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WO2000050077A1 - Immunogenes comprenant un peptide et support derive de haemophilius influenzae - Google Patents

Immunogenes comprenant un peptide et support derive de haemophilius influenzae Download PDF

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Publication number
WO2000050077A1
WO2000050077A1 PCT/EP2000/001457 EP0001457W WO0050077A1 WO 2000050077 A1 WO2000050077 A1 WO 2000050077A1 EP 0001457 W EP0001457 W EP 0001457W WO 0050077 A1 WO0050077 A1 WO 0050077A1
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WO
WIPO (PCT)
Prior art keywords
peptide
protein
immunogen
carrier
peptides
Prior art date
Application number
PCT/EP2000/001457
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English (en)
Other versions
WO2000050077A9 (fr
Inventor
Michel Coste
Yves Lobet
Marcelle Paulette Van-Mechelen
Christophe Verriest
Original Assignee
Smithkline Beecham Biologicals S.A.
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
Priority claimed from GBGB9904408.3A external-priority patent/GB9904408D0/en
Priority claimed from GBGB9904412.5A external-priority patent/GB9904412D0/en
Priority claimed from GBGB9904405.9A external-priority patent/GB9904405D0/en
Priority claimed from GBGB9919260.1A external-priority patent/GB9919260D0/en
Application filed by Smithkline Beecham Biologicals S.A. filed Critical Smithkline Beecham Biologicals S.A.
Priority to JP2000600687A priority Critical patent/JP2002537354A/ja
Priority to CA002363118A priority patent/CA2363118A1/fr
Priority to EP00909235A priority patent/EP1156825A2/fr
Priority to AU31589/00A priority patent/AU3158900A/en
Publication of WO2000050077A1 publication Critical patent/WO2000050077A1/fr
Publication of WO2000050077A9 publication Critical patent/WO2000050077A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C07K16/4283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an allotypic or isotypic determinant on Ig
    • C07K16/4291—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an allotypic or isotypic determinant on Ig against IgE
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6425—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a receptor, e.g. CD4, a cell surface antigen, i.e. not a peptide ligand targeting the antigen, or a cell surface determinant, i.e. a part of the surface of a cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/14—Decongestants or antiallergics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/285—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pasteurellaceae (F), e.g. Haemophilus influenza
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6068—Other bacterial proteins, e.g. OMP
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • 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 immunogens comprising a peptide and a carrier, in particular when the carrier is derived from Haemophilus Influenzae Protein 5 D.
  • the immunogens of the present invention may be used in pharmaceutical compositions for the prophylaxis or therapy of disease, especially in the form of vaccines.
  • the invention further relates to their production, pharmaceutical compositions containing them, and their use in medicine.
  • the peptides which are commonly used may be the full length native immunogen, for example human peptidic hormones, or may be fragments of a larger antigen derived from a given pathogen, or from a large self-protein.
  • immunoprophylaxis of allergy may beneficially comprise the use of short peptides of IgE (EP 0477231 Bl), whereas the use of IgE itself as the immunogen may induce anaphylactic shock.
  • IgE whole length Gonadotrophin hormone releasing hormone
  • a ⁇ has both a prophylactic and therapeutic potential.
  • peptides per se are poor immunogens.
  • sequence of the peptides are chosen such that they include a B-cell epitope to provide a target for the generation of anti-peptide antibody responses, but because of their limited size rarely encompass sufficient T-cell epitopes in order to provide the necessary cytokine help in the induction of strong B-cell responses.
  • Strategies which have been designed to overcome this lack of immunogenicity include the linking of the peptide to large highly immunogenic protein carriers, which provide bystander T-cell help, and/or the use of strong adjuvants in the vaccine formulation.
  • Examples of these highly immunogenic carriers which are currently commonly used for the production of peptide immunogens include the Diptheria and Tetanus toxoids (DT and TT respectively), Keyhole Limpet Haemocyanin (KLH), and the purified protein derivative of Tuberculin (PPD).
  • DT and TT Diptheria and Tetanus toxoids
  • KLH Keyhole Limpet Haemocyanin
  • PPD purified protein derivative of Tuberculin
  • a number of problems are associated with each of these commonly used carriers, including both problems in production of GMP constructs and also in immunological characteristics of the constructs.
  • the present invention provides a new carrier for use in the preparation of peptide-based immunogen constructs, that does not suffer from the disadvantages associated with such large highly immunogenic carriers.
  • KLH is known as potent immunogen and has already been used as a carrier for peptides derived from IgE (EP 047723 IB 1) in human clinical trials. However, some adverse reactions (DTH-like reactions or IgE sensitisation) as well as antibody responses against KLH which could compete with the anti-decapeptide antibody have been observed.
  • a carrier protein for a peptide based vaccine will require a balance between the necessity to use a carrier working in all patients (broad MHC recognition) and the induction of high levels of anti-peptide antibody responses and of low antibody response against the carrier.
  • the present invention provides a protein D from Haemophilus Influenzae, or fragments thereof, as a carrier for peptide based vaccines which induces high anti- peptide immune responses with a moderate or low anti-carrier response.
  • the peptide is a non-Haemophilius Influenzae derived peptide.
  • IgE The role of IgE in the mediation of allergic responses, such as asthma, food allergies, type-I hypersensitivity and sinus inflammation, is well known.
  • B cells On encountering an antigen, such as pollen or dust mite allergens, B cells commence the synthesis of allergen specific IgE.
  • the allergen specific IgE then binds to its Fc ⁇ RI receptor (the high affinity receptor) on basophils and mast cells. Any subsequent encounter with allergen leads to the triggering of histamine release from the mast cells or basophils, and cross-linking of neighbouring IgE/ Fc ⁇ RI complexes (EP 0 477 231 Bl).
