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Document AL3 
Appl.No. 09/848,616 



j 



WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




PCX 

INTERNATIONAL APPUCATIQM PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent Classification ^ ; 
C07K 16/00, A61K 39/395 



Al 



(11) Internationa) Publication Number: WO 97^1948 

(43) International Publication Date: 4 September 1997 (04.09.97) 



(21) International Application Number: PCT/EP97/01013 

(22) International FUing Date: 28 February 1997 (28.02.97) 



(30) Priority Data: 
9604412.8 
9617702.7 



1 March 1996 (01.03.96) GB 
22 August 1996 (22.08.96) GB 



(71) Applicant (for all designated States except US): NOVARTIS 

AG (CH/CHl; Schwarzwaldallec 215, CH-4058 Basel (CH). 

(72) Inventors; and 

(75) Inventors/AppUcants (for US only): KRICEK, Franz (AT/ AT]; 
Ulmcnweg 9, A-2362 Bicdermannsdorf (AT). STADLER, 
Bcda fCH/CH]; Maricnstrasse 37, CH-3005 Bern (CH). 

(74) Agent: VALLET, Lucien; Novartis AG, Patents & Trademarks 
DepL CH, Lichistrassc 35, CH-4002 Basel (CH). 



(81) Designated States: AL. AM. AT. AU, AZ. BA. BB. BG, BR. 
BY. CA. CH. CN. CU CZ. DE. DK. EE. ES. FI. GB. GE. 
HU. IL. IS. JP. KE. KG. KP. KR. ICZ. LC, LK. LR, LS 
LT. LU. LV. MD. MG. MK, MN. MW. MX. NO. NZ PL 
PT. RO. RU. SD. SB. SG. SI. SK. TJ. TM, TR. TT. UA* 
UG. US. UZ. VN, ARIPO patent (GH. KE. LS. MW. SD. 
SZ. UG). Eurasian patent (AM. AZ. BY. KG. KZ. MD, RU. 
TJ. TM). European patent (AT. BE. CH. DE. DK. ES, FI. 
FR. GB, GR. IE. IT, LU. MC. NL. PT. SE). OAPI patent 
(BF. BJ. CF, CG, CI. CM, OA. GN. 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) Title: PEPTIDE IMMUNOGENS FOR VACCINATION AGALNST AND TREATMENT OF ALLERGY 
(57) Abstract 

Immunogenic molecules comprising (a) at least one moiety of a peptide mimicking the natural epitope on human IgE recognized by 
the monoclor^l anti-human IgE antibody BSW17. and (b) a moiety capable of eliciting an immune response against that peptide. They are 
uscfiil m, or for the preparation of. phannaccutical compositions, particularly for the treatment of IgE-mcdiated diseases such as allcreics 
and atopic demiatitis. e.g. vaccines against allergy. * 



FOR THE PURPOSES OF INFORMATION ONLY 



Codes used to identify 
applications under the PCT. 



AM 


Annenia 


AT 


Aiutru 


AU 


Australia 


BB 


Bartwkia 


BB 


Belgium 


BF 


Buitiaa Fuo 


BG 


Bulgsria 


BJ 


Benin 


BR 


Brazil 


BY 


Belarus 


CA 


Canada 


CF 


Central African Republic 


CG 


Congo 


CH 


Swtizerliiid 


CI 


C^ dM voire 


CM 


Cameroon 


CN 


China 


cs 


Czechoslovikla 


cz 


Czech Republic 


DE 


Germany 


DK 


Denmark 


EE 


Estonia 


ES 


Spain 


n 


Failand 


FR 


France 


GA 


Gabon 



party to the PCT on the front pages of 



GB 


United Kingdom 


GE 


Geaisia 


GN 


Guinea 


GR 


Greece 


HU 


Hungary 


IE 


Ireland 


IT 


Italy 


JP 


Japan 


K£ 


Kenya 


KG 


Kyrgyslan 


KP 


Democratic People's Republic 




of Korea 


KR 


Republic of Korea 


KZ 


Kazakhstan 


U 


Liechtenstein 


LK 


Sri Lanka 


LR 


Liberia 


LT 


Uthuania 


LU 


Luxembourg 


LV 


Litvia 


MC 


Mooaco 


MD 


Republic of Moldova 


MG 


Madigascar 


ML 


K4ali 


MN 


Mongolia 


MR 


Mauritania 



publishing international 



MW 


Malawi 


MX 


Mexico 


NE 


Niger 


NL 


Netherlands 


NO 


Norway 


NZ 


New Zealand 


PL 


Poland 


PT 


Portugal 


RO 


Romania 


RU 


Russian Federation 


SD 


Sudan 


SE 


Sweden 


SG 


Singapore 


SI 


Slovenia 


SK 


Skivakia 


SN 


Senegal 


SZ 


Swaziland 


TD 


Chad 


TG 


Togo 


TJ 


Tajikistan 


TT 


Trinidad and Tobago 


UA 


Ukraine 


UG 


Uganda 


US 


United States of America 


VZ 


Uzbekistan 


VN 


Viet Nam 



wo 97/31948 PCT/EP97/01013 



PEPTIDE IMMUNOGENS FOR VACCINATION AGAINST AND TREATMENT OF ALLERGY 
1. Field 

The present invention relates to peptide immunogens. and is directed to inhibition of 
interactions which would normally cause the triggering of mast cells and basophils induced by 
cell-bound IgE linked to an allergen resulting in the release of pharmacologically active 
mediators as well as the de novo synthesis of cytokines involved in the regulation of allergic 
and inflammatory reactions. It concerns immunogenic molecules including a moiety of a 
BSW17 mimotope peptide and their use. 

2. Background 

Allergic symptoms are brought about through the release of pharmacologically active 
mediators, notably histamine, leukotrienes and enzymes, from cells into surrounding tissue and 
vascular structures. These mediators are normally stored or synthesized de novo in special cells 
known as mast cells and basophil granulocytes. Mast cells are dispersed throughout animal 
tissue whilst basophils circulate within the vascular system. These cells synthesize and store 
mediators within the cell, unless a specialized sequence of events occurs to trigger its release. 

The role of immunoglobulin E (IgE) antibodies in mediating allergic reactions is well 
known. IgE is a complex arrangement of polypeptide chains which, as in other 
immunoglobulins consists of two light and two heavy chains linked together by disulphide 
bonds in a "Y" shaped configuration. Each light chain has two domains, one variable (VJ 
domain linked to a domain with a relatively invariant amino acid sequence termed a constant 
domain (Cj. Heavy chains, by contrast, have one variable domain (V„) and in the case of IgE. 
four constant domains (ChL Ch2, Ch3, C„4. also known as Cel. Ce2. Ce3, Ce4). The two 
• arms" of the antibody are responsible for antigen binding, having regions where the 
polypeptide stnicture varies, and are termed Fab' fragments or F(ab')2. which respresents two 



V 



wo 97/31948 



PCT/EP97/01013 



-2- 

Fab' arms linked together by disulphide bonds. The "tail" or central axis of the antibody 
contains a fixed or constant sequence of peptides and is termed the Fc fragment. The 
Fc fragment contains interactive sites which enable the antibody to communicate with other 
immune system molecules or ceils by binding to their Fc receptors. 

Fc receptors are molecules which bind specifically to active molecular sites within 
immunoglobulin Fc regions. Fc receptors may exist as integral membrane proteins within a 
cell's outer plasma membrane or may exist as free "soluble" molecules which freely circulate in 
blood plasma or other body fluids. In the human system, high affinity binding of IgE to the 
receptor FceRI is accomplished by a complex protein-protein interaction involving various 
parts of the third heavy chain constant region domain (C£3) of IgE, and the 
membrane-proximal, immunoglobulin-like domain (oc2) of the FceRIa subunii. 

Although residues within the Ct3 domain of the IgE heavy chain constant region, and 
regions belonging lb the oc2 domain of the FceRIa receptor, have been identified which are 
important for binding, the detailed mechanism of the binding process are still obscure. 
Experimental evidence has been provided by fluorescence energy transfer measurements as 
well as X-ray and neutron scattering that human IgE adopts a bent structure which is speculated 
to conlribuie to the uniquely high affinity of IgE for FceRI (Kd - 10**° M). Moreover, this bent 
structure is also postulated to be responsible for the equimolar complex between IgE and cell 
bound or soluble FceRIa, although the IgE molecule would provide identical epitopes on the 
two C£3 domains for receptor binding. This monovalency is a functional necessity if receptor 
triggering in the absence of allergen is to be avoided (Figure 1). Interactive sites, depending on 
their function, may already be exposed and therefore able to bind to cellular receptors. 
Alternatively, they may be hidden until the antibody binds to the antigen, whereupon the 
antibody may change in structure and subsequently expose other active sites which can then 
trigger a specific immune activity. Based on data obtained from circular dichroism spectra, a 
conformational rearrangement affecting Ce3 upon receptor binding has been proposed as an 
explanation for the 1:1 stoichiomeiry of the Fce/FceRl complex on the cellular surface. 

For allergic (immunological) release of histamine within the organism from mast cells 
and basophils, an IgE molecule must lock onto or attach itself with its Fc portion to the cellular 
Fc receptor site, thus securing the IgE molecule to the mast cell or basophil. The Fab' portions 



wo 97/31948 PCT/EP97/01013 



-3- 

of the cell-bound IgE molecules must be cross-linked by a particular compatible antigen (the 
allergen). When such an interaction occurs, the mast cell or basophil is automatically triggered 
to release histamine to the local environment, manifesting familiar allergic symptoms. Other 
biochemical events follow in a late phase reaction, resulting in de novo synthesis and release of 
cytokines and other mediators [Ravetch. J.V., and J.P. Kinet. Ann. Rev. Immunol 9 (1991) 
457-492]. 

Conventional approaches to allergy treatment have involved systemic therapy with 
anti-histamines or steroids or attempts to desensitize patients; these approaches are not adressed 
to the basic IgE-mast cell^asophil interaction. Other approaches have been concerned with the 
production of polypeptide chains capable of blocking the binding of the IgE antibody to the 
Fc receptors on the cell surfaces and displacing IgE from binding sites upon which IgE is 
already bound. Moreover, investigations have been carried out in order to define the nature of a 
putative "effector" site within the IgE Fc region, which was speculated to provide an 
immunological signal which triggers mast cells/basophils for mediator release. 

Using recombinant IgE fragments as immunogens for the generation of a protective 
anti-lgE vaccine has also been tried and shown to be effective. The main argument against such 
a vaccine results from the fear that using large IgE fragments for immunization could initiate 
not only the production of inhibitory antibodies but also generate crosslinking and thereby 
anaphylactogenic antibodies in the patients (Figure 2). 

A strategy to overcome this problem would aim at the identification of the smallest IgE 
fragment possible, ideally consisting of the receptor binding site only, which is buried within 
the IgE/ FceRI complex after binding and therefore no longer accessible for crosslinking by the 
vaccine-generated immune response. Attempts to reconstruct such a complex molecular entity 
seem unlikely to be succe.ssful in view of the spatial distances of the various Ce3 regions 
involved in IgE/FceRI interaction. 



WO 97/31948 



PCT/EP97/0IO13 



3. Summary of the invention 

It has now been found that the problems intrinsicaJly linked to the "classical" vaccine 
approach are overcome by using BSW17 mimotopes for active immunization, either as 
chemically synthesized peptides coupled to appropriate carriers, or as recombinant fusion 
constructs (e.g. with ovalbumin, IgG, etc.). 

BSW17 is a monoclonal antibody which recognizes a conformational epitope on Fee 
with at least part of it residing within Cz3. The hybridoma cell line producing monoclonal 
antibody BSW17 has been deposited on December 19, 1996 with ECACC under the provisions 
of the Budapest Treaty on the deposit of microorganisms, under deposit number 96121916. 
This antibody displays an interesting profile of biological activities, as summarized in Figure 3. 
BSW17 or BSW17-like antibodies circulating within the vascular system protect from allergic 
reactions by 

a) inhibiting the triggering of mast cells and basophils through competitive inhibition of the 
IgE/IgERI interaction and 

b) lowering serum IgE levels through downregulaiion of IgE synthesis at the B ceil level. 

