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Document AP4 
Appl. No. 09/848,616 



WORLD rNTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




PCX 

INTERNATIQNAL APPUCATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent Classification 7 : 

C07K 16/00, 16/42, A61K 39/00, 39/385, 
39/395, GOIN 33/577, 33/68, A61P 37/08 



Al 



(11) International PubUcation Number: WO 00/50461 

(43) International Publication Date: 31 August 2000 (31.08.00) 



(21) International Application Number: PCT/EPOO/01456 

(22) International FUing Date: 22 February 2000 (22.02.00) 



(30) Priority Data: 






9904408.3 


25 February 1999 (25.02,99) 


GB 


9917144.9 


21 July 1999 (21.07.99) 


GB 


9918598.5 


7 August 1999 (07.08.99) 


GB 


9918599.3 


7 August 1999 (07.08.99) 


GB 


9918601.7 


7 August 1999 (07.08.99) 


GB 


9918604.1 


7 August 1999 (07.08.99) 


GB 


9918606.6 


7 August 1999 (07.08.99) 


GB 


9925618.2 


29 October 1999 (29.10.99) 


GB 



(71) Applicants (for all designated States except US): SMITHK- 

LINE BEECHAM BIOLOGICALS S.A. PE/BE]; Rue de 
rinstitut 89, B-1330 Rixensart (BE). PEPTIDE THER- 
APEUTICS LIMITED [GB/GB]; Peterhouse Technology 
Park. 100. Fulboum Road, Cambridge CBl 9PT (GB). 

(72) Inventors; and 

(75) Inventors/Applicants (for US only): FRIEDH, Martin 
[GB/GB]; Corporate Intellectual Property, SmithKIine 
Beecham, 2 New Horizons Court, Brentford. Middlesex 
TW8 9EP (GB). MASON. Sean [GB/GB]; Peptide Thera- 
peutics Limited, Peterhouse Technology Park, 100 Fulboum 



Road, Cambridge CBl 9PT (GB). TURNELL, William, 
Gordon [GB/GB]; Peptide Therapeutics Limited. Peterhouse 
Technology Park, 100 Fulboum Road, Cambridge CBl 9PT 
(GB). VAN MECHELEN, Marcelle, Paulette [GB/GB]; 
SmithKIine Beecham Biologicals S.A., rue de Tlnstitut 
89, B-1330 Rixensart (GB). VINALS Y DE BASSOLS, 
Carlota [BE/BE]; Smithkline Beecham Biologicals S.A., 
Rue de Tlnstitut 89. B-1330 Rixensart (BE). 

(74) Agent: DALTON, Marcus, Jonathan, William; SmithKIine 
Beecham, Two New Horizons Court, Brentford, Middlesex 
TW8 9EP (GB). 



(81) Designated States: AE, AL, AM, AT, AU. AZ, BA, BB. BG, 
BR. BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, Ee! 
ES. FI, GB. GD, GE. GH. GM, HR. HU. ID, XL, IN, IS. JP, 
KE. KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, 
MD, MG, MK. MN. MW, MX, NO, NZ, PL. PT, RO, RU. 
SD, SE, SG, SI, SK. SL, TJ, TM, TR, TT. UA, UG. US, 
UZ, VN. YU. ZA. ZW, ARIPO patent (GH, GM, KE, LS, 
MW. SD, SL. SZ, TZ, UG. ZW), Eurasian patent (AM, AZ. 
BY. KG, KZ, MD, RU, TJ. TM). European patent (AT, BE, 
CH. CY. DE, DK, ES, FI. FR. GB, GR, IE, IT, LU, MC, 
NL, PT. SE), OAPI patent (BF. BJ, CF. CG, CI, CM, GA 
GN. GW, ML, MR, NE, SN, TD, TG). 



Published 

With international search report. 

Before the expiration of the time limit for amending the 
claims and to be republished in the event of the receipt of 
amendments. 



(54) Titie: EPITOPES OR MIMOTOPES DERIVED FROM THE C-EPSILON-3 OR C-EPSILON-4 DOMAINS OF IGE ANTAGO- 
NISTS THEREOF, AND THEIR THERAPEUTIC USES 

(57) 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 epitopes or mimotopes derived from the Ce3 or C€4 domains of IgE. These novel regions may be 
the target for both passive and active immunoprophylaxis or immunotherapy. The invention further relates to methods for production of the 
medicaments, pharmaceutical compositions containing them and their use in medicine. Also forming an aspect of the present invention are 
ligands, especially monoclonal antibodies, which are capable of binding the IgE regions of the present invention, and their use in medicine 
as passive immunotherapy or immunoprophylaxis. 



FOR THE PURPOSES OF INFORMATION ONLY 
Codes used to identify States party to the PCX on the front pages of pamphlets publishing international applications under the PCT. 



AL 


Albania 


ES 


Spain 


LS 


Lesotho 


SI 


Slovenia 


AM 


Armenia 


FI 


Finland 


LT 


Lithuania 


SK 


Slovakia 


AT 


Austria 


FR 


France 


LU 


Luxembourg 


SN 


Senegal 


AU 


Australia 


GA 


Gabon 


LV 


Latvia 


sz 


Swaziland 


AZ 


Azerbaijan 


GB 


United Kingdom 


MC 


Monaco 


TD 


Chad 


BA 


Bosnia and Herzegovina 


GE 


Georgia 


MD 


Republic of Moldova 


TG 


Togo 


BB 


Barbados 


GH 


Ghana 


MG 


Madagascar 


TJ 


Tajikistan 


BE 


Belgium 


GN 


Guinea 


MK 


The former Yugoslav 


TM 


Turkmenistan 


BF 


Burkina Faso 


GK 


Greece 




Republic of Macedonia 


TR 


Turkey 


BG 


Bulgaria 


HU 


Hungary 


ML 


Mali 


TT 


Trinidad and Tobago 


BJ 


Benin 


IE 


Ireland 


MN 


Mongolia 


UA 


Ukraine 


BR 


Brazil 


IL 


Israel 


MR 


Mauritania 


VG 


Uganda 


BY 


Belarus 


IS 


Iceland 


MW 


Malawi 


US 


United States of America 


CA 


Canada 


IT 


Italy 


MX 


Mexico 


uz 


Uzbekistan 


CF 


Central African Republic 


JP 


Japan 


NE 


Niger 


VN 


Viet Nam 


CG 


Congo 


KE 


Kenya 


NL 


Netherlands 


vu 


Yugoslavia 


CH 


Switzerland 


KG 


Kyrgyzstan 


NO 


Norway 


zw 


Zimbabwe 


CI 


C6te d*Ivoirc 


KP 


Democratic People's 


NZ 


New Zealand 






CM 


Cameroon 




Republic of Korea 


PL 


Poland 






CN 


China 


KR 


Republic of Korea 


PT 


Portugal 






cu 


Cuba 


KZ 


Kazakstan 


RO 


Romania 






cz 


Czech Republic 


LC 


Saint Lucia 


RU 


Russian Federation 






DE 


Germany 


LI 


Liechtenstein 


SD 


Sudan 






DK 


Denmark 


LK 


Sri Lanka 


SE 


Sweden 






EE 


Estonia 


LR 


Liberia 


SG 


Singapore 







wo 00/50461 PCT/EPOO/01 456 

EPITOPES OR MIMOTOPES DERIVED FROM THE C-EPSILON-3 OR C-EPSILON-4 DOMAINS OF IGE, ANTAGO- 
NISTS THEREOF, AND THEIR THERAPEUTIC USES 

The present invention relates to the provision of novel medicaments for the 
treatment, prevention or amelioration of allergic disease. In particular, the novel 
5 medicaments are epitopes or mimotopes derived from the Ce3 or Ce4 domains of IgE. 
These novel regions may be the target for both passive and active immunoprophylaxis 
or immunotherapy. The invention further relates to methods for production of the 
medicaments, pharmaceutical compositions containing them and their use in 
medicine. Also forming an aspect of the present invention are ligands, especially 

10 monoclonal antibodies, which are capable of binding the IgE regions of the present 
invention, and their use in medicine as passive iimnunotherapy or 
immunoprophylaxis. 

In an allergic response, the symptoms commonly associated with allergy are 
brought about by the release of allergic mediators, such as histamine, from immune 

15 cells into the surrounding tissues and vascular structures. Histamine is normally stored 
in mast cells and basophils, until such time as the release is triggered by interaction 
with allergen specific IgE. The role of IgE in the mediation of allergic responses, such 
as asthma, food allergies, atopic dermatitis, type-I hypersensitivity and allergic 
rhinitis, is well known. On encoimtering an antigen, such as pollen or dust mite 

20 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 
triggering of histamine release from the mast cells or basophils, by cross-linking of 
neighbouring IgE/ FceRI complexes (Sutton and Gould, Nature, 1993, 366: 421-428; 

25 EP 0 477 231 Bl). 

IgE, like all immunoglobulins, comprises two heavy and two light chains. The 
e heavy chain consists of five domains: one variable domain (VH) and four constant 
domains (Cel 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 

30 discussed in Padlan and Davis (Mol. Immunol., 23, 1063-75, 1986) and Helm et al., 
(2IgE model structure deposited 2/10/90 with PDB (Protein Data Bank, Research 
Collabarotory for Structural Bioinformatics; http:\pdb-browsers.ebi.ac.uk)). Each of 



1 

SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 PCT/EPOD/01456 

the IgE domains consists of a squashed barrel of seven anti-parallel strands of 
extended (p-) polypeptide segments, labelled a to f, grouped into two (J-sheets. Four 
P-strands {a,b,d & e) form one sheet that is stacked against the second sheet of three 
strands (c/& g) (see FIG 8). The shape of each p-sheet is maintained by lateral 
5 packing of amino acid residue side-chains from neighbouring anti-parallel strands 
within each sheet (and is further stabilised by main-chain hydrogen-bonding between 
these strands). Loops of residues, forming non-extended (non-P-) conformations, 
connect the anti-parallel P-strands, either within a sheet or between the opposing 
sheets. The connection from strand a to strand b is labelled as the A-B loop, and so 

10 on. The A-B and d-e loops belong topologically to the four-stranded sheet, and loop f- 
g to the three-stranded sheet. The interface between the pair of opposing sheets 
provides the hydrophobic interior of the globular domain. This water-inaccessible, 
mainly hydrophobic core results from the close packing of residue side-chains that 
face each other from opposing P-sheets. 

15 In the past, a number of passive or active immunotherapeutic approaches 

designed to interfere with IgE-mediated histamine release mechanism have been 
investigated. These approaches include interfering with IgE or allergen/IgE complexes 
binding to the FceRI or FcsRII (the low affinity IgE receptor) receptors, with either 
passively administered antibodies, or with passive administration of IgE derived 

20 peptides to competitively bind to the receptors. In addition, some authors have 
described the use of specific peptides derived from IgE in active immunisation to 
stimulate histamine release inhibiting immune responses. 

In the course of their investigations, previous workers in this field have 
encountered a number of considerations, and problems, which have to be taken into 

25 account when designing new anti-allergy therapies. One of the most dangerous 
problems revolves around the involvement of IgE cross-linking in the histamine 
release signal. It is most often the case that the generation of anti-IgE antibodies 
during active vaccination, are capable of triggering histamine release per se, by the 
cross-linking of neighbouring IgE-receptor complexes in the absence of allergen. This 

30 phenomenon is termed anaphylactogenicity. Indeed many commercially available 

anti-IgE monoclonal antibodies which are normally used for IgE detection assays, are 



2 

SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 PCT/EPOO/01456 

anaphylactogenic, and consequently useless and potentially dangerous if administered 
to a patient. 

Whether or not an antibody is anaphylactogenic, depends on the location of 
the target epitope on the IgE molecule. However, based on the present state of 
knowledge in this area, and despite enormous scientific interest and endeavour, there 
is little or no predictability of what characteristics any antibody or epitope may have 
and whether or not it might have a positive or negative clinical effect on a patient. 

Therefore, in order to be safe and effective, the passively administered, or 
vaccine induced, antibodies must bind in a region of IgE which is capable of 
interfering with the histamine triggering pathway, without being anaphylactic perse. 
The present invention achieves all of these aims and provides medicaments which are 
capable of raising non-anaphylactic antibodies which inhibit histamine release. These 
medicaments may form the basis of an active vaccine or be used to raise appropriate 
antibodies for passive immunotherapy, or may be passively administered themselves 
for a therapeutic effect. 

Much work has been carried out by those skilled in the art to identify specific 
anti-IgE antibodies which do have some beneficial effects against IgE-mediated 
allergic reaction (WO 90/15878, WO 89/04834, WO 93/05810). Attempts have also 
been made to identify epitopes recognised by these useful antibodies, to create peptide 
mimotopes of such epitopes and to use those as immunogens to produce anti-IgE 
antibodies. 

WO 97/31948 describes an example of this type of work, and further describes 
IgE peptides from the C£3 and Cs4 domains conjugated to carrier molecules for active 
vaccination purposes. These immunogens may be used in vaccination studies and are 
said to be capable of generating antibodies which subsequently inhibit histamine 
release in vivo . In this work, a monoclonal antibody (BSW17) was described which 
was said to be capable of binding to IgE peptides contained within the Ce3 domain 
which are useful for active vaccination purposes. 

EP 0 477 231 Bl describes immunogens derived from the Cs4 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 23 1 BL 



3 

SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 PCT/EPOO/01456 

Other approaches are based on the identification of peptides derived from Ce3 
or Ce4, which themselves compete for IgE binding to the high or low affinity 
receptors on basophils or mast cells (WO 93/04173, WO 98/24808, EP 0 303 625 Bl, 
EP0 341 290). 

5 The present invention is the identification of novel sequences of IgE which are 

used in active or passive immunoprophylaxis or therapy. These sequences have not 
previously been associated with anti-allergy treatments. The present invention 
provides peptides, per se, that incorporate specific isolated epitopes fi-om continuous 
portions of IgE which have been identified as being surface exposed, and further 
10 provides mimotopes of these newly identified epitopes. These peptides or mimotopes 
may be used alone in the treatment of allergy, or may be used vaccines to induce auto 
anti-IgE antibodies during active immunoprophylaxis or immunotherapy of allergy to 
limit, reduce, or eliminate allergic symptoms in vaccinated subjects. 

Surprisingly, the anti-IgE antibodies induced by the peptides of the present 
15 invention are non-anaphy lactogenic and are capable of blocking IgE-mediated 
histamine release from mast cells and basophils. 

The regions of human IgE which are peptides of the present invention, and 
which may serve to provide the basis for peptide modification are: 

20 Table 1 



Peptide 


Sequence 


Location sequence 


SEQ 






and IgE Domain 


ID NO. 


P5 


RASGKPVNHSTRKEEKQRNGTL 


Ce3 


1 


P6 


GTRDWffiGE 


Ce3 


2 


P7 


PHLPRALMRSTTKTSGPRA 


Ce3/Ce4 


3 


P8 


PEWPGSRDKRT 


Ce4 (Pro451-Thr461) 


4 


P9 


EQKDE 


Ce4 


5 


P200 


LSRPSPFDLFIRKSPTITC 


Ce3 


6 


P210 


WLHNEVQLPDARHSTTQPRKT 


Ce4 


7 


1-90N 


LFIRKS 


Ce3 


81 


2-90N 


PSKGTVN 


Cs3 


82 


3-90N 


LHNEVQLPDARHSTTQPRKTKGS 


Ce4 


83 


4-90N 


SVNPGK 


Ce4 


84 



Peptides that incorporate these epitopes form a preferred aspect of the present 
invention. Mimotopes which have the same characteristics as these epitopes, and 

4 



SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 PCT/EPOO/01456 
immunogens comprising such mimotopes which generate an immune response which 
cross-react with the IgE epitope in the context of the IgE molecule, also form part of 
the present invention. 

The present invention, therefore, includes isolated peptides encompassing 
these IgE epitopes themselves, and any mimotope thereof The meaning of mimotope 
is defined as an entity which is sufficiently similar to the native IgE epitope so as to 
be capable of being recognised by antibodies which recognise the native IgE epitope; 
(Gheysen, H.M., et al., 1986, Synthetic peptides as antigens. Wiley, Chichester, Ciba 
foundation symposium 1 19, pi 30- 149; Gheysen, H.M., 1986, Molecular 
Immunology, 23,7, 709-715); or are capable of raising antibodies, when coupled to a 
suitable carrier, which antibodies cross-react with the native IgE epitope. 

