EP1155037A1 - Epitopes ou mimotopes derives du domaine c-epsilon-2 de l'immoglobuline e, leurs antagonistes et leurs applications therapeutiques - Google Patents

Epitopes ou mimotopes derives du domaine c-epsilon-2 de l'immoglobuline e, leurs antagonistes et leurs applications therapeutiques

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Publication number
EP1155037A1
EP1155037A1 EP00905073A EP00905073A EP1155037A1 EP 1155037 A1 EP1155037 A1 EP 1155037A1 EP 00905073 A EP00905073 A EP 00905073A EP 00905073 A EP00905073 A EP 00905073A EP 1155037 A1 EP1155037 A1 EP 1155037A1
Authority
EP
European Patent Office
Prior art keywords
ige
peptide
mimotope
ptmabool
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Withdrawn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
EP00905073A
Other languages
German (de)
English (en)
Inventor
Michael Peptide Therapeutics Ltd. Dyson
Martin Corporate Intellectual Property Friede
Judith Peptide Therapeutics Ltd. Greenwood
Ellen Peptide Therapeutics Ltd. Hewitt
Alan Peptide Therapeutics Ltd. Lamont
Sean Peptide Therapeutics Ltd. Mason
Roger Peptide Therapeutics Ltd. Randall
William Gordon Peptide Therapeutics Ltd. Turnell
Marcelle Paulette Van Mechelen
Carlota Vinals Y De Bassols
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.)
GlaxoSmithKline Biologicals SA
Sanofi Pasteur Holding Ltd
Original Assignee
SmithKline Beecham Biologicals SA
Peptide Therapeutics 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
Priority claimed from GBGB9904405.9A external-priority patent/GB9904405D0/en
Priority claimed from GBGB9907151.6A external-priority patent/GB9907151D0/en
Priority claimed from GBGB9910538.9A external-priority patent/GB9910538D0/en
Priority claimed from GBGB9910537.1A external-priority patent/GB9910537D0/en
Priority claimed from GBGB9918594.4A external-priority patent/GB9918594D0/en
Priority claimed from GBGB9918603.3A external-priority patent/GB9918603D0/en
Priority claimed from GBGB9921047.8A external-priority patent/GB9921047D0/en
Priority claimed from GBGB9921046.0A external-priority patent/GB9921046D0/en
Priority claimed from GBGB9925619.0A external-priority patent/GB9925619D0/en
Priority claimed from GBGB9927698.2A external-priority patent/GB9927698D0/en
Application filed by SmithKline Beecham Biologicals SA, Peptide Therapeutics Ltd filed Critical SmithKline Beecham Biologicals SA
Publication of EP1155037A1 publication Critical patent/EP1155037A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • 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
    • A61P37/00—Drugs for immunological or allergic disorders
    • 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
    • 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
    • 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

Definitions

  • the present invention relates to the provision of novel medicaments for the treatment, prevention or amelioration of allergic disease.
  • the novel medicaments are isolated peptides incorporating epitopes or mimotopes of surface exposed regions of the C ⁇ 2 domain of IgE.
  • the inventors have found that these novel regions may be the target for both passive and active immunoprophylaxis or immunotherapy.
  • the invention further relates to methods for production of the medicaments, pharmaceutical compositions containing them and their use in medicine.
  • ligands, especially monoclonal antibodies which are capable of binding the surface exposed IgE regions of the present invention, and their use in medicine as passive immunotherapy or in immunoprophylaxis.
  • Non-peptidic mimotopes are also an embodiment of the present invention.
  • allergen specific IgE In an allergic response, the symptoms commonly associated with allergy are brought about by the release of allergic mediators, such as histamine, from immune cells into the surrounding tissues and vascular structures. Histamine is normally stored in mast cells and basophils, until such time as the release is triggered by interaction with allergen specific IgE.
  • IgE The role of IgE in the mediation of allergic responses, such as asthma, food allergies, atopic dermatitis, type-I hypersensitivity and allergic rhinitis, 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 the Fc ⁇ RI receptor (the high affinity IgE receptor) on basophils and mast cells.
  • IgE like all immunoglobulins, comprises two heavy and two light chains.
  • the ⁇ heavy chain consists of five domains: one variable domain (VH) and four constant domains (C ⁇ l to C ⁇ 4).
  • the molecular weight of IgE is about 190,000 Da, the heavy chain being approximately 550 amino acids in length.
  • the structure of IgE is discussed in Padlan and Davis (Mol. Immunol., 23, 1063-75, 1986) and Helm et al., (2IgE model structure deposited 2/10/90 with PDB (Protein Data Bank, Research Collabarotory for Structural Bioinformatics; http ⁇ pdb-browsers.ebi.ac.uk)).
  • the second domain, C ⁇ 2 approximately comprises amino acids 226-328 of IgE (Flanagan J.G. and Rabbitts, T.H., 1982, EMBO J., 1, 655-660; Kenten et al., 1982, Proc.Natl.Acad.Sci., USA, 79, 6661-6665), but may encompass additional amino acids.
  • IgE Longagan J.G. and Rabbitts, T.H., 1982, EMBO J., 1, 655-660; Kenten et al., 1982, Proc.Natl.Acad.Sci., USA, 79, 6661-6665
  • anti-IgE antibodies generated during active vaccination are capable of triggering histamine release per se, by the cross-linking of neighbouring IgE-receptor complexes in the absence of allergen. This phenomenon is termed anaphylactogenicity. Indeed many commercially available anti-IgE monoclonal antibodies which are normally used for IgE detection assays, are anaphylactogenic, and consequently useless and potentially dangerous if administered to a patient.
  • an antibody is anaphylactogenic depends on the location of the target epitope on the IgE molecule. However, based on the present state of knowledge in this area, and despite enormous scientific interest and endeavour, there is little or no predictability of what characteristics any antibody or epitope may have and whether or not it might have a positive or negative clinical effect on a patient.
  • the passively administered, or vaccine induced, antibodies must bind in a region of IgE which is capable of interfering with the histamine triggering pathway, without being anaphylactic per se.
  • the present invention achieves all of these aims and provides medicaments which are capable of raising non-anaphylactic antibodies which inhibit histamine release. These medicaments can form the basis of an active vaccine or be used to raise appropriate antibodies for passive immunotherapy, or may be passively administered themselves for a therapeutic effect.
  • WO 97/31948 describes an example of this type of work, and further describes IgE peptides from the C ⁇ 3 and C ⁇ 4 domains conjugated to carrier molecules for active vaccination purposes. These immunogens may be used in vaccination studies and are said to be capable of generating antibodies which subsequently inhibit histamine release in vivo .
  • a monoclonal antibody (BSW17) was described which was said to be capable of binding to IgE peptides contained within the C ⁇ 3 domain which are useful for active vaccination purposes.
  • EP 0 477 231 Bl describes immunogens derived from the C ⁇ 4 domain of IgE
  • the present invention identifies novel surface exposed epitopes of the C ⁇ 2 domain of IgE, which may be used as the target of active or passive immunoprophylaxis or therapy of allergic disease states.
  • the present invention provides peptides incorporating the isolated epitopes per se, and further provides mimotopes of these newly identified epitopes, which may be used per se in the treatment of allergy, or may be used in immunogens in active vaccination immunoprophylaxis or therapy.
  • the isolated epitopes or mimotopes of the present invention are preferably used in immunogens for active vaccination protocols to induce auto anti-IgE antibodies, which themselves limit, reduce, or eliminate allergic responses or symptoms in vaccinated subjects.
  • the mimotopes or the immunogens of the present invention may be passively administered to a patient to limit, reduce, or eliminate allergic responses or symptoms in vaccinated subjects.