BSW 1 7 "mimotope" peptides have now been identified by random peptide phage 
display library screening, i.e. peptides which mimic at least part of the complex conformational 
epitope on the IgE molecule. Chemically synthesized mimotope peptides coupled to an 
inrmiunogenic carrier protein can be used e.g. as vaccines for the specific generation of 
antibodies in an allergic host which inhibit mast cell/basophil triggering by blocking 
IgE/FcERIa binding and/or IgE synthesis. As mimotopes of an anti-IgE antibody they induce 
an immune response which results in the production of BSW17-like antibodies in the host. 
Since BSW 1 7 has been shown to be non-anaphylaclogenic, inhibitory to IgE/ FceRI binding 
and IgE synthesis on B cells, these antibodies raised against the BSW 17 mimotope-based 
vaccines have analogous protective properties. The immune response is very specific since, in 
contrast to the "classical vaccine approach", no IgE-derived protein fragments are present 
which could generate crosslinking antibodies in the immunized patients (Figure 4). 



wo 97A31948 



PCT/EP97/01013 



-5- 



The invention thus concerns inununogenic molecules comprising 

(a) at least one moiety of a BS W 1 7 mimotope peptide and 

(b) a moiety capable of eliciting an immune response against that peptide, 
hereinafter briefly named "the immunogens of the invention". 

Component (a) preferably consists of up to five, preferably one or two, especially one 
moiety of a BSW 1 7 mimotope peptide. Component (b) preferably is a convenUonal 
immunogenic carrier as set out hereunder, especially BSA or KLH. 

The BSW 17 mimotope peptide in component (a) preferably is up to about 15 amino 
acids altogether, it is e.g. one of the sequences (A) to (Q) (Seq.ld.no. 1 to no. 17) hereafter. 
However, it may appropriately include further components for hapten-carrier binding, e.g. to 
facilitate coupling to component (b) or further processing. Thus, when the BSW 1 7 mimotope 
peptide is cyclic, the two ends can e.g. be held together by two additional cystein residues 
forming a disulfide bridge, or the ends can be chemically crosslinked, e.g. with lysine; or when 
the BSW 1 7 mimotope peptide is linear, the carboxy terminal amino acid may conveniently be 
blocked by amidation, and/or the amino terminal amino acid may conveniently be blocked by 
acetylation. Funher. the BSW17 mimotope peptide moieties in component (A), e.g. the 
preferred moieties (A) to (Q) hereafter, may be flanked in the immunogens of the invention by 
a few, preferably one or two. additional ancillary groups, such as acetyl, cysteine or lysine, 
and/or an additional coupling group, such as DC or BSS. e.g. as set out for the specific 
immunogens of the invention disclosed in Example 8 hereunder as conjugates (2) to (4). (6) to 
(ll).(13)and{l4). 

The antibodies elicited by the immunogens of the invention, in contrast to the 
antibodies produced by hybridoma BSW17. will be endogenous and thus, in a patient, human; 
they may be used for prophylactic treatment. 

They may be prepared by appropriately coupling components (a) and (b) as defined 

above. 



wo 97/31948 PCT/EP97/01013 

-6- 

4. Detailed explanation 

The immunogens of the invention are e.g. in the form of a polymeric peptide or a 
recombinant fusion protein, whereby a monomeric component of the polymeric peptide, or one 
partner of the fusion protein, constitutes a moiety of a BSW17 mimotope peptide (a) and the 
remainder of the polymeric peptide or fusion protein constitutes the immune response - 
eliciting moiety (b). 

They especially are in the form of a conjugate of at least one BSW 17 mimotope peptide 
moiety (a) and an immunogenic carrier moiety (b). 

Preferred BSW 17 mimotope peptide moieties, i.e. component (a), of the immunogens 



of the invention essentially consist of or contain 


an amino acid sequence selected from 


Ue- Asn-His-Arg-Gly-Tyr-Trp-Val 


(A) 


(Seq.id.no. 1), 


Arg-Asn-His-Arg-Gly-Tyr-Trp-Val 


(B) 


(Seq.id.no. 2), 


Arg-Ser-Arg-Ser-Gly-Gly-Tyr-Trp-Leu-Trp 


(C) 


(Seq.id.no. 3), 


Val-Asn-Leu-Thr-Trp-Ser-Arg-Ala-Ser-Gly 


(D) 


(Seq.id.no. 4), 


Val-Asn-Leu-Pro-Trp-Ser-Arg-AJa-Ser-Gly 


(E) 


(Seq.id.no. 5), 


Val-Asn-Leu-Thr-Trp-Ser-Phe-Gly-Leu-Glu 


(F) 


(Seq.id.no. 6), 


Val-Asn-Leu-Pro-Trp-Ser-Phe-Gly-Leu-Glu 


(G) 


(Seq.id.no. 7), 


Val-Asn-Arg-Pro-Trp-Ser-Phe-Gly-Leu-Glu 


(H) 


(Seq.id.no. 8), 


Val-Lys-Leu-Pro-Trp-Arg-Phe-Tyr-Gln-Val 


(I) 


(Seq.id.no. 9). 


Val-Trp-Thr-Ala-Cys-Gly-Tyr-Gly-Arg-Met 


(J) 


(Seq.id.no. 10), 


Gly-Thr-Val-Ser-Thr-Leu-Ser 


(K) 


(Seq.id.no. 11), 


Leu-Leu-Asp-Ser-Arg-Tyr-Trp 


(L) 


(Seq.id.no. 12), 


Gin-Pro-Ala-His-Ser-LcL* ly 


(M) 


(Seq.id.no. 13), 


Leu-Trp-Gly-Met-Gln-Gi> - Arg 


(N) 


(Seq.id.no. 14), 


Leu-Thr-Leu-Ser-His-Pro-His-Trp-Val-Leu-Asn-His-Phe-Val-Ser (O) (Seq.id.no. 15). 


Ser-Met-Gly-Pro-Asp-Gln-Thr-Leu-Arg 


(P) 


(Seq.id.no. 16), or 


Val-Asn-Uu-Thr-Trp-Ser 


(Q) 


(Seq.id.no. 17). 



wo 97/31948 PCT/EP97/01013 



-7- 



More preferred are (A), (D) and (G) above, especially (A) and (D). 

The invention also concerns pharmaceutical compositions, especially vaccines, 
comprising immunogen molecules as defined above and an adjuvant. 

It also concerns ligands, i.e. antibodies or fragments derived therefrom directed against 
BSWI7 mimolope peptides used in "passive immunization" (see below), whereby the antibody 
or antibody fragments also recognize the natural epitope for BSW17 on human IgE; namely, it 
concerns ligands comprising an antibody domain specific for a moiety of a BSW17 mimolope 
peptide as defined above, whereby the antibody domain is reactive also with the sequence of 
amino acids on the heavy chain of IgE which comprises the natural epitope recognized by 
BSW 1 7. Such ligands can be generated in mammals as polyclonal or monoclonal antibodies; 
they preferably are in the form of monoclonal antibodies, preferably in the form of an 
Fab' fragment or an F(ab*)2 fragment thereof. 

It further concerns a process for the preparation of an immunogen of the invention, 
comprising covalently coupling 

a J at least one moiety of a BSW17 mimotope peptide with 

b) a moiety capable of eliciting an immune response against that peptide. 

It also concerns immunogenic molecules as defined above, for use as a pharmaceutical, 
e.g. in the ireaimeni of IgE-mediated diseases such as allergy and atopic dermatitis. 

It further concerns the use of immunogenic molecules as defined above in the 
preparation of pharmaceutical compositions, especially vaccines, against IgE-mediated 
diseases, in particular allergy and atopic dermatitis. 

It further concerns a method of prophylactic or curative immunization against 
IgE-mediated diseases such as allergies and atopic dermatitis comprising the administration of 
a therapeutically effective amount of immunogenic molecules as defined above to a patient in 
need of such treatment. 

The immunogens of the invention, while being substantially incapable of mediating 
non-cytolytic histamine release, are capable of eliciting antibodies with strong serological 
cross-reactivity with the target amino acid sequences of the Fc region of IgE. They are thus 
useful in. or as, vaccines. 



wo 97/3I948 



PCT/EP97/01013 



-8- 

The initial dose of immunogen (e.g. from about 0.2 mg to about 5 mg. especially about 
1 mg) is for example administered intra-muscularly, followed by repeat (booster) doses of the 
same 14 to 28 days later. Doses will of course depend to some extent on the age» weight and 
general health of the patient. Inimunization may be "active" or "passive". 

In "active" immunization the patient receives immunogen of the invention and thereby 
an anti-hlgE response is actively induced by the patient's immune system. 

"Passive" immunization is achieved by administering anii-BSW17 mimotope 
antibodies, either polyclonal or monoclonal, to a patient suffering from IgE-mediated disease, 
preferably by injection. 

Polyclonal anti - BSW17 mimotope antibodies can be prepared by administering 
immunogen of the invention, preferably using an adjuvant, to a non-human mammal and 
collecting the resultant antiserum. Improved titres can be obtained by repeated injections over a 
period of time. There is no particular limitation to the species of mammals which may be used 
for eliciting antibodies; it is generally preferred to use rabbits or guinea pigs, but horses, cats, 
dogs, goats, pigs, rats, cows, sheep, etc., can also be used. In the production of antibodies, a 
definite amount of immunogen of the invention is e.g. diluted with physiological saline 
solution to a suitable concentration and the resulting diluted solution is mixed with complete 
Freund's adjuvant to prepare a suspension. The suspension is administered to manmials, e.g. 
intrapcritoneally. e.g. to a rabbit, using from about 50 pg to about 2500 pg immunogen of the 
invention per administration. The suspension is preferably administered about every two 
weeks over a period of up to about 2-3 months, preferably about 1 month, to effect 
immunization. Antibody is recovered by collecting blood from the inununized animal after the 
passage of 1 to 2 weeks subsequently to the last administration, centrifuging the blood and 
isolating serum from the blood. 

Monoclonal anti - BS W 1 7 mimotope antibodies may e.g. be human or murine. 
Preferably, the patient will be treated with an Fab' fragment preparation from murine 
monoclonal antibody or a chimeric human-mouse antibody (comprising human Fc region and 
mouse Fab' region) so as to minimize any adverse reaction to the foreign animal 
inimunoglobulin. Murine monoclonal antibodies may be prepared by the method of Kohler 
and Milstein (Kohler. G. and Milstein, C. Nature 256 [1975] 495). e.g. by fusion of spleen 



wo 97/31948 



PCT/EP97/01013 



-9- 

cells of hyperimmunized mice with an appropriate mouse myeloma cell line. Numerous 
methods may be utilized to raise human monoclonal antibodies, including production by: 

( 1 ) Epstein-Barr virus (EB V) - transformed B-cells; 

(2) cell line for B lymphocyte hybridization; 

(3) human-murine hybridomas; 

(4) human-human hybridomas; 

(5 ) human x human-mouse heterohybridomas; and 
(6) repertoire cloning (phage display). 

Human x human-mouse heterohybridomas are the most preferred, and involve combining 
favourable characteristics of both human and murine parental cell types. Human-mouse 
heterohybridoma cell lines have been rendered suitable for B cell fusion (Teng, N.N.M. et al., 
Proc. Natl. Acad. Sci. USA 80 [19831 7308). 

When used for immunization, antibody can be introduced into the host most 
conveniently by intramuscular injection. Any conventional liquid or solid vehicle may be 
employed which is acceptable to the host and does not have adverse side effects on the host nor 
deirimenial effects on the vaccine. Phosphate-buffered saline (PBS), at a physiological pH, e.g. 
about pH 6.8 to 7.2, preferably about pH 7.0, may be used as a vehicle, alone or with a suitable 
adjuvant, such as an aluminium hydroxide - based adjuvant. The concentration of 
immunogenic antigen may vary from about 50 pg to about 500 pg, preferably from about 
200 pg to about 300 pg per injection, in a volume of solvent generally of from about 0.25 ml to 
about 1 ml, preferably about 0.5 ml. Multiple injections will be required after the initial 
injection and may be given e.g. at annual intervals. 

