Kg per day of a 4:1 mixture of lucerne chaff and rolled barley until 6 weeks of age, at which point the experiment was concluded and the ewes and lambs were turned out to pasture.
  • the ewes which were to be immunised were injected with 3ml of FEEDMIZA subcutaneously in the flank at 90, 110 and 132 days of pregnancy.
  • the liveweights of the ewes were recorded at the time of first immunisation, at birth and at 6 weeks of age "weaning". Liveweights of the lambs were recorded at birth and at weaning. Wool samples (100 square centimetres) were collected as mid side patches as described by Wynn et al (1988) at time of first immunisation, lambing and weaning of the ewes, and at weaning for the lambs.
  • the follicle density and types were assessed by histological examination of core samples collected at birth, 1 week of age and at weaning.
  • Milk production was measured one day of each week for the first 6 weeks of lactation by depriving the lambs access to their mothers for a period of 6 hours . After the 6 hours of lamb deprivation the ewes were handmilked with the total weight of milk being recorded. A 10ml sample of this milk was taken for assay, and the remainder was fed to the lambs by hand using teats attached to the pen draining a pouch containing the milk.
  • the weights of the ewes at first immunisation were 57.3 ⁇ 2.4 kg for the group to be immunised, and 57.5 ⁇ 2.7 kg for the group to remain as non-immunised controls.
  • the immunised ewes weighed 53.2 ⁇ 3.0 kg while the non-immunised group weighed 51.8 ⁇ 2.7 kg.
  • the immunised ewes weighed 55.2 ⁇ 3.1 kg and the non-immunised group weighed 52.7 kg. There were no stastistically significant differences between the weights of the ewes at any stage of the experiment, nor was there any significant differences in the birthweights of the lambs.
  • the milk yields recorded for the ewes are presented in Figure 16 and show that the milk production of the immunised ewes was approximately 20% greater at each observation. There was no stastistically significant differences in the milk fat, milk lactose or milk protein levels measured in the samples of milk collected from the treatment groups at each milk collection. There were slight differences in the analyses from week to week.
  • the yields of wool were approximately 10% heavier from the immunised ewes than from the non- immunised ewes during pregnancy, and during lactation. Yields of wool from the lambs consuming milk from the immunised ewes was 20% greater than that produced by the lambs drinking milk from the non-immunised ewes. In addition the wool which was harvested from the lambs from the immunisd ewes was significantly finer than that which was observed from the non-immunised lambs. The lambs on the immunised ewes had greater densities of secondary and primary follicles.
  • a line of fifty Hereford x Angus heifers was selected and placed onto actively growing pasture.
  • the heifers were oestrus synchronised by injecting with Lutalyse (Upjohn, Rydalmere, Australia) and naturally mated to Hereford bulls .
  • the animals were randomly divided into two groups at the time of synchronisation; one group of 20 animals was to be the immunised group, and a second group of 20 animals was the non-immunised heifers.
  • Animals to be immunised were injected with 3ml of emulsion subcutaneously in the neck at mating, 3 months of pregnancy and two weeks prior to parturition. The animals were weighed at the time of mating, at parturition and at 3 months after parturition. The calves produced were weighed at birth and each 28 days thereafter until 3 months old.
  • Mean birth weight of the non-immunised calves was 35 ⁇ 3.5 kg compared with 40 ⁇ 4.5 kg. (an average difference of 14%) .
  • Mean weights of the calves three months after birth were 116.2 ⁇ 6.4 kg for the non- immunised control group, and 145.0 ⁇ 7.5 kg for the immunised group (an average difference of 20%) .
  • the average gain of the non-immunised calves for days 1-84 was 81.2 kg, and for the immunised group was 105 kg (an average difference of 23.8 kg, or 29.3%), as shown in Figure 18.
  • Total net gain of liveweight (cow plus calf) from mating to 84 days after calving was 131.2 Kg for the non-immunised group, and 205 Kg for the immunised group.
  • BSA bovine serum albumin
  • the growth responses measured over an 84 day period were:
  • the affinities of antibodies to SRIF, the cyclosised form of the preferred sequence (SEQ ID Nos 2 to 3) and the linear form (SEQ ID No. 4) were determined for hyperimmune sheep and swine sera produced against each of the three presentations .
  • Pregnant pigs which were immunized twice in the last trimester of pregnancy with SEQ ID Nos 3, 4,5 and SRIF 14 (native somatostatin) conjugate presented with the preferred oil all produced anti-SRIF antibodies in colostrum and milk of the sow and in the blood and gut scrapings of the piglets at 3 days and 21 days of age.
  • the absolute growth rates of the piglets from birth to weaning were:
  • Piglets from sows immunised 275 g per day n 42 with SEQ ID No:3
  • Piglets from sows immunised 240 g per day n 44 with SEQ ID No:4
  • the type of antibody produced in response to the cyclic peptide delivered in the preferred oil has a bearing upon the growth of the immunised animals compared to SRIF 14 conjugate delivered in the same manner.
  • Sows immunized twice in the last trimester of pregnancy with SEQ ID No 3 produced piglets which were on average 27% heavier at weaning (at 21 days of age) than the offsprings of sows immunised with SRIF 14 conjugate vaccine.
  • the levels of anti-SRIF antibodies measured are given below: TREATMENT DAY 3 DAY 21
  • the preferred form of antigen delivery involves the immunogens being mixed, suspended or preferably emulsified in a non-macrophage stimulating system injected either subcutaneously or intraperitoneally.
  • An important feature of the preferred delivery system is that it facilitates the expression of the humoral immune response to multiple antigens as if each was presented alone.
  • the antibodies are extremely potent (high titres and affinity) and involve the whole complement of isotypes, especially those associated with mucosal surfaces (IgA and IgM) .
  • the mechanisms by which the invention improves animal productivity are extremely complex and not understood completely.
  • the antibodies alter the metabolism of particularly somatostatin, gastrin, insulin, and glucogon; and augment the circulating concentrations of insulin-like growth factor (IGF-I and II) .
  • the endocrine ramifications associated with altering the matabolism of these hormones are vast and include changes in the circulating concentrations of, at least, gastrin, cholecystokinin, motilin, secretin, the thyroid hormones, glucagon, insulin, IGF I and II, somatostatin, prostaglandins, histamine, and vasoactive intestinal peptide. It has been demonstrated from the results obtained from our laboratory that immunisation against the peptides induce changes in both gastrointestinal and metabolic functions. For example the rate of the secretion of gastric acid and the activity of many of the gastric proteases are retarded significantly in response to both chemical and physiological stimulation following immunisation.
  • the motility of digesta through various segments of the gastrointestinal tract are altered significantly and consequently the absorption and metabolism of key metabolites are enhanced.
  • the antibodies allow many cells particularly those associated with the endocrine system to be more responsive to Ca 2 " ions, thus increasingly the secretion of hormones associated with the somatotropic axis and gastrointestinal tract.
  • the effect of this is that mRNA production within cells, especially muscle fibres, mammotrophs and somatotrophs are enhanced.
  • Intraperitoneal immunisation 104 ⁇ 2.79 12 625 ⁇ 21.0
  • the heaviest group was those which had been immunised subcutaneously. They were significantly heavier than all other treatment groups (p ⁇ 0.05) .
  • the pigs immunised by the intraperitoneal route were significantly heavier than those immunised intramuscularly and those given placebo injections (p ⁇ 0.05) . There was no significant difference between the intramuscularly immunised and the placebo groups. Table 6
  • Heaviest liveweight was recorded in the group which had been immunised by the subcutaneous route. This was significantly heavier (p ⁇ 0.05) than t he weight of the group immunised intramuscularly. The weights of both of these groups were significantly heavier (p ⁇ 0.05) than the placebo injected group. There were no significant differences between the weights of the other treatments.
  • ADDRESSEE GRIFFITH HACK
  • STREET 509 ST KILDA ROAD
  • ATTORNEY/AGENT INFORMATION (A) NAME: SANTER, VIVIEN (C) REFERENCE/DOCKET NUMBER: JMW/SW:FP4656