  • the peptides, which incorporate the isolated epitopes of the present invention are immunogenic, when suitably presented (e.g. on a carrier), and are capable of inducing auto anti-IgE antibodies which are non-anaphylactogenic, and function in ameliorating allergic responses in vivo.
  • the epitopes or mimotopes of the present invention are preferably exclusively derived from C ⁇ 2 domain, in that they are not derived from any other domain, i.e. they are not found within the C ⁇ l, C ⁇ 3 or C ⁇ 4 domains. In particular, as a preferred embodiment they are derived from the domain encoded by Ser222-Ala329 of human IgE.
  • epitopes of the C ⁇ 2 domain which have been found to be particularly suitable for use in the mimotopes or immunogens of the present invention are those which have been found by the present inventors to be surface exposed.
  • the surface exposure of a region of IgE may be determined from its modelled structure. (Padlan and Davies, Mol. Immunol., 23, 1063-75, 1986; Helm et al., 2IgE model structure deposited 2/10/90 with PDB (Protein Data Bank, Research Collabarotory for Structural Bioinformatics)).
  • PDB Protein Data Bank, Research Collabarotory for Structural Bioinformatics
  • the present inventors have designed a method for providing other suitable epitopes, those being epitopes having highly accessible regions calculated over a sliding window of five residues.
  • the inventors have found that preferred regions of the C ⁇ 2 domain have an accessible surface calculated over a sliding window of 5 residues using the Molecular Simulations Software (MSI) of greater than 50 A 2 , and preferably greater than 8 ⁇ A 2 .
  • MSI Molecular Simulations Software
  • Mimotopes which have the same characteristics as these epitopes, and immunogens comprising such mimotopes which generate an immune response which cross-react with the IgE C ⁇ 2 epitope in the context of the IgE molecule, also form part of the present invention.
  • the present invention therefore, includes the isolated peptides encompassing the native IgE epitopes themselves, and any mimotope thereof.
  • mimotope is defined as an entity which is sufficiently similar to the native IgE epitope so as to be capable of being recognised by antibodies which recognise the native IgE epitope; (Gheysen, H.M., et al., 1986, Synthetic peptides as antigens. Wiley, Chichester, Ciba foundation symposium 119, pl30-149; Gheysen, H.M., 1986, Molecular Immunology, 23,7, 709-715); or are capable of raising antibodies, when coupled to a suitable carrier, which antibodies cross-react with the native IgE epitope.
  • the mimotopes of the present invention may be peptidic or non-peptidic.
  • a peptidic mimotope of the surface exposed IgE epitopes identified above may have a sequence which differs from the native epitope but may also be of exactly the same sequence as the native epitope.
  • Such a molecule is described as a mimotope of the epitope, because although the two molecules share the same sequence, the mimotope will not be presented in the context of the whole C ⁇ 2 domain structure, and as such the mimotope may take a slightly different conformation to that of the native IgE epitope.
  • a mimotope of PI when in the tertiary structure of IgE, lie adjacent to other regions that may be distant in the primary sequence of IgE.
  • a mimotope of PI may be continuous or discontinuous, in that it comprises or mimics segments of PI and segments made up of these distant amino acid residues.
  • Preferred surface exposed regions which may be used in the present invention contain regions which are associated with a loop structure.
  • the peptides or mimotopes of the present invention may comprise, therefore, a loop with N or C terminal extensions which may be the natural amino acid residues from neighbouring ⁇ -sheets.
  • this PI contains the C-D loop
  • P2 contains the D-E loop
  • P3 contains the E-F loop
  • P4 contains the F-G loop
  • P5 contains the A-B loop
  • P6 contains the B-C loop of the C ⁇ 2 domain of IgE. Accordingly, mimotopes of these loops form an aspect of the present invention.
  • Particularly preferred medicaments are based on the epitope PI, and mimotopes thereof.
  • Peptides incorporating this epitope, and mimotopes thereof, when coupled to a carrier, are potent in inducing anti-IgE immune responses which are capable of inhibiting histamine release from human basophils. Moreover, these immune responses are non-anaphylactogenic.
  • Mimotopes of PI are described primarily as any entity which when formulated into an immunogen, is capable of inducing an immune response, which response is capable of recognising PI when in the context of C ⁇ 2 domain of IgE. PI corresponds to the C-D loop of the C ⁇ 2 domain.
  • the C-D loop structure of immunoglobulin folds corresponds to the linking chain between the end of the C beta- strand and the beginning of the D beta-strand (Introduction to protein Structure, page 304, 2 nd Edition, Branden and Tooze, Garland Publishing, New York, ISBN 0 8153 2305-0), corresponding approximately to amino acid residue numbers Trp268-Ser280 of the IgE molecule. Accordingly, mimotopes of the C-D loop of IgE C ⁇ 2, and ligands that are capable of binding to the C-D loop of IgE C ⁇ 2, form a preferred aspect of the present invention.
  • Peptide mimotopes of the above-identified IgE epitopes may be designed for a particular purpose by addition, deletion or substitution of elected amino acids.
  • the peptides of the present invention may be modified for the purposes of ease of conjugation to a protein carrier.
  • the peptides may be altered to have an N-terminal cysteine and a C-terminal hydrophobic amidated tail.
  • the addition or substitution of a D-stereoisomer form of one or more of the amino acids may be performed to create a beneficial derivative, for example to enhance stability of the peptide.
  • modified peptides could be a wholly or partly non-peptide mimotope wherein the constituent residues are not necessarily confined to the 20 naturally occurring amino acids.
  • these may be cyclised by techniques known in the art to constrain the peptide into a conformation that closely resembles its shape when the peptide sequence is in the context of the whole IgE molecule.
  • Examples of preferred cyclised peptides which contain a pair of cysteine residues to allow the formation of a disulphide bridge are PT1079 (SEQ ID NO. 14), PT1079GS (SEQ ID NO.15), PT1078 (SEQ ID NO.16), and P15q (SEQ ID NO. 11).
  • mimotopes or immunogens of the present invention may be longer than the isolated epitopes, and may comprise the sequences disclosed herein. Accordingly, the mimotopes of the present invention may consist of addition of N and/or C terminal extensions of a number of other natural residues at one or both ends.
  • the peptide mimotopes may also be retro sequences of the natural IgE sequences, in that the sequence orientation is reversed; or alternatively the sequences may be entirely or at least in part comprised of D-stereo isomer amino acids (inverso sequences). Also, the peptide sequences may be retro-inverso in character, in that the sequence orientation is reversed and the amino acids are of the D-stereoisomer form.
  • retro or retro-inverso peptides have the advantage of being non-self, and as such may overcome problems of self-tolerance in the immune system (for example P 15r - see below).
  • peptide mimotopes may be identified using antibodies which are capable themselves of binding to the IgE epitopes of the present invention using techniques such as phage display technology (EP 0 552 267 Bl). This technique, generates a large number of peptide sequences which mimic the structure of the native peptides and are, therefore, capable of binding to anti-native peptide antibodies, but may not necessarily themselves share significant sequence homology to the native IgE peptide.
  • This approach may have significant advantages by allowing the possibility of identifying a peptide with enhanced immunogenic properties (such as higher affinity binding characteristics to the IgE receptors or anti-IgE antibodies, or being capable of inducing polyclonal immune response which binds to IgE with higher affinity), or may overcome any potential self-antigen tolerance problems which may be associated with the use of the native peptide sequence. Additionally this technique allows the identification of a recognition pattern for each native-peptide in terms of its shared chemical properties amongst recognised mimotope sequences.
  • Preferred examples of modified peptide mimotopes and examples of bacteriophage derived mimotopes include:
  • amino acid residues of PI, P2, P3, P4, P4, P5, P6 or P7 can each individually be replaced by the amino acid that most closely resembles that amino acid.