As regards "active" immunization, this is preferred for human use, but other 
mammalian species may be treated similarly, using analogous mimotopes corresponding to the 
IgE of these specie, as e.g. in the dog. The term "immunogenic carrier" herein includes those 
materials which have the property of independently eliciting an inununogenic response in a 
host animal and which can be covalently coupled to polypeptide either directly via formation of 
peptide or ester bonds between free carboxyl, amino or hydroxyl groups in the polypeptide and 
corresponding groups on the immunogenic carrier material, or alternatively by bonding through 
a conventional bifunctional linking group, or as a fusion protein. 



wo 97/31948 PCT/EP97/01013 

-10- 

Examples of such carriers include: albumins, such as BSA; globulins; thyroglobuhns; 
hemoglobins; hemocyanins (particularly Keyhole Limpet Hemocyanin [KLH]); proteins 
extracted from ascaris. e.g. ascaris extracts such as those described in J. Immun . Ill [1973] 
260-268, J. Immun . 122 [ 1979] 302-308. J. Immun . 98 ( 1 967] 893-900, and 
Am. J. Physiol. 199 [1960] 575-578 or purified products thereof; polylysine; polygluiamic acid; 
lysine-glutamic acid copolymers; copolymers containing lysine or ornithine; etc. Recently, 
vaccines have been produced using diphteria toxoid or tetanus toxoid as immunogenic carrier 
material fLepow M. L. et al., J. of Infectious Diseases J50.[1984] 402-406: Coen Beuvery, 
E- et al. Infection and Immunity 40 [1983] 39-45) and these toxoid materials can also be used 
in the present invention. The purified protein derivative of tuberculin (PPD) is particularly 
preferred for utilization in the "active" immunization scheme since (1) it does not induce a T- 
cell response itself (i.e. it is in effect a "T-cell hapten"), and yet behaves as a fully processed 
antigen and is recognized by T-cells as such; (2) it is known to be one of the most powerful 
hapten "carriers" in the linked recognition mode; and (3) it can be used in humans without 
further testing. 

As hapien-carrier binding agents, those conventionally employed in the preparation of 
antigens can be employed, e.g. those set out above, or in the Examples hereunder 

The process of the invention for covalently coupling component (a) to moiety (b) can be 
effected in known manner. Thus, for example, for direct covalent coupling it is preferred to 
utilize bis-N-succinimidyl derivatives, more preferably bis(sulfosuccinimidyl)suberale (BSS) as 
coupling agent. Glutaraldehyde or carbodiimide, more preferably dicyclohexyl-carbodiimide 
(DC) or l-ethyl-3-(3-dimeihylaniinopropyl) carbodiimide may also be used for covalent 
coupling of peptide (a) to immunogenic carrier material (b). 

The amounts of hapten and hapien-carrier binding agent [i.e. component (a)] and carrier 
[ i.e. component (b)] can be readily ascertained in conventional manner. It is preferred that the 
carrier be employed in an amount of about ! to about 6 times, preferably about 1 to about 5 
times the weight of the hapten, and the hapten-carrier binding agent be employed in an amount 
of about 5 to about 10 limes the molar equivalent of the hapten. After reaction, the carrier is 
bound to the hapten via the hapien-carrier binding agent to obtain the desired antigen composed 



wo 97/31948 



PCT/EP97/01013 



-11- 



of a peptide-caiTier complex. The resultant immunogen of the invention can be readily isolated 
in conventional manner, e.g. by dialysis, gel filtration, fractionation precipitation, etc. 

The preparation of the starting materials may be effected in conventional manner. 
Appropriate peptides for use as component (a) may e.g. be identified by screening of random 
pepude phage display libraries, and readily synthesized e.g. by conventional solid phase 
procedures, e.g.. for cyclic peptides, by the solid phase procedure employing the we!i-kno^vn 
"F-moc" procedure, or may alternatively be identified using a peptidomimetic strategy by 
screening of randonily synthesized peptides. 



wo 97/31948 PCT/EP97/01013 

-12- 

Explanation of the Figures : 

Figure 1 : Interaction between IgE and its high affinity receptor IgERI 
Figure 2 : "Classical" anti-IgE vaccine approach 
Figure 3 : Biological activity profile of BS W 1 7 
Figure 4 : BSW 1 7 minaotope-based immunotherapy 

Figure 5 : Phage-displayed BSW 1 7 mimotopes specifically recognize BSW 1 7 

Figure 6 : Phage-displayed BSW 1 7 mimotopes inhibit IgE/BS W 1 7 binding 

Figure 7 : Binding of a chemically synthesized BSW 1 7 mimotope peptide to BSW 1 7 

Figure 8a : Recognition of cyclic BSW 1 7 mimotope GEFCINHRG YW VCGDPA - KLH 

(BSS) conjugate (SDS 236) and Fct (500-509) - KLH (BSS) conjugate (SDS 237) 
by BSW 17 

Figure 8b : Recognition of various BSW 1 7 mimotope conjugates by BSW 1 7 

Figure 9a : Specific recognition of BSW 1 7 mimotope conjugates [BSA (DC)] and 
Fc£ (500-509) conjugate [KLH (glutaraldehyde)] by BSW 17 

Figure 9b : Specific recognition of BSW 1 7 mimotope conjugates [KLH (DC); Lys] 
by BSW 17 



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PCT/EP97/01013 



-13- 

Figure 10a: Anti-human IgE immune response induced in rabbits after immunization with 
BS W 1 7 mimotope conjugates ( 1 ) 

Figure 10b : Anti-human IgE immune response induced in rabbits after immunization with 
BSWI 7 mimotope conjugates (2) 

^'g"'"^" - Serum titers of anti BSW 1 7 mimotope antibodies generated in rabbits by 
immunization 

Figure 12: Isotype specificity of the anti-human IgE response in BSW 1 7 
mimotope-immunized rabbits 

Figure 13 : Competition of anti BSW 1 7 mimotope serum with BSW 1 7 for IgE binding 

Binding of affmity-purified anti BSW 1 7 mimotope antibodies to hIgE 

^'g"*"^ '^^^^ of rabbit anti BSW 1 7 mimoiope sera and affinity-purified anti BSW 1 7 
mimotope antibodies for anaphylactogenicity on human blood cells: 

- R2/0. R4/0: pre immune sera of rabbits R2 and R4 prior to BSWl 7 mimotope vaccination; 

- R2/SDS2 14. R4/SDS213: sera of rabbits R2 and R4 65 days after primary immunization; 

- antiSDS213. antiSDS2l4: affinity-purified anti - BSW 1 7 mimoiope antibodies; 

- R2/0 PC, R4/0 PC: posiiive triggering control (PC) in the presence of rabbit serum. 
Sample concentrations: 

A = 0. 1 Jig anti - BSW ] 7 mimotope antibodies (or equivalents in complete rabbit sera) per mi 
B = 1 .0 Jig anti - BSW 1 7 mimotope antibodies (or equivalents in complete rabbit sera) per ml 
C = 5.0 iLg anti - BSW17 mimotope antibodies (or equivalents in complete rabbit .sera) per ml. 



wo 97/31948 



PCr/EP97/01013 



Abbreviations : 

Ac 

AMS 

BSA 

BSS 

BSW17 



BSWI7 mimotope peptide 



cfu 

DC 

EBV 

ELISA 

FceRl 

PCS 

gam 

gar 

h 

HEPES 

HRP 

HSA 

IgE 

IPTG 

KLH 

U27 

LB-plates 



.14- 

acetyl 

ammonium sulphate 

bovine serum albumin 

bis(sulfosuccinimidyl)suberate 

an IgG monoclonal antibody directed against the 
CH3 epitope of native IgE (J.Knutti-MQUer et al., 
Allergy 41 [1986] 457-465; S.Miescheret al., 
Int.Arch.Allergv Immunol 105 [ 1 994] 75 ) 

a peptide mimicking the natural epitope on human IgE 
recognized by the monoclonal anti-human IgE antibody 
BSW17 

colony-forming units 

dihexylcaii>odiimide 

Epstein-Barr virus 

enzyme-linked immunosorbent assay 

high-affinity receptor 1 for the constant region of IgE 

fetal calf serum 

goal anii -mouse 

goat anti-rabbit 

human 

N-2-hydroxyethylpiperazine-N*-2-ethanesulfonic acid 

horse raddish peroxidase (= POX) 

human serum albumin 

immunoglobulin E 

isopropyl-B-D-thiogalactoside 

keyhole limpet hemocyanin 

a monoclonal anti-human IgE antibody (Allergy [1986], 
supra; Int. Arch. Allergy Immunol . [1994] supra) 
Luria-Bertani medium plates 



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-15- 



mimotope peptide a peptide which mimicks at least pan of the 

conformational epitope of an antibody molecule 



PBS 


phosphate-buffered saline 


PEG 


polyethyleneglycol 


POX 


horse radish peroxidase {= HRP) 


PPD 


purified protein derivative of tubercuhn 


rhIL-3 


recombinant human interleukin-3 


RIA plates 


radioimmunoassay plates 


RPMII640 


standard cell culture medium (Sigma, St. Louis, USA) 


SB-medium 


Sodium - Bacto medium (Pharmacia, Uppsala, Sweden) 


sLi 


soluble leukotriene 


TBS 


Tris-buffered saline 


VCS 


VCS Ml 3 helper phage (Straiagene, USA). 



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.16- 

5, Examples 

In the following Examples, which illustrate the invention but in no way limit its scope, 
references to temperature are in degrees Celsius. 

Example 1: Anti-allergic potenUal of the anti>hIgE monocionfll antibody BSW17 

As a model for testing the anti-allergic capacity of BSW17 and BSW17-like antibodies 
generated in human patients by active inununization with BSW17 mimoiope peptides, the 
effect of BSW17 on the histamine release of human basophil-like cells, obtained from bone 
marrow cells cultured with rhE--3, is shown. 

Mononuclear cells are prepared from bone marrow from patients requiring femur bone 
head replacement by Ficoll-Hypaque density sedimentation (1 .077 g/mi; 400 g), 5x 1 0^ cells/ml 
are cultured in RPMI1640 medium containing 15% FCS and 2 ng/ml human rhIL-3. After 6 
days of culture at 37"^ in 5% CO2, an equal volume of medium containing rhIL-3 is added. On 
day 12, cells are harvested and used for passive sensitization and histamine release assay. 
Approximately 5x 10*^ cultured bone manrow cells are incubated in HA buffer (20 mM HEPES; 
pH=7.4; 0.3 mg/mi HSA) with 500 ng/ml of human IgE in the presence or absence of a 50- fold 
excess of monoclonal anti-IgE antibody in a total volume of 1 nil. After incubation for 2 hours 
at 37°, cell supematants are used to measure histamine during passive sensitization. Cell pellets 
are used to trigger histamine release. To determine the extent of direct histamine release, the 
passively sensitized bone marrow cells are resuspended in 0.3 ml of HCM buffer (HA buffer 
containing 0.6 M CaCh and 1 mM MgCb and incubated with 0. 1 pg/ml of the 
anaphylaciogenic monoclonal anii-lgE antibody Le27. The amount of histamine in the 
supernatant and in the cellular sediment is measured in a Technicon Autoanalyzer D 
(Technicon, Tarrytown. New York, USA). Percentage of histamine release is calculated 
[according to the formula: histamine release {%) = histamine in the supernatant divided by 
histamine in the supernatant + histamine in the cell pellet, multiplied by 100]. Percentage of 
histamine release during passive sensitization is calculated {according to the formula: histamine 
release {%) = histamine in the supematant (after passive sensitization) divided by histamine in 
the supematant (after triggering) + histamine in the supematant (after sensitization) + histamine 



wo 97/31948 



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-17- 

in the cell pellet, multiplied by 100]. Anti-IgE antibody - specific histamine release is 
calculated [according to the formula: specific histamine release (%) = % total histamine release, 
minus % spontaneous histamine release]. 

