Skip to main content

Full text of "USPTO Patents Application 09848616"

See other formats


WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




id 

ATONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



Document AL4 
Appl.No. 09/848,616 



>- 
Q. 

O 

o 

—J 

m 



/tional Patent Classification 6 : 
16/46, A61K 39/44 



Al 



(11) International Publication Number: WO 99/67293 

(43) International Publication Date: 29 December 1999 (29.12.99) 



j, / ■ 

/temational ApplicaUon Number: PCT/US99/ 13959 

/international Ffling Date: 21 June 1999 (21 .06.99) 



(30) Priority Data: 
09/100,287 



20 June 1998 (20.06.98) 



US 



(63) Related by Continuation (CON) or Continuation-in-Part 
(CIP) to Earlier Application 

US 09/100,287 (CIP) 

Filed on 20 June 1998 (20.06.98) 



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

BIOMEDICAL INC. [US/US]; 25 Davids Drive, Haup- 
pauge. NY 1 1788 (US). 

(72) Inventors; and 

(75) Inventors/Applicants (for US only): WANG, Chang, Yi 
[US/US]; 47 Snake Hill Road, Cold Spring Harbor, NY 
11724 (US). WALFIELD, Alan, M. [US/US]; 45 Schiller 
Avenue, Huntington Station, NY 11746 (US). 

(74) Agent: LIN, Maria, C, H.; Morgan & Finnegan, L.L.P., 345 
Park Avenue, New York, NY 10154 (US). 



(81) Designated States: AE, AL, AM, AT, AU, AZ, BA, BB, BG, 
BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, 
GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, 
KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, 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, 
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. 



(54) Title: PEPTIDE COMPOSITION AS IMMUNOGEN FOR THE TREATMENT OF ALLERGY 



(57) Abstract 

The invention provides peptides comprising a sequence homologous to a portion of the third constant domain of the epsilon heavy 
chain of IgE, covalently linked to either (1) a carrier protein, or (2) a helper T cell epitope, and optionally to other immunostimulatory 
sequences as well. The invention provides for the use of such peptides as immunogens to elicit the production in mammals of high titer 
polyclonal antibodies, which are specific to a target effector site on the epsilon heavy chain of IgE. The peptides are expected to be useful 
in pharmaceutical compositions, to provide an immunotherapy for IgEr-mediated allergic diseases. 



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



AL 


Albania 


ES 


Spain 


LS 


Lesotho 


SI 


Slovenia 


AM 


Armenia 


FI 


Finland 


LT 


Lithuania 


SK 


Slovakia 


AT 


Austria 


FR 


France 


LU 


Luxembourg 


SN 


Senegal 


AU 


Australia 


GA 


Gabon 


LV 


Latvia 


sz 


Swaziland 


AZ 


Azerbaijan 


GB 


United Kingdom 


MC 


Monaco 


TD 


Chad 


BA 


Bosnia and Herzegovina 


GE 


Georgia 


MD 


Republic of Moldova 


TG 


Togo 


DB 


Barbados 


GH 


Ghana 


MG 


Madagascar 


TJ 


Tajikistan 


BE 


Belgium 


GN 


Guinea 


MK 


The former Yugoslav 


TM 


Turkmenistan 


BF 


Burkina Faso 


GR 


Greece 




Republic of Macedonia 


TR 


Turkey 


BC 


Bulgaria 


IIU 


Hungary 


ML 


Mali 


TT 


Trinidad and Tobago 


BJ 


Benin 


IE 


Ireland 


MN 


Mongolia 


UA 


Ukraine 


BR 


Brazil 


IL 


Israel 


MR 


Mauritania 


UG 


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 


YU 


Yugoslavia 


CH 


Switzerland 


KG 


Kyrgyzstan 


NO 


Norway 


ZW 


Zimbabwe 


ci 


Cote d' I voire 


KP 


Democratic People's 


iNZ 


New Zealand 






CM 


Cameroon 




Republic of Korea 


PL 


Poland 






CN 


China 


KK 


Republic of Korea 


PT 


Portugal 






cu 


Cuba 


KZ 


Kazakstan 


RO 


Romania 






cz 


Czech Republic 


LC 


Saint Lucia 


RVJ 


Russian Federation 






DE 


Germany 


LI 


Liechtenstein 


SD 


Sudan 






DK 


Denmark 


LK 


Sri Lanka 


SE 


Sweden 






EE 


Estonia 


LR 


Liberia 


SG 


Singapore 







WO 99/67293 



PCT/US99/13959 



PEPTIDE COMPOSITION AS IMMUNOGEN 
FOR THE TREATMENT OF ALLERGY 

FIELD OF THE INVENTION 

5 The present invention relates to the use of 

peptide conjugate compositions as an immunogen, with each 
peptide conjugate contained therein comprising a target 
antigenic site on the third constant domain (CH3) of the 

epsilon (e) heavy chain of IgE, with said target antigenic 

10 u 

site covalently linked to (1) a carrier protein through 

chemical coupling, or (2) a helper T cell epitope and 
other immunostimulatory sequences through chemical 
coupling or through direct synthesis, for the treatment of 
15 allergy. 

More particularly, the present invention* relates 
to the use of such peptide conjugate compositions as an 
immunogen to elicit the production, in mammals including 
2 Q humans, of high titer polyclonal antibodies specific to a 
target effector site on the CH3 domain of the e heavy 
chain of IgE, and to the use of such composition as a 
pharmaceutical to provide an immunotherapy for IgE- 
mediated allergic diseases. 

25 

BACKGROUND OF THE INVENTION 

In the immune system of humans and other mammals, 
30 IgE mediates type I hypersensitivities. These are the 
allergic responses to certain foods, drugs, and 
environmental allergens which are manifested by such 
symptoms as allergic rhinitis, asthma, allergic 



35 



WO 99/67293 PCT/US99/13959 



- 2 - 

dermatitis, and anaphylaxis. Existing strategies to treat 
allergic diseases are of limited utility, consisting of 
attempts to either desensitize the atopic individual to an 
identified allergen or to ameliorate an ongoing allergic 
5 reaction with therapeutic compounds. Limitations to 
allergen-based desensitization immunotherapy include 
difficulties in identifying the allergen involved and the 
adverse reactions frequently caused by the use of the 
identified allergen (World Health Organization and 

^ International Union of Immunological Societies Working 

Group, Lancet, 1989; i:259-261). Other treatments for the 
relief of allergies employ therapeutic compounds to block 
the acute inflammatory cascade that is responsible for 

15 allergic reactions. These compounds include anti- 
histamines, decongestants, P2 agonists, and 
corticosteroids. Anti-histamines , decongestants, and p 2 
agonists act on events downstream of IgE in the allergic 
cascade, making them palliative remedies which address 
allergic symptoms rather than preventative treatments 
which must act on events closer to the initiation of IgE- 
mediated allergic reactions. These palliative remedies 
provide relief that is short term and partial, frequently 

25 accompanied by adverse side effects. Many patients with 
severe allergies are effectively treated with 
corticosteroids. Steroid therapy reduces inflammation but 
is broadly immunosuppressive. 

30 To avoid the shortcomings of the known therapeutic 

drugs, it would be more desirable to prevent allergic 
responses by selective intervention targeted to IgE. In 
common with the other immunoglobulins, IgE has two heavy 



35 



10 



WO 99/67293 PCT/US99/13959 



- 3 - 

chains and two light chains. The e heavy chain has five 
domains, a variable VH domain and constant domains CHI to 
CH4. The constant domains from both e chains of an IgE 
molecule combine to comprise the constant or Fc region of 
IgE. IgE circulates and becomes attached to effector 
cells such as basophils and mast cells through a site on 
the IgE Fc region, becoming bound to a high affinity FceRI 
receptor on the cell surface. In an allergic response, 
allergens (e.g., pollen, dust mite proteins, flea 
antigens) bind to the antigen-binding sites on the 
variable region of mast cell or basophil-bound IgE. This 
action crosslinks the IgE molecules and the underlying 
FceRI receptors. The IgE-allergen complexes thereby 
signal the degranulation of mast cells and basophils with 
the concomitant release of histamine and the other 
inflammatory mediators. These mediators produce the 
symptoms of allergy, up-regulate the production of IgE, 
20 and result in heightened sensitivity to the allergen 

(Davis et al., Springer Semin Immunopathol, 1993; 15: 51- 
73) . 

It has been suggested that allergic diseases may 
be treated by interventions which inhibit the binding of 
IgE to mast cells and basophils. For example, synthetic 
peptides corresponding to various sites on the Fc of IgE 
have been studied as competitive inhibitors for the 
binding of IgE to the FceRI receptor. The presumption of 
the investigators has been that such peptides act as 
antagonists for sites on IgE that participate in the 
binding of IgE to the FceRI receptor, and serve to map 
portions of the binding site. 



15 



25 



30 



35 



WO 99/67293 



PCT/US99/13959 



10 



15 



- 4 - 

The amino acid residues of the competitively 
inhibiting IgE peptides and of all IgE peptides to follow, 
including non-human IgE peptide homologues, are indexed in 
accordance with the numbering for human IgE given by 
Dorrington and Bennich (Immunol Rev, 1978; 41: 3-25, also 
accessible at internet location 

http : /www . pdb . bnl . gov/pdb . bin/pdbids ) . That human 
sequence is listed here as SEQ ID NO:l and is numbered as 
shown in Table 1. The homologous dog, rat and mouse 
sequences for IgE (Patel et al., Immunogenetics, 1995; 41: 
282-286; Steen et al., J Mol Biol, 1984; 177: 19-32; and, 
Ishida et al., EMBO, 1982; 1: 1117-1123) are also shown in 
Table 1 and listed as SEQ ID NOS: 2, 3, and 4 
respectively. The animal sequences are shown in register 
with human IgE. Individual amino acid positions in human 
IgE, and in homologues from other species, are identified 
herein according to the numbering system for the amino 
acid sequences shown in Table 1, unless otherwise 
specified. 