  • A may be substituted by V, L or I, as described in the following table.
  • Such ligands are capable of being used in passive prophylaxis or therapy, by administration of the ligands into a patient, for the amelioration of allergic disease.
  • useful ligands include monoclonal or polyclonal antibodies.
  • antibodies induced in one animal may be purified and passively administered to another animal for the prophylaxis or therapy of allergy.
  • the peptides of the present invention may also be used for the generation of monoclonal antibody hybridomas (using known techniques e.g.
  • ligands capable of binding to the surface exposed epitopes of the C ⁇ 2 domain of IgE are antibodies (or Fab fragments). Such antibodies may be used in passive immunoprophylaxis or immunotherapy, or may be used themselves in the identification of IgE peptide mimotopes.
  • antibody herein is used to refer to a molecule having a useful antigen binding specificity.
  • Preferred ligands are monoclonal antibodies.
  • Particularly preferred ligands are ligands of PI, and are preferably monoclonal antibodies.
  • PTmAbOOl 1 is the reference name for a mouse IgGl-type monoclonal antibody deposited as Budapest Treaty patent deposit at ECACC (European Collection of Cell Cultures, Vaccine Research and Production Laboratory, Public Health Laboratory Service, Centre for Applied Microbiology Research, Porton Down, Salisbury, Wiltshire, SP4 OJG, UK) on 8 ,h March 1999 under Accession No. 99030805.
  • PTmAbOOl 1 recognises the C-D loop of C ⁇ 2, and is itself capable of recognising IgE when bound to its high affinity receptor on human basophils without causing degranulation, moreover it is able to block the passive sensitisation of non-allergic basophils by preventing the binding of IgE to Fc ⁇ RI ⁇ , and inhibits LolPl -triggered histamine release in allergic basophils.
  • Another monoclonal antibody which recognises the C-D loop of C ⁇ 2 is PTmAb0005 (available from Sigma Chemicals Catalogue number 16510, clone number GE-1). The present invention provides this monoclonal antibody in a pharmaceutical composition.
  • PI ligands of PI
  • a monoclonal antibody which is capable of recognising PI, bound bacteriophages expressing the following sequences:
  • peptide mimotopes of the C-D loop of C ⁇ 2 IgE have been identified by bacteriophage panning with PTmAbOOl 1 and PTmAb0005. Examples of such mimotopes include:
  • mimotopes of Ig ⁇ C ⁇ 2 that are capable of binding to PTmAb0005 or PTmAbOOl 1, and immunogens comprising these mimotopes form an important aspect of the present invention.
  • Vaccines comprising mimotopes that are capable of binding to PTmAb0005 or PTmAbOOl 1 are useful in the treatment of allergy.
  • PI mimotopes Without limiting the broader definition of PI mimotopes, from these and other phage-sequences, a core pattern has been identified for a sub-set of a PI -like peptide.
  • This pattern is a sub-set of PI mimotopes, and describes its mimotopes in terms of the chemical properties of the amino acids in each position which are desirable for recognition to that particular anti-P monoclonal antibody: y h x d h h a n a n x y wherein: v v Can be cyclised.
  • Hydrophobic cys;pro;gly;ala;val;ile;leu;trp;met;phe).
  • mimotopes of PI may be described by the general core feature y h x d h h a n a n x y described above.
  • the peptide PI or mimotope thereof may be optionally flanked by other amino acids at either end to aid conjugation or for any other purpose.
  • a particularly preferred mimotopes of PI is PI 5s (SEQ ID NO. 17), whose Q,
  • PI 5s in which the non-essential residues were replaced by similar amino acids (as outlined above) would be: Q, X travel M, D, X tract X 2 , X 3 wherein X, is selected from V, I, L, M, F or A; X 2 is selected from D or E; and X 3 is selected from L, I, V, M, A or F.
  • PTmAb0005 and PTmAbOOl 1 are used in the identification of novel mimotopes of IgE, for subsequent use in allergy therapy.
  • this ligand does not form a composition of the present invention, however, pharmaceutical compositions comprising PTmAb0005, and its use in the identification of PI mimotopes, form two important aspects of the present invention.
  • Mimotopes of P2, P3, P4 and P5 also form an important aspect of the present invention.
  • PI 6 and PI 7 are mimotopes of P2 and P3 respectively.
  • These peptides when suitably presented on carriers, are both capable of inducing strong anti-IgE antibody responses which are non anaphylactogenic.
  • the peptides incorporating the above identified epitopes or peptidic or non-peptidic mimotopes of the present invention will be of a small size, such that they mimic a region selected from the whole C ⁇ 2 domain. It is envisaged that peptidic mimotopes, therefore, should be less than 100 amino acids in length, preferably shorter than 75 amino acids, more preferably less than 50 amino acids, and most preferable within the range of 4 to 25 amino acids long. Specific examples of preferred peptide mimotopes are PT1079 and P15q, which are respectively 21 and 13 amino acids long. Non-peptidic mimotopes are envisaged to be of a similar size, in terms of molecular volume, to their peptidic counterparts.
  • the putative mimotope can be assayed to ascertain the immunogenicity of the construct, in that antisera raised by the putative mimotope cross-react with the native IgE molecule, and are also functional in blocking allergic mediator release from allergic effector cells.
  • the specificity of these responses can be confirmed by competition experiments by blocking the activity of the antiserum with the mimotope itself or the native IgE, and/or specific monoclonal antibodies that are known to bind the surface exposed epitope within C ⁇ 2 of IgE.
  • monoclonal antibodies for use in the competition assays include, for example, PTmAb0005 and PTmAbOOl 1, which would confirm the status of the putative mimotope as a mimotope of the C-D loop of the C ⁇ 2 domain of IgE.
  • At least one peptide as hereinbefore described, incorporating an IgE epitope or mimotope is linked to carrier molecules to form immunogens for vaccination protocols.
  • the carrier molecules are not related to the native IgE molecule.
  • the peptides or mimotopes may be linked via chemical covalent conjugation or by expression of genetically engineered fusion partners, optionally via a linker sequence.
  • the covalent coupling of the peptide to the immunogenic carrier 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 types of carriers used in the immunogens of the present invention will be readily known to the man skilled in the art.
  • the function of the carrier is to provide cytokine help in order to help induce an immune response against the IgE peptide.
  • a non-exhaustive list of carriers which may be used in the present invention include: Keyhole limpet Haemocyanin (KLH), serum albumins such as bovine serum albumin (BSA), inactivated bacterial toxins such as tetanus or diptheria toxins (TT and DT), or recombinant fragments thereof (for example, Domain 1 of Fragment C of TT, or the translocation domain of DT), or the purified protein derivative of tuberculin (PPD).
  • KLH Keyhole limpet Haemocyanin
  • BSA bovine serum albumin
  • TT and DT inactivated bacterial toxins
  • TT and DT inactivated bacterial toxins
  • recombinant fragments thereof for example, Domain 1 of Fragment C of TT, or the translocation domain of DT
  • PPD purified protein derivative of tubercul
  • the mimotopes or epitopes may be directly conjugated to liposome carriers, which may additionally comprise immunogens capable of providing T-cell help.
  • liposome carriers which may additionally comprise immunogens capable of providing T-cell help.
  • the ratio of peptides to carrier is in the order of 1 : 1 to 20: 1, and preferably each carrier should carry between 3-15 peptides.
  • a preferred carrier is Protein D from Haemophilus influenzae (EP 0 594 610 Bl).
  • Protein D is an IgD-binding protein from Haemophilus influenzae and has been patented by Forsgren (WO 91/18926, granted EP 0 594 610 Bl).