The results of three independent experiments are summarized in Table 1: 

Table 1 : Effect of anti-IgE antibodies on histamine release of sensitized bone 



marrow cells 





Specific histamine release (%)** 




Experiment 1 


Experiment 2 


Experiment 3 


Sensitized with* 


A 


B 


A 


B 


A 


B 


IgE(contro)) 


3.0 


10.3 


4.3 


11.0 


1.5 


36.5 


+BSW17 


3.6 


3.1 


3.0 


1.5 


4.4 


0.9 


+ Le27 


11.0 


2.7 


8.7 


1.7 


15.7 


3.8 



*) Human bone marrow cells cultured with rhIL-3 (2 ng/ml ) are passively sensitized with IgE 
(0.5 ^ig/ml ) or with IgE and anti-IgE antibodies ( 0.25 |ig/ml ) 

**) A: Histamine release during sensitization 

B: Histamine release after triggering with the monoclonal anti-IgE antibody Le27 
(0.1 \ig/ml y 

The data in Table 1 clearly shows that bone marrow cells incubated with IgE and 
BSW17 do not release histamine during sensitization but inhibit subsequent triggering with 
Le27. Bone marrow cells incubated with IgE and Le27 are Uiggered already during passive 
sensitization to an extent that no more histamine can be released during the second incubation 
with this anaphylactogenic monoclonal antibody. Bone marrow cells passively sensitized in the 
absence of either monoclonal anti-IgE antibody, however, effectively release histamine after 
subsequent triggering with Le27. 



wo 97/315M8 



PCT/EP97/01013 



-18- 

It can be concluded thai a) BSW17 by itself is non-anaphyiactogenic and b) BSW17 
protects human basophils from histamine release induced by triggering agents. Therefore, the 
generation of BSW17-like antibodies in allergic patients by active immunization with BSW17 
mimotope peptides can be expected to protect immunized hosts from the development of 
allergic reactions. 

Example 2: Random pe ptide phage display library screening and selection of 
positive clones 

Bacteriophage particles specifically recognized by BSW17 are identified by biopanning 
of phage libraries displaying linear or circular random peptides from 6 to 1 5 amino acid 
residues in length fused to phage peptides pHl and pVffl, respectively. For amplification of 
phage libraries. 2 ml of a liquid culture of E.coli XL-I blue grown in SB medium to 
OD600 = 1.0 are incubated with lO'^ phages for 15 minutes at room temperature. 10 ml of SB 
medium containing 10 mg/ml of tetracycline and 20 [ig/ml of carbenicillin are added and 10 pi, 
1 ^l and 0. 1 III respectively, are plated on LB plates containing 100 [ig/ml of carbenicillin. The 
culture is incubated at 37° with vigorous shaking for one hour, then 100 ml of SB containing 
10 |ig/ml of tetracycline and 50 (ig/mJ of carbenicillin are added and incubation is continued for 
one hour. 10*^ cfu of VCS M 13 helper phage are added. After 2 hours with vigorous shaking at 
37°, kanamycin is added to a final concentration of 70 ^g/ml and incubation continued 
overnight. After centrifugaiion at 4000 g and 4° for 20 minutes, supematants are mixed with 
38 ml of an ice-cold sterile filtered solution of 12 % NaCl and 16 % PEG 8000, cooled on ice 
for 30 minutes and centrifuged for 30 minutes at 10000 g at 4°. The phage pellet is solubilized 
in 2 ml of TBS containing 1 .5 % casein and stored at 4°. For biopanning, Costar RIA plates 
(Costar 3690) are coated over-night at 4*^ with 20 ng/ml of BSW17 in 0. 1 M carbonate buffer, 
pH 9.6, and thereafter blocked with TBS containing 1.5 % casein. 2 x lO" cfu of phages are 
added and incubated at 37"* for 2 hours, then washed ten times with PBS / 0. 1 % Tween 20. The 
wells are rinsed with water and the bound phages eluted with a total of 200 pJ of O.I M HCl, 
pH 2.2 for 10 minutes. Eluted phages are neutralized with 2 M Tris base and amplified as 
described above. 



wo 97/31948 



PCT/EP97/01013 



-19- 

For the selection of positive clones 50 ^il of a Ecoli XL-I blue liquid culture grown in 
SB-medium to OD600 = 1-0 are incubated with 1 fil of a 10 ® dilution of amplified phages after 
the 3rd round of panning for 15 minutes at room temperature, then plated on LB plates 
containing 100 ^ig/ml of carbenicillin and grown overnight. Colonies are randomly picked and 
plated on LB plates containing 100 |ig/ml of carbenicillin. After 4 hours at 37°, nitrocellulose 
fillers soaked with 10 mM of IPTG are put on top and incubation continued overnight at 32°. 
Filters are removed and incubated at 37° for 30 minutes in a CHCb-atmosphere. Bacterial 
debris are removed by incubating the filters in 50 mM Tris, pH 8. 150 mM NaCl, 
5 mM MgCb, 3 % BSA, 100 U DNAasel and 40 mg lysozyme per 100 ml for 1 hour, blocked 
in TBS containing 1.5 % casein and incubated over night with BSW17-POX (BSW17 coupled 
to POX) in TBS containing 1.5 % casein. Filters are washed with TBS, TBS/0.5 % Tween 20 
and then TBS for 10 minutes each. For staining the strips are incubated in 600 |ig of 4-chloro- 
1-naphthol per ml and 0.042 % of hydrogen peroxide in TBS. 

Example 3: Characterization of phage particles displaying peptides which mimick the 
natural BSW17 epitope (BSW17 mimotope peptides) 

Various phage particles displaying circular peptides consisting of seven, eight or nine 
amino acids and linear peptides consisting often and fifteen amino acid residues, respectively, 
have been found to bind to BSWI7. The nucleotide sequence of the DNA encoding these 
peptides was determined. According to their homology among each other as well as to 
homologous regions within Fee, 3 groups of phages displaying peptides recognized by BSW17 
could be deduced from the DNA sequence. An overview is shown in Table 2: 



wo 97/31948 



-20- 



PCT/EP97/010I3 



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wo 97/31948 



-21- 



PCT/EP97/01013 



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wo 97/31948 



PCT/EP97/01013 



.22- 

As can be seen from Table 2, the carboxy-temiinal parts of the peptides from group A 
are highly conserved and the amino-terminal parts, though more degenerated, contain 
positively-charged, polar amino acids. This charge distribution seems to be essential for 
BSW17 recognition, since one clone which differs in the amino-terminus from this feature 
proves to only very weakly bind to BSW17. No consecutive stretch of amino acids with 
significant sequence homology can be found in Fee. Alignment by pattern similarity, however, 
allows allocation of these peptides to a stretch of amino acids ranging from position 500 to 508 
within the Ce4 domain. The decapeptide Fee 500-509 has been postulated by 
Stanworth et al. [Lancet 336 (1990)1279] to be involved in mast cell triggering by receptor- 
bound IgE. 

Peptides belonging to group B also are highly homologous among each other. 
Furthermore, their amino-terminal part is almost identical with amino acid positions 370-375 of 
Fee. This sequence is part of Ce3 and contains or is adjacent to a region which has been shown 
to be involved in binding of IgE to its high affinity receptor. This finding explains the inhibiton 
of the IgE/IgERI interaction by BSW17. 

It can be concluded that the complex conformational epitope recognized by BSW17 
contains conformational structures represented by amino acid stretches 500-508 (within Ce4) 
and 370-375 (within Ce3) of the IgE molecule (numbering according to E. A.Padian and 
D.R.Davies, Molecul.lmmunol . 23 (1986) 1063). Antibodies generated in an individual by 
immunization with mimotopes from groups A and B above coupled to an immunogenic carrier 
molecule will thus protect from allergic reactions by blocking the binding of IgE to its high 
affinity receptor and/or triggering the degranulalion of target cells. 

Peptides summarized in group C of the table exhibit no significant homologies among 
each other nor with Fee. Therefore, they represent "genuine mimotopes" mimicking the three 
dimensional structure recognized by BSW17. Using these mimotopes as immunogens thus 
will also result in the generation of anti-allergic, BSW17-like antibodies in the immunized host. 

The generation of a BSW 17-like immune response specifically directed against the 
BSW17 epitope on human IgE is shown for selected mimotopes in the following Examples 4 
to 6: 



wo 97/31948 



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-23- 



Example4 : Bacteriophages displaying BS W17 iniinotop> peptides an. 5iiw.rmr«Hy 
recognized bv BSW17 

Binding of mimotope peptides displayed by phage particles (group A of Table 2: 
first clone = clone 1 in Fig. 5 = Cys-Arg-Aig-His-Asn-Tyr-Gly-Phe-Trp-Val-Cys = 
Seq.id.no. 18; second clone = clone 18 in Fig. 5 = Cys-De-Asn-His-Arg-Gly-Tyr-Trp-Val-Cys 
= Seq,id.no. 19) to the anUgen-specific hypervariable regions of BSW17 is demonstrated by 
ELIS A. 1 0 ^g of BSW 1 7 and various monoclonal antibodies are coated to the wells of 
COSTAR plates following standard ELBA procedures. After blocking with BSA. the wells are 
incubated with 100 ^ of standard ELISA incubation buffer containing bacteriophage panicles 
obtained from phage-infected bacterial culture supematants with a phage titer of lO'cfu/ml. 
After washing, the plates are incubated with 5 ng of horse raddish peroxidase (HRP) - coupled 
anti-M13 antiserum (Pharmacia. Uppsala. Sweden) in incubation buffer. All incubations are 
carried out at room temperature for 1 hour. After fmal washing, phage panicles bound to the 
coated antibodies are visualized via the bound HRP-anti M13 senim by chromogenic substrate 
development following standard ELISA procedure. Figure 5 shows that mimotope phages 
very specifically bind to BSWI7 and to no other antibodies, such as 8E7, 3G9 and 5H10 which 
are also monoclonal anti-CE3 antibodies and which have been generated by using recombinant 
Ce3 as immunogen. nor to 5H5/F8 which represents another monoclonal antibody which is 
directed against the extracellular portion of human FceRIa. As negative non-antibody control, 
recombinant FceRI a-chain and as positive control, wells coated with polyclonal anti-M13 
antiserum are included. 

^^^Sffifiki: Bacteriophages disnlavin. R«;wi7 nentirf.c ..».petitivelv inhiKU 

the binding of BSW17 tn I pF 

The specificity of the mimotope - BSWl 7 interaction is further demonstrated by 
showing ihai the attachment of mimotope-displaying phage particles interferes with the 
BSW 1 7/IgE binding. Bacterial cells are grown in SB containing 1 0 ,xg/ml of tetracycline and 
50 Mg/ml of carbenicillin to OD.00 = 0.4. then VCS M13 helper phage is added and incubation 



wo 97/31948 



PCT/EP97/01013 



-24- 

continued for 2 hours at 37°. Then kanamycin is added to a final concentration of 70 ^g/ml and 
incubation continued overnight. Phage particles are precipitated with polyelhyleneglycol. Soft 
RIA plates are coaled with 20 ^ig/ml of BSW17 in 0. 1 M carbonate buffer pH 9.6 and then 
blocked with TBS containing 1.5 % casein. Human IgE is '^^I-labelled by the 
chloramine-T method and 100000 cpm per well are used for binding to BSWI7 coated to the 
RIA plates in the presence of increasing concentrations of non-labelled IgE (standard curve) or 
phage particles, respectively. All experiments are performed at room termperature and plates 
are washed 3 times with 0.9 % NaCl + 0.05 % Tween 20, cut in pieces and each well was 
measured in a y-counter for 1 minute. 

Mimoiope phages (PhBSW.6-9 in Fig. 6 = clone 1 of Fig. 5 = CRRHNYGFWVC = 
Seqad.no. 18; PhBSW.29-8 in Fig. 6 = clone 18 of Fig. 5 = CINHRGYWVC = Seq.id.no. 19) 
(see Example 4 above) inhibit the binding of BSWI7 to IgE. Figure 6 shows the binding of 
*"^I-labelled IgE to BSW17 in the presence of BSW17 mimotope - displaying phage clones. 
Closed circles show a standard inhibition curve by unlabelled IgE to allow an estimation of 
inhibition of labeled IgE binding in the presence of 10*' cfu/ml of phage particles. The 
inhibition achieved with the phage clones is equal to non-labelled IgE concentrations of I 
\ig/m\ and 1.7 jUg/ml, respectively. 