Helm et al. (Mature, 1988; 331:180-183) have shown 
that a 76 amino acid long recombinant polypeptide, 
spanning the C-terminal CH2 and N-terminal CH3 region of 
25 human IgE, from amino acids 301-376, reduces binding of 

IgE to human mast cells by competitive inhibition-. Other 
studies reported that only the CH3 domain is involved with 
binding to FccRI . For example, a rat sequence peptide 
corresponding to amino acids 401-415 of the human sequence 
(Table 1)' inhibited the binding of rat IgE to rat mast 
cells (Burt and Stanworth, Eur J Immunol, 1987; 17:437- 
440). A peptide of residues 419 to 463 from human IgE 
prevented the sensitization of rat mast cells (Nio et al., 



20 



30 



WO 99/67293 PCT/US99/13959 



- 5 - 

FEBS Lett, 1992; 314: 229-231). Jardieu and Presta (WO 
93/04173) reported on peptides homologous to the CH3 and 
CH4 regions which may include amino acids 373-390, 420- 
428, 446-453, and adjacent regions, which differentially 
5 bind to the FceRI receptor. However, high concentrations 
of all such peptides were required to achieve effective 
inhibition of IgE binding. These high concentrations are 
predictive of excessively large doses for significant 
physiological effect, and are not therapeutically 

10 

practical . 

Anti-IgE antibodies have also been applied as a 
method for mapping sites on IgE that participate in 
binding to the FceRI receptor. Studies with mouse 

15 monoclonal antibodies directed against various domains of 
IgE Fc revealed that anti-IgE monoclonal antibodies with 
specificities for the CH3 domain inhibit the binding of 
IgE to its high affinity receptor (Baniyash et al., Molec 

20 Immunol, 1988; 25: 705-711; and, Stadler et al., Immunol 
Cell Biol, 1996; 74: 195-200). These monoclonal antibody 
studies are in agreement with earlier studies that used 
polyclonal antipeptide antibodies to map sites that are 
apparently involved in receptor binding. For example, 

25 rabbit antibodies with specificities for IgE amino acid 
positions 401-415 (Burt et al., Molec Immunol, 1987; 24: 
379-389), and 355-368 (Robertson and Liu, Molec Immunol, 
1988; 25:103-113) showed specificity for unbound IgE but 

30 reacted poorly with receptor-bound IgE. 

A canine IgE peptide fragment containing at least 
five continuous amino acids from dog IgE amino acids 356- 
479 is useful for the preparation of antibodies for 



35 



WO 99/67293 PCT/US99/13959 



- 6 - 

diagnosis of allergy in dogs (JP 9179795, 1997). Those 
results are suggestive of surface-exposed effector sites 
in the CH3 domain of the dog e chain, but no such effector 
site is taught nor is a therapeutic application disclosed 
5 for the anti-IgE antibodies. 

These epitope mapping studies demonstrate most 
consistently that the CH3 domain of the e heavy chain can 
be targeted for interventions aimed at inhibiting the 

IQ binding of IgE to basophils and mast cells. However, the 
various studies are quite inconsistent on precise 
locations for sites on CH3 that are most useful. ' Also, 
results from cross-inhibition studies on IgE, with site- 
specific antibodies (e.g., Burt et al., 1987) have 

15 frequently been over-interpreted to signify that they have 
defined a precise location for the FcsRl binding site on 
the e chain. Interpretation of such cross-inhibition 
studies is limited because it cannot be assumed that an 

20 antibody recognition site is equivalent to a ligand 

binding site. Antibodies may inhibit by directly binding 
to the desired target site, but they can also occupy non- 
continuous effector sites and inhibit ligand binding 

2j through steric hindrance or induction of conformational 
change. 

Therefore, the epitope mapping studies have 
provided empirical observations but have not resolved the 
binding site for the high affinity receptor within the CH3 

OA 

domain. In the absence of a defined binding site, no 
means is available for the reliable prediction of 
potentially therapeutic synthetic immunogens with 
immunologic crossreactivities for effector sites that 



35 



10 



20 



WO 99/67293 PCT/US99/13959 



- 7 - 

participate directly or indirectly in binding to FceRl . 

Furthermore, in the absence of X-ray 
crystallography data for IgE, the available structural 
models for IgE are not sufficient for the reliable 
prediction of the sites on IgE that are suitable for anti- 
IgE interventions. Conflicting structures based on the 
divulged three-dimensional structure of IgG have been 
modeled for IgE and for the CH2/CH3 region of IgE that is 
associated with the interaction between IgE and its high 
affinity receptor. These models propose various 
conformational^ dependent structures for the site, 
involving contact with linearly non-adjacent residues of 
the IgE molecule. Some models for the site suggest 
interactions between non-contiguous sites on the same e 
chain mediated by intramolecular disulfide bonded loops 
(Helm et al., Eur J Immunol, 1991; 21:1543-1548) or 
intramolecular loops maintained by electrostatic 
interactions (Presta et al.,JBiol Chem, 1994; 269: 26368- 
26373) . Other models propose intermolecular interactions 
between segments of the dimerized e chains of an IgE 
molecule (McDonnell et al., Biochem Soc Trans, 1997; 25: 
387-392). In fact, experimental observations show that 
potential contact points comprise several scattered and 
discontinuous sites on the CH3 domain of the £ chain and 
make it clear that the three-dimensional structure of the 
FceRl binding site cannot be readily resolved by modeling 
(Helm et al., 1988; Baniyash et al., 1988; and, Presta et 
al., 1994). Therefore, the identification of useful 
synthetic peptide antagonists and immunogens that mimic 
effector sites on IgE has not been disclosed by 



.15 



25 



30 



35 



WO 99/67293 PCT/US99/139S9 



- 8 - 

theoretical modeling. In the absence of a structure for 
IgE resolved by X-ray crystallography, such useful peptide 
sites can only be arrived at by empirical experimentation. 
The concept of treating allergic diseases with 
5 anti-IgE antibodies, of specificities that inhibit the 

binding of IgE to the high affinity receptor on basophils 
and mast cells, also has been known (Stadler et al., 1996; 
Davis et al., 1993). Such anti-IgE antibodies are either 
anaphylactogenic (crosslinking) or non-anaphylactogenic 

10 (non-crosslinking) . Most such anti-IgE antibodies are 

anaphylactogenic. They will bind and crosslink IgE on the 
surface of basophils and mast cells and trigger the 
release of the pharmacologic mediators of allergy. This 

15 crosslinking could lead to anaphylaxis and death. 

It is therefore crucial that anti-IgE antibodies 
for treatment be non-anaphylactogenic. Certain non- 
anaphylactogenic antibodies retain specificity for the CH3 

20 domain of the e chain and do not crosslink cell-bound IgE, 
while displaying inhibitory activity for IgE-mediated 
histamine release (Davis et al., 1993; Stadler et al., 
1996). Rup and Kahn (U.S. 4,940,782) report such a non- 
anaphylactogenic monoclonal antibody that reacts with free 

25 rat IgE and rat IgE bound to B cells, but not IgE bound to 

the rat mast cell FceRl receptor. Most significantly, it 
inhibits the sensitization of rat mast cells. The non- 
anaphylactogenic antibodies with homologous specificities 

30 for human IgE also inhibit sensitization by the same 

action mode. These anti-human IgE antibodies bind free 
serum IgE, bind to B cell-bound IgE, fail to bind to IgE 
attached to the basophil and mast cell high affinity 



35 



WO 99/67293 PCT/US99/13959 



- 9 - 

receptor and prevent sensitization of human cells. These 
antibodies are presumed to act by specificity for the site 
on IgE that binds to the FceRl receptor (Rup and Kahn, 
U.S. 4,940,782; Davis et al., 1993; Chang, U.S. 5,420,251; 

5 Presta et al., J Immunol, 1993; 151: 2623-2632). In 

addition, a non-anaphylactogenic anti-human IgE monoclonal 
antibody with a different specificity has been found that 
also neutralizes free IgE (Rudolf et al., J Immunol, 1996; 
157: 5646-5652). This anti-IgE does not directly bind 

10 with the receptor binding site because it also recognizes 
FceRl-bound IgE. Apparently, it functions to reduce 
sensitization of basophils by altering the thermodynamic 
balance of receptor-bound versus free IgE. 

15 Thus, anti-IgE antibodies that directly bind to 

the FceRl binding site and anti-IgE antibodies that 
interfere with FceRl binding at other effector sites, both 
serve to block the sensitization of mast cells and 

20 basophils by free IgE. These potentially 

immunotherapeutic antibodies identify CH3 as the domain of 
IgE that interacts with the high affinity IgE Fc receptor, 
in agreement with the previous mapping studies. However, 
a more precise identification of the binding site and 
alternative useful effector sites such as that described 
by Rudolf et al. remain elusive. Rudolf et al. have also 
used a phage display library to identify mimotope peptides 
which bind to their anti-IgE monoclonal antibody; however, 

30 the peptide mimotopes did not show homology to the primary 
amino acid sequence of human IgE (Rudolf et al., J. 
Immunol., 1998; 160: 3315-3321). 

A humanized monoclonal anti-IgE antibody with 



35 



WO 99/67293 PCT/US99/13959 



- 10 - 

apparent specificity for the FcsRl receptor site is under 
clinical study in humans for the treatment of allergy by 
passive immunotherapy (MacGlashan et al., J Immunol, 1997; 
158:1438-1445). It has been found that infusion with that 

5 antibody, rhuMAb-E25, reduces the serum concentration of 
IgE in patients, down-regulates the expression of IgE 
receptor on effector cells, reduces allergic sensitivities 
to challenge by allergen, and improves the symptoms of 
asthma and allergic rhinitis. The antibody displays a 

10 good safety profile. The clinical trial results establish 
the feasibility of an anti-IgE approach for the treatment 
of allergic diseases. But this treatment mode is 
problematical: Immunotherapy by the anti-IgE invention is 

15 accomplished by passive immunization, i.e., by infusion of 
the antibody. The antibody must be delivered in doses 
high enough and at frequencies often enough, via 
inconvenient intravenous or subcutaneous routes, to 
achieve a continuous pharmacologically effective 

20 

concentration of antibody. The effective dose is 
determined by patient body weight, baseline level of free 
IgE in circulation, and by route of administration. In 
recent clinical trials, the steady-state concentration 
25 required for therapeutic efficacy was achieved by two 

weekly doses and maintained thereafter by biweekly doses. 
A full course of treatment for a typical allergy patient 
would expend a total of 2000-3000 mg of humanized antibody 
and requires seven to 10 inconvenient intravenous 
administrations (MacGlashan et al., 1997; Boulet et al., Am 
J Respir Crit Care Med, 1997; 155:1835-1840). The cost 
for this amount of antibody and the expense and 
inconvenience of multiple infusions in a hospital setting 



35 



WO 99/67293 PCT/US99/13959 



- 11 - 

suggest that this treatment is too expensive for all but a 
small proportion of the patient population. 