  • fragments of protein D for example Protein D l/3 rd (comprising the N-terminal 100-110 amino acids of protein D (GB 9717953.5)).
  • IgE peptides of the present invention is in the context of a recombinant fusion molecule.
  • EP 0 421 635 B describes the use of chimeric hepadnavirus core antigen particles to present foreign peptide sequences in a virus-like particle.
  • immunogens of the present invention may comprise IgE peptides presented in chimeric particles consisting of hepatitis B core antigen.
  • the recombinant fusion proteins may comprise the mimotopes of the present invention and a carrier protein, such as NS1 of the influenza virus.
  • the nucleic acid which encodes said immunogen also forms an aspect of the present invention.
  • 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, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989).
  • the immunogens of the present invention may comprise the peptides as previously described, including mimotopes, or may be immunologically cross- reactive derivatives or fragments thereof. Also forming part of the present invention are portions of nucleic acid which encode the immunogens of the present invention or peptides, mimotopes or derivatives thereof.
  • the immunogens of the present invention may comprise more than one type of epitope, i.e. PI and P2, in the same immunogen, or the mimotope itself may comprise more than one type of epitope.
  • the present invention therefore, provides the use of novel peptides encompassing the epitopes or mimotopes of the present invention (as defined above), in the manufacture of pharmaceutical compositions for the prophylaxis or therapy of allergies.
  • Immunogens comprising the mimotopes or peptides of the present invention, and carrier molecules are also provided for use in vaccines for the immunoprophylaxis or therapy of allergies. Accordingly, the mimotopes, peptides or immunogens of the present invention are provided for use in medicine, and in the medical treatment or prophylaxis of allergic disease. Accordingly, there is provided a method of treatment of allergy comprising the administration to a patient suffering from or susceptible to allergy, of a vaccine or medicament of the present invention.
  • Vaccines of the present invention may advantageously also include an adjuvant.
  • Suitable adjuvants for vaccines of the present invention comprise those adjuvants that are capable of enhancing the antibody responses against the IgE peptide immunogen.
  • 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 (for example hydroxide or phosphate salts).
  • Other adjuvants include saponin adjuvants such as QS21 (US 5,057,540) and 3D-MPL (GB 2220 211).
  • the vaccines of the present invention will be generally administered for both priming and boosting doses. It is expected that the boosting doses will be adequately spaced, or preferably given yearly or at such times where the levels of circulating antibody fall below a desired level.
  • Boosting doses may consist of the peptide in the absence of the original carrier molecule. Such booster constructs may comprise an alternative carrier or may be in the absence of any carrier.
  • 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.
  • a preferred route of administration is via the transdermal route, for example by skin patches.
  • 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.
  • Pharmaceutical compositions comprising the ligands, described above, also form an aspect of the present invention. Also provided are the use of the ligands in medicine, and in the manufacture of medicaments for the treatment of allergies.
  • aspects of the present invention may also be used in diagnostic assays.
  • panels of ligands which recognise the different peptides of the present invention may be used in assaying titres of anti-IgE present in serum taken from patients.
  • the peptides may themselves be used to type the circulating anti- IgE. It may in some circumstances be appropriate to assay circulating anti-IgE levels, for example in atopic patients, and as such the peptides and poly/mono-clonal antibodies of the present invention may be used in the diagnosis of atopy.
  • the peptides may be used to affinity remove circulating anti-IgE from the blood of patients before re-infusion of the blood back into the patient.
  • Also forming part of the present invention is a method of identifying peptide immunogens for the immunoprophylaxis or therapy of allergy comprising using a computer model of the structure of IgE, and identifying those peptides of the IgE which are surface exposed. These regions may then be formulated into immunogens and used in medicine. Accordingly, the use of PTmAb0005 and PTmAbOOl 1 in the identification of peptides for use in allergy immunoprophylaxis or therapy forms part of the present invention.
  • Vaccine preparation is generally described in New Trends and Developments in Vaccines, edited by Voller et al., University Park Press, Baltimore, Maryland,
  • FIG 1 IgE amino acid surface exposure using the Padlan and Davies 1986 model.
  • FIG 2 Chemistry Scheme 1 , solid phase peptide synthesis.
  • FIG 3 Chemistry Scheme 2 and Scheme 3, Modified carrier preparation.
  • A Anti-plate bound IgE reactivity of serum from Balb C mice immunised with 25 ⁇ g BSA-IgE C67-8 (conjugated using PTL chemistry) or 3 ⁇ g HepB core-IgE C67-8 construct.
  • B Anti-receptor bound IgE reactivity of serum from Balb C mice immunised with 25 ⁇ g BSA-IgE C67-8 (conjugated using PTL chemistry) or 3 ⁇ g HepB core-IgE C67-8 construct.
  • FIG 8 Anti-receptor bound IgE reactivity of serum from Balb C mice immunised with 25 ⁇ g BSA- 1079 (conjugated using PTL chemistry) or 3 ⁇ g HepB core- 1079 construct.
  • FIG 8 Competition assay with soluble IgE and PT1079 peptide.
  • FIG 10 Competition assay with soluble IgE and PT1078 peptide.
  • Sera from BSA- 1078 immunised mice were pre-incubated with soluble IgE (lO ⁇ g/ml) or PT1078 peptide (25 ⁇ M) or the irrelevant peptide PT326 (25 ⁇ M) and added to IgE-coated ELISA plates.
  • FIG. 11 PT1079gs Anti-IgE Data.
  • A Anti-plate bound IgE reactivity of serum from Balb C mice immunised with 3 ⁇ g HBC-1079gs
  • B Anti-receptor bound IgE reactivity of serum from Balb C mice immunised with 3 ⁇ g HBC-1079gs.
  • FIG 12 Competition assay with soluble IgE and PT1079 peptide.
  • FIG 13 Inhibitory Activity of Mouse BSA-C67-8 induced Antisera.
  • FIG 15 Inhibitory Activity of Mouse Antisera induced by HBC-C67-8, HBC-1078, HBC-1079 and HBC-1079gs.
  • Cells from a LolPl -sensitive donor were treated with mouse serum (HBC wild type (wt) and HBC-IgEC67-8 antisera diluted 1/50; HBC- 1079 and HBC-1079gs antisera diluted 1/1250) and then triggered to release histamine with LolPl .
  • FIG 16 shows the concentration dependent binding of antibody PTmAb0005 and PTmAbOOl 1 to IgE.
  • FIG 17, shows the concentration dependent inhibition of IgE binding to an
  • FIG 18 shows the concentration dependent inhibition of IgE binding to clipped ectodomain of Fc ⁇ RI ⁇ -bound directly to plastic plates, by antibody PTmAb0005, compared to control.
  • FIG 19 shows IgE binding to Fc ⁇ RII (CD23) by antibody PTmAb0005 (GE-1) and PTmAbOOl 1.
  • FIG 20 shows the concentration-dependent blocking of histamine release from allergic human blood basophils with antibody PTmAb0005 and PTmAbOOl 1 compared to control.
  • FIG 21 inhibition of LolPl triggered histamine release in allergic human basophils by both PTmAb0005 and PTmAbOO 11.
  • FIG 22 PTmAbOOl 1 binding to different IgE;
  • A PTmAbOOl 1 Binding to Chimaeric IgE;
  • B PTmAbOOl 1 Binding to Myeloma IgE;
  • C PTmAbOOl 1 Binding to Antigen Orientated IgE;
  • D PTmAbOOl 1 Binding to Heat Denatured IgE.
  • FIG 23 Inhibition of IgE Binding to Fc ⁇ RI ⁇ by PTmAbOOl 1.
  • FIG 24 Binding of PTmAbOOl 1 to Receptor Bound IgE.
