CA2363637A1 - Epitopes or mimotopes derived from the c-epsilon-2 domain 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, the novel medicaments are isolated peptides incorporating epitopes or mimotopes of surface exposed regions of the C.epsilon.2 domain of IgE. The inventors have found that these novel regions may be the target for both passive and active immunoprophylaxis or immunotherapy. The invention further relates to methods for production of the medicaments, pharmaceutical compositions containing them and their use in medicine. Also forming an aspect of the present invention are ligands, especially monoclonal antibodies, which are capable of binding the surface exposed IgE regions of the present invention, and their use in medicine as passive immunotherapy or in immunoprophylaxis.

Description

IGE, ANTAGONISTS THEREOF, AND T,HEI~ TH~RA~~EUTIC US~S
The present invention relates to the provision o nove me icaments for a treatment, prevention or amelioration of allergic disease. In particular, the novel medicaments are isolated peptides incorporating epitopes or mimotopes of surface exposed regions of the Cs2 domain of IgE. The inventors have found that these novel regions may be the target for both passive and active immunoprophylaxis or immunotherapy. The invention further relates to methods for production of the medicaments, pharmaceutical compositions containing them and their use in to medicine. Also forming an aspect of the present invention are ligands, especially monoclonal antibodies, which are capable of binding the surface exposed IgE
regions of the present invention, and their use in medicine as passive immunotherapy or in immunoprophylaxis. Non-peptidic mimotopes are also an embodiment of the present invention.
15 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. The role of IgE in the mediation of allergic responses, such 2o 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 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 encounter with allergen leads to the 25 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;
EP 0 477 231 B 1 ).
IgE, like all immunoglobulins, comprises two heavy and two light chains. The g heavy chain consists of five domains: one variable domain (VH) and four constant 3o domains (Cs 1 to CE4). The molecular weight of IgE is about 190,000 Da, the heavy chain being approximately 550 amino acids in length. The structure of IgE is discussed in Padlan and Davis (Mol. Immunol., 23, 1063-75, 1986) and Helm et al., (2IgE model structure deposited 2/10/90 with PDB (Protein Data Bank, Research Collabarotory for Structural Bioinformatics; http:\pdb-browsers.ebi.ac.uk)).
The second domain, CE2, approximately comprises amino acids 226-328 of IgE
(Flanagan J.G. and Rabbitts, T.H., 1982, EMBO J., 1, 655-660; Kenten et al., 1982, Proc.Natl.Acad.Sci., USA, 79, 6661-6665), but may encompass additional amino acids. By comparison with the known structure of IgGI, the start point of Cs3 domain is deduced to be Ser337.
In the past, a number of passive or active immunotherapeutic approaches designed to interfere with IgE-mediated histamine release mechanism have been to investigated with variable success. These approaches include interfering with IgE or allergen/IgE complexes binding to the FcsRI or FcsRII (the low affinity IgE
receptor) receptors, with either passively administered antibodies, or with passive administration of IgE derived 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.
It has been reported that the IgE domains involved in the binding of IgE to its receptor are Cs3 and Cg4 (Sutton, B.J. and Gould, H.J.; Nature, 1993, 366: 421-428;
WO 97/31948), and as such the previous therapeutic strategies have focussed on portions of these two domains.
. 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 anti-IgE antibodies generated during active vaccination, are capable of triggering histamine release per se, by the cross-linking of neighbouring IgE-receptor complexes in the absence of allergen. This phenomenon is termed anaphylactogenicity. Indeed many commercially available anti-IgE
monoclonal antibodies which are normally used for IgE detection assays, are anaphylactogenic, and consequently useless and potentially dangerous if administered 3o 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 can form the basis of an active vaccine or be used to raise appropriate to 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 Cs3 and Cs4 domains conjugated to carrier molecules for active 2o 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 (BS W 17) was described which was said to be capable of binding to IgE peptides contained within the Cs3 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 B 1.
Other approaches are based on the identification of peptides which themselves 3o 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 l, EP 0 341 290).

The present invention identifies novel surface exposed epitopes of the Cs2 domain of IgE, which may be used as the target of active or passive immuno-prophylaxis or therapy of allergic disease states. The present invention provides peptides incorporating the isolated epitopes per se, and further provides mimotopes of these newly identified epitopes, which maybe used per se in the treatment of allergy, or may be used in immunogens in active vaccination immunoprophylaxis or therapy.
The isolated epitopes or mimotopes of the present invention are preferably used in immunogens for active vaccination protocols to induce auto anti-IgE
antibodies, which themselves limit, reduce, or eliminate allergic responses or symptoms in to vaccinated subjects. Alternatively, the mimotopes or the immunogens of the present invention may be passively administered to a patient to limit, reduce, or eliminate allergic responses or symptoms in vaccinated subjects.
The peptides, which incorporate the isolated epitopes of the present invention are immunogenic, when suitably presented (e.g. on a carrier), and are capable of inducing auto anti-IgE antibodies which are non-anaphylactogenic, and function in ameliorating allergic responses in vivo. The epitopes or mimotopes of the present invention are preferably exclusively derived from CE2 domain, in that they are not derived from any other domain, i. e. they are not found within the CE 1, Ce3 or Cs4 domains. In particular, as a preferred embodiment they are derived from the domain 2o encoded by Ser222-A1a329 of human IgE.
Specific epitopes of the CE2 domain which have been found to be particularly suitable for use in the mimotopes or immunogens of the present invention are those which have been found by the present inventors to be surface exposed. The surface exposure of a region of IgE may be determined from its modelled structure.
(Padlan and Davies, Mol. Immunol., 23, 1063-75, 1986; Helm et al., 2IgE model structure deposited 2/10/90 with PDB (Protein Data Bank, Research Collabarotory for Structural Bioinformatics)). The present inventors have found that the epitopes useful in the present invention, are also found to be highly surface exposed. From this observation the present inventors have designed a method for providing other suitable 3o epitopes, those being epitopes having highly accessible regions calculated over a sliding window of five residues. The inventors have found that preferred regions of the CE2 domain have an accessible surface calculated over a sliding window of residues using the Molecular Simulations Software (MSI) of greater than 50 ~2, and preferably greater than 80~z.
Examples of such surface exposed CE2 IgE epitopes are:
Peptide Sequence Location sequence SEQ ID NO.
Name and IgE Domain P 1 EDGQVMDVD Cs2 (G1u270-Asp278)1 P2 STTQEGEL Cs2 (Ser283-Leu290)2 P3 SQKHWLSDRT Cs2 (Ser300-Thr309)3 P4 GHTFEDSTKK CE2 (G1y318-Lys327)4 PS GGGHFPPT Cs2 (G1y245-Thr250)5 P6 PGTINI Cs2 (Pro262-I1e267)6 P7 FTPPT Cs2 (Phe231-Thr235)7 Peptides incorporating such epitopes form a preferred aspect of the present invention. 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 Cs2 epitope in the context of the IgE molecule, also form part of the present invention.
to The present invention, therefore, includes the isolated peptides encompassing the native 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.
15 Wiley, Chichester, Ciba foundation symposium 119, p130-149; Gheysen, H.M., 1986, Molecular Immunology, 23,7, 709-715); or are capable of raising antibodies, when coupled to a suitable carrier, which antibodies cross-react with the native IgE epitope.
The mimotopes of the present invention may be peptidic or non-peptidic. A
peptidic mimotope of the surface exposed IgE epitopes identified above, may have a 2o sequence which differs from the native epitope but may also be of exactly the same sequence as the native epitope. Such~a molecule is described as a mimotope of the epitope, because although the two molecules share the same sequence, the mimotope will not be presented in the context of the whole Cs2 domain structure, and as such the mimotope may take a slightly different conformation to that of the native IgE
25 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 adjacent to other regions that may be distant in the primary sequence of IgE. As such, for example, a mimotope of P 1 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.
Preferred surface exposed regions which may be used in the present invention contain regions which are associated with a loop structure. The peptides or mimotopes of the present invention may comprise, therefore, a loop with N or C terminal extensions which may be the natural amino acid residues from neighbouring ~3-sheets.
As examples of this P 1 contains the C-D loop, P2 contains the D-E loop, P3 contains the E-F loop, P4 contains the F-G loop, PS contains the A-B loop, and P6 contains the l0 B-C loop of the Cs2 domain of IgE. Accordingly, mimotopes of these loops form an aspect of the present invention.
Particularly preferred medicaments are based on the epitope P1, and mimotopes thereof. Peptides incorporating this epitope, and mimotopes thereof, when coupled to a carrier, are potent in inducing anti-IgE immune responses which are capable of inhibiting histamine release from human basophils. Moreover, these immune responses are non-anaphylactogenic. Mimotopes of P 1 are described primarily as any entity which when formulated into an immunogen, is capable of inducing an immune response, which response is capable of recognising P 1 when in the context of Cc2 domain of IgE.
2o P 1 corresponds to the C-D loop of the Cs2 domain. The C-D loop structure of immunoglobulin folds corresponds to the linking chain between the end of the C
beta-strand and the beginning of the D beta-strand (Introduction to protein Structure, page 304, 2"° Edition, Branden and Tooze, Garland Publishing, New York, ISBN

2305-0), corresponding approximately to amino acid residue numbers Trp268-Ser280 of the IgE molecule. Accordingly, mimotopes of the C-D loop of IgE Ce2, and ligands that are capable of binding to the C-D loop of IgE Cs2, form a preferred aspect of the present invention.
Peptide mimotopes of the above-identified IgE epitopes may be designed for a particular purpose by addition, deletion or substitution of elected amino acids. Thus, the peptides of the present invention may be modified for the purposes of ease of conjugation to a protein earner. 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 to create a beneficial derivative, for example to enhance stability of the peptide. Those 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 occun-ing 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.
Examples of preferred cyclised peptides which contain a pair of cysteine residues to allow the formation of a disulphide bridge are PT1079 (SEQ ID NO.
14), PT1079GS (SEQ ID NO.15), PT1078 (SEQ ID N0.16), and PlSq (SEQ ID NO. 11).
2o Further, those skilled in the art will realise that mimotopes or immunogens of the present invention may be longer than the isolated epitopes, and may comprise the sequences disclosed herein. Accordingly, the mimotopes of the present invention may consist of addition of N and/or C terminal extensions of a number of other natural residues at one or both ends. The peptide mimotopes may also be retro sequences of the natural IgE sequences, in that the sequence orientation is reversed; or alternatively the sequences may be entirely or at least in part comprised of D-stereo isomer amino acids (inverso sequences). Also, the peptide sequences may be retro-inverso in character, in that the sequence orientation is reversed and the amino acids are of the D-stereoisomer form. Such retro or retro-inverso peptides have the advantage of being 3o non-self, and as such may overcome problems of self tolerance in the immune system (for example P 1 Sr - see below).

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 B 1). This technique, generates a large number of peptide sequences which mimic the structure of the native peptides and are, therefore, capable of binding to anti-native peptide antibodies, but may not necessarily themselves share significant sequence homology to the native IgE
peptide. This approach may have significant advantages by allowing the possibility of identifying a peptide with enhanced immunogenic properties (such as higher affinity binding characteristics to the IgE receptors or anti-IgE antibodies, or being capable of inducing polyclonal immune response which binds to IgE with higher affinity), or may overcome any potential self antigen tolerance problems which may be associated with the use of the native peptide sequence. Additionally this technique allows the identification of a recognition pattern for each native-peptide in terms of its shared chemical properties amongst recognised mimotope sequences.
Preferred examples of modified peptide mimotopes and examples of bacteriophage derived mimotopes include:

Peptide Sequence DescriptionSEQ
ID NO.

P15 CLEDGQVMDVDLL-NHz Pl mimotope8 PlSr LLDVDMVQGDELC-NH, P1 retro 9 mimotope PlSp WLEDGQVMDVDLC Pl mimotope10 PlSq CLEDGQVMDVDLC Pl mimotope11 C67/8 CFINKQMADLELCPRE P1 mimotope12 C67 CFMNKQLADLELCPRE P 1 mimotope13 PT1079 CLEDGQVMDVDLCPREAAEGDK P1 mimotope14 PT1079GS CLEDGQVMDVDLGGGSSGGP Pl mimotope15 PT1078 CLEDGQVMDVDCPREAAEGDK Pl mimotope16 PlSs QVMDVDL P1 mimotope17 EEC39-I KCREVWLGESETIMDCE P1 mimotope18 EEC39-J ACREVWLGESETIMDCD Pl mimotope19 EEC39-10 SCREVWLGESETVMDCG P1 mimotope20 EEC40-9 NCQDLMLREDAGCWSKM Pl mimotope21 EEC47-3 DCEEPMCSPVLLQQLKL P1 mimotope22 PlSt LEDGQVMDVD P1 mimotope23 P16 CSTTQEGELA-NHz P2 mimotope24 P2sh TTQEGE P2 mimotope25 P 17 CSQKHWLSDRT- NHZ P3 mimotope26 P4ex TYQGHTFEDSTKKCADSNPRGV P4 mimotope27 PSsh GGHFPP PS mimotope28 PSlongl CSSCDGGGHFPPTIQC PS mimotope192 PSlong2 CLQSSCDGGGHFPPTIQLLC PS mimotope193 In other mimotopes, the amino acid residues of P1, P2, P3, P4, P4, P5, P6 or P7 can each individually 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 P, A A

H N, Q,K,R R

I L, V, M,A,F L

L I, V, M,A,F I

K R, Q, N R

M L, F, I L

F L, V, I, A, L
Y

P A A

S T T

T S S

W Y, F Y

Y - W, F,T,S F

V I, L, M, F, L- _ A- _[

Ligands which are capable of binding to, the surface exposed Cs2 IgE
epitopes, and pharmaceutical compositions comprising them, form part of the present invention. Such ligands are capable of being used in passive prophylaxis or therapy, to by administration of the ligands into a patient, for the amelioration of allergic disease.
Examples of such useful ligands include monoclonal or polyclonal antibodies.
For example, antibodies induced in one animal may be purified and passively administered to another animal for the prophylaxis or therapy of allergy. The peptides of the present invention may also be used for the generation of monoclonal antibody 15 hybridomas (using known techniques e.g. Kohler and Milstein, Nature, 1975, 256, p495), humanised monoclonal antibodies or CDR grafted monoclonals, by techniques known in the art. Accordingly, in a related aspect of the present invention are ligands capable of binding to the surface exposed epitopes of the Cs2 domain of IgE.
Example of such ligands are antibodies (or Fab fragments). Such antibodies may be used in passive immunoprophylaxis or immunotherapy, or may be used themselves in the identification of IgE peptide mimotopes.
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 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 1o herein.
Preferred ligands are monoclonal antibodies. Particularly preferred ligands are ligands of P 1, and are preferably monoclonal antibodiesw For example, PTmAb0011 is the reference name for a mouse IgGI-type monoclonal antibody deposited as Budapest Treaty patent deposit at ECACC (European Collection of Cell Cultures, 15 Vaccine Research and Production Laboratory, Public Health Laboratory Service, Centre for Applied Microbiology Research, Porton Down, Salisbury, Wiltshire, OJG, UK) on 8'" March 1999 under Accession No. 99030805.
For example, PTmAb0011 recognises the C-D loop of CE2, and is itself capable of recognising IgE when bound to its high affinity receptor on human 2o basophils without causing degranulation, moreover it is able to block the passive sensitisation of non-allergic basophils by preventing the binding of IgE to FcsRla, and inhibits LolPl-triggered histamine release in allergic basophils. Another monoclonal antibody which recognises the C-D loop of CE2 is PTmAb0005 (available from Sigma Chemicals Catalogue number I6510, clone number GE-1). The present 25 invention provides this monoclonal antibody in a pharmaceutical composition.
The ligands of P1 have been used in bacteriophage panning techniques to identify new P 1 mimotopes. For example a monoclonal antibody which is capable of recognising P1, bound bacteriophages expressing the following sequences:
SE ID Se uence 29 C F I N K M. A D L E L C