Example 6 : Induction of an epitope-specific immune reaction by immunization of 
rabbits with mimotope-displaving phage clones 

To test the feasibility of a BSW17 mimolope-based vaccine approach, rabbits are 
immunized with phage panicles displaying BSWI7 mimoiope peptides and helper phage 
VCS M 13 as control respectively. lO'' cfu of freshly prepared phage particles are dialyzed 
against PBS at 4"*. 1 ml is emulsified with complete Freund's adjuvant for the first 
immunization, or with 1 ml incomplete Freund s adjuvant for boosting. Immunization is 
repealed subcutaneously every 14 days. After the third boost, 12 ml of blood are taken, clotted 
for 4 hours ai room temperature in glass vials and centrifuged for 10 minutes at 2000 g. The 
supematants are tested for the presence of anti-human IgE antibodies by ELIS A. Human IgE 
from three different individuals (SUSl 1-IgE; PS-IgE; WT-IgE) is diluted in PBS at a 



wo 97/31948 



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-25- 

concentration of 50 ng/ml and 1 pJ aliquots are dotted onto nitrocellulose. Nitrocellulose is 
blocked in TBS containing 1 .5 % casein for 1 hour. Rabbit serum is diluted 1 :200 in TBS 
containing 1.5 % casein and incubated overnight. Washing is performed in TBS. TBS-0.05 % 
Tween 20 and TBS for 1 0 minutes each. Developing goat anti-rabbit-HRP is 1 : 1 .000 diluted in 
TBS containing 1 .5 % casein and incubated for 4 hours. Washing and staining is performed as 
described. As shown in Table 3. the control rabbits immunized with VCS M13 phage, as well 
as the non-immunized rabbits show a weak reaction against human IgE in their sera, 
presumably reflecting naturally occuring anti-rabbit IgE autoantibodies crossreacting with 
human IgE. However, in addition to a massive immune response to the bacteriophage pVm 
coat protein, sera of rabbits immunized with BSW17 mimotope phages also contain strongly 
elevated levels of antibodies which specifically recognize human IgE: 



Table 3: Anti-IgE response in rabbits immunized with BSW17 mimotope displaying 
phage 



Recognition of IgE (relative OD) 


Rabbit serum 


SUSll-IgE 


PS-IgE 


WT-IgE 


nonimmune 


2.0 ± 0.3 


2.0 ± 0.2 


2.0 ± 1.0 


VCS M13 helper phage 


2.0 ± 0.6 


2.0 ± 1.0 


3.0 ± 0.5 


PhBSW.6-9 f= clone 1) 


5.0 ± 0.2 


8.0 ± 0.6 


21.0 ± 1.0 


PhBSW.29-8(= clone 18) 


16.5 ± 0.7 


9.0 ± 0.4 


25.0 ± 1.0 



BSWl 7 epitope specificity of these antisera can be demonstrated in a competition 
ELISA: nitrocellulose strips are prepared as described above and incubated with rabbit serum 
at a dilution of 1:50 in TBS containing 1.5 % casein overnight. After washing, mAb BSWl 7 
labeled with horse raddish peroxidase (BSWH-HRP) is added in a 1:50000 dilution in 
TBS containing 1.5 % casein for 4 hours. Subsequent washing and staining is performed a.s 
described. As can be seen from Table 4. the binding of BS W 1 7-HRP to IgE preparations from 
various sources is inhibited by sera from the rabbits immunized with BSWl 7 mimotope 
phages, while serum from rabbits immunized with helper phage do not show any inhibitory 
effect: 



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Table 4: Inhibition of BSW17 / IgE Interaction with sera of rabbits immunized with 



BSW17 mimotope phages 





Recognition of different IgE (% inhibition) 




SUSll-IgE 


WT-IgE 


PS-IgE 


Zavasal-IgE 


VCSM 13 helper phage 


0 


0 


0 


0 


PhBSW.6-9 


34 


65 


63 


51 


PhBSW.29.8 


52 


65 


48 


47 



In the following Examples 7 and 8, the feasibility of using chemically synthesized 
numoiope peptides coupled to carrier protein as immunogen for the preparation of an 
anti-allergy vaccine is shown: 

Example 7 : A chemically synthesized BSW17 mimotope peptide binds to BSW17 with 
high affinity 

To confirm that the bacieriophage-derived portion of the BSW17 mimotope phages can 
be omitted without destroying the biological activity of the mimotope peptide sequence, the 
following peptide is chemically synthesized, comprising the cyclic BSW17 mimotope 
octapeptide (A) [shown in linear form in Table 2, Group A, second clone as peptide (A) with 
two additional Cys (Seq.id.no. 19), and containing here the 7 additional adjacent bacteriophage- 
derived amino acid residues Gly-Glu-Phe- and -Gly- Asp-Pro-Ala as flanking sequences 
included to facilitate correct folding of the circular mimotope peptide]: 

Giy-Glu-Phe-Cys-Ile-Asn-His-Arg-Gly-Tyr-Trp-Val-Cys-Gly-Asp-Pro-Ala fSeq.id.no. 27) 

After synthesis by standard technique this peptide is cyclized via the two cysteins and 
fluorescence-iabelled with Rhodol Green. Binding to increasing concentrations of BSW17 
immobilized in microtiier plate wells under ELISA conditions is determined by fluorescence 
measurement and is shown in Figure 7. 



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^2S!SE!e8: Chemically synthesized BSW17 mi nHitoDe n«.nfiH., ^upjed to a r^fji.r 
protein are spgcifi rallv recnpni^ed bv BSWI7 

Various mimotope peptides are cliemically synthesized and coupled to immunogenic 
carrier proteins. The coupling reactions are perfomied at a 1 : 1 mass ratio of peptide and carrier 
protein, following standard procedures (Shan S. Wong, Chemistn^of Prnr.in ConinP.rinn .n^ 
Cross-linking , CRC Press f 1993]). Conjugates obtained from the following coupling variants 
are prepared: 

- glutaraidehyde coupling 

- EXT (dihexylcarbodiimide) coupling 

- BSS [bis(sulfosuccinimidyl)suberate] coupling 

- lysine crosslinking 

The resultant mimotope conjugates are: 

( 1 ) P 1 = linear KTKGSGFFVF - BS A (glutaraidehyde) 

(2) P4 = linear AcINHRGYWVC - BSA (DC) 

(3) P5 = linear AcRSRSGGYWLWC - BSA (DC) 

(4) SAFl -iCLH = cyclic GEFCINHRGYWVCGDPA - KLH (IX:) 

(5) SAF2-KLH = linear KTKGSGFFVF - KLH (DC) 

(6) SAF3-KLH = linear VNLPWSFGLE - KLH (DC) 

(7) SAF3-Lys = linear VNLPWSFGLE - lysine crosslinking 

(8) SAF4-KLH = linear VNLTWSRASG - KLH (DC) 

(9) SAF5-KLH = VNLTWS - KLH (DC) 

(10) SDS2 14 = cyclic GEFCRRHN YGFWVCGDPA - KLH (BSS) 

(11) SDS213. SDS227. SDS236. SDS252 = cychc GEFCINHRGYWVCGDPA - KLH (BSS) 

(12) SDS237, SDS253 = linear KTKGSGFFVF - KLH (BSS) 

(13) SDS242. SDS254 = linear VNLPWSFGLE - KLH (BSS) and 

(14) SDS243 = linear VNLTWSRASG - KLH (BSS). 



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-28. 

whereby (1). (5) and (12) are reference immunogen molecules prepared by, respectively, 
glutaraldehyde, DC and BSS coupling; thus: 

( 1 i.e. Pljs amino acids 500-509 of the human IgE, SeqJd.no. 28, see The Lancet [1990] 
supra, coupled to BSA by glutaraldehyde coupling; 

(5), i.e. SAF2-KLH. is that same peptide, Seq.id.no. 28, coupled to KLH by DC coupling; and 
(12), i.e. SDS237 and SDS253, is that same peptide, Seq.id.no. 28, coupled to KLH by BSS 
coupling. 

The other peptides are inununogens of the invention, namely: 

- (2) (P4) is N-aceiylated peptide (A) (Seq.id.no. 1) with an additional Cys at the C-tenninus 
(Seq.id.no. 29), coupled to BSA by DC coupling; 

- (3) (P5) is N-aceiylated peptide (C) (Seq.id.no. 3) with an additional Cys at the C-lerminus 
(Seq.id.no. 30), coupled to BSA by DC coupling; 

- (4) (SAFl-KLH) is the peptide ofExample 7 (Seq.id.no. 31), cyclized via a disulfide bridge 
formed by the two flanking Cys, and coupled to KLH by DC coupling; 

- (6) (SAF3-KLH) is peptide (G) (Seq.id.no. 7), coupled to KLH by DC coupling; 

- (7) (SAF3-Lys) is peptide (G) (Seq.id.no. 7), coupled by lysine crosslinking; 

- (8) (SAF4-KLH) is peptide (D) (Seq.id.no. 4), coupled to KLH by DC coupling; 

- (9) (SAF5-KLH) is peptide (Q) (Seq.id.no. 17) coupled to KLH by DC coupling; 

- ( 10) (SDS2I4) is the first peptide of group A in Table 2, with the same 7 adjacent 
bacteriophage-derived amino acid residues as the peptide in Example 7 (Seq.id.no. 32), 
cyclized, and coupled to KLH by BSS coupling; 

- (1 1) (SDS213, SDS227, SDS236, SDS252) is the same peptide as (4) above (Seq.id.no. 31), 
cyclized, and coupled to KLH by BSS coupling; 

- (13) (SDS242, SDS254) is peptide (G) (Seq.id.no. 7), coupled to KLH by BSS coupling; and 

- ( 14) (SDS243) is peptide (D) (Seq.id.no. 4), coupled to KLH by BSS coupling. 

Figure 8a shows that both the cyclic BSW17 mimolope - KLH (BSS) conjugate (11). 
i.e. SDS236. and the Fee-derived **originar* epitope motif (12), i.e. SDS237, i.e. Fc£ (500-509) 
coupled as linear peptide to KLH (BSS), are recognized by BSW17 in a dose-dependent 



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-29- 

manner, while free KLH shows no binding. Microliter plate wells are coated with 1 each of 
SDS236, SDS237 or free KLH and incubated with increasing concentrations of BSW17. 
Bound antibody is detected with goat anti-mouse IgG-HRP (gamlgG-HRP). The data shown 
represents means of duplicates minus background binding to uncoated (BSA-blocked) wells. 

Figure 8b summarizes BUS A data obtained with various BSWI7 mimotope 
conjugates coupled to the carrier protein using several chemical coupling procedures. 
Microtiter plate wells are coated with 5 fig each of the mimotope conjugates, or free KLH, and 
incubated with 10 ng of BSWI7. Bound antibody is detected with gamlgG-HRP. The data 
shown represents means of duplicates minus background binding to uncoated (BSA-blocked) 
wells. In contrast to free KLH. each BSWI7 mimotope conjugate is found to be recognized by 
BSWI7. However, from repeated experiments carried out over several weeks it becomes clear 
that the KLH conjugates coupled via DC are less stable and gradually lose their binding 
capacity for BSWI7. In contrast. BSW17 mimotope conjugates obtained by BSS coupling 
prove to be stable even at +4°. 

In Figures 9a and 9b it is shown that BSW17 mimotopes coupled to KLH or BSA are 
specifically recognized by BSW17 and not by the non-related monoclonal antibodies 309 
(anti-human Ce3) and 5H5/F8 (anti-human RIa). Again, microtiter plate wells are coated with 
5 Mg each of the mimotope conjugates shown and incubated with 10 ^g of the corresponding 
monoclonal antibody. Bound antibody is detected with gamlgG-HRP. The data shown 
represents means of duplicates minus background binding to uncoated (BSA-blocked) wells. 