The clinical effectiveness of the monoclonal 
antibody rhuMAb-E25 establishes the feasibility of 
5 immunotherapy by passively administered anti-IgE. It also 
provides the rationale for an alternative anti-IgE 
approach by active immunization, if and when such 
immunogens can be designed. 

An anti-IgE treatment affected by active 

10 

immunization with an IgE immunogen, i.e., by "vaccination" 
against endogenous IgE, would be preferable on the basis 
of cost and convenience. "Vaccination" against IgE offers 
advantages over passive immunization: small amounts of 

15 inexpensive immunogen, infrequent and conveniently 

administered intramuscular injections, and no need to 
customize murine antibodies for compatibility with the 
subject species, i.e., to "humanize" antibodies for use in 

2Q humans, since the procedure uses the patient's own immune 
system to produce antibodies. However, while the 
desensitizing monoclonal antibodies cited above may be 
suggestive of the desirability of IgE immunogens, they do 
not disclose the identity of safe and effective 

^ immunogens. Such immunogens must mimic relevant IgE 

effector sites with fidelity sufficient to evoke cross- 
inhibitory antibodies, while retaining site-specificity 
sufficient to avoid induction of anaphylactogenic 

30 antibodies. Moreover, effective IgE immunogens must be 
highly immunostimulatory . There remains a need for such 
immunogens, of relevant and safe site-specificity, and of 
sufficient immunopotency . 



35 



WO 99/67293 



PCT/US99/13959 



- 12 - 

IgE immunogens for immunotherapy of allergy must 
be immunostimulatory so as to evoke levels of anti-IgE 
sufficient to reduce IgE-mediated sensitization. Such 
immunogens must be designed to overcome the strong 
tolerance exhibited towards self molecules. Haba and 
Nisonoff (Proc Natl Acad Sci USA, 1990; 87:3363-3367) 
induced an effective anti-IgE response in mice only by 
immunizations with IgE during a short neonatal window of 
development, from birth to day 10. Vaccinations initiated 
beyond this time failed to induce the desired autoimmune 
response unless the IgE used to immunize the mice had been 
covalently coupled to a foreign carrier protein, keyhole 
limpet hemocyanin (KLH) . Similarly, a desensitizing anti- 
IgE response was evoked in rats by a recombinant protein 
comprising the CH2-CH3 s chain domains fused to the 
glutathione-S-transferase protein of Schistosoma japonicum 
(Hellman, Eur J Immunol, 1994; 24:415-420). 

Other investigators have been concerned with 
minimizing the risk of evoking anaphylactogenic anti-IgE 
antibodies that crosslink IgE already bound to the surface 
of mast cells and basophils by seeking peptide IgE 
immunogens of finer site specificity. For example, a 
peptide corresponding to a site in the CH4 domain of IgE 
(residues 497-506 of SEQ ID N0:1) was coupled to KLH and 
used to induce polyclonal antibodies that were effective 
in suppressing IgE-mediated signal transduction in rat 
mast cells. However, the peptide-KLH conjugate displayed 
poor immunostimulatory capabilities which necessitated 
demonstration of efficacy by passive immunization of rats 
with peak immune rabbit antiserum (Stanworth et al., 
Lancet, 1990; 336:1279-1281). The CH4 immunogen of 



WO 99/67293 



PCT/US99/13959 



- 13 - 

Stanworth et al. was later produced, by the work of the 
present inventor, as a series of wholly synthetic 
immunogens by synthesis that provided covalent linkage to 
promiscuous human T helper epitopes. Immunogenicity of 

5 these peptides was improved over that of the original KLH- 
peptide conjugate, but no evidence was provided for the 
efficacy of resultant anti-IgE CH4 antibodies (Wang, WO 
95/26365) . Furthermore, as shown herein in Example 1 
(Table 2, entry 34), anti-peptide antibodies with 

10 specificity for the previously disclosed CH4 effector site 
(Stanworth et al., 1990) had no crossreactivity to human 
IgE. The earlier antipeptide studies of Burt and 
Stanworth (1987) targeted to the IgE-CH3 401-415 peptide 

15 also provided evidence of evoking desensitizing cross- 
reactivity, but this too required selected peak rabbit 
antiserum and use of an ill-defined peptide-carrier 
protein conjugate to observe effects by passive 
immunization in a rat model. No synthetic peptides have 

20 

ever been demonstrated to be effective in eliciting the 
production in immunized hosts of polyclonal antisera 
capable of inhibition of histamine release. 

The improvement of the prior art immunogens 

25 discussed above is necessary before a synthetic peptide 

immunogen of immunogenicity and specificity sufficient for 
efficacy and safety can be attained. The present 
invention accomplishes these improvements through 

3Q incorporation of a collection of additional methods for 
the identification and design of synthetic peptide 
immunogens. These methods include: (1) an effective 
procedure for the identification of an effective target 
epitope; (2) the means to augment the immunogenicity of a 

35 



WO 99/67293 



PCT/US99/13959 



- 14 - 

B cell target epitope by combining it with a peptide 
comprising broadly reactive promiscuous T helper cell (Th) 
epitope; (3) the means of enlarging the repertoire of T 
cell epitopes by application of combinatorial peptide 
5 chemistry and thereby further accommodate the variable 
immune responsiveness of an outbred population; and (4) 
the stabilization of conformational features by the 
introduction of cyclic constraints, so as to maximize 
cross-reactivity to the native molecule. 

10 Synthetic peptides have been used for "epitope 

mapping" to identify immunodominant determinants or 
epitopes on the surface of proteins, for the development 
of new vaccines and diagnostics. Epitope mapping employs 

15 a series of overlapping peptides corresponding to regions 

on the protein of interest to identify sites which 

participate in antibody-immunogenic determinant 

interaction. Most commonly, epitope mapping employs 

peptides of relatively short length to precisely detect 
20 _ 

linear determinants. A fast method of epitope mapping 

known under the trademark U PEPSCAN" is based on the 

simultaneous synthesis of hundreds of overlapping 

peptides, of lengths of 8 to 14 amino acids, coupled to 

25 solid supports. The coupled peptides are tested for their 
ability to bind antibodies. The PEPSCAN approach" is 
effective in localizing linear determinants, but not for 
the identification of epitopes needed for mimicry of 

3 q discontinuous effector sites such as the FcsRl binding 

site (Meloen et al., Ann Biol Clin, 1991; 49:231-242). An 
alternative method relies on a set of nested and 
overlapping peptides of multiple lengths ranging from 15 
to 60 residues. These longer peptides can be reliably 



WO 99/67293 



PCT/US99/13959 



- 15 - 

synthesized by a laborious series of independent solid- 
phase peptide syntheses, rather than by the rapid and 
simultaneous PEPSCAN syntheses. The resulting set of long 
nested and overlapping peptides can then be used for 
5 analyses of antibody binding in systems such as 

experimental immunizations and natural infections, to 
identify long peptides which best present immunodominant 
determinants, including simple discontinuous epitopes . 
This method is exemplified by the studies of Wang for the 

^ mapping of immunodominant sites from HTLV I/II (US 

5,476,765) and HCV (US 5,106,726); and it was used for the 
selection of a precise position on the gpl20 sequence for 
optimum presentation of an HIV neutralizing epitope (Wang 

15 et al. f Science, 1991; 254:285-288). 

Peptide immunogens are generally more flexible 
than proteins and tend not to retain any preferred 
structure. Therefore it is useful to stabilize a peptide 

2Q immunogen by the introduction of cyclic constraints. A 
correctly cyclized peptide immunogen can mimic and 
preserve the conformation of a targeted epitope and 
thereby evoke antibodies with cross-reactivities for that 
site on the authentic molecule (Moore, Chapter 2 in 

25 Synthetic Peptides: A User's Guide, ed Grant, WH Freeman 
and Company: New York, 1992, pp 63-67). 

Another important factor affecting the 
immunogenicity of an IgE-derived peptide for an allergy 

30 pharmaceutical is its presentation to the immune system by 
T helper cell epitopes that react with a host's T-helper 
cell receptors and Class II MHC molecules (Babbitt et al., 
Nature, 1985; 317: 359-361). These are often provided by 
carrier proteins with concomitant disadvantages due to the 

35 



WO 99/67293 



PCT/US99/13959 



- 16 - 

difficulties for the manufacture of well-defined peptide- 
carrier conjugates, misdirection of most antibody response 
to the carrier, and carrier-induced epitopic suppression 
(Cease, Intern Rev Immunol. , 1990; 7: 85-107; Schutze et 
5 al., J Immunol., 1985; 135: 2319-2322). Alternatively, T- 
helper cell epitopes (Th) may also be supplied by 
synthetic peptides comprising Th sites. Thus, Th epitopes 
termed promiscuous Th evoke efficient T cell help and can 
be combined with synthetic B cell epitopes that by 

^ themselves are poorly immunogenic to generate potent 
peptide immunogens (US 5,759,551). Well-designed 
promiscuous Th/B cell epitope chimeric peptides are 
capable of eliciting Th responses and resultant antibody 

15 responses in most members of a genetically diverse 

population expressing diverse MHC haplotypes. Promiscuous 
Th can be provided by specific sequences derived from 
potent foreign antigens, such as for example measles virus 
F protein, hepatitis B virus surface antigen, and 

on 

Chlamydia trachomatis major outer membrane protein (MOMP) . 
Many known promiscuous Th, taken from viral and bacterial 
pathogens, have been shown to be effective in potentiating 
a poorly immunogenic peptide corresponding to the 

25 decapeptide hormone LHRH (US 5,759,551) 

Promiscuous Th epitopes derived from foreign 
pathogens may include, but are not limited to, hepatitis B 
surface and core antigen helper T cell epitopes (HB S Th 

3Q and HB C Th) , pertussis toxin helper T cell epitopes (PT 

Th), tetanus toxin helper T cell epitopes (TT. Th) , measles 
virus F protein helper T cell epitopes (MV F Th) , Chlamydia 
trachomatis major outer membrane protein helper T cell 
epitopes (CT Th) , diphtheria toxin helper T cell epitopes 



WO 99/67293 



PCT/US99/139S9 



- 17 - 

(DT Th) , Plasmodium falciparum circumsporozoite helper T 
cell epitopes (PF Th) , Schistosoma mansoni triose 
phosphate isomerase helper T cell epitopes (SM Th), and 
Escherichia coli TraT helper T cell epitopes (TraT Th) . 
The pathogen-derived Th were listed as SEQ ID NOS:2-9 and 
42-52 in US 5,759,551; as Chlamydia helper site Pll in 
Stagg et al. t Immunology, 1993; 79;l-9; and as HBc peptide 
50-69 in Ferrari et al., J Clin Invest, 1991; 88: 214-222. 