Other peptide mimotopes of the C-D loop of Cs2 IgE have been identified by bacteriophage panning with PTmAb0011 and PTmAb0005. Examples of such mimotopes include:
imotope (PTmAb0011 phage panning) SEQ NO.
~ Peptide PI m _ SEQ ID No. 32 HCQQVFFPQDYLWCQRG ~

SCREVWLGGSEMIMDCE SEQ ID No. 33 ECNQNLSGSLRHVDLNC SEQ ID No. 34 DCEEPMCSPVLLQKLKP SEQ ID No. 35 SCREVWLGGSEMIMDCE SEQ ID No. 36 RCDQQLPRDSYTFCMMS SEQ ID No. 37 SCPAFPREGDLCAPPTV SEQ ID No. 38 FCPEPICSPPLSRMTLS SEQ ID No. 39 VCDECVSRELAL SEQ ID No. 40 WCLEPECAPGLL SEQ ID No. 41 VCDECVSRELAL SEQ ID No. 42 DCLSKGQMADLC SEQ ID No. 43 SCQGREVRRECW SEQ ID No. 44 WCREVWLGESETIMDCE SEQ ID No. 45 ACREVWLGESETIMDCD - SEQ ID No. 46 GCAEPKCWQALHQKLKP - SEQ ID No. 47 Peptide PI mimotope (PTmAb0005 phageSEQ NO.
panning ECRGPNMQMQDHCPTTD SEQ ID No. 48 QCNAVLEGLQMVDHCWN SEQ ID No. 49 CCVADPETQMTPSSEMF SEQ ID No. 50 HCKNEFKKGQWTYSCSD SEQ ID No. 51 QCRQFVMNQSEKEFGQC SEQ ID No: 52 NCFMNKQLADLELCPRE SEQ ID No. 53 SCAYTAQRQCSDVPNPG SEQ ID No. 54 GCFMNKQMADLELCPRTAA SEQ ID No. 55 ACFMNKQMADLELCPRVAA SEQ ID No. 56 GCFINKQLADLELCPRVAA SEQ ID No. 57 GCFMNKQLADWELCPRAAA SEQ ID No. 58 ECFMNKQLADSELCPRVAA SEQ ID No. 59 GCFMNKQLADPELCPREAE SEQ ID No. 60 GCFMNKQLVDLELCPRGAA SEQ ID No. 61 GCFMNKQLADLELCPREAA SEQ ID No. 62 GCFMNKQQADLELCPRGAA SEQ ID No. 63 GCFINKQMADLELCPREAA SEQ ID No. 64 Therefore, mimotopes of IgE Cs2 that are capable of binding to PTmAb0005 or PTmAb0011, and immunogens comprising these mimotopes, form an important aspect of the present invention. Vaccines comprising mimotopes that are capable of binding to PTmAb0005 or PTmAb0011 are useful in the treatment of allergy.
Without limiting the broader definition of P 1 mimotopes, from these and other phage-sequences, a core pattern has been identified for a sub-set of a P 1-like peptide.
This pattern is a sub-set of P 1 mimotopes, and describes its mimotopes in terms of the chemical properties of the amino acids in each position which are desirable for recognition to that particular anti-P 1 monoclonal antibody:
y h x d h h a n a n x y wherein:
to y......y Can be cyclised.
h Hydrophobic (cys;pro;gly;ala;val;ile;leu;trp;met;phe).
d Ionic bond donating (arg;lys;his;gln;asn;trp;tyr;thr;ser).
a Acidic (asp;glu).
n Ionically neutral/ non-polar (all except asp,glu,lys,arg).
x Any amino acid (n=0 - 3).
Accordingly, in one embodiment, mimotopes of Pl may be described by the general core feature y h x d h h a n a n x y described above. The peptide P1 or mimotope thereof may be optionally flanked by other amino acids at either end to aid conjugation or for any other purpose.
2o A particularly preferred mimotopes of P 1 is P 15s (SEQ ID NO. 17), whose Q, M, and first D residues have been shown to be critical for PTmAb0011 and PTmAb0005 binding activity (see examples). Hence a mimotope formula for PlSs, in which the non-essential residues were replaced by similar amino acids (as outlined above) would be:
Q, X,, M, D, X,, X2, X3 wherein X, is selected from V, I, L, M, F or A; Xz is selected from D or E;
and X3 is selected from L, I, V, M, A or F.
Also forming an important aspect of the present invention is the use of PTmAb0005 and PTmAb0011 in the identification of novel mimotopes of IgE, for 3o subsequent use in allergy therapy. As PTmAb000~ is commercially available, this ligand does not form a composition of the present invention, however, pharmaceutical compositions comprising PTmAb0005, and its use in the identification of P 1 mimotopes, form two important aspects of the present invention.
Mimotopes of P2, P3, P4 and PS also form an important aspect of the present invention. For example P16 and P17, are mimotopes of P2 and P3 respectively.
These peptides, when suitably presented on carriers, are both capable of inducing strong anti-IgE antibody responses which are non anaphylactogenic.
In a preferred embodiment, the peptides incorporating the above identified epitopes or peptidic or non-peptidic mimotopes of the present invention will be of a small size, such that they mimic a region selected from the whole Cs2 domain.
It is l0 envisaged that peptidic mimotopes, therefore, should be less than 100 amino acids in length, preferably shorter than 75 amino acids, more preferably less than 50 amino acids, and most preferable within the range of 4 to 25 amino acids long.
Specific examples of preferred peptide mimotopes are PT1079 and P 15q, which are respectively 21 and 13 amino acids long. Non-peptidic mimotopes are envisaged to be of a similar size, in terms of molecular volume, to their peptidic counterparts.
It will be apparent to the man skilled in the art that techniques may be used to confirm the status of a specific construct as a mimotope. Such techniques include the following: 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 2o native IgE molecule, and are also functional in blocking allergic mediator release from allergic effector cells. The specificity of these responses can be confirmed by competition experiments by blocking the activity of the antiserum with the mimotope itself or the native IgE, and/or specific monoclonal antibodies that are known to bind the surface exposed epitope within CE2 of IgE. Specific examples of such monoclonal antibodies for use in the competition assays include, for example, PTmAb0005 and PTmAb0011, which would confirm the status of the putative mimotope as a mimotope of the C-D loop of the Cs2 domain of IgE.
In one embodiment of the present invention at least one peptide as hereinbefore described, incorporating an IgE epitope or mimotope, is linked to carrier molecules to form immunogens for vaccination protocols. Preferably the carrier molecules are not related to the native IgE molecule. The peptides or mimotopes may be linked via chemical covalent conjugation or by expression of genetically engineered fusion partners; optionally via a linker sequence.
The covalent coupling of the peptide to the immunogenic carrier can be carried out in a manner well known in the art. 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 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.
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).
Alternatively the mimotopes or epitopes may be directly conjugated to liposome carriers, which may additionally comprise immunogens capable of providing T-cell 2o help. Preferably the ratio of peptides to carrier is in the order of 1:1 to 20:1, and preferably each carrier should carry between 3-15 peptides.
In an embodiment of the invention a preferred carrier is Protein D from Haemophilus influenzae (EP 0 594 610 Bl). Protein D is an IgD-binding protein from Haemophilus influenzae and has been patented by Forsgren (WO 91/18926, granted EP 0 594 610 B1). 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 chimeric 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 chimeric particles consisting of 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.
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 to solid phase procedure employing the well-known "F-moc" procedure and polyamide resin in the fully automated apparatus. Alternatively, those skilled in the art will know the necessary laboratory procedures to perform the process manually.
Techniques and procedures for solid phase synthesis are described in 'Solid Phase Peptide Synthesis:
A Practical Approach' by E. Atherton and R.C. Sheppard, published by IRL at Oxford 15 University Press (1989). Alternatively, 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 20 cloning, a laboratory manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989).
The immunogens of the present invention may comprise the peptides as previously described, including mimotopes, or may be immunologically cross-reactive derivatives or fragments thereof. Also forming part of the present invention 25 are portions of nucleic acid which encode the immunogens of the present invention or peptides, mimotopes or derivatives hereof. In addition, the immunogens of the present invention may comprise more than one type of epitope, i.e. P l and P2, in the same immunogen, or the mimotope itself may comprise more than one type of epitope.
30 The present invention, therefore, provides the use of novel peptides encompassing the epitopes or mimotopes of the present invention (as defined above), in the manufacture of pharmaceutical compositions for the prophylaxis or therapy of allergies. Immunogens comprising the mimotopes or peptides of the present invention, and carrier molecules are also provided for use in vaccines for the immunoprophylaxis or therapy of allergies. Accordingly, the mimotopes, peptides or immunogens of the present invention are provided for use in medicine, and in the medical treatment or prophylaxis of allergic disease. Accordingly, there is provided a method of treatment of allergy comprising the administration to a patient suffering from or susceptible to allergy, of a vaccine or medicament of the present invention.
Vaccines of the present invention, may advantageously also include an adjuvant. Suitable adjuvants for vaccines of the present invention comprise those 1o 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 15 aluminium or calcium salts (for example hydroxide or phosphate salts).
Other adjuvants include saponin adjuvants such as QS21 (US 5,057,540) and 3D-MPL (GB
2220 211 ).
The vaccines of the present invention will be generally administered for both priming and boosting doses. It is expected that the boosting doses will be adequately 20 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 Garner or may be in the absence of any carrier.
In a further aspect of the present invention there is provided a vaccine as 25 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 30 via mucosal administration to the oral/alimentary, respiratory, genitourinary tracts. A
preferred route of administration is via the transdermal route, for example by skin patches.

The amount of protein in each vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccinees. Such amount will vary depending upon which specific immunogen is employed and how it is presented. Generally, it is expected that each dose will comprise 1-1000 p,g of protein, preferably 1-500 p.g, preferably 1-100 pg, of which 1 to SOp,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.
to Pharmaceutical compositions comprising the ligands, described above, also form an aspect of the present invention. Also provided are the use of the ligands in medicine, and in the manufacture of medicaments for the treatment of allergies.
Aspects of the present invention may also be used in diagnostic assays. 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, 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, 2o the peptides may be used to affinity remove circulating anti-IgE from the blood of patients before re-infusion of the blood back into the patient.
Also forming part of the present invention is a method of identifying peptide immunogens for the immunoprophylaxis or therapy of allergy comprising using a computer model of the structure of IgE, and identifying those peptides of the IgE
which are surface exposed. These regions may then be formulated into immunogens and used in medicine. Accordingly, the use of PTmAb0005 and PTmAb0011 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.

The numbering system for IgE amino acid residues is often that described by Dorrington KJ and Bennicb H (1978) Immunol Rev 41 3-25; and also Bennicb H and Bahr-Lindastrom, H von (1978) Prog Immunol 11 49-58. However, subsequent determination of the gene and cDNA sequence of human IgE (Max, E.E. et al 1982, Cell 29 691-699; Flanagan J.G. and Rabbitts, T.H., 1982, supra; Kenten, J.H.
et al 1982, supra) revealed an extra leucine at position 273 (Kabat numbering) in which was not reported in the earlier papers. The numbering scheme used by the present inventors may, therefore, differ from that used by Dorrington KJ and Bennicb.
Description of drawings FIG 1, IgE amino acid surface exposure using the Padlan and Davies 1986 model.
FIG 2, Chemistry Scheme 1, solid phase peptide synthesis.
FIG 3, Chemistry Scheme 2 and Scheme 3, Modified carrier preparation.
FIG 4, Chemistry Scheme 4, Peptide/carner conjugation.
FIG 5, C67-8 Anti-IgE Data. (A) Anti-plate bound IgE reactivity of serum from Balb C mice immunised with 25p,g BSA-IgE C67-8 (conjugated using PTL chemistry) or 3p,g HepB core-IgE C67-8 construct. (B) Anti-receptor bound IgE reactivity of serum from Balb C mice immunised with 25p,g BSA-IgE C67-8 (conjugated using PTL
chemistry) or 3p.g HepB core-IgE C67-8 construct.
2o FIG 6, Competition assay with soluble IgE and IgE C67-8 peptide. Sera from BSA-IgE C67-8 or HBC-IgEC67-8 immunised mice were pre-incubated with soluble IgE
(lOp,g/ml) or IgE C67-8 peptide (25p.M) or the irrelevant peptide PT326 (25p,M) and added to IgE-coated ELISA plates. Data are mean ~ S.E.M (n = 10).
FIG 7, PT1079 Anti-IgE Data. (A) Anti-plate bound IgE reactivity of serum from Balb C mice immunised with 25~.g BSA-PT1079 (conjugated using PTL chemistry) or 3~g HepB core-1079 construct. (B) Anti-receptor bound IgE reactivity of serum from Balb C mice immunised with 25p,g BSA-1079 (conjugated using PTL
chemistry) or 3pg HepB core-1079 construct.
FIG 8, Competition assay with soluble IgE and PT1079 peptide. Sera from BSA-or HBC-1079 immunised mice were.pre-incubated with soluble IgE (lOpg/ml) or PT1079 peptide (25p.M) or the irrelevant peptide PT326 (25p.M) and added to IgE-coated ELISA plates. Data are mean ~ S.E.M (n = 10).

FIG 9, PT1078 Anti-IgE Data. (A) Anti-plate bound IgE reactivity of serum from Balb C mice immunised with 25pg BSA-PT1078 (conjugated using PTL chemistry.
(B) Anti-receptor bound IgE reactivity of serum from Balb C mice immunised with 25p,g BSA-1078 (conjugated using PTL chemistry).
FIG 10, Competition assay with soluble IgE and PT1078 peptide. Sera from BSA-1078 immunised mice were pre-incubated with soluble IgE (lOp,g/ml) or PT1078 peptide (25pM) or the irrelevant peptide PT326 (25pM) and added to IgE-coated ELISA plates. Data are mean ~ S.E.M (n = 10).
FIG. 11, PT1079gs Anti-IgE Data. (A) Anti-plate bound IgE reactivity of serum from l0 Balb C mice immunised with 3p,g HBC-1079gs, (B) Anti-receptor bound IgE
reactivity of serum from Balb C mice immunised with 3p.g HBC-1079gs.
FIG 12, Competition assay with soluble IgE and PT1079 peptide. Sera from HBC-1079gs immunised mice were pre-incubated with soluble IgE (lOp.g/ml) or PT1079 peptide (25p.M) or the irrelevant peptide PT326 (25pM) and added to IgE-coated ELISA plates. Data are mean ~ S.E.M (n = 10).
FIG 13, Inhibitory Activity of Mouse BSA-C67-8 induced Antisera. Cells from a LolPl-sensitive donor were treated with mouse serum (diluted 1/50) and then triggered to release histamine with LoIP 1. Data are mean t S.E.M. (n = 10).
FIG 14, Inhibitory Activity of Mouse Antisera induced by BSA-1078 and BSA
1079.
Cells from a LolPl-sensitive donor were treated with mouse serum (BSA and BSA-1078 anti-sera diluted 1/50; BSA-1079 antiserum diluted 1/1250) and then triggered to release histamine with LoIP 1. Data are mean t S.E.M. (n = 10).
FIG 15, Inhibitory Activity of Mouse Antisera induced by HBC-C67-8, HBC-1078, HBC-1079 and HBC-1079gs. Cells from a LolPl-sensitive donor were treated with mouse serum (HBC wild type (wt) and HBC-IgEC67-8 antisera diluted 1/50; HBC
1079 and HBC-1079gs antisera diluted 1/1250) and then triggered to release histamine with LoIP 1. Data are mean ~ S.E.M. (n = 10).
FIG 16 shows the concentration dependent binding of antibody PTmAb0005 and PTmAb0011to IgE.
3o FIG 17, shows the concentration dependent inhibition of IgE binding to an FcERIa/IgG construct with antibody PTmAb0005 and PTmAb0011 compared to control.