The mimotopes of the present invention may be peptidic or non-peptidic. A 
peptidic mimotope of the surface exposed IgE epitopes identified above, may also be 
of exactly the same sequence as the native epitope.' Such a molecule is described as a 
mimotope of the epitope, because although the two molecules share the same 
sequence, the mimotope will not be presented in the context of the whole IgE domain 
structure, and as such the mimotope may take a slightly different conformation to that 
of the native IgE epitope. It will also be clear to the man skilled in the art that the 
above identified linear sequences (PI to P7), when in the tertiary structure of IgE, lie 
adjacent to other regions that may be distant in the primary sequence of IgE. As such, 
for example, a mimotope of PI may be continuous or discontinuous, in that it 
comprises or mimics segments of PI and segments made up of these distant amino 
acid residues. 

The mimotopes of the present invention mimic the surface exposed regions of 
the IgE structure, however, within those regions the dominant aspect is thought by the 
present inventors to be those regions within the surface exposed area which correlate 
to a loop structure. The structure of the domains of IgE are described in "Introduction 
to protein Structure" (page 304, Edition, Branden and Tooze, Garland Publishing, 
New York, ISBN 0 8153 2305-0) and take the form a p-barrel made up of two 
opposing anti-parallel P-sheets (see FIG. 8). The mimotopes may comprise, therefore, 
a loop with N or C terminal extensions which may be the natural amino acid residues 
from neighbouring sheets. As examples of this, PI 00 contains the A-B loop of Ce3, 

5 



SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 PCT/EPOD/01456 

P8 contains the A-B loop of Cs4, P5 contains the C-D loop of Ce3 and PI 10 contains 
the C-D loop of Ce4. Accordingly, mimotopes of these loops form an aspect of the 
present invention. Particularly preferred loops are the C-D loops of Cs3 or Cs4, and 
the A-B loop of Ce4. 

5 Peptide mimotopes of the above-identified IgE epitopes may be designed for a 

particular purpose by addition, deletion or substitution of elected amino acids. Thus, 
the peptides of the present invention may be modified for the purposes of ease of 
conjugation to a protein carrier. For example, it may be desirable for some chemical 
conjugation methods to include a terminal cysteine to the IgE epitope. In addition it 

10 may be desirable for peptides conjugated to a protein carrier to include a hydrophobic 
terminus distal fi-om the conjugated terminus of the peptide, such that the fi-ee 
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 

15 closely resembles that of the IgE peptide as found in the context of the whole IgE 
molecule. For example, the peptides may be altered to have an N-terminal cysteine 
and a C-terminal hydrophobic amidated tail. Alternatively, the addition or substitution 
of a D-stereoisomer form of one or more of the amino acids may be performed to 
create a beneficial derivative, for example to enhance stability of the peptide. Those 

20 skilled in the art will realise that such modified peptides, or mimotopes, could be a 
wholly or partly non-peptide mimotope wherein the constituent residues are not 
necessarily confined to the 20 naturally occurring amino acids. In addition, these may 
be cyclised by techniques known in the art to constrain the peptide into a 
conformation that closely resembles its shape when the peptide sequence is in the 

25 context of the whole IgE molecule. A preferred method of cyclising a peptide 
comprises the addition of a pair of cysteine residues to allow the formation of a 
disulphide bridge. 

Further, those skilled in the art will realise that mimotopes or immunogens of 
the present invention may be larger than the above-identified epitopes, and as such 
30 may comprise the sequences disclosed herein. Accordingly, the mimotopes of the 
present invention may consist of addition of N and/or C terminal extensions of a 
number of other natural residues at one or both ends. The peptide mimotopes may also 



SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 PCT/EPOO/01456 

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 (inverse sequences). Also, the peptide sequences may 
be retro-inverso in character, in that the sequence orientation is reversed and the 

5 amino acids are of the D-stereoisomer form. Such retro or retro-inverso peptides have 
the advantage of being non-self, and as such may overcome problems of self-tolerance 
in the immune system (for example P14c). 

Alternatively, peptide mimotopes may be identified using antibodies which are 
capable themselves of binding to the IgE epitopes of the present invention using 

10 techniques such as phage display technology (HP 0 552 267 Bl). This technique, 

generates a large number of peptide sequences which mimic the structure of the native 
peptides and are, therefore, capable of binding to anti -native peptide antibodies, but 
may not necessarily themselves share significant sequence homology to the native IgE 
peptide. This approach may have significant advantages by allowing the possibility of 

15 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 

20 identification of a recognition pattern for each native-peptide in terms of its shared 
chemical properties amongst recognised mimotope sequences. 



7 



SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 PCT/EPOO/01456 
Examples of such mimotopes are: 



Table 2 



Peptide 


Sequence 


Description 


SEQ ID NO. 


Pll 


CRASGKPVNHSTRKEEKQRNGLL 


P5 mimotope 


8 


Plla 


(Ac) GKPVNHSTGGC 


P5 mimotope 


9 


Pllb 


(Ac) GKPVNHSTRKEEKQRNGC 


P5 mimotope 


10 


Flic 


CGKPVNHSTRKEEKQRNGLL (NH^) 


P5 mimotope 


11 


Plld 


(Ac) RASGKPVNHSTGGC 


P5 mimotope 


12 


P12 


CGTRDWIEGLL 


P6 mimotope 


13 


P12a 


CGTRDWIEGETL (NH,) 


P6 mimotope 


14 


P12b 


(Ac) GTRDWIEGETGC 


P6 mimotope 


15 


P13 


CHPHLPRALMLL 


P7 mimotope 


16 


P13a 


CGTHPHLPRALM (NH^) 


P7 mimotope 


17 


P13b 


(Ac) THPHLPRALMRSC 


P7 mimotope 


18 


P13c 


(Ac) GPHLPRALMRSSSC 


P7 mimotope 


19 


P14 


APEWPGSRDKRTC 


P8 mimotope 


20 


P14a 


(Ac) APEWPGSRDKRTLAGGC 


P8 mimotope 


21 


P14b 


CGGATPEWPGSRDKRTL (NHj) 


P8 mimotope 


22 


P14c 


CTRKDRSGPWEPA (NHj) 


P8 retro 


23 


P14d* 
(cyclic) 


(Ac) APCWPGSRDCRTLAG 


P8 mimotope 


24 


P14d 
(cyclic) 


(Ac) ACPEWPGSRDRCTLAG 


P8 mimotope 


25 


C-1C14 


CATPEWPGSRDKRTLCG 


P8 mimotope 


26 


C-1C13 


CATPEWPGSRDKRTCG 


P8 mimotope 


27 


C3C12 


TPCWPGSRDKRCG 


P8 mimotope 


28 


P9a 


CGAEWEQKDEL (NHj) 


P9 mimotope 


29 


P9b 


(Ac) AEVVEQKDEFIC 


P9 mimotope 


30 


P9b* 


(Ac) GEQKDEFIC 


P9 mimotope 


31 


P9a* 


CAEGEQKDEL (NH^) 


P9 mimotope 


32 


Carll 


CPEWPGCRDKRTG 


P8 mimotope 


85 


Carl2 


TPEWPGCRDKRCG 


P8 mimotope 


86 



Alternatively, peptide mimotopes may be generated with the objective of 
increasing the immunogenicity of the peptide by increasing its affinity to the anti-IgE 
peptide polyclonal antibody, the effect of which may be measured by techniques 
known in the art such as (Biocore experiments) . In order to achieve this the peptide 
sequence may be electively changed following the general rules: 

* To maintain the structural constraints, prolines and glycines should not be 

replaced 



8 



SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 PCT/EPOO/01456 

* Other positions can be substituted by an amino acid that has similar 
physicochemical properties. 

As such, each amino acid residue can be replaced by the amino acid that most 
closely resembles that amino acid. For example, A may be substituted by V, L or I, as 
described in the following table. 



Original residue 


Exemplary 


Preferred 




substitutions 


substitution 


A 


V. L,I 


V 


R 


K,Q,N 


K 


N 


Q, H, K, R 


Q 


D 


E 


E 


C 


S 


S 


Q 


N 


N 


E 


D 


D 


G 


A 


A 


H 


N, Q, K, R 


N 


I 


L, V, M, A, F 


L 


L 


I. V, M, A, F 


I 


K 


R,Q,N 


R 


M 


L,F,I 


L 


F 


L, V. I. A, Y,W 


W 


P 


A 


A 


S 


T 


T 


T 


S 


S 


W 


Y,F 


Y 


Y 


W, F, T, S 


F 


V 


I, L, M, F, A 


L 



Particularly preferred IgE peptides are P8 and variants thereof (such as P 14 or 
PI 4a). These peptides, when coupled to a carrier are potent in inducing anti-IgE 
immune responses, which responses are capable of inhibiting histamine release from 
human basophils. Variants, or mimotopes, of P8 are described primarily as any 
peptide based immunogen which is capable of inducing an immune response, which 
response is capable of recognising P8. Without being limiting to the scope of the 
present invention, some variants of P8 may be described by a general formula in 
which certain amino acids may be replaced by their closest counterparts. Using this 
technique, P8 peptide mimotopes may be described by the general formula: 

P, X|, Xj, P, X3, X4, X5, Xg, X5, X5 

or, 

9 



SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 PCT/EPOO/01456 
P, X,, X2, P, G, X4, R, D, Xj, X5 

wherein; X, is an amino acid selected from E, D, N, or Q; X2 is an amino acid 
selected from W, Y, or F; X3 is an amino acid selected from G or A, X4 is an amino 
acid selected from S, T or M; X5 is an amino acid selected from R or K; and X^ is an 

5 amino acid selected from D or E. 

P8 mimotopes may also be identified using antibodies which are capable 
themselves of binding to P8, using techniques such as phage display technology (EP 0 
552 267 Bl). Monoclonal antibodies such as P14/23, P14/31 and P14/33 are 
particularly suitable in this regard. 

10 The present invention, therefore, provides novel epitopes, and mimotopes 

thereof, and their use in the manufacture of pharmaceutical compositions for the 
prophylaxis or therapy of allergies. Immunogens comprising at least one of the 
epitopes or mimotopes of the present invention and carrier molecules are also 
provided for use in vaccines for the immunoprophylaxis or therapy of allergies. 

15 Accordingly, the epitopes, mimotopes, or immunogens of the present invention are 
provided for use in medicine, and in the medical treatment or prophylaxis of allergic 
disease. Preferred immunogens and vaccines of the present invention comprise the 
IgE epitope P8, or mimotopes thereof, including P14. - 

The present inventors have shown that different methods by which the epitope 

20 or mimotope is presented has significant effects upon binding to monoclonal 

antibodies and to the immune response after vaccination. For example, when using 
cyclised peptides, altering the length and phase of the loop may have significant 
effects on the binding activity of the cyclised mimotopes to the P14 monoclonal 
antibodies (P14/23, P14/31 or P14/33). As such the present inventors have developed 

25 a novel system which selects the sites of cyclisation, thereby increasing the 

probability that the cyclised peptides are held in the correct loop structure, which 
comprises the correct amino acid residues. In this way, the peptide is likely to be 
constrained in a conformation that most closely resembles that which the peptides 
would normally adopt if they were in the context of the whole IgE domain. Hence, 

30 without limiting the present invention the cyclised mimotopes which follow these new 
rules form one preferred aspect of the present invention. 



10 



SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 PCT/EPOO/01 456 

Putative mimotope sequences that are not consistent with these rules may still 
raise useful antisera (for example P14 and Pll), as such the following examples are 
only a sub-set of the types of mimotopes of the present invention. 

Examples of preferred peptides that follow these newly defined structural rules 

are: 



Table 3 





iVMiifgUHifli: Uj 




CSRPSPFDLFIRKSPTITC 


A-B loop of Ce3 


33 


CSRPSPFDLFIRKSPTC 


A-B loop of Ce3 


35 


CPSPFDLFIRXSPTITC 


A-B loop of Ce3 


41 


CPSPFDLFIRKSPC 


A-B loop of Ce3 


43 


CTWSRASGKPVNHSTC 


C-D loop of Ce3 


58 


CTWSRASGKPVNHC 


C-D loop of Ce3 


60 


CSRASGKPVNHSTC 


C-D loop ofCe3 


66 


CSRASGKPVNHC 


C-D loop of Ce3 


68 


CYAFATPEWPGSRDKRTLAC 


A-B loop of Ce4 


45 


CYAFATPEWPGSRDKRTC 


A-B loop of Ce4 


47 


CFATPEWPGSRDKRTLAC 


A-B loop ofCe4 


53 


CFATPEWPGSRDKRTC 


A-B loop of Ce4 


55 


CQWLHNEVQLPDARHC 


C-D loop ofCe4 


70 


CQWLHNEVQLPDAC 


C-D loop of Ce4 


72 


CLHNEVQLPDARHC 


C-D loop ofC84 


78 


CLHNEVQLPDAC 


C-D loop ofCe4 


80 



It is envisaged that the mimotopes of the present invention will be of a small 
size, such that they mimic a region selected from the whole IgE domain in which the 
native epitope is found. Peptidic mimotopes, therefore, should be less than 100 amino 
acids in length, preferably shorter than 75 amino acids, more preferably less than 50 
amino acids, and most preferable within the range of 4 to 25 amino acids long. 
Specific examples of preferred peptide mimotopes are P 14 and Pll, which are 
respectively 13 and 23 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 which techniques may be used 
to confirm the status of a specific construct as a mimotope which falls within the 
scope of the present invention. Such techniques include, but are not restricted to, the 
following. The putative mimotope can be assayed to ascertain the immunogenicity of 
the construct, in that antisera raised by the putative mimotope cross-react with the 



SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 PCT/EPOO/01456 
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 
5 the epitope within IgE. Specific examples of such monoclonal antibodies for use in 
the competition assays include P14/23, P14/31 or P14/33, which would confirm the 
status of the putative mimotope as a mimotope of P8. 

In one embodiment of the present invention at least one IgE epitope or 
mimotope are linked to carrier molecules to form immunogens for vaccination 
10 protocols, preferably wherein the carrier molecules are not related to the native IgE 
molecule. The mimotopes may be linked via chemical covalent conjugation or by 
expression of genetically engineered fiision partners, optionally via a linker sequence. 
As one embodiment, the peptides of the present invention are expressed in a fiision 
molecule with the fiision partner, wherein the peptide sequence is found within the 
15 primary sequence of the fiision partner. 

The covalent coupling of the peptide to the immunogenic carrier can be carried 
out in a manner well known in the art. Thus, for example, for direct covalent coupling 
it is possible to utilise a carbodiimide, glutaraldehyde or (N-[y-maleimidobutyryloxy] 
succinimide ester, utilising common commercially available hetcrobifimctional 
20 linkers such as CDAP and SPDP (using manufacturers instructions). After the 

coupling reaction, the immunogen can easily be isolated and purified by means of a 
dialysis method, a gel filtration method, a fractionation method etc. 

In a preferred embodiment the present inventors have found that peptides, 
particularly cyclised peptides may be conjugated to the carrier by preparing 
25 Acylhydrazine peptide derivatives. 

The peptides/protein carrier constructs can be produced as follows. 
Acylhydrazine peptide derivatives can be prepared on the solid phase as shown in the 
following scheme 1 Solid Phase Peptide Synthesis: 



12 



SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 

Scheme 1 



PCT/EPOO/01456 



Rink-Resin 



Solid Phase Pq)tide Synthesis 



X-AA,...C(Trt)....C(Trt)....AA„-Lys(Dde)-Rink.Resin 



Hydrazine hydrate 



X-AA,...C(Trt)....C(Trt)....AA„-Lys(NH2)-Rink-Resin 

(i) Succinic anhydride 
(iO HBTU/HOBt/NNIM/NsHj 



X-AA,...C(Trt)....C(Trt)...J\A„-Lys-Rink-Resin Z= ""^NHN^ 

N 



TFA 



X-AA, ...C(-SH)....C(-SH)....AA„-Lys-CONH2 



1 



I 

N HZ- 
H2P2 oxidation 



X-AA, ...C....C... AA„-Lys-C0NH2 



N H2 



2= >*^^Nm4 



These peptide derivatives can be readily prepared using the well-known 
5 'Fmoc' procedure, utilising either polyamide or polyethyleneglycol-polystyrene 
(PEG-PS) supports in a fully automated apparatus, through techniques well known 
the art [techniques and procedures for solid phase synthesis are described in 'Solid 

13 



in 



SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 PCT/EPOO/01456 
Phase Peptide Synthesis: A Practical Approach' by E. Athenon and R,C. Sheppard, 
published by IRL at Oxford University Press (1989)]. Acid mediated cleavage 
afforded the linear, deprotected, modified peptide. This could be readily oxidised and 
purified to yield the disulphide-bridged modified epitope using methodology outlined 
in 'Methods in Molecular Biology, Vol. 35: Peptide Synthesis Protocols (ed. M.W. 
Pennington and B.M. Dunn), chapter 7, pp91-171 by D. Andreau et al. 

