CA2363641A1 - Epitopes or mimotopes derived from the c-epsilon-3 or c-epsilon-4 domains of ige, antagonists thereof, and their therapeutic uses - Google Patents

Abstract

The present invention relates to the provision of novel medicaments for the treatment, prevention or amelioration of allergic disease. In particular, th e novel medicaments are epitopes or mimotopes derived from the C.epsilon.3 or C.epsilon.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. Also forming an aspe ct of the present invention are ligands, especially monoclonal antibodies, whic h are capable of binding the IgE regions of the present invention, and their u se in medicine as passive immunotherapy or immunoprophylaxis.

Description

IGE, ANTAGO-NISTS THEREOF, AND THEIR THERAPEUTIC USES
The present invention relates to the provision of novel medicaments for the treatment, prevention or amelioration of allergic disease. In particular, the novel s medicaments are epitopes or mimotopes derived from the Cs3 or Cs4 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. Also forming an aspect of the present invention are ligands, especially 1o monoclonal antibodies, which are capable of binding the IgE regions of the present invention, and their use in medicine as passive immunotherapy or immunoprophylaxis.
In an allergic response, the symptoms commonly associated with allergy are brought about by the release of allergic mediators, such as histamine, from immune 15 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. 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. On encountering an antigen, such as pollen or dust mite 2o allergens, B-cells commence the synthesis of allergen specific IgE. The allergen specific IgE then binds to the FcsRI receptor (the high affinity IgE receptor) on basophils and mast cells. Any subsequent encounterwith allergen leads to the triggering of histamine release from the mast cells or basophils, by cross-linking of neighbouring IgE/ FcsRI complexes (Sutton and Gould, Nature, 1993, 366: 421-428;
2s EP 0 477 231 B 1 ).
IgE, like all immunoglobulins, comprises two heavy and two light chains. The E heavy chain consists of five domains: one variable domain (VH) and four constant domains (CE1 to Cs4). 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 3o 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 SUBSTITUTE SHEET (RULE 26) the IgE domains consists of a squashed barrel of seven anti-parallel strands of extended ((3-) polypeptide segments, labelled a to f, grouped into two (3-sheets. Four ~3-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 (3-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). Loops of residues, forming non-extended (non-~3-) conformations, connect the anti-parallel ~i-strands, either within a sheet or between the opposing sheets. The connection from strand a to strand b is labelled as the A-B loop, and so on. The A-B and d-a loops belong topologically to the four-stranded sheet, and loop f 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 ~i-sheets.
In the past, a number of passive or active immunotherapeutic approaches designed to interfere with IgE-mediated histamine release mechanism have been investigated. These approaches include interfering with IgE or allergen/IgE
complexes binding to the FcsRI or FcERII (the low affinity IgE receptor) receptors, with either passively administered antibodies, or with passive administration of IgE
derived 2o peptides to competitively bind to the receptors. In addition, some authors have described the use of specific peptides derived from IgE in active immunisation to stimulate histamine release inhibiting immune responses.
In the course of their investigations, previous workers in this field have encountered a number of considerations, and problems, which have to be taken into account when designing new anti-allergy therapies. One of the most dangerous problems revolves around the involvement of IgE cross-linking in the histamine release signal. It is most often the case that the generation of anti-IgE
antibodies 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 3o phenomenon is termed anaphylactogenicity. Indeed many commercially available anti-IgE monoclonal antibodies which are normally used for IgE detection assays, are SUBSTITUTE SHEET (RULE 26) anaphylactogenic, and consequently useless and potentially dangerous if administered to a patient.
Whether or not 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.
Much work has been carried out by those skilled in the art to identify specific anti-IgE antibodies which do have some beneficial effects against IgE-mediated allergic reaction (WO 90/15878, WO 89/04834, WO 93/05810). Attempts have also been made to identify epitopes recognised by these useful antibodies, to create peptide mimotopes of such epitopes and to use those as immunogens to produce anti-IgE
antibodies.
WO 97/31948 describes an example of this type of work, and further describes IgE peptides from the C~3 and CE4 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 . In this work, a monoclonal antibody (BSW 17) was described which was said to be capable of binding to IgE peptides contained within the CE3 domain which are useful for active vaccination purposes.
EP 0 477 231 B 1 describes immunogens derived from the CE4 domain of IgE
(residues 497-506, also known as the Stanworth decapeptide), conjugated to Keyhole Limpet Haemocyanin (KLH) used in active vaccination immunoprophylaxis. WO
96/14333 is a continuation of the work described in EP 0 477 231 B1.
SUBSTITUTE SHEET (RULE 26) Other approaches are based on the identification of peptides derived from Cs3 or Cs4, 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 B
1, 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 1o 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.
Surprisingly, the anti-IgE antibodies induced by the peptides of the present 15 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:
20 Table 1 PeptideSequence Location sequenceSEQ
and IgE Domain ID NO.

PS RASGKPVNHSTRKEEKQRNGTL Cs3 1 P7 PHLPRALMRSTTKTSGPRA Cs3/Cs4 3 P8 PEWPGSRDKRT CE4 (Pro451-Thr461)4 P9 EQKDE Cs4 P210 WLHNEVQLPDARHSTTQPRKT Cs4 7 1-90N LFIRKS CE3 g 1 2-90N PSKGTVN Cs3 g2 3-90N LHNEVQLPDARHSTTQPRKTKGS Cs4 g3 4-90N SVNPGK CE4 g4 Peptides that incorporate these epitopes form a preferred aspect of the present invention. Mimotopes which have the same characteristics as these epitopes, and SUBSTITUTE SHEET (RULE 26) 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. The meaning of 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, p130-149; Gheysen, H.M., 1986, Molecular to 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 15 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. It will also be clear to the man skilled in the art that the above identified linear sequences (P1 to P7), when in the tertiary structure of IgE, lie 2o adjacent to other regions that may be distant in the primary sequence of IgE. As such, for example, a mimotope of P1 may be continuous or discontinuous, in that it comprises or mimics segments of P 1 and segments made up of these distant amino acid residues.
The mimotopes of the present invention mimic the surface exposed regions of 25 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"d Edition, Branden and Tooze, Garland Publishing, New York, ISBN 0 8153 2305-0) and take the form a (3-barrel made up of two 30 opposing anti-parallel ~3-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. As examples of this, P 100 contains the A-B loop of Cs3, SUBSTITUTE SHEET (RULE 26) P8 contains the A-B loop of CE4, PS contains the C-D loop of Cs3 and P 110 contains the C-D loop of Cs4. Accordingly, mimotopes of these loops form an aspect of the present invention. Particularly preferred loops are the C-D loops of Cs3 or Cs4, and the A-B loop of CE4.
Peptide mimotopes of the above-identified IgE epitopes may be designed for a particular purpose by addition, deletion or substitution of elected amino acids. Thus, the peptides of the present invention may be modified for the purposes of ease of conjugation to a protein carrier. For example, it may be desirable for some chemical conjugation methods to include a terminal cysteine to the IgE epitope. In addition it may be desirable for peptides conjugated to a protein carrier to 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. For example, the peptides may be altered to have an N-terminal cysteine and a C-terminal hydrophobic amidated tail. Alternatively, 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. Those 2o skilled in the art will realise that such 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. In addition, 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.
Further, those skilled in the art will realise that 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 SUBSTITUTE SHEET (RULE 26) 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. Such 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 Pl4c).
Alternatively, 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 BI). 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 2o identification of a recognition pattern for each native-peptide in terms of its shared chemical properties amongst recognised mimotope sequences.

SUBSTITUTE SHEET (RULE 26) Examples of such mimotopes are:
Table 2 Peptide Sequence Description SEQ ID
NO.