  • Fc ⁇ RI receptor the high affinity receptor
  • a number of carriers for the IgE peptides are suggested including KLH, Tetanus toxoid, Diptheria toxoid, albumins, haemocyanins such as Keyhole Limpet Haemocyanin (KLH), polymers of amino acids, and preferably purified protein derivative of tuberculin (PPD).
  • KLH Keyhole Limpet Haemocyanin
  • PPD purified protein derivative of tuberculin
  • WO 95/26365 further continues investigation of the C ⁇ 4 (497-506) peptide and describes immunogens which is rendered substantially free of carrier protein by the addition of universal T-helper epitopes.
  • the immunogens of WO 95/26365 are in the general formula: (A) n -(Th) m -(B) 0 -(Stan) p where A is an amino acid; Th is a T-helper epitope; B is an amino acid; and Stan is the Stanworth decapeptide.
  • WO 98/24808 describes oligopeptides derived from C ⁇ 3, which interact with the high or low affinity receptors of IgE. These oligopeptides are expressed as fusion proteins together with the expression partner Glutathione-S-transferase (GST).
  • GST Glutathione-S-transferase
  • WO 97/31948 describes IgE peptide immunogens conjugated to a protein carrier, the carriers described are TT, DT, KLH and PPD.
  • GnRH Gonadotrophin hormone releasing hormone
  • DT Diptheria toxoid
  • WO99/27944 contemplates conjugating the peptide to a conventional carrier protein.
  • the present invention overcomes these problems associated with peptide carriers of the prior art.
  • the problems are surprisingly overcome by the use of Protein
  • Protein D is an IgD-binding protein from Haemophilus influenzae and has been patented by Forsgren WO 91/18926 (EP 0 594 610 Bl). The patent describes the cloning, expression, and protein sequence of Protein D. It suggests its uses could be methods of detecting IgD using its selective affinity for IgD (as for Protein A and
  • EP 0 594 610 Bl further describes the Protein D gene, and suggests that it may be fused to other genes and expressed to form fusion proteins.
  • the present invention provides the use of protein D as a carrier for peptide immunogens. Said immunogens have the advantage of inducing high levels of anti- peptide immune responses whilst inducing a moderate humoral response against itself.
  • protein D as a carrier has the additional advantage of inducing low anti-carrier IgE responses in comparison to known carriers, such as KLH.
  • protein D for example, protein D l/3 rd (comprising the N-terminus 100-110 amino acids of protein D (GB 9717953.5)). Accordingly there is provided by the present invention protein D or fragment thereof and a peptide immunogen for use in the manufacture of an immunogen.
  • the immunogens of the present invention therefore, comprise a peptide conjugated to protein D as a carrier.
  • the peptides are preferably chemically conjugated.
  • the term "peptide" within the meaning of the present invention is a polymer of amino acids containing a sequence of amino acids less than 100 amino acids in length preferably between 2-50 amino acids in length, and more preferably between 2- 45 amino acids in length more preferably 2-30, and most preferably between 5-25 amino acids in length.
  • the peptides of the present invention may be either lipidated or non-lipidated. It will be appreciated however that peptide residues may be joined in any convenient chemical way, for example via an ester linkage and the like, but typically will be joined by a peptide bond.
  • the peptides may be full-length sequences of naturally occurring peptides like peptide hormones.
  • the peptides may be derived from naturally occurring self proteins or proteins derived from pathogens.
  • non-natural amino acids such as D-amino acids.
  • the peptide also preferably include the naturally occurring peptide of between 39 and 43 amino acids in length.
  • the preferred sequences correspond to the naturally occurring forms i.e. those sequence corresponding to amino acids 1 to 39, 1 to 40, 1 to 41 1 to 42, 1 to 43 of the amyloid precursor protein and which are disclosed in Hardy et al ., TINS 20, 155 - 158, 1997.
  • Immunogenic fragments of A ⁇ 43 can also be coupled to Protein D in accordance with the invention.
  • Preferred fragments include peptides incorporating residues selected from the group: A ⁇ 1-5; 1-12, 13 -28; 17 -28 and 33-42. Such constructs find utility in the treatment or prevention of Alzheimer's disease.
  • Peptide mimotopes of A ⁇ 43 or fragments thereof are also contemplated.
  • GnRH has the sequence EHWSYGLRPG (SEQ ID NO. 1).
  • tandem repeats, of GnRH such as E-H- W-S-Y-G-L-R-P-G-S-C-S-E-H-W-S-Y-G-L-R-P-G-NH2 (SEQ ID NO. 2) may be conjugated to a protein D molecule, typically through a central cysteine.
  • Tandem dimers are also contemplated by the present invention such as E-H-W-S-Y-G-L-R-P-G-Q-H-W-S- Y-G-L-R-P-G-S-C-E-H-W-S-Y-G-L-R-P-G-Q-H-W-S-Y-G-L-R-P-G-NH2 (SEQ ID NO. 3).
  • These advantageously are conjugated to Protein D via the central Cysteine.
  • Such constructs find utility, inter alia, in the treatment of Prostate cancers.
  • GnRH may be linked to Protein D via a peptide spacer.
  • Typical spacers includes Cys(Pro) 4 Ser Ser.
  • the peptides are derived from mammalian IgE, such that the immunogens are capable of stimulating a non- anaphy lactic anti-IgE antibody response in a vaccinated individual.
  • mammalian IgE such that the immunogens are capable of stimulating a non- anaphy lactic anti-IgE antibody response in a vaccinated individual.