FIG 18, shows the concentration dependent inhibition of IgE binding to clipped ectodomain of FcsRIa-bound directly to plastic plates, by antibody PTmAb0005, compared to control.
FIG 19, shows IgE binding to FcsRII (CD23) by antibody PTmAb0005 (GE-1) and PTmAb0011.
FIG 20, shows the concentration-dependent blocking of histamine release from allergic human blood basophils with antibody PTmAb0005 and PTmAb0011 compared to control.
FIG 21, inhibition of LolP1 triggered histamine release in allergic human basophils by to both PTmAb0005 and PTmAb0011.
FIG 22, PTmAb0011 binding to different IgE; (A) PTmAb0011 Binding to Chimaeric IgE; (B) PTmAb0011 Binding to Myeloma IgE; (C) PTmAb0011 Binding to Antigen Orientated IgE; (D) PTmAb0011 Binding to Heat Denatured IgE.
FIG 23, Inhibition of IgE Binding to FcgRla by PTmAb0011.
15 FIG 24, Binding of PTmAb0011 to Receptor Bound IgE.
FIG 25, (A) The effect of PTmAb001 lon IgE binding to FcgRII on RPMI 8866 cells.
RPMI 8866 cells (1x106/ml) were incubated for an hour on ice with chimaeric IgE (1 p.g/ml) and anti-IgE mAb (10 to 0 ~g/ml). The IgE and anti-IgE were pre-incubated for an hour at room temperature prior to addition to the cells. Bound IgE was detected 2o with FITC-goat anti-human IgE. The results show the mean channel fluorescence (MCF) of duplicate samples as determined by flow cytometric analysis of 10,000 live gated events. (B) Non P 1 specific antibody PTmAb0017.
FIG 26, The effects of PTmAb0011 on IgE binding to FcERII on primary human B
cells. Peripheral blood mononuclear cells ( 1 x106/ml) were incubated for an hour on 25 ice with chimaeric IgE (1 p.g/ml) and anti-IgE mAb (10 to 0 ~g/ml; open) or equivalent concentrations of isotype matched control mAb (solid). The IgE and anti-IgE were pre-incubated for an hour at room temperature prior to addition to the cells.
Bound IgE was detected with FITC-goat anti-human IgE and the primary B-cells were elucidated with PE-conjugated anti-CD19. The results show the mean channel 3o fluorescence (MCF) of duplicate samples as determined by flow cytometric analysis of 5,000 live gated events.
FIG 27, Effects of PTmAb0011 on IgE secretion from primary human B-cells.

Peripheral blood mononuclear cells (2x105/well) were cultured in medium supplemented with IL-4 ( 1 Ong/ml) and anti-CD40 antibody ( 1 p,g/ml).
PTmAb0011 or an isotype matched control mAb were added ( 1 ug/ml) for 14 days and then cell supernatant harvested and analysed for total IgE content by ELISA. The results are expressed as a percentage of the amount of IgE secreted in the absence of any antibody.
FIG 28, Anaphylactogenicity of anti-human IgE monoclonal antibodies in allergic (A) and non-allergic (B) human basophils. PBMC from allergic donors or from non-allergic donors passively sensitised with 1 pg/ml chimeric IgE were treated with mAbs to for 30 min. at 37°C. Histamine release was determined by specific EIA. Data are mean of 3 separate experiments each with different donors.
FIG 29, Anaphylactogenicity of anti-human IgE antibodies in sensitised (A) and non-sensitised (B) human lung mast cells. Sensitised or non-sensitised crude human lung mast cell suspensions were treated with antibodies for 45 min. at 37°C.
Tryptase release in supernatants was determined by colorimetric assay. Data are means of duplicate determinations from a single representative experiment.
FIG 30, Anaphylactogenicity of anti-human IgE antibodies in RBL J41 cells through human FcERI (A) and mouse FcsRl (B). RBL J41 cells were sensitised either with chimeric human IgE or with mouse IgE and treated with antibodies for 30 min.
at 37°C. (3-hexosaminidase release was determined in supernatants by colorimetric assay.
Data are means of triplicate determinations from a single representative experiment.
FIG 31, Inhibition of allergen-triggered histamine release in human basophils by PTmAb0011. PBMC were incubated with PTmAb0011 either directly (allergic assay (A)) or together with IgE (blocking assay (B)) for 30 min. at 37°C.
Cells were subsequently triggered with antigen for 30 min. at 37°C and histamine release determined by specific EIA. Data are mean t s.e.m. from 3 separate experiments from different donors.
FIG 32, Inhibition of passive cutaneous anaphylaxis in Monkey skin by PTmAb0011 and PTmAb0005. Monoclonal antibody Dec7B (stanworth decapeptide) was used as a 3o control.

The present invention is illustrated by but not limited to the following examples.
Part 1 Mimotopes and immunogens of the present invention Example 1.
1.1 Surface exposed epitope identification, chemical conjugation and serological methods The surface exposed epitopes of the Cs2 domain of IgE were identified using the modelled structure of human IgE described by Padlan and Davies (Mol. Immunol., 23, l0 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 80~r2.
The 15 results of the test are shown in figure 1.
Results From figure 1, and also from repeats of the same procedure using the 1990 Helm et al.
model (2IgE model structure deposited 2/10/90 with PDB (Protein Data Bank, 20 Research Collabarotory for Structural Bioinformatics)), there are a number of native peptides which may be used as immunogens for raising antibodies against IgE.
Table 1, Native surface exposed and continuous IgE peptides PeptideSequence Location sequence SEQ ID NO.
Name and IgE Domain P1 EDGQVMDVD Cs2 (G1u270-Asp278)1 P2 STTQEGEL Cs2 (Ser283-Leu290)2 P3 SQKHWLSDRT Cs2 (Ser300-Thr309)3 P4 GHTFEDSTKK Cs2 (G1y318-Lys327)4 PS GGGHFPPT C2 (G1y245-Thr250) 5 P6 - PGTINI CE2 (Pro262-I1e267)6 P7 FTPPT Cs2 (Phe231-Thr235)7 These peptides, or mimotopes thereof, were synthesised and either conjugated to Garner proteins, or put into Hepatitis Core antigen constructs to form recombinant peptide expressing virus-like particles.
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.
The to Maleimide group is a cysteine-binding site. Therefore, for the purpose of the following examples, the IgE peptides to be conjugated require the addition of an N-terminal cysteine.
The coupling reagent is a selective heterobifunctional cross-linker, one end of the compound activating amino group of the protein carrier by an succinimidyl ester and the other end coupling sulhydryl group of the peptide by a maleimido group.
The reaction scheme is as the following a. Activation of the protein by reaction between lysine and succinimidyl ester So3- /~ -Protein~NH2 + CN-OC-(CH2)3 I I
0 .;.
O O
O
,;
~Protein\\~N~'C-(CHZ)~-b. Coupling between activated protein and the peptide cysteine by reaction with the maleimido group Protei \\ NH-C-(CH2)3 N~ ~ + SH~ww~,w~.
Peptide O
O~~ O
.-._ . ~S .niw,aw~
r'Protein N~~~ -(CH2)~-N/
C
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 -1o 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 table 1 or derivatives or mimotopes thereof) is dissolved at 4 mg/ml in 0.1 M acetic acid to avoid di-sulfide bond formation. A molar ratio of between 2 to 20 peptides per 1 molecule of activated Protein D is used for the coupling. The peptide solution is slowly added to the protein and the mixture is incubated 1 h at 25°C. The pH is kept at a value of 6.6 during the coupling phase. A quenching step is performed 2o by addition of cysteine (0.1 mg cysteine per mg of activated PD dissolved at 4 mg/ml in acetic acid 0.1 M), 30 minutes at 25°C and a pH of 6.5. Two dialyses against NaCI
150 mM Tween 80 0.1 % are performed to remove the excess of cysteine or peptide.
The last step is sterilising filtration on a 0.22 ~tm 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.

In an analogous fashion the peptides of the present invention may be conjugated to other carriers including BSA.
A mimotope of P1 was synthesised CLEDGQVMDVDLL (P1~, SEQ ID NO. 8) which was 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-carrier immune responses were investigated using an ELISA
to technique outlined below. Microtiterplates (Nunc) are coated with the specific antigen in PBS (4° overnight) with either: Streptavidin at 2p,g/ml (followed by incubation with biotinylated peptide (1~M) 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 SX. 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 ~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 PTmAb0011 is included on each plate so that the anti-IgE reactivity in serum samples can be calculated in qg/ml.

Method for the Detection ofAnti-Human Receptor-Bound IgE Reactivity in Mouse Serum ELISA plates are coated with recombinant human FcsRla at O.Spg/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 low fat milk powder for 1 hour at 37°C. Human IgE at 1 p,g/ml is then added for 1 hour at 37°C Serial dilutions of mouse serum in PBS/0.05% Tween-20/1% w/v BSA/4% New 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 to Streptavidin-HRP (1/1000). Conjugated antibody is detected 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 p.g/ml.
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 2o 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).
1.5 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 3o monoclonal antibodies.

Blood is collected by venepuncture from allergic donors into tubes containing 0.1 volumes 2.7% EDTA, pH 7Ø It is then diluted 1/2 with an equal volume of HBH
medium containing 0.1% human serum albumin (HBH/HSA). The resulting cell suspension is layered over 50% volume Ficoll-Paque and centrifuged at 4008 for minutes at room temperature. The peripheral blood mononuclear cell (PBMC) layer at the interface is collected and the pellet is discarded. The cells are washed once in HBH/HSA, counted, and re-suspended in HBH/HSA at a cell density of 2.0 x 106 per ml. 100p.1 cell suspension are added to wells of a V-bottom 96-well plate containing 100p,1 diluted test sample or monoclonal antibody. Each test sample is tested at a to range of dilutions with 6 wells for each dilution. Well contents are mixed briefly using a plate shaker, before incubation at 37°C for 30 minutes with shaking at 120 rpm.
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 15 anaphylactogenicity. Well contents are again mixed briefly using a plate shaker, before incubation at 37°C for a further 30 minutes with shaking at 120 rpm.
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 containing cells without test sample are routinely 20 included to determine spontaneous and triggered release. Wells containing cells +
0.05% Igepal detergent are also included to determine total cell histamine.
The results are expressed as following:
Anaphylactogenesis assay 25 Histamine release due to test samples =
histamine release from test sample treated cells - % spontaneous histamine release.
Blocking assay The degree of inhibition of histamine release can be calculated using the formula:
3o % 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 PI S conjugates (PI S-BSA or PI S -PD) induces production of anti-human IgE antibodies.
The conjugates comprising the mimotope P15 (25p,g protein/dose), described in 1.4, were administered into groups of 10 BalbC mice, adjuvanted with and oil in water emulsion containing QS21 and 3D-MPL described in WO 95/17210 . Boosting was be performed on day 21 and on day 42 and sera can be harvested on day 42 and 56.
to The immune response anti-peptide and anti-plate bound IgE was followed using methods described in Example 1.
Results The results for anti peptide and anti-IgE responses measured at day 14 post third vaccination are shown in table 2.
Table 2, P 15 Immnuogenicity results MimotopeAnti-peptide Anti-IgE
responses responses conjugate(Mid (p.g/ml point (PTmAb0011)) titre) Average Std Dev. Geomean Average Std Dev.Geomean P15-PD 41391 26858 36154 1.6 4.5 0.3 (n=16) P15-BSA 49591 9259 48719 2.2 2.5 1.0 (n=10) Example 3, Anti-IgE induced in mice after immunisation with conjugate are non anaphylactogenic Several dilutions of complete sera or IgG purified from conjugate immunised mice can be tested in presence of basophils from freshly harvested peripheral blood from allergic patients.
The anaphylactogenicity can be evaluated by the measuring of the histamine released induced by the antibodies to be tested as described below:

~ Erythrocytes are removed from peripheral blood on glucose dextran gradient ~ Cells are washed and plated with samples to be tested (for example allergen, antibodies, allergen plus antibodies,...) ~ After incubation , supernatants are collected and histamine release is measured according to manufacturer's instructions (Immunotech , histamine enzyme immunoassay kit) Neither antiserum generated with P 15-BSA or P 15-PD was shown to be anaphylactogenic.
Example 4, Anti-IgE induced in mice after immunisation with conjugate are capable of blocking IgE mediated histamine release induced by allergen triggering of basophil from allergic patient.
Histamine release can be measured in basophil samples triggered with various concentrations of allergen in presence or absence of several dilutions of complete sera or IgG purified from conjugate immunised mice. Blocking activity of anti-P15 antibodies in the antiserum was evaluated by the measuring of the inhibition of the histamine release induced by the allergen. Histamine release and inhibition was measured as described in example 3. As P15 is a mimotope of P1, PTmAb0011 was 2o used as a control as it is.known to bind to the same epitope (P1). The results are shown in table 3.
Table 3, Histamine release inhibition from allergic human basophils Antiserum Dilution % inhibition of histamine release P15-PD (mouse 4.12)1/30 79 P15-PD (mouse 4.5) 1/30 57 P15-BSA (mouse 7.3)1/30 67 P15-BSA (mouse 7.5)1/30 ~ 57 PTmAb0011 0.1 pg/ml 56 PTmAb0011 1 p,g/ml 90 anti-BSA serum 1/30 40 anti-PD serum 1/30 40 Example 5, Immunogenicity of Mimotopes of P2 and P3 The following mimotopes were conjugated to BSA using techniques described in example 1.2, and mice were immunised with the conjugates using the same formulation and schedule as that described in example 2.
P 16 CSTTQEGELA- NHZ P2 mimotope SEQ ID NO. 24 P17 CSQKHWLSDRT- NHz P3 mimotope SEQ ID NO. 26 The mice were bled after the last immunisation and tested for anti-IgE
reactivity in the IgE plate bound ELISA. The individual, average (Av), geomean (GM), results are summarised below (SD = standard deviation).
Table 4, P 16 and P 17 immunogenicity results Anti peptide immune responses/mouse ( days after third vaccination), Mid point titres.