  • FIG 25, (A) The effect of PTmAbOOl Ion IgE binding to Fc ⁇ RII on RPMI 8866 cells.
  • RPMI 8866 cells (lxlOVml) were incubated for an hour on ice with chimaeric IgE (1 ⁇ g/ml) and anti-IgE mAb (10 to 0 ⁇ g/ml).
  • the IgE and anti-IgE were pre-incubated for an hour at room temperature prior to addition to the cells.
  • Bound IgE was detected with FITC-goat anti-human IgE. The results show the mean channel fluorescence
  • FIG 26 The effects of PTmAbOOl 1 on IgE binding to Fc ⁇ RII on primary human B- cells.
  • Peripheral blood mononuclear cells (lxl0 ⁇ /ml) were incubated for an hour on ice with chimaeric IgE (1 ⁇ g/ml) and anti-IgE mAb (10 to 0 ⁇ g/ml; open) or equivalent concentrations of isotype matched control mAb (solid).
  • the IgE and anti- IgE were pre-incubated for an hour at room temperature prior to addition to the cells.
  • Bound IgE was detected with FITC-goat anti-human IgE and the primary B-cells were elucidated with PE-conjugated anti-CD 19.
  • the results show the mean channel fluorescence (MCF) of duplicate samples as determined by flow cytometric analysis of 5,000 live gated events.
  • FIG 27, Effects of PTmAbOOl 1 on IgE secretion from primary human B-cells.
  • Peripheral blood mononuclear cells (2xl ⁇ Vwell) were cultured in medium supplemented with IL-4 (lOng/ml) and anti-CD40 antibody (1 ⁇ g/ml).
  • PTmAbOOl 1 or an isotype matched control mAb were added (1 ⁇ g/ml) for 14 days and then cell supernatant harvested and analysed for total IgE content by ELISA. The results are expressed as a percentage of the amount of IgE secreted in the absence of any antibody.
  • FIG 28 Anaphylactogenicity of anti-human IgE monoclonal antibodies in allergic (A) and non-allergic (B) human basophils.
  • FIG 29 Anaphylactogenicity of anti-human IgE antibodies in sensitised (A) and non- sensitised (B) human lung mast cells. Sensitised or non-sensitised crude human lung mast cell suspensions were treated with antibodies for 45 min. at 37°C. Tryptase release in supematants was determined by colorimetric assay. Data are means of duplicate determinations from a single representative experiment.
  • FIG 30, Anaphylactogenicity of anti-human IgE antibodies in RBL J41 cells through human Fc ⁇ RI (A) and mouse Fc ⁇ RI (B). RBL J41 cells were sensitised either with chimeric human IgE or with mouse IgE and treated with antibodies for 30 min. at 37°C.
  • FIG 31 Inhibition of allergen-triggered histamine release in human basophils by PTmAbOOl 1.
  • PBMC were incubated with PTmAbOOl 1 either directly (allergic assay (A)) or together with IgE (blocking assay (B)) for 30 min. at 37°C.
  • Cells were subsequently triggered with antigen for 30 min. at 37°C and histamine release determined by specific EIA.
  • Data are mean ⁇ s.e.m. from 3 separate experiments from different donors.
  • FIG 32 Inhibition of passive cutaneous anaphylaxis in Monkey skin by PTmAbOOl 1 and PTmAb0005.
  • Monoclonal antibody Dec7B (stan worth decapeptide) was used as a control.
  • the present invention is illustrated by but not limited to the following examples.
  • the surface exposed epitopes of the C ⁇ 2 domain of IgE were identified using the modelled structure of human IgE described by Padlan and Davies (Mol. Immunol., 23, 1063-75, 1986). Peptides were identified which were both continuous and solvent exposed. This was achieved by using Molecular Simulations software (MSI) to calculate the accessibility for each IgE amino acid, the accessible surface was averaged over a sliding window of five residues, and thereby identifying regions of the IgE peptides which had an average over that 5-mer of greater than 8 ⁇ A 2 . The results of the test are shown in figure 1.
  • MSI Molecular Simulations software
  • peptides or mimotopes thereof, were synthesised and either conjugated to carrier proteins, or put into Hepatitis Core antigen constructs to form recombinant peptide expressing virus-like particles.
  • Protein D may be conjugated directly to IgE 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.
  • the Maleimide group is a cysteine-binding site. Therefore, for the purpose of the following examples, the IgE peptides to be conjugated require the addition of an N- terminal cysteine.
  • 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 reaction 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 reaction 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 peptides (as identified in table 1 or derivatives or mimotopes thereof) is dissolved at 4 mg/ml in 0.1 M acetic acid to avoid di-sulfide bond formation.
  • a molar ratio of between 2 to 20 peptides per 1 molecule of 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 25°C.
  • the pH is kept at a value of 6.6 during the coupling phase.
  • a quenching step is performed by addition of cysteine (0.1 mg cysteine 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 dialyses against NaCl 150 mM Tween 80 0.1 % are performed to remove the excess of cysteine or peptide.
  • the last step is sterilising 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 may be determined by amino acid analysis.
  • the peptides of the present invention may be conjugated to other carriers including BSA.
  • a mimotope of PI was synthesised CLEDGQVMDVDLL (PI 5, SEQ ID NO. 8) which was conjugated to both Protein D and BSA using techniques described above.
  • 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 Bom 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.
  • ELISA plates are coated with recombinant human Fc ⁇ Rl ⁇ at 0.5 ⁇ g/ml in pH 9.6 carbonate/T-ticarbonate coating buffer for 1 hour at 37°C or overnight at 4°C.
  • Non-specific binding sites are blocked with PBS/0.05% Tween-20 containing 5% w/v Marvel low fat milk powder for 1 hour at 37°C.
  • Human IgE at 1 ⁇ g/ml is then added for 1 hour at 37°C
  • Serial dilutions of mouse serum in PBS/0.05% Tween-20/1% w/v BSA/4% New Bom 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.
  • PTmAbOOl 1 for IgE binding mixtures of serum and PTmAbOOl 1-biotin are added to IgE-coated ELISA plates. PTmAbOOl 1 binding is detected with Streptavidin-HRP (1/1000).
  • HBA human basophils
  • the peripheral blood mononuclear cell (PBMC) layer at the interface is collected and the pellet is discarded.
  • the cells are washed once in HBH/HSA, counted, and re-suspended in HBH/HSA at a cell density of 2.0 x 10 6 per ml.
  • 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 with shaking at 120 rpm.
  • the degree of inhibition of histamine release can be calculated using the formula: % inhibition
  • the conjugates comprising the mimotope PI 5 (25 ⁇ g protein/dose), described in 1.4, 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 be 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 in Example 1.
  • the anaphylactogenicity can be evaluated by the measuring of the histamine released induced by the antibodies to be tested as described below: ⁇ Erythrocytes are removed from peripheral blood on glucose dextran gradient
  • Anti-IgE induced in mice after immunisation with conjugate are capable of blocking IgE mediated histamine release induced by allergen triggering of basoph ⁇ l from 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 table 3.
  • mice were immunised with the conjugates using the same formulation and schedule as that described in example 2.
  • mice were bled after the last immunisation and tested for anti-IgE reactivity in the IgE plate bound ELISA.
  • Example 6 Production of mimotopes of PI, and immunogenicity/functional activity thereof
  • Mimotopes of PI were derived either by phage display techniques or by rational design by molecular modelling of the C-D loop of C ⁇ 2 domain of IgE.
  • the following peptides were synthesised and formulated into both BSA-peptide conjugates and also into HepB core antigen recombinant constructs.
