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
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/08—Antiallergic agents
• • 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 epitopes or mimotopes derived from the C ⁇ 3 or C ⁇ 4 domains of IgE. 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.
• 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)).
• Each of the IgE domains consists of a squashed barrel of seven anti-parallel strands of extended ( ⁇ -) polypeptide segments, labelled a to f, grouped into two ⁇ -sheets.
• Four ⁇ -strands (a,b,d & e) form one sheet that is stacked against the second sheet of three strands (c,f& g) (see FIG 8).
• the shape of each ⁇ -sheet is maintained by lateral packing of amino acid residue side-chains from neighbouring anti-parallel strands within each sheet (and is further stabilised by main-chain hydrogen-bonding between these strands).
• the connection from strand a to strand b is labelled as the A-B loop, and so on.
• the A-B and d-e loops belong topologically to the four-stranded sheet, and loop 1 g to the three-stranded sheet.
• the interface between the pair of opposing sheets provides the hydrophobic interior of the globular domain. This water-inaccessible, mainly hydrophobic core results from the close packing of residue side-chains that face each other from opposing ⁇ -sheets.
• 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. Therefore, in order to be safe and effective, 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 may 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 B 1 describes immunogens derived from the C ⁇ 4 domain of IgE (residues 497-506, also known as the Stanworth decapeptide), conjugated to Keyhole Limpet Haemocyanin (KLH) used in active vaccination immunoprophylaxis.
• KLH Keyhole Limpet Haemocyanin
• WO 96/14333 is a continuation of the work described in EP 0 477 231 Bl.
• Other approaches are based on the identification of peptides derived from C ⁇ 3 or C ⁇ 4, which themselves compete for IgE binding to the high or low affinity receptors on basophils or mast cells (WO 93/04173, WO 98/24808, EP 0 303 625 Bl, EP 0 341 290).
• the present invention is the identification of novel sequences of IgE which are used in active or passive immunoprophylaxis or therapy. These sequences have not previously been associated with anti-allergy treatments.
• the present invention provides peptides, per se, that incorporate specific isolated epitopes from continuous portions of IgE which have been identified as being surface exposed, and further provides mimotopes of these newly identified epitopes. These peptides or mimotopes may be used alone in the treatment of allergy, or may be used vaccines to induce auto anti-IgE antibodies during active immunoprophylaxis or immunotherapy of allergy to limit, reduce, or eliminate allergic symptoms in vaccinated subjects.
• the anti-IgE antibodies induced by the peptides of the present invention are non-anaphylactogenic and are capable of blocking IgE-mediated histamine release from mast cells and basophils.
• the regions of human IgE which are peptides of the present invention, and which may serve to provide the basis for peptide modification are:
• 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 epitope in the context of the IgE molecule, also form part of the present invention.
• the present invention therefore, includes isolated peptides encompassing these 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 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 IgE domain structure, and as such the mimotope may take a slightly different conformation to that of the native IgE epitope.
• the above identified linear sequences (PI to P7) 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.
• the mimotopes of the present invention mimic the surface exposed regions of the IgE structure, however, within those regions the dominant aspect is thought by the present inventors to be those regions within the surface exposed area which correlate to a loop structure.
• the structure of the domains of IgE are described in "Introduction to protein Structure” (page 304, 2 nd Edition, Branden and Tooze, Garland Publishing, New York, ISBN 0 8153 2305-0) and take the form a ⁇ -barrel made up of two opposing anti-parallel ⁇ -sheets (see FIG. 8).
• the mimotopes may comprise, therefore, a loop with N or C terminal extensions which may be the natural amino acid residues from neighbouring sheets.
• PI 00 contains the A-B loop of C ⁇ 3
• P8 contains the A-B loop of C ⁇ 4
• P5 contains the C-D loop of C ⁇ 3
• PI 10 contains the C-D loop of C ⁇ 4.
• mimotopes of these loops form an aspect of the present invention.
• Particularly preferred loops are the C-D loops of C ⁇ 3 or C ⁇ 4, and the A-B loop of C ⁇ 4.
• 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.