1 2 3 4 5 6 7 8 9 10 Av SD GM

~

P 100 434928503434 6133223150852991 ~307ogg74544635154656 I

Example 6, Production of mimotopes of P1, and immunogenicitylfunctional IS activity thereof 6.1 Production of immunogens Mimotopes of P 1 were derived either by phage display techniques or by rational design by molecular modelling of the C-D loop of Cs2 domain of IgE. The following peptides were synthesised and formulated into both BSA-peptide conjugates and also into HepB core antigen recombinant constructs.
Name of Sequence SEQ ID NO.
peptide C67/8 CFINKQMADLELCPRE P 1 mimotope12 PT1079 CLEDGQVMDVDLCPREAAEGD P1 mimotope 14 PT1079GS CLEDGQVMDVDLCGGSSGGP P1 mimotope 15 PT1078 CLEDGQVMDVDCPREAAEGDK P1 mimotope 16 The peptides/protein Garner constructs were produced as follows. Acylhydrazine peptide derivatives were prepared on the solid phase as shown in scheme 1 (Figure 2).
These peptide derivatives can be readily prepared using the well-known 'Fmoc' procedure, utilising either polyamide or polyethyleneglycol-polystyrene (PEG-PS) supports in a fully automated apparatus, through techniques well known in the art l0 [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 15 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 (in this case Bovine Serum Albumin, BSA) using the following technique:
6.2 Modified Carrier Synthesis Introduction of the aryl aldehyde functionality utilised the succinimido active ester (BAL-OSu) prepared as shown in scheme 2 (FIG 3, see WO 98/17628 for further details). Substitution of the amino functions of BSA (bovine serum albumin) to ~50%
gave routinely soluble modified protein. Greater substitution of the BSA led to insoluble constructs. BSA and BAL-OSu were mixed in equimolar concentration in DMSO/buffer (see scheme 3, FIG. 3) for 2 hrs. This experimentally derived protocol gave ~50% substitution of BSA as judged by the Fluorescamine test for free amino groups.
6.3 Peptide-BSA construct.' Simple combination of modified peptide and derivatised BSA afforded peptide-BSA
constructs readily isolated by dialysis (scheme 4, FIG. 4). SDS-PAGE was used to confirm an increase in molecular weight.
6.4 Hepatitis Core antigen constructs Hepatitis Core antigen recombinant constructs (HBC) were also prepared, using to molecular biology techniques described in EP 0 421 635 B. In these HBC
experiments PT1079 was modified to remove the terminal lysine.
Peptide Sequence SEQ ID NO.

The expression of the P1-mimotope peptides was confirmed by BIAcore experiments with PTmAb0005 and PTmAb0011. The immunogenicity results were generated using doses of only 3 p,g/dose of HBC.
6.5 Immunogenicity studies The mimotope/HBC and mimotopeBSA constructs were purified and formulated into vaccines and adjuvanted with an oil in water emulsion containing QS21 and 3D-MPL
2o described in WO 95/17210 the (25~g BSA conjugate dose). These vaccines were administered into groups of 10 BalbC mice, and boosting was be performed on day 14 and on day 28 and sera was harvested on day 42. The immune response to anti-plate bound IgE and receptor orientated IgE, was then followed using the techniques described in example 1.4. Also, the activity of the antiserum in the inhibition of histamine release from allergic basophils was measured in the techniques described in 1.5.
6.6 Results All BSA and HBC constructs induced high titres of anti-IgE antibodies, when the IgE
was bound directly to the ELISA plate, and when orientated on the high affinity receptor. Moreover, all of these responses were confirmed to be specific, in that they were competed by free IgE and the mimotope itself, and not by non-specific peptides.
The anti-IgE induced by these immunogens were capable of inhibiting histamine release from human basophils derived from an allergic donor (rye grass, LOLP 1 ).
For the results for C67-8 see figures 5, 6, 13 and 15. For the results for PT1078 see figures 9, 10, 14 and 15. For the results for PT1079 see figures 7, 8, 14 and 15. For the to results for PT1079GS see figures 11, 12 and 15.
Moreover, the immune responses generated by these peptide mimotopes were not anaphylactogenic.
Table 5, Anaphylactogenicity of the PI mimotope antisera Immunogen Serum % Histamine Release Dilution Spontaneous Release 0.25 t 0.06 Naive serum 1/50 1.9 t 0.4 BSA 1/50 2.15 t 0.65 BSA-IgE C67-8 1/50 2.9 ~ 1.1 BSA-1078 1/50 5.00 ~ 1.40 BSA-1079 1/1250 0.43 ~ 0.04 HBCwt 1/50 3.5 ~ 1.0 '~

HBC-1079 1/1250 0.12 ~ 0.04 HBC-1079gs 1/120 0.02 ~ 0.02 HBC-IgE C67-8 1/50 2.14 ~ 0.26 Footnote to table, Cells from a LolPl-sensitive donor were treated with diluted mouse serum for 30 mins. Released histamine was determined by a commercially available histamine specific EIA. Data are mean t S.E.M. (n = 10).
Part 2 Ligands that bind to the epitopes and mimotopes of the present invention 2o Peptide immunogens are described in part l, which after administered to a mammal in the form of a vaccine, induce immune responses which (a) recognise IgE, and (b) are capable of inhibiting histamine release in vitro. Part 2 describes ligands that are capable of binding to the epitopes or mimotopes of the present invention, and describes their function. Two monoclonal antibodies have been identified, PTmAb0005 and PTmAb001 l, which recognise the c-d loop of Cs2 of IgE.
Mimotopes of this peptide have been shown in part 1 to be immunogenic and functional in active vaccination. This section describes the characterisation of these monoclonal antibodies and provides evidence of their utility in passive vaccination.
The target epitope of the antibodies was identified using phage panning techniques, namely sequence alignment of multiple bacteriophage targets, and subsequently refined and confirmed by domain mapping and site directed mutagenesis. The to functional activity of the antibodies has been confirmed not only in vitro by assaying for anti-IgE recognition and inhibition of allergic mediator release; but also in vivo in monkey Passive Cutaneous Anaphylaxis (PCA) studies.
Example 7, TS 7.1 Phage mapping of monoclonal antibody target Phage display libraries were used to map the binding sites of the monoclonal antibodies using three different phage libraries, displaying either the XCX,S, XCX,o or XAX,o peptide sequence (where X is any amino acid) at the N-terminus of the phage gVIIIp. Tables 6 and 7 show the results of selecting for peptide ligands with the anti 2o human IgE monoclonal antibodies PTmAb0005 and respectively. Amino acid pattern similarities between the peptides and human IgE revealed a strong homology match with the c-d loop in the Cs2 domain of IgE. The homology pattern produced from the phage returns was: Q h h a h a h (where h = hydrophobic amino acid and a =, acidic amino acid) and this aligned to the sequence QVMDVDL (SEQ ID NO. 17) in the C-25 D loop of the human IgE CE2 domain.
IgEC67, the peptide derived from phage panning experiments and which had the highest affinity to PTmAb0005 was also epitope mapped. This was performed by introducing random mutations by PCR mutagenesis and sub-cloning into the Fuse 3o vector for minor filamentous phage protein gIIIp display. The IgEC67 mutants were ranked in order of binding to PTmAb0005, as shown in Table 8. These, and other results, confirmed the importance of the amino acids within IgEC67 which aligned with the Cs2 epitope. For example the LBP, D10G, L11M, E12G and L13R mutants all reduced binding to the anti-IgE PTmAb0005 (Data not shown). Mutations in other sites had little effect on the affinity to PTmAb0005.
Random sub-libraries were made of the highest affinity PTmAb0005 and PTmAb0011 phage display derived peptides to enhance the affinity of the peptide to the antibodies by adapting methods described previously (Yu, J. and Smith, G.
P.
(1996) "Affinity maturation of phage-displayed peptide ligands." Methods in 1o Enzymology, 267, 3-27). This involved a DNA sub-cloning transfer from the major coat protein (gVIIIp) filamentous phage display vector to a lower copy number minor phage coat protein (gIIIp) display vector with a random PCR step. Sub-libraries were made of several phage sequences including the highest affinity PTmAb0005 ligand IgEC67 and IgE C67-8. Affinity matured sequences for C67 and C67-8 are shown in 15 tables 8 and 9 respectively. Included in the tables are ranking orders and also BIAcore affinities where available. IgEC67-8 capable of inducing an anti-human IgE
response in mice when the peptide expressing phage was used as an immunogen.
7.2 Confirmation of target by domain mapping 2o A number of constructs were generated in order to map the binding specificities of PTmAb0005 and PTmAb0011 with respect to the IgE constant domains. The following constructs were generated: Cg2'-4, CE2-3, C~3-4, Cs3-4L (CE3-4 plus linker sequence between domains Cs2 and Cs3) and CE2 alone.
25 Fragments encompassing various domains) of human IgE Fc were cloned using cDNA derived from the hybridoma line JW8/5/I3, which expresses a chimaeric human IgE (Neuberger, MS et al (1985) Nature 314 268-270; Bruggemann, M et al (1987) J Exp Med 166 1351-61). The IgE Fc fragments were amplified using appropriate primer pairs and JW8/5/3 cDNA as template. The cs2-4 fragment encodes 3o amino acids (aa) S225-K547. The cE3-4 fragment encodes as 6335-547. The cg3-fragment (domains 3 - 4 plus the linker sequence that joins cs2 to cs3) encodes as E322-K547. The cE2-3 fragment encodes as 5225-6436. The cs2 fragment encodes as S225-5324. All constructs contain a COOH terminal hexahistidine tail for detection and purification purposes. These fragments were cloned into a eukaryotic expression vector in frame with a CD33 derived leader encoding sequence to direct secretion of the expressed fragment. This enabled expression in mammalian cell lines. The vector was derived from pcDNA3.1+ (Invitrogen). To express the cloned fragments, the appropriate clones were transfected into COS-7 cells and the resulting conditioned medium harvested 48-60 hours post transfection.
The binding of PTmAb0005 and PTmAb0011 to the expressed IgE domains was 1o investigated by ELISA assay, by binding the constructs to an ELISA plate followed by incubation with the monoclonal antibodies, and revelation with an anti-mouse antibody. Also, binding to denatured constructs was investigated by the well known technique of Western blot.
15 The results for PTmAb0005 showed strong binding to Cs2-4, Cg2-3 and Cs2 in their native forms, and also bound to Cs2-4 and Cg2 after denaturation in western blot. No binding to CE3-4 or CE3-4L was observed in either assay.
PTmAb0011 also bound to Cs2-4, Cs2-3 and CE2 in their native form; and also bound 20 to CE2-4 and CE2 in their denatured forms.
It is clear therefore that both antibodies recognised a target epitope present in the Cg2 domain of IgE.
25 7.3 Confirmation of target by site directed mutagenesis Domain mapping studies demonstrated that both mAbs were able to bind to the Cs2 domain alone. Analysis of sequences derived from biopanning of phage displayed peptide libraries revealed that PTmAb0005 derived sequences showed striking similarity to Pl. This region forms a~loop between the C-D (3 strands of CE2 in the IgE
30 model structure. Site-directed mutagenesis studies were undertaken to validate this sequence as the epitope for PTmAb0005 and PTmAb0011.

Analysis of the panned phage sequences and a comparison of the IgE model structure (Helm et al 1990, supra) with the known structure of human IgGI Fc (Deisenhoffer, J., 1981, Biochemistry, 20, 2361-2370) led to the identification of three residues that were likely to be involved in antibody recognition. These residues are glutamine (Q) 273, methionine (M) 275 and aspartate (D) 276. Each of these was changed to alanine (A) and at least one other amino acid residue as shown below.
Q273: A and E (glutamate) M275: A; Q and K (lysine) D276: A and N (asparagine) to The alanine mutations changed both the structure and chemistry of the target residue whilst the other mutations maintained structure (as closely as possible) but altered the charge, e.g Q273E. Here, glutamate has essentially the same structure as glutamine but is negatively charged instead of neutral.
Each mutation was generated independently in a CE2-4 construct. Each mutant polypeptide was expressed to a similar level as the wild type (WT) Cs2-4 and each was able to bind to recombinant FcERIa ectodomain as efficiently as the WT cs2-product in ELISA based assays. Together, these data demonstrated that the mutations had no effect on the production/secretion of the polypeptides in the expression system and did not grossly affect the structure of the cs2-4 fragment.
2o All mutations essentially abrogated binding to both PTmAb0005 and PTmAb0011 except D276N which reduced binding to PTmAb0005 by only ~50% (Table 10).
Mutation of an alternative glutamine residue within CE2, Q317, was carried out to act as a control in these experiments. Q317E and Q317K mutants were generated and found to have no affect on the ability of PTmAb0005 and PTmAb0011 to recognise z5 Cs2-4. Similarly, recognition of FcsRla was not affected.
Thus, the binding activities of PTmAb0005 and PTmAb0011 are specifically affected by mutations within the C-D loop of Cs2.
In summary, the sequence P1 comprises the major binding determinant for both PTmAb0005 and PTmAb0011.

Table 10, recognition of IgE domain constructs by PTmAb0005 and PTmAb0011.
Mutation Recognition Recognition Recognition key by by PTmAb0005 PTmAb0011 FcsRla ectodomain WT cE2-4 ++++ ++++ ++++

Q273A - - ++++

Q273E - - ++++

M275A +/- +/- ++++

M275Q +/- +/- ++++

M275K +/- +/- ++++

D276A - - ++++

D276N ++ - ++-~+

Q317E ++++ ~ ++++ ++++

Q317K ++++ ++++ ++~-~

7.4 Refrned modelling of the C-D loop of IgE Cs2 As the exact structure of human IgE has not been determined yet (although a model is available) there are likely to be errors in this model structure after inspection at a detailed level. The present inventors, therefore, have refined this model of the Ce2 loop region of IgE by mapping this loop onto the equivalent region of Cy2 of human IgGI (Deisenhoffer J 1981 supra).
to From this new information about the confines of the structural features, a cyclised peptide was designed which when synthesised should adopt a conformation which closely resembles that of the C-D loop of C82 in the context of the full IgE
molecule.
This peptide, Ac-CLEDGVQMDVDLCPREAAEGDK(Ac)-NH2, was named PT1079 (SEQ ID NO. 14).

The affinity of PT1079 to both PTmAb0005 and PTmAb0011 was measured using a BIAcore technique and was found to exhibit very strong recognition to both of these monoclonal antibodies (recognised by both PTmab0005 and PTmAb0011 with apparent affinities of ~20nM and ~250nM respectively). Control, derivative peptides of PT1079, where the site of cyclisation was shifted by only one amino acid residue, thereby decreasing the length of peptide between the cyclisation sites by one amino acid residue (PT1078), reduced the binding of the peptide to either PTmab0005 or PTmAb0011. Also, PT1078 was modified such that an additional residue was added so that the loop region had the same number of residues as PT1079, however this 1o modification failed to restore binding to PTmAb0005 or PTmAb0011. Thus indicating the importance of correct presentation of the peptides of the present invention to adopt a shape which closely resembles the native target in the context of the whole IgE molecule.
-..',~ sp' 1 pos .

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-*- C67-8 p,, ~C ' M NN K ~ '~> A, ,I~ L ,.~ I~C -*- C67 ~ K C E WYIi'''G ;E S ,E."~'T= I M -*- EEC39-I
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Dnderlined - has been verified bY site-directed mutagenesis.

Summary The work described herein shows that the monoclonal antibodies PTmAb0005 and PTmAb0011 specifically recognise P 1. These antibodies have been used in phage display studies to identify mimotopes of the c-d loop of the Cs2 domain of IgE
which are recognised by the monoclonal antibodies with high affinity.