The peptides thus synthesised can then be conjugated to protein carriers using 
the following technique: 

Introduction of the aryl aldehyde functionality utilised the succinimido active 
ester (BAL-OSu) prepared as shown in scheme 2 (see WO 98/17628 for further 
details). Substitution of the amino functions of a carrier eg BS A (bovine serum 
albumin) to -50% routinely give soluble modified protein. Greater substitution of the 
BSA leads to insoluble constructs. BSA and BAL-OSu were mixed in equimolar 
concentration in DMSO/buffer (see scheme) for 2 hrs. This experimentally derived 
protocol gives -50% substitution of BSA as judged by the Fluorescamine test for free 
amino groups in the following Scheme 2/3 - Modified Carrier Preparation: 



14 



SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 

Scheme 2 



PCT/EPOO/01456 




Scheme 3 



BSAv'w-(NH2L + mBAL-OSu 



40% DMSO/buffer 

(pH 7.25,0.2 MNaOAc) 

2hr. 



BSA^^NH-CO-pH2)4 
BSA-BAL 



Simple combination of modified peptide and derivatised carrier affords 
peptide carrier constructs readily isolated by dialysis - Scheme 4 - Peptide/carrier 
5 conjugate: 



15 



SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 

Scheme 4 



PCT/EPOO/01456 



BSAW^O 



+ 3mf X-AAi...C....C....AAj,-Lys-CONH2 



m 



BSA-BAL 



NH-Z 



50% DMSO/buffer 

(pH 3.5, 0.1 MNaHCOs) 

-8-16 hr. 




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 

5 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 
(BS A), 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 

10 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 
help. Preferably the ratio of mimotopes to carrier is in the order of 1 : 1 to 20: 1 , and 
preferably each carrier should carry between 3-15 peptides, 

15 In an embodiment of the invention a preferred carrier is Protein D from 

Haemophilus influenzae (EP 0 594 610 Bl), Protein D is an Igl>-binding protein from 
Haemophilus influenzae and has been patented by Forsgren (WO 91/18926, granted 

16 



SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 PCT/EPOO/01456 
EP 0 594 610 Bl). In some circumstances, for example in recombinant immunogen 
expression systems it may be desirable to use fragments of protein D, for example 
Protein D 1/3"* (comprising the N-terminal 100-1 10 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 0 421 
635 B describes the use of chimaeric hepadnavirus core antigen particles to present 
foreign peptide sequences in a virus-like particle. As such, immunogens of the present 
invention may comprise IgE peptides presented in chimaeric particles consisting of 
hepatitis B core antigen. Additionally, the recombinant fusion proteins may comprise 
the mimotopes of the present invention and a earner protein, such as NSl 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 
solid phase procedure employing the well-known "F-moc" procedure and polyamide 
resin in the fully automated apparatus. Alternatively, those skilled in the art will know 
the necessary laboratory procedures to perform the process manually. Techniques and 
procedures for solid phase synthesis are described in 'Solid Phase Peptide Synthesis: 
A Practical Approach' by E. Atherton and R.C. Sheppard, published by IRL at Oxford 
University Press (1989). 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 
cloning, a laboratory manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold, 
Spring Harbor, New York (1989). 

The immunogens of the present invention may comprise the peptides as 
previously described, including mimotopes or analogues thereof, or may be 
immunologically cross-reactive derivatives or fragments thereof Also forming part of 

17 



SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 PCT/EPOO/01456 
the present invention are portions of nucleic acid which encode the immunogens of 
the present invention or peptides, mimotopes or derivatives thereof. 

The present invention, therefore, provides the use of novel epitopes or 
mimotopes (as defined above) in the manufacture of pharmaceutical compositions for 
the prophylaxis or therapy of allergies. Immunogens comprising the mimotopes or 
peptides of the present invention, and carrier molecules are also provided for use in 
vaccines for the immunoprophylaxis or therapy of allergies. Accordingly, the 
mimotopes, peptides or immunogens of the present invention are provided for use in 
medicine, and in the medical treatment or prophylaxis of allergic disease. 

Vaccines of the present invention, may advantageously also include an 
adjuvant. Suitable adjuvants for vaccines of the present invention comprise those 
adjuvants that are capable of enhancing the antibody responses against the IgE peptide 
immunogen. Adjuvants are well known in the art (Vaccine Design - The Subunit and 
Adjuvant Approach, 1995, Pharmaceutical Biotechnology, Volume 6, Eds. Powell, 
M.F., and Newman, M.J., Plenum Press, New York and London, ISBN 0-306-44867- 
X). Preferred adjuvants for use with immunogens of the present invention include 
aluminium or calcium salts (hydroxide or phosphate). 

The vaccines of the present invention will be generally administered for both 
priming and boosting doses. It is expected that the boosting doses will be adequately 
spaced, or preferably given yeariy or at such times where the levels of circulating 
antibody fall below a desired level. Boosting doses may consist of the peptide in the 
absence of the original carrier molecule. Such booster constructs may comprise an 
alternative carrier or may be in the absence of any carrier. 

In a further aspect of the present invention there is provided an immunogen or 
vaccine as herein described for use in medicine. 

The vaccine preparation of the present invention may be used to protect or 
treat a mammal susceptible to, or suffering from allergies, by means of administering 
said vaccine via systemic or mucosal route. These administrations may include 
injection via the intramuscular, intraperitoneal, intradermal or subcutaneous routes; or 
via mucosal administration to the oral/alimentary, respiratory, genitourinary tracts. A 
preferred route of administration is via the transdermal route, for example by skin 
patches. Accordingly, there is provided a method for the treatment of allergy, 

18 



SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 PCT/EP00/014S6 
comprising the administration of a peptide, immunogen, or ligand of the present 
invention to a patient who is suffering from or is susceptible to allergy. 

The amount of protein in each vaccine dose is selected as an amount which 
induces an immunoprotective response without significant adverse side effects in 
typical vaccinees. Such amount will vary depending upon which specific immunogen 
is employed and how it is presented. Generally, it is expected that each dose will 
comprise 1-1000 ^g of protein, preferably 1-500 ^g, more preferably 1-100 ^ig, of 
which 1 to 50|ig is the most preferable range. An optimal amount for a particular 
vaccine can be ascertained by standard studies involving observation of appropriate 
immune responses in subjects. Following an initial vaccination, subjects may receive 
one or several booster immunisations adequately spaced. 

In a related aspect of the present invention are ligands capable of binding to 
the peptides of the present invention. Example of such ligands are antibodies (or Fab 
fragments). Also provided are the use of the ligands in medicine, and in the 
manufacture of medicaments for the treatment of allergies. The term "antibody" 
herein is used to refer to a molecule having a usefiil 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 ftinctionality. Such antibody fragments or derivatives are intended to 
be encompassed by the term antibody as used herein. 

Particularly preferred ligands are monoclonal antibodies. For example, P 14/23, 
PI 4/31 or PI 4/33 are monoclonal antibodies which recognise P8 (which were raised 
by vaccination with a PI 4 immunogen). The hybridomas of these antibodies were 
deposited as Budapest Treaty patent deposit at ECACC (European Collection of Cell 
Cultures, Vaccine Research and Production Laboratory, Public Health Laboratory 
Service, Centre for Applied Microbiology Research, Porton Down, Salisbury, 
Wiltshire, SP4 OJG, UK) on 26 January 2000 under Accession No.s 00012610, 
0001261 1, 00012612 respectively. Also forming an important aspect of the present 
invention is the use of these monoclonal antibodies in the identification of novel 
mimotopes of IgE, for subsequent use in allergy therapy, and the use of the antibodies 
in the manufacture of a medicament for the treannent or prophylaxis of allergy. All of 
these monoclonal antibodies fiinction in vitro in inhibiting histamine release from 

19 



SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 PCT/EP00/014S6 
human basophils, and also PI 4/23 and PI 4/31 have been shown to inhibit passive 
cutaneous anaphylaxis in vivo. 

Therefore, mimotopes of IgE C84 that are capable of binding to P14/23, 
PI4/31 or P14/33, and immunogens comprising these mimotopes, form an important 
aspect of the present invention. Vaccines comprising mimotopes that are capable of 
binding to P14/23, P14/31 or P14/33 are useful in the treatment of allergy. 

Additionally, antibodies induced in one animal by vaccination with die 
peptides or immunogens of the present invention, may be purified and passively 
administered to another animal for the prophylaxis or therapy of allergy. The peptides 
of the present invention may also be used for the generation of monoclonal antibody 
hybridomas (using know techniques e.g, K5hler and Milstein, Nature, 1975, 256, 
p495), humanised monoclonal antibodies or CDR grafted monoclonals, by techniques 
known in the art. Such antibodies may be used in passive immunoprophylaxis or 
immimotherapy, or be used in the identification of IgE peptide mimotopes. 

As the ligands of the present invention may be used for the prophylaxis or 
treatment of allergy, there is provided pharmaceutical compositions comprising the 
ligands of the present invention. Preferred pharmaceutical compositions for the 
treatment or prophylaxis of allergy comprise the monoclonal antibodies PI 4/23, 
P14/31 orP14/33. 

Aspects of the present invention may also be used in diagnostic assays. For 
example, panels of ligands which recognise the different peptides of the present 
invention may be used in assaying titres of anti-IgE present in serum taken from 
patients. Moreover, the peptides may themselves be used to type the circulating anti- 
IgE. 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, 
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 

20 



SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 PCT/EPOO/01 456 

and used in medicine. Accordingly, the use of PI 4/23, PI 4/3 1 or PI 4/33 in the 
identification of peptides for use in allergy immunoprophylaxis or therapy forms part 
of the present invention. 

Vaccine preparation is generally described in New Trends and Developments 
in Vaccines, edited by Voller et al., University Park Press, Baltimore, Maryland, 
U.S.A. 1978. Conjugation of proteins to macromolecules is disclosed by Likhite, U.S. 
Patent 4,372,945 and by Armor et aL, U.S. Patent 4,474,757. 

Description of drawings 

FIG 1, Surface exposure of Ce3 an C84 of human IgE as calculated fi-om the Padlan 
and Davis model 1986, 

FIG 2, Histamine release inhibition and anaphylactogenicity of P14 antiserum. 
Monoclonal Antibodies, PTmAb0005 and PTmAbOOll, which were used as positive 
controls, were added at 1 |ig/ml to anti-BSA sera diluted 1/100 and 1/500 (final). The 
anti-P14 antisera were added at 1/100 and 1/500 final dilution. Cells were taken fi-om 
an allergic patient sensitive to grass pollen, histamine release was triggered by 
incubation with this grass pollen allergen, 

FIG 3, Histamine release inhibition and anaphylactogenicity of anti-P14 antiserum. 
The P 14 antiserum fi-om different mice, was added at different dilutions (SOX or 40X) 
to contain approximately l|ig/ml of anti-IgE antibody as measured by IgE receptor- 
bound ELISA, Three negative controls were used: Anti-BSA antiserum, non-specific 
IgGl and a mixture of non-specific IgGl diluted in anti-BSA antiserum. mAbl 1 is a 
monoclonal antibody known to inhibit histamine release and was used as a positive 
control (added at 2^g/ml). 

FIG 4, Histamine release inhibition and anaphylactogenicity of anti-P14 antiserum. 
Anti-P14 Antisera fi-om different mice were added at a 1/50 final dilution. 
Monoclonal Abs were added at 2 ^ig/ml either in assay buffer or in anti-BSA sera 
dilution 1/50. Three negative controls were used: Anti-BSA antiserum, non-specific 
IgGl and a mixture of non-specific IgGl diluted in anti-BSA antiserum, mAbl 1 is a 
monoclonal antibody known to inhibit histamine release and was used as a positive 
control (added at 2p.g/ml), 

21 



SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 PCT/EPOO/01456 
FIG 5, Antibody response anti-Pl I . Peptide PI I is coated at I fig/ml in carbonate 
buffer at +4°C overnight. After saturation of plates, two-fold serial dilution of sera are 
added and incubated for Ih at ST^C. Bound IgG is detected with a biotinylated anti- 
mouse Ab followed by streptavidin-POD and TMB substrate. Time points measured 
A. days 14 post vaccination 1, and day 14 post v2; B, Day 14 post v3. 
FIG 6, Anti-Pl 1 IgG anti-human IgE titres. Human IgE was coated at 1 |ag/ml. Two- 
fold serial dilutions of sera ("BSA pool" is a pool of the control group) or 
PTmAbOOOS (a positive control monoclonal antibody) were incubated for Ih at 37°C. 
Bound IgG is detected with a biotinylated anti-mouse Ab. 

FIG 7, Histamine release inhibition studies with anti-P14 monoclonal antibodies, on 
allergic basophils donated by dustmite allergic patients (A 10 and All) and from grass 
pollen allergic patients (08 and G4). PTI 1 (PTmAbOOl 1) was used as a positive 
control, and non-specific IgG2a was used as an isotype control for the P14/23, P14/31 
and PI 4/33. 

FIG 8, IgE domain structure. (A) Each domain is composed of two facing P-sheets. 
shoAvn in outline, one of 4 anti-parallel p-strands (labelled 4) and the other of 3 anti- 
parallel p-strands (labelled 3). (B) The seven strands are shown topographically as 
block arrows labelled a to f, partitioned between the two sheets as shown. The loop- 
connectivity of the strands is shown topologically with curved arrows: solid arrows 
are /«/ra-sheet loops and dashed arrows are inter-sheet loops. In the IgGl Fc domain 
strucmres a short c' strand forms part of the C-D loop, as is predicted for IgE Fc. 
FIG 9, (A) Predicted stnictural alignment of the A-B loop sequences of human IgE 
domains Ce2, 3 & 4 with the equivalent segments from the crystallographically 
determined stnicnire of human IgGl Fc (domains Cy2 & Cy3). p-strands in the IgGl 
structure are underlined and labelled a and b; amino acid residues at the ends of each 
sequence segment are numbered. Vertical arrows below the block of sequences point 
to predicted optimal cyclisation positions, labelled and connected by dashed or solid 
lines as shown in FIG 10b. (B) Predicted strucmral alignment of the c_d loops of 
human IgE Ce2,3 & 4 with human IgGl Fc. P-strands in the IgGl structure are 
underlined and labelled c. c ■ and d; amino acid residues at the ends of each sequence 
segment are numbered. Residues highlighted by the shaded boxes form (Cy2 & Cy3) 
or are predicted to form (Ce2, by homology model refinement and experiment, Ce3, 



22 



SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 PCT/EPOO/01 456 

Ce4, by homology-modelling) a protected core within the loop. Residues within the 
plain bold boxes are predicted to be involved in recognition by receptors and/or 
antibodies. Vertical arrows below the block of sequences point to predicted optimal 
cyclisation positions, labelled and connected by dashed or solid lines as shown in FIG 
5 lib. 

FIG 10, (A) The schematic structure of the A-B hairpin at the sheet-sheet interface of 
Ig constant domains. Adjacent anti-parallel P-strands are shown as solid arrows, 
labelled a and b. Residues along strand a are labelled i, those along strand b are 
labelled j. Residues i+n & j+m, where both n and m are zero or even, form part of the 
10 sheet-sheet interface within a domain. Residues i+n & j+m, where both n and m are 
odd, form part of the solvent-exposed surface of a domain. The A-B loop is shown as 
a black arrow. (B) The schematic structure of the A-B hairpin as in figure 3a, with 
residue positions optimal for cyclisation connected by dashed or solid dumbbells. 
FIG 1 1, (A) The schematic structure of the C-D hairpin (loop plus supporting p- 
15 strands) at the edge of the sheet-sheet interface of Ig constant domains. Opposing anti- 
parallel P-strands are shown as solid arrows, labelled c and d. Residues along strand c 
are labelled i, those along strand d are labelled j. Residues i+n & j+m, where n is odd 
but m is even, form part of the sheet-sheet interface within a domain. Residues i+n & 
j+m, where n is zero or even but m is odd, form part of the solvent-exposed surface of 
20 a domain. The c_d loop, containing the short c' strand, is shown as a black arrow, (B) 
The schematic structure of the c_d hairpin, with residue positions optimal for 
cyclisation connected by dashed or solid dumbbells. 

The present invention is illustrated by but not limited to the following examples, 

25 

Part 1» Active vaccination studies 

Examples 

LI Peptide identification 

30 

The peptides were identified by the following technique. 