• 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. This reduces the conformational degrees of freedom of the peptide, and thus increases the probability that the peptide is presented in a conformation which most closely resembles that of the IgE peptide as found in the context of the whole IgE molecule.
• 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, or mimotopes 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.
• a preferred method of cyclising a peptide comprises the addition of a pair of cysteine residues to allow the formation of a disulphide bridge.
• mimotopes or immunogens of the present invention may be larger than the above-identified epitopes, and as such 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).
• 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 PI 4c).
• 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.
• peptide mimotopes may be generated with the objective of increasing the immunogenicity of the peptide by increasing its affinity to the anti-IgE peptide polyclonal antibody, the effect of which may be measured by techniques known in the art such as (Biocore experiments) .
• the peptide sequence may be electively changed following the general rules:
• prolines and glycines should not be replaced * Other positions can be substituted by an amino acid that has similar physicochemical properties.
• each amino acid residue can 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.
• IgE peptides are P8 and variants thereof (such as P14 or PI 4a). These peptides, when coupled to a carrier are potent in inducing anti-IgE immune responses, which responses are capable of inhibiting histamine release from human basophils.
• Variants, or mimotopes, of P8 are described primarily as any peptide based immunogen which is capable of inducing an immune response, which response is capable of recognising P8.
• some variants of P8 may be described by a general formula in which certain amino acids may be replaced by their closest counterparts. Using this technique, P8 peptide mimotopes may be described by the general formula:
• X is an amino acid selected from E, D, N, or Q
• X 2 is an amino acid selected from W, Y, or F
• X 3 is an amino acid selected from G or A
• X 4 is an amino acid selected from S, T or M
• X 5 is an amino acid selected from R or K
• X 6 is an amino acid selected from D or E.
• P8 mimotopes may also be identified using antibodies which are capable themselves of binding to P8, using techniques such as phage display technology (EP 0 552 267 Bl). Monoclonal antibodies such as P14/23, P14/31 and P14/33 are particularly suitable in this regard.
• the present invention therefore, provides novel epitopes, and mimotopes thereof, and their use in the manufacture of pharmaceutical compositions for the prophylaxis or therapy of allergies.
• Immunogens comprising at least one of the epitopes or mimotopes of the present invention and carrier molecules are also provided for use in vaccines for the immunoprophylaxis or therapy of allergies.
• the epitopes, mimotopes, or immunogens of the present invention are provided for use in medicine, and in the medical treatment or prophylaxis of allergic disease.
• Preferred immunogens and vaccines of the present invention comprise the IgE epitope P8, or mimotopes thereof, including PI 4.
• the present inventors have shown that different methods by which the epitope or mimotope is presented has significant effects upon binding to monoclonal antibodies and to the immune response after vaccination. For example, when using cyclised peptides, altering the length and phase of the loop may have significant effects on the binding activity of the cyclised mimotopes to the P14 monoclonal antibodies (P14/23, P14/31 or P14/33). As such the present inventors have developed a novel system which selects the sites of cyclisation, thereby increasing the probability that the cyclised peptides are held in the correct loop structure, which comprises the correct amino acid residues.
• the peptide is likely to be constrained in a conformation that most closely resembles that which the peptides would normally adopt if they were in the context of the whole IgE domain.
• the cyclised mimotopes which follow these new rules form one preferred aspect of the present invention.
• Putative mimotope sequences that are not consistent with these rules may still raise useful antisera (for example P14 and PI 1), as such the following examples are only a sub-set of the types of mimotopes of the present invention.
• the mimotopes of the present invention will be of a small size, such that they mimic a region selected from the whole IgE domain in which the native epitope is found.
• 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 P 14 and PI 1, which are respectively 13 and 23 amino acids long.
• ⁇ on-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 epitope within IgE.
• Specific examples of such monoclonal antibodies for use in the competition assays include P14/23, P14/31 or P14/33, which would confirm the status of the putative mimotope as a mimotope of P8.
• At least one IgE epitope or mimotope are linked to carrier molecules to form immunogens for vaccination protocols, preferably wherein the carrier molecules are not related to the native IgE molecule.