7. S Functional characteristics of characteristics of PTmAb0005 and PTmAb0011 The following experiments describe the functional characteristics of PTmAb0005 and PTmAb0011. Accordingly, the use of the targets of these antibodies will induce PTmAb0005 and PTmAb0011 like immune responses. The vaccination using these peptide based immunogens will, therefore, have the same functional characteristics.
Example 8, to 8.1 Materials and Methods 8.1. I FcERIa binding assay (Protein A plates) In this assay, a recombinant form of the ectodomain of the alpha chain of the high affinity receptor for IgE (alpha ectodomain) is utilised to bind chimaeric IgE. The 15 carboxyl terminus of the alpha ectodomain is fused to a human IgGI Fc sequence.
This enables the recombinant molecule to be bound to protein A coated microtitre plates via the Fc region. Hence, the majority of the alpha ectodomain molecules should be available for binding ligand and provides a system for the analysis of IgE -receptor interactions. The format described below is aimed at detecting the (high 20 affinity) receptor blocking activity of anti-IgE antibodies.
8.1.2 ELISA protocol for detection of binding of IgE to the alpha chain ectodomain of the high affinity receptor Coat protein A plates with 100p.1/well a.-ecto-Ig fusion protein diluted to 0.25p,g/ml in 25 blocking buffer (PBS/5% BSA/0.05% Tween-20). Incubate 1 hour at 37°C. Dilute chimaeric IgE to 0.03125p,g/ml in 10% pig serum. Dilute anti-IgE antibody to appropriate test concentrations) in this IgE solution. Incubate 1 hour at room temperature. Wash plates three times with PBS/0.05% Tween-20 using plate washer.
Add 100p.1/well of IgE:anti-IgE solution (quadruplicates of each anti-IgE
3o concentration are assayed). Incubate 1 hour at 37°C. Wash plates three times with PBS/0.05% Tween-20 using plate washer. Add 100p1/well of goat anti-mouse lambda chain HRPO conjugated antibody diluted 1:6000 dilution in blocking buffer.
Incubate 1 hour at 37°C. Wash plates three times with PBS/0.05% Tween-20 using plate washer. Add 200p,1/well of OPD substrate and incubate at room temperature in the dark for 2-10 minutes. Stop the reaction by the addition of 25p125% HZS04. Mix stopped reactions on plateshaker - SLOW speed. Read OD at 490nm.
A figure for the percentage of inhibition of binding of IgE to its receptor can be calculated. A maximum binding value for IgE is determined from the average of a set of wells that contained IgE in 10% pig serum alone (i.e no anti-IgE).
to The % inhibition value is calculated thus:
(max IgE value - average of anti-IgE replicates)/max IgE value x 100 8.1.3 FcERla binding assay (Clipped ectodomain) 15 This assay is essentially identical to the previous assay except that the FcsRIoc ectodomain/IgG construct is treated with the proteolytic enzyme Factor X to cleave the two moieties. The IgG Fc moiety is removed using protein A beads, and the Factor X is removed using strepatavidin beads, thus leaving an essentially pure alpha chain ectodomain product. In this assay format, the alpha ectodomain is bound directly to 2o plastic microtitre plates, all other assay details are as described above.
8.1.4 CD23-binding assay (FcERII, low affinity receptor).
This assay was performed on either RPMI 8866 cells or primary human B-cells;
two formats may be used, one for the detection of mAbs that bind to IgE associated with 25 FcsRII, and a second that analysed whether the mAbs interfered with IgE
association with FcERII. For the first assay cells were loaded with chimaeric IgE (1 p,g/ml) for an hour on ice in PBS, 1% FBS, 0.1% NaN3. Excess IgE was removed and anti-IgE mAb added. Bound mAb was elucidated with FITC-conjugated rat anti-mouse IgG, antibody. For the second assay, chimaeric IgE (1 p.g/ml) was pre-incubated with anti-30 IgE mAb for an hour at room temperature, with gentle mixing, prior to addition to the cells. The mixture was incubated with the cells for an hour on ice and then washed to remove unbound IgE. Bound IgE was detected with FITC-goat anti-human IgE or bound anti-IgE mAb was detected with FITC-conjugated rat anti-mouse IgG, antibody. Where studies were performed on PBMCs, constituent B-cells were identified with a PE-conjugated anti-CD19 antibody. Samples were analysed by flow cytometry.
8.2 Results The results for both PTmAb0005 and PTmAb0011 are shown in figures 16 to 21.
Figure 16 shows the concentration dependent binding of monoclonal antibody to plate bound IgE. Figure 17 shows the concentration dependent inhibition of IgE
binding to to an FcsRla/IgG construct with PTmAb0005 and PTmAb0011. Figure 18 shows the inhibition of IgE binding to clipped ectodomain of FcERIa-bound directly to plastic plates, by antibody PTmAb0005 and PTmAb0011. Figure 19 shoal s the lack of inhibition of IgE binding to FcsRII (CD23) by antibody PTmAb0005 (clone GE-1) and PTmAb0011. Figure 20 and 21, shows the concentration-dependent blocking of 15 histamine release from allergic human blood basophils with antibody PTmAb0005 and PTmAb0011.
PTmAb0011 is a mouse monoclonal antibody with specificity for human IgE, showing no cross-reactivity with other human Ig isotypes or rat/mouse IgE.
2o PTmAb0011 binds to both native and heat-treated IgE, when bound to an ELISA
plate in a random orientation, indicating that its recognition site on IgE is not heat labile.
PTmAb0011 also recognises IgE when bound via antigen to the ELISA plate.
Importantly this mAb can completely block the interaction between human IgE
and the a-chain binding component of the high affinity IgE receptor (FcERI).
However, 25 this mAb still recognises human IgE when pre-bound to FcERI, indicating that the mAb binding site is not lost upon receptor binding.
Example 9, 9.1 Analysis of IgE binding properties of PTmAb0011 by normal and Antigen 30 orientated ELISA
As described in Example l, the normal IgE binding ELISA method was performed by coating plates with human chimaeric IgE, myeloma IgE, human Ig isotypes or rodent IgE (lpg/ml in pH 9.6 carbonate/bicarbonate coating buffer). For antigen orientated ELISAs, NP-BSA was coated at a saturating concentration prior to the addition of chimaeric IgE (l~,g/ml). Alternatively, soluble human FcERI a-chain was coated (0.25~g/ml) followed by chimaeric IgE. The remaining ELISA was carried out as described in Experiment 1 (ELISA protocol for the detection of mouse anti-human IgE mAbs).
9.2 Results Figure 22 illustrates that PTmAb0011 binds to both human/mouse chimaeric IgE
and human myeloma IgE when bound to an ELISA plate in a random orientated manner.
Similarly, binding to antigen orientated IgE (i.e IgE bound to plate bound NP-BSA) is dose dependent. PTmAb0011 was also analysed for its ability to recognise chimaeric IgE following heat treatment at 56°C for a range of time periods.
Figure 22 also shows that the binding capacity of PTmAb0011 for IgE is unaffected by heat treatment.
The mAb characterisation was further extended to determine whether PTmAb0011 was able to inhibit the interaction of IgE with the a-chain component of the high affinity IgE receptor (Fig 23). Pre-incubation of IgE with PTmAb0011 prior to addition to plate bound FcsRI a-chain, resulted in a dose dependent inhibition of the interaction of IgE with FcERI a-chain. PTmAb0011 was also (Figure 24) recognises FcsRI a-chain associated IgE in a dose dependent manner.
Example 10 1 D.1 Analysis of IgE secretion from primary human B-cells PBMCs were plated at 2x105 cells per well in 96 U-well plates, in medium supplemented with both IL-4 and anti-CD40. PTmAb0011 or isotype matched control mAb was added and cells incubated for 14 days prior to harvesting of supernatants for IgE analysis. Total IgE levels were measured by coating ELISA plates with rabbit 3o anti-human IgE antibody (lOpg/ml) in O.SM carbonatelbicarbonate buffer (pH9.6).
Washed plates were blocked with PBS, 0.05% Tween 20, 5% BSA. Both cell supernatants and IgE standard were incubated with saturating amounts of PTmAb0011 (lOpg/ml) for an hour at room temperature prior to addition to the ELISA plate to allow for IgE/anti-IgE complexes to be formed. Following incubation and washing steps, bound IgE was detected with HRP-sheep anti-human IgE, followed by OPD substrate. Levels of IgE in the cell supernatants were then estimated relative to the standard curve.
10.2 Results IgE was pre-incubated with PTmAb0011 over a dose range from 10 p.g/ml to 0.5 pg/ml and examined for its effect on subsequent IgE binding to FcsRII on the human 1o B-cell line RPMI8866. Figure 25 illustrates that pre-incubation of IgE with PTmAb0011 enhances IgE binding to FcsRII. A non P 1 specific monoclonal antibody (PTmAb0017) did not enhance the binding of IgE to the FcBRII receptor.
PTmAb0011 also enhances IgE binding to Fc~RII on primary B-cells (Figure 26).
15 10. 3 Effects of PTmAb0011 on IgE secretion from primary human B-cells Peripheral blood mononuclear cells were cultured with PTmAb001 l, in the presence of additional IL-4 and anti-CD40 antibody to promote B-cell isotype switch to IgE
secretion. An ELISA assay was developed that allowed for measurement of total IgE
levels, that is free IgE and PTmAb0011 complexed IgE. To achieve such quantitation 2o secreted IgE was pre-incubated with saturating levels of PTmAb0011 to allow for all of the IgE to be complexed. The total IgE within the tissue culture supernatant was quantitated relative to a standard curve of IgE that had also been complexed with saturating levels of PTmAb0011. Figure 27, illustrates that in three different donors, incubation of primary B-cells with PTmAb0011 ( 1 ~g/ml) resulted in a significant 25 reduction in the total levels of secreted IgE. No such inhibition was seen with the isotype matched control antibody.
10. 4 Determination of histamine release from human basophils Two. assay formats were adopted. PBMC from non-allergic donors were passively 3o sensitised with 1 p,g/ml chimeric IgE for 30min at 37°C, washed and treated with monoclonal antibodies for 30min at 37°C. Alternatively PBMC from LolPl-sensitive donors were treated directly with monoclonal antibodies for 30min at 37°C. Reactions were terminated by centrifugation. Histamine release in cell supernatants was determined by specific immunoassay (Immunotech 2562). Total cellular histamine content was determined in cells lysed with 0.5% Igepal detergent.
10.5 Basophil blocking assay The ability of anti-IgE antibodies to block binding of chimeric IgE to FcsRl on human basophils was determined by incubation of PBMC from non-allergic donors with chimeric IgE in the presence of monoclonal antibodies and IL-3 for 30min at l0 37°C. Cells were washed and histamine release was triggered with NP-BSA for a further 30min at 37°C. Reactions were terminated by centrifugation and histamine release measured as above.
10.6 Allergic basophil inhibition assay 15 The ability of anti-IgE antibodies to inhibit allergen-triggered degranulation was investigated by pre-incubating PBMC from LolP1-sensitive donors with monoclonal antibodies for 30min at 37°C prior to triggering with LolPl.
10. 7 Determination of tryptase release from human lung mast cells 2o Crude mast cell suspensions were prepared from human lung tissue by enzymatic digestion with a cocktail comprising hyaluronidase, pronase and DNAse. Cells were either used directly or pre-sensitised with chimeric IgE prior to treatment with anti-IgE antibodies. Mast cell degranulation was determined by colorimetric assay of the granule enzyme tryptase.
10.8 Determination of ~3-hexosaminidase release from RBL cells transfected with human FceRl cr Transfected cell line RBL J41 was obtained from Dr B. Helm, University of Sheffield.
Cells were passively sensitised with either mouse monoclonal IgE anti-DNP or human chimeric IgE anti-NP and triggered with anti-human IgE antibodies.
Degranulation was measured by the colorimetric assay of ~3-hexosaminidase release.
10.9 Results 10.9. I Anaphylactogenicity of anti-IgE monoclonal antibodies in human basophils A number of different anti-IgE monoclonal antibodies were assayed for their ability to trigger histamine release from both allergic and non-allergic basophils (figure 28). In contrast to the other antibodies, PTmAb0011 was consistently unable to generate to significant histamine release.
10.9.2 Anaphylactogenicity of anti-IgE monoclonal antibodies in human lung mast cells PTmAb0011 was also unable to release significant amounts of tryptase in both sensitised and non-sensitised human lung mast cells (figure 29). Polyclonal anti-human IgE gave 60-70% release in these cells.
10.9.3 Anaphylactogenicity of anti-IgE monoclonal antibodies in RBL cells transfected with human FcERI a 2o RBL J41 cells, passively sensitised with chimeric human IgE anti-NP, could be triggered with antigen NP-BSA and with polyclonal anti-human IgE but not with PTmAb0011 (figure 30). In contrast, when cells were sensitised with mouse IgE
anti-DNP, both anti-human IgE antibodies were without effect. The cells could still be triggered by antigen DNP-BSA.
10.9.4 Basophil blocking assay PTmAb0011 was able to block the binding of IgE to FcsRl in non-allergic basophils and thus to inhibit subsequent triggering with NP-BSA antigen. The ICso value of this activity was around 60ng/ml (figure 31 ). PTmAb0011 was also able to potently inhibit LolPl-triggered histamine release from allergic basophils with an ICSO
value of 40ng/ml (figure 31).
Example 11, Monkey passive cutaneous anapylalaxis studies PTmAb0005 and PTmAb0011 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-1o 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)). 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. Dec7B, is described in EP 0 477 231 B, which recognises a peptide 496-X06 in the C84 domain of human IgE, was used as a positive control.
Amount Mean diameter of of oedema sample (mm) to be tested (lag) mAb0005 mAb0011 Dec7B IgE a-CMV IgE a-HIV
control control 20 0 0 0 19.5 21 10 0 0 20 20.7 22 1 4 4.5 25 22.7 23 0.1 14.8 15.7 20 21.8 22.5 0.05 17.8 18.7 22.5 21.5 22.8 PBS 23.2 28.2 26 24.5 22.5 The percentage inhibition of anaphylaxis are shown in Figure 32.

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SUBSTITUTE SHEET (RULE 26) applicant'soragent's -- ~lnternationalapplicauon~o.
fitrreference RE/B45172 INDICATIONS RELATtiYC TO DEPOSITED rIICROORCANtSrI
OR OTHER BIOLOGICAL VL~TERIAL
(PCT Rule l3bis) a. The indications made below relate to the deposited microorganism or ocher biological mate: ial rear-ed to in the description on page 1I . line 12-17 B. IDENTIFICATION OF DEPOSIT
Further deposits are identified on an additional sheet Yamr of depository institution European Collection of Cell Cultures address of depository institution (inctudirtg postal code and corurtrr~

Vaccine Research and Production Laboratory Public Health Laboratory Service Centre For Applied Microbiology Research Porton Down, Salisbury Wiltshire SP4 OJG, GB

Oate of deposit accession ~fumbrr 08 March 1999 C. ADDCfIONAL INDICATIONS (leave btattk ifnot applicable) This information is continued on an additional sheet 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 e:cpert nominated by the person requesting the sample.