23 



SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 PCT/EPOO/01456 
The modelled structure of human IgE has been described Padlan and Davies {MoL 
Immunol., 23, 1063-75, 1986). Peptides were identified which were both continuous 
and solvent exposed. This was achieved by using Molecular Simulations software 
(MSI) to calculate the accessibility for each IgE amino acid, the accessible surface 
5 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 A^. 

The results of the test are shown in FIG 1. 

10 Results 

From figure 1 there are a number of native peptides which may be used as 
immimogens for raising antibodies against IgE. 

Table 4, Native surface exposed and continuous IgE peptides using the 1986 Padlan 
15 and Davies model. 



Peptide 


Sequence 


Location sequence 


SEQ 






and IgE Domain 


ID NO. 


P5 


RASGKPVNHSTRKEEKQRNGTL 


Ce3 


1 


P6 


GTRDWIEGE 


Ce3 


2 


P7 


PHLPRALMRSTTKTSGPRA 


CE3/Ce4 


3 


P8 


PEWPGSRDKRT 


Ce4(Pro451-Thr461) 


4 


P9 


EQKDE 


C84 


5 


P200 


LSRPSPFDLFIRKSPTITC 


Ce3 


6 


P210 


WLHNEVQLPDARHSTTQPRKT 


Ce4 


7 



In addition to those peptides identified above, the following peptides have been 
identified using the same selection criteria with the Helm et al. IgE model (2IgE 
model structure deposited 2/10/90 with PDB (Protein Data Bank, Research 
20 Collabarotory for Structural Bioinformatics; httpApdb-browsers.ebi, ac.uk)). 



Table 5, Peptides identified using the Helm et al. 1990 model. 



Name 


Sequence 


Location 


SEQ ID NO. 


1-90N 


LFIRKS 


Ce3 


81 


2-90N 


PSKGTVN 


Ce3 


82 


3-90N 


LHNEVQLPDARHSTTQPRKTKGS 


Ce4 


83 


4-90N 


SVNPGK 


Ce4 


84 



24 



SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 



PCT/EPOO/01456 



These peptides, or mimotopes thereof, were synthesised and conjugated to carrier 
proteins for use in immunogenicity studies. 

5 1.2 Synthesis oflgE peptide/Protein 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 NHj activation of carrier residues by fixing a succinimide group. 
10 Maleimide groups is a cysteine-binding site. Therefore, for the purpose of the 

following examples, the IgE peptides to be conjugated require the addition of an N- 
terminal cysteine. 

The coupling reagent is a selective heterobifunctional cross-linker, one end of the 
15 compound activating amino group of the protein carrier by an succinimidyl ester and 
the other end coupling sulhydryl group of the peptide by a maleimido group. The 
reactional scheme is as the following : 

a. Activation of the protein by reaction between lysine and succinimidyl ester : 



20 




b. Coupling between activated protein and the peptide cysteine by reaction with 
the maleimido group : 



25 



SUBSTITUTE SHEET (RULE 2B) 



wo 00/50461 



PCT/EPOO/01456 



o 




5 vWWWVii. 



o 

Conjugue 



].3 Preparation of IgE peptide-Protein D conjugate 

5 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-nialeimidobutyryloxy] succinimide ester - 
GMBS) is dissolved at 102.5 mg/ml in DMSO and added to the protein solution. 
1.025 mg of GMBS is used for 1 mg of Protein D. The reaction solution is incubated 
1 hour at room temperature. The by-products are removed by a desalting step onto a 

10 sephacryl 200HR permeation gel. The eluant used is a phosphate buffer saline Tween 
80 0. 1 % pH 6.8, The activated protein is collected and pooled. The peptides (as 
identified in tables 4 or 5, or derivatives or mimotopes thereof) is dissolved at 4 
mg/ml in 0. 1 M acetic acid to avoid di-sulfure bond formation. A molar ratio of 
between 2 to 20 peptides per 1 activated Protein D is used for the coupling. The 

15 peptide solution is slowly added to the protein and the mixture is incubated 1 h at 

25°C. The pH is kept at a value of 6.6 during the coupling phase. A quenching step is 
performed by addition of cysteine (0.1 mg cysteine per mg of activated PD dissolved 
at 4 mg/ml in acetic acid 0.1 M), 30 minutes at 25°C and a pH of 6.5. Two dialysis 
against NaCl 1 50 mM Tween 80 0.1 % are performed to remove the excess of 

20 cysteine or peptide. 

The last step is sterile filtration through a 0,22 \im 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. 

25 



26 



SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 PCT/EPOO/01456 
In an analogous fashion the peptides of the present invention may be conjugated to 

other carriers including BSA. A pre-activated BSA may be purchased commercially 

from Pierce Inc. 



5 Mimotopes of P8 (P14, SEQ ID NO. 20; CLEDGQVMDVDLL) and P5 (Pll, SEQ 
ID NO. 8; CRASGKPVNHSTRKEEKQRNGLL) were synthesised which were 
conjugated to both Protein D and BSA using techniques described above. 

L4 ELISA methods 

1 0 A nti-peptide or A nti-peptide carrier ELISA 

The anti-peptide and anti-carrier immune responses were investigated using an ELISA 
technique outlined below. Microtiterplates (Nome) are coated with the specific antigen 
in PBS (4° overnight) with either: Streptavidin at 2|ig/ml (followed by incubation 
with biotinylated peptide (l^M) for 1 hour at 37°C), Wash 3X PBS-Tween 20 0.1%. 

15 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 S?*'. 
Wash 3X. Add 2° anti-mouse Ig (or anti-mouse isotype specific monoclonal antibody) 
coupled to HRP. Incubate I hr at 37°. Wash 5X. Reveal with TMB (BioRad) for 10 
minutes at room temperature in the dark. Block reaction with 0.4N H2SO4. 

20 

Method for the Detection of Anti-Human IgE Reactivity in Mouse Serum (IgE plate 
bound EUSA) 

ELISA plates are coated with human chimaeric IgE at I|ig/ml in pH 9.6 
carbonate/bicarbonate coating buffer for 1 hour at 3TC or overnight at 4°C. Non- 
25 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 Bom Calf serum are then added for 1 
hour at 37°C. Polyclonal serum binding is detected with goat anti-mouse IgG-Biotin 
(1/2000) followed by Streptavidin-HRP (1/1000). Conjugated antibody is detected 
30 with TMB substrate at 450nm. A standard curve of PTmAbOOl 1 is included on each 
plate so that the anti-IgE reactivity in serum samples can be calculated in ng/ml. 



27 

SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 PCT/EP00/0I456 

Competition oflgE Binding with Mimotope Peptides, Soluble IgE or PTmAbOOll 
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 'iTC and then added to IgE-coated ELISA plates 
5 for 1 hour at 37°C. Polyclonal serum binding is detected with goat anti-mouse IgG- 
Biotin (1/2000) followed by Streptavidin-HRP (1/1000). Conjugated antibody is 
detected with TMB substrate at 450nm. For competition between serum and 
PTmAbOOl 1 for IgE binding, mixtures of serum and PTmAbOOl 1 -biotin are added to 
IgE-coated ELISA plates. PTmAbOOl 1 binding is detected with Streptavidin-HRP 
10 (1/1000). 

L5 Human Basophil Assays 

Two types of assay were performed with human basophils (HBA), one to determine 
the anaphylactogenicity of the monoclonal antibodies, consisting of adding the 
15 antibodies to isolated PBMC; and a second to measure the inhibition of Lot PI (a 
strong allergen) triggered histamine release be pre-incubation of the HBA with the 
monoclonal antibodies. 

Blood is collected by venepuncture from allergic donors into tubes containing 
20 heparin, and the non-erythrocytic cells were purified. The cells are washed once in 
. HBH/HSA, counted, and re-suspended in HBH/HSA at a cell density of 2.0 x 10* per 
ml. 100^1 cell suspension are added to wells of a V-bottom 96-well plate containing 
100^1 diluted test sample or monoclonal antibody. Each test sample is tested at a 
range of dilutions with 6 wells for each dilution. Well contents are mixed briefly using 
25 a plate shaker, before incubation at 37°C for 30 minutes. 

For each serum dilution 3 wells are triggered by addition of 10^1 Lol p I extract (final 
dilution 1/10000) and 3 wells have 10^1 HBH/HSA added for assessment of 
anaphylactogenicity. Well contents are again mixed briefly using a plate shaker, 
30 before incubation at 37°C for a further 30 minutes. Incubations are terminated by 

centrifligation at 500g for 5 min. Supematants are removed for histamine assay using 
a commercially available histamine EIA measuring kit (Immunotech). Control wells 



SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 PCT/EPOO/01456 
containing cells without test sample are routinely included to determine spontaneous 
and triggered release. Samples of cells were lysed by 2X freeze/thawing to assay total 
histamine contained in the cells. 

The results are expressed as following: 

Anaphylactogenesis assay 

Histamine release due to test samples = 

% histamine release from test sample treated cells - % spontaneous histamine release. 
Blocking assay 

The degree of inhibition of histamine release can be calculated using the formula: 
% inhibition 

= 1 - (histamine release from test sample treated cells*) x 100 
(histamine release from antigen stimulated cells*) 

Values corrected for spontaneous release. 

Example 2. Immunisation of mice with P14 conjugates (P14-BSA, P14 -BSA) induces 
production of anti-human IgE antibodies. 

The conjugates comprising the mimotope P14 (25fig protein/dose), described in 
example 1, were administered into groups of 10 BalbC mice, adjuvanted with and oil 
in water emulsion containing QS21 and 3D-MPL described in WO 95/17210 . 
Boosting was be performed on days 14, 24 and 72, sera was harvested 14 days after 
each immunisation. 

The immune responses anti-peptide and anti-plate bound IgE was followed using 
ELISA methods described in Example 1. The antiserum was then tested for 
anaphylactogenicity and functional activity in the inhibition of histamine release from 
human allergic basophils (methods as described in example 1), 



29 



SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 PCT/EPOO/01 456 

Immunogenicity Results 

Both conjugates, PD-P14 and BSA-P14, were capable of inducing anti-P14 and anti- 
IgE immune responses. The results for anti peptide and anti-IgE responses, induced by 
the BSA-P14 conjugates, as measured at day 14 post third and fourth vaccination, are 
5 shown in table 6. PTmAbOOll is a monoclonal antibody which is known to bind to 
the C82 domain of IgE, and was used to quantify the anti-IgE responses in |ig/ml. 



Table 6, Immunogenicity results for BSA-P14 conjugates 



Anti-peptide responses 
(14 days post 3) Mid 
point titre 


Anti-IgE responses (14 
days post 3) (^g/ml 
(PTmAbOOll)) 


Anti-IgE responses (14 
days post 4) (^g/mI 
(PTmAbOOll)) 


AV 


SD 


GM 


AV 


SD 


GM 


AV 


SD 


GM 


25974 


22667 


15492 


9.9 


2.18 


0.7 


22.9 


33.5 


4.8 



Table footnotes: AV (average), SD (standard deviation), GM (geomean) 



10 

Mice vaccinated with BSA alone as controls did not generate any detectable anti- 
peptide or anti-IgE responses. 

Functional activity results 

15 

The antiserum raised by the P14 vaccination was found to be functional, in that it was 
potent in the inhibition of histamine release from allergic human basophils after 
triggering with allergen (see FIGS. 2, 3 and 4). Moreover, the antiserum was not 
found to be anaphylactogenic (FIGs. 2, 3 and 4). 

20 

Summary 

P14 (mimotope of P8) was shown to be capable of raising high titres of anti-P14 and 
anti-IgE antibodies in mice. These antibodies were subsequently shown to be 
functional, in that they inhibited histamine release from allergic human basophils, and 
25 were not anaphylactogenic. P14 and P8, therefore, may be used in the treatment or 
prophylaxis of allergy. 

Example 3, Immunisation of mice with Pll conjugates (Pll-BSA, Pll -BSA) induces 
production of anti-human IgE antibodies, 

30 



SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 



PCT/EPOO/01456 



Human IgE epitope peptide PI 1 was coupled to maleimide-activated BSA (Pierce) 
(BSA-CRASGKPVNHSTRKEEKQRNGLL). 25 ^ig of conjugate formulated in 
SBAS2 was injected IM into 8 female BALB/c mice at days 0, 14 and 28. One control 
5 group of mice was injected with BSA/SBAS2. Blood samples were taken 14 days 
after each injection (a fourth bleeding was performed at day 24 post 3 to increase the 
availability of sera). Anti-peptide and anti-IgE antibodies raised by vaccination were 
measured by ELISA, as described in Example L 

10 Results 

A homogeneous IgG anti-Pl 1 response could be detected already after one injection, 
but increased ftirther after the second and third injection (FIG. 5a and 5b). All mice 
showed an anti-IgE response (ranging from 28 - 244 jig/ml as expressed in mAbOOS 
15 equivalents) after a third injection (FIG, 6). 

Part 2, Functional activity of epitope specific monoclonal antibodies 

Example 4, Functional activity of monoclonal antibodies raised against PI 4 

20 

Monoclonal antibodies have been generated that recognise specifically P8 and 
mimotopes thereof, using techniques known in the art. Briefly, the P14-BSA 
conjugate described in part 1 of these examples, was injected into groups of Balb/C 
mice with the o/w adjuvant containing QS21 and 3D-MPL. Spleen cells were taken 

25 and fiised with SP2/0 B-cell tumour cell line, and supematants were screened for 
reactivity against both P14 peptide and IgE. Several cell lines were generated, 
amongst which were P 14/23, P 14/31 and P14/33 which were deposited as Budapest 
Treaty patent deposit at ECACC on 26/1/00 under Accession No.s 00012610, 
0001261 1, 00012612 respectively. All three monoclonal antibodies were confirmed to 

30 bind to IgE, and specifically to PI 4, by ELISA binding assays, and P14 competition 
assays against monoclonal antibody binding to IgE. 



31 



SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 PCT/EPOO/01456 
The functional activity of these monoclonal antibodies was assayed in the human 

basophil histamine release inhibition assay as described in Example 1 . 
Results 

5 

All of the P14 monoclonal antibodies were tested on basophils taken from four 
different allergic patients (A patients were allergic to dust mite antigen, G patients 
were allergic to grass pollen). PTI 1 (PTmAbOOl 1) was included as a positive control 
antibody which is known to inhibit histamine release in vitro. All of the three P14 
10 monoclonal antibodies (23, 31, and 33) were potent in inhibiting histamine release 
from allergic basophils (See FIG. 7). 

Example 5, Anti-IgE induced in mice after immunisation with conjugate are capable 
of blocking local allergic response in the Monkey Cutaneous Anaphylaxis model. 

15 

PI 4/23 and PI 4/31 have also been tested for /n vivo activity. Briefly, the local skin 
mast cells of African green monkeys were shaved and sensitised with intradermal 
administration of lOOng of anti-NP IgE (human IgE anti-nitrophenylacetyl (NP) 
purchased from Serotech) into both arms. After 24 hours, a dose range of the 

20 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) 

25 was administered by intravenous injection. After 15-30 minutes, the control animals 
develop a readily observable roughly circular oedema from the anyphylaxis, which is 
measurable in millimeters. Results are expressed in either the mean oedema diameter 
of groups of three monkeys or as a percentage inhibition in comparison to PBS 
controls. PTmAbOOl 1, is a monoclonal antibody was used as a positive control. 

30 SBmAb0006 was used as a negative control. 



32 



SUBSnrUTE sheet (rule 26) 



wo 00/50461 
Table 7, PI 4/23 results 



PCT/EPOO/01456 



Amount of sample 
to be tested (ng) 


Mean diameter of oedema (mm) 


P14/23 


mAbOOU 


mAbOOOd 


20 


0 


ND 


12/15 


10 


0 


0 


17/19 


1 


15/13 


0 


20/20 


0.1 


15/12 


ND 


ND 


0.05 


15/15 


ND 


ND 


0 


15/15 


ND 


17/17 



ND = Not done. 



Table 8, PI 4/31 results 



Amount of 
sample to 
he tested 


Mean diameter of oedema (mm) 


P14/31 


mAbOOU 


mAbOOOd 


20 


0 


ND 


15/15 


10 


0 


0 


15/15 


1 


22/25 


0 


20/20 


0.1 


22/25 


ND 


ND 


0.05 


25/25 


ND 


ND 


0 


20/25 


ND 


20/25 



As complete inhibition of anaphylaxis was observed with higher doses of monoclonal 
antibody, these antibodies are not anaphylactogenic per se when administered in vivo. 



Example 6, Structural aspects of IgE mimotopes 

The present inventors have shown that the conformation in which the epitopes or 
mimotopes of the present invention is important for both anti-mimotope antibody 
recognition, and also for the ability of the peptides to generate a strong anti-IgE 
immune responses. As such the present inventors have developed structural rules 
which predict the optimal sites for peptide cyclisation. Peptides that use these sites of 
cyclisation form one prefered aspect of the present invention. 