  • the stanworth decapeptides as described in EP 0 477 231 B 1 for example KTKGSGFFVF (SEQ ID NO. 4) or mimotopes thereof.
  • Other IgE peptides are described in WO 97/31948 and WO 96/14333, and are useful in immunogens of the present invention.
  • Preferred epitopes from IgE are for example, those which are surfaced exposed and have the following sequences:
  • P4 GHTFEDSTKKCADSNPRGV 19 C2/C3 (SEQ ID NO. 8) PI and P2 are located within the C2 domain of IgE, within a region which has not previously been reported as being useful for the active vaccination based immunoprophylaxis of allergy.
  • the present invention includes the native peptides themselves, and any mimotope thereof.
  • mimotope is defined as a peptide sequence which is sufficiently similar to the native peptide (sequentially or structurally), which is capable of being recognised by antibodies which recognise the native peptide; or are capable of raising antibodies, when coupled to a suitable carrier, which antibodies are capable of recognising the native peptide.
  • Peptide mimotopes may be designed for a particular purpose by addition, deletion or substitution of elected amino acids.
  • the peptides may be modified for the purposes of ease of conjugation to a protein carrier. For example, it may be desirable for some chemical conjugation methods to include a terminal cysteine.
  • peptides conjugated to a protein carrier may include a hydrophobic terminus distal from the conjugated terminus of the peptide, such that the free unconjugated end of the peptide remains associated with the surface of the carrier protein. Thereby presenting the peptide in a conformation which most closely resembles that of the peptide as found in the context of the whole native molecule.
  • modified peptides include:
  • modified peptides useful as anti allergy medicament include:
  • the immunogens of the present invention may alternatively contain a peptide capable of eliciting an immune response against a human pathogen.
  • peptides may be derived from the following group: an HIV antigen (such as tat, nef, gpl20 or gpl60), a human herpes virus antigen (such as gD or derivatives thereof or Immediate Early protein such as ICP27 from HSV1 or HSV2), a cytomegalovirus antigen (such as gB or derivatives thereof), a Rotavirus antigen, an Epstein Barr virus antigen (such as gp350), Varicella Zoster Virus antigens (such as gpl, II and IE63), or from a hepatitis virus such as hepatitis B virus (for example Hepatitis B Surface antigen), hepatitis A virus antigen, hepatitis C virus and hepatitis E virus, or from other viral pathogens, such as paramyxo
  • Neisseria spp including N. gonorrhea and N. meningitidis (for transferrin-binding proteins, lactoferrin binding proteins, PilC, adhesins); Streptococcus spp, including S. pneumoniae (streptolysin or choline-binding proteins), S. pyogenes (for example M proteins, C5 A protease), S. agalactiae, S. mutans; Haemophilus spp, including H.
  • Neisseria spp including N. gonorrhea and N. meningitidis (for transferrin-binding proteins, lactoferrin binding proteins, PilC, adhesins); Streptococcus spp, including S. pneumoniae (streptolysin or choline-binding proteins), S. pyogenes (for example M proteins, C5 A protease), S. agalactiae, S.
  • influenzae type B non typeable H. influenzae (for example OMP26, high molecular weight adhesins, P5, P6), H. ducreyi; Moraxella spp, including M catarrhalis, also known as Branhamella catarrhalis (for example high and low molecular weight adhesins and invasins,); Bordetella spp, including B. pertussis (for example pertactin, pertussis toxin, filamenteous hemagglutinin, adenylate cyclase, fimbriae), B. parapertussis and B. bronchiseptica; Mycobacterium spp., including M.
  • B. pertussis for example pertactin, pertussis toxin, filamenteous hemagglutinin, adenylate cyclase, fimbriae
  • Mycobacterium spp.
  • tuberculosis for example ESAT6, Antigen 85A, -B or -C
  • M. bovis for example ESAT6, Antigen 85A, -B or -C
  • M. leprae for example, M. avium
  • M. paratuberculosis for example, M. smegmatis
  • Legionella spp including L. pneumophila
  • Escherichia spp including enterotoxic E. coli (for example colonization factors, heat- labile toxin or derivatives thereof, heat-stable toxin or derivatives thereof), enterohemorragic E. coli, enteropathogenic E. coli (for example shiga toxin-like toxin or derivatives thereof); Vibrio spp, including V. cholera (for example cholera toxin or derivatives thereof); Shigella spp, including S. sonnei, S. dysenteriae, S. flexnerii; Yersinia spp, including Y. enterocolitica (for example a Yop protein) , Y. pestis, Y.
  • enterotoxic E. coli for example colonization factors, heat- labile toxin or derivatives thereof, heat-stable toxin or derivatives thereof
  • enterohemorragic E. coli enteropathogenic E. coli (for example shiga toxin-like toxin or derivatives
  • Campylobacter spp including C. jejuni (for example toxins, adhesins and invasins) and C. coli; Salmonella spp, including S. typhi, S. paratyphi, S. choleraesuis, S. enter itidis; Lister ia spp., including L. monocytogenes; Helicobacter spp, including H. pylori (for example urease, catalase, vacuolating toxin);
  • Pseudomonas spp including P. aerugino ' sa; Staphylococcus spp., including S. aureus, S. epidermidis; Enterococcus spp., including E. faecalis, E. faecium; Clostridium spp., including C. tetani (for example tetanus toxin and derivative thereof), C. botulinum (for example botulinum toxin and derivative thereof), C. difficile (for example clostridium toxins A or B and derivatives thereof); Bacillus spp., including B.