Promiscuous Th epitopes range in size from about 
15 to about 50 amino acid residues in length (US 
5,759,551) and often share common structural features and 
may contain specific landmark sequences. For example, a 
common feature is amphipathic helices, which are alpha- 
helical structures with hydrophobic amino acid residues 
dominating one face of the helix and with charged and 
polar resides dominating the surrounding faces (Cease et 
al., Proc Natl Acad Sci USA, 1987; 84:4249-4253). Th 
epitopes frequently contain additional primary amino acid 
patterns such as a Gly or charged residue followed by two 
to three hydrophobic residues, followed in turn by a 
charged or polar residue. This pattern defines what are 
called Rothbard sequences. Also, Th epitopes often obey 
the 1, 4, 5, 8 rule, where a positively charged residue is 
followed by hydrophobic residues at the fourth, fifth and 
eighth positions after the charged residue, consistent 
with an amphipathic helix having positions 1, 4, 5, and 8 
located on the same face. Since all of these structures 
are composed of common hydrophobic, charged and polar 
amino acids, each structure can exist simultaneously 
within a single Th epitope (Partidos et al., J Gen Virol, 
1991; 72:1293-1299). Most, if not all, of the promiscuous 



WO 99/67293 



PCT/US99/13959 



10 



- 18 - 

T cell epitopes fit at least one of the periodicities 
described above. These features may be incorporated into 
the designs of ''idealized artificial Th sites". 

Useful Th sites may also include combinatorial Th 
that incorporate selected degenerate sites into the design 
of the idealized Th sites. In Wang et al. (WO 95/11998), a 
particular class of a combinatorial epitope was designated 
as a "Structured Synthetic Antigen Library" or SSAL. A Th 
constructed as an SSAL epitope is composed of positional 
substitutions organized around a structural framework of 
invariant residues. The sequence of the SSAL is 
determined by aligning the primary amino acid sequence of 
a promiscuous Th, retaining relatively invariant residues 
15 at positions responsible for the unique structure of the 
Th peptide and providing degeneracy at the positions 
associated with recognition of the diverse MHC restriction 
elements. Lists of variable and preferred amino acids are 
available for MHC-binding motifs (Meister et al., Vaccine, 
1995; 13: 581-591; Alexander et al., Immunity, 1994, 
1:751-761) . 

All members of the SSAL are produced 
simultaneously in a single solid-phase peptide synthesis 
in tandem with the targeted B cell epitope and other 
sequences. The Th library sequence maintains the binding 
motifs of a promiscuous Th and accommodates reactivity to 
a wider range of haplotypes. For example, the degenerate 
30 Th epitope described in WO 95/11998 as "SSAL1TH1" was 
modeled after a promiscuous epitope taken from the F 
protein of measles virus (Partidos et al., 1991). 
SSAL1TH1 was designed to be used in tandem with an LHRH 
target peptide. Like the measles epitope, SSAL1TH1 



20 



25 



WO 99/67293 



PCT/US99/I3959 



- 19 - 

follows the Rothbard sequence and the 1, 4, 5, 8 rule: 

1 5 10 15 

Asp-Leu-Ser-Asp-Leu-Lys-Gly-Leu-Leu-Leu-His-Lys-Leu-Asp-Gly- 

Leu 

. 5 Glu He Glu He Arg He He He Arg He Glu 

He 

Val Val Val Val Val Val Val 
Phe Phe Phe Phe Phe Phe Phe 
Charged residues Glu or Asp are added at position 
1 to increase the charge surrounding the hydrophobic face 
of the Th. The hydrophobic face of the amphipathic helix 
is then maintained by hydrophobic residues at 2, 5, 8, 9, 
10, 13 and 16, with variability at 2, 5, 8, 9, 10, 13, and 

15 16 to provide a facade with the capability of binding to a 
wide range of MHC restriction elements. The net effect of 
the SSAL feature is to enlarge the range of immune 
responsiveness to an artificial Th (WO 95/11998) . 

20 Peptide immunogens that have been designed with 

the peptide technologies and peptide design elements 
discussed above, i.e., precise epitope mapping, cyclic 
constraint, and the incorporation of promiscuous Th 
epitopes or idealized promiscuous Th, and idealized SSAL 

25 Th epitopes, are the basis for the effective synthetic 
peptide IgE immunogens of the present invention. Such 
peptides are preferred for appropriate targeting and 
safety due to effective presentation of the IgE effector 

30 site by optimized positioning and cyclization, and for 

immunopotency due to broadly reactive Th responsiveness. 



WO 99/67293 



PCT/US99/13959 



- 20 - 

SUMMARY OF THE INVENTION 

The present invention provides new synthetic 
peptide conjugate compositions for the treatment of IgE- 
mediated allergic diseases by active immunization. The 
immunization induces the production of high titer non- 
anaphylactogenic polyclonal antibodies specific to an 
effector site of IgE in an immunized host. This in turn 
prevents the triggering and activation of mast 
cells/basophils and down-regulates IgE synthesis. 

Treatment is effected by immunization of the host 
with the peptide composition, with each peptide contained 
therein comprising a target antigenic peptide sequence 
(referred to herein as an "IgE-CH3 domain antigen" or 
"IgE-CH3 domain antigen peptide") modified from a segment 
of the CH3 domain of the epsilon (e) heavy chain of human 
IgE (e.g., amino acids 413-435 of SEQ ID No : 1 or SEQ ID 
NO: 5) or the homologous sequence from other species (e.g. 
SEQ ID NOS: 6-8 and 84) . 

In general, the IgE-CH3 domain antigen is a 
peptide sequence between about 25 and about 29 amino acids 
in length, is substantially homologous to the above 
segment of the CH3 domain of the epsilon heavy chain of a 
mammalian IgE antibody, and contains two cysteine' residues 
separated by about 23 amino acid residues. In the present 
context, substantially homologous means that in addition 
to the two cysteine residues, which may be introduced by 
insertion or substitution, up to about four other amino 
acid substitutions (preferably conservative substitutions) 
may also be made. 

Preferably, the target site is modified from that 



WO 99/67293 PCT/US99/13959 



- 21 - 

of the naturally occurring IgE sequences as follows: 

(1) by the insertion of a cysteine residue to the 
N-terminus side of position 413 or homologous position, 
unless cysteine is already present at positions 413 or 414 

5 in the natural sequence; 

(2) by the conservative substitution (preferably 
of serine) for any native cysteines from positions 415 to 
434 of the natural target sequence; 

jq (3) by the insertion of cysteine at the C-terminus 

side of position 435 or homologous position unless 
cysteine is already present at positions 435 or 436 in the 
natural sequence; and 

(4) by the formation of a disulfide bond between 

^ the retained cysteines so as to produce a cyclic 

structure. The structures may also comprise 1 to 5 
additional amino acids taken from either terminus of the 
413-435 segment of IgE, provided that the single disulfide 

20 looped structure is preserved. 

An optimized IgE-CH3 domain antigen peptide for 
human IgE, having the sequence Cys-Gly-Glu-Thr-Tyr-Gln- 
Ser-Arg-Val-Thr-His-Pro-His-Leu-Pro-Arg-Ala-Leu-Met-Arg- 

2 5 Ser-Thr-Thr-Lys-Cys (SEQ ID NO: 5) is provided by the 

present invention. The human IgE target site is cyclized 
through the unnatural terminal cysteines and a serine 
residue substitutes for the cysteine residue of the 
natural sequence. Antibody that is evoked by peptide 

30 immunogens comprising this IgE-CH3 domain antigen is 
crossreactive with human IgE and inhibits the 
sensitization of human basophils by human IgE. 

Likewise, corresponding target sites for IgE of 



35 



WO 99/67293 PCT/US99/13959 



- 22 - 

O 

other species can be derived from the homologous e chain 
segment of the relevant species. For example, such target 
sequences can be taken from the dog, rat and mouse e 
sequences shown in Table 1 (SEQ ID NOS: 2, 3 and 4), or 
5 the horse IgE-CH3 sequence provided by Navarro et al., 

Molec. Immunol., 1995, 32:1-8. Additional IgE-CH3 domain 
antigen peptides (SEQ ID NOS: 6, 7, 8, and 84), may be 
derived from these sequences. 

10 Preferably, the IgE-CH3 domain antigens of the 

invention are rendered more immunogenic via covalent 
linkage to a carrier protein through chemical coupling, or 
more preferably via covalent linkage to synthetic 
immunostimulatory elements, such as promiscuous Th 

* 5 epitopes, through direct synthesis. Specific examples of 
carrier protein and immunostimulatory elements are 
provided, e.g., Keyhole Limpet Hemocyanin (KLH) carrier, 
an artificial Th (SEQ ID N0:9), artificial SSAL Th (SEQ ID 

20 NOS: 10 and 11), a pathogen-derived Th (SEQ ID NO: 12), and 
an immunostimulatory invasin peptide (Inv) taken from 
Yersinia (SEQ ID NO: 13). 