D. DESIGNATED STATES FOR WHICH
INDICATIONS ARE MADE (if the indications art not for all designated States E. SEPARATE FURNISHING OF INDICATIONS
(leave btarrk ifrrot applicable) The indications listed below will besubmuted to the Intrrttauonal Bureau later(ipecifvrhe3enerelnaaurofrheindiccrionse ':accession "

.g..
iVumoer of Deposit ) . -~ ~---.-...5 ~~~.~.. u~c ~nty For lntemational Bureau use only This sheet was received with the international application a This sheet was received by the International Bureau on:
Authorized officer Authorized officer Farts PCTIRn/1311JuIv199Q1 SUBSTITUTE SHEET (RULE 26) SEQUENCE LISTING
<110> SmithKline Beecham Biologicals s.a.
Peptide Therapeutics <120> Vaccine <130> B45172 <160> 193 <170> FastSEQ for WindcwsVersion 3.0 <210> 1 <211> 9 <212> PRT

<213> Human and artificialsequences <400> 1 Glu Asp Gln Val Met Asp Asp Gly Val <210> 2 <211> 8 <212> PRT

<213> Human and artificialsequences <400> 2 Ser Thr Gln Glu Gly Glu Thr Leu <210> 3 <211> 10 <212> PRT

<213> Human and artificialsequences <400> 3 Ser Gln His Trp Leu Ser Arg Thr Lys Asp <210> 4 <211> 10 <212> PRT

<213> Human and artificialsequences <400> 4 Gly His Phe Glu Asp Ser Lys Lys Thr Thr <210> 5 <211> 8 <212> PRT

<213> Human and artificialsequences <400> 5 Gly Gly His Phe Pro Pro Gly Thr <210> 6 <211> 6 <212> PRT

<213> Human and artificialsequences <400> 6 Pro Gly Thr Ile Asn Ile <210> 7 <211> 5 <212> PRT

<213> Human and artificialsequences <400> 7 Phe Thr Pro Thr Pro <210> 8 <211> 13 <212> PRT

<213> Human and artificialsequences <400> 8 Cys Leu Asp Gly G1n Val Asp Val Leu Leu Glu Met Asp <210> 9 <211> 13 <212> PRT

<213> Human and artificialsequences <400> 9 Leu Leu Val Asp Met Va1 Gly Asp Leu Cys Asp Gln Glu <210> 10 <211> 13 <212> PRT

<213> Human and artificialsequences <400> 10 .

Trp Leu Asp Gly Gln Val Asp Val Leu Cys Glu Met Asp <210> 11 <211> 13 <212> PRT

<213> Human and artificialsequences <400> 11 Cys Leu Asp Gly Gln Val Asp Val Leu Cys Glu Met Asp <210> 12 <211> 16 <212> PRT

<213> Human and artificialsequences <400> 12 SS Cys Phe Asn Lys Gln Met Asp Leu Leu Cys Pro Arg Ile Ala Glu Glu <210> 13 <211> 16 <212> PRT

<213> Human and artificial sequences <400> 13 Cys Phe Asn Lys Gln Leu Asp Leu Leu Cys Pro Arg Met Ala Glu Glu <210> 14 <211> 22 <212> PRT
<213> Human and artificial sequences <400> 14 Cys Leu Asp Gly Gln Val Met Asp Leu Pro Glu Glu Val Asp Cys Arg Ala Ala Gly Asp Lys Glu <210> 15 <211> 20 <212> PRT

IS <213> Human and artificial sequences <400> 15 Cys Leu Asp Gly Gln Val Met Asp Leu Gly Ser Glu Val Asp Cys Gly Ser Gly Pro Gly <210> 16 <211> 21 <212> PRT

<213> Human and artificial sequences <400> 16 Cys Leu Asp Gly Gln Val Met Asp Cys Arg Ala Glu Val Asp Pro Glu Ala Glu Asp Lys Gly <210> 17 <211> 7 <212> PRT

<213> Human and artificial sequences <400> 17 Gln Val Asp Val Asp Leu Met <210> 18 <211> 17 <212> PRT

<213> Human and artificial sequences <400> 18 Lys Cys Glu Val Trp Leu Gly Glu Thr Met Cys Arg Ser Glu Ile Asp 1 5 l0 15 Glu <210> 19 <211> 17 <212> PRT

<213> Human and artificial sequences <400> 19 Ala Cys Glu Val Trp Leu Gly G1u Thr Met Cys Arg Ser G1u Ile Asp Asp <210> 20 <211> 17 <212> PRT

<213> Human and artificialsequences <400> 20 Ser Cys Glu Val Trp Leu Glu Ser Thr Val Met Asp Cys Arg Gly Glu Gly <210> 21 <211> 17 <212> PRT

<213> Human and artificialsequences <400> 21 Asn Cys Asp Leu Met Leu G1u Asp Gly Cys Trp Ser Lys Gln Arg Ala Met <210> 22 <211> 17 <212> PRT

<213> Human and artificialsequences <400> 22 Asp Cys Glu Pro Met Cys Pro Val Leu Gln Gln Leu Lys Glu Ser Leu Leu <210> 23 <211> 10 ' <212> PRT

<213> Human and artificialsequences <400> 23 Leu Glu Gly Gln Va1 Met Val Asp Asp Asp <210> 24 <211> 10 <212> PRT

<213> Human and artificialsequences <400> 24 Cys Ser Thr Gln Glu Gly Leu Ala Thr Glu <210> 25 <211> 6 <212> PRT

<213> Human and artificialsequences <400> 25 Thr Thr Glu Gly Glu Gln <210> 26 <211> 11 <212> PRT

<213> Human and artificialsequences <400> 26 Cys Ser Lys His Trp Leu Asp Arg G1n Ser Thr <210> 27 <211> 22 <212> PRT

<213> Human and artificial sequences <400> 27 Thr Tyr Gly His Thr Phe Glu Asp Lys Lys Cys Ala Gln Ser Thr Asp Ser Asn Arg Gly Val Pro <210> 28 <211> 6 <212> PRT

<213> Human and artificial sequences <400> 28 Gly Gly Phe Pro Pro His <210> 29 <211> 13 <212> PRT

<213> Human and artificial sequences <400> 29 Cys Phe Asn Lys G1n Met Ala Asp Leu Cys Ile Leu Glu <210> 30 <211> 13 <212> PRT

<213> Human and artificial sequences <400> 30 Cys Phe Asn Lys Gln Leu Ala Asp Leu Cys Met Leu Glu <210> 31 <211> 16 <212> PRT

<213> Human and artificial sequences <400> 31 Lys Cys Glu Val Trp Leu Gly Glu Thr Ile Met Asp Arg Ser Glu Cys <210> 32 <211> 17 <212> PRT

<213> Human and artificial sequences <400> 32 His Cys Gln Val Phe Phe Pro Gln Leu Trp Cys G1n Gln Asp Tyr Arg Gly <210> 33 <211> 17 <212> PRT
<213> Human and artificial sequences <400> 33 Ser Cys Arg Glu Val Trp Leu Gly Gly Ser Glu Met Ile Met Asp Cys Glu <210> 34 <211> 17 <212> PRT

<213> Human and artificialsequences <400> 34 Glu Cys Gln Asn Leu Ser Ser Leu His Val LeuAsn Asn Gly Arg Asp Cys <210> 35 <211> 17 <212> PRT

<213> Human and artificialsequences <400> 35 Asp Cys Glu Pro Met Cys Pro Val Leu Gln LeuLys Glu Ser Leu Lys Pro <210> 36 <211> 17 <212> PRT

<213> Human and artificialsequences <400> 36 Ser Cys Glu Val Trp Leu Gly Ser Met Ile AspCys Arg Gly Glu Met Glu <210> 37 <211> 17 <212> PRT

<213> Human and artificialsequences <400> 37 Arg Cys Gln Gln Leu Pro Asp Ser Thr Phe MetMet Asp Arg Tyr Cys Ser <210> 38 <211> 17 <212> PRT

<213> Human and artificialsequences <400> 38 Ser Cys Ala Phe Pro Arg Gly Asp Cys Ala ProThr Pro Glu~ Leu Pro Val <210> 39 <211> 17 <212> PRT

<213> Human and artificialsequences <400> 39 Phe Cys G1u Pro Ile Cys Pro Pro Ser Arg ThrLeu Pro Ser Leu Met Ser <210> 40 <211> 12 <212> PRT

<213> Human and artificialsequences <400> 40 Val Cys Glu Cys Val Ser Glu Leu Leu Asp Arg Ala <210> 41 <211> 12 <212> PRT

<213> Human and artificialsequences <400> 41 Trp Cys Glu Pro Glu Cys Pro Gly Leu Leu Ala Leu <210> 42 <211> 12 <212> PRT

<213> Human and artificialsequences <400> 42 Va1 Cys Glu Cys Val Ser Glu Leu Leu Asp Arg Ala <210> 43 <211> 12 <212> PRT

<213> Human and artificialsequences <400> 43 Asp Cys Ser Lys Gly Gln Ala Asp Cys Leu Met Leu <210> 44 <211> 12 <212> PRT

<213> Human and artificialsequences <400> 44 Ser Cys Gly Arg Glu Va1 Arg Glu Trp Gln Arg Cys <210> 45 <211> 17 <212> PRT

<213> Human and artificialsequences <400> 45 Trp Cys Glu Val Trp Leu Glu Ser Thr Ile Met Asp Cys Arg Gly Glu Glu <210> 46 <211> 17 <212> PRT

<213> Human and artificial sequences <400> 46 Ala Cys Arg Glu Val Trp Leu Gly Glu Ser Glu Thr Ile Met Asp Cys Asp <210> 47 <211> 17 <212> PRT
<213> Human and artificial sequences <400> 47 Gly Cys Ala Glu Pro Lys Cys Trp Gln Ala Leu His Gln Lys Leu Lys Pro <210> 48 <211> 17 <212> PRT
<213> Human and artificial sequences <400> 48 Glu Cys Arg Gly Pro Asn Met Gln Met G1n Asp His Cys Pro Thr Thr Asp <210> 49 <211> 17 <212> PRT
<213> Human and artificial sequences <400> 49 Gln Cys Asn Ala Val Leu Glu Gly Leu Gln Met Val Asp His Cys Trp Asn <210> 50 <211> 17 <212> PRT
<213> Human and artificial sequences <400> 50 Cys Cys Val Ala Asp Pro Glu Thr Gln Met Thr Pro Ser Ser Glu Met Phe <210> 51 <211> 17 <212> PRT
<213> Human and artificial sequences <400> 51 His Cys Lys Asn Glu Phe Lys Lys Gly Gln Trp Thr Tyr Ser Cys Ser Asp <210> 52 <211> 17 <212> PRT
<213> Human and artificial sequences <400> 52 Gln Cys Arg Gln Phe Val Met Asn Gln Ser Glu Lys Glu Phe Gly Gln Cys <210> 53 <211> 17 <212> PRT
<213> Human and artificial sequences <400> 53 Asn Cys Phe Met Asn Lys Gln Leu Ala Asp Leu Glu Leu Cys Pro Arg Glu <210> 54 <211> 17 <212> PRT
<213> Human and artificial sequences <400> 54 Ser Cys Ala Tyr Thr A1a Gln Arg G1n Cys Ser Asp Val Pro Asn Pro Gly <210> 55 <211> 19 <212> PRT
<213> Human and artificial sequences <400> 55 Gly Cys Phe Met Asn Lys Gln Met Ala Asp Leu Glu Leu Cys Pro Arg Thr Ala Ala <210> 56 <211> 19 <212> PRT
<213> Human and artificial sequences <400> 56 Ala Cys Phe Met Asn Lys Gln Met Ala Asp Leu Glu Leu Cys Pro Arg Val Ala Ala <210> 57 <211> 19 <212> PRT
<213> Human and artificial sequences <400> 57 Gly Cys Phe Ile Asn Lys Gln Leu Ala Asp Leu Glu Leu Cys Pro Arg Val Ala Ala <210> 58 <211> 19 <212> PRT
<213> Human and artificial sequences <400> 58 Gly Cys Phe Met Asn Lys Gln Leu Ala Asp Trp Glu Leu Cys Pro Arg Ala Ala Ala <210> 59 <211> 19 <212> PRT
<213> Human and artificial sequences <400> 59 Glu Cys Phe Met Asn Lys Gln Leu Ala Asp Ser Glu Leu Cys Pro Arg Val Ala Ala <210> 60 <211> 19 <212> PRT
<213> Human and artificial sequences <400> 60 Gly Cys Phe Met Asn Lys Gln Leu Ala Asp Pro Glu Leu Cys Pro Arg Glu Ala Glu <210> 61 <211> 19 <212> PRT
<213> Human and artificial sequences <400> 61 Gly Cys Phe Met Asn Lys Gln Leu Val Asp Leu Glu Leu Cys Pro Arg Gly Ala Ala <210> 62 <211> 19 <212> PRT
<213> Human and artificial sequences <400> 62 Gly Cys Phe Met Asn Lys Gln Leu Ala Asp Leu Glu Leu Cys Pro Arg Glu Ala Ala <210> 63 <211> 19 <212> PRT
<213> Human and artificial sequences <400> 63 Gly Cys Phe Met Asn Lys Gln Gln Ala Asp Leu Glu Leu Cys Pro Arg Gly Ala Ala <210> 64 <211> 19 <212> PRT
<213> Human and artificial sequences <400> 64 Gly Cys Phe Ile Asn Lys G1n Met Ala Asp Leu Glu Leu Cys Pro Arg Glu Ala Ala <210> 65 <211> 20 <212> PRT

<213> Human and artificial sequences <400> 65 Cys Leu Asp Gly Gln Val Met Asp Cys Pro GluAla Glu Val Asp Arg Ala Glu Asp Gly <210> 66 <211> 21 <212> PRT

<213> Human and artificial sequences <400> 66 Cys Leu Asp Gly Gln Val Met Asp Leu Cys ArgGlu Glu Val Asp Pro Ala Ala Gly Asp Glu <210> 67 <211> 17 <212> PRT

<213> Human and artificial sequences <400> 67 Gln Cys Ala Val Leu Glu Gly'Leu Val Asp CysTrp Asn Gln Met His Asn <210> 68 <211> 17 <212> PRT

<213> Human and artificial sequences <400> 68 Cys Cys Ala Asp Pro Glu Thr Gln Pro Ser GluMet Val Met Thr Ser Phe <210> 69 <211> 17 <212> PRT

<213> Human and artificial sequences <400> 69 Glu Cys Lys Ile G1u Gln Gln Cys Ile Val I1ePro Leu Ala Asp G1u Arg <210> 70 <211> 17 <212> PRT