  • anthracis for example botulinum toxin and derivatives thereof
  • Corynebacterium spp. including C. diphtheriae (for example diphtheria toxin and derivatives thereof); Borrelia spp., including B. burgdorferi (for example OspA, OspC, DbpA, DbpB), B. garinii (for example OspA, OspC, DbpA, DbpB), B. afzelii (for example OspA, OspC, DbpA, DbpB B. andersonii (for example OspA, OspC, DbpA, DbpB), B.
  • C. trachomatis for example MOMP, heparin-binding proteins
  • C. pneumoniae for example MOMP, heparin-binding proteins,
  • Leptospira spp. including L. interrogans
  • Plasmodium spp. including P. falciparum
  • Toxoplasma spp. including T gondii (for example SAG2, SAG3, Tg34); Entamoeba spp., including E. histolytica
  • Babesia spp. including B. microti
  • Trypanosoma spp. including T. cruzi
  • Giardia spp. including G. lamblia
  • Leshmania spp. including L. major
  • Pneumocystis spp. including P. carinii
  • Trichomonas spp. including T. vaginalis
  • Schisostoma spp. including S. mansoni, or derived from yeast such as Candida spp., including C. albicans
  • Cryptococcus spp. including C. neoformans.
  • Vaccines of the present invention further comprise peptides derived from parasites that cause Malaria.
  • Peptides may be derived from the circumsporozoite (CS) protein of P, falciparum, or TRAP antigens (WO 90/01496).
  • CS circumsporozoite
  • TRAP antigens WO 90/01496
  • Other plasmodia antigens that are likely candidates to be donors of peptides for a multistage Malaria vaccine are P.
  • the immunogens of the present invention may also contain an anti-tumour peptide and be useful for the immunotherapeutic treatment cancers.
  • the adjuvant formulation finds utility with tumour rejection antigens such as those for prostrate, breast, colorectal, lung, pancreatic, renal or melanoma cancers.
  • Exemplary antigens include MAGE 1 and MAGE 3 or other MAGE antigens for the treatment of melanoma or MAGE expressing tumour (particularly the peptide EVDPIGHLY ( Seq
  • an immunogen comprising a peptide derived from a tumour rejection antigen coupled to protein D.
  • Peptides used in the present invention can be readily synthesised by solid phase procedures well known in the art. Suitable syntheses may be performed by utilising "T-boc” or "F-moc” procedures. Cyclic peptides can be synthesised by the solid phase procedure employing the well-known "F-moc” procedure and polyamide resin in the fully automated apparatus. Alternatively, those skilled in the art will know the necessary laboratory procedures to perform the process manually. Techniques and procedures for solid phase synthesis are described in 'Solid Phase Peptide Synthesis: A Practical Approach' by E. Atherton and R.C. Sheppard, published by IRL at Oxford University Press (1989).
  • the peptides may be produced by recombinant methods, including expressing nucleic acid molecules encoding the mimotopes in a bacterial or mammalian cell line, followed by purification of the expressed mimotope.
  • Techniques for recombinant expression of peptides and proteins are known in the art, and are described in Maniatis, T., Fritsch, E.F. and Sambrook et al., Molecular cloning, a laboratory manual, 2 nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989).
  • the ratio of peptide to protein D -carrier is typically in the order of between 1 : 1 to 1 :20, but preferably between 2 to 10 peptides per Protein D carrier, typically 3 to 8.
  • Different peptides may be coupled to the same carrier molecule to provide a hybrid immunogen.
  • the covalent coupling of the peptide to Protein D or fragment thereof can be carried out in a manner well known in the art.
  • a carbodiimide, glutaraldehyde or (N-[ ⁇ - maleimidobutyryloxy] succinimide ester utilising common commercially available heterobifunctional linkers such as CDAP and SPDP (using manufacturers instructions).
  • the immunogen can easily be isolated and purified by means of a dialysis method, a gel filtration method, a fractionation method etc.
  • the vaccines of the present invention are preferably used for the prophylaxis or therapy of allergy.
  • Such vaccines may comprise allergen specific peptide (for example from Der pi) or allergen non-specific peptides (for example the stanworth decapeptide or PI to P4 or mimotope thereof).
  • Vaccines of the present invention comprise an immunogen as described above and an adjuvant. Suitable adjuvants are well known in the art (Vaccine Design - The Subunit and Adjuvant Approach, 1995, Pharmaceutical Biotechnology, Volume 6, Eds. Powell, M.F., and Newman, M.J., Plenum Press, New York and London, ISBN 0-306-44867-X).
  • Preferred adjuvants for use with immunogens of the present invention include: aluminium or calcium salts (eg hydroxide or phosphate), oil in water emulsions (WO 95/17210, EP 0 399 843), or particulate carriers such as liposomes (WO 96/33739).
  • Immunologically active saponin fractions e.g. Quil A
  • QS21 an HPLC purified fraction derivative of Quil A
  • 3 De-O-acylated monophosphoryl lipid A is a well known adjuvant manufactured by Ribi Immunochem, Montana. It can be prepared by the methods taught in GB 2122204B.
  • a preferred form of 3 De-O-acylated monophosphoryl lipid A is in the form of an emulsion having a small particle size less than 0.2 ⁇ m in diameter (EP 0 689 454 B 1 ).
  • Adjuvants also include, but are not limited to, muramyl dipeptide and saponins such as Quil A, bacterial lipopolysaccharides such as 3D-MPL (3-O-deacylated monophosphoryl lipid A), or TDM.
  • the protein can be encapsulated within microparticles such as liposomes, or in non-particulate suspensions of polyoxyethylene ether (UK Patent Application No. 9807805.8).