Completely synthetic peptide conjugates of the 

2j invention may be represented by the formulas: 

(A) n - (IgE-CH3 domain antigen) - (B) 0 _ (Th) m -X. 

or 

(A) n -(Th) m -(B) 0 -(IgE-CH3 domain antigen)-X 

30 

(A) n -(B)o-(Th) m -(B)o-(IgE-CH3 domain antigen)-X 

or 

(IgE-CH3 domain antigen) - (B) Q - (Th) m - (A) n -X 



35 



WO 99/67293 PCT/US99/13959 



- 23 - 

O 

or 

(Th)m- (B)o- (IgE-CH3 domain antigen) - (A) n ~X 
wherein 

each A is independently an amino acid or a general 
^ immunostimulatory sequence; 

each B is chosen from the group consisting of amino 
acids, 

-NHCH (X) CH 2 SCH 2 CO-, -NHCH (X) CH 2 SCH 2 CO (s-N) Lys-, 
10 -NHCH (X) CH 2 S-succinimidyl (s-N) Lys-, and 

-NHCH (X) CH 2 S- (succinimidyl) -; 
each Th is independently a sequence of amino acids 

that constitutes a helper T cell epitope, or an 
15 immune enhancing analog or segment thereof; 

IgE-CH3 domain antigen is a peptide between about 25 
and about 29 amino acids in length, is 
substantially homologous to one of the segments 

represented by SEQ ID NOS:5-8 and 84 of the CH3 
20 ^ 

domain of the epsilon heavy chain of a mammalian 

IgE antibody, and contains two cysteine residues 

separated by about 23 amino acid residues; 

X is an amino acid a-COOH or a-C0NH 2 ; 

25 

n is from 0 to about 10; 

m is from 1 to about 4; and 

o is from 0 to about 10. 

More specifically, IgE-CH3 domain antigen is selected 
30 from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ 
ID NO: 7, SEQ ID NO: 8, homologous sequences from the 
epsilon heavy chain of mammalian IgE-CH3 antibodies, and 
crossreactive and immunologically functional analogs 



35 



WO 99/67293 PCT/US99/13959 



- 24 - 

thereof. 

The peptide compositions of the present invention 
comprises peptide immunogens from about 25 to about 100 
amino acid residues, preferably from about 25 to about 80 
5 amino acid residues and more preferably from about 45 to 

about 65 amino acid residues. 

Also provided are adjuvants and/or delivery 
vehicles and other ingredients routinely incorporated with 
vaccine formulations, and instructions for dosage such 

10 that immunotherapeutic antibodies directed against the 

targeted IgE effector site are generated. This in turn 
inhibits the sensitization by circulatory IgE of basophils 
and mast cells, and thereby prevents the triggering and 

15 activation of mast cells/basophils by IgE-allergen 

complexes. The inhibitory mechanism, mediated by the 
antibodies and induced by the peptide composition of the 
present invention, will specifically reduce or eliminate 
the IqE-mediated pathology while leaving the defensive 

20 

components of the immune system, e.g. IgG, unaffected. 

DETAILED DESCRIPTION OF THE INVENTION 

25 This invention is directed to novel peptide and 

peptide conjugate compositions for the generation of high 
titer polyclonal antibodies with specificity for a target 
effector site on the third domain of the Fc portion of 

30 IgE, i.e., the CH3 domain of the e chain. 

For convenience, the term "peptide conjugate" as 
used herein refers to molecules which comprise Th epitopes 
covalently linked to IgE-CH3 domain antigen peptides, 



35 



WO 99/67293 



PCT/US99/13959 



- 25 - 

0 whether through conventional peptide bonds so as to form a 
single larger peptide, or through other forms of covalent 
linkage. 

The high site-specificity of the compositions of 
5 this invention minimizes the generation of anti-IgE 

antibodies that can crosslink the bivalent IgE bound to 
FceRI on the basophil/mast cell surface, and thereby evoke 
the production of non-anaphylactogenic anti-IgE 
antibodies. Therefore, the invention is further directed 

10 to a safe method for the treatment of IgE-mediated 

allergic diseases in mammals, including humans. 

The targeted antigenic sequence was determined by 
a thorough screening of candidate sites on the CH2 and CH3 

15 domains of human IgE for useful immunoreactivities . CH2 

and CH3 sites were selected for synthesis as peptide 
immunogens based on the disclosures by Helm et al. (1988) 
and Presta et al. (1994) that a long region which begins 

2 q in the carboxyl terminus region of the CH2 domain of IgE 

and proceeds through the CH3 domain contains potential 
effector sites. Potential loop structures in the 
conformation of IgE were deduced from a theoretical model 
for the three dimensional structure of human IgE made 

25 available by the Brookhaven National Laboratory at 

internet address http://www.pdb.bnl.gov/pdb.bin/pdbids and 
reported in Helm et al. (Eur J Immunol, 1991; 21: 1543- 
154 8) . Disulf ide-bonded loops were incorporated into the 

30 design of selected peptide immunogens so as to mimic the 

positions of predicted loops, so as to maximize the 
possibility of crossreactivity between the designed target 
antigenic peptides and the native IgE molecule. Potential 
target antigenic sites were synthesized and made 



WO 99/67293 



PCT/US99/13959 



- 26 - 

° immunogenic either by chemical conjugation to KLH 

following solid-phase peptide synthesis, or by covalent 
attachment to promiscuous Th epitopes and other 
immunostimulatory sequences by continuous synthesis (Table 
^ 2) . Several sites were synthesized as cyclic peptides, 

with the incorporation of specific disulfide bonds, so as 
to stabilize the mobile peptides into conformations that 
resemble predicted IgE loop structures. Potentially 
useful effector target sites were then identified by the 

^ preparation of hyperimmune sera and testing of the 

antiserum for crossreactivity to human IgE. Antibodies 
from sera with high crossreactivity to human IgE were 
purified and evaluated for ability to inhibit the IgE- 

l5 mediated sensitization of human basophils in an in vitro 

assay for histamine release. Anti-peptide antibodies 
evoked by peptides, SEQ ID NOS: 14 and 15 comprising SEQ 
ID NO: 5, displayed strong crossreactivity for IgE (Table 
2), and most consistently displayed high inhibitory 

20 activity in the histamine release assay (Table 3) . The 

target epitope common to the peptides of SEQ ID NOS: 14 and 
15 corresponds to a segment of the IgE CH3 domain shown in 
Table 1. Table 1 shows the amino acid sequence of CH2, 

25 CH3 and CH4 domains of the e heavy chain of the human IgE 

aligned with the homologous sequences taken from the dog, 
rat, and mouse. The target site on the human e chain 
sequence that was determined to be useful for 

30 representation as the IgE-CH3 domain antigens of the 

invention is underlined in Table 1 and includes human e 
chain residues 413-435. Homologous target sequences in 
the dog, rat, and mouse proteins are also underlined in 
Table 1. The homologous sequence in the horse is residues 



WO 99/67293 



PCT/US99/13959 



- 27 - 

° 296-318 in the amino acid sequence of Navarro et al., 

Molec. Immunol., 1995, 32:1-8. 

The underlined target IgE CH3 effector sites, and 
the derived IgE-CH3 domain antigen peptides of this 
5 invention, are short peptide sequences which, when 

synthesized by themselves, are usually weakly or non- 
immunogenic, more so for being self -antigens . These short 
peptides can be immunopotentiated by chemically coupling 
to a carrier protein, for example, keyhole limpet 

10 hemocyanin (KLH) . A disadvantage of such "IgE-CH3 domain 

antigen-carrier protein" based immunogens is the weak 
immunogenicity of the antigen compared to the large 
carrier protein, an inherent problem associated with 

15 peptide-carrier protein conjugates. The majority of 

antibodies generated by such a conjugate are non- 
functional antibodies directed against the carrier 
protein. The preferred immunogens of the present 
invention are wholly synthetic peptides which minimize the 

20 

generation of irrelevant antibodies, and thereby elicit 
immune responses more focused to the target IgE-CH3 domain 
antigens, e.g., SEQ ID NOS:5-8 and 84. 

However, because the short IgE-CH3 domain antigen 

25 peptides of the present invention (e.g., SEQ ID NOS:5-8 

and 84) are non-immunogenic T cell-dependent epitopes, 
they are dependent for immunogenicity on extrinsic Th 
epitopes. These are provided for the preferred peptides 

3 q of the invention as covalently linked promiscuous Th 

epitopes. The immunogens of the invention elicit site- 
specific immunoreactivity to provide precise targeting of 
the effector site and thus produce non-crosslinking anti- 
IgE antibodies. The resultant site-specific antibodies 



WO 99/67293 PCT/US99/13959 



- 28 - 

inhibit sensitization and allergic response but do not 
induce spontaneous degranulation. 

Specific examples are provided in the present 
invention as embodiments of the immunogenic peptide 
conjugates of the invention. These examples provide for 
the linkage of synthetic immunostimulatory elements to 
IgE-CH3 domain antigen peptides (e.g., SEQ ID NOS:5-8 and 
84) such that potent crossreactive antibodies are broadly 
generated, in a genetically diverse host population, 
against the targeted site on the IgE CH3 domain. These 
anti-IgE antibodies are non-anaphylactogenic and 
specifically directed against IgE (Examples 2 and 3) . 
These antibodies, in turn, lead to inhibition of histamine 
release and diminished IgE-mediated responses, thus 
resulting in effective treatment and/or prevention of IgE- 
mediated allergic diseases. 

For active immunization, the term "immunogen" 
referred to herein relates to a peptide conjugate 
composition which is capable of inducing antibodies 
against an effector site present on the third domain of 
the e-heavy chain of IgE (e.g., SEQ ID NOS:5-8 and 84), 
leading to inhibition or suppression of IgE-mediated 
basophil and mast cell degranulation. The peptide 
compositions of the present invention include IgE-CH3 
domain antigen peptides, preferably linked to carrier 
proteins via chemical coupling, more preferably IgE-CH3 
domain antigen peptides linked to promiscuous helper T 
cell epitopes (Th epitopes) via chemical coupling, and 
most preferably wholly synthetic peptides which contain 
IgE-CH3 domain antigen sequences and promiscuous helper T 
cell epitope (Th epitope) sequences. 