• the mimotopes may be linked via chemical covalent conjugation or by expression of genetically engineered fusion partners, optionally via a linker sequence.
• the peptides of the present invention are expressed in a fusion molecule with the fusion partner, wherein the peptide sequence is found within the primary sequence of the fusion partner.
• 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.
• peptides particularly cyclised peptides may be conjugated to the carrier by preparing Acylhydrazine peptide derivatives.
• the peptides/protein carrier constructs can be produced as follows. Acylhydrazine peptide derivatives can be prepared on the solid phase as shown in the following scheme 1 Solid Phase Peptide Synthesis: Scheme 1
• 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. This could be readily oxidised and purified to yield the disulphide-bridged modified epitope using methodology outlined in 'Methods in Molecular Biology, Vol. 35: Peptide Synthesis Protocols (ed. M.W. Pennington and B.M. Dunn), chapter 7, pp91-171 by D. Andreau et al.
• PEG-PS polyethyleneglycol-polystyrene
• the peptides thus synthesised can then be conjugated to protein carriers using the following technique:
• succinimido active ester BAL-OSu
• BSA bovine serum albumin
• BSA and BAL-OSu were mixed in equimolar concentration in DMSO/buffer (see scheme) for 2 hrs. This experimentally derived protocol gives ⁇ 50% substitution of BSA as judged by the Fluorescamine test for free amino groups in the following Scheme 2/3 - Modified Carrier Preparation:
• 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 diptheria toxins
• PPD purified protein derivative of tuberculin
• 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 mimotopes 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
• 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). In some circumstances, for example in recombinant immunogen expression systems it may be desirable to use 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)). Another preferred method of presenting the IgE peptides of the present invention is in the context of a recombinant fusion molecule.
• EP 0 421 635 B describes the use of chimaeric 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 chimaeric 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 NSI 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.
• 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 analogues thereof, 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 present invention therefore, provides the use of novel epitopes or mimotopes (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.
• 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.
• 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 (hydroxide or phosphate).
• 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.
• 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.
• a preferred route of administration is via the transdermal route, for example by skin patches. Accordingly, there is provided a method for the treatment of allergy, comprising the administration of a peptide, immunogen, or ligand of the present invention to a patient who is suffering from or is susceptible to allergy.
• 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. Generally, it is expected that each dose will comprise 1-1000 ⁇ g of protein, preferably 1-500 ⁇ g, more 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.
• ligands capable of binding to the peptides of the present invention.
• ligands capable of binding to the peptides of the present invention.
• Example of such ligands are antibodies (or Fab fragments).
• antibody herein is used to refer to a molecule having a useful antigen binding specificity. Those skilled in the art will readily appreciate that this term may also cover polypeptides which are fragments of or derivatives of antibodies yet which can show the same or a closely similar functionality. Such antibody fragments or derivatives are intended to be encompassed by the term antibody as used herein.
• Particularly preferred ligands are monoclonal antibodies.
• P14/23, P14/31 or P14/33 are monoclonal antibodies which recognise P8 (which were raised by vaccination with a P14 immunogen).
• the hybridomas of these antibodies were 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 26 January 2000 under Accession No.s 00012610, 00012611, 00012612 respectively.
• Also forming an important aspect of the present invention is the use of these monoclonal antibodies in the identification of novel mimotopes of IgE, for subsequent use in allergy therapy, and the use of the antibodies in the manufacture of a medicament for the treatment or prophylaxis of allergy. All of these monoclonal antibodies function in vitro in inhibiting histamine release from human basophils, and also PI 4/23 and PI 4/31 have been shown to inhibit passive cutaneous anaphylaxis in vivo.
• mimotopes of IgE C ⁇ 4 that are capable of binding to PI 4/23, P14/31 or P14/33, and immunogens comprising these mimotopes, form an important aspect of the present invention.
• Vaccines comprising mimotopes that are capable of binding to PI 4/23, PI 4/31 or PI 4/33 are useful in the treatment of allergy.
• antibodies induced in one animal by vaccination with the peptides or immunogens of the present invention 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 know techniques e.g. K ⁇ hler and Milstein, Nature, 1975, 256, p495), humanised monoclonal antibodies or CDR grafted monoclonals, by techniques known in the art.