  • Particularly preferred adjuvants are combinations of 3D-MPL and QS21 (EP 0 671 948 Bl), oil in water emulsions comprising 3D-MPL and QS21 (WO 95/17210, PCT/EP98/05714), 3D-MPL formulated with other carriers (EP 0 689 454 Bl), or QS21 formulated in cholesterol containing liposomes (WO 96/33739), or immunostimulatory oligonucleotides (WO 96/02555).
  • Suitable pharmaceutically acceptable excipients include water, phosphate buffered saline, isotonic buffer solutions.
  • the vaccines of the present invention may also be administered via the oral route.
  • the pharmaceutically acceptible excipient may also include alkaline buffers, or digestible capsules.
  • the formulations of the present invention maybe used for both prophylactic and therapeutic purposes. Accordingly, the present invention provides for a method of treating a mammal susceptible to or suffering from an infectious disease or chronic disorders such as cancer, allergy, or autoimmune disease.
  • compositions (depending on the peptide used) of the present invention are useful in the treatment and prevention of Alzheimer's, Allergy and Prostate Carcinoma.
  • a vaccine as herein described for use in medicine. Vaccine preparation is generally described in New Trends and Developments in Vaccines, edited by Voller et al., University Park Press, Baltimore, Maryland, U.S.A. 1978.
  • an immunogen or vaccine as herein described for use in medicine.
  • the vaccine preparation of the present invention may be used to protect or treat a mammal susceptible to, or suffering from allergies, by means of administering said vaccine via systemic or mucosal route.
  • administrations may include injection via the intramuscular, intraperitoneal, intradermal or subcutaneous routes; or via mucosal administration to the oral/alimentary, respiratory, genitourinary tracts.
  • the amount of protein in each vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccinees. Such amount will vary depending upon which specific immunogen is employed and how it is presented.
  • each dose will comprise 1-1000 ⁇ g of protein, preferably 1-500 ⁇ g, preferably 1-100 ⁇ g, of which 1 to 50 ⁇ g is the most preferable range.
  • An optimal amount for a particular vaccine can be ascertained by standard studies involving observation of appropriate immune responses in subjects. Following an initial vaccination, subjects may receive one or several booster immunisations adequately spaced.
  • Vaccine preparation is generally described in New Trends and Developments in Vaccines, edited by Voller et al., University Park Press, Baltimore, Maryland, U.S.A. 1978. Conjugation of proteins to macromolecules is disclosed by Likhite, U.S.
  • Protein D may be produced and purified using methods described in EP 0 594 610 Bl.
  • IgE Peptides for conjugation to the carrier
  • the peptides which have been used in these examples are peptides derived from human or rodent IgE C ⁇ 4. These immunogens have previously been shown to be capable of stimulating antibody responses which are capable of inhibiting histamine release from IgE bearing mast cells (EP 0477 231 Bl).
  • the peptides were stored dry and frozen at -20°C until used in the conjugation procedure.
  • Protein D may be conjugated directly to peptides to form antigens of the present invention by using a maleimide-succinimide cross-linker.
  • This chemistry allows controlled NH 2 activation of carrier residues by fixing a succinimide group.
  • Maleimide groups is a cystein-binding site. Therefore, for the purpose of the following examples, the IgE peptides to be conjugated required the addition of an N- terminal cystein (PEP2 and PEP3).
  • the coupling reagent is a selective heterobifunctional cross-linker, one end of the compound activating amino group of the protein carrier by an succinimidyl ester and the other end coupling sulhydryl group of the peptide by a maleimido group.
  • the reactional scheme is as the following :
  • the protein D is dissolved in a phosphate buffer saline at a pH 7.2 at a concentration of 2.5 mg/ml.
  • the coupling reagent N-[ ⁇ -maleimidobutyryloxy] succinimide ester - GMBS
  • DMSO dimethyl sulfoxide
  • GMBS 1.025 mg of GMBS is used for 1 mg of Protein D.
  • the reactional solution is incubated 1 hour at room temperature.
  • the by-products are removed by a desalting step onto a sephacryl 200HR permeation gel.
  • the eluant used is a phosphate buffer saline Tween 80 0.1 % pH 6.8.
  • the activated protein is collected and pooled.
  • the peptide is dissolved at 4 mg/ml in 0.1 M acetic acid to avoid di-sulfure bond formation.
  • a molar ratio of between 2 to 4 peptides per 1 activated Protein D is used for the coupling.
  • the peptide solution is slowly added to the protein and the mixture is incubated 1 h at
  • a quenching step is performed by addition of cystein (0.1 mg cystein per mg of activated PD dissolved at 4 mg/ml in acetic acid 0.1 M), 30 minutes at 25°C and a pH of 6.5. Two dialysis against NaCl 150 mM Tween 80 0.1 % are performed to remove the excess of cystein or peptide.
  • the last step is a sterile filtration on a 0.22 ⁇ m membrane.
  • the final product is a clear filtrable solution conserved at 4°C.
  • the final ratio of peptide/PD in the construct is determined by Amino acid analysis.
  • the activated PD is prepared as in example 1.4.
  • the activated Protein D is passed through a 0.22 ⁇ m membrane.
  • Peptide is also diluted in acetic acid 0.1 M and filtrated on a 0.22 ⁇ m membrane.
  • the two solutions are then slowly mixed to obtain a final molar peptide/Protein D ratio of 8.
  • the pH is adjusted to 6.5. After 1 h at 25°C, the solution is conserved at 4°C.
  • the figure 1 shows the evolution of protein concentration according to increasing initial peptide / Protein D ratio.
  • the majority of the conjugate is soluble and is, therefore, found in the supernatant.