WO 99/67293 PCT/US99/13959 



- 29 - 

° The carrier proteins are covalently attached to 

the IgE-CH3 domain antigen peptides, preferably with a 
spacer (e.g., Lys-Lys-Lys) , via chemical coupling. The Th 
peptides (e.g., SEQ ID NOS:9-12) are covalently attached 
to the IgE-CH3 domain antigen peptides (e.g., SEQ ID 
NOS:5-8 and 84) either via chemical coupling or preferably 
via direct synthesis, preferably with a spacer (e.g., Gly- 
Gly) , so as to be adjacent to either the N- or Oterminus 
of the IgE-CH3 domain antigen sequences, in order to evoke 

10 efficient antibody responses. The immunogen optionally 

may also comprise a general immunostimulatory amino acid 
sequence, for example one corresponding to a domain of an 
invasin protein from the bacteria yersinia spp (Brett et 

15 al. f Eur J Immunol, 1993, 23: 1608-1614) (SEQ ID NO:13). 

The general immunostimulatory sequence may comprise an 
optional spacer through which it is attached to a Th 
peptide. 

The completely synthetic peptides of this 

20 

invention can be represented by the formulas: 

(A) n - (IgE-CH3 domain antigen) - (B) 0 ~ (Th) m -X 

or 

(A) n -(Th) n -(B) 0 -(IgE-CH3 domain antigen)-X 

25 

or 

(A) n -(B)o-(Th) m -(B)o-(IgE-CH3 domain antigen) -X 

or 

(IgE-CH3 domain antigen) - (B) 0 _ (Th) m - (A) n -X 
(Th)m- (B)o- (IgE-CH3 domain antigen) - (A) n -X 

wherein 

each A is independently an amino acid or a general 



35 



WO 99/67293 



PCT/US99/13959 



10 



15 



- 30 - 

immunostimulatory sequence; 

each B is chosen from the group consisting of amino acids, 
-NHCH (X) CH 2 SCH 2 CO-, -NHCH (X) CH 2 SCH 2 CO (e-N) Lys-, 
-NHCH (X) CH 2 S-succinimidyl (e-N) Lys-, and -NHCH (X) CH 2 S- 
( succinimidy 1 ) - ; 

each Th is independently a sequence of amino 
acids that constitutes a helper T cell epitope, or an 
immune enhancing analog or segment thereof; 

IgE-CH3 domain antigen represents the sequence of 
an IgE-CH3 domain antigen peptide as defined herein (or a 
crossreactive and immunologically functional analog 
thereof) ; 

n is from 0 to about 10; 
m is from 1 to about 4; and 
o is from 0 to about 10. 

The peptide immunogen of the present invention 
comprises from about 25 to about 100 amino acid residues, 
preferably from about 25 to about 80 amino acid residues 
and more preferably from about 25 to about 65 amino acid 
residues . 

When A is an amino acid, it can be any non- 
naturally occurring or any naturally occurring amino acid. 
Non-naturally occurring amino acids include, but are not 
limited to, D-a-amino acids, ft-alanine, ornithine, 
norleucine, norvaline, hydroxyproline, thyroxine, y-amino 
30 butyric acid, homoserine, citrulline and the like. 

Naturally-occurring amino acids include alanine, arginine, 
asparagine, aspartic acid, cysteine, glutamic acid, 
glutamine, glycine, histidine, isoleucine, leucine, 

35 



20 



25 



WO 99/67293 



PCT/US99/13959 



- 31 - 

° lysine, methionine, phenylalanine, proline, serine, 

threonine, tryptophan, tyrosine and valine. Moreover, 
when n is greater than one, and two or more of the A 
groups are amino acids, then each amino acid may be 
^ independently the same or different. 

When A is an invasin domain, it is an immune 
stimulatory epitope from the invasin protein of a Yersinia 
species. This immune stimulatory property results from 
the capability of this invasin domain to interact with the 

10 (Jl integrin molecules present on T cells, particularly 

activated immune or memory T cells. The specific sequence 
for an invasin domain found to interact with the pi 
integrins has been described by Brett et al. {Eur J 

15 Immunol, 1993) . A preferred embodiment of the invasin 

domain (Inv) for linkage to a promiscuous Th epitope has 
been previously described in US 5,759,551 which is 
incorporated herein by reference. The Inv domain has the 

2Q sequence Thr-Ala-Lys-Ser-Lys-Lys-Phe-Pro-Ser-Tyr-Thr-Ala- 

Thr-Tyr-Gln-Phe (SEQ ID NO: 13) or is an immune stimulatory 
homologue thereof from the corresponding region in another 
Yersinia species invasin protein. Such homologues thus 
may contain substitutions, deletions or insertions of 

^ amino acid residues to accommodate bacterial strain 

variation, provided that the homologues retain immune 
stimulatory properties. An immune stimulatory homologue 
may also comprise an optional spacer through which it is 

30 attached to a Th epitope. 

In one embodiment, n is 3 and (A) 3 is an invasin 
domain (Inv), glycine and glycine, in that order. 

(B) 0 is an optional spacer and comprises amino 



35 



WO 99/67293 PCT/US99/13959 



- 32 - 

acids which can be naturally occurring or the non- 
naturally occurring amino acids as described above. Each 
B is independently the same or different. The carrier 
proteins are covalently attached to the peptides with a 
spacer (e.g., Lys-Lys-Lys) via chemical coupling. The 
amino acids of B can also provide a spacer, e.g., Gly-Gly 
or (D-N)Lys, between the promiscuous Th epitope (e.g., SEQ 
ID NO: 9) and the IgE-CH3 peptide (e.g., SEQ ID NO: 5) and 
crossreactive and functional immunological analogs 
thereof. In addition to physically separating the Th 
epitope from the B cell epitope, i.e., the IgE-CH3 peptide 
(e.g., SEQ ID NO: 5) and immunological analogs thereof, the 
spacer can disrupt any artifactual secondary structures 
created by the joining of the Th epitope with the IgE-CH3 
peptide (e.g., SEQ ID NO: 5) and crossreactive and 
functional immunological analogs thereof and thereby 
eliminate interference between the Th and/or B cell 
responses. The amino acids of B can also form a spacer 
which acts as a flexible hinge that enhances separation of 
the Th and IgE domains. Examples of sequences encoding 
flexible hinges are found in the immunoglobulin heavy 
chain hinge region. Flexible hinge sequences are often 
proline rich. One particularly useful flexible hinge is 
provided by the sequence Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID 
NO: 16), where Xaa is any amino acid, and preferably 
aspartic acid. The conformational separation provided by 
the amino acids of B permits more efficient interactions 
between the presented peptide immunogen and the 
appropriate Th cells and B cells and thus enhances the 
immune responses to the Th epitope and the antibody- 
eliciting epitope and their crossreactive and functional 



35 



WO 99/67293 



PCT/US99/13959 



- 33 - 

° immunological analogs thereof. 

Th is a sequence of amino acids (natural or non- 
natural amino acids) that comprises a Th epitope. A Th 
epitope can consist of a continuous or discontinuous 

5 epitope. Hence not every amino acid of Th is necessarily 

part of the epitope. Accordingly, Th epitopes, including 
analogs and segments of Th epitopes, are capable of 
enhancing or stimulating an immune response to the IgE-CH3 
antigen peptides (e.g., SEQ ID NOS:5-8 and 84, and 
10 immunological analogs thereof) . Th epitopes that are 

immunodominant and promiscuous are highly and broadly 
reactive in animal and human populations with widely 
divergent MHC types (Partidos et al., 1991; US 5,759,551). 
15 The Th domain of the subject peptides has from about 10 to 

about 50 amino acids and preferably from about 10 to about 
30 amino acids. When multiple Th epitopes are present 
(i.e. m > 2), then each Th epitope is independently the 
same or different. Th segments are contiguous portions of 

20 

a Th epitope that are sufficient to enhance or stimulate 
an immune response to the IgE-CH3 peptide (e.g., SEQ ID 
NO: 5) and immunological analogs thereof. 

Th epitopes of the present invention include as 
examples, but are not limited to, pathogen-derived 
hepatitis B surface and core antigen helper T cell 
epitopes (HBs Th and HBc Th) , pertussis toxin helper T 
cell epitopes (PT Th), tetanus toxin helper T cell 
30 epitopes (TT Th) , measles virus F protein helper T cell 

epitopes (MVF Th) , Chlamydia trachomatis major outer 
membrane protein helper T cell epitopes (CT Th) , 
diphtheria toxin helper T cell epitopes (DT Th) , 
Plasmodium falciparum circumsporozoite helper T cell 

35 



WO 99/67293 PCT/US99/13959 



- 34 - 

° epitopes (PF Th) , Schistosoma mansoni triose phosphate 

isomerase helper T cell epitopes (SM Th) , and Escherichia 
coli TraT helper T cell epitopes (TraT Th) . The pathogen- 
derived Th were listed as SEQ ID NOS:2-9 and SEQ ID 
5 NOS: 42-52 in US 5,759,551; as Chlamydia helper site Pll in 

Stagg et al., Immunology, 1993; 79:1-9 (also listed here 
as SEQ ID N0:12); and as HBc peptide 50-69 in Ferrari et 
al., J Clin Invest, 1991; 88: 214-222, and are 
incorporated herein by reference. 

10 Exemplary Th sites of the invention also include 

the artificial Th site termed "Syn Th (1,2,4)" (SEQ ID 
N0:9), artificial SSAL Th sites "(1,4,9 PALINDROMIC) Th", 
M IS (1,4,9 PALINDROMIC) LF Th" and "IS (1, 4, 9 

15 PALINDROMIC) LF simplified Th" (SEQ ID NOS:10, 11 and 60), 

and immunologically functional analogs thereof. 
Functional Th analogs include immune-enhancing analogs, 
crossreactive analogs and segments of any of these Th 
epitopes. Functional Th analogs further include 

20 

conservative substitutions, additions, deletions and 
insertions of from one to about 10 amino acid residues in 
the Th epitope which do not essentially modify the Th- 
stimulating function of the Th epitope. 
25 The synthetic peptide of this invention are 

generally about 50 to about 90 amino acids, and comprise 

(a) an immunostimulatory invasin domain, 

(b) a helper T cell (Th) epitope, and 
30 ( C) an igE-CH3 domain antigen peptide. 