  • the peptides/protein carrier constructs were produced as follows. Acylhydrazine peptide derivatives were prepared on the solid phase as shown in scheme 1 ( Figure 2). These peptide derivatives can be readily prepared using the well-known 'Fmoc' procedure, utilising either polyamide or polyethyleneglycol-polystyrene (PEG-PS) supports in a fully automated apparatus, through techniques well known in the art [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)]. Acid mediated cleavage afforded the linear, deprotected, modified peptide.
  • PEG-PS polyethyleneglycol-polystyrene
  • the peptides thus synthesised can then be conjugated to protein carriers (in this case Bovine Serum Albumin, BSA) using the following technique:
  • protein carriers in this case Bovine Serum Albumin, BSA
  • BSA succinimido active ester
  • Hepatitis Core antigen recombinant constructs were also prepared, using molecular biology techniques described in EP 0 421 635 B. In these HBC experiments PT1079 was modified to remove the terminal lysine.
  • the expression of the PI -mimotope peptides was confirmed by BIAcore experiments with PTmAb0005 and PTmAbOOl 1.
  • the immunogenicity results were generated using doses of only 3 ⁇ g/dose of HBC.
  • Peptide immunogens are described in part 1, which after administered to a mammal in the form of a vaccine, induce immune responses which (a) recognise IgE, and (b) are capable of inhibiting histamine release in vitro.
  • Part 2 describes ligands that are capable of binding to the epitopes or mimotopes of the present invention, and describes their function.
  • Two monoclonal antibodies have been identified, PTmAb0005 and PTmAbOOl 1, which recognise the c-d loop of C ⁇ 2 of IgE. Mimotopes of this peptide have been shown in part 1 to be immunogenic and functional in active vaccination. This section describes the characterisation of these monoclonal antibodies and provides evidence of their utility in passive vaccination.
  • the target epitope of the antibodies was identified using phage panning techniques, namely sequence alignment of multiple bacteriophage targets, and subsequently refined and confirmed by domain mapping and site directed mutagenesis.
  • the functional activity of the antibodies has been confirmed not only in vitro by assaying for anti-IgE recognition and inhibition of allergic mediator release; but also in vivo in monkey Passive Cutaneous Anaphylaxis (PCA) studies.
  • Phage display libraries were used to map the binding sites of the monoclonal antibodies using three different phage libraries, displaying either the XCX 15 , XCX 10 or XAX 10 peptide sequence (where X is any amino acid) at the N-terminus of the phage gVIIIp.
  • Tables 6 and 7 show the results of selecting for peptide ligands with the anti human IgE monoclonal antibodies PTmAb0005 and respectively. Amino acid pattern similarities between the peptides and human IgE revealed a strong homology match with the c-d loop in the C ⁇ 2 domain of IgE.
  • IgEC67 the peptide derived from phage panning experiments and which had the highest affinity to PTmAb0005 was also epitope mapped. This was performed by introducing random mutations by PCR mutagenesis and sub-cloning into the Fuse 5 vector for minor filamentous phage protein glllp display. The IgEC67 mutants were ranked in order of binding to PTmAb0005, as shown in Table 8. These, and other results, confirmed the importance of the amino acids within IgEC67 which aligned with the C ⁇ 2 epitope. For example the L8P, D10G, LI 1M, E12G and L13R mutants all reduced binding to the anti-IgE PTmAb0005 (Data not shown). Mutations in other sites had little effect on the affinity to PTmAb0005.
  • Sub-libraries were made of several phage sequences including the highest affinity PTmAb0005 ligand IgEC67 and IgE C67-8. Affinity matured sequences for C67 and C67-8 are shown in tables 8 and 9 respectively. Included in the tables are ranking orders and also
  • IgEC67-8 capable of inducing an anti-human IgE response in mice when the peptide expressing phage was used as an immunogen.
  • Fragments encompassing various domain(s) of human IgE Fc were cloned using cDNA derived from the hybridoma line JW8/5/13, which expresses a chimaeric human IgE (Neuberger, MS et al (1985) Nature 314 268-270; Bruggemann, M et al (1987) J Exp Med 166 1351-61).
  • the IgE Fc fragments were amplified using appropriate primer pairs and JW8/5/3 cDNA as template.
  • the c ⁇ 2-4 fragment encodes amino acids (aa) S225-K547.
  • the c ⁇ 3-4 fragment encodes aa G335-547.
  • the c ⁇ 3-4L fragment (domains 3 - 4 plus the linker sequence that joins c ⁇ 2 to c ⁇ 3) encodes aa E322-K547.
  • the c ⁇ 2-3 fragment encodes aa S225-G436.
  • the c ⁇ 2 fragment encodes aa S225-S324. All constructs contain a COOH terminal hexahistidine tail for detection and purification purposes.
  • These fragments were cloned into a eukaryotic expression vector in frame with a CD33 derived leader encoding sequence to direct secretion of the expressed fragment. This enabled expression in mammalian cell lines.
  • the vector was derived from pcDNA3.1+ (Invitrogen). To express the cloned fragments, the appropriate clones were transfected into COS-7 cells and the resulting conditioned medium harvested 48-60 hours post transfection.
  • PTmAb0005 and PTmAbOOl 1 The binding of PTmAb0005 and PTmAbOOl 1 to the expressed IgE domains was investigated by ELISA assay, by binding the constmcts to an ELISA plate followed by incubation with the monoclonal antibodies, and revelation with an anti-mouse antibody. Also, binding to denatured constructs was investigated by the well known technique of Western blot.
  • PTmAbOOl 1 also bound to C ⁇ 2-4, C ⁇ 2-3 and C ⁇ 2 in their native form; and also bound to C ⁇ 2-4 and C ⁇ 2 in their denatured forms.
  • Q273 A and E (glutamate) M275: A; Q and K (lysine) D276: A and N (asparagine)
  • the alanine mutations changed both the stmcture and chemistry of the target residue whilst the other mutations maintained stmcture (as closely as possible) but altered the charge, e.g Q273E.
  • glutamate has essentially the same stmcture as glutamine but is negatively charged instead of neutral.
  • Each mutation was generated independently in a C ⁇ 2-4 construct. Each mutant polypeptide was expressed to a similar level as the wild type (WT) C ⁇ 2-4 and each was able to bind to recombinant Fc ⁇ RI ⁇ ectodomain as efficiently as the WT c ⁇ 2-4 product in ELISA based assays. Together, these data demonstrated that the mutations had no effect on the production/secretion of the polypeptides in the expression system and did not grossly affect the stmcture of the c ⁇ 2-4 fragment.
  • WT wild type
  • binding activities of PTmAb0005 and PTmAbOOl 1 are specifically affected by mutations within the C-D loop of C ⁇ 2.
  • sequence PI comprises the major binding determinant for both PTmAb0005 and PTmAbOOl 1.
  • Table 10 recognition of IgE domain constmcts by PTmAb0005 and PTmAbOOl 1.
  • PT1078 was modified such that an additional residue was added so that the loop region had the same number of residues as PT1079, however this modification failed to restore binding to PTmAb0005 or PTmAbOOl 1.
  • a recombinant form of the ectodomain of the alpha chain of the high affinity receptor for IgE (alpha ectodomain) is utilised to bind chimaeric IgE.
  • the carboxyl terminus of the alpha ectodomain is fused to a human IgGl Fc sequence.
  • This enables the recombinant molecule to be bound to protein A coated microtitre plates via the Fc region.
  • the majority of the alpha ectodomain molecules should be available for binding ligand and provides a system for the analysis of IgE - receptor interactions.
  • the format described below is aimed at detecting the (high affinity) receptor blocking activity of anti-IgE antibodies.
  • a figure for the percentage of inhibition of binding of IgE to its receptor can be calculated.
  • a maximum binding value for IgE is determined from the average of a set of wells that contained IgE in 10% pig serum alone (i.e no anti-IgE).