PI1 CRASGKPVNHSTRKEEKQRNGLL PS mimotope 8 Pl la (Ac) GKPV~1HSTGGC PS mimotope 9 PI Ib (Ac) GKPVNHSTRKEEKQRNGC PS mimotope 10 P1 lc CGKPVNHSTRKEEKQRNGLL (NH,) PS mimotope I I

PI Id (Ac) RASGKPVNHSTGGC PS mimotope 12 P 12 CGTRDWIEGLL P6 mimotope 13 P 12a CGTRDWIEGETL (NHS) P6 mimotope 14 P 12b (Ac) GTRDWIEGETGC P6 mimotope 15 P 13 CHPHLPRALMLL P7 mimotope I 6 Pl3a CGTHPHLPRALM (NHZ) P7 mimotope 17 Pl3b (Ac) THPHLPRALMRSC P7 mimotope 18 Pl3c (Ac) GPHLPRALMRSSSC P7 mimotope 19 P14 APEWPGSRDKRTC P8 mimotope 20 Pl4a (Ac) APEWPGSRDKRTLAGGC P8 mimotope 21 Pl4b CGGATPEWPGSRDKRTL (NHz) P8 mimotope 22 Pl4c CTRKDRSGPWEPA (NHz) P8 retro 23 Pl4d* (Ac) APCWPGSRDCRTLAG P8 mimotope 24 (cyclic) Pl4d (Ac) ACPEWPGSRDRCTLAG P8 mimotope 25 (cyclic) C-I C CATPEWPGSRDKRTLCG P8 mimotope 26 C-1 C CATPEWPGSRDKRTCG P8 mimotope 27 C3C12 TPCWPGSRDKRCG P8 mimotope 28 P9a CGAEWEQKDEL (NHz) P9 mimotope 29 P9b (Ac) AEWEQKDEFIC P9 mimotope 30 P9b* (Ac) GEQKDEFIC P9 mimotope 31 P9a* CAEGEQKDEL (NHZ) P9 mimotope 32 Carll CPEWPGCRDKRTG P8 mimotope 85 Carl2 TPEWPGCRDKRCG P8 mimotope 86 Alternatively, 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) . In order to achieve this the peptide sequence may be electively changed following the general rules:
* To maintain the structural constraints, prolines and glycines should not be 1o replaced SUBSTITUTE SHEET (RULE 26) * Other positions can be substituted by an amino acid that has similar physicochemical properties.
As such, each amino acid residue can be replaced by the amino acid that most closely resembles that amino acid. For example, A may be substituted by V, L
or I, as described in the following table.
Original residueExemplary Preferred substitutions substitution A V, L, I V

R K,Q,N K

N Q, H,K,R Q

D E E

C S S

Q N N

E D D

G A A

H N, Q,K,R N

I L, V, M, A, L
F

L I, V, M, A, I
F

K R, Q, N R

M L, F, I L

F L, V, I, A, W
Y,W

P A A

S T T

T I S S

W Y~ F Y

Y W, F,T,S F

V I, L, M, F, L
A

Particularly preferred IgE peptides are P8 and variants thereof (such as P14 or P 14a). These peptides, when coupled to a carrier are potent in inducing anti-IgE
immune responses, which responses are capable of inhibiting histamine release from 1o 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. Without being limiting to the scope of the present invention, 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:
P~ Xu Xz~ P~ xs~ X4~ Xs~ X6~ xs~ Xs or, SUBSTITUTE SHEET (RULE 26) P, X~, Xz, P, G, X4, R, D, Xs, Xs wherein; X, is an amino acid selected from E, D, N, or Q; X, is an amino acid selected from W, Y, or F; X3 is an amino acid selected from G or A, X4 is an amino acid selected from S, T or M; Xs is an amino acid selected from R or K; and X6 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 B 1 ). Monoclonal antibodies such as P 14/23, P 14/31 and P 14/33 are particularly suitable in this regard.
1o 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.
15 Accordingly, 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 P14.
The present inventors have shown that different methods by which the epitope 20 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 25 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. In this way, 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.
Hence, 3o without limiting the present invention the cyclised mimotopes which follow these new rules form one preferred aspect of the present invention.
SUBSTITUTE SHEET (RULE 26) Putative mimotope sequences that are not consistent with these rules may still raise useful antisera (for example P 14 and P 11 ), as such the following examples are only a sub-set of the types of mimotopes of the present invention.
Examples of preferred peptides that follow these newly defined structural rules are:
Table 3 Peptide sequence Mimotope of SEQ ID NO.

CSRPSPFDLFIRKSPTITC A-B loop of 33 CSRPSPFDLFIRKSPTC A-B loop of 35 CPSPFDLFIRKSPTITC A-B loop of 41 Cs3 CPSPFDLFIRKSPC A-B loop of 43 CTWSRASGKPVNHSTC C-D loop of 58 Cs3 CTWSRASGKPVNHC C-D loop of 60 CSR.ASGKPVNHSTC C-D loop of 66 Cs3 CSRASGKPVNHC C-D loop of 68 Cs3 CYAFATPEWPGSRDKRTLAC A-B loop of 45 Cs4 CYAFATPEWPGSRDKRTC A-B loop of 47 Cs4 CFATPEWPGSR.DKRTLAC A-B loop of 53 Ce4 CFATPEWPGSRDKRTC A-B loop of 55 C~4 CQWLHNEVQLPDARHC C-D loop of 70 CQWLHNEVQLPDAC C-D loop of 72 Cs4 CLHNEVQLPDARHC C-D loop of 78 Cs4 CLHNEVQLPDAC C-D loop of 80 It is envisaged that 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 1o 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 P 1 l, which are respectively 13 and 23 amino acids long. Non-peptidic mimotopes are envisaged to be 15 of a similar size, in terms of molecular volume, to their peptidic counterparts.
It will be apparent to the man skilled in the art which techniques may be used to confirm the status of a specific construct as a mimotope which falls within the scope of the present invention. Such techniques include, but are not restricted to, the following. The putative mimotope can be assayed to ascertain the immunogenicity of 20 the construct, in that antisera raised by the putative mimotope cross-react with the SUBSTITUTE SHEET (RULE 26) 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 P 14/23, P 14/31 or P 14/33, which would confirm the status of the putative mimotope as a mimotope of P8.
In one embodiment of the present invention at least one IgE epitope or mimotope are linked to Garner molecules to form immunogens for vaccination 1o protocols, preferably wherein the Garner 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.
As one embodiment, the peptides of the present invention are expressed in a fusion molecule with the fusion partner, wherein the peptide sequence is found within the 15 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. Thus, for example, for direct covalent coupling it is possible to utilise a carbodiimide, glutaraldehyde or (N-[y-maleimidobutyryloxy]
succinimide ester, utilising common commercially available heterobifunctional 20 linkers such as CDAP and SPDP (using manufacturers instructions). After the coupling reaction, the immunogen can easily be isolated and purified by means of a dialysis method, a gel filtration method, a fractionation method etc.
In a preferred embodiment the present inventors have found that peptides, particularly cyclised peptides may be conjugated to the carrier by preparing 25 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:

SUBSTITUTE SHEET (RULE 26) Scheme 1 Rink-Resin Solid Phase Peptide Synthesis X-AAL..C(Trt)....C(Trt)....AAn-Lys(Dde)-Rink-Resin Hydrazine hydrate X-AAi...C(Trt)....C(Trt)....AAr,-Lys(NH~-Rink-Resin (i) Succinic anhydride (ii) HBTU/HOBt/NnRVI/NZH4 O
X AAI...C(Trt)....C(Trt)....AA"-Lys-Rink-Resin Z= ~N ~ ~
t N I-TFA
O
~ N t-~t ~
X-AAA...C(-SH)....C(-SH)....AA~,-Lys-CONHZ Z = O
t N I-a H2O2 oxidation n X-AA~...C....C....AA,~-Lys-CONH2 t N I-a 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 SUBSTITUTE SHEET (RULE 26) 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.
The peptides thus synthesised can then be conjugated to protein carriers using the following technique:
Introduction of the aryl aldehyde functionality utilised the succinimido active 1o ester (BAL-OSu) prepared as shown in scheme 2 (see WO 98/17628 for further details). Substitution of the amino functions of a carrier eg BSA (bovine serum albumin) to ~SO% routinely give soluble modified protein. Greater substitution of the BSA leads to insoluble constructs. 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:

SUBSTITUTE SHEET (RULE 26) Scheme 2 HO O
H /~
O O-(CHZ)a-COZH + ~N=C=N--( ) + HO-N
O
Dioxan HO
H / \ O O
O OWC~)a O-N BAL-OSu O
Scheme 3 BSA~(NI-i~)m + mBAL-OSu 40% DMSO/buffer (pH 7.25,0.2 M NaOAc) 2 hr.
OH
O
BSA~MNH~CO-(CHZ)a-O ~ ~ H
BSA-BAL
Simple combination of modified peptide and derivatised carrier affords peptide carrier constructs readily isolated by dialysis - Scheme 4 -Peptide/carrier s conjugate:
IS
SUBSTITUTE SHEET (RULE 26) Scheme 4 OH
O
BSA O . \ / H + 3 m ~ X-AA1...C....C....AA°-Li s-CONHZ
NH-Z
m BSA-BAL
50% DMSO/buffer (pH 3.5, 0.1 M NaHC02) ~8-16 hr.
H
BSA H O
N-N
'' NH
O
H2N~ Lys...AA~...C.........C...AA~-X
O m 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 Garner 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 1o translocation domain of DT), or the purified protein derivative of tuberculin (PPD).
Alternatively the mimotopes or epitopes may be directly conjugated to liposome Garners, which may additionally comprise immunogens capable of providing T-cell help. Preferably the ratio of mimotopes to carrier is in the order of 1:1 to 20: l, and preferably each carrier should carry between 3-15 peptides.
In an embodiment of the invention a preferred carrier is Protein D from Haemophilus influenzae (EP 0 594 610 B 1). Protein D is an IgD-binding protein from Haemophilus influenzae and has been patented by Forsgren (WO 91/18926, granted SUBSTITUTE SHEET (RULE 26) EP 0 594 610 B 1 ). In some circumstances, for example in recombinant immunogen expression systems it may be desirable to use fragments of protein D, for example Protein D 1/3~d (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. For example, EP

635 B describes the use of chimaeric hepadnavirus core antigen particles to present foreign peptide sequences in a virus-like particle. As such, immunogens of the present invention may comprise IgE peptides presented in chimaeric particles consisting of 1o hepatitis B core antigen. Additionally, the recombinant fusion proteins may comprise the mimotopes of the present invention and a carrier protein, such as NS 1 of the influenza virus. For any recombinantly expressed protein which forms part of the present invention, the nucleic acid which encodes said immunogen also forms an aspect of the present invention.
15 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 2o 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). Alternatively, the peptides may be produced by recombinant methods, including expressing nucleic acid molecules encoding the mimotopes in a 25 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).
3o 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 SUBSTITUTE SHEET (RULE 26) 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. 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.
1o 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 2o 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 Garner molecule. Such booster constructs may comprise an alternative carrier or may be in the absence of any carrier.
In a further aspect of the present invention there is provided 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. These administrations may include injection via the intramuscular, intraperitoneal, intradermal or subcutaneous routes; or 3o 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, SUBSTITUTE SHEET (RULE 26) 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.
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. Generally, it is expected that each dose will comprise 1-1000 pg of protein, preferably 1-500 fig, more preferably 1-100 pg, of which 1 to SO~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.
In a related aspect of the present invention are ligands capable of binding to the peptides of the present invention. Example of such ligands are antibodies (or Fab fragments). Also provided are the use of the ligands in medicine, and in the manufacture of medicaments for the treatment of allergies. The term "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 teen 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 2o be encompassed by the term antibody as used herein.
Particularly preferred ligands are monoclonal antibodies. For example, P14/23, P 14/31 or P 14/33 are monoclonal antibodies which recognise P8 (which were raised by vaccination with a P 14 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 3o 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 SUBSTITUTE SHEET (RULE 26) human basophils, and also P 14/23 and P 14/31 have been shown to inhibit passive cutaneous anaphylaxis in vivo.
Therefore, mimotopes of IgE CE4 that are capable of binding to P 14/23, P 14/31 or P 14/33, and immunogens comprising these mimotopes, form an important aspect of the present invention. Vaccines comprising mimotopes that are capable of binding to P 14/23, P 14/31 or P 14/33 are useful in the treatment of allergy.
Additionally, 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 to of the present invention may also be used for the generation of monoclonal antibody hybridomas (using know techniques e.g. Kohler 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.
15 As the ligands of the present invention may be used for the prophylaxis or treatment of allergy, there is provided pharmaceutical compositions comprising the ligands of the present invention. Preferred pharmaceutical compositions for the treatment or prophylaxis of allergy comprise the monoclonal antibodies P
14/23, P 14/31 or P 14/33.
20 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. It may in some circumstances be appropriate to assay circulating anti-IgE
levels, 25 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. In addition, 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 30 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 SUBSTITUTE SHEET (RULE 26) and used in medicine. Accordingly, the use of P 14/23, P 14/31 or P 14/33 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, U.S.A. 1978. Conjugation of proteins to macromolecules is disclosed by Likhite, U.S.
Patent 4,372,945 and by Armor et al., U.S. Patent 4,474,757.
Description of drawings 1o FIG 1, Surface exposure of Cs3 an Ce4 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 PTmAb001 l, which were used as positive controls, were added at 1 pg/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-P 14 antiserum.
The P 14 antiserum from different mice, was added at different dilutions (80X
or 40X) 2o to contain approximately 1 pg/ml of anti-IgE antibody as measured by IgE
receptor-bound ELISA. Three negative controls were used: Anti-BSA antiserum, non-specific IgGl and a mixture of non-specific IgGI 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 2pg/ml).
FIG 4, Histamine release inhibition and anaphylactogenicity of anti-P 14 antiserum.
Anti-P14 Antisera from different mice were added at a 1/50 final dilution.
Monoclonal Abs were added at 2 pg/ml either in assay buffer or in anti-BSA
sera dilution 1/50. Three negative controls were used: Anti-BSA antiserum, non-specific IgGI and a mixture of non-specific IgGl diluted in anti-BSA antiserum. mAbl l is a 3o monoclonal antibody known to inhibit histamine release and was used as a positive control (added at 2~g/ml).

SUBSTITUTE SHEET (RULE 26) FIG 5, Antibody response anti-P 11. Peptide P 11 is coated at 1 pg/ml in carbonate buffer at +4°C overnight. After saturation of plates, two-fold serial dilution of sera are added and incubated for lh at 37°C. Bound IgG is detected with a biotinylated anti-mouse Ab followed by streptavidin-POD and TMB substrate. Time points measured A. days 14 post vaccination 1, and day 14 post v2; B, Day 14 post v3.
FIG 6, Anti-P11 IgG anti-human IgE titres. Human IgE was coated at 1 pg/ml.
Two-fold 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.
1 o FIG 7, Histamine release inhibition studies with anti-P 14 monoclonal antibodies, on allergic basophils donated by dustmite allergic patients (A 10 and A 11 ) and from grass pollen allergic patients (G8 and G4). PT11 (PTmAb0011) was used as a positive control, and non-specific IgG2a was used as an isotype control for the P14/23, and P14/33.
FIG 8, IgE domain structure. (A) Each domain is composed of two facing (3-sheets, shown in outline, one of 4 anti-parallel (3-strands (labelled 4) and the other of 3 anti-parallel (3-strands (labelled 3). (B) The seven strands are shown topographically as block arrows labelled a to f, partitioned between the two sheets as shown. The loop-connectivity of the strands is shown topologically with curved arrows: solid arrows 2o are intra-sheet loops and dashed arrows are inter-sheet loops. In the IgGl Fc domain structures a short c' strand forms part of the C-D loop, as is predicted for IgE Fc.
FIG 9, (A) Predicted structural alignment of the A-B loop sequences of human IgE
domains Cs2, 3 & 4 with the equivalent segments from the crystallographically determined structure of human IgGI Fc (domains C~y2 & Cy3). [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. 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 Ob. (B) Predicted structural alignment of the c d loops of human IgE Cs2,3 & 4 with human IgGI Fc. (3-strands in the IgGI structure are 3o underlined and labelled c, c ~ and d; amino acid residues at the ends of each sequence segment are numbered. Residues highlighted by the shaded boxes form (Cy2 &
Cy3) or are predicted to form (Cs2, by homology model refinement and experiment, Cs3, SUBSTITUTE SHEET (RULE 26) CE4, by homology-modelling) a protected core within the loop. 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 lb.
FIG 10, (A) The schematic structure of the A-B hairpin at the sheet-sheet interface of Ig constant domains. Adjacent anti-parallel ~i-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 1o 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. (B) The schematic structure of the A-B hairpin as in figure 3a, with residue positions optimal for cyclisation connected by dashed or solid dumbbells.
FIG 11, (A) The schematic structure of the C-D hairpin (loop plus supporting (3-strands) at the edge of the sheet-sheet interface of Ig constant domains.
Opposing anti-parallel ~3-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, forni part of the sheet-sheet interface within a domain.
Residues i+n &
j+m, where n is zero or even but m is odd, form part of the solvent-exposed surface of 2o 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 present invention is illustrated by but not limited to the following examples.
Part 1, Active vaccination studies Exam les 1.1 Peptide identification The peptides were identified by the following technique.