  • a ratio 8/1 nearly all of protein content is precipitated.
  • the conjugate is precipitated and the excess of unconjugated peptide is found in the supernatant.
  • oil in water emulsion adjuvant formulations used in the subsequent examples were each made comprising the following oil in water emulsion component: 5% Squalene, 5% ⁇ -tocopherol, 2.0% polyoxyethylene sorbitan monooleate (TWEEN 80).
  • the emulsion was prepared as follows as a 2 fold concentrate. All examples used in the immunological experiments are diluted with the addition of extra components and diluents to give either a lx concentration (equating to a squalene:QS21 ratio (w/w) of
  • the TWEEN 80 is dissolved in phosphate buffered saline (PBS) to give a 2% solution in the PBS.
  • PBS phosphate buffered saline
  • 5ml of DL alpha tocopherol and 5ml of squalene are vortexed to mix thoroughly.
  • 95ml of PBS/TWEEN solution is added to the oil and mixed thoroughly.
  • the resulting emulsion is then passed through a syringe needle and finally microfluidised by using an Ml 1 OS Micro fluidics machine.
  • the resulting oil droplets have a size of approximately 145-180 nm (expressed as z av. measured by PCS) and is termed SB62.
  • the other adjuvant/vaccine components (QS21, 3D-MPL or antigen) are added to the emulsion in simple admixture.
  • Alum adjuvant may be bought from Superfos.
  • the anti-peptide and anti-carrier immune responses were investigated using an ELISA technique outlined below.
  • a monoclonal anti-human decapeptide antibody (Dec7B) was used as reference in deca/dodeca ELISA. This makes it possible to calculate anti-decapeptide antibody responses either in ⁇ g specific antibody per 1ml or serum ( ⁇ g/ml), or as a midpoint litre. Anit-Protein D and anti-KLH responses are calculated as midpoint titers.
  • Peptide-KLH conjugates were made according to manufacturer's instructions.
  • mice Three groups of 10 Balb/C mice were vaccinated with the PEP2 human decapeptide - protein D conjugates produced according to the methods described in example 1.
  • the vaccines comprised 25 ⁇ g of antigen in lOO ⁇ l volumes, and were given s.c on three occasions on days 0, 21, and 42 (Nl, V2, and V3).
  • the ratio's of Mid-point titres of anti-peptide:anti-carrier IgG responses were measured (results see table 4). This ratio is a measure of the relative levels of anti- peptide or anti-carrier immune response which were generated by vaccination. A high ratio (>1) indicates that strong anti-peptide response is being generated with respect to that induced against the carrier.
  • IgE peptide conjugated to Protein D was capable of inducing strong anti-peptide immune responses when adjuvanted with alum.
  • the protein D conjugates induced greater anti-peptide responses compared with those induced by the KLH conjugates, whilst inducing less anti-carrier responses that that induced by the KLH conjugates.
  • ProteinD-IgE peptide conjugates with a ratio of (8:1 peptide: carrier) performed better that those with a ratio of 2: 1, in both the magnitude of the anti peptide IgG response and the relative proportions of peptide:carrier IgG responses.
  • the conjugates with a ratio of 2:1 (peptide:carrier) nevertheless performed better than KLH in these respects.
  • the vaccines comprised 25 ⁇ g of antigen formulated with an oil in water emulsion adjuvant (produced as described in example 1.8) in 1 OO ⁇ l volumes, and were given s.c on three occasions on days 0, 21, and 42 (VI, V2, and V3).
  • the ratio's of Mid-point titres of anti-peptide: anti-carrier IgG responses were measured (results see table 7). This ratio is a measure of the relative levels of anti- peptide or anti-carrier immune response which were generated by vaccination. A high ratio (>1) indicates that strong anti-peptide response is being generated with respect to that induced against the carrier.
  • IgE peptide conjugated to Protein D was capable of inducing strong anti-peptide immune responses when adjuvanted with oil in water emulsions.
  • the protein D conjugates induced greater anti-peptide responses compared with those induced by the KLH conjugates, whilst inducing less anti-carrier responses that that induced by the
  • PEP3 The dodeca-peptide used in this example (PEP3) is the rodent equivalent of the human IgE peptide used previously (PEP2). This example, therefore, examines the use of ProteinD-IgE peptide conjugates in a self-antigen model.
  • the conjugates were formulated as described in examples 1.4 and 1.5 above.
  • the vaccines comprised 25 ⁇ g of antigen formulated with an alum adjuvant in lOO ⁇ l volumes, and were given s.c. on three occasions on days 0, 21, and 42 (VI, V2, and V3).
  • ProteinD-dodeca peptide conjugates adsorbed onto alum are capable of inducing anti- peptide immune responses in the context of a self-antigen model. In this respect the high ratio conjugate performed better than the low ratio construct.
  • PEP3 The dodeca-peptide used in this example (PEP3) is the rodent equivalent of the human IgE peptide used previously (PEP2). This example, therefore, examines the use of ProteinD-IgE peptide conjugates in a self-antigen model.
  • the conjugates were formulated as described in examples 1.4 and 1.5 above.
  • the vaccines comprised 25 ⁇ g of antigen formulated with an oil in water emulsion adjuvant (produced according to example 1.8) in lOO ⁇ l volumes, and were given s.c. on three occasions on days 0, 21, and 42 (VI, V2, and V3).
  • ProteinD-dodeca peptide conjugates adsorbed onto alum are capable of inducing anti- peptide immune responses in the context of a self-antigen model. In this respect the high ratio conjugate performed better than the low ratio construct.