^'^^iBEkl: Immunization of rabbit, with RSW1 7 mimotonp rnni .. pat« re^nlt. in .h. 
in vivo generation of ant j human IgF. antibodies 

To confirm the specificity of the immunogenicity of the BSW17 mimotope conjugates, 
rabbits are immunized with a set of mimotope/carrier preparations as outlined below: 



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Rabbit No. Immunogen Conjugate type 



Rl (10) SDS214 

R2 (10) SDS214 Ce4 mimotopes 

R3 (11) SDS213 

R4 (11) SDS213 

1 KLH (Pierce) 

2 KLH-BSS linker KLH control 

4 (12) SDS237 

11 (12) SDS237 Ce4(500 - 509) 

7 (6) SAF3-KLH 

13 (7) SAF3-Lys 

14 (13) SDS242 C£3 mimotope 

16 (13) SDS242 

17 (13) SDS254 

18 (7) SAF3-Lys 

5 (8) SAF4-KLH C£3 (370-379) 

8 (8) SAF4-KLH 

19 (14) SDS243 

20 (14) SDS243 

12 (4) SAFl-KLH 

15 (4) SAFl-KLH Ce4 mimotope 



Rabbits are immunized with 200 ng of the corresponding conjugate preparation 
dissolved in a total volume of 500 jil of phosphate-buffered saline (PBSdef.) by s.c. injection. 
For the firsl immunization samples are mixed 1:1 with complete Freund's adjuvant 
(rabbits Rl - R4) or TiterMax (Sigma; rabbits 1 - 20). Prior to the initial immunization. 5 ml of 
blood samples are taken. Boosting is performed on days 21 and 28 after the first injection using 
incomplete Freund's adjuvant. Blood samples are taken on day 28 and 49 and serum is 
prepared from ail samples. 

The generation of an anti-human IgE immune response in the immunized rabbits is 
monitored by ELIS A: microtiter plate wells are coated with 5 \ig of human IgE (SUS- 1 1 ; JW8) 
and incubated with 100 jil of serum samples, diluted 1 :50 with ELISA incubation buffer. 
Rabbit antibodies bound by the immobilized human IgE are delected by incubation with goat 



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anti-rabbit IgG-HRP (garlgG-HRP). The measured OD405 values are coirccted by the read-out 
obtained with pre-iramune serum from each corresponding rabbit, diluted 1 :50. The data given 
in Figures 10a and 10b represents mean values of duplicate measurements. Figures 10a and 
10b clearly demonstrate the induction of antibodies in rabbits immunized with various BSW17 
mimotope conjugates which are directed against human IgE. 

Serum titers of the newly generated antibodies with respect to the KLH carrier, the 
peptide used as immunogen and human IgE, respectively, are also determined by ELISA. using 
serial serum dilutions for incubation with inunobilized KLH, mimotope peptide - BSA 
conjugate and human IgE. respectively. Two examples of such serum titer determinations are 
shown in Figure 11. for (1 1). i.e. SDS 213, and for (10), i.e. SDS 214, respectively. 

The above Example demonstrates the applicability of BSW17 mimotope conjugates as 
immunogens for the induction of an anti - human IgE response. 

Example 10 : The anti-hlgE response induc ed in rabhite immunized with RSW17 

mimotope conjugates is hotvpe-SDecifif and cam^ t^ with RSWI7 fnr 
IgE binding 

The isotype specificity of the anti - human IgE immune response in the immunized 
rabbits .s monitored by EUSA: microtiter plate wells are each coated with 3 ^ig of human IgE 
(SUS-1 1 ), IgA, IgG. and IgM. respectively, and incubated with 100 jil of serum samples, 
diluted 1:50 with ELISA incubation buffer. Rabbit antibodies bound by the immobilized 
human antibodies are detected by incubation with goat garlgG-HRP. The measured 
OD.05 values are corrected by the read-out obtained with 1 :50 diluted pre-immune serum of 
each corresponding rabbit, diluted 1 :50. The data given in Figu.^ 12 represents mean values of 
duplicate measurements. As can be seen, the immune response generated m rabbits by 
immunization with BSW 1 7 mimotope conjugates is specific for IgE. apart from a partial 
recognition of human IgM by the serum of the rabbit immunized with (10), i.e. SDS2I4. 

In a competition ELISA it is further shown that rabbit anti-SDS2 14 antiserum is able to 
partially compete with BSW 1 7 for IgE binding. Microtiter plate wells are each coated with 



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1 fig of human IgE (SUS-1 1 ) and either incubated with 5 \ig of BSW17 or incubation buffer 
without BSW17. After washing, lOOfU of anti-SDS213 antiserum, diluted 1:50 with ELISA 
incubation buffer, is added for a second incubation. Rabbit antibodies bound by untreated 
immobilized human IgE and IgE preincubated with BSWl 17 are delected by incubation with 
goat garlgG-HRP. The data given in Figure 13 represent mean values of duplicate 
measurements. 

Example 11 ; The anti^hlcE antibodies generated in rabbits bv immunization with 

BSW17 mimotope conjugates can be purified bv affinity chromatography 
using mimotope peptide coupled to Sepharose as affinity reagent 

This Example demonstrates that the anti-hlgE response induced in rabbits by 
immunization with BSW17 mimotope conjugates is identical with the specific 
anii-mimotope peptide response. 

Immunoaffinity purification of rabbit polyclonal anti-BSWI7 mimotofje antibodies: 

a) Ammonium sulfate precipitation : 

To 22 ml of rabbit antiserum (60 mg/ml of total protein according to Bradford, 
BSA-Standard, BIO-RAD) 5.5 g of solid AMS (25% w/v) is added, the mixture is stirred for 
3 hours and incubated overnight at room temperature. The precipitate is removed by 
centrifugation ai 18(XX) rpm (Sorvall) for 45 minutes and washed with 25 ml of 25 % AMS 
aqueous solution (w/v). The washed precipitate is centrifuged again under the same conditions, 
dissolved in 10 ml of PBS, 0.05 % NaNi, pH 7.2, and dialysed against 5 1 of the same buffer 
overnight at + 4°. 

b) Immunoaffinity chromatograt)hv: 

The dialysaie is filtered on a 0.2 |am Millex-GV filter unit (Millipore) and applied to a 
Pharmacia XK 16/30 column filled with 10 ml of CH-Sepharose 4B covalently coupled to 5 mg 
of BSWl 7 mimotope peptide SDS227 at a flow rate of I ml/min with PBS, 0.05% NaN3 as 
running buffer. After application and elution of the unbound protein fraction, specifically 
bound antibody is eiuted with 0.1 M glycine/HCl buffer pH 2.8. The eluaie (fractions #12-20 in 
9 ml) is trapped under stirring for immediate neutralization to pH 7.4 with 3.3 M TRIS/HAc, 
0.8% NaN3 pH 8.0 (50 Ml/ml) and finally dialysed against 3 1 of PBS, 0.05% NaN3, pH 7.2 



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ovemight at 4". Total protein is about 1 mg/ml according to Bradford, BIgG Standard (BIO- 
RAD). The recovery from total protein applied is about 0.7%. 

Affinity - purified IgG fractions are tested for hIgE binding by ELISA. Microliter plate 
wells are coated with increasing concentrations of human IgE (JW8) and incubated with 5 ng 
of the purified anti-SDS-213 antiserum and anti SDS-214 antiserum, i^spectively. Bound 
antibody is detected with garlgO-HRP. The data shown in Figure 14 i^presents mean values 
of duplicates. As can be seen from the Figure, the IgG antibodies purified on the anti - BSWl 7 
mimotope affinity column are dose-dependently recognized by human IgE. Only low 
background binding activity is observed with samples of the column flow through. This data 
demonstrates that the anti-hlgE activity induced in the rabbits is identical with the antibodies 
directed against the mimotope peptide. 

Example 12 : The anti human IgE antibo dies generatgrf in rabbits after immunizatinn 
with BSW17 mimotope conjugates are non-anaphvlactogenic on human 
blood ceils 

Ii is shown here that the anti-IgE antibodies generated in rabbits by immunization with 
BSW17 mimotope conjugates are non-anaphylactogenic on human blood basophils. As test 
system, the commercially available CAST sU ELISA kit (Buhlmann AG. Allschwil. 
Switzerland) is used. In this assay the release of soluble leukotriene (sLt) from human basophils 
as a consequence of triggering the cells by anaphylactogenic agents is deteimined following the 
experimental protocol provided by the supplier. The read-out of the assay is given as pg sLt 
present per ml of human white blood cell supernatant after incubation of the cells with the test 
sample, as determined by comparison with a standard curve. The standard curve is obtained 
using a serial dilution of standard sLt in a competition ELISA. sLt background levels resulting 
from spontaneous sLt release from the blood cell preparation (PB = "patient blank") and 
maximum sLt that can be released from a given blood cell preparation (PC = "patient control" ; 
obtained after triggering of the cell samples with a crosslinking anti-IgERIa antibody; are 
included in each test as negative and positive controls, respectively. 

Complete sera from rabbits immunized with BSWl 7 mimotope conjugates as well as 
the affmity-purified anti-BSW17 mimotope antibodies described in Example 1 1 are tested for 



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the presence of basophil triggering and thus for anaphylactogenic anti-hlgE antibodies. As 
source for basophils, freshly taken whole human blood of a healthy donor is used and processed 
strictly according to the protocol of the CAST ELISA kit. The results are summarized in 
Figure 15 and show thai neither non-purified rabbit serum containing the anti-hlgE antibodies 
generated by immunization with BSW17 mimotope conjugates, nor the affinity-purified 
anti - BSW17 mimotope antibodies, have the capability of uiggering human blood cells for 
leukotriene release; they are thus non-anaphylactogenic. This is an absolute prerequisite for the 
application of BSW17 mimotopes in a human anti-allergy vaccine. 



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Seouence listing 



( 1 ) GENERAL INFORMATION: 

(i) APPUCANT: 

(A) NAME: Novartis AG 

(B) STREET: Schwarzwaldallee 215 

(C) CITY: Basle 

(E) COUNTRY: Switzerland 

(F) POSTAL CODE (ZIP): CH.4058 

(G) TELEPHONE: 61-324 5269 

(H) TELEFAX: 61-322 7532 

(ii) TITLE OF INVENTION: PEPTIDE IMMUNOGENS 

(iii) NUMBER OF SEQUENCES: 32 

(iv) COMPUTER READABLE FORM: 

(A) MEDIUM TYPE: Floppy disk 

(B) COMPUTER: IBM PC compatible 

(C) OPERATING SYSTEM: PC-DOS/MS-DOS 

(D) SOFTWARE: Patentin Release #1.0, Version #1.25 (EPO) 

(V) CURRENT APPLICATION DATA: 

APPLICATION NUMBER: WO PCT/EP97/.... 

fvi) PRIOR APPLICATION DATA: 

(A) APPLICATION NUMBER: GB 9604412.8 

(B) FILING DATE: Ol-MAR-1996 
<vi) PRIOR APPLICATION DATA: 

(A) APPLICATION NUMBER: GB 9617702.7 

(B) FILING DATE: 22-AUG-1996 



(2) INFORMATION FOR SEg ID NO: 1: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 8 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(iii) HYPOTHETICAL: YES 
(iii) ANTI-SENSE: NO 

(v) FRAGMENT TYPE: internal 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: 



lie Asn His Arg Gly Tyr Trn Val 
1 5 



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(2) INFORMATION FOR SSg ID NO: 2: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 8 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(iii) HYPOTHETICAL: YES 
(iii) ANTI-SENSE: NO 

(v) FRAGMENT TYPE: internal 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: 

Arg Asn His Arg Gly Tyr Trp Val 
1 5 

(2) INFORMATION FOR SBQ ID NO: 3: 

(i) SEQUENCE CHARACTERISTICS; 

(A) LENGTH: 10 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(iii) HYPOTHETICAL: YES 
(iii) ANTI-SENSE: NO 

(v) FRAGMENT TYPE: internal 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: 

Arg Ser Arg Ser Gly Gly Tyr Trp Leu Trp 
15 10 

(2) INFORBflATION FOR SEQ ID NO: 4: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 10 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(iii) HYPOTHETICAL: YES 
(iii) ANTI-SENSE: NO 

(v) FRAGMENT TYPE: internal 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: 

Val Asn Leu Thr Trp Ser Arg Ala Ser Gly 
15 10 

(2) INFORMATION FOR SEQ ID NO: 5: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 10 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 



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(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 
(ill) HYPOTHETICAL: YES 

(iii) ANTI-SENSE: NO 

(v) FRAGMENT TYPE: internal 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: 