<213> Human and artificial sequences <400> 70 Ser Cys Ala Tyr Thr Ala Gln Arg Gln Cys Ser Asp Val Pro Asn Pro Gly <210> 71 <211> 17 <212> PRT
<213> Human and artificial sequences <400> 71 Glu Cys Arg Gly Pro Asn Met Gln Met Gln Asp His Cys Pro Thr Thr Asp IS
<210> 72 <211> 17 <212> PRT
<213> Human and artificial sequences <400> 72 Glu Cys Leu Val Tyr Gly Gln Met Ala Asp Cys Ala Ala Gly Gly Trp Pro <210> 73 <211> 17 <212> PRT
<213> Human and artificial sequences <400> 73 Gln Cys Arg Gln Phe Val Met Asn Gln Ser Glu Lys Glu Phe Gly Gln Cys <210> 74 <211> 17 <212> PRT
<213> Human and artificial sequences <400> 74 His Cys Lys Asn Glu Phe Lys Lys~Gly Gln Trp Thr Tyr Ser Cys Ser Asp <210> 75 <211> 17 <212> PRT
<213> Human and artificial sequences <400> 75 Cys Cys Val Thr Asp Val Gln Thr Thr Asn Met Asp Val Pro Ala Gly Gln <210> 76 <211> 17 <212> PRT
<213> Human and artificial sequences <400> 76 Thr Cys Cys Val Thr Asp Ile Pro Pro Pro Asp Tyr Glu Gln Ser Leu Gly S
<210> 77 <211> 17 <212> PRT
<213> Human and artificial sequences <400> 77 Cys Cys Glu Ser Asp Ile Pro Leu Asn G1u Leu His Ala Leu Ala Asp Pro <210> 78 <211> 17 <212> PRT
<213> Human and artificial sequences <400> 78 Cys Cys Lys Ser Asp Ile Pro Ser Pro Val Thr Gln Phe Asn Thr Met 2S Lys <210> 79 <211> 17 <212> PRT
<213> Human and artificial sequences <400> 79 Cys Cys Gln Ser Asp Val Pro His Gln Pro Gly Ile Asn Asp Leu His Val <210> 80 <211> 17 <212> PRT
<213> Human and artificial sequences <400> 80 4S Cys Cys Met Ser Asp Thr Pro Asp Ile Ser Arg Leu Pro Val Pro Asp Ser SO <210> 81 <211> 17 <212> PRT
<213> Human and artificial sequences SS <400> 81 Cys Cys Met Ser Asp Ser Pro Ala~Asp Pro Asn Arg Gly Leu Pro Ile Trp <210> 82 <211> 14 <212> PRT
<213> Human and artificial sequences <400> 82 Cys Cys Leu Ser Asp Asp Ala Pro Thr Leu Pro Val Arg Arg <210> 83 <211> 17 <212> PRT
<213> Human and artificial sequences <400> 83 Cys Cys Ile Thr Asp Val Pro Gln Gly Va1 Met Tyr Lys Gly Ser Pro Asp <210> 84 <211> 17 <212> PRT
<213> Human and artificial sequences <400> 84 Glu Cys Lys Val Asp Gly Gln Leu Ser Asp Ser Pro Leu Leu Arg Asn Asn <210> 85 <211> 17 <212> PRT
<213> Human and artificial sequences <400> 85 Cys Cys Met Thr Asp Asp Pro Met Asp Pro Asn Ser Thr Trp Ala Ile Arg <210> 86 <211> I7 <212> PRT
<213> Human and artificial sequences <400> 86 Cys Cys Met Thr Asp Asp Pro Met Tyr Thr Asn Ser Thr Trp Ala Ile Arg <210> 87 <211> 17 <212> PRT
<213> Human and artificial sequences <400> 87 Cys Cys Val Asp Asp Thr Pro Asn Ser Gly Leu Ala Met Arg Val Ser Lys <210> 88 <211> 17 <212> PRT
<213> Human and artificial sequences <400> 88 Cys Cys Glu Val Asp Asp Phe Pro Thr His His Pro Gly Trp Thr Leu Arg <210> 89 <211> 17 <212> PRT

<213> Human and artificial sequences <400> 89 Ser Cys Leu Asn His Gln Ser Cys Pro Pro Val Lys Asn Asp Ile Gln Ile <210> 90 <211> 17 <212> PRT

<213> Human and artificial sequences <400> 90 Cys Cys Ala Asp Gln Glu Leu Asp His Asn Ala A1a Met Leu Gly Asn Ala <210> 91 <211> 12 <212> PRT

<213> Human and artificial sequences <400> 91 Cys Cys Met Asp Leu Glu Leu Ala Phe Val Ser Gly <210> 92 <211> 12 <212> PRT

<213> Human and artificial sequences <400> 92 Cys Cys Met Asp I1e Glu Val Arg Ala Val Gly Ser <210> 93 <211> 12 <212> PRT

<213> Human and artificial sequences <400> 93 Cys Cys Arg Asp Val Glu Leu Val Ser Gln Phe Gly <210> 94 <211> 12 <212> PRT

<213> Human and artificial sequences <400> 94 Cys Cys Ala Asp Phe Glu Val Gly Gly Arg Asn Gly <210> 95 <211> 12 <212> PRT

<213> Human and artificial sequences <400> 95 Cys Cys Val Ser Asp Glu Pro Ala Gly Val Arg Asp <210> 96 <211> 12 <212> PRT

<213> Human and artificialsequences <400> 96 Gly Ala Trp Gln Glu Lys Lys G1u Arg Gly Asp_ Leu <210> 97 <211> 12 <212> PRT

<213> Human and artificialsequences <400> 97 Gly Ala Thr Ala Gly Gln Ser Asp Pro Met Leu Leu <210> 98 <211> 12 <212> PRT

<213> Human and artificialsequences <400> 98 Val Ala Gly Gln Val Val Arg Glu Lys Gly Asp Leu <210> 99 <211> 12 <212> PRT

<213> Human and artificialsequences <400> 99 Lys Ala Glu Gln Ala Met Met Glu Arg Gly Asp Leu <210> 100 <211> 11 <212> PRT

<213> Human and artificialsequences <400> 100 Arg Gly Asn Gln Ile Met Leu Glu Arg Asp Ile <210> 101 <211> 11 <212> PRT

<213> Human and artificialsequences <400> 101 Gln Ile Arg Gln Ile Thr Thr Leu Asp Asp Leu <210> 102 <211> 11 <212> PRT

<213> Human and artificialsequences <400> 102 Arg Glu Gln Ile Ser Asp Pro Arg Gln Val Val <210> 103 <211> 12 <212> PRT

<213> Human and artificialsequences <400> 103 Cys Gln Met Asp A1a Glu Leu Asn Gln Ala I1e Val <210> 104 <211> 11 <212> PRT

<213> Human and artificialsequences <400> 104 Gly Gln Met Asp Thr Glu Leu Asn Arg Met Leu <210> 105 <211> 11 <212> PRT

<213> Human and artificialsequences <400> 105 Ser Met Gly Gln Val Arg Ile Gln Va1 Glu Asp_ <210> 106 <211> 11 <212> PRT

<213> Human and artificialsequences <400> 106 Tyr Gln Arg Asp Leu Glu Leu Ala Glu Gln Leu <210> 107 <211> 11 <212> PRT

<213> Human and artificialsequences <400> 107 Ser Met Gln Lys Val Asp Glu Leu Val Gly Arg <210> 108 <211> 11 <212> PRT

<213> Human and artificial sequences <400> 108 Ser Met Gln Glu Val Asp Glu Leu Val Gly Arg <210> 109 <211> 11 <212> PRT

<213> Human and artificial sequences <400> 109 Ala Glu Asp Gln Met Val Trp Glu Ile Asn Asp <210> 110 <211> 11 <212> PRT

<213> Human and artificial sequences <400> 110 Gly Gly Gln Glu Ser Asp Ile Pro Trp Gly Arg <210> 111 <211> 11 <212> PRT

<213> Human and artificial sequences <400> 111 Gly Gly Gln Glu Lys Asp Lys Glu Trp Leu Arg <210> 112 <211> 12 <212> PRT

<213> Human and artificial sequences <400> 112 His Cys Arg I1e Asp Arg Glu Val Ala Cys Ser Gly <210> 113 <211> 12 <212> PRT

<213> Human and artificial sequences <400> 113 Cys Ala Gly Met Gly Cys Trp Glu Lys Pro Ser Val <210> 114 <211> 17 <212> PRT

<213> Human and artificial sequences <400> 114 Ser Cys Glu Val Trp Leu Gly Gly Met Ile Met Asp Cys Arg Ser Glu Glu <210> 115 <211> 17 <212> PRT

<213> Human and artificial sequences <400> 115 Ser Cys Ala Phe Pro Arg Glu Gly Cys Ala Pro Pro Thr Pro Asp Leu Val <210> 116 <211> 17 <212> PRT

<213> Human and artificial sequences <400> 116 Phe Cys Glu Pro Ile Cys Ser~Pro Ser Arg Met Thr Leu Pro Pro Leu Ser <210> 117 <211> 17 <212> PRT
<213> Human and artificial sequences <400> 117 Glu Cys Asn Gln Asn Leu Ser Gly Ser Leu Arg His Val Asp Leu Asn Cys <210> 118 <211> 17 <212> PRT
<213> Human and artificial sequences <400> 118 Arg Cys Asp Gln Gln Leu Pro Arg Asp Ser Tyr Thr Phe Cys Met Met Ser <210> 119 <211> 17 <212> PRT
<213> Human and artificial sequences <400> 119 His Cys Gln Gln Val Phe Phe Pro Gln Asp Tyr Leu Trp Cys Gln Arg Gly <210> 120 <211> 17 <212> PRT
<213> Human and artificial sequences <400> 120 Asp Cys Glu Glu Pro Met Cys Ser Pro Val Leu Leu Gln Lys Leu Lys Pro <210> 121 <211> 17 <212> PRT
<213> Human and artificial sequences <400> 121 Asn Cys Gln Asp Gln Met Leu Arg Glu Asp Ala Gly Cys Trp Ser Lys Ile <210> 122 <211> 17 <212> PRT
<213> Human and artificial sequences <400> 122 His Cys Glu Glu Pro Glu Tyr Ser Pro Ala Thr Arg Val Phe Cys Gly Arg <210> 123 <211> 17 <212> PRT
<213> Human and artificial sequences <400> 123 Asp Cys Asp Trp Ile Asn Pro Pro Asp Pro Pro His Phe Trp Lys Asp 1 5 ' 10 15 Thr <210> 124 <211> 17 <212> PRT
<213> Human and artificial sequences <400> 124 Ala Cys Phe Ser Arg Asn Gly Gln Val Thr Asp Val Pro His Ser Cys Tyr <210> 125 <211> 17 <212> PRT
<213> Human and artificial sequences <400> 125 Lys Cys Pro Thr Tyr Pro Lys Pro Asn Asp Arg Cys Leu Trp Pro Val Pro <210> 126 <211> 17 <212> PRT
<213> Human and artificial sequences <400> 126 Tyr Cys Pro Lys Tyr Pro Leu Glu Gly Asp Cys Leu Leu Asp Asn Asp Tyr <210> 127 <211> 17 <212> PRT
<213> Human and artificial sequences <400> 127 Arg Cys Glu Glu Trp Leu Cys Ile Pro Pro Ala Pro Ala Phe Ala Pro Pro <210> 128 <211> 17 <212> PRT
<213> Human and artificial sequences <400> 128 Thr Cys Gly Gln Ser Glu Leu Arg Cys Ala Ser Leu Glu Thr His His Val <210> 129 <211> 16 <212> PRT

<213> Human and artificialsequences <400> 129 Asn Cys Asp Asn Pro Met Asp Cys Pro Ala Trp Ser Asn Leu Met Ser <210> 130 <211> 12 <212> PRT

<213> Human and artificialsequences <400> 130 Asp Ala Asp Glu Arg Ala Arg Ala Ala Leu Trp Arg <210> 131 <211> 12 <212> PRT

<213> Human and artificialsequences <400> 131 Ser Cys Gly Arg Glu Val Arg Glu Trp Gln Arg Cys <210> 132 <211> 12 <212> PRT

<213> Human and artificialsequences <400> 132 Val Cys Glu Cys Val Ser Glu Leu Leu Asp Arg Ala <210> 133 <211> 12 <212> PRT

<213> Human and artificialsequences <400> 133 Trp Cys Glu Pro Glu Cys Pro Gly Leu Leu Ala Leu 1 5 . 10 <210> 134 <211> 12 <212> PRT

<213> Human and artificialsequences <400> 134 Asp Cys Ser Lys Gly Gln Ala Asp Cys Leu Met Leu <210> 135 <211> 12 <212> PRT

<213> Human and artificialsequences <400> 135 Val Cys Glu Cys Val Ser Glu Leu Leu Asp Arg Ala <210> 136 <211> 12 <212> PRT

<213> Human and artificial sequences <400> 136 Gly Cys Thr Trp Pro Arg Val.Gly Cys Pro Asp His <210> 137 <211> 12 <212> PRT

<213> Human and artificial sequences <400> 137 Arg Cys Ser Ala Arg Val Val Pro Trp Gln Glu Cys <210> 138 <211> 12 <212> PRT

<213> Human and artificial sequences <400> 138 Ser Cys Pro Ser Gly Asp Cys Gly Gly Ala Tyr Lys <210> 139 <211> 12 <212> PRT

<213> Human and artificial sequences <400> 139 Gly Cys Met Trp Pro Gln Pro Asp Cys Pro Asp Glu <210> 140 <211> 12 <212> PRT

<213> Human and artificial sequences <400> 140 Glu Cys Arg Trp Pro Leu Met Gly Cys Pro Asp Gly <210> 141 <211> 12 <212> PRT

<213> Human and artificial sequences.

<400> 141 Gly Cys Val G1y Glu Leu Val Trp G1u Gln Cys Arg <210> 142 <211> 12 <212> PRT

<213> Human and artificial sequences <400> 142 Gln Cys Arg Asp Gly Thr Arg Lys Met Val Val Cys <210> 143 <211> 12 <212> PRT

<213> Human and artificial sequences <400> 143 Thr Cys Leu Val Asp Arg Gln Glu Ser Asp Val Cys <210> 144 <211> 12 <212> PRT

<213> Human and artificialsequences <400> 144 Asp Cys Val Asp Gly Asp Leu Val Leu Val Arg Cys <210> 145 <211> 12 <212> PRT

<213> Human and artificialsequences <400> 145 Arg Cys Gln Gly Ala Leu Cys Val Glu Glu Arg Gly <210> 146 <211> 12 <212> PRT

<213> Human and artificialsequences <400> 146 Val Cys Pro Gly Trp Lys Leu Gly Asn Pro Asn Cys <210> 147 <211> 12 <212> PRT

<213> Human and artificialsequences <400> 147 Met Cys Gly Trp Glu Ile Ser Glu Trp Gln Val Cys <210> 148 <211> 25 <212> PRT

<213> Human and artificialsequences <400> 148 Ala Asp A1a Gly Cys Phe Asn Lys Met Ala Asp Leu Gly Met Gln Glu Leu Cys Arg Glu Ala Ala Ala Pro Glu <210> 149 <211> 25 <212> PRT

<213> Human and artificialsequences <400> 149 Ala Asp Ala Gly Cys Phe Asn Lys Met Ala Asp Leu Gly Met Gln Glu Leu Cys Arg Thr Ala Ala Ala Pro Glu <210> 150 <211> 25 <212> PRT

<213> Human and artificial sequences <400> 150 Ala Asp AlaAla Cys Phe AsnLys Met Ala LeuGlu Gly Met Gln Asp Leu Cys ArgVal Ala Ala Ala Pro Glu <210> 151 <211> 25 <212> PRT

<213> Human sequences and artificial <400> 151 Ala Asp AlaGly Cys Phe AsnLys Leu Ala LeuGlu Gly Ile Gln Asp Leu Cys ArgVal Ala Ala Ala Pro Glu <210> 152 <211> 25 <212> PRT

<213> Human sequences and artificial <400> 152 Ala Asp AlaGly Cys Phe AsnLys Leu Ala LeuGlu Gly Ile Gln Asp Leu Cys ArgGlu A1a Ala Ala Pro Glu~

<210> 153 <211> 25 <212> PRT

<213> Human sequences and artificial <400> 153 Ala Asp AlaGly Cys Phe AsnLys Leu Ala LeuGlu Gly Met Gln Asp Met Cys ArgAsp Asp Ala Ala Pro Glu <210> 154 <211> 25 <212> PRT

<213> Human sequences and artificial <400> 154 Ala Asp AlaGly Cys Phe AsnLys Leu Ala ProGlu Gly Met Gln Asp Leu Cys ArgGlu Ala Glu Ala Pro Glu <210> 155 <211> 25 <212> PRT

<213> Human and artificial sequences <400> 155 Ala Asp AlaGly Cys Phe AsnLys Leu Val LeuG1u Gly Met Gln Asp Leu Cys ArgGly Ala Ala Ala Pro Glu <210> 156 <211> 25 <212> PRT

<213> Human and artificial sequences <400> 156 Ala Asp Ala Gly Cys Phe Met Asn Leu Ala Trp Glu Gly Asn Gln Asp Leu Cys Arg Ala Ala Ala Glu Ala Pro <210> 157 <211> 25 <212> PRT