  • mice were vaccinated in order to further investigate the benefits of the Protein D carrier over the use of KLH. Briefly, groups of 10 mice were vaccinated on three occasions with on days 0, 21 and 42 with vaccines conjugated, formulated and inoculated according to Table 1.
  • Groups 1 -2 and 3-4 were produced as described in examples 1.5 and 1.4.
  • Group 5-6 was produced as described in example 1.9
  • the vaccine comprised 250 ⁇ g of antigen in 1ml and were given on three occasions on day 0, 28 and 56. Bleeds were taken on day 28, 42 and 70.
  • the anti-peptide responses were investigated using an ELISA described in section 1.8
  • Protein D was evaluated as a carrier for successive boost using KLH as a positive control.
  • KLH-decapeptide EKLH00B
  • IM intramuscular
  • SC subcutaneous
  • Bleedings were made at day 14 after injection and on day 150 before injection.
  • the anti-carrier and anti-peptide antibody responses were measured in ELISA. There is a striking difference between the antibody responses made from KLH-deca and
  • Protein D-deca (PD-deca) constructs (figure 7).
  • KLH-deca induced a very strong anti-carrier response, whereas the anti-decapeptide response was quite low.
  • the opposite can be said for PD-deca.
  • IgE was therefore measured ( Figure 8). As for total Ig, the IgE anti-KLH levels were much higher than IgE anti-PD.
  • Protein D allows a higher anti-decapeptide antibody response than KLH, while keeping a moderate level of anti-carrier antibodies, both total Ig and IgE.
  • KLH has a molecular weight of +/- 390 000 against the 40 000 for PD.
  • 25 ⁇ g of construct (the injected dose) will contain around 0.5 ⁇ g or 2 ⁇ g of decapeptide if it is KLH-deca or PD-deca respectively.
  • PD can be said to be a "better" carrier than KLH, since more peptide can be administered with the same amount of construct.
  • the IM route for injection appears preferable. It induces higher Ig levels and somewhat lower levels of IgE.
  • a mimotope of PI was synthesised CLEDGQVMDVDLL (PI 5) (SEQ ID No. 10) which was conjugated to Protein D using techniques described above in Example 1.
  • ELISA plates are coated with human chimaeric IgE at 1 ⁇ g/ml in pH 9.6 carbonate/bicarbonate coating buffer for 1 hour at 37°C or overnight at 4°C.
  • Nonspecific binding sites are blocked with PBS/0.05% Tween-20 containing 5% w/v Marvel milk powder for 1 hour at 37°C.
  • Serial dilutions of mouse serum in PBS/0.05% Tween-20/1 % w/v BSA/4% New Born Calf serum are then added for 1 hour at 37°C.
  • Polyclonal serum binding is detected with goat anti-mouse IgG-Biotin (1/2000) followed by Streptavidin-HRP (1/1000).
  • Conjugated antibody is detected with TMB substrate at 450nm.
  • a standard curve of PTmAbOOl 1 is included on each plate so that the anti-IgE reactivity in serum samples can be calculated in ⁇ g/ml.
  • HB A human basophils
  • HBH/HSA human serum albumin
  • lOO ⁇ l cell suspension are added to wells of a V-bottom 96-well plate containing lOO ⁇ l diluted test sample or monoclonal antibody. Each test sample is tested at a range of dilutions with 6 wells for each dilution. Well contents are mixed briefly using a plate shaker, before incubation at 37°C for 30 minutes.
  • the degree of inhibition of histamine release can be calculated using the formula: % inhibition
  • Example 10 Immunisation of mice with PI 5 conjugates (P15 -PD) induces production of anti-human IgE antibodies.
  • the Protein D conjugate comprising the mimotope PI 5 (25 ⁇ g protein/dose), were administered into groups of 10 BalbC mice, adjuvanted with and oil in water emulsion containing QS21 and 3D-MPL described in WO 95/17210 . Boosting was performed on day 21 and on day 42 and sera can be harvested on day 42 and 56. The immune response anti-peptide and anti-plate bound IgE was followed using methods described above.
  • results The results for anti peptide and anti-IgE responses measured at day 14 post third vaccination are shown in the table below.
  • the anaphylactogenicity can be evaluated by the measuring of the histamine released induced by the antibodies to be tested as described below:
  • Anti-IgE induced in mice after immunisation with conjugate are capable of blocking IgE mediated histamine release induced by allergen triggering of basophilfrom an allergic patient. Histamine release can be measured in basophil samples triggered with various concentrations of allergen in presence or absence of several dilutions of complete sera or IgG purified from conjugate immunised mice. Blocking activity of anti-P15 antibodies in the antiserum was evaluated by the measuring of the inhibition of the histamine release induced by the allergen. Histamine release and inhibition was measured as described in example 3. As PI 5 is a mimotope of PI, PTmAbOOl 1 was used as a control as it is known to bind to the same epitope (PI). The results are shown in the table below.
  • Example 1 GnRH peptide EHWSYGLRPG (SEQ ID NO. 1) was synthesised with an N terminal linker CPPPPSS (SEQ ID NO. 18) and conjugated to protein D via a succinimide - maleimide cross-linker. Four formulations were evaluated to assess anti GnRH antibodies and Testerone levels in male rabbits.
  • Non typeable Haemopbilus influenzae are encapsulated gram-negative bacteria which are common human upper respiratory tracts inhabitants frequently associate with otitis media, bronchitis and other infections of mucosal surfaces.
  • Colonisation of the respiratory tract mucosa is the first and next step the presumed pathogenesis of NTHi infection.
  • NTHi strains contain bacterial fimbriae defined as non flagellar proteinaceous surface appendages.
  • H.influenzae 1128 was isolated, cloned and sequenced by L. Bakaletz.