Val Asn Leu Pro Trp Ser Arg Ala Ser Glv 

1 5 10 

(2) INFORMATION FOR SEQ ID NO: 6: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 10 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(iii) HYPOTHETICAL: YES 
(iii) ANTI-SENSE: NO 

(V) FRAGMENT TYPE: internal 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: 

Val Asn Leu Thr Trp Ser Phe Gly Leu Glu 
1 5 10 

(2) INFORMATION FOR SEQ ID NO: 7: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 10 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(iii) HYPOTHETICAL: YES 
(iii) ANTI-SENSE: NO 

(V) FRAGMENT TYPE: internal 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: 

Val Asn Leu Pro Trp Ser Phe Gly Leu Glu 

. 5 10 

(2) INFORMATION FOR SEQ ID NO: 8: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 10 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(iii) HYPOTHETICAL: YES 
(iii) ANTI-SENSE: NO 

(V) FRAGMENT TYPE: internal 



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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: 

Val Asn Arg Pro Trp Ser Phe Gly Leu Glu 
1 5 10 

(2) INFORMATION FOR SEQ ID NO: 9: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 10 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(iii) HYPOTHETICAL: YES 
(iii) ANTI-SENSE: NO 

(v) FRAGMENT TYPE: internal 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: 

Val Lys Leu Pro Trp Arg Phe Tyr Gin Val 
15 10 

(2) INFORUAHON for SEQ id NO: 10: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 10 amino adds 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(iii) HYPOTHETICAL: YES 
(iii) ANTI-SENSE: NO 

(v) FRAGMENT TYPE: internal 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: 

Val Trp Thr Ala Cys Gly Tyr Gly Arg Met 
15 10 

(2) INFORMATION FOR SEQ ID NO: 11: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 7 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(iii) HYPOTHETICAL: YES 
(iii) ANTI-SENSE: NO 

(V) FRAGMENT TYPE: internal 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: 



Gly Thr Val Ser Thr Leu Ser 
1 5 



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(2) INFORMATION FOR SE9 ID NO: 12: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 7 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(iii) HYPOTHETICAL: YES 
(iii) ANTI-SENSE: NO 

(v) FRAGMENT TYPE: internal 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: 

Leu Leu Asp Ser Arg Tyr Trp 
1 5 

(2) INFORMATION FOR SBQ ID NO: 13: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 7 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(iii) HYPOTHETICAL: YES 
(iii) ANTI-SENSE: NO 

(v) FRAGMENT TYPE: internal 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: 

Gin Pro Ala His Ser Leu Gly 
1 5 

(2) INFORMATION FOR SEQ ID NO: 14: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 7 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(iii) HYPOTHETICAL: YES 
(iii) ANTI-SENSE: NO 

(v) FRAGMENT TYPE: internal 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: 

Leu Trp Gly Met Gin Gly Arg 
1 5 

(2) INFORMATION FOR SBg ID NO: 15: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 15 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 



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(ii) MOLECULE TYPE: peptide 

(iii) HYPOTHETICAL: YES 
(iii) ANTI-SENSE: NO 

(v) FRAGMENT TYPE: internal 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: 

Leu Thr Leu Ser His Pro His Trp Val Leu Asn His Phe 
15 10 
Val Ser 
15 

(2) INFORBIATION FOR SEQ W NO: 16: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 9 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(iii) HYPOTHETICAL: YES 
(iii) ANTI-SENSE: NO 

(v) FRAGMENT TYPE: internal 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: 

Ser Met Gly Pro Asp Gin Thr Leu Arg 
1 5 

(2) INFORMATION FOR SB9 ID NO: 17: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 6 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(iii) HYPOTHETICAL: YES 
(iii) ANTI-SENSE: NO 

(V) FRAGMENT TYPE: internal 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: 

Val Asn Leu Thr Trp Ser 
1 5 

(2) INFORMATION FOR SEg ID NO: 18: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 11 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(iii) HYPOTHETICAL: YES 
(iii) ANTI-SENSE: NO 



wo 97/31948 



PCT/EP97/01013 



-41- 

(v) FRAGMENT TYPE: internal 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: 

Cys Arg Arg His Asn Tyr Gly Phe Trp Val Cys 
15 10 

(2) Bm>RMATION FOR SB9 ID NO: 19: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 10 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(iii) HYPOTHETICAL: YES 
(iii) ANTI-SENSE: NO 

(v) FRAGMENT TYPE: internal 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: 

Cys lie Asn His Arg Gly Tyr Trp Val Cys 
15 10 

(2) INFORMATION FOR SBg ID NO: 20: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 11 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(iii) HYPOTHETICAL: YES 
(iii) ANTI-SENSE: NO 

(v) FRAGMENT TYPE: internal 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: 

Cys Thr Arg Leu His Thr Gly Tyr Trp Val Cys 
15 10 

(2) INFORMATION FOR SE9 ID NO: 21: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 11 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(iii) HYPOTHETICAL: YES 
(iii) ANTI-SENSE: NO 

(v) FRAGMENT TYPE: internal 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: 



Cys Thr Leu Ser Val Phe Gly Tyr Trp Val Cys 
15 10 



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PCT/EP97/01013 



-42- 



(2) INFORMATION FOR SBQ ID NO: 22: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 11 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(iii) HYPOTHETICAL: YES 
(iii) ANTI-SENSE: NO 

(v) FRAGMENT TYPE: internal 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: 

Cys Ser Met Gly Pro Asp Gin Thr Leu Arg Cys 
1 5 10 

(2) INFORMATION FOR SBQ ID NO: 23: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 9 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(iii) HYPOTHETICAL: YES 
(iii) ANTI-SENSE: NO 

(v) FRAGMENT TYPE: internal 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: 

Cys Leu Leu Asp Ser Arg Tyr Trp Cys 
1 5 

(2) INFORMATION FOR SBg ID NO: 24: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 9 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(iii) HYPOTHETICAL: YES 
(iii) ANTI-SENSE: NO 

(v) FRAGMENT TYPE: internal 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: 

Cys Gin Pro Ala His Ser Leu Gly Cys 

1 5 

(2) INFORMATION FOR SBQ ID NO: 25: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 9 amino acids 

(B) TYPE: amino acid 



wo 97/3X948 PCT/EP97/01013 



-43- 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(iii) HYPOTHETICAL: YES 
(iii) ANTI-SENSE: NO 

(v) FRAGMENT TYPE: internal 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25: 

Cys Leu Trp Gly Met Gin Gly Arg Cys 
1 5 

(2) INFORMATION FOR SEQ ID NO: 26: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 9 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(iii) HYPOTHETICAL: YES 
(iii) ANTI-SENSE: NO 

(v) FRAGMENT TYPE: internal 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: 

Cys Gly Thr Val Ser Thr Leu Ser Cys 
1 5 

(2) INFORMATION FOR SEg ID NO: 27: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 17 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(iii) HYPOTHETICAL: YES 
(iii)AOTI-SENSE: NO 

(v) FRAGMENT TYPE: internal 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: 

Gly Glu Phe Cys lie Asn His Arg Gly Tyr Trp Val Cys 
15 10 
Gly Asp Pro Ala 
15 

(2) INFORMATION FOR SEg ID NO: 28: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 10 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(iii) HYPOTHETICAL: NO 



wo 97/31948 



PCT/EP97/01013 



-44- 

(iii)ANTI.SENSE:NO 
(v) FRAGMENT TYPE: internal 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: 

Lys Thr Lys Gly Ser Gly Phe Phe Val Phe 
1 5 10 

(2) INFORMATION FOR SBQ ID NO: 29: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 9 amino acids 

(B) TYPE: amino add 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(iii) HYPOTHETICAL: YES 
(iii) ANTI-SENSE: NO 

(v) FRAGMENT TYPE: internal 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: 

lie Asn His Arg Gly Tyr Trp Val Cys 
1 5 

(2) INFORMATION FOR SEQ ID NO: 30: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 11 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(iii) HYPOTHETICAL: YES 
(iii) ANTI-SENSE: NO 

(V) FRAGMENT TYPE: internal 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30: 

Arg Ser Arg Ser Gly Gly Tyr Trp Leu Trp Cys 
15 10 

(2) INFORMATION FOR SBQ ID NO: 31: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 17 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: both 

(ii) MOLECULE TYPE: peptide 

(iii) HYPOTHETICAL: YES 
(iii) ANTI-SENSE: NO 

(v) FRAGMENT TYPE: internal 



wo 97/31948 



PCT/EP97/01013 



-45- 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: 

Gly Glu Phe Cys He Asn His Arg Gly Tyr Trp Val Cys 
^5 10 
Gly Asp Pro Ala 
15 



(2) INFORMATION FOR S£g ID NO: 32: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 18 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: both 

(ii) MOLECULE TYPE: peptide 

(iii) HYPOTHETICAL: YES 
(iii)ANTI.SENSE:NO 

(v) FRAGMENT TYPE: internal 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: 

Gly Glu Phe Cys Arg Arg His Asn Tyr Gly Phe Trp Val 
^5 10 
Cys Gly Asp Pro Ala 
15 



wo 97/31948 



PCT/EP97/01013 



-46- 

Claims 

1 . An immunogenic molecule comprising 

(a) at least one moiety of a BSW17 mimotope peptide and 

(b) a moiety capable of eliciting an inunune response against that peptide. 

2. An immunogenic molecule according to claim 1 wherein the BSW17 mimotope peptide 
is cyclic, whereby the ends are held together by two additional cystein residues forming a 
disulfide bridge, or the ends are chemically crosslinked. or wherein the BSW17 mimotope 
peptide is linear, whereby the carboxy terminal amino acid may conveniently be blocked by 
amidation and/or the amino terminal amino acid may conveniently be blocked by acetylaiion. 

3. An immunogenic molecule according to claim I in the form of a polymeric peptide or a 
recombinant fusion protein, whereby one monomeric compound of the polymeric peptide, or 
one partner of the fusion protein, constitutes the moiety of a BSW17 mimotope peptide, and the 
remainder of the polymeric peptide or fusion protein constitutes the immune response-eliciting 
moiety, 

4. An immunogenic molecule according lo claim I in the form of a conjugate of a BS W 1 7 
mimotope peptide and an immunogenic carrier. 

5. An inmiunogenic molecule according to claim 1 wherein the BSW17 mimotope peptide 
moiety essentially consists of or contains an amino acid sequence selected from 



Ue-Asn-His-Arg-Gly-Tyr-Trp-Val 


(A). 


Arg-Asn-His-Arg-Gly-Tyr-Trp-Val 


(B), 


Arg-Ser-Arg-Ser-Gly-Gly-Tyr-Trp-Leu-Trp 


(C). 


Val-Asn-Leu-Thr-Trp-Ser-Arg-Ala-Ser-Gly 


(D). 


Val-Asn-Leu-Pro-Trp-Ser-Arg-Ala-Ser-Gly 


(E). 


Val-Asn-Leu-Thr-Trp-Ser-Phe-GIy-Leu-Glu 


(F). 


Val-Asn-Leu-Pro-Trp-Ser-Phe-Gly-Leu-Glu 


(G). 


Val-Asn-Arg-Pro-Trp-Ser-Phe-Gly-Leu-Glu 


(H), 



wo 97/31948 



PCT/EP97/01013 



-41' 

Val-Lys-Leu-Pro-Trp-Arg-Phe-Tyr-Gln-Val (I), 

Va! -Trp-Thr- Ala-Cys-Gly-Tyr-Gly- Arg-Met ( j ), 

Gly-Thr-Val-Ser-Thr-Leu-Ser (K). 

Leu-Leu-Asp-Ser-Arg-Tyr-Trp (L), 

Gln-Pro-Ala-His-Ser-Leu-Gly (M), 

Leu-Trp-Gly-Met-Gln-Gly-Arg (N). 
Leu-Thr-Leu-Ser-His-Pro-His-Trp-Val-Leu-Asn-His-Phe-Val-Ser (O), 

Ser-Met-Gly-Pro-Asp-Gln-Thr-Leu-Arg (P), and 

Val-Asn-Leu-Thr-Trp-Ser (Q). 



6. A pharmaceutical composition comprising an immunogenic molecule according to any 
one of claims 1 to 5 and an adjuvant. 