• Such antibodies may be used in passive immunoprophylaxis or immunotherapy, or be used in the identification of IgE peptide mimotopes.
• compositions comprising the ligands of the present invention.
• Preferred pharmaceutical compositions for the treatment or prophylaxis of allergy comprise the monoclonal antibodies PI 4/23, P14/31 or P14/33.
• Aspects of the present invention may also be used in diagnostic assays. For example, 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. Moreover, the peptides may themselves be used to type the circulating anti- IgE.
• 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 P14/23, P14/31 or P14/33 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 Surface exposure of C ⁇ 3 an C ⁇ 4 of human IgE as calculated from the Padlan and Davis model 1986.
• FIG 2 Histamine release inhibition and anaphylactogenicity of P14 antiserum.
• Monoclonal Antibodies, PTmAb0005 and PTmAbOOl 1 which were used as positive controls, were added at 1 ⁇ g/ml to anti-BSA sera diluted 1/100 and 1/500 (final).
• the anti-P14 antisera were added at 1/100 and 1/500 final dilution.
• Cells were taken from an allergic patient sensitive to grass pollen, histamine release was triggered by incubation with this grass pollen allergen.
• FIG 3 Histamine release inhibition and anaphylactogenicity of anti-P14 antiserum.
• Three negative controls were used: Anti-BSA antiserum, non-specific IgGl and a mixture of non-specific IgGl diluted in anti-BSA antiserum.
• mAbl 1 is a monoclonal antibody known to inhibit histamine release and was used as a positive control (added at 2 ⁇ g/ml).
• FIG 4 Histamine release inhibition and anaphylactogenicity of anti-P14 antiserum.
• Anti-P14 Antisera from different mice were added at a 1/50 final dilution. Monoclonal Abs were added at 2 ⁇ g/ml either in assay buffer or in anti-BSA sera dilution 1/50. Three negative controls were used: Anti-BSA antiserum, non-specific IgGl and a mixture of non-specific IgGl diluted in anti-BSA antiserum.
• mAbl 1 is a monoclonal antibody known to inhibit histamine release and was used as a positive control (added at 2 ⁇ g/ml).
• Peptide PI 1 is coated at 1 ⁇ g/ml in carbonate buffer at +4°C overnight.
• FIG 6 Anti-Pi 1 IgG anti-human IgE titres. Human IgE was coated at 1 ⁇ g/ml. Twofold serial dilutions of sera ("BSA pool” is a pool of the control group) or PTmAb0005 (a positive control monoclonal antibody) were incubated for lh at 37°C. Bound IgG is detected with a biotinylated anti-mouse Ab.
• FIG 7 Histamine release inhibition studies with anti-P14 monoclonal antibodies, on allergic basophils donated by dustmite allergic patients (A 10 and Al l) and from grass pollen allergic patients (G8 and G4).
• PT11 (PTmAbOOl 1) was used as a positive control, and non-specific IgG2a was used as an isotype control for the PI 4/23, PI 4/31 and PI 4/33.
• Each domain is composed of two facing ⁇ -sheets, shown in outline, one of 4 anti -parallel ⁇ -strands (labelled 4) and the other of 3 anti- parallel ⁇ -strands (labelled 3).
• B The seven strands are shown topographically as block arrows labelled a to f, partitioned between the two sheets as shown.
• FIG 9 (A) Predicted structural alignment of the A-B loop sequences of human IgE domains C ⁇ 2, 3 & 4 with the equivalent segments from the crystallographically determined structure of human IgGl Fc (domains C ⁇ 2 & C ⁇ 3). ⁇ -strands in the IgGl structure are underlined and labelled a and b; amino acid residues at the ends of each sequence segment are numbered.
• Residues within the plain bold boxes are predicted to be involved in recognition by receptors and/or antibodies.
• Vertical arrows below the block of sequences point to predicted optimal cyclisation positions, labelled and connected by dashed or solid lines as shown in FIG l ib.