<213> Human and artificial sequences <400> 157 ~

Ala Asp Asn Lys Gln Met Ala Trp Glu Gly Ala Gly Cys Phe Met Asp Met Cys Arg Ala Ala Ala Glu Ala Pro <210> 158 <211> 25 <212> PRT

<213> Human and artificial sequences <400> 158 Ala Asp Ala Gly Cys Phe Met Asn Gln A1a Leu Glu Gly Lys Gln Asp Leu Cys Arg Gly Ala Ala Glu Ala Pro <210> 159 <211> 25 <212> PRT

<213> Human and artificial sequences <400> 159 Ala Asp Ala G1u Cys Phe Met Asn Leu Ala Ser Glu Gly Lys Gln Asp Leu Cys Arg Val Ala Ala Glu Ala Pro 20 ~25 <210> 160 <211> 25 <212> PRT

<213> Human and artificial sequences <400> 160 Ala Asp Ala Gly Cys Phe Met Asn Leu Ala Leu Glu Gly Lys Gln Asp Leu Cys Arg Glu Ala Ala G1u Ala Pro <210> 161 <211> 25 <212> PRT

<213> Human and artificial sequences <400> 161 Ala Asp Ala Gly Cys Phe Ile Asn Met Ala Gln Glu G1y Met Gln Asp Leu Cys Arg Ala Ala Ala Glu Ala Pro <210> 162 <211> 25 <212> PRT

<213> Human and artificial sequences <400> 162 Ala Asp Phe Ile Lys Met Ser PheGlu Gly Asn Gln Asp Ala Gly Cys Leu Cys Arg Glu Gly Glu Pro Ala Ala <210> 163 <211> 25 <212> PRT

<213> Human and artificial sequences <400> 163 Ala Asp Ala Gly Phe Ile Lys Met Ala LeuGlu Gly Cys Asn Gln Asp Leu Cys Arg G1u Ala Glu Thr Ala Ala <210> 164 <211> 25 <212> PRT

<213> Human and artificial sequences <400> 164 Ala Asp Ala Gly Phe Ile Lys Met Ala LeuGlu Gly Cys Asn Gln Asp Leu Cys Arg Gln Ala Glu Pro Ala Ala <210> 165 <211> 25 <212> PRT

<213> Human and artificial sequences <400> 165 Ala Asp Ala Gly Phe Ile Asn Met Ala LeuGlu Gly Cys Asn Gln Asp Leu Cys Arg Gly Ala Glu Pro Gly Ala <210> 166 <211> 25 <212> PRT

<213> Human and artificial sequences <400> 166 Ala Asp Ala Gly Phe Ile Lys Met Ala TrpGlu Gly Cys Asn Gln Asp Leu Cys Arg Glu Ala Glu Pro Gly Ala 20 ~ 25 <210> 167 <211> 25 <212> PRT

<213> Human rtificial quences and a se <400> 167 Ala Asp Ala Gly Phe Ile Lys Met Ala LeuGlu Gly Cys Asn Gln Asp Leu Cys Ser G1n Ala G1u Pro Ala Ala <210> 168 <211> 25 <212> PRT

<213> Human rtificial quences and a se <400> 168 Ala Asp Ala Cys Phe Ile Lys Met Ala LeuGlu Gly Gly Asn Gln Asp Leu Cys Arg Gly Ala Glu Pro Glu Ala <210> 169 <211> 25 <212> PRT

<213> Human and artificial sequences <400> 169 Ala Asp Ala Cys Phe Ile Lys Met Ala SerGlu Gly Gly Asn Gln Asp Leu Cys Arg Pro Ala G1u Pro Glu A1a <210> 170 <211> 25 <212> PRT

<213> Human and artificial sequences <400> 170 Ala Asp Ala Cys Phe Ile Lys Met Ala LeuGlu Gly Gly Lys Gln Asp Leu Cys Arg Ala Trp Glu Pro Glu Ala <210> 171 <211> 25 <212> PRT

<213> Human and artificial sequences <400> 171 Ala Asp Ala Cys Phe Ile Lys Met Ala ArgGlu Gly Glu Asn Gln Asp Leu Cys Arg Val Ala Glu Ala Glu Ala <210> 172 <211> 25 <212> PRT

<213> Human and artificial sequences <400> 172 Ala Asp Ala Cys Phe Ile Lys Met Ala LeuGlu Gly Gly Asp Gln Asp Leu Cys Arg Ala Ala Glu Pro Ala Ala <210> 173 <211> 25 <212> PRT

<213> Human and artificial sequences <400> 173 Ala Asp Ala Cys Phe Ile Lys Met Ala LeuGlu Gly Gly Asn G1n Asp Leu Cys Arg Ala Gly Glu Arg Glu Ala 20 '25 <210> 174 <211> 25 <212> PRT

<213> Human nd artificialquences a se 2?

<400> 174 Ala Asp Gly Ala Gly Cys Phe Lys Asn Met Val SerGlu Lys Gln Asp Leu Cys Ala Arg Gln Ala Ala Glu Ala <210> 175 <211> 25 <212> PRT

<213> Human and artificial sequences <400> 175 Ala Asp Gly Ala Gly Cys Phe Gln Asn Met Ala LeuGlu Lys Gln Asp Leu Cys Pro Arg Glu Ala Ala Glu Ala <210> 176 20 <211> 25 <212> PRT

<213> Human and artificial sequences <400> 176 Ala Asp Gly Ala Glu Cys Phe Ile Asn Arg Ala LeuGlu Lys Gln Asp Leu Cys Pro Gly Glu Ala Ala Glu Ala <210> 177 <211> 25 <212> PRT

<213> Human and artificial sequences <400> 177 Ala Asp Gly Ala Gly Cys Phe Ile Asn Met Ala SerGlu Lys Gln Asp Leu Cys Pro Ala Ala Ala Ala Glu Ala <210> 178 <211> 25 <212> PRT

<213> Human and artificial sequences <400> 178 .

Ala Asp Gly Ala Gly Cys Phe Ile Asn Met Ala ProGlu Arg Gln Asp Leu Cys Pro Arg Glu Ala Ala G1u Ala <210> 179 <211> 25 <212> PRT

<213> Human and artificial sequences <400> 179 Ala Asp Gly Ala Gly Cys Phe Ile Glu Met Ala MetGlu Lys Gln Asp Leu Cys Gln Ala Arg Ala Ala Glu Ala <210> 180 <211> 25 6~ <212> PRT

<213> Human and artificial sequences <400> 180 Ala Asp Ala Gly Cys Phe Ile Asn Met Ala TrpGlu Gly Lys Gln Asp Leu Cys Arg Glu Ala Ala Glu Ala Pro <210> 181 <211> 25 <212> PRT

<213> Human and artificial sequences <400> 181 Ala Asp Ala Gly Cys Phe Ile Asn Met Ala LeuGlu Gly Asn Gln Asp Leu Cys Arg Glu Ala Ala Glu Ala Pro <210> 182 <211> 25 <212> PRT

<213> Human and artificial sequences <400> 182 Ala Asp Ala Gly Cys Phe Ile Glu Met Ala MetGlu Gly Lys Gln Asp Leu Cys Arg Glu Thr Ala Glu Ala Gln <210> 183 <211> 25 <212> PRT ' <213> Human and artificial sequences <400> 183 Ala Asp Ala Gly Cys Phe Ile Asn Met A1a MetGlu Gly Lys Gln Asp Leu Cys Arg Glu Ala Ala Glu Ala Pro <210> 184 <211> 25 <212> PRT

<213> Human and artificial sequences <400> 184 Ala Asp Ala Gly Cys Phe Ile Asn Met Ala LeuGlu Gly Lys Gln Asp Leu Cys Arg Glu A1a Ala Glu Ala Pro <210> 185 <211> 25 <212> PRT

<213> Human and artificial sequences <400> 185 ~

Ala Asp Asn Lys Gln Met Ala LeuGlu Gly Ala Gly Cys Phe Arg Asp Leu Cys Arg Glu Ala Ala Glu Ala Pro <210> 186 <211> 25 <212> PRT

<213> Human and artificial sequences <400> 186 Ala Asp Ala Gly Cys Phe Ile Lys Met Ala LeuGlu Gly Asn Gln Asp Leu Cys Ala Arg Ala Ala Glu Pro Ala <210> 187 <211> 25 <212> PRT

<213> Human and artificial sequences <400> 187 Ala Asp Ala Gly Cys Phe Ile Arg Leu Ala MetGlu Gly Asn Gln Asp Leu Cys Arg Gly Ala Ala Glu Ser Ala 20 ,25 <210> 188 <211> 25 <212> PRT

<213> Human and artificial sequences <400> 188 Ala Asp Ala G1u Cys Phe Ile Arg Met Ala LeuGlu Gly Asn Gln Asp Leu Cys Arg Glu Ala Ala Glu Gly Ala <210> 189 <211> 25 <212> PRT

<213> Human and artificial sequences <400> 189 Ala Asp Ala Gly Cys Phe Ile Pro Leu Ala TrpLys Gly Ser Gln Asp Arg Cys Arg Glu Ala Ala Glu Met Ala <210> 190 <211> 25 <212> PRT ' <213> Human and artificial sequences <400> 190 Ala Asp Ala Gly Cys Ser Ile Thr Met Ala TrpGlu Gly His Gln Asp Arg Cys Arg Glu Gly Ala Glu Leu Ala <210> 191 <211> 25 <212> PRT

<213> Human and artificial sequences <400> 191 Ala Asp Ala Gly Cys Ser Ile Arg Met Ala TrpGlu Gly His Gln Asp Arg Cys Arg Glu Gly Ala Glu Leu Ala <210> 192 <211> 16 <212> PRT

<213> Human and artificialquences se <400> 192 Cys Ser Ser Cys Asp Gly Gly Gly'His Lys Pro Pro Thr Ile Gln Cys <210> 193 <211> 20 <212> PRT
<213> Human and artificial sequences <400> 193 Cys Leu Gln Ser Ser Cys Asp Gly G1y G1y His Phe Pro Pro Thr Ile Gln Leu Leu Cys

Claims (41)

Claims

1. A peptide comprising an isolated surface exposed epitope of the CE2 domain of Ig E, or mimotope thereof.

2. A peptide as claimed in claim 1 wherein the surface exposed epitope of CE2 is P1 (SEQ ID No. 1), or mimotope thereof.

3. A peptide as claimed in claim 1 wherein the surface exposed epitope of CE2 is P2 (SEQ ID No. 2), or mimotope thereof.

4. A peptide as claimed in claim 1 wherein the surface exposed epitope of CE2 is P3 (SEQ ID No. 3), or mimotope thereof.

5. A peptide as claimed in claim 1 wherein the surface exposed epitope of CE2 is P4 (SEQ ID No. 4), or mimotope thereof.

6. A peptide as claimed in claim 1 wherein the surface exposed epitope of CE2 is P5 (SEQ ID No. 5), or mimotope thereof.

7. A peptide as claimed in claim 1 wherein the surface exposed epitope of CE2 is P6 (SEQ ID No. 6), or mimotope thereof.

8. A peptide as claimed in claim 1 wherein the surface exposed epitope of CE2 is P7 (SEQ ID No. 7), or mimotope thereof.

9. A mimotope as claimed in any one of claims 1 to 8 wherein the mimotope is a peptide.

10. A peptide as claimed in claim 1 wherein the isolated epitope is derived from a loop structure of the CE2 domain of Ig E.

11. A peptide as claimed in claim 10 wherein the loop structure of the CE2 domain of Ig E is the A-B or the C-D loop.

12. A peptide as claimed in claim 2 wherein the mimotope of P1 is a peptide of the general formula: h x d h h a n a n x y;
wherein: h is a hydrophobic amino acid residue; d is an ionic bond donating amino acid residue; a is an acidic amino acid residue; n is an ionically neutral/
non-polar amino acid residue; and x is an amino acid.

13. A peptide as claimed in claim 2, wherein the mimotope of P1 is a peptide of the general formula:

wherein X, is selected from V, I, L, M, F or A; X2 is selected from D or E;
and X3 is selected from L, I, V, M, A or F.

14. A peptide as claimed in claim 2 wherein the mimotope of P1 is selected from the group comprising Pl5q (SEQ ID No. 11), PT1079 (SEQ ID No. 13), PT1079GS
(SEQ ID No. 15), PT1078 (SEQ ID No. 16), PT15 (SEQ ID No. 8).

15. A peptide as claimed in claim 3, wherein the mimotope of P2 is P16 (SEQ ID
No.
24).

16. A peptide as claimed in claim 4 wherein the mimotope of P3 is P17 (SEQ ID
No.
26).

17. An immunogen for the treatment of allergy comprising a peptide or mimotope as claimed in any one of claims 1 to 16, additionally comprising a carrier molecule.

18. An immunogen as claimed in claim 17, wherein the carrier molecule is selected from Protein D or Hepatitis B core antigen.

19. An immunogen as claimed in claim 17 or 18, wherein the immunogen is a chemical conjugate of the peptide or mimotope as claimed in claims 1 to 16, or wherein the immunogen is expressed as a fusion protein.

20. An immunogen as claimed in any one of claims 17 to 19, wherein the peptide or peptide mimotope is presented within the primary sequence of the carrier.

21. A vaccine for the treatment of allergy comprising an immunogen as claimed in any one of claims 17 to 20, further comprising an adjuvant.

22. A ligand which is capable of recognising a surface exposed epitope of the domain of Ig E, characterised in that the ligand is not PTm Ab 0005.

23. A ligand as claimed in claim 22, wherein the ligand is PTm Ab 0011 deposited under the Budapest Treaty patent deposit at ECACC on 8th March 1999 under Accession No. 99030805.

24. A pharmaceutical composition comprising a ligand which is capable of recognising a surface exposed epitope of the CE2 domain of Ig E.

25. A pharmaceutical composition as claimed in claim 24 wherein the ligand is capable of recognising the C-D Loop of the CE2 domain of Ig E.

26. A pharmaceutical composition as claimed in claim 25, wherein the ligand is a monoclonal antibody selected from PTm Ab 0005 or PTm Ab 0011.

27. A peptide as claimed in any one of claims 1 to 16 for use in medicine.

28. A vaccine as claimed in claim 21 for use in medicine.

29. An immunogen as claimed in any one of claims 17 to 20, for use in medicine.

30. Use of a peptide as claimed in any one of claims 1 to 16 in the manufacture of a medicament for the treatment or prevention of allergy.

31. A ligand which is capable of recognising a surface exposed epitope of the domain of Ig E for use in medicine.

32. Use of a ligand which is capable of recognising a surface exposed epitope of the Cs2 domain of Ig E in the manufacture of a medicament for the treatment of allergy.

33. Use of a ligand as clamed in claim 31 or 32, wherein the ligand is PTm Ab 0005 or PTm Ab 0011.

34. Use of PTm Ab 0005 or PTm Ab 0011 in the identification of mimotopes of P1.

35. A peptide which is capable of being recognised by PTm Ab 0005 or PTm Ab 0011.

36. An immunogen comprising a peptide as claimed in claim 35.

37. Use of a peptide as claimed in claims 1-16 in diagnostics or in the affinity purification of circulating anti-IgE antibodies from blood.

38. A method of manufacturing a vaccine comprising the manufacture of an immunogen as claimed in any one of claims 17 to 20, and formulating the immunogen with an adjuvant.

39. A method for treating a patient suffering from or susceptible to allergy, comprising the administration of a peptide as claimed in any one of claims 1 to 16, to the patient.

40. A method for treating a patient suffering from or susceptible to allergy, comprising the administration of a vaccine as claimed in claim 21 to the patient.

41. 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 24 to 26, to the patient.