  • One constitutive peptide of the fimbrin: LB1 was identified.
  • the amino acid sequence shows one important constitutive peptide (19 aa): LB1 with presumed B-cell determinants.
  • LB 1 is derived from the N-terminal half of the mature P5-f ⁇ mbrin protein.
  • a vaccine including two NTHi antigens (LB1 and PD) known to play a role in pathogenesis of otitis media could be interesting.
  • a possible approach was to synthesize LB1 conjugates using protein D as carrier.
  • the coupling reagent is a selective heterobifunctional crosslinker, one end of the compound activating amino group of the protein carrier by a succinimidyl ester and the other end coupling sulfhydryl group of the peptide by a maleimido group.
  • the reactional scheme is described previously.
  • the maleimide group is most selective for sulfhydryl groups when the pH of the reaction mixture is kept between 6.5 and 7.5. At pH 7, the rate of reaction of maleimides with sulfhydryl is 1000-fold faster than with amines. A stable thioether linkage between the maleimide group and the reacted sulfhydryl is formed which cannot be cleaved under physiological conditions.
  • LBlgrl peptide was synthesized by Eurogentec. The following amino acid sequence was coupled though an additional C-terminal cysteine via maleimide to protein D (PD).
  • PD-LB1 conjugates were prepared as follows:
  • the activated PD was purified by gel filtration (Toyopearl HW-40, XK 16/40, elution with 100 mM phosphate buffer pH 6.8).
  • LB1 peptide (2.5 mg/ml) was dissolved in acetic acid 0.1M and added to the activated protein solution.
  • the conjugation reaction is allowed to continue for one hour at 25°C and followed by a quenching step with cysteine during 30 minutes at the same temperature. During the conjugation step, a slight precipitation was observed.
  • the conjugate solution was dialysed against NaCl 150 mM or 100 mM phosphate buffer pH 6.8 with or without Tween 80 0.1%.
  • the PD-LB1 conjugate solution is then filtered through a sterile 0.22 ⁇ m membrane.
  • Presence of LBl peptide on the carrier was confirmed by SDS-PAGE followed by coomassie blue staining or transfer on nitrocellulose followed by immunodectection with anti-LBl antibodies.

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Abstract

L'invention concerne des immunogènes peptidiques liés à un support, ledit support étant dérivé de la protéine D de Haemophilius Influenzae ou de ses fragments.
PCT/EP2000/001457 1999-02-25 2000-02-22 Immunogenes comprenant un peptide et support derive de haemophilius influenzae WO2000050077A1 (fr)

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JP2000600687A JP2002537354A (ja) 1999-02-25 2000-02-22 ペプチド、及びＨ．ｉｎｆｌｕｅｎｚａｅのプロテインＤ由来の担体を含む免疫原
CA002363118A CA2363118A1 (fr) 1999-02-25 2000-02-22 Immunogenes comprenant un peptide et support derive de haemophilius influenzae
EP00909235A EP1156825A2 (fr) 1999-02-25 2000-02-22 Immunogenes comprenant un peptide et support derive de haemophilius influenzae
AU31589/00A AU3158900A (en) 1999-02-25 2000-02-22 Immunogens comprising a peptide and a carrier derived from h.influenzae protein

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EP1701968B1 (fr) * 2003-12-17 2015-06-03 Wyeth LLC Conjugues porteurs de peptides immunogenes et procedes de production associes
WO2018017020A1 (fr) * 2016-07-21 2018-01-25 Vac4All Pte. Ltd. Protéines de biofusion en tant que vaccins antipaludiques

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US7749507B2 (en) 2002-11-12 2010-07-06 Statens Serum Institut Malaria vaccine
US7732568B2 (en) 2003-01-14 2010-06-08 Affiris Forschungs-Und Entwicklungs Gmbh Methods for preventing and treating Alzheimer's disease
AU2011200785B2 (en) * 2003-12-17 2012-12-06 Janssen Alzheimer Immunotherapy AB immunogenic peptide carrier conjugates and methods of producing same
US9095536B2 (en) 2003-12-17 2015-08-04 Janssen Sciences Ireland Uc Aβ immunogenic peptide carrier conjugates and methods of producing same
NO340432B1 (no) * 2003-12-17 2017-04-24 Wyeth Llc Fremgangsmåte for å konjugere et peptid-immunogen, peptid-immunogen-protein/polypeptidbærerkonjugat og immunogen blanding
EP2336147A3 (fr) * 2003-12-17 2011-07-27 Janssen Alzheimer Immunotherapy Conjugués transporteur de peptides bêta immunogènes et leurs procédés de production
US9125847B2 (en) 2003-12-17 2015-09-08 Janssen Sciences Ireland Uc A-β immunogenic peptide carrier conjugates and methods of producing same
US8227403B2 (en) 2003-12-17 2012-07-24 Wyeth Llc A-β immunogenic peptide carrier conjugates and methods of producing same
EP1699810A4 (fr) * 2003-12-17 2009-10-21 Elan Pharm Inc Conjugues porteurs de peptide immunogene a beta les techniques de production de ces conjugues
EP1699810A2 (fr) * 2003-12-17 2006-09-13 Elan Pharmaceuticals, Inc. Conjugues porteurs de peptide immunogene a beta les techniques de production de ces conjugues
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EP1156825A2 (fr) 2001-11-28
AU3158900A (en) 2000-09-14
AR022758A1 (es) 2002-09-04
WO2000050077A9 (fr) 2001-08-16
CA2363118A1 (fr) 2000-08-31
JP2002537354A (ja) 2002-11-05

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