SUBSTiITUTE SHEET (RULE 26) 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 802.
The results of the test are shown in FIG 1.
Results From figure 1 there are a number of native peptides which may be used as immunogens for raising antibodies against IgE.
Table 4, Native surface exposed and continuous IgE peptides using the 1986 Padlan and Davies model.
PeptideSequence Location sequenceSEQ
and IgE Domain ID NO.

P6 GTRDWIEGE Cs3 2 P7 PHLPRALMRSTTKTSGPRA CE3/Cs4 3 P8 PEWPGSRDKRT CE4 (Pro451-Thr461)4 P200 LSRPSPFDLFIRKSPTITC Cs3 6 P210 WLHNEVQLPDARHSTTQPRKT Cs4 7 In addition to those peptides identified above, the following peptides have been identified using the same selection criteria with the Helm et al. IgE model (2IgE
model structure deposited 2/10/90 with PDB (Protein Data Bank, Research 2o Collabarotory for Structural Bioinformatics; http:\pdb-browsers.ebi.ac.uk)).
Table 5, Peptides identified using the Helm et al. 1990 model.
Name Sequence LocationSEQ ID NO.

I-90N LFIRKS Cs3 81 2-90N PSKGTVN Cs3 82 3-90N LHNEVQLPDARHSTTQPRKTKGS Cs4 83 4-90N SVNPGK C~4 84 SUBSTITUTE SHEET (RULE 26) These peptides, or mimotopes thereof, were synthesised and conjugated to carrier proteins for use in immunogenicity studies.
1.2 Synthesis of IgE peptidelProtein D conjugates using a succinimide-maleimide cross-linker 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 NHz activation of carrier residues by fixing a succinimide group.
to 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 15 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 a. Activation of the protein by reaction between lysine and succinimidyl ester S O '~ - O \
Protein NH2 + ~ N OC-(CH2)3-N
I I
O O ~/
O
O
Protein N~~~ -(CH2)3-O //
b. Coupling between activated protein and the peptide cysteine by reaction with the maleimido group SUBSTfNTE SHEET (RULE 26) Protein N~'IC-(CH2)~~~ + gH
O // Pep ide O O
S ,.wwvw Protein'' N~~~ -(CH2)3-~~
O //
O
Conjugue 1.3 Preparation of IgE peptide-Protein D conjugate 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-[y-maleimidobutyryloxy] succinimide ester -GMBS) is dissolved at 102.5 mg/ml in DMSO and added to the protein solution.
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 NaCI 150 mM Tween 80 0.1 % are performed to remove the excess of 2o 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.

SUBSTITUTE SHEET (RULE 26) In an analogous fashion 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 (P 14, SEQ ID NO. 20; CLEDGQVMDVDLL) and PS (P 1 l, SEQ
ID NO. 8; CRASGKPVNHSTRKEEKQRNGLL) were synthesised which were conjugated to both Protein D and BSA using techniques described above.
1.4 ELISA methods Anti peptide or Anti peptide carrier ELISA
The anti-peptide and anti-Garner immune responses were investigated using an ELISA
technique outlined below. Microtiterplates (Nunc) are coated with the specific antigen in PBS (4° overnight) with either: Streptavidin at 2pg/ml (followed by incubation with biotinylated peptide (1pM) for 1 hour at 37°C), Wash 3X PBS-Tween 20 0.1%.
Saturate plates with PBS-BSA 1%-Tween 20 0.1% (Sat buffer) for 1 hr at 37°. Add 1°
antibody = sera in two-step dilution (in Sat buffer), incubate 1 hr 30 minutes at 37°.
Wash 3X. Add 2° anti-mouse Ig (or anti-mouse isotype specific monoclonal antibody) coupled to HRP. Incubate 1 hr at 37°. Wash 5X. Reveal with TMB (BioRad) for 10 minutes at room temperature in the dark. Block reaction with 0.4N HZS04.
Method for the Detection of Anti-Human IgE Reactivity in Mouse Serum (IgE
plate bound ELISA) ELISA plates are coated with human chimaeric IgE at 1 pg/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/O.OS% Tween-20 containing 5% w/v Marvel milk powder for 1 hour at 37°C. Serial dilutions of mouse serum in PBS/O.OS% 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 3o with TMB substrate at 450nm. A standard curve of PTmAb0011 is included on each plate so that the anti-IgE reactivity in serum samples can be calculated in gg/ml.

SUBSTfNTE SHEET (RULE 26) Competition of IgE Binding with Mimotope Peptides, Soluble IgE or PTmAb0011 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. For competition between serum and PTmAb0011 for IgE binding, mixtures of serum and PTmAb0011-biotin are added to IgE-coated ELISA plates. PTmAb0011 binding is detected with Streptavidin-HRP
(1/1000).
L5 Human Basophil Assays Two types of assay were performed with human basophils (HBA), one to determine the anaphylactogenicity of the monoclonal antibodies, consisting of adding the antibodies to isolated PBMC; and a second to measure the inhibition of Lol P I
(a strong allergen) triggered histamine release be pre-incubation of the HBA with the monoclonal antibodies.
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 106 per ml. 100p1 cell suspension are added to wells of a V-bottom 96-well plate containing 1001 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.
For each serum dilution 3 wells are triggered by addition of 10.1 Lol p I
extract (final dilution 1/10000) and 3 wells have lOpl HBH/HSA added for assessment of anaphylactogenicity. Well contents are again mixed briefly using a plate shaker, 3o before incubation at 37°C for a further 30 minutes. Incubations are terminated by centrifugation at 500g for 5 min. Supernatants are removed for histamine assay using a commercially available histamine EIA measuring kit (Immunotech). Control wells SUBSTITUTE SHEET (RULE 26) containing cells without test sample are routinely included to determine spontaneous and triggered release. Samples of cells were lysed by 2X freeze/thawing to assay total histamine contained in the cells.
The results are expressed as following:
Anaphylactogenesis assay Histamine release due to test samples =
histamine release from test sample treated cells - % spontaneous histamine release.
to Blocking assay The degree of inhibition of histamine release can be calculated using the formula:
inhibition = 1 -(histamine release from test sample treated cells*) x 100 (histamine release from antigen stimulated cells*) Values corrected for spontaneous release.
Example 2, Immunisation of mice with Pl4 conjugates (Pl4-BSA, PI4 -BSA) induces production ojanti-human IgE antibodies.
The conjugates comprising the mimotope P14 (25ug protein/dose), described in example 1, 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 days 14, 24 and 72, sera was harvested 14 days after each immunisation.
The immune responses anti-peptide and anti-plate bound IgE was followed using ELISA methods described in Example 1. The antiserum was then tested for anaphylactogenicity and functional activity in the inhibition of histamine release from human allergic basophils (methods as described in example 1).

SUBSTITUTE SHEET (RULE 26) Immunogenicity Results Both conjugates, PD-P 14 and BSA-P 14, were capable of inducing anti-P 14 and anti-IgE immune responses. The results for anti peptide and anti-IgE responses, induced by the BSA-P14 conjugates, as measured at day 14 post third and fourth vaccination, are shown in table 6. PTmAb0011 is a monoclonal antibody which is known to bind to the Cs2 domain of IgE, and was used to quantify the anti-IgE responses in p.g/ml.
Table 6, Immunogenicity results for BSA-P14 conjugates Anti-peptide Anti-IgE Anti-IgE
responses responses responses (14 (14 (14 days days days post post post 3) 4) 3) (pg/ml (pg/ml Mid point (PTmAb0011)) (PTmAb0011)) titre AV SD GM AV SD GM AV SD GM

25974 22667 15492 9.9 2.18 0.7 22.9 33.5 4.8 fable jootnotes: AV (average), SD (standard deviation), GM (geomean) Mice vaccinated with BSA alone as controls did not generate any detectable anti-peptide or anti-IgE responses.
Functional activity results 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).
Summary 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 (PII-BSA, Pll -BSA) induces production of anti-human IgE antibodies.
SUBSTITUTE SHEET (RULE 26) Human IgE epitope peptide P11 was coupled to maleimide-activated BSA (Pierce) (BSA-CRASGKPVNHSTRKEEKQRNGLL). 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.
Results A homogeneous IgG anti-P11 response could be detected already after one injection, but increased further after the second and third injection (FIG. Sa and 5b).
All mice showed an anti-IgE response (ranging from 28 - 244 ~g/ml as expressed in mAb005 equivalents) after a third injection (FIG. 6).
Part 2, Functional activity of epitope specific monoclonal antibodies Example 4, Functional activity of monoclonal antibodies raised against P14 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 tumour cell line, and supernatants were screened for reactivity against both P14 peptide and IgE. Several cell lines were generated, amongst which were P 14/23, P 14/31 and P 14/33 which were deposited as Budapest Treaty patent deposit at ECACC on 26/1/00 under Accession No.s 00012610, 00012611, 00012612 respectively. All three monoclonal antibodies were confirmed to 3o bind to IgE, and specifically to P 14, by ELISA binding assays, and P 14 competition assays against monoclonal antibody binding to IgE.