• FIG 10 (A) The schematic structure of the A-B hairpin at the sheet-sheet interface of Ig constant domains. Adjacent anti-parallel ⁇ -strands are shown as solid arrows, labelled a and b. Residues along strand a are labelled i, those along strand b are labelled j. Residues i+n & j+m, where both n and m are zero or even, form part of the sheet-sheet interface within a domain. Residues i+n & j+m, where both n and m are odd, form part of the solvent-exposed surface of a domain.
• the A-B loop is shown as a black arrow.
• FIG 11 (A) The schematic structure of the C-D hairpin (loop plus supporting ⁇ - strands) at the edge of the sheet-sheet interface of Ig constant domains. Opposing anti- parallel ⁇ -strands are shown as solid arrows, labelled c and d. Residues along strand c are labelled i, those along strand d are labelled j. Residues i+n & j+m, where n is odd but m is even, form part of the sheet-sheet interface within a domain.
• Residues i+n & j+m form part of the solvent-exposed surface of a domain.
• the c_d loop containing the short c' strand, is shown as a black arrow.
• (B) The schematic structure of the c_d hairpin, with residue positions optimal for cyclisation connected by dashed or solid dumbbells.
• the peptides were identified by the following technique.
• the modelled structure of human IgE has been described 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 .
• MSI Molecular Simulations software
• 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.
• Maleimide groups 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 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 methacrylate
• 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 tables 4 or 5, or derivatives or mimotopes thereof) 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 20 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 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 dialysis against NaCl 150 mM Tween 80 0.1 % are performed to remove the excess of cysteine or peptide.
• the last step is sterile filtration through 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 pre-activated BSA may be purchased commercially from Pierce Inc.
• Mimotopes of P8 (P14, SEQ ID NO. 20; CLEDGQVMDVDLL) and P5 (PI 1, SEQ ID NO. 8; CRASGKPVNHSTRKEEKQRNGLL) were synthesised which were 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.
• Non-specific 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.
• Competition of IgE Binding with Mimotope Peptides, Soluble IgE or PTmAbOOl 1 Single dilutions of polyclonal mouse serum are mixed with single concentrations of either mimotope peptide or human IgE in a pre-blocked polypropylene 96-well plate. Mixtures are incubated for 1 hour at 37°C and then added to IgE-coated ELISA plates 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.
• TMB substrate for competition between serum and 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
• Blood is collected by venepuncture from allergic donors into tubes containing heparin, and the non-erythrocytic cells were purified.
• 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.
• the degree of inhibition of histamine release can be calculated using the formula:
• PTmAbOOl 1 is a monoclonal antibody which is known to bind to the C ⁇ 2 domain of IgE, and was used to quantify the anti-IgE responses in ⁇ g/ml.
• mice vaccinated with BSA alone as controls did not generate any detectable anti- peptide or anti-IgE responses.
• the antiserum raised by the P 14 vaccination was found to be functional, in that it was potent in the inhibition of histamine release from allergic human basophils after triggering with allergen (see FIGS. 2, 3 and 4). Moreover, the antiserum was not found to be anaphylactogenic (FIGs. 2, 3 and 4).
• P14 (mimotope of P8) was shown to be capable of raising high titres of anti-P14 and anti-IgE antibodies in mice. These antibodies were subsequently shown to be functional, in that they inhibited histamine release from allergic human basophils, and were not anaphylactogenic. P14 and P8, therefore, may be used in the treatment or prophylaxis of allergy.
• Example 3 Immunisation of mice with Pll conjugates (Pll-BSA, Pll -BSA) induces production of anti-human IgE antibodies.
• Human IgE epitope peptide Pl l was coupled to maleimide-activated BSA (Pierce) (BSA-CRASGKPVNHSTPvKEEKQRNGLL).
• 25 ⁇ g of conjugate formulated in SBAS2 was injected IM into 8 female BALB/c mice at days 0, 14 and 28.
• One control group of mice was injected with BSA SBAS2. Blood samples were taken 14 days after each injection (a fourth bleeding was performed at day 24 post 3 to increase the availability of sera).
• Anti-peptide and anti-IgE antibodies raised by vaccination were measured by ELISA, as described in Example 1.