More specifically, the synthetic peptides of this 
invention are described by the formulas 

<A)n-(Th) m -(B) 0 -(IgE-CH3 domain antigen) -X, 



35 



WO 99/67293 PCT/US99/13959 



- 35 - 

(A)„-(B) 0 -(Th) B -(B)o-(IgE-CH3 domain antigen) -X, 
(A) n -dgE-CH3 domain antigen) - (B) c - (Th) m -X, 
(IgE-CH3 domain antigen) - (B) D - (Th) m - (A) n -X, and 
(Th) m -(B) 0 - (IgE-CH3 domain antigen) - (A) n ~X. 
5 The Th epitope is attached, optionally through 

spacer B, to either the N terminus or C terminus of the 
IgE-CH3 peptide and crossreactive and functional 
immunological analogs thereof. Preferred peptide 
10 immunogens of this invention are the peptides containing 

the IgE-CH3 domain antigen peptides (e.g., SEQ ID NO: 5) 
(or immunological analogs thereof) and Th peptides, and 
optionally Inv (SEQ ID NO: 13). In a more preferred 
embodiment the Th epitope is an HBs Th, HBc Th, MV F Th, 
15 pT Th ^ TT Th ^ CT Th (e.g., SEQ ID NO: 12) or artificial Th 

(SEQ ID N0S:9-11 and 60), or functional immunogenic 
analogue thereof, and optionally, A is Inv (SEQ ID NO: 13) 
attached through a (B) G spacer such as Gly-Gly or (□- 
20 N)Lys. 

The structure of the IgE-CH3 domain antigen 
comprises a peptide sequence taken from the CH3 domain of 
human IgE (amino acids 413-435 of SEQ ID No:l) or the 
homologous sequences from other species (e.g., SEQ ID 

25 

NOS:6-8 and 84) and subjected to the following 
modifications : 

the target site is modified from that of the 
naturally occurring IgE sequences by the insertion of a 
30 cysteine residue to the N-terminus side of position 413 or 

homologous position unless cysteine is already present at 
positions 413 or 414 in the natural sequence, 

the substitution for the native cysteine of 



35 



WO 99/67293 



PCT/US99/13959 



- 36 - 

° position 418 or corresponding position of an homologous 
non-human sequence or any other cysteine of the native 
target sequence by serine (unless said native cysteines 
are present at positions 413 or 414 and 435 or 436), 
j the insertion of cysteine at C-terminus side of 

position 435 or homologous position unless cysteine is 
already present at positions 435 or 436 in the natural 
sequence, and 

the formation of a disulfide bond between the 

^ retained cysteines so as to produce a cyclic structure. 

Said cyclic structures also comprise 1 to 5 
additional amino acids taken from either terminus of the 
413-435 segment of IgE provided that the single disulfide 

15 looped structure is preserved. An optimized target 

antigen for human IgE of sequence Cys-Gly-Glu-Thr-Tyr-Gln- 
Ser-Arg-Val-Thr-His-Pro-His-Leu-Pro-Arg-Ala-Leu-Met-Arg- 
Ser-Thr-Thr-Lys-Cys (SEQ ID NO: 5) is provided by the 
present invention. The human IgE target antigen is 
cyclized through the unnatural terminal cysteines and the 
first serine residue substitutes for the cysteine residue 
of the natural sequence. Antibody that is evoked by 
peptide immunogens comprising this IgE-CH3 domain antigen 

^ is crossreactive with human IgE and inhibits the 

sensitization of human basophils by human IgE. 

Likewise, corresponding IgE-CH3 domain antigen 
sequences for IgE of other species can be derived from the 

30 homologous e chain segment of the relevant species. For 

example, such target sequences can be taken from the dog, 
rat and mouse e chain sequences shown in Table 1 as SEQ ID 
NOS:2, 3 and 4, and the equine sequence published by 



35 



WO 99/67293 



PCT/US99/1J959 



- 37 - 

° Navarro et ai., and IgE-CH3 domain antigen sequences such 
as SEQ ID N0S:6, 7, 8 and 84 can be derived. 

Crossreactive and immunologically functional 
analogs of the IgE-CH3 domain antigen peptides (e.g., SEQ 
5 ID NOS:5-8 and 84) according to the invention, may further 

comprise conservative substitutions, additions, deletions, 
or insertions of from one to about four amino acid 
residues, provided that the resulting peptide analogs are 
capable of eliciting immune responses crossreactive with 
10 the IgE-CH3 peptides (e.g., SEQ ID NOS:5-8 and 84). The 

conservative substitutions, additions, and insertions can 
be accomplished with natural or non-natural amino acids as 
defined herein. 

15 Peptide compositions which contain mixtures of the 

subject peptide immunogens with two or more of the Th 
epitopes may enhance immunoef f icacy in a broader 
population and thus provide an improved immune response to 

2Q the IgE-CH3 domain antigen (e.g., SEQ ID NOS:5-8 and 84). 

The peptide immunogens of this invention can be 
made by chemical synthesis methods which are well known to 
the ordinarily skilled artisan. See, for example, Fields 
et ai., Chapter 3 in Synthetic Peptides: A User's Guide, 

25 ed. Grant, W. H. Freeman & Co., New York, NY, 1992, p. 77. 

When a peptide immunogen includes a SSAL Th, the coupling 
of the alternative amino acids at a given variable 
position is accomplished by providing a mixture of the 

30 amino acids specified for that position. Hence, peptides 

can be synthesized using the automated Merrifield 
techniques of solid phase synthesis with the a-NH 2 
protected by either t-Boc or Fmoc chemistry using side 



35 



10 



WO 99/67293 PCT/US99/13959 



- 38 - 

chain protected amino acids on, for example, an Applied 
Biosystems Peptide Synthesizer Model 430A or 431. 

After complete assembly of the desired peptide 
immunogen, the resin is treated according to standard 
procedures to cleave the peptide from the resin and 
deblock the functional groups on the amino acid side 
chains. The free peptide is purified, for example by 
HPLC, and characterized biochemically, for example, by 
amino acid analysis, mass spectrometry, and/or by 
sequencing. Purification and characterization methods for 
peptides are well known to those of ordinary skill in the 
art . 

Other chemical means to generate the synthetic 
15 peptide constructs of the invention containing IgE and Th 

sites include the ligation of haloacetylated and 
cysteinylated peptides through the formation of a 
"thioether" linkage. For example, a cysteine can be added 
to the C terminus of a Th-containing peptide and the thiol 
group of cysteine may be used to form a covalent bond to 
an electrophilic group such as an N chloroacetyl-modif ied 
or a maleimide-derivatized a- or e-NH 2 group of a lysine 
residue attached to the N-terminus . of an IgE-CH3 peptide 
(e.g., SEQ ID NO: 5) or crossreactive and functional 
immunological analogs thereof. In this manner, a 
construct with' Th- ( IgE-CH3 domain antigen) or its reverse, 
(IgE-CH3 domain antigen) -Th, may be obtained. 
30 The subject immunogen may also be polymerized. 

Polymerization can be accomplished for example by reaction 
of the immunogen with a cross-linking agent, for example 
by reaction between glutaraldehyde and the -NH 2 groups of 



20 



25 



35 



WO 99/67293 PCT/US99/13959 



- 39 - 

0 lysine residues, using routine methodology. By another 

method, a synthetic immunogen, such as for example "A-Th m - 
spacer- (IgE-CH3 domain antigen)", can be polymerized or 
co-polymerized with another immunogen by utilization of an 
5 additional cysteine added to the N-terminus of the 

synthetic immunogen. The thiol group of the N-terminal 
cysteine can be used for the formation of a "thioether" 
bond with haloacetyl-modif ied amino acid or a maleimide- 
derivatized <x-NH 2 or e-NH 2 group of a lysine residue that 

10 is attached to the N-terminus of a branched poly-lysyl 

core molecule (e.g., K 2 K, K 4 K 2 K or K 8 K 4 K 2 K) . The subject 
immunogen may also be prepared as a branched polymer 
through synthesis of the desired peptide construct 

15 directly onto a branched poly-lysyl core resin (Wang et 

ai., Science, 1991; 254: 285-288). 

Alternatively, the longer synthetic peptide 
immunogens can be synthesized by well-known recombinant 
DNA techniques. Many standard manuals on molecular 
cloning technology provide detailed protocols to produce 
the peptides of the invention by expression of recombinant 
DNA and RNA. To construct a gene encoding a peptide of 
this invention (e.g., immunogenic peptides comprising SEQ 

25 ID NOS:5-8 and 84, and other species-specific homologs), 

the amino acid sequence is reverse translated into a 
nucleic acid sequence, preferably using optimized codon 
usage for the organism in which the gene will be 

2q expressed. Next, a gene encoding the peptide is made, 

typically by synthesizing overlapping oligonucleotides 
which encode the peptide and necessary regulatory 
elements. The synthetic gene is assembled and inserted 
into the desired expression vector. The synthetic nucleic 



35 



WO 99/67293 



PCT/US99/13959 



- 40 - 

° acid sequences encompassed by this invention include those 

which encode the peptides of the invention, immunologicaly 
functional homologs, and nucleic acid constructs 
characterized by changes in the non-coding sequences that 
5 do not alter the immunogenic properties of the peptide 

encoded thereby. Nucleic acids which comprise sequences 
that encode the peptides of this invention are also 
provided. The synthetic gene is inserted into a suitable 
cloning vecor and recombinants are obtained and 

10 characterized. The peptide is then expressed under 

conditions appropriate for the selected expression system 
and host. The peptide is purified and characterized by 
standard methods. 

15 The nucleic acids of this invention may themselves 

be useful as components of so-called " DNA vaccines". In 
this embodiment of the invention, expression of the 
immunogenic peptides of the invention is induced in the 
patient's own cells, by introduction into those cells of 

20 

nucleic acids which encode the peptides. Methods of 
making and using DNA vaccines are disclosed in US Patents 
5,580,859, 5,589,466, and 5,703,055; see also WO 97/02840 
and W. McDonnell and F, Askari, New Engl. J. Med., 1996, 

25 334:2-45, all of which are incorporated herein by 

reference. Such methods of making and using the peptides 
and peptide conjugates of this invention are contemplated 
to be within the scope of this invention. 