SUBSTITUTE SHEET (RULE 26) The functional activity of these monoclonal antibodies was assayed in the human basophil histamine release inhibition assay as described in Example 1.
Results All of the P 14 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 (PTmAb0011) was included as a positive control antibody which is known to inhibit histamine release in vitro. All of the three P14 l0 monoclonal antibodies (23, 31, and 33) were potent in inhibiting histamine release from allergic basophils (See FIG. 7).
Example 5, Anti-IgE induced in mice after immunisation with conjugate are capable of blocking local allergic response in the Monkey Cutaneous Anaphylaxis model.
P 14/23 and P 14/31 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 100ng of anti-NP IgE (human IgE anti-nitrophenylacetyl (NP) .
purchased from Serotech) into both arms. After 24 hours, a dose range of the 2o 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 (HN)).
After 5 hours, 10 mg of 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. PTmAb0011, is a monoclonal antibody was used as a positive control.
3o SBmAb0006 was used as a negative control.

SUBSTITUTE SHEET (RULE 26) Table 7, P 14/23 results Amount of sampleMean diameter to be tested of oedema (,u~ (mm) P14/23 mAb001 I mAb0006 0.1 15/ 12 ND ND

0.05 15/ 15 ND ND

1VL - lVUI 11U11G.

5 Table 8, P 14/31 results Amount Mean diameter of of oedema (mm) sample to be tested (f~~

P14/31 mAb0011 mAb0006 0.1 22/25 ND ND

0.05 25/25 ND ND

As complete inhibition of anaphylaxis was observed with higher doses of monoclonal antibody, these antibodies are not anaphylactogenic per se when administered in vivo.
10 Example 6, Structural aspects of IgE mimotopes 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 15 which predict the optimal sites for peptide cyclisation. Peptides that use these sites of cyclisation form one prefered aspect of the present invention.
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) SUBSTITUTE SHEET (RULE 26) using the known structure of Cy2 and Cy3 of IgGI (Deisenhofer J., 1981.Biochemistry, 20, 2361-2370). In addition, models of the CE2 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 infra-sheet (A-B loop, FIG 9A) and inter-sheet loops in IgE
Fc domains (C-D loop, FIG 9B). Having defined the predicted IgE Fc A-B and C-D
loops together with their supporting (3-strands, 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 ~i-strands. Cyclisation is 1o preferably realised by the formation of a disulphide bond between terminal cysteines which therefore combine to become a cystine.
Based upon our structural alignments (FIG 9A & 9B) we have derived simple predictive rules in order to enhance the probability that the conformations adopted by ~5 a mimotope, after conjugation to a suitable carrier molecule, are similar to those of the parent epitope.
Rule 1 The hydrophobic cystine group should replace WT ~3-strand residues that belong to 2o the water-inaccessible core of the Ig constant domain, formed by the interface between the two (3-sheets.
Rule 2i For infra-sheet loops (e.g. the A-B loop) the cystine group should replace WT
25 residues that are from adjacent anti-parallel (3-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 IOB.
3o Rule 2ii For inter-sheet loops (e.g. the C-D loop) the cystine group should replace WT
residues on anti-parallel ~3-strands, one strand from each sheet. Following rule 1, the SUBSTITUTE SHEET (RULE 26) residues forming the optimal pair pack together from facing (3-sheet surfaces, so forning part of the interface between the sheets. The structural derivation of this rule for the C-D loops is shown schematically in FIG. 11 A and FIG. 11 B. 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 lOB (for A-B loops) and in FIG. 11B (for C-D loops).
Using the sequence alignment as shown in FIG 9A and 9B, together with the above to rules, the present inventors have designed the following peptides listed in tables 9 to 12. The peptides which are underlined (in solid or dotted lines) are the optimal peptides according to the above identified rules, the same lines are shown in and FIG 11B. Non-underlined sequences are mimotopes.
Table 9 , IgE CE3 A-B loop sequences Peptide SEQ ID NO.
sequence (solid and dotted underlined are optimal) C S R P SP F D L F I R K S P T I T
C

_C _S _R_P_S_PF D L F I R K S P T _C 35 C R P SP F D L F I R K S P C

C R P SP F D L F I R K S P T C

3g C R P SP F D L F I R K S P T I C

C R P SP F D L F I R K S P T I T
C

_CP SP F D L F I R K S P T I T
C

_ __ _ _ _ _ _ _ _ _ _ _ _ _ 41 C P SP F D L F I R K S P T I C

SUBST1ITUTE SHEET (RULE 26) Table 1 D , IgE Ce4 A-B loop sequences Peptide SEQ ID NO.
sequence (solid and dotted underlined are optimal) _C Y A F A T P E W P G S R D K R T C 4~

C Y A F A T P E W P G S R D K R C 4g _C_F_A_T_P_E_W_P_G_S_R_D_K_R_T_LA _C 53 C F A T P E W P G S R D K R C

SUBSTITUTE SHEET (RULE 26) Table ll, IgE CE3 C-D loop sequences Peptide SEQ ID NO.
sequence (solid and dotted underlined are optimal) -___ C T W S R A S G K P V N H S TC 5g C _S_RA__S_G_K_P_V_N_H_S_TC 66 C S R A S G K P V N H S C

C S R A S G K P V N H C

Table 12 , IgE Cs4 C-D loop mimotope sequences Peptide SEQ ID NO.
sequence (solid and dotted underlined are optimal) -A H

A H

A C

C Q W L HN E V Q _LP D C 72 A

A

A C

A H

A H

A H

_C_L _H_N_E_VQ _L_P_D _R C 7g _A _H

C L HN E V Q L P D R
A C

C L HN E Q L P D C
V A

SUBSTITUTE SHEET (RULE 26) IYDICATIO:YS RELATI:'1G TO DEPOSITED t1-tICROORGA:VISVt OR OTHER BIOLOGICAL :yIATERIAL
(PCT Rule I ibis) A. ihc indic~tions made below reiate to the depa~ued mtcroor~antsm or other btolog:cal mate::al ,~te:red ;o w the dcsc::peon on pace 1 R
:line 7-13 B. IDEVTIFICATION OF DEPOSIT Further deposits ue ideatitied on an additional sheer ~amc of dc;,ositary institution European Collection of Cell Cultures address of dcposituy institution (including postal code errd couan-r~
vaccine Research and Production Laboratory Public Health Laboratory Service Centre for Applied Microbiology Research Porton Down, Salisbury Wiltshire SP4 OJG, GB
Date of deposit ~ Accession i'lumber 00012610, 00012611 26 January 2000 (26/01/00) and 00012612 C. ADDfTIOi (,aL fYDICATIONS (leave blank ijnor applicable This inlottnation is continued on an additional shit Q
In respect of those designations where a European Patent is sought, a sample of the deposited microorganism will be made available until the publication of the mention of the grant of the European Patent or until the date on which the application has been refused or withdrawn, only by issue of such a sample to an expert nominated by the person requesting the sample.
D. DESICi'1ATED STATES FOR WHICH INDICATIONS ARE D2iDE (ijrhe irtdicatiorts ore nor jor all designated Srctes~
E. SEP.iR.~ITE FURi'lISHL'!C OF INDICATIONS (leave blank ijnor applicable) The indications listed below will be submitted to the lntemational Bureau lacer Ispecifvchegenercl ncrueojtheindicertorere.g.. ;~ccrssion r~umbv ojDeaosir'~
For receiving Office use only For lncernational Bure_u use only This sheet was received with the inte~ational application ~ This sheet wu received by the lnte:national Burau on:
Authorized officer Form pCTIR()/ I 3.t t Julv I 99Q1 Authorized office-.