• mice showed an anti-IgE response (ranging from 28 - 244 ⁇ g/ml as expressed in mAb005 equivalents) after a third injection (FIG. 6).
• Monoclonal antibodies have been generated that recognise specifically P8 and mimotopes thereof, using techniques known in the art. Briefly, the P14-BSA conjugate described in part 1 of these examples, was injected into groups of Balb/C mice with the o/w adjuvant containing QS21 and 3D-MPL. Spleen cells were taken and fused with SP2/O B-cell rumour cell line, and supematants were screened for reactivity against both P14 peptide and IgE.
• P14 monoclonal antibodies were tested on basophils taken from four different allergic patients (A patients were allergic to dust mite antigen, G patients were allergic to grass pollen).
• PT11 PTmAbOOl 1
• All of the three P14 monoclonal antibodies 23, 31, and 33 were potent in inhibiting histamine release from allergic basophils (See FIG. 7).
• PI 4/23 and PI 4/31 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 1 OOng 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 Virus (HIV)).
• PBS phosphate buffered saline
• CMV Human Cytomegalovirus
• HAV Human Immunodeficiency Virus
• a BSA-NP conjugate (purchase from Biosearch Laboratories) was administered by intravenous injection. After 15-30 minutes, the control animals develop a readily observable roughly circular oedema from the anyphylaxis, which is measurable in millimeters. Results are expressed in either the mean oedema diameter of groups of three monkeys or as a percentage inhibition in comparison to PBS controls.
• PTmAbOOl 1 is a monoclonal antibody was used as a positive control.
• SBmAb0006 was used as a negative control. Table 7, PI 4/23 results
• the present inventors have shown that the conformation in which the epitopes or mimotopes of the present invention is important for both anti-mimotope antibody recognition, and also for the ability of the peptides to generate a strong anti-IgE immune responses. As such the present inventors have developed structural rules which predict the optimal sites for peptide cyclisation. Peptides that use these sites of cyclisation form one prefered aspect of the present invention.
• IgE Fc As the full structure of IgE Fc has not been determined, the present inventors have refined the currently available models (Helm et al. supra, Padlan and Davis supra) using the known structure of C ⁇ 2 and C ⁇ 3 of IgGl (Deisenhofer J., 1981. Biochemistry, 20, 2361-2370). In addition, models of the C ⁇ 2 domain have been built by comparison with known Ig folding-unit structures. The present inventors have designed these homology models of IgE Fc and thereby predicted the termini and the gross structure of intra-sheet (A-B loop, FIG 9A) and inter-sheet loops in IgE Fc domains (C-D loop, FIG 9B).
• mimotopes of the loops may be derived from the wild-type (WT) primary sequence of each loop by covalent cyclisation between chosen specific residues along the adjoining ⁇ -strands. Cyclisation is preferably realised by the formation of a disulphide bond between terminal cysteines which therefore combine to become a cystine.
• the hydrophobic cystine group should replace WT ⁇ -strand residues that belong to the water-inaccessible core of the Ig constant domain, formed by the interface between the two ⁇ -sheets.
• the cystine group should replace WT residues that are from adjacent anti-parallel ⁇ -strands (see FIG. 8) and that pack laterally together on the same side of the sheet. Following rule 1, this will be on the domain-interior side of the sheet.
• the structural derivation of this rule for the A-B loops is shown schematically in FIG 10 A and 10B.
• the cystine group should replace WT residues on anti-parallel ⁇ -strands, one strand from each sheet.
• the residues forming the optimal pair pack together from facing ⁇ -sheet surfaces, so forming part of the interface between the sheets.
• FIG.11 A and FIG.1 IB The structural derivation of this rule for the C-D loops is shown schematically in FIG.11 A and FIG.1 IB.
• FIG.11 A and FIG.1 IB In the tables of putative mimotope sequences that follow, designs predicted to be optimal are underlined. Below each block of sequences the dotted and solid lines link the residue positions chosen for optimal cyclisation, which are also shown in the same way in FIG 10B (for A-B loops) and in FIG.1 IB (for C-D loops).

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