3 q The efficacy of any peptide composition of the 

present invention can be established by in vitro assay in 
which a host animal is immunized with a peptide 
composition of the invention and the resulting antibodies 
are shown to inhibit the sensitization of basophils and 



WO 99/67293 PCT/US99/13959 



- 41 - 

° mastcells by IgE, as shown in Examples 2 and 6. Efficacy 
can be established in vivo by injecting a host with a 
species-appropriate peptide composition (for example, 
immunizing mice with a formulation of immunogens 
5 comprising SEQ ID NOS:24 and/or 25) followed by monitoring 

the humoral immune response to the IgE-CH3 peptide and 
crossreactive and functional immunological homologues 
thereof, as detailed in Example 5. 

Another aspect of this invention provides a 

^ peptide composition comprising an immunologically 

effective amount of one or more of the peptide immunogens 
of this invention in a pharmaceutical^ acceptable 
delivery system. Accordingly, the subject peptides can be 

15 formulated as a pharmaceutical composition using 

adjuvants, pharmaceutically acceptable carriers, or other 
ingredients routinely provided in vaccine compositions. 
Among the ingredients that can be used in this invention 
are adjuvants or emulsifiers including alum, incomplete 

20 

Freund's adjuvant, liposyn, saponin, squalene, L121, 
emulsigen, monophosphoryl lipid A (MPL) , QS21, ISA51, 
ISA35, ISA 206, and ISA 720, as well as other known 
efficacious adjuvants and emulsifiers. The formulations 
25 include formulations for immediate release and/or for 

sustained release, and for induction of systemic immunity 
and/or induction of localized mucosal immunity, which may 
be accomplished by, for example, immunogen entrapment by 
or coadministration with microparticles . Such 

30 

formulations are readily determined by one of ordinary 
skill in the art, and methods for the preparation, 
preservation, and sterilization of such formulations are 
known to those skilled in the art. 



35 



WO 99/67293 



PCT/US99/13959 



- 42 - 

0 The present pharmaceuticals can be administered by 

any convenient route including subcutaneous, oral, 
intramuscular, or other parenteral or enteral route. 
Similarly the pharmaceuticals can be administered as a 
single dose or multiple doses. Immunization schedules are 
readily determined by the ordinarily skilled artisan. 

The pharmaceutical composition of the instant 
invention contain an effective amount of one or more of 
the peptide immunogens of the present invention and a 

10 pharmaceutical^ acceptable carrier. Such a composition 

in a suitable dosage unit form generally contains about 
0.5 *xg to about 1 mg of the immunogen per kg body weight. 
When delivered in multiple doses, it may be conveniently 

15 divided into an appropriate amount per dosage unit form. 

For example, an initial dose, e.g. 0.2-2.5 mg; preferably 
1 mg, of immunogen represented as a peptide composition of 
the present invention, may be administered by injection, 
preferably intramuscularly, followed by repeat (booster) 

20 

doses. Dosage will depend on the age, weight and general 
health of the patient as is well known in the vaccine and 
therapeutic? arts. 

The immune response to synthetic IgE-CH3 peptide 

25 immunogens may be improved by delivery through entrapment 

in or on biodegradable microparticles of the type 
described by O'Hagan et al. (Vaccine, 1991; 9:768-771). 
The immunogens can be encapsulated with or without an 

30 adjuvant in biodegradable microparticles, to potentiate 

immune responses, including localized mucosal immunity 
which may be especially applicable to mucosally localized 
allergic reactions, and to provide time-controlled release 
for sustained or periodic responses, for oral 



WO 99/67293 



PCT/US99/13959 



- 43 - 

administration, and for topical administration (O'Hagan et 
al., 1991; Eldridge et al., Molec. Immunol., 1991; 28: 
287-294) . 

The pharmaceutical compositions of this invention 
are used in a manner similar to that of vaccines, for the 
prevention of atopic allergic reactions including allergic 
rhinitis, those of food allergies, asthma, anaphylaxis, 
flea allergy dermatitis, and other IgE-mediated 
hypersensitivities . 

All patents and literature references referenced 
hereinabove are incorporated herein by reference. 

Specific peptide and peptide conjugate immunogens 
are provided in the following examples to illustrate the 
invention. These examples are for purpose of illustration 
only, and are not to be construed as limiting the scope of 
the invention in any manner. 



20 EXAMPLE 1 

IDENTIFICATION OF POTENTIAL EFFECTOR 
SITES ON THE HUMAN IqE MOLECULE 

Peptide De sign 
25 — c 

Sites within the CH2 and CH3 domains of z heavy 
chain of human IgE were selected for mimicry by peptides, 
in accordance with the disclosures of Helm et al. (1988) 

and Presta et al. (1994) that a long segment of the e 

30 . . 

chain which overlaps both these domains participates in 

binding IgE to the FceRl receptor. The sequences of such 

sites were synthesized as target site peptides and 

rendered into antigens by (1) attaching them through 



35 



WO 99/67293 PCT/US99/13959 



15 



- 44 - 

° chemical coupling to large carrier proteins such as KLH or 
(2) constructing peptides where promiscuous Th and Inv 
(SEQ ID NO: 13) were linked to the amino terminal of the 
target sites. Specific sites within these domains were 
5 selected as peptides for cyclization based on predictions 
by the Brookhaven 3-dimensional model for human IgE 
( http : www . pdb . bnl . gov/pdb . bin/pdbids ) of surface-exposed 
loops. Specified cyclic constraints were installed into 
the design of those peptides so as to maximize the 
10 crossreactions between the antigens and the native IgE 
molecule. Accordingly, several of the synthetic 
constructs were synthesized with introduced cysteines not 
found in the native sequence to produce disulfide bond 
loops of specified position, in mimicry of loop structures 
predicted by the Brookhaven model. In some cases 
naturally occurring cysteines were substituted with 
serines so as to prevent the formation of conformations 
not favored by the model. 

The constructs are listed in Table 2. Peptides 
marked by * in the description column of Table 2 are 
cyclized by cysteine disulfide bonds. Cysteine residues 
that have been inserted into the native sequence for 
25 cyclization are denoted in the amino acid sequences of 

Table 2 by parentheses, other residues that have been 
inserted, substituted for a native residue, or are natural 
cysteines that participate in disulfide bonds are 
indicated in the amino acid sequences of Table 2 by 
underlining. Other peptides are linear. Peptides labeled 
by "a" in the third column represent the IgE-CH2/3 or -CH3 
antigen peptide, chemically linked to KLH carrier protein 
by conventional glutaraldehyde or MBS (m-Maleimidobenzoyl- 



20 



30 



35 



WO 99/67293 PCT/US99/13959 



- 45 - 

N-hydroxysuccinimide ester, Pierce Chemical Co., catalogue 
No. 22510) coupling reactions. Peptides marked by "b" in 
the third column were synthesized as IgE antigen peptides 
in tandem with the Th sites shown. Th sites used include 
5 the HBS19-32 Th taken from hepatitis B virus, the MVf Th 

taken from measles virus, and PTi 49 -i46 Th taken from 
pertussis toxin as referenced in US 5,759,551, the CT Th 
termed Pll (Stagg et al., 1993) and novel artificial Th 
sites termed "1,4,9 PALINDROMIC Th" (SEQ ID NO:10), 

10 W IS(1,4,9 PALINDROMIC) LF Th" (SEQ ID NO:ll), tt IS(l,4,9 

PALINDROMIC) LF simplified Th" (SEQ ID NO:60), and "Syn Th 
(1,2,4)" (SEQ ID NO: 9). Peptides marked by "c" are 
variants of the "b" constructs synthesized in tandem with 

15 the Inv domain immunostimulatory peptide (SEQ ID NO: 13). 

The "b" and "c" constructs were also synthesized 
with Gly-Gly spacers for separation of the IgE-CH2/3 or - 
CH3 target antigen site from the Th site, and separation 
of the Th from the Inv immunostimulatory site. The "b" 

20 

and "c" constructs in Table 2 had the Th and/or Inv 
domains attached to the amino terminal of the IgE target 
site. The peptide immunogens of Table 2 were screened as 
candidate target antigenic peptides for the treatment of 
25 allergy, by the hyperimmunization of animals followed by 

testing of the hyperimmune sera for crossreactivity to 
human IgE. 

Specific Procedures for the Screening of Candidate Target 
30 Antigenic Peptides : 

1. Synthesis of IqE-CH3 domain antigen Peptides and 
Conjugates . 

Peptides listed in Table 2 were synthesized by the 



35 



WO 99/67293 PCT/US99/13959 



- 46 - 

0 Merrifield solid-phase synthesis technique on Applied 
Biosystems automated peptide synthesizers using Fmoc 
chemistry. When a peptide immunogen included a SSAL Th, 
the coupling of one of the alternate amino acids at a 
5 given variable position was accomplished by providing a 

mixture of amino acids at equivalent molar ratios. After 
complete assembly of the desired peptide, the resin was 
treated according to standard procedure using 
trifluoroacetic acid to cleave the peptide from the resin 

10 and deblock the protecting groups on the amino acid side 
chains. For cyclic peptides, the cleaved peptides were 
dissolved in 15% DMSO in water for 48 hours to facilitate 
intradisulfide bond formation between cysteines. 

15 2. Experimental Immunizations, 

Rats or guinea pigs were immunized intramuscularly 
with experimental peptide immunogens . The dose was 100 \iq 
of peptide suspended in a volume of 0.5 ml. The first 

2Q dose was administered with Complete Freunds Adjuvant. 

Subsequent doses were administered in Incomplete Freunds 
Adjuvant. Animals received injections on weeks 0, 3, 6, 
and 10 or 0, 2, 4, and 8. Test bleeds were taken at 
biweekly intervals and reactivities were determined by IgE 

25 peptide ELISA and crossreactivities by human IgE ELISA. 

3. ELISA Assays. 

Peptide ELISAs for determination of anti-IgE 
peptide reactivity were conducted in peptide-coated 96- 

30 well microtiter plates coated by 1 hr incubation at 37°C 

with an appropriate "a" target antigen site peptide 
without carrier at 0.5 ^ig/mL using 100 jiL per well in 10 
mM NaHC0 3 buffer, pH 9.