SUBSTfTUTE SHEET (RULE 26) FOR THE PURPOSES OF INFORMATION ONLY
Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.
AL Albania ES Spain LS Lesotho SI Slovenia AM Armenia FI Finland LT Lithuania SK Slovakia AT Austria FR France LU Luxembourg SN Senegal AU Australia GA Gabon LV Latvia SZ Swaziland AZ Azerbaijan GB United KingdomMC Monaco TD Chad BA Bosnia and GE Georgia MD Republic of TG Togo Herzegovina Moldova BB Barbados GH Ghana MG Madagascar TJ Tajikistan BE Belgium GN Guinea MK The former TM Turkmenistan Yugoslav BF Burkina Faso GR Greece Republic of TR Turkey Macedonia BG Bulgaria HU Hungary ML Mali TT Trinidad and Tobago BJ Benin IE Treland MN Mongolia UA Ukraine BR Brazil IL Israel MR Mauritania UG Uganda BY Belarus IS Tceland MW Malawi US United States of America CA Canada IT Ttaly MX Mexico UZ Uzbekistan CF Central African Re ublic n NE Niger VN Viet Nam p CG Con o KE Ken NL Netherlands YU Yugoslavia g a Y

CH Switzerland KG Kyrgyzstan NO Norway ZW Zimbabwe CI C&te d'TvoireKP Democratic NZ New Zealand People's CM Cameroon Republic PL Poland of Korea CN China KR Republic PT Portugal of Korea CU Cuba KZ Kazakstan RO Romania -CZ Czech RepublicLC Saint Lucia RU Russian Federation DE Germany LI LiechtensteinSD Sudan DK Denmark LK Sri Lanka SE Sweden EE Estonia LR Liberia SG Singapore SEQUENCE LISTING
<110> SmithKline Beecham Biolicals s.a.
Peptide Therapeutics Ltd <120> Vaccine <130> B45173 <160> 86 <170> FastSEQ for Windows Version 3.0 <210> 1 <211> 22 <212> PRT
<213> Human and artificial sequence <400> 1 Arg Ala Ser Gly Lys Pro Val Asn His Ser Thr Arg Lys Glu Glu Lys 1 5 ~ 10 15 Gln Arg Asn Gly Thr Leu <210> 2 <211> 9 <212> PRT
<213> Human and artificial sequence <400> 2 Gly Thr Arg Asp Trp Ile Glu Gly Glu <210> 3 <211> 19 <212> PRT
<213> Human and artificial sequence <400> 3 Pio His Leu Pro Asg Ala Leu Met Arg Ser Thr Thr Lys Thr Ser Gly Pro Arg Ala <210> 4 <211> 11 <212> PRT
<213> Human and artificial sequence <400> 4 Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg Thr <210> 5 <211> 5 <212> PRT
<213> Human and artificial sequence <400> 5 Glu Gln Lys Asp Glu <210> 6 <211> 19 <212> PRT

<213> Human and artificial sequence <400> 6 Leu Ser Pro Ser Pro Phe Asp Leu Arg Ser Thr Arg Phe Ile Lys Pro Ile Thr Cys <210> 7 <211> 21 <212> PRT

<213> Human and artificial sequence <400> 7 Trp Leu Asn Glu Val Gln Leu Pro Arg Ser Thr His Asp Ala His Thr Gln Pro Lys Thr Arg <210> 8 <211> 23 <212> PRT

<213> Human and artificial sequence <400> 8 Cys Arg Ser Gly Lys Pro Val Asn Thr Lys Glu Ala His Ser Arg Glu Lys Gln Asn Gly Leu Leu Arg <210> 9 <211> 11 <212> PRT

<213> Human and artificial sequence <400> 9 Gly Lys Val Asn His Ser Thr Gly Pro Gly Cys <210> 10 <211> 18 <212> PRT

<213> Human and artificial sequence <400> 10 Gly Lys Val Asn His Ser Thr Arg Glu Gln Asn Pro Lys Glu Lys Arg Gly Cys <210> 11 <211> 20 <212> PRT

<213> Human and artificial sequence <400> 11 Cys Gly Pro Val Asn His Ser Thr Glu Lys Arg Lys Arg Lys Glu G1n 1 5 ~ 10 15 Asn Gly Leu Leu <210> 12 <211> 14 <212> PRT

<213> Human and artificial sequence <400> 12 Arg Ala Gly Lys Pro Val Asn His Gly Gly Cys Ser Ser Thr <210> 13 <211> 11 <212> PRT

<213> Human and artificial sequence <400> 13 Cys Gly Arg Asp Trp Ile Glu Gly Thr Leu Leu <210> 14 <211> 12 <212> PRT

<213> Human and artificial sequence ' <400> 14 Cys Gly Arg Asp Trp Ile Glu Gly Leu Thr Glu Thr <210> 15 <211> 12 <212> PRT

<213> Human and artificial sequence <400> 15 Gly Thr Asp Trp Ile Glu Gly Glu Cys Arg Thr Gly <210> 16 <211> 12 <212> PRT

<213> Human and artificial sequence <400> 16 Cys His His Leu Pro Arg Ala Leu Leu Pro Met Leu <210> 17 <211> 12 <212> PRT

<213> Human and artificial sequence <400> 17 Cys Gly His Pro His Leu Pro Arg Met Thr Ala Leu <210> 18 <211> 13 <212> PRT

<213> Human and artificial sequence <400> 18 Thr His His Leu Pro Arg Ala Leu Ser Cys Pro Met Arg <210> 19 <211> 14 <212> PRT

<213> Human and artificial sequence <400> 19 Gly Pro His Leu Pro Arg Ala Leu Met Arg Ser Ser Ser Cys <210> 20 <211> 13 <212> PRT

<213> Human and artificial sequence <400> 20 Ala Pro Trp Pro Gly Ser Arg Asp Thr Glu Lys Arg Cys <210> 21 <211> 17 <212> PRT

<213> Human and artificial sequence <400> 21 Ala Pro Trp Pro Gly Ser Arg Asp Thr Ala Gly Glu Lys Arg Leu Gly Cys <210> 22 <211> 17 <212> PRT

<213> Human and artificial sequence <400> 22 Cys Gly Ala Thr Pro Glu Trp Pro Arg Lys Arg Gly Gly Ser Asp Thr 1 5 ' 10 15 Leu <210> 23 <211> 13 <212> PRT

<213> Human and artificial sequence <400> 23 Cys Thr Lys Asp Arg Ser Gly Pro Pro Arg Trp Glu Ala <210> 24 <211> 15 <212> PRT

<213> Human and artificial sequence <400> 24 Ala Pro Trp Pro Gly Ser Arg Asp Thr Ala Gly Cys Cys Arg Leu <210> 25 <211> 16 <212> PRT

<213> Human and artificial sequence <400> 25 Ala Cys Glu Trp Pro Gly Ser Arg Cys Leu Ala Pro Asp Arg Thr Gly <210> 26 <211> 17 <212> PRT

<213> Human and artificial sequence <400> 26 Cys Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg Thr Leu Cys Gly <210> 27 <211> 16 <212> PRT

<213> Human and artificialsequence <400> 27 Cys Ala Pro Glu Trp Pro Ser Arg Asp Lys Arg Thr Thr Gly Cys Gly <210> 28 <211> 13 <212> PRT

<213> Human and artificialsequence <400> 28 Thr Pro Trp Pro Gly Ser Asp Lys Arg Cys Gly Cys Arg <210> 29 <211> 11 <212> PRT

<213> Human and artificialsequence <400> 29 Cys Gly Glu Trp Glu Gln Asp Glu Leu Ala Lys <210> 30 <211> 11 <212> PRT

<213> Human and artificialsequence <400> 30 Ala Glu Glu Gln Lys Asp Phe Ile Cys Trp Glu <210> 31 <211> 9 <212> PRT

<213> Human and artificialsequence <400> 31 Gly Glu Lys Asp Glu Phe Cys Gln Ile <210> 32 <211> 10 <212> PRT

<213> Human and artificialsequence <400> 32 Cys Ala Gly Glu Gln Lys Glu Leu Glu Asp -<210> 33 <211> 19 <212> PRT

<213> Human and artificialsequence <400> 33 Cys Ser Arg Pro Ser Pro Phe Asp Leu Phe Ile Arg Lys Ser Pro Thr Ile Thr Cys <210> 34 <211> 18 <212> PRT

<213> Human and artificial sequence <400> 34 Cys Ser Pro Ser Pro Phe Asp Leu Arg Lys Ser Thr Arg Phe Ile Pro Ile Cys <210> 35 <211> 17 <212> PRT