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Abstract

L'invention se rapporte à des peptides immunogènes, que l'on ne rencontre pas à l'état naturel, et à des molécules réagissant immunologiquement avec ces peptides, qui modulent l'activité d'hormones ou de récepteurs d'hormones. Ces peptides sont basés sur des parties de somatostatine, sur des récepteurs de somatostatine et sur une protéine de liaison du facteur de croissance proche de l'insuline. L'invention se rapporte également à des procédés visant à moduler l'activité hormonale chez un animal ainsi qu'à des compositions utilisées à cet effet.
EP97921529A 1996-05-22 1997-05-22 Modulation de l'activite d'hormones ou de leurs recepteurs - peptides, anticorps, vaccins et utilisations correspondantes Withdrawn EP1012171A1 (fr)

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AUPN9990A AUPN999096A0 (en) 1996-05-22 1996-05-22 Peptides, antibodies, vaccines & uses thereof
AUPN999096 1996-05-22
PCT/AU1997/000312 WO1997044352A1 (fr) 1996-05-22 1997-05-22 Modulation de l'activite d'hormones ou de leurs recepteurs - peptides, anticorps, vaccins et utilisations correspondantes

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CN (1) CN1226896A (fr)
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BR (1) BR9709038A (fr)
CA (1) CA2255888A1 (fr)
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US7217796B2 (en) 2002-05-24 2007-05-15 Schering Corporation Neutralizing human anti-IGFR antibody
ES2527871T3 (es) 2003-05-01 2015-02-02 Imclone Llc Anticuerpos completamente humanos dirigidos contra el receptor del factor de crecimiento 1 similar a la insulina humana
EP1694850B1 (fr) 2003-11-12 2011-06-29 Schering Corporation Systeme de plasmide pour l'expression multigenique
PE20050928A1 (es) 2003-11-21 2005-11-08 Schering Corp Combinaciones terapeuticas de anticuerpo anti-igfr1
FR2874156B1 (fr) * 2004-08-13 2007-04-06 Univ Rennes 1 Etablissement Pu Complement alimentaire pour animaux d'elevage et procede de mise en oeuvre de celui-ci
ES2356830T3 (es) 2004-12-03 2011-04-13 Schering Corporation Biomarcadores para preselección de pacientes para terapia de anti-igf-1r.
CA2678008C (fr) 2005-06-17 2013-07-30 Imclone Systems Incorporated Antagonistes de recepteur pour le traitement de cancer osseux metastatique
EP1981512B1 (fr) * 2006-01-20 2022-11-02 Starpharma Pty Limited Macromolecule modifiee
CN101484587B (zh) 2006-02-03 2014-02-12 英克隆有限责任公司 Igf-ir拮抗剂作为辅药用于前列腺癌的治疗
MX2010014078A (es) * 2008-06-25 2011-04-11 Braasch Biotech Llc Composiciones y metodos para inmunogenecidad mejorada de somatostatina.
US11053316B2 (en) 2013-01-14 2021-07-06 Xencor, Inc. Optimized antibody variable regions
EP3620473A1 (fr) 2013-01-14 2020-03-11 Xencor, Inc. Nouvelles protéines hétérodimères
US10858417B2 (en) 2013-03-15 2020-12-08 Xencor, Inc. Heterodimeric proteins
UA119167C2 (uk) 2014-03-28 2019-05-10 Зенкор, Інк. Біспецифічне антитіло, яке зв'язується з cd38 та cd3
US10259887B2 (en) 2014-11-26 2019-04-16 Xencor, Inc. Heterodimeric antibodies that bind CD3 and tumor antigens
EP3223845B1 (fr) 2014-11-26 2021-05-19 Xencor, Inc. Anticorps hétérodimériques se liant à l'antigène cd3 et l'antigène cd20
KR20180085800A (ko) 2015-12-07 2018-07-27 젠코어 인코포레이티드 Cd3 및 psma에 결합하는 이종이합체성 항체
MA45255A (fr) 2016-06-14 2019-04-17 Xencor Inc Anticorps inhibiteurs de points de contrôle bispécifiques
AU2017290086A1 (en) * 2016-06-28 2019-01-24 Xencor, Inc. Heterodimeric antibodies that bind somatostatin receptor 2
US11919956B2 (en) 2020-05-14 2024-03-05 Xencor, Inc. Heterodimeric antibodies that bind prostate specific membrane antigen (PSMA) and CD3
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AUPN999096A0 (en) 1996-06-13
BR9709038A (pt) 2000-01-04
CA2255888A1 (fr) 1997-11-27
JP2000512130A (ja) 2000-09-19
CN1226896A (zh) 1999-08-25
NZ332926A (en) 2000-08-25

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