  • the % inhibition value is calculated thus:
  • This assay is essentially identical to the previous assay except that the Fc ⁇ Rl ⁇ ectodomain/IgG construct is treated with the proteolytic enzyme Factor X to cleave the two moieties.
  • the IgG Fc moiety is removed using protein A beads, and the Factor X is removed using strepatavidin beads, thus leaving an essentially pure alpha chain ectodomain product.
  • the alpha ectodomain is bound directly to plastic microtitre plates, all other assay details are as described above.
  • This assay was performed on either RPMI 8866 cells or primary human B-cells; two formats may be used, one for the detection of mAbs that bind to IgE associated with Fc ⁇ RII, and a second that analysed whether the mAbs interfered with IgE association with Fc ⁇ RII.
  • cells were loaded with chimaeric IgE (1 ⁇ g/ml) for an hour on ice in PBS, 1% FBS, 0.1% NaN 3 . Excess IgE was removed and anti-IgE mAb added. Bound mAb was elucidated with FITC-conjugated rat anti-mouse IgG, antibody.
  • chimaeric IgE (1 ⁇ g/ml) was pre-incubated with anti- IgE mAb for an hour at room temperature, with gentle mixing, prior to addition to the cells. The mixture was incubated with the cells for an hour on ice and then washed to remove unbound IgE. Bound IgE was detected with FITC-goat anti-human IgE or bound anti-IgE mAb was detected with FITC-conjugated rat anti-mouse IgG, antibody. Where studies were performed on PBMCs, constituent B-cells were identified with a PE-conjugated anti-CD 19 antibody. Samples were analysed by flow cytometry.
  • FIG. 16 shows the concentration dependent binding of monoclonal antibody to plate bound IgE.
  • Figure 17 shows the concentration dependent inhibition of IgE binding to an Fc ⁇ Rl ⁇ /IgG constmct with PTmAb0005 and PTmAbOOl 1.
  • Figure 18 shows the inhibition of IgE binding to clipped ectodomain of Fc ⁇ RI ⁇ -bound directly to plastic plates, by antibody PTmAb0005 and PTmAbOOl 1.
  • Figure 19 shows the lack of inhibition of IgE binding to Fc ⁇ RII (CD23) by antibody PTmAb0005 (clone GE-1) and PTmAbOOl 1.
  • Figure 20 and 21 shows the concentration-dependent blocking of histamine release from allergic human blood basophils with antibody PTmAb0005 and PTmAbOOl 1.
  • PTmAbOOl 1 is a mouse monoclonal antibody with specificity for human IgE, showing no cross-reactivity with other human Ig isotypes or rat/mouse IgE. PTmAbOOl 1 binds to both native and heat-treated IgE, when bound to an ELISA plate in a random orientation, indicating that its recognition site on IgE is not heat labile.
  • PTmAbOOl 1 also recognises IgE when bound via antigen to the ELISA plate.
  • this mAb can completely block the interaction between hum,an IgE and the ⁇ -chain binding component of the high affinity IgE receptor (Fc ⁇ RI). However, this mAb still recognises human IgE when pre-bound to Fc ⁇ RI, indicating that the mAb binding site is not lost upon receptor binding.
  • the normal IgE binding ELISA method was performed by coating plates with human chimaeric IgE, myeloma IgE, human Ig isotypes or rodent IgE (1 ⁇ g/ml in pH 9.6 carbonate/bicarbonate coating buffer).
  • NP-BSA was coated at a saturating concentration prior to the addition of chimaeric IgE (1 ⁇ g/ml).
  • soluble human Fc ⁇ RI ⁇ -chain was coated (0.25 ⁇ g/ml) followed by chimaeric IgE.
  • the remaining ELISA was carried out as described in Experiment 1 (ELISA protocol for the detection of mouse anti-human IgE mAbs).
  • Figure 22 illustrates that PTmAbOOl 1 binds to both human/mouse chimaeric IgE and human myeloma IgE when bound to an ELISA plate in a random orientated manner. Similarly, binding to antigen orientated IgE (i.e IgE bound to plate bound NP-BSA) is dose dependent. PTmAbOOl 1 was also analysed for its ability to recognise chimaeric IgE following heat treatment at 56°C for a range of time periods. Figure 22 also shows that the binding capacity of PTmAbOOl 1 for IgE is unaffected by heat treatment.
  • PTmAbOOl 1 was able to inhibit the interaction of IgE with the ⁇ -chain component of the high affinity IgE receptor (Fig 23).
  • PTmAbOOl 1 was also ( Figure 24) recognises Fc ⁇ RI ⁇ -chain associated IgE in a dose dependent manner.
  • PBMCs were plated at 2x10 5 cells per well in 96 U-well plates, in medium supplemented with both IL-4 and anti-CD40.
  • PTmAbOOl 1 or isotype matched control mAb was added and cells incubated for 14 days prior to harvesting of supematants for IgE analysis.
  • Total IgE levels were measured by coating ELISA plates with rabbit anti-human IgE antibody (lO ⁇ g/ml) in 0.5M carbonate/bicarbonate buffer (pH9.6). Washed plates were blocked with PBS, 0.05% Tween 20, 5% BSA.
  • Both cell supematants and IgE standard were incubated with saturating amounts of PTmAbOOl 1 (lO ⁇ g/ml) for an hour at room temperature prior to addition to the ELISA plate to allow for IgE/anti-IgE complexes to be formed. Following incubation and washing steps, bound IgE was detected with HRP-sheep anti-human IgE, followed by OPD substrate. Levels of IgE in the cell supematants were then estimated relative to the standard curve.
  • IgE was pre-incubated with PTmAbOOl 1 over a dose range from 10 ⁇ g/ml to 0.5 ⁇ g/ml and examined for its effect on subsequent IgE binding to Fc ⁇ RII on the human B-cell line RPMI8866.
  • Figure 25 illustrates that pre-incubation of IgE with
  • PTmAbOOl 1 enhances IgE binding to Fc ⁇ RII.
  • a non PI specific monoclonal antibody did not enhance the binding of IgE to the Fc ⁇ RII receptor.
  • PTmAbOOl 1 also enhances IgE binding to Fc ⁇ RII on primary B-cells ( Figure 26).
  • Peripheral blood mononuclear cells were cultured with PTmAbOOl 1, in the presence of additional IL-4 and anti-CD40 antibody to promote B-cell isotype switch to IgE secretion.
  • An ELISA assay was developed that allowed for measurement of total IgE levels, that is free IgE and PTmAbOOl 1 complexed IgE.
  • To achieve such quantitation secreted IgE was pre-incubated with saturating levels of PTmAbOOl 1 to allow for all of the IgE to be complexed.
  • the total IgE within the tissue culture supernatant was quantitated relative to a standard curve of IgE that had also been complexed with saturating levels of PTmAbOOl 1.
  • Figure 27 illustrates that in three different donors, incubation of primary B-cells with PTmAbOOl 1 (1 ⁇ g/ml) resulted in a significant reduction in the total levels of secreted IgE. No such inhibition was seen with the isotype matched control antibody.
  • PBMC from non-allergic donors were passively sensitised with 1 ⁇ g/ml chimeric IgE for 30min at 37°C, washed and treated with monoclonal antibodies for 30min at 37°C.
  • PBMC from LolPl -sensitive donors were treated directly with monoclonal antibodies for 30min at 37°C. Reactions were terminated by centrifugation. Histamine release in cell supematants was determined by specific immunoassay (Immunotech 2562). Total cellular histamine content was determined in cells lysed with 0.5% Igepal detergent.