<213> Human and artificial sequence <400> 35 Cys Ser Pro Ser Pro Phe Asp Leu Arg Lys Ser Thr Arg Phe Ile Pro Cys <210> 36 <211> 16 <212> PRT

<213> Human and artificial sequence <400> 36 Cys Ser Pro Ser Pro Phe Rsp Leu Arg Lys Ser Cys Arg Phe Ile Pro <210> 37 <211> 15 <212> PRT

<213> Human and artificial sequence <400> 37 Cys Arg Ser Pro Phe Asp Leu Phe Lys Ser Pro Pro Ile Arg Cys <210> 38 <211> 16 <212> PRT

<213> Human and artificial sequence <400> 38 Cys Arg Ser Pro Phe Asp Leu Phe Lys Ser Pro Cys Pro Ile Arg Thr <210> 39 <211> 17 <212> PRT

<213> Human and artificial sequence <400> 39 Cys Arg Ser Pro Phe Asp Leu Phe Lys Ser Pro Ile Pro Ile Arg Thr Cys <210> 40 <211> 18 <212> PRT

<213> Human and artificial sequence <400> 40 Cys Arg Ser Pro Phe Asp Leu Phe Lys Ser Thr Ile Pro Ile Arg Pro Thr Cys <210> 41 <211> 17 <212> PRT

<213> Human and artificial sequence <400> 41 Cys Pro Pro Phe Asp Leu Phe Ile Ser Pro Ile Thr Ser Arg Lys Thr Cys <210> 42 <211> 16 <212> PRT

<213> Human and artificial sequence <400> 42 Cys Pro Pro Phe Asp Leu Phe Ile Ser Pro Ile Cys Ser Arg Lys Thr <210> 43 <211> 15 <212> PRT

<213> Human and artificial sequence <400> 43 Cys Pro Pro Phe Asp Leu Phe Ile Ser Pro Cys Ser Arg Lys Thr <210> 44 <211> 14 <212> PRT

<213> Human and artificial sequence <400> 44 Cys Pro Pro Phe Asp Leu Phe Ile Ser Pro Ser Arg Lys Cys <210> 45 <211> 20 <212> PRT

<213> Human and artificial sequence <400> 45 Cys Tyr Phe Ala Thr Pro Glu Trp Ser Arg Lys Arg Ala Pro Gly Asp Thr Leu Cys Ala <210> 46 <211> 19 <212> PRT

<213> Human and artificial sequence <400> 46 Cys Tyr Phe Ala Thr Pro Glu Trp Ser Arg Lys Arg Ala Pro Gly Asp Thr Leu Cys <210> 47 <211> 18 <212> PRT
<213> Human and artificial sequence <400> 47 Cys Tyr Ala Phe Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg Thr Cys <210> 48 <211> 17 <212> PRT

<213> Human and artificial sequence <400> 48 Cys Tyr Phe Ala Thr Pro Glu Trp Ser Arg LysArg Ala Pro Gly Asp Cys ' <210> 49 <211> 16 <212> PRT

<213> Human and artificial sequence <400> 49 Cys Ala Ala Thr Pro Glu Trp Pro Arg Asp ArgCys Phe Gly Ser Lys <210> 50 <211> 17 <212> PRT

<213> Human and artificial sequence <400> 50 Cys Ala Ala Thr Pro Glu Trp Pro Arg Asp ArgThr Phe Gly Ser Lys Cys <210> 51 <211> 18 <212> PRT

<213> Human and artificial sequence <400> 51 Cys Ala Ala Thr Pro Glu Trp Pro Arg Asp ArgThr Phe Gly Ser Lys Leu Cys <210> 52 <211> 19 <212> PRT

<213> Human and artificial sequence <400> 52 Cys Ala Ala Thr Pro Glu Trp Pro Arg Asp ArgThr Phe Gly Ser Lys Leu Ala Cys g <210> 53 <211> 18 <212> PRT
<213> Human and artificial sequence <400> 53 Cys Phe Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg Thr Leu 1 5 ~ 10 15 Ala Cys <210> 54 <211> 17 <212> PRT
<213> Human and artificial sequence <400> 54 Cys Phe Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg Thr Leu Cys <210> 55 <211> 16 <212> PRT
<213> Human and artificial sequence <400> 55 Cys Phe Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg Thr Cys <210> 56 <211> 15 <212> PRT
<213> Human and artificial sequence <400> 56 Cys Phe Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg Cys <210> 57 <211> 17 <212> PRT
<213> Human and artificial sequence <400> 57 Cys Thr Trp Ser Arg Ala Ser Gly Lys Pro Val Asn His Ser Thr Arg Cys <210> 58 <211> 16 <212> PRT
<213> Human and artificial sequence <400> 58 Cys Thr Trp Ser Arg Ala Ser Gly Lys Pro Val Asn His Ser Thr Cys <210> 59 <211> 15 <212> PRT
<213> Human and artificial sequence <400> 59 Cys Thr Trp Ser Arg Ala Ser Gly Lys Pro Val Asn His Ser Cys <210> 60 <211> 14 <212> PRT

<213> Human and artificial sequence <400> 60 Cys Thr Ser Arg Ala Ser Gly Lys Asn His Cys Trp Pro Val IS <210> 61 <211> 13 <212> PRT

<213> Human and artificial sequence <400> 61 Cys Trp Arg Ala Ser Gly Lys Pr0 His Cys Ser Val Asn <210> 62 <211> 14 <212> PRT

<213> Human and artificial sequence <400> 62 Cys Trp Arg Ala Ser Gly Lys Pro His Ser Cys Ser Val Asn <210> 63 <211> 15 <212> PRT

<213> Human and artificial sequence <400> 63 Cys Trp Arg Ala Ser Gly Lys Pro His Ser Thr Cys Ser Val Asn <210> 64 <211> 16 <212> PRT

<213> Human and artificial sequence <400> 64 Cys Trp Arg Ala Ser Gly Lys Pro His Ser Thr Arg Ser Val Asn Cys <210> 65 <211> 15 <212> PRT

<213> Human and artificial sequence <400> 65 Cys Ser Ala Ser Gly Lys Pro Val Ser Thr Arg Cys Arg Asn His <210> 66 <211> 14 <212> PRT

<213> Human and artificial sequence <400> 66 Cys Ser Ala Ser Gly Lys Pro Val Ser Thr Cys Arg Asn His <210> 67 <211> 13 <212> PRT

<213> Human and artificial sequence <400> 67 Cys Ser Ala Ser Gly Lys Pro Val Ser Arg Asn His Cys <210> 68 <211> 12 <212> PRT

<213> Human and artificial sequence <400> 68 Cys Ser Ala Ser Gly Lys Pro Val Cys Arg Asn His <210> 69 <211> 17 <212> PRT

<213> Human and artificial sequence <400> 69 Cys Gln Leu His Asn Glu Val Gln Asp Ala Arg His Ser Trp Leu Pro Cys <210> 70 ' <211> 16 <212> PRT

<213> Human and artificial sequence <400> 70 Cys Gln Leu His Asn Glu Val Gln Asp Ala Arg His Cys Trp Leu Pro <210> 71 <211> 15 <212> PRT

<213> Human and artificial sequence <400> 71 Cys Gln Leu His Asn Glu Val Gln Asp Ala Arg Cys Trp Leu Pro <210> 72 <211> 14 <212> PRT

<213> Human and artificial sequence <400> 72 Cys Gln Leu His Asn Glu Val Gln Asp Ala Cys Trp Leu Pro <210> 73 <211> 13 <212> PRT

<213> Human and artificial sequence <400> 73 Cys Trp His Asn Glu Val Gln Leu Ala Cys Leu Pro Asp <210> 74 <211> 14 <212> PRT

<213> Human and artificial sequence <400> 74 Cys Trp His Asn Glu Val Gln Leu AlaArg Cys Leu Pro Asp <210> 75 <211> 15 <212> PRT

<213> Human and artificial sequence <400> 75 Cys Trp His Asn Glu Val Gln Leu AlaArg His Cys Leu Pro Asp <210> 76 <211> 16 <212> PRT

<213> Human and artificial sequence <400> 76 Cys Trp His Asn Glu Val Gln Leu AlaArg His Ser Cys Leu Pro Asp <210> 77 <211> 15 <212> PRT