7. A ligand comprising an antibody domain specific for a moiety of a BSW 1 7 mimotope 
peptide as defined in any one of claims 1 to 5, whereby the antibody domain is reactive also 
with the sequence of amino acids on the heavy chain of IgE which comprise the natural epitope 
recognized by BSW 17. 

8. A ligand according to claim 7 which is in the form of a monoclonal antibody or an 
Fab'or F(ab*)2 fragment thereof 

9. A process for the preparation of an immunogenic molecule according to claim 1 
comprising appropriately coupling (a) a moiety of a BSW 17 mimotope peptide with (b) a 
moiety capable of eliciting an immune response against that peptide. 

10. An immunogenic molecule as defined in any one of claims 1 to 5, for use as a 
pharmaceutical in the treatment of IgE-mediated diseases. 

11. Use of an immunogenic molecule as defined in any one of claims 1 to 5 in the preparation 
of a vaccine against allergy. 



wo 97/31948 



PCT/EP97/01013 



1/20 



Figure 1: Interaction between IgE and its high affinity receptor IgERI 




r X 



SUBSTITUTE SHEET (RULE 26) 



m * 



WO 97/31948 



PCT/EP97/01013 



2/20 



Figure 2: ^^ClassicaF' anti-IgE vaccine approach 




A 



. ,. . n non inhibitory 

neutral.2.ng ^ anaphylactogenic ? 



wo 97/31948 



PCT/EP97/01013 



3/20 



Figure 3: Biological activity profile of BSWI7 




1. Non-ana- 
phylactogentc 

2. Removal of 
surface IgE 

3. Neutralization 
of IgE 

4. Inhibition of 
IgE synthesis 



wo 97/31948 



PCT/EP97/01013 



4/20 



Figure 4: BSW17 mimotope-based immunotherapy 




wo 97/31948 PCT/EP97/01013 



5/20 



Figure 5: Phage-displayed BSW17 mimotopes specifically recognize BSW17 




SUBSTITUTE SHEET (RULE 26) 



wo 97/31948 PCT/EP97/010I3 



6/20 



Figure 6: Phage-displayed BSW17 mimotopes inhibit IgE / BSW17 binding 




cold competftion with lgE-SUS11 (ng/ml) 



wo 97/31948 



PCT/EP97/01013 



7/20 



Figure 7: Binding of a chemicaliy synthesized BSW17 mimotope peptide to 
BSWI7 




ooooooooooo 



tuu 0C9/00S=Wa/X3 '%^\ Jonij % 

SUBSnrUTE SHEET (RULE 26) 



wo 97/31948 



PCT/EP97/01013 



8/20 

Figure 8a: Recognition of cyclic BSW17 mimotope GEFCINHRGYWVCGDPA - KLH 
(BSS) conjugate (SDS 236) and Fee (500-509) • KLH (BSS) 
coiyugate (SDS 237) by BSW17 



-trr 






CD 


CO 




CsJ 


CM 




(0 


CD 


X 


Q 


Q 


-J 


cn 

+ 


CO 






1 



o 
■* 

D 
O 




SUBSTITUTE SHEET (RULE 26) 



wo 97/31948 



PCT/EP97/01013 



9/20 




SUBSTITUTE SHEET (RULE 26) 



wo 97/31948 



PCT/EP97/01013 



10/20 



Figure 9a: Specific recognition of BSW17 mimotope coi^fugates [BSA (DC)] and 
FcE (500-509) conjugate [KLH (glutaraldehyde)] by BSW17 



O.D.405 
1.7 - 
1.6 



P5 

PI = KTKGSGFFVF-BSA; P4 = AclNHRGYWVC-BSA ; P5 = AcRSRSGGYWLWC-BSA 




SUBSmWE SHEET (RULE 26) 



wo 97/31948 



PCT/EP97/01013 



11/20 



Figure 9b: SpeciHc recognotion of BSW17 mimotope coiuugates [KLH (DC); LysJ by 
BSW17 



O.D. 405 



1.2- 
1.1- 
1 - 
0.9- 
0.8 
0.7-h 
0.6 
0.5-h 
0.4 
0.3- 
0.2 - 
0.1 - 
0 






SAFl-KLH 




SAF2-KLH 




SAF3-KLH 


1 1 


SAF3-Lys 




SAF4-KLH 




SAF5-KLH 



n.d.n.d. iiil ^n.d 




BSW 17 



3G9 



5H5/F8 



n.d. - not determined 



SUBSTITUTE SHEET (RULE 26) 



wo 97/31948 



PCT/EP97/01013 



12/20 



Figure 10a: Anti human IgE immune response induced in rabbits after immunization 
with BSW17 mimotope conjugates (1) 




CO 
(N 
CO 

Q 

CO 




SUBSnrUTE SHEET (RULE 26) 



wo 97/31948 



PCT/EP97/010I3 



13/20 



Figure 10b: Anti human IgE immune response induced in rabbits after 
immunization with BSW17 mimotope conjugates (2) 



1 

as 
o.(, 
an 




^11 T 6 1i 15 13 it -(6 U> 

SDS 237 SAF3KLH SAF4KLH SAFIKLH SAF3Lys SDS242 



SDS243 



SUBSTITUTE SHEET (RULE 26) 



wo 97/31948 



PCT/EP97/010I3 



14/20 




SUBSmUTE SHEET (RULE 26) 




SUBSnrUTE SHEET (RULE 26) 



wo 97/31948 PCT/EP97/01013 



16/20 



Figure 12: Isotype specificity of the anti human IgE response in BSW17 mimotope' 
inununized rabbits 

OD405 

1,2 T 




R2 (SDS 214) R4 (SDS 213) 



SUBSTTTUTE SHEET (RULE 26) 



wo 97/31948 



PCT/EP97/01013 



17/20 



Figure 13: Competition of anti BSW17 mimotope serum with BSWI7 for IgE 
binding 



OD405 
0,5 



0,4 



0,3 



0,2 



0,1 



E30ugBSW17 
5ugBSWl7 




SUS-U IgE - R2 (SDS 214) serum 1:50) 



SUBSTTTUTE SHEET (RULE 26) 



wo 97/31948 



PCT/EP97/010U 



18/20 



Figure 14: Binding of affinity - purified anti BSWI7 miniotope antibodies to hIgE 
OD 40B 




M€hIgE(JW8) 



SUBSTTTUTE SHEET (RULE 26) 



wo 97/31948 



PCT/EP97/01013 



19/20 



Figure 15: Test of rabbit anti BSW17 mimotope sera and affinity-purined anti BS WIT 
mimotope antibodies for anaphylactogenicity on human biood cells 



O 
O 



n4 

CO 

1 



o 
o 

oo 



o 




< ea u 
o o o 

2 2 2 



< « 

s g g 



< 03 
o o 



2 



a 
o 



en CO 



CO 



2 2 2 





\m\ 




< 




o 


CO 


CO 


CO 


r-* 












CO 


CO 


CO 


a 


O 


a 


CO 


CO 


CO 














OS 







SUBSTITUTE SHEET (RULE 26) 



wo 97/31948 



PCT/EP97/01O13 




20/20 



O 
O 




SUBSTITUTE SHEET (RULE 26) 



INTERNATIONAL SEARCH REPORT 



Inter nal ApplicaDon No 

PCT/EP 97/01G13 



A. CLASSIFICATION OF SUBJECT MATTER 

IPC 6 C07K16/OO A61K39/395 

According to International Patent Qasafication (IPO or to both rianonal clasgficaaon and IPC 

B. FIELDS SEARCHED 

Minimum documentanon searched (dassirication system followed by classification symbols) 

IPC 6 C07K A61K 



Documentation searched other than minimum documentation to the extent that such documents are included in the Gelds searched 



Electronic data base consutud dunng the mtcmaDonal search (name of data base and, where practical, search terms used) 



C. DOCUMENTS CONSIDERED TO BE RELEVANT 



CatcKoty ' 


Citabon of document, with mdication, where appropriate, of the relevant passages 


Relevant to claim No. 


X 


wo 95 14779 A (31 RES EXPL LTD ;CELLTECH 
THERAPEUTICS LTD (GB) ; GOULD HANNAH JANE) 
1 June 1995 

see SeqID 1, pos 146 ff 
see claims; examples 


1,5.10 


X 


WO 95 20606 A (PASTEUR MERIEUX SERUMS VACC 
;HURPIN CHRISTIAN MARCEL (FR); UTOUR) 3 
August 1995 
see claims; examples 


1.5.6.11 


A 


WO 95 26365 A (UNITED BIOMEDICAL INC ;WAN6 
CHANG YI (US)) 5 October 1995 
see claims; examples 


1.10,11 


A 


WO 93 05810 A (HELLMAN LARS T) 1 April 
1993 

see claims; examples 

-/" 


1.10.11 



Further documents are listed in the continuation of box C. 



|)( [ Pitcnt family members are listed in annex. 



* Special categories of cited documents : 

'A' document definiog the general state of the art which is not 
considered to be of particular relevance 

'E' earlier document but published on or after the international 
filing dale 

'L' document which may throw doubts on priority ctaim<s) or 
which is cited to estaUtth the publication dau of another 
dtalion or other special reason (as specified) 

'O* document referring to an oral disdosure, use, exhibition or 
other mcMM 

'P* document published pnor to the international filing date taut 
later than the priority date claimed 



"T later document published after the mlcmationil filing date 
or pnon^ date and not m conflict with the application but 
atcd to urukrstand the principle or theory underlying the 
invention 

'X' document of particular relevance; the claimed invention 
caimot be considered novel 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 u combined with one or more other such docu* 
ments, such combination being obvious to a person skilled 
m the art. 

document member of the same patent family 



Date of the actual completion of the international scaKh 

9 June 1997 


Date of mailing of the intcmabonal search report 

25. 06.97 


Name and mailing address of the ISA 

European Patent Office. P.8. 5St8 Patentlaan 2 
NL - 2280 HV Riiswi}k 
Td.C-^ 31-70) 140-204O. Tx. 31 6S1 epo nl, 
Fax: ('^ 31-70)340-3016 


Authonzed officer 

Fuhr, C 



Form PCT.1SAOI0 (weoad itiMl) (iuly 



page 1 of 2 



INTERNATIONAL SEARCH REPORT 



Intet 'oai ApplicaOon No 

PCT/EP 97/01G13 



C.(Coimmiation) DOCUMKNTS CONSIDERED TO BE RELEVANT 



Category ' Citation of doctiment, with indication, where appropriate, of the relevant paiu^ 



Relevant to claim No. 



o.P, 

X 



21st Collegium Internationale 
Allergologicuin - Allergy A Disease of 
Modern Society; September 6-11, 1996, 
Salzburg. Austria 
XP002032664 

& INTERNATIONAL ARCHIVES OF ALLERGY AND 
IMMUNOLOGY. 

vol. 113, no. 1-3, May 1997 - July 1997, 
BASEL, CH. 

pages 216-218, XPe0O674655 

B.M. STADLER ET AL.: "Can Active 

Imnunization Redirect an Anti-IgE Inmune 

Response" 

see the whole document 



1-11 



Form PCT,1SA.'2lO (cxwUnMbon of teoood ihMl> (July 1993) 



page 2 of 2 



INTERNATIONAL SEARCH REPORT 

[nfomution on patent funiiy members 



Inu ofuU Applicatton No 

PCT/EP 97/01013 



Patent documcni 


Publication 




Patent family 




cited in search report 


date 




mcmber(s) 


date 


wo 9514779 A 


01-06-95 


AU 


1072395 A 


13-06-95 






EP 


0730649 A 


11-09-96 


WO 952G606 A 


03-08-95 


FR 


2715304 A 


28-07-95 






AU 


1538895 A 


15-08-95 


WO 9526365 A 


05-10-95 


AU 


2195395 A 


17-10-95 






CA 


2186595 A 


05-10-95 






CN 


1146772 A 


02-04-97 


WO 9305810 A 


01-04-93 


AU 


677573 B 


01-05-97 






AU 


2676592 A 


27-04-93 






EP 


0666760 A 


16-08-95 






FI 


941193 A 


14-03-94 






HU 


69782 A 


28-09-95 






JP 


5510768 T 


01-12-94 






NO 


941096 A 


25-03-94 






SE 


9102808 A 


27-03-93