As the full structure of IgE Fc has not been determined, the present inventors have 
refined the currently available models (Helm et al. supra, Padlan and Davis supra) 

33 

SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 PCT/EPOO/01456 
using the known structure of Cyl and Cy3 of IgGl (Deisenhofer J., 
1981. Biochemistry, 20, 2361-2370), In addition, models of the Ce2 domain have been 
built by comparison with known Ig folding-unit structures. The present inventors 
have designed these homology models of IgE Fc and thereby predicted the termini 
and the gross structure of intra-sheet (A-B loop, FIG 9A) and inter-sheet loops in IgE 
Fc domains (G-D loop, FIG 9B). Having defined the predicted IgE Fc A-B and C-D 
loops together with their supponing P-strands, mimotopes of the loops may be derived 
from the wild-type (WT) primary sequence of each loop by covalent cyclisation 
between chosen specific residues along the adjoining p-strands. Cyclisation is 
preferably realised by the formation of a disulphide bond between terminal cysteines 
which therefore combine to become a cystine. 

Based upon our structural alignments (FIG 9A & 9B) we have derived simple 
predictive rules in order to enhance the probability that die conformations adopted by 
a mimotope, after conjugation to a suitable carrier molecule, are similar to those of the 
parent epitope. 

Rule 1 

The hydrophobic cystine group should replace WT P-strand residues that belong to 
the water-inaccessible core of the Ig constant domain, formed by die interface 
between the two P-sheets. 

Rule 2i 

For intra-sheet loops (e.g. the A-B loop) the cystine group should replace WT 
residues that are fi-om adjacent anti-parallel P-strands (see FIG. 8) and that pack 
laterally together on the same side of the sheet. Following rule 1, this will be on the 
domain-interior side of the sheet. The structural derivation of this rule for the A-B 
loops is shown schematically in FIG 10 A and 1GB. 

Rule 2ii 

For inter-sheet loops (e.g. the C-D loop) the cystine group should replace WT 
residues on anti-parallel p-strands, one strand fi-om each sheet. Following rule 1, the 

34 



SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 PCT/EPOO/01456 
residues forming the optimal pair pack together from facing p-sheet surfaces, so 
forming part of the interface between the sheets. The structural derivation of this rule 
for the C-D loops is shown schematically in FIG. II A and FIG. IIB. In the tables of 
putative mimotope sequences that follow, designs predicted to be optimal are 
5 underlined. Below each block of sequences the dotted and solid lines link the residue 
positions chosen for optimal cyclisation, which are also shown in the same way in 
FIG lOB (for A-B loops) and in FIG. 1 IB (for C-D loops). 



Using the sequence alignment as shown in FIG 9A and 9B, together with the above 
10 rules, the present inventors have designed the following peptides listed in tables 9 to 
12. The peptides which are underlined (in solid or dotted lines) are the optimal 
peptides according to the above identified rules, the same lines are shown in FIG lOB 
and FIG 11 B. Non-underlined sequences are mimotopes. 



1 5 Table 9 , IgE CaS A-B loop sequences 



Peptide sequence (solid and dotted underlined are optimal) 



SEQ ID NO. 



341 

C S R P S P F D L F I 



C 

c 
c 



357 

RK S P T I T C 



R 
R 
R 
R 
R 
R 
R 



P 
P 
P 
P 
P 
P 
P 



D 
D 
D 
D 
D 
D 
D 



F 
F 
F 
F 
F 
F 
F 



R K 

R K 

R K 

R K 

R K 

R K 

R K 



T 
T 
C 

c 

T 
T 
T 



C 

T C 



£f§ZZDLFIRKSPTITC 



CPSPFDLFI 
C P S P F D L F I 



R K S P T I C 
R K S P T C 



CPSPFDLFIRKSPC 



33 
34 
35 
36 
37 
38 
39 
40 
41 
42 
43 
44 



35 



SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 

Table 10 , IgE C£4 A-B loop sequences 



PCT/EPOO/01456 



Peptide sequence (solid and dotted underlined 


are optimal) 






SEQ ID NO. 


446 














463 






C 


Y A 


FAT 


PEW 


P G 


S 


R D K R 


T 




c 


45 


c 


Y A 


FAT 


PEW 


P G 


S 


R D K R 


T 


L C 




46 


c 


Y A 


FAT 


PEW 


P G 


S 


R D K R 


T 


C 




47 


c 


Y A 


FAT 


PEW 


P G 


S 


R D K R 


C 






48 




C A 


FAT 


PEW 


P G 


S 


R D K R 


c 






49 




C A 


FAT 


PEW 


P G 


S 


R D K R 


T 


c 




50 




C A 


FAT 


PEW 


P G 


S 


R D K R 


T 


L C 




51 




C A 


FAT 


PEW 


P G 


S 


R D K R 


T 


L A 


c 


52 




C 


FAT 


PEW 


P G 


S 


R D K R 


T 


L A 


c 


53 
54 
55 
56 




c 


FAT 


PEW 


P G 


S 


R D K R 


T 


L C 






c 


FAT 


PEW 


P G 


S 


R D K R 


T 


C 






c 


FAT 


PEW 


P G 


S 


R D K R 


C 







36 



SUBSnrUTE sheet (rule 26) 



wo 00/50461 



PCT/EPOO/01456 



Table 11, IgE CeS C-D loop sequences 



Peptide sequence (solid and dotted underlined are optimal) 



SEQ ID NO. 



373 

CTWSRASGK 
CTWSRASGK 



387 

PVNHSTRC 
P V N H S T C 



C T 
C T 
C 

c 
c 
c 



w 
w 
w 
w 
w 
w 
c 
c 



S R A S G K 
S R A S G K 



P V N H S 
P V N H C 



R A S 

R A S 

R A S 

R A S 

R A S 

R A S 



c s 
c s 



K 
K 
K 
K 
K 

K 

R A S G K 
R A S G K 



P V N H C 

P V N H S C 

P V N H S T 

P V N H S T 

P V N H S T 

P V N H S T 

P V N H S C 

P V N H C 



C 
R 
R 
C 



C 

c 



57 
58 
59 
60 
61 
62 
63 
64 
65 
66 
67 
68 



Table 12 , IgE Ce4 C-D loop mimotope sequences 



Peptide sequence (solid and dotted underlined are optimal) 



SEQ ID NO. 



477 491 
CQWLHNEVQLPDARHSC 
CQWLHNEVQLPDARHC 
CQWLHNEVQLPDARC 
CQWLHNEVQLPDAC 
CWLHNEVQLPDAC 
CWLHNEVQLPDARC 
CWLHNEVQLPDARHC 
CWLHNEVQLPDARHSC 
CLHNEVQLPDARHSC 
£iiliMIYQI;fDARHC 
CLHNEVQLPDARC 
CLHNEVQLPDA C 



69 
70 
71 
72 
73 
74 
75 
76 
77 
78 
79 
80 



37 



SUBSITTUTE SHEET (RULE 26) 



wo 00/50461 



PCT/EPOO/01456 



iNDtCATlOiNS RELAThNC TO DEPOSITED iMICROORCa.MSM 
OR OTHER BIOLOGICAL MATERIAL 



(PCT Rule \jdis) 



A. The indicitions made below reiatit ta rh* h— ■. ^ . !~ ! ^ ~ [ ■ — - 

<iwc ta tne deposited microorganism or other biological material mcn-ed ;o m the descripuon 



onpage _IR _ 7.I3 



8. rDE;NTinCATION OF DEPOSIT ^ u m ' Z — === 
* rurther deposits are identitied on an additional sheet I — J 

Name ofdcpositary insticution ' 

European Collection of Cell Cultures 



Address of depositary insticution (inciuduxs poual code andcountry) 

Vaccine Research and Production Laboratory 

Public Health Laboratory Service 

Centre for Applied Microbiology Research 

Porton Down, Salisbury 

Wiltshire SP4 QJG, GB 



Date of deposit 

26 January 2000 
C 26/01/00) 



Accession Number 



00012610, 00012611 
and 00012612 



C ADDnrtONAL INDICATIONS (Ua^ ,ia^ y.^, appiicai>icj This information is continued 00 an additional sheet R' 



^'t/lV'^V Of those designations where a European Patent is sought a s^mni^ 

Che application hJ befn r^fnf w ^"^"P^" ""^ ^^'^ °° "f^i^h 

CO an Lperc no„.^cXlL"^;L'on'^.SSSA.°^^^^ llX^^ ^"'^^ ^ ^^"^^^ 



0. DESICNATED STATES FOR WHICH .ND.CATIONS AR£ .VtADE nf,,e U«aca,io„ or. no. for aU .^^S,^j 



E. SEPARATE rmwrSHLNC Of IND.CATIONS rUcr^ blank if.o, cppli^abl,) 



J^7^T » iatemaciona. Bureau .^.yyU„^„a.„ojO,e^.^,.,_ -^o. 



For receiving OfTtce use only 



I I *M HKsived with the incerr 



incemational application 



Authorized offtcer 



Form PCT^RO/Ija (Ju(v|99S^ 



For International Bureau use onN 



I I This sheet was received bv 



y the Inte.Tiationai Surrau on: 



Authorized ofTicer 



38 



SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 PCT/EPOO/01 456 

Claims 

1. A peptide comprising an isolated surface exposed epitope of the Ce3 domain of 
IgE, wherein the peptide is P5 (SEQ ID No. 1), or mimotope thereof. 

2. A peptide comprising an isolated surface exposed epitope of the Ce3 domain of 
IgE, wherein the peptide is P6 (SEQ ID No. 2), or mimotope thereof. 

3. A peptide comprising an isolated surface exposed epitope of the region spanning 
Ce3 and Ce4 domains of IgE, wherein the peptide is P7 (SEQ ID No. 3), or mimotope 
thereof. 

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

5. A peptide comprising an isolated surface exposed epitope of the Ce4 domain of 
IgE, wherein the peptide is P9 (SEQ ID No. 5), or mimotope thereof. 

6. A peptide comprising an isolated surface exposed epitope of the Ce3 domain of 
IgE, wherein the peptide is P200 (SEQ ID No. 6), or mimotope thereof. 

7. A peptide comprising an isolated surface exposed epitope of the Ce3 domain of 
IgE, wherein the peptide is P210 (SEQ ID No. 7), or mimotope thereof 

8. A peptide comprising an isolated surface exposed epitope of the Ce3 domain of 
IgE, wherein the peptide is 2-90N (SEQ ID No. 82), or mimotope thereof. 

9. A peptide comprising an isolated surface exposed epitope of the Cs4 domain of 
IgE, wherein the peptide is 3-90N (SEQ ID No. 83), or mimotope thereof. 

10. A peptide comprising an isolated surface exposed epitope of the Cs4 domain of 
IgE, wherein the peptide is 4-90N (SEQ ID No. 84), or mimotope thereof. 

11. A mimotope as claimed in any one of claims 1 to 10 wherein the mimotope is a 
peptide. 

12. A peptide as claimed in claim 4, wherein the mimotope of P8 is a peptide of the 
general formula: 

P, Xi, Xj, P, X3, X4, X5, X^, X5, X5 

wherein; X, is an amino acid selected from E, D, N, or Q; X^ is an amino acid selected 
from W, Y, or F; X3 is an amino acid selected from G or A, X^ is an amino acid 
selected from S, T or M; X, is an amino acid selected from R or K; and X^ is an amino 
acid selected from D or E. 



39 



SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 PCT/EPOO/01456 

13. A peptide as claimed in claim 12, wherein the mimotope of P8 is a peptide of the 

general formula P, X„ X2, P, G, X4, R, D, X5, X5 

wherein; X, is an amino acid selected from E, D, N, or Q; X, is an amino acid selected 
from W, Y, or F; X4 is an amino acid selected from S, T or M; X5 is an amino acid 
5 selected from R or K; and X^ is an amino acid selected from D or E. 

14. An immunogen for the treatment of allergy comprising a peptide or mimotope as 
claimed in any one of claims 1 to 13, additionally comprising a carrier molecule. 

15. An immunogen as claimed in claim 14, wherein the carrier molecule is selected 
from Protein D or Hepatitis B core antigen. 

10 16. An immunogen as claimed in claim 14 or 15, wherein the immunogen is a 
chemical conjugate of the peptide or mimotope, or wherein the immunogen is 
expressed as a fusion protein. 

17. An immunogen as claimed in any one of claims 14 to 16, wherein the peptide or 
peptide mimotope is presented within the primary sequence of the carrier. 
15 18. A vaccine for the treatment of allergy comprising an immunogen as claimed in 
any one of claims 14 to 17, further comprising an adjuvant. 

19. A ligand which is capable of recognising the peptides as claimed in any one of 
claims 1 to 13. 

20. A ligand as claimed in claim 19, wherein the ligand is selected from PI 4/23, 

20 PI 4/31 or PI 4/33; which are deposited as Budapest Treaty patent deposit at ECACC 
on 26/1/00 under Accession No.s 00012610, 0001261 1, 00012612 respectively. 

21. A pharmaceutical composition comprising a ligand as claimed in claim 19. 

22. A pharmaceutical composition comprising a ligand as claimed in claim 20. 

23. A peptide as claimed in any one of claims 1 to 13 for use in medicine. 
25 24. A vaccine as claimed in claim 18 for use in medicine. 

25. An immunogen as claimed in any one of claims 14 to 17, for use in medicine. 

26. Use of a peptide as claimed in any one of claims 1 to 13 in the manufacture of a 
medicament for the treatment or prevention of allergy. 

27. A ligand which is capable of recognising a peptide as claimed in any one of claims 
30 1 to 13, for use in medicine. 

28. Use of a ligand which is capable of recognising a peptide as claimed in any one of 
claims 1 to 13, in the manufacture of a medicament for the treatment of allergy. 

40 



SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 PCT/EPOO/01456 

29. Use of P14/23, P14/31 or P14/33; which are deposited as Budapest Treaty patent 
deposit at ECACC on 26/1/00 under Accession No.s 00012610, 0001261 1, 00012612 
respectively, in the identification of mimotopes of P8. 

30. A peptide which is capable of being recognised by P14/23, P14/31 or P14/33; 

5 which are deposited as Budapest Treaty patent deposit at ECACC on 26/1/00 under 
Accession No.s 00012610, 0001261 1, 00012612 respectively. 

31. A vaccine comprising a peptide as claimed in claim 30. 

32. A method of manufacturing a vaccine comprising the manufacture of an 
immunogen as claimed in any one of claims 14 to 17, and fomiulating the immxmogen 

10 with an adjuvant. 

33. 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 13, to 
the patient. 

34. A method for treating a patient suffering from or susceptible to allergy, 

15 comprising the administration of a vaccine as claimed in claim 24 or 3 1 to the patient. 

35. 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 21 or 
22, to the patient. 



41 



SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 



PCT/EPO0/OI456 



Fiq, 1 


igt amino 


Residue 


Surface Area 


PRO_108 


70.754211 


ARGJ09 


205.863144 


6LYJ10 


48.697884 


: VAL 111 


14.094634 


:. SERJ12 


45.00526 


ALA 113 


13786729 


TYR_n4 


113.018738 


LEU_115 


20.160578 


SERJ16 


49507282 


ARGJ17 


148 687027 


PR0_118 


13.401789 


SER 119 


65.402878 


PROJ20 


55.390755 


PHE_121 


42.690605 


ASPJ22 


24.277737 


LEUJ23 


18993639 


PHEJ24 


67 098251 


ILE_125 


158 574051 


ARGJ26 


215.537506 


LYSJ27 


98.602692 


SER 128 


96506714 


PRO_129 


8 909876 


Ce3 THRJ30 


32 048698 


ILEJSl 


0 


THR 132 


28.179621 


CYSJ33 


0 


LEUJ34 


56 904575 


VALJ35 


0.847608 


VALJ36 


43.135212 


ASP_137 


46.897842 


LEUJ38 


0 


ALA_139 


14.162636 


PROJ40 


101.686447 


SERJ41 


72.739418 


LYSJ42 


71.689812 


GLYJ43 


19.275223 


THR 144 


94.73616 


VAL 145 


16.920198 


ASNJ46 


90.04982 


LEU 147 


27.456902 


THR 148 


56.718086 


TRP 149 


7.372435 


SER 150 


18.283966 


ARGJ51 


102.064598 



1/15 

surface exposure. 



verage/5 >50 >80 



99.7733764 


1 


89.6122724 


1 


76.8830266 


1 


65.4895302 


1 


46.920649 


0 


41.2131878 


0 


48.2957174 


0 


69.0320708 


1 


68.9550828 


1 


59.4319108 


1 


66.4779462 


1 


65.1146108 


1 


40.2327528 


0 


41.3511228 


0 


41.6901974 


0 


62.3268566 


1 


96.8962368 


1 


111.7612278 


1 


127.2638428 


1 


115.6261678 


1 


90.3210972 


1 


47.213596 


0 


33.1289818 


0 


13.827639 


0 


23.4265788 


0 


17.1863608 


0 


25.8134032 


0 


29.5570474 


0 


29.5570474 


0 


21.0086596 


0 


41.1764274 


0 


47.0972686 


0 


52.0556626 




55.9107072 




72.025412 




55.0721622 




58.5342426 




49.6876606 


0 


57.1762332 


1 


39.7034882 


0 


39.9762418 


0 


42.3791974 


0 


48.0311764 


0 


61.6391216 


1 



1 
1 



0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 




0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 



SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 



PCT/EPOO/01456 



2/15 



ALAJ52 


55.716797 


68 756572A 

UU*/ mJlMJi £U 






SERJ53 


124 757812 


98 5954416 






GLYJ54 


42.95969 


89 71 7AAQA 




1 ''M 


LYS 155 


167 128311 

1 Ur • 1 ^UO 1 1 


99 7866798 

7Z./00U/ ^0 




1 


Pr6^156 


23 025738 


99 86971 89 




1 


VAL 157 


106 06181? 

1 UU.UU 1 U 1 w 


100 8787409 




, 


ASN 158 


125 138039 


81 9094iOA 




1 


HIS 159 


83 037*^ 


81 8A410d9 




1 = 


SER160 


68 749311 


100 109dA1i 






THR 161 


36 3350S6 


101 M8^Aifl 




1 


ARG 162 


187 252609 


10*5 U7«;779 

1 UJ. IQ/ J/ /Z 




1 


LYS 163 


132 868546 

1 v^«UUU«/^U 


111 ld8117i 






GLU 164 


100 61936? 


1 91 707';09A 


- 




GLU 165 


108 653114 


1171 989088 

II/. IZOZUOO 






LYS 166 


89 130889 

U7. 1 tJXJOQi. 


1 1 8 1814Qid 






GIN 167 


154 3S614 


191 aoo9';id 






ARG 168 


139 144974 


1 1 'i 0/i147AA 

1 1 J.UOuH/ DO 






ASN 169 


197 761147 

14./ ,/\)\ 1 *f / 


101 19«;i099 
1 U 1 .OZ JU7ZZ 






GIY 170 


64 99494 


79 *i7A840R 

/Z.J/ 00470 




0 

u 


THR 171 


90 4404A 


i7 000d9^A 

4/.77 74ZO0 


n 
u 


n 
u 


IFU 177 


10 613498 


91 IROAAAft 
ZO.OO7OOOO 


u 


n 
u 


THR 171 


16 957843 


1 9A1Q9dd 

1 J.70j7Z44 


n 
u 


n 
u 


VAL 174 


4 719363 


11 914i9AA 

1 1 .7 1 *t*TZUO 


n 
u 


u 


THR 17S 
III i\ 1 / J 


27 795598 


1 7 8898S66 

1 / .UU7UJOU 


n 
u 


n 
u 


SER 176 


0 192971 

U* 1 1 1 1 


14 6768899 

i ~*u/ uuu^^ 


n 
u 


n 
u 


THR 177 
1 1 1 1\ 1 # / 


40 490578 

~U."T 7UJ / U 


23 6673198 

^U.UU/ %J 1 £U 


n 
u 


n 

u 


LEU 178 


0 192971 


19 1496968 


n 
u 


n 

u 


PRO 179 


49 664516 


25 6114614 


n 
u 


n 

u 


VAL 180 

T 1 UU 


5 172098 

«/. 1 / £v / U 


23 0737176 

^vi.ur U/ 1 / U 


n 
u 


n 
u 


GLY 181 

1 1 U 1 


32 537144 


63 9982649 




n ■ 


THR 182 


27 801859 


61 8714416 

u 1 .U/ 1 i u 




n ''■ '•■'•■^ 

u 


ARG 183 


204 815704 

AtV^*U 1 ^/ U~ 


A<k 0i^017i 
Oj.74 jUO/ 4 




0 ' 
u 


ASP 184 

f^Ji 1 UT 


39 030403 


A7 9107R14 
O/^OU/ 0J4 




0 PR 


TRP 185 


25 540077 


90 ^OHQ'iR 

7U.JU 1 /7JU 




1 V. " -: 


ILeT86 

1 L u u 


38 965874 


'I'i 8891974 
JJ.OOZ 1 7/4 


- 


u 


GLU 187 

L. u 1 u/ 


144 156921 


60 7A9811 

UU./ UZOJ 1 




0 
u 


GLY 188 

w L. 1 ^1 U U 


31 717712 


A8 4Q01Q4A 

00.47UJ740 




U .V 


GLu"l89 


63 433571 


AO 77440R4 
OU./ / 44U04 




u 


THR 190 

1 1 1 1\ 1 7U 


64 1 77895 

U^. 1/7 U7j 


49 187199 
4Z.I0/ JZZ 


n 
u 


A 

u 


TYRJ91 


0 385943 


36 3067554 

«^U>UUU/ JJ*1 


0 
u 


u 


GLN 192 


51.221489 


49.1596714 


0 


0 


as 193 


2.314879 


36.401281 


0 


0 


ARG_194 


127.698151 


48.626639 


0 


0 


VAL 195 


0.385943 


38.7900666 


0 


0 


THR 196 


61.512733 


56.894756 


1 


0 


HIS 197 


2.038627 


45.6995426 


0 


0 


PROJ98 


92.838326 


51.6998584 


1 


0 


His 199 


71.722084 


59.4304494 


1 


0 



SUBSTITUTE SHEET (RULE 26) 



BEST AVAILABLE COPY 



wo 00/50461 



PCT/EPOO/01456 



LEUJOO 

PROJOl 

ARG_202 

ALA_203 

LEU_204 

METJ05 

ARG_206 

SER_207 

THRJOB 

THR_209 

LYSJIO 

THRJll 

SER_212 

GLYJIS 

PROJM 

ARG_215 

ALA_216 

ALA_217 

PRO_218 

GLU_219 

VAL_220 

TYR_221 

ALA_222 

PHE_223 

AU_224 

THR_225 

PRO_226 

GLU_227 

TRP_228 

PRO_229 

GLY_230 

SER_231 

ARG_232 

ASP_233 

LYS_234 

ARG_235 

THR_236 

LEU_237 

AU238 

aSJ39 

LEU_240 

ILE_241 

GLN_242 

ASN_243 

PHE_244 

MEL245 

PRO_246 

GLU 247 



30.387522 
100.165688 
210.511932 
35.168427 
70.497948 
140.638672 
138.608749 
87.617599 
15.800501 
55.303375 
25.067995 
51.407516 
111i70686 
41.888771 
115.529221 
110.744141 
37.145763 
54.781963 
8.161689 
31.599922 
1.970238 
6.712473 
14.550046 
10.871778 
28.272247 
12.634302 
21.980837 
157.767822 
135.485703 
1.040579 
19.028141 
109.766861 
126.577293 
116.259117 
113.302795 
30.729465 
3.757154 
0 

2.7016 
0 
0 

1.157829 
23.797855 
72.443329 

2.921356 
47.595181 

9.738265 
96.778259 



3/15 

101.1251104 
89.5911306 
89.3463034 
111.3965334 
119.0851456 

94.506279 
90.6326938 
87.5937792 
64.4796438 
47.0393972 
51.8300146 
57.0476686 
69.0928378 

86.228067 
83.3757164 
72.0179718 
65.2725554 
48.4866956 

26.731915 

20.645257 
12.5988736 
13.1408914 
12.4753564 
14.6081692 

17.661842 
46.3053972 
71.2281822 
65.7818486 
67.0606164 
84.6178212 
78.3797154 
74.5343982 
96.9868414 
99.3271062 
78.1251648 
52.8097062 
30.0982028 
7.4376438 
1.2917508 
0.7718858 
5.5314568 
19.4798026 
20.0640738 

29.58311 
31.2991972 
45.895278 
41.159651 
45.6910524 



0 
0 
0 
0 
0 
0 
0 
0 
0 



0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 



0 
0 
0 
0 
0 
1 
1 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 

1 

0 
0 

1 
1 

0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 




P8 



i 

m 

i 

m 

- ma 



SUBSTITUTE 



SHEET (RULE 



26) 



wo 00/50461 PCT/EPOO/0 1 456 



4/15 





4876S194 

*tU./ UJ 1 7*T 


40 00676Q6 
4U.7UO/000 


n 
u 


n 
u 


II F ?49 


75 578^6^ 


41 641 
40.04 1 /33Z 


n 
U 


n 
U 




71 671717 


44 9091 7R 
44.Z7ZI / O 


n 
u 


n 


VAL 751 


71411771 

LO**i 1 OL£,0 


17 46Q60Q6 
0/.4070U00 


n 
u 


u 


GIN ?S? 


1 nn nifli7i 


40 7Ra<;qa 

4 U./ 00300 


U 


U 


TRP 9*;^ 


1 4 659179 


07.7 1 4*H*JZ 


n 
U 


u 


I FN 9*14 


47 161075 


QAA7AAR 
33.000/<fOO 




n 
U 


Cad HK 95^ 


10 11197ft 
17.01 JZ/0 


A4 OA^^RflR 
04.110440110 




A 

U 


A^N 956 


1 no 674716 

1 UU.O/ 1/ JO 


R7 R099QQA . 

o/.oy/zooo 






fil II 957 


141 510041 


74.7030044 






VAI 958 


111 701116 


19't TIliX^KA 
IZ3.///U334 






ni N 95Q 


77 170070 

// .0/77/7 


117 44Q7nnA 

1 I/.440/UU4 






1 Fll 7An 


171 5909^^ 
1 / O.JZUZOO 


inn ^R90iQ9 

IUU.4QZ7I0Z 






PRO 7A1 


50 007061 

J7.UU/70 1 


flft 7^1 1A19 
00./3 1 lOiZ 






A^P 7A7 


58 71 5107 
JO./ 1 JIU/ 


7/.UOUOZ30 






Al A 7A^ 


75 119576 
/ J. IOZj/0 


A4 ^lA9fl9 
04.0 lOZoZ 




A 
U 


ARR 7A4 


118 0757';i 

1 1 0.7ZJ/ J 1 


7n 1 <;9QR0i! 
/U. 13/7074 




A 
U 


HK 7A5 


0 fiOOOl ^ 


Al ^7779^4 
0 I.3///Zj4 




A 
U 


^FR 9AA 


ftft 10140R 

00. 17 IH70 


CO OQOOQOO 

37.7OZ77Z0 




A 
U 


THP 7A7 


1 *; R1R7R7 
13.000/0/ 


^7 ^^9QQ1>1 
0/.33Z0ol4 




A 

D 


1 ni\__f 00 


66 OOAOI^ 
OO.7UO7 1 0 


^1 A0A09CA 
3 1 .0707Z30 




A 
0 


f^l M 9A0 


7 099604 
/.UZZO74 


LC 771 0 AO 
03.// lOUO 




A 
U 


ppn 77n 
rivu_Z/U 


flO 599716 
OU.jZZ/00 


RO RA71 n4 A 
07,00/ 1U40 








1 J 0.3000 00 


fl9 91 CQQRR 
OZ.Z 130000 






lY^ 779 


116 11770*; 
100.0 1//7J 


194 9QAftQlA 
IZ4.ZOO00 14 






THR 77^ 


98 6500R4 

ZO.OJUUO'r 


1 99 1A0W9 
1 ZZ.OO737Z 






1 974 


917 110157 
Z I/.I0UI3/ 


11^ 7';9^9^ 
1 I0./3Z3Z3 






Rl Y 975 


71 186519 

/I.I UOJ07 


80 17105(;4 
07, 1 / 07334 






^FR 97A 


1 1 5 47805 


87 755050A 
0/./33U370 






ni Y 977 


1 1 494047 

IO.*tZ47H/ 


44 40A91AR 
44.4U0Z 1 00 


A 
U 


A 
U 


PHF 978 


71 55560^; 

Z 1 .3330U3 


10 111101 
0U.00107 1 


A 
U 


A 
U 


PHF 970 


0 1R5041 
U.003740 


7 9157R1 

/ .Z03/ 0 1 


A 
U 


A 
U 


VAI 780 


0 ft1941 

U.O IZ4 1 


4 R171A19 
4.00/ 10 IZ 


A 
U 


A 
U 


PHF 781 


n 
u 


1 705A808 

O.Z730070 


A 
U 


n 
U 


5FR 787 


1 411848 

1. to 1040 


1 7185019 

O.Z 1 OJUIZ 


A 
U 


n 
U 


ARfr78^ 


1 1 84R94R 

lO-OtOZHO 


1 0 974010A 
1 U.Z/4U0U0 


A 
0 


A 
U 


1 Fl 1 784 


n 


10 A0ft'^A7R 
1 11.07030/ 0 


0 


A 
0 


ni II 9A5 


16 000057 

0U.U7UU3/ 


lA 7101179 
10./ 17 lO/Z 


A 

0 


A 
0 


VAL 286 


2 122686 


37 9263888 


n 

U 


n 
u 


THR 287 


31.534695 


44.9612932 


0 


0 


ARG288 


119.884506 


47.621104 


0 


0 


: I- ALA 289 


35.174522 


48.1620878 


0 


0 


GLU 290 


49.389111 


53.3908726 


1 


0 


TRP 291 


4.827605 


46.7616094 


0 


0 


: GLU 292 


57.678619 


74.1783896 


1 


0 


: GLN 293 


86.73819 


74.2797528 


1 


0 


• , : : • LYS 294 


172.258423 


95.33479 


1 


1 


ASP 295 


49.895927 


84.3393862 


1 


1 



SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 



5/15 



PCT/EPOO/01456 



GLU 296 


110.102791 


77.3154726 


1 


0 


PHE 297 


2.7016 


42.863788 


0 


0 


ILE 298 

ILL. Lt\3 


51 618622 


50.8319354 


1 


0 


CYS 299 


0 


28 8885658 


0 


0 


ARG 300 


89 736664 


28 5866504 

^ w « w u w www* 


0 


0 


ALA 301 


0 385943 


19.2429276 


0 


0 


VAL 302 


1.192023 


26.9888008 


0 


0 


Hisl03 


4 900008 


9 4758236 


0 


0 


GLU_304 


38.729366 


26.6422386 


0 


0 


ALAJOS 


2.171778 


35.9697824 


0 


0 


ALA 306 


86.218018 


55.9203442 




0 


SER 307 


47.829742 


65.2374516 




0 


PR0_308 


104.652817 


74.870169 




0 


SER 309 


85.314903 


64.5256652 




0 


GLN 310 


50.335365 


61.2491192 




0 


THRJll 


34.495499 


57.5090542 




0 


VAL 312 


31.447012 


52.1974324 




0 


GLN_313 


85.952492 


53.3657582 




0 


ARG 314 


58.756794 


47.6630814 




0 


ALAJIS 


56.176994 


54.8599582 




0 


VAL 316 


5.982115 


51.1280902 




0 


SER 317 


67.431396 


63.6157024 




0 


VAL 318 


67.293152 


62.0941464 




0 


ASN_319 


121.194855 


78.2977722 




0 


PRO_320 


48.569214 


119.372577 




1 


GLY 321 


87.000244 








LYS 322 


272.80542 









SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 



6/15 



PCT/EPOO/01456 



Fig. 2 

Histamine release inhibition and anaphy lactogen icity of anti-P14 antiserum 



% inhibition of Grass-allergen induced histamine release 



@ 1/100 
O 1/500 




25 
20 
15 
10 
5 
0 



mAb005 Ipg/ml mAbOll 1 pg/ml P14.2 

Anaphylaxis (as % of allergen) 



BSA 



















1 





























mAbOOS Ipg/ml mAbOll 1 pg/ml 



PI 4.2 



BSA 



SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 



7/15 



PCT/EPOO/01456 



Fig. 3 

Histamine release inhibition and anaphylactogenicity of anti-P14 antiserum. 
% inhibition of allergen-induced histamine release 




PI 4.2 SOX P14.7 40X BSA 40X 




mAbI 
2Mg/ml 



mAbll mAbll 
2pg/ml + 2pg/ml + 
BSA 40X BSA BOX 



IgGl 
2pg/ml 



IgGl 
2|jg/ml + 
BSA 40X 



Anaphylactogenicity (% of allergen) 



100 
90 - 
80 
70 
60 
50 
40 
30 
20 
10 
0 



P14.2 80X P14.7 40X BSA 40X 



mAbll 
2pg/ml 



mAbll 
2pg/ml + 
BSA 40X 



mAbll 
2|jg/ml + 
BSA SOX 



IgGl 
2(jg/ml 



IgGl 
2pg/ml + 
BSA 40X 



SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 PCT/EPOO/01 456 

8/15 



Fig. 4 

Histamine release inhibition and anaphylactogenicity of anti-P14 antiserum 



Inhibition of allergen-induced histamine release 





P14.13 



PI 4.2 



1 — I — — \ r 

BSA mAbOII mAbOII/BSA IgGI IgGI/BSA 



100 
90 
80 
70 
60 

; 50 
40 
30 
20 
10 
0 



Anaphylactogenicity 



P14.13 



PI 4.2 



BSA 



mAbOn mAbOn/BSA IgGI 



IgGI/BSA 



SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 PCT/EPOO/01456 



9/15 



F i C| . S Antibody response anti-Pi 1 . 



A. 



Figure 1a. IgG anti-Pi 1, 14 post 1 and 2 ^ 




100 1000 
Serum dilution 



10000 



100000 



1 14 pi 

2 

3 

4 

5 

6 

7 

8 

1 14 p2 

2 

3 

4 

5 

6 

7 

8 



- o- 2 

3 

-X- 4 

-r- 5 

^ 6 
7 

— 8 



anti-BSA 



B. 



Figure 1b. IgG anti-Pi 1, 14 post 3 




1000 



10000 100000 
Serum dilution 



1000000 10000000 



1 
2 
3 
4 
5 
6 
7 
8 



SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 



10/15 



PCT/EPOO/01456 



Fig. 6 



Anti-P1 1 IgG anti-human IgE titres. 



IgG anti IgE 24 Post 3 




Dilution (80pg/ml] 



SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 



11/15 



PCT/EPOO/01456 



Fig. 7 

Histamine release inliibltion activity of anti-P14 monoclonal antibodies. 





SUBSTTTUTE SHEET (RULE 26) 



12/15 



PCT/EPOO/01456 



8 



IgE domain structure. 



A. IgE Domain viewed face-on 





\ ^. ^ 



B. IgE Domain viewed topologically 



SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 



PCT/EPOO/01456 



13/15 



Fig. 9A A-B 



loops 



...a 
2U1 



b... 



261 



L?_ PPKPKDTLMI SRTP^VTC 

235 



Ce2 
Ce3 



I LQSSCDGGGHFPPTIQLLC 
LSRPSPFDLF-I RKSPT I TC 

Ce4 VYAFATPEWPGSRDKRTLAC 
350 

^^^3 T_L PPS-REEM-TKN Q V S L T C 

t t 

i+2 i+4 



254 
C 

358 
C 

464 
C 

367 



IL 



t t 

j i+2 
JJ 



Fig. 9B 



C-D loop 
...c 



C72 

Ce2 

C€3 

C€4 
C73 



275. 



266 

w 

372 
476. 
379 



W; 

M 



w 
w 



i+1 i+3 
II 



V D G V 



V 


Q 




Q 


G 


K 


V 


Q 


Q 


P 



i 



H N 

M D 

V N 

P D 

N N 



i 

A 
I 

i 



K 
D 
S 
R 
K 



(33 295 
K P R E Q Q 



^ 392 
p R K E E K Q 

|STTQP^^^ 

^ 399 
^ T P P V L D 



f t 

j+2 j+4 

I I <l 



L' ! 

1 



SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 



14/15 



PCT/EPOO/01456 



Fig. 10 



A. B. 




SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 PCT/EPOO/01456 

15/15 



Fig. 11 



A. B. 




SUBSTITUTE SHEET (RULE 26) 



wo 00/50461 



PCT/EPOO/01456 



SEQUENCE LISTING 

<110> SmithKiine Beechain Biolicals s.a. 
Peptide Therapeutics Ltd 

<120> Vaccine 

<130> 845173 
<160> 86 

<170> FastSEQ for Windows Version 3.0 

<210> 1 
<211> 22 
<212> PRT 

<213> Human and artificial sequence 
<400> 1 

Arg Ala Ser Gly Lys Pro Val Asn His Ser Thr Arg Lys Glu Glu Lys 

1 5 * 10 15 

Gin Arg Asn Gly Thr Leu 
20 

<210> 2 
<211> 9 
<212> PRT 

<213> Human and artificial sequence 
<400> 2 

Gly Thr Arg Asp Trp lie Glu Gly Glu 
1 5 

<210> 3 
<211> 19 
<212> PRT 

<213> Human and artificial sequence 
<400> 3 

Pro His Leu Pro Arg Ala Leu Met Arg Ser Thr Thr Lys Thr Ser Glv 

1 5 10 15 

Pro Arg Ala 

<210> 4 
<211> 11 
<212> PRT 

<213> Human and artificial sequence 
<400> 4 

Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg Thr 
1 5 10 

<210> 5 
<211> 5 
<212> PRT 

<213> Human and artificial sequence 

<400> 5 
Glu Gin Lys Asp Glu 
1 5 

<210> 6 



1 



wo 00/50461 PCT/EPOO/01456 





<£. 1 1> 


1 Q 












<212> 


PRT 












<^ 1 J> 


Human and artificial sequence 












<4 00> 


b 










Leu 


Ser Arg 


Pro Ser Pro Phe Asp Leu Phe lie 


Arg 


Lys 


Ser 


Pro 


1 




5 10 








15 


lie 


Thr Cys 
















/ 












<211> 


21 












<212> 


PRT 












<213> 


Human and artificial sequence 












<400> 


7 










Trp 


Leu His 


Asn Glu Val Gin Leu Pro Asp Ala 


Arg 


His 


Ser 


Thr 


1 




5 10 








15 


Gin 


Pro Arg 


Lys Thr 




























0 
0 












<211> 


23 












<212> 


PRT 












<213> 


Human and artificial sequence 












<400> 


8 










Cys 


Arg Ala 


Ser Gly Lys Pro Val Asn His Ser 


Thr 


Arg 


Lys 


Glu 


1 




5 10 








15 


Lys 


Gin Arg 


Asn Gly Leu Leu 














20 












<210> 


9 












<211> 


11 












<212> 


PRT 












<213> 


Human and artificial sequence 












<400> 


9 










Gly 


Lys Pro 


Val Asn His Ser Thr Gly Gly Cys 










1 




5 10 












<210> 


10 












<211> 


18 












<212> 


PRT 












<213> 


Human and artificial sequence 












<400> 


10 










Gly 


Lys Pro 


Val Asn His Ser Thr Arg Lys Glu 


Glu 


Lys 


Gin 


Arg 


1 




5 10 








15 



10 



15 



20 



25 



30 



35 



40 



45 



50 Gly Cys 

<210> 11 

<211> 20 

55 <212> PRT 

<213> Human and artificial sequence 

<400> 11 

Cys Gly Lys Pro Val Asn His Ser Thr Arg Lys Glu Glu Lys Gin Arg 
60 1 5 • 10 15 

Asn Gly Leu Leu 
20 

<210> 12 

65 <211> 14 

<212> PRT 



2 



20 



WO 00/50461 PCT/EPOO/01 456 

<213> Human and artificial sequence 

<400> 12 

Arg Ala Ser Gly Lys Pro Val Asn His Ser Thr Gly Gly Cys 
5 15 10 

<210> 13 
<211> 11 
<212> PRT 

10 <213> Human and artificial sequence 

<400> 13 

Cys Gly Thr Arg Asp Trp lie Glu Gly Leu Leu 
15 10 

15 

<210> 14 
<211> 12 
<212> PRT 

<213> Human and artificial sequence 
<400> 14 

Cys Gly Thr Arg Asp Trp lie Glu Gly Glu Thr Leu 
15 10 

25 <210> 15 

<211> 12 
<212> PRT 

<213> Human and artificial sequence 

30 <400> 15 

Gly Thr Arg Asp Trp lie Glu Gly Glu Thr Gly Cys 
15 10 

<210> 16 
35 <211> 12 

<212> PRT 

<213> Human and artificial sequence 
<400> 16 

40 Cys His Pro His Leu Pro Arg Ala Leu Met Leu Leu 
15 10 

<210> 17 
<211> 12 
45 <212> PRT 

<213> Human and artificial sequence 

<400> 17 

Cys Gly Thr His Pro His Leu Pro Arg Ala Leu Met 
50 1 5 10 

<210> 18 
<211> 13 
<212> PRT 

55 <213> Human and artificial sequence 

<400> 18 

Thr His Pro His Leu Pro Arg Ala Leu Met Arg Ser Cys 
15 10 



60 



65 



<210> 19 
<211> 14 
<212> PRT 

<213> Human and artificial sequence 
<400> 19 



3 



wo 00/50461 PCT/EPOO/01456 

Gly Pro His Leu Pro Arg Ala Leu Met Arg Ser Ser Ser Cys 
15 10 

<210> 20 

5 <211> 13 

<212> PRT 

<213> Human and artificial sequence 

<400> 20 

10 Ala Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg Thr Cys 
15 10 

<210> 21 

<211> 17 

15 <212> PRT 

<213> Human and artificial sequence 

<4 00> 21 

Ala Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg Thr Leu Ala Gly Gly 
20 1 5 10 15 

Cys 



<210> 22 

25 <211> 17 

<212> PRT 

<213> Human and artificial sequence 

<400> 22 

30 Cys Gly Gly Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg Thr 
1 5 '10 15 

Leu 



35 <210> 23 

<211> 13 

<212> PRT 

<213> Human and artificial sequence 

40 <400> 23 

Cys Thr Arg Lys Asp Arg Ser Gly Pro Trp Glu Pro Ala 
15 10 

<210> 24 

45 <211> 15 

<212> PRT 

<213> Human and artificial sequence 

<400> 24 

50 Ala Pro Cys Trp Pro Gly Ser Arg Asp Cys Arg Thr Leu Ala Gly 
15 10 15 

<210> 25 

<211> 16 

55 <212> PRT 

<213> Human and artificial sequence 

<400> 25 

Ala Cys Pro Glu Trp Pro Gly Ser Arg Asp Arg Cys Thr Leu Ala Gly 
60 1 5 10 15 

<210> 26 

<211> 17 

<212> PRT 

65 <213> Human and artificial sequence 



4 



20 



WO 00/50461 PCT/EPOO/01456 

<400> 26 

Cys Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg Thr Leu Cys 

15 10 15 

Gly 

5 

<210> 27 
<211> 16 
<212> PRT 

10 <213> Human and artificial sequence 

<400> 27 

Cys Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg Thr Cys Gly 
15 10 15 

15 

<210> 28 
<211> 13 
<212> PRT 

<213> Human and artificial sequence 
<400> 28 

Thr Pro Cys Trp Pro Gly Ser Arg Asp Lys Arg Cys Gly 
15 10 

25 <210> 29 

<211> 11 
<212> PRT 

<213> Human and artificial sequence 

30 <400> 29 

Cys Gly Ala Glu Trp Glu Gin Lys Asp Glu Leu 
15 10 

<210> 30 

35 <211> 11 

<212> PRT 

<213> Human and artificial sequence 

<400> 30 

40 Ala Glu Trp Glu Gin Lys Asp Glu Phe lie Cys 
15 10 

<210> 31 

<211> 9 

45 <212> PRT 

<213> Human and artificial sequence 

<400> 31 

Gly Glu Gin Lys Asp Glu Phe lie Cys 
50 1 5 

<210> 32 
<211> 10 
<212> PRT 

55 <213> Human and artificial sequence 

<400> 32 

Cys Ala Glu Gly Glu Gin Lys Asp Glu Leu 
15 10 



60 



65 



<210> 33 
<211> 19 
<212> PRT 

<213> Human and artificial sequence 
<400> 33 



5 



wo 00/50461 PCT/EPOO/01456 

Cys Ser Arg Pro Ser Pro Phe Asp Leu Phe lie Arg Lys Ser Pro Thr 

15 10 15 

lie Thr Cys 

<210> 34 

<211> 18 

<212> PRT 

<213> Human and artificial sequence 

<400> 34 

Cys Ser Arg Pro Ser Pro Phe Asp Leu Phe lie Arg Lys Ser Pro Thr 

15 10 15 

lie Cys 

<210> 35 
<211> 17 
<212> PRT 

<213> Hioman and artificial sequence 
<400> 35 

Cys Ser Arg Pro Ser Pro Phe Asp Leu Phe lie Arg Lys Ser Pro Thr 

15 10 15 

Cys 

<210> 36 
<211> 16 
<212> PRT 

<213> Human and artificial sequence 
<400> 36 

Cys Ser Arg Pro Ser Pro Phe Asp Leu Phe lie Arg Lys Ser Pro Cys 
15 10 15 

<210> 37 
<211> 15 
<212> PRT 

<213> Hioman and artificial sequence 
<400> 37 

Cys Arg Pro Ser Pro Phe Asp Leu Phe He Arg Lys Ser Pro Cys 
15 10 15 

<210> 38 
<211> 16 
<212> PRT 

<213> Human and artificial sequence 
<400> 38 

Cys Arg Pro Ser Pro Phe Asp Leu Phe He Arg Lys Ser Pro Thr Cys 
1 5 10 15 

<210> 39 
<211> 17 
<212> PRT 

<213> Human and artificial sequence 
<400> 39 

Cys Arg Pro Ser Pro Phe Asp Leu Phe He Arg Lys Ser Pro Thr He 

15 10 15 

Cys 



<210> 40 



10 



15 



20 



35 



WO 00/50461 PCT/EPOO/01456 

<211> 18 
<212> PRT 

<213> Human and artificial sequence 
<400> 40 

Cys Arg Pro Ser Pro Phe Asp Leu Phe lie Arg Lys Ser Pro Thr lie 

15 10 15 

Thr Cys 

<210> 41 
<211> 17 
<212> PRT 

<213> Human and artificial sequence 
<400> 41 

Cys Pro Ser Pro Phe Asp Leu Phe lie Arg Lys Ser Pro Thr lie Thr 

15 10 15 

Cys 



<210> 42 
<211> 16 
<212> PRT 

25 <213> Human and artificial sequence 

<400> 42 

Cys Pro Ser Pro Phe Asp Leu Phe lie Arg Lys Ser Pro Thr lie Cys 
15 10 15 

30 

<210> 43 
<211> 15 
<212> PRT 

<213> Human and artificial sequence 
<400> 43 

Cys Pro Ser Pro Phe Asp Leu Phe He Arg Lys Ser Pro Thr Cys 
15 10 15 

40 <210> 44 

<211> 14 
<212> PRT 

<213> Human and artificial sequence 

45 <400> 44 

Cys Pro Ser Pro Phe Asp Leu Phe lie Arg Lys Ser Pro Cys 
15 10 

<210> 45 

50 <211> 20 

<212> PRT 

<213> Human and artificial sequence 

<400> 45 

55 Cys Tyr Ala Phe Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg 
15 10 15 

Thr Leu Ala Cys 
20 

60 <210> 46 

<211> 19 

<212> PRT 

<213> Human and artificial sequence 

65 <400> 46 

Cys Tyr Ala Phe Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg 



7 



wo 00/50461 

1 

Thr Leu Cys 



5 



10 



PCT/EPOO/01456 

15 



<210> 47 
<211> 18 
<212> PRT 

<213> Human and artificial sequence 
<400> 47 

Cys Tyr Ala Phe Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg 

15 10 15 

Thr Cys 



<210> 48 

<211> 17 

<212> PRT 

<213> Human and artificial sequence 

<400> 48 

Cys Tyr Ala Phe Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg 

15 10 15 

Cys 



<210> 49 
<211> 16 
<212> PRT 

<213> Human and artificial sequence 
<400> 49 

Cys Ala Phe Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg Cys 
15 10 15 

<210> 50 
<211> 17 
<212> PRT 

<213> Human and artificial sequence 
<400> 50 

Cys Ala Phe Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg Thr 

15 10 15 

Cys 

<210> 51 
<211> 18 
<212> PRT 

<213> Human and artificial sequence 
<400> 51 

Cys Ala Phe Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg Thr 

15 10 15 

Leu Cys 



<210> 52 
<211> 19 
<212> PRT 

<213> Human and artificial sequence 



<400> 52 

Cys Ala Phe Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg Thr 
15 10 15 



Leu Ala Cys 



8 



wo 00/50461 PCT/EPOO/0 1 456 

<210> 53 
<211> 18 
5 <212> PRT 

<213> Human and artificial sequence 

<400> 53 

Cys Phe Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg Thr Leu 
10 1 5 -10 15 

Ala Cys 

<210> 54 
15 <211> 17 

<212> PRT 

<213> Human and artificial sequence 
<400> 54 

20 Cys Phe Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg Thr Leu 
15 10 15 

Cys 

25 <210> 55 

<211> 16 
<212> PRT 

<213> Human and artificial sequence 

30 <400> 55 

Cys Phe Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg Thr Cys 
15 10 15 

<210> 56 
35 <211> 15 

<212> PRT 

<213> Human and artificial sequence 
<400> 56 

40 Cys Phe Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg Cys 
15 10 15 

<210> 57 
<211> 17 
45 <212> PRT 

<213> Human and artificial sequence 

<400> 57 

Cys Thr Trp Ser Arg Ala Ser Gly Lys Pro Val Asn His Ser Thr Arg 
50 1 5 10 15 

Cys 

<210> 58 

55 <211> 16 

<212> PRT 

<213> Human and artificial sequence 

<400> 58 

60- Cys Thr Trp Ser Arg Ala Ser Gly Lys Pro Val Asn His Ser Thr Cys 
1 5 - 10 15 

<210> 59 
<211> 15 
65 <212> PRT 

<213> Human and artificial sequence 



9 



10 



wo 00/50461 PCT/EPOO/01 456 

<400> 59 

Cys Thr Trp Ser Arg Ala Ser Gly Lys Pro Val Asn His Ser Cys 
1 5 10 15 

<210> 60 
<211> 14 
<212> PRT 

<213> Human and artificial sequence 
<400> 60 

Cys Thr Trp Ser Arg Ala Ser Gly Lys Pro Val Asn His Cys 
15 10 

15 <210> 61 

<211> 13 

<212> PRT 

<213> Human and artificial sequence 

20 <400> 61 

Cys Trp Ser Arg Ala Ser Gly Lys Pro Val Asn His Cys 
15 10 

<210> 62 

25 <211> 14 

<212> PRT 

<213> Human and artificial sequence 

<400> 62 

30 Cys Trp Ser Arg Ala Ser Gly Lys Pro Val Asn His Ser Cys 
15 10 

<210> 63 

<211> 15 

35 <212> PRT 

<213> Human and artificial sequence 

<400> 63 

Cys Trp Ser Arg Ala Ser Gly Lys Pro Val Asn His Ser Thr Cys 
40 1 5 10 15 

<210> 64 
<211> 16 
<212> PRT 

45 <213> Human and artificial sequence 

<400> 64 

Cys Trp Ser Arg Ala Ser Gly Lys Pro Val Asn His Ser Thr Arg Cys 
15 10 15 

50 

<210> 65 
<211> 15 
<212> PRT 

<213> Human and artificial sequence 
<400> 65 

Cys Ser Arg Ala Ser Gly Lys Pro Val Asn His Ser Thr Arg Cys 
15 10 15 

60 <210> 66 

<211> 14 
<212> PRT 

<213> Human and artificial sequence 

65 <400> 66 

Cys Ser Arg Ala Ser Gly Lys Pro Val Asn His Ser Thr Cys 



55 



10 



wo 00/50461 

1 5 



10 



PCT/EPOO/01456 



<210> 67 
<211> 13 
<212> PRT 

<213> Human and artificial sequence 
<400> 67 

Cys Ser Arg Ala Ser Gly Lys Pro Val Asn His Ser Cys 
1 5 10 

<210> 68 
<211> 12 
<212> PRT 

<213> Human and artificial sequence 
<400> 68 

Cys Ser Arg Ala Ser Gly Lys Pro Val Asn His Cys 
15 10 

<210> 69 
<211> 17 
<212> PRT 

<213> Human and artificial sequence 
<400> 69 

Cys Gin Trp Leu His Asn Glu Val Gin Leu Pro Asp Ala Arg His Ser 

15 10 15 

Cys 



<210> 70 
<211> 16 
<212> PRT 

<213> Human and artificial sequence 
<400> 70 

Cys Gin Trp Leu His Asn Glu Val Gin Leu Pro Asp Ala Arg His Cys 
15 10 15 

<210> 71 
<211> 15 
<212> PRT 

<213> Human and artificial sequence 
<400> 71 

Cys Gin Trp Leu His Asn Glu Val Gin Leu Pro Asp Ala Arg Cys 

10 15 

<210> 72 
<211> 14 
<212> PRT 

<213> Human and artificial sequence 
<400> 72 

Cys Gin Trp Leu His Asn Glu Val Gin Leu Pro Asp Ala Cys 
15 10 

<210> 73 
<211> 13 
<212> PRT 

<213> Human and artificial sequence 
<400> 73 

Cys Trp Leu His Asn Glu Val Gin Leu Pro Asp Ala Cys 
15 10 



11 



10 



15 



60 



WO 00/50461 PCT/EPOO/01456 

<210> 74 

<211> 14 

<212> PRT 

<213> Human and artificial sequence 

<400> 74 

Cys Trp Leu His Asn Glu Val Gin Leu Pro Asp Ala Arg Cys 
1 5 • 10 



<210> 75 

<211> 15 

<212> PRT 

<213> Human and artificial sequence 

<400> 75 

Cys Trp Leu His Asn Glu Val Gin Leu Pro Asp Ala Arg His Cys 
15 10 15 

20 <210> 76 

<211> 16 

<212> PRT 

<213> Human and artificial sequence 

25 <400> 76 

Cys Trp Leu His Asn Glu Val Gin Leu Pro Asp Ala Arg His Ser Cys 
1 5 10 • 15 

<210> 77 
30 <211> 15 

<212> PRT 

<213> Human and artificial sequence 
<400> 77 

35 Cys Leu His Asn Glu Val Gin Leu Pro Asp Ala Arg His Ser Cys 
15 10 15 

<210> 78 
<211> 14 
40 <212> PRT 

<213> Human and artificial sequence 

<400> 78 

Cys Leu His Asn Glu Val Gin Leu Pro Asp Ala Arg His Cys 
45 1 5 10 

<210> 79 
<211> 13 
<212> PRT 

50 <213> Human and artificial sequence 

<400> 79 

Cys Leu His Asn Glu Val Gin Leu Pro Asp Ala Arg Cys 
15 10 

55 

<210> 80 
<211> 12 
<212> PRT 

<213> Human and artificial sequence 
<400> 80 

Cys Leu His Asn Glu Val Gin Leu Pro Asp Ala Cys 
15 10 

65 * <210> 81 

<211> 6 



12 



wo 00/50461 



PCT/EPOO/01456 



<212> PRT 

<213> Human and artificial sequence 



Leu 
1 



<400> 81 
Phe lie Arg Lys Ser 
5 



<210> 82 

<211> 7 

<212> PRT 

<213> Human and artificial sequence 

<400> 82 
Pro Ser Lys Gly Thr Val Asn 
1 5 

<210> 83 
<211> 23 
<212> PRT 

<213> Human and artificial sequence 
<400> 83 

Leu His Asn Glu Val Gin Leu Pro Asp Ala Arg His Ser Thr Thr Gin 

15 10 15 

Pro Arg Lys Thr Lys Gly Ser 



<210> 84 
<211> 6 
<212> PRT 

<213> Human and artificial sequence 

<400> 84 
Ser Val Asn Pro Gly Lys 
1 5 

<210> 85 
<211> 13 
<212> PRT 

<213> Human and artificial sequence 
<400> 85 

Cys Pro Glu Trp Pro Gly Cys Arg Asp Lys Arg Thr Gly 
15 10 

<210> 86 
<211> 13 
<212> PRT 

<213> Human and artificial sequence 
<400> 86 

Thr Pro Glu Trp Pro Gly Cys Arg Asp Lys Arg Cys Gly 



20 



1 



5 



10 



13 



INTERNATIONAL SEARCH REPORT 


national Application No 

PCT/EP 00/01456 


A. CLASSIFICATION OF SUBJECT MATTER , . . , 

IPC 7 C07K16/00 C07K16/42 A61K39/00 A61K39/385 A61K39/395 
G01N33/577 G01N33/68 A61P37/08 


According to Intemational Patent Classification (IPC) or to both national classification and IPC 






B. FIELDS SEARCHED 


Minimum documentation searched (classification system followed by classification symbols) 

IPC 7 C07K A61K 


Documentation searched other ttian minimum documentation to the extent that such documents are included in the fields searched 


Electronic data t>ase consulted during the intemational search (name of data base and. \where practical, search temis used) 

STRAND, CHEM ABS Data, MEDLINE, LIFESCIENCES, CANCERLIT, AIDSLINE, EMBASE, 
SCISEARCH, EPO-Internal , BIOSIS, WPI Data, PAJ 


C. DOCUMENTS CONSIDERED TO BE RELEVANT 


Categoiy • 


Citation of document, with indcation. where appropriate, of the relevant passages 


Relevant to claim No. 


X 


WO 98 24808 A (UNITED STATES DEPT. OF 
HEALTH AND HUMAN SERVICES, USA;PADLAN, 
EDUARDO) 11 June 1998 (1998-06-11) 

page 10 seq.ID 3 100% identity in 18aa 
overlap with seq.ID 6 
page 3, line 10-24 
page 4, line 21-23 
page 11, line 16-29 
page 28, line 32 -page 29, line 12 
claims 




6,14, 

16-19, 

21. 

23-28, 
32-35 






/-- 






1 y [ Further documents are listed in the continuation of box C. 


j Patent family members are listed in annex. 


o Special categories of cited documents : 

"A" document defining the general state of the art which is not 
considered to be of particular relevarx;e 

"E" earlier document but published on or after the intemational 
filing date 

"L" document which may throw doubts on priority c!aim(s) or 
which is cited to establish the publication date of another 
citation or other special reason (as specified) 

"0' document referring to an oral disclosure, use. exhibition or 
other means 

"P" document putjtished prior to the intemational filing date txjt 
later than the priorit/ date claimed 


T* later document published after die intemational filing date 
or priority date and not in conflict with the application but 
cited to understand the principle or theory urKterfying the 
invention 

'X" document of particular relevance; the claimed invention 
cannot be considered rK}vel or cannot be considered to 
involve an inventive step when the document is taken alone 

'Y" document of particular relevance; the claimed invention 

cannot be considered to involve an inventive step when the 
document is combined with one or more other such docu- 
ments, such combination being obvious to a person skilled 
in the art. 

*&' document member of the same patent family 


Date of the actual completion of the international search 


Date of malting of the intemational search report 


26 July 2000 


02/08/2000 




Name and mailing address of the ISA 

European Patent Office, P.B. 5818 Patentiaan 2 
NL - 2280 HV Riiswijk 
Tel. (+31-70) 340-2040. Tx. 31 651 epo nl. 
Fax: (+31-70) 340-3016 


Authorized officer 

Covone, M 



Form PCT/(SA/210 (second sheet) (July 1892) 



page 1 of 3 



INTERNATIONAL SEARCH REPORT 



t national Application No 

PCT/EP 00/01456 



C.(Contlnuatton) OOCUMEMTS CONSIDERED TO BE REUEVANT 



Category 



Citation ot document, with indication, where appropriate* ol the relevant passages 



Relevant to claim No. 



o.x 



STADLER, B.M. ET AL: "Mimotope and 
Anti -Idiotypic Vaccines to Induce an 
Anti-IgE Response" 

INTERNATIONAL ARCHIVES OF ALLERGY AND 
IMMUNOLOGY 'INT. ARCH. ALLERGY IMMUNOL.!, 
(19990000) VOL. 118, NO. 2-4, PP. 119-121. 
ISSN: 1018-2438., 

September 1998 (1998-09), XP000929243 
the whole document 

PATENT ABSTRACTS OF JAPAN 

vol. 014, no. 543 (C-0783), 

30 November 1990 (1990-11-30) 

-& JP 02 229200 A (DAINIPPON PHARMACEUT CO 

LTD; OTHERS: 01), 

11 September 1990 (1990-09-11) 

abstract 

column 1 line 18 100% identity in 18aa 
overlap with seq. ID 7 and 100% identity 
in 18aa overlap with seq. ID 83 

WO 93 04173 A (GENENTECH INC) 

4 March 1993 (1993-03-04) 

page 57 table 6 seq. ID 14 100% identity 

in lOaa overlap with seq.IDl, seq. ID 16 

100% identity in 9aa overlap with seq. ID 2 

WO 97 31948 A (CIBA GEIGY AG ;KRICEK FRANZ 

(AT); STADLER BEDA (CH)) 

4 September 1997 (1997-09-04) 

page 4, line 6 -page 5, line 9 

page 8, line 5-9 

claims 

figure 4 

LAFFER S ET AL: "Characterization of 
non-anaphylactogenic anti human IgE 
antibody that completes with the IgE-high 
affinity Fee-receptor interaction." 
ALLERGY (COPENHAGEN), 

vol. 53, no. SUPPL. 43, 1998, pages 14-15, 
XP000929220 

Annual Meeting of the European Academy of 

Allergology and Clinical 

Immunol ogy;Birmingham, England, UK; June 

21-26, 1998 

ISSN: 0105-4538 

the whole document 

-/-- 



1-14, 

16-19, 

21. 

23-28, 
32-35 



7,9,23, 
26,33 



1,2 



1-35 



1-35 



Form PCT/ISA/210 (continuation of second sheet) (July 1992) 



page 2 of 3 



INTERNATIONAL SEARCH REPORT 



I latlonal Application No 

PCT/EP 00/01456 



C.(Contlnuatlon) DOCUMEhTTS CONSIDERED TO BE RELEVANT 



Category * Citation of document, with indication, where appropriate, of the relevant passages 



Relevant to claim No. 



HEUSSER C ET AL: "Therapeutic potential 
of anti-IgE antibodies." 
CURRENT OPINION IN IMMUNOLOGY, (1997 DEC) 
9 (6) 805-13, REF: 76 , 

1997, XP002125679 
page 805, right-hand column, paragraph 2 
page 807, left-hand column, paragraphs 3,4 
page 811, right-hand column, paragraph 3 

WO 99 67293 A (UNITED BIOMEDICAL INC, 
USA) 29 December 1999 (1999-12-29) 



page 73 seq.ID 57 100% Identity in 15aa 

overlap with sed.ID 1 

page72 seq.ID 49 seq.ID 57 100% identity 

in 9aa overlap with sed.ID 2, seq.ID 53 

100% identity in 7aa overlap with sed.ID 

82 

page 21 seq.ID 5 100% identity in 13aa 

overlap with sed.ID 3 

page 71 seq.ID 47 100% identity in 18aa 

overlap with sed.ID 6 

page 8, line 4-17 

page 41, line 10 -page 43, line 18 



1-35 



1-3,6,8, 
11,14, 
16-19, 
21, 

23-28, 
32-35 



Form PCT/lSAffilO (continuation of second sheet) (July 1992) 



page 3 of 3 



International Application No. PCT£P 00 i0l456 



FURTHER INFORMATION CONTINUED FROM PCT/ISA/ 210 



Continuation of Box I.l 

Although claim 29 (partially) is directed to a diagnostic method 
practised on the human/animal body, and claims 33-35 (completely) are 
directed to a method of treatment of the human/animal body, the search 
has been carried out and based on the alleged effects of the 
compound/compos i t i on , 



INTERNATIONAL SEARCH REPORT 

Information on patent family members 


ti latlonal Application No 

PCT/EP 00/01456 


Patent document 
cited in search report 


Publication 
date 


Patent family 
memt3er(s) 


Publication 
date 


UO 9824808 A 


11-06-1998 




AU , 6532498 A 


29-06-1998 


JP 02229200 A 


11-09-1990 




NONE 







WO 9304173 A 04-03-1993 AU 2498192 A 16-03-1993 

AU 706584 B 17-06-1999 

AU 7038096 A 16-01-1997 

EP 0602126 A 22-06-1994 

JP 6509944 T 10-11-1994 

US 5965709 A 12-10-1999 

US 5994514 A 30-11-1999 



WO 9731948 



04-09-1997 



WO 9967293 



29-12-1999 



AU 


719609 


B 


11-05-2000 


AU 


1879697 


A 


16-09-1997 


BR 


9707819 


A 


27-07-1999 


CN 


1213380 


A 


07-04-1999 


CZ 


9802771 


A 


16-12-1998 


EP 


0885244 


A 


23-12-1998 


HU 


9901109 


A 


28-07-1999 


JP 


2000502571 


T 


07-03-2000 


NO 


983999 


A 


02-11-1998 


PL 


328858 


A 


01-03-1999 


SK 


118198 


A 


10-03-1999 


AU 


4580299 


A 


10-01-2000 



Form PCT/ISA/210 (patent family annex) (July 1992)