<213> Human and artificial sequence <400> 77 Cys Leu Asn Glu Val Gln Leu Pro ArgHis Ser Cys His Asp Ala <210> 78 <211> 14 <212> PRT

<213> Human and artificial sequence <400> 78 Cys Leu Asn Glu Val Gln Leu Pro ArgHis Cys His Asp Ala <210> 79 <211> 13 <212> PRT

<213> Human and artificial sequence <400> 79 Cys Leu Asn Glu Val Gln Leu Pro ArgCys His Asp Ala <210> 80 <211> 12 <212> PRT

<213> Human and artificial sequence <400> 80 Cys Leu Asn Glu Val Gln Leu Pro Cys His Asp Ala <210> 81 <211> 6 <212> PRT

<213> Human and artificial sequence <400> 81 Leu Phe Arg Lys Ser Ile <210> 82 <211> 7 <212> PRT

<213> Human and artificial sequence <400> 82 Pro Ser Gly Thr Val Asn Lys <210> 83 <211> 23 <212> PRT

<213> Human and artificial sequence <400> 83 Leu His Glu Val Gln Leu Pro Asp Ala Arg His Ser Thr Thr Asn Gln Pro Arg Thr Lys Gly Ser Lys <210> 84 <211> 6 <212> PRT

<213> Human and artificial sequence <400> 84 Ser Val Pro Gly Lys Asn <210> 85 <211> 13 <212> PRT

<213> Human and artificial sequence <400> 85 Cys Pro Trp Pro Gly Cys Arg Asp Lys Arg Thr Gly Glu <210> 86 <211> 13 <212> PRT

<213> Human and artificial sequence <400> B6 Thr Pro Trp Pro Gly Cys Arg Asp Lys Arg Cys Gly Glu

Claims (39)

Claims

1. A peptide comprising an isolated surface exposed epitope of the region spanning C.epsilon.3 and C.epsilon.4 domains of IgE, wherein the peptide is P7 (SEQ ID No.3), or mimotope thereof.

2. A peptide comprising an isolated surface exposed epitope of the C.epsilon.4 domain of IgE, wherein the peptide is P8 (SEQ ID No.4), or mimotope thereof.

3. A peptide comprising an isolated surface exposed epitope of the C.epsilon.4 domain of IgE, wherein the peptide is P9 (SEQ ID No.5), or mimotope thereof.

4. A peptide comprising an isolated surface exposed epitope of the C.epsilon.4 domain of IgE, wherein the peptide is 4-90N (SEQ ID No.84), or mimotope thereof.

5. A peptide as claimed in claim 2, wherein the mimotope of P8 is a peptide of the general formula:
P, X1, X2, P, X3, X4, X5, X6, X5, X5 wherein; X1 is an amino acid selected from E, D, N, or Q; X2 is as amino acid selected from W, Y, or F; X3 is an amino acid selected from G or A, X4 is an amino acid selected from S, T or M; X, is as amino acid selected from R or K; and X6 is an amino acid selected from D or E.

6. A peptide as claimed in claim 5, wherein the mimotope of P8 is a peptide of the general formula P, X1, X2, P, G, X4, R, D, X5, X5 wherein; X1 is an amino acid selected from E, D, N, or Q; X2 is an amino acid selected from W, Y, or F; X4 is an amino acid selected from S, T or M; X5 is an amino acid selected from R or K; and X6 is an amino acid selected from D or E.

7. A mimotope as claimed in any one of claims 1 to 6 wherein the mimotope is a peptide.

8. An immunogen for the treatment of allergy composing a peptide or mimotope as claimed in any one of claims 1 to 7, additionally comprising a carrier molecule.

9. An immunogen as claimed in claim 8, wherein the carrier molecule is selected from Protein D or Hepatitis B core antigen.

10. An immunogen as claimed in claim 8 or 9, wherein the immunogen is a chemical conjugate of the peptide or mimotope, or wherein the immunogen is expressed as a fusion protein.

11. An immunogen as claimed in any one of claims 8 to 10, wherein the peptide or peptide mimotope is presented within the primary sequence of the carrier.

12. A vaccine for the treatment of allergy comprising a peptide or immunogen as claimed in any one of claims 1 to 11, further comprising an adjuvant.

13. A vaccine for the treatment of allergy comprising a peptide comprising an isolated surface exposed epitope of the C.epsilon.3 domain of IgE, wherein the peptide is P5 (SEQ ID No.
1), or mimotope thereof, and an adjuvant.

14. A vaccine for the treatment of allergy comprising a peptide comprising an isolated surface exposed epitope of the C.epsilon.3 domain of IgE, wherein the peptide is P6 (SEQ ID No.
2), or mimotope thereof, and an adjuvant.

15. A vaccine for the treatment of allergy comprising a peptide comprising an isolated surface exposed epitope of the C.epsilon.3 domain of IgE, wherein the peptide is P200 (SEQ ID
No.6), or mimotope thereof, and an adjuvant.

16. A vaccine for the treatment of allergy comprising a peptide comprising an isolated surface exposed epitope of the C.epsilon.3 domain of IgE, wherein the peptide is 2-90N (SEQ ID
No.7), or mimotope thereof, and an adjuvant.

17. A vaccine for the treatment of allergy comprising a peptide comprising an isolated surface exposed epitope of the C.epsilon.3 domain of IgE, wherein the peptide is 2-90N (SEQ ID
No.82), or mimotope thereof, and an adjuvant.

18. A vaccine for the treatment of allergy comprising a peptide comprising an isolated surface exposed epitope of the C.epsilon.4 domain of IgE, wherein the peptide is 3-90N (SEQ ID
No.83), or mimotope thereof, and an adjuvant.

19. A vaccine as claimed in any one of claims 13 to 18, wherein the peptide is linked to a carrier molecule to form an immunogen.

20. A vaccine as claimed in claim 19, wherein the immunogen carrier molecule is selected from Protein D Hepatitis B core antigen.

21. A vaccine as claimed in claim 19 or 20, wherein the immunogen is a chemical conjugate of the peptide or mimotope, or wherein the immunogen is expressed as a fusion protein.

22. A vaccine as claimed in any one of claims 19 to 21, wherein the peptide or peptide mimotope is presented within the primary sequence of the carrier.

23. A ligand which is capable of recognising the peptides as claimed in any one of claims 1 to 6.

24. A ligand as claimed in claim 23, wherein the ligand is selected from P14/23, P14/31 or P14/33; which are deposited as Budapest Treaty patent deposit at ECACC on under Accession No.s 00012610, 00012611, 00012612 respectively.

25. A pharmaceutical composition comprising a ligand as claimed in claim 23.

26. A pharmaceutical composition comprising a ligand as claimed in claim 24.

27. A peptide as claimed in any one of claims 1 to 6 for use in medicine.

28. A vaccine as claimed in claim any one of claims 12 to 22 for use in medicine.

29. An immunogen as claimed is any are of claims 8 to 11, for use in medicine.

30. Use of a peptide as claimed in any one of claims 1 to 6 in the manufacture of a medicament for the treatment or prevention of allergy.

31. A ligand which is capable of recognising a peptide as claimed in any one of claims 1 to 6, for use in medicine.

32. Use of a ligand which is capable of recognising a peptide as claimed in any one of claims 1 to 6, in the manufacture of a medicament for the treatment of allergy.

33. Use of P14/23, P14/31 or P14/33; which are deposited as Budapest Treaty patent deposit at ECACC on 26/1/00 under Accession No.s 00012610, 00012611, 00012612 respectively, in the identification of mimotopes of P8.

34. A peptide which is capable of being recognised by P14/23, P14/31 or P14/33; which are deposited as Budapest Treaty patent deposit at ECACC on 26/1/00 under Accession No.s 00012610, 00012611, 00012612 respectively.

35. A vaccine comprising a peptide as claimed in claim 34.

36. A method of manufacturing a vaccine comprising the manufacture of an immunogen as claimed in any one of claims 8 to 11, and formu1ating the immunogen with an adjuvant.

37. A method for treating a patient suffering from or susceptible to allergy, comprising the administration of a vaccine as claimed in any one of claims 12 to 22, to the patient.

38. A method of treating a patient suffering from or susceptible to allergy comprising administration of a pharmaceutical composition as claimed in any one of claims 25 or 26, to the patient.

39. Use of any one of peptides SEQ ID No.s 1, 2, 3, 4, 5, 6, 7, 82, 83 and 84 in the manufacture of a vaccine for the treatment of allergy.