  • anti-IgE antibodies to block binding of chimeric IgE to Fc ⁇ RI on human basophils was determined by incubation of PBMC from non-allergic donors with chimeric IgE in the presence of monoclonal antibodies and IL-3 for 30min at 37°C. Cells were washed and histamine release was triggered with NP-BSA for a further 30min at 37°C. Reactions were terminated by centrifugation and histamine release measured as above.
  • Cmde mast cell suspensions were prepared from human lung tissue by enzymatic digestion with a cocktail comprising hyaluronidase, pronase and DNAse. Cells were either used directly or pre-sensitised with chimeric IgE prior to treatment with anti- IgE antibodies. Mast cell degranulation was determined by colorimetric assay of the granule enzyme tryptase.
  • Transfected cell line RBL J41 was obtained from Dr B. Helm, University of Sheffield. Cells were passively sensitised with either mouse monoclonal IgE anti-DNP or human chimeric IgE anti-NP and triggered with anti-human IgE antibodies. Degranulation was measured by the colorimetric assay of ⁇ -hexosaminidase release.
  • PTmAbOOl 1 was also unable to release significant amounts oftryptase in both sensitised and non-sensitised human lung mast cells (figure 29). Polyclonal anti- human IgE gave 60-70% release in these cells.
  • PTmAbOOl 1 was able to block the binding of IgE to Fc ⁇ RI in non-allergic basophils and thus to inhibit subsequent triggering with NP-BSA antigen.
  • the IC 50 value of this activity was around 60ng/ml (figure 31).
  • PTmAbOOl 1 was also able to potently inhibit LolPl -triggered histamine release from allergic basophils with an IC 50 value of 40ng/ml (figure 31).
  • PTmAb0005 and PTmAbOOl 1 have also been tested for in vivo activity. Briefly, the local skin mast cells of African green monkeys were shaved and sensitised with intradermal administration of lOOng of anti-NP IgE (human IgE anti- nitrophenylacetyl (NP) purchased from Serotech) into both arms. After 24 hours, a dose range of the monoclonal antibodies to be tested were injected at the same injection site as the human IgE on one arm. Control sites on the opposite arm of the same animals received either phosphate buffered saline (PBS) or non-specific human IgE (specific for Human Cytomegalovirus (CMV) or Human Immunodeficiency Vims (HIV)).
  • PBS phosphate buffered saline
  • CMV Human Cytomegalovirus
  • HAV Human Immunodeficiency Vims

Abstract

La présente invention concerne de nouveaux médicaments destinés au traitement, à la prévention ou à l'atténuation des allergies. Ces nouveaux médicaments sont notamment des peptides isolés comprenant des épitopes ou des mimotopes des régions exposées en surface du domaine Cε2 des IgE. Les inventeurs ont découvert que ces nouvelles régions peuvent servir de cible à l'immunoprophylaxie ou à l'immunothérapie, active ou passive. L'invention concerne également des procédés de préparation de médicaments, de compositions pharmaceutiques les contenant et leur utilisation en médecine. L'un des aspects de la présente invention couvre des ligands, notamment des anticorps monoclonaux, qui sont capables de lier les régions exposées en surface des IgE de la présente invention, et leur utilisation en médecine et en immunoprophylaxie ou en immunothérapie passive.
EP00905073A 1999-02-25 2000-02-22 Epitopes ou mimotopes derives du domaine c-epsilon-2 de l'immoglobuline e, leurs antagonistes et leurs applications therapeutiques Withdrawn EP1155037A1 (fr)

Applications Claiming Priority (21)

Application Number Priority Date Filing Date Title
GBGB9904405.9A GB9904405D0 (en) 1999-02-25 1999-02-25 Vaccine
GB9904405 1999-02-25
GBGB9907151.6A GB9907151D0 (en) 1999-03-29 1999-03-29 Therapeutic antibody composition and use
GB9907151 1999-03-29
GBGB9910538.9A GB9910538D0 (en) 1999-05-07 1999-05-07 Therapeutic antibody, Immunogenic compositions ansd uses
GB9910537 1999-05-07
GB9910538 1999-05-07
GBGB9910537.1A GB9910537D0 (en) 1999-05-07 1999-05-07 Therapeutic antibody, Immunogenic compositions and uses
GB9918594 1999-08-07
GBGB9918594.4A GB9918594D0 (en) 1999-08-07 1999-08-07 Novel peptides
GB9918603 1999-08-07
GBGB9918603.3A GB9918603D0 (en) 1999-08-07 1999-08-07 Novel peptides
GBGB9921047.8A GB9921047D0 (en) 1999-09-07 1999-09-07 Therapeutic antibody, immunogenic compositions & uses
GBGB9921046.0A GB9921046D0 (en) 1999-09-07 1999-09-07 Therapeutic antibody composition & use
GB9921046 1999-09-07
GB9921047 1999-09-07
GB9925619 1999-10-29
GBGB9925619.0A GB9925619D0 (en) 1999-10-29 1999-10-29 Vaccine
GB9927698 1999-11-23
GBGB9927698.2A GB9927698D0 (en) 1999-11-23 1999-11-23 Therapeutic antibody
PCT/EP2000/001455 WO2000050460A1 (fr) 1999-02-25 2000-02-22 Epitopes ou mimotopes derives du domaine c-epsilon-2 de l'immoglobuline e, leurs antagonistes et leurs applications therapeutiques

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GB9913327D0 (en) * 1999-06-08 1999-08-11 Smithkline Beecham Biolog Vaccine
US6787524B2 (en) 2000-09-22 2004-09-07 Tanox, Inc. CpG oligonucleotides and related compounds for enhancing ADCC induced by anti-IgE antibodies
EP1497318A4 (fr) * 2001-04-18 2006-03-01 Dyax Corp Molecules de liaison pour les polypeptides de zone fc
GB0209878D0 (en) * 2002-04-30 2002-06-05 Glaxosmithkline Biolog Sa Vaccine
AR065368A1 (es) * 2007-02-15 2009-06-03 Astrazeneca Ab Anticuerpos para moleculas de ige
AU2008215926B2 (en) * 2007-02-15 2012-07-19 Astrazeneca Ab Binding members for IgE molecules
WO2010128265A2 (fr) 2009-05-07 2010-11-11 Stallergenes S.A. Utilisation d'immunoglobulines igg1 et/ou de ligands du récepteur cd32 pour le traitement de maladies et manifestations inflammatoires par voie mucosale
CA3113604A1 (fr) * 2018-09-21 2020-03-26 Riken Anticorps anti-ige se liant de maniere specifique a un anticorps ige lie a une membrane de lymphocytes b produisant un anticorps ige et methode de diagnostic et de traitement de s ymptomes allergiques l'utilisant

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US4171299A (en) * 1975-04-04 1979-10-16 The Regents Of The University Of California Polypeptide agents for blocking the human allergic response
AU6532498A (en) * 1996-12-06 1998-06-29 United States Of America, Represented By The Secretary, Department Of Health And Human Services, The Inhibition of ige-mediated allergies by a human ige-derived oligopeptide
JP2001516009A (ja) * 1997-07-17 2001-09-25 ルードヴィッヒ インスティテュート フォー キャンサー リサーチ ガン関連核酸及びポリペプチド

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AR029336A1 (es) 2003-06-25
CO5210906A1 (es) 2002-10-30
JP2004538238A (ja) 2004-12-24
WO2000050460A1 (fr) 2000-08-31
KR20020007313A (ko) 2002-01-26
NO20014130D0 (no) 2001-08-24
CA2363637A1 (fr) 2000-08-31
CN1348466A (zh) 2002-05-08
PL350993A1 (en) 2003-02-24
NZ513679A (en) 2001-09-28
NO20014130L (no) 2001-09-13
TR200102506T2 (tr) 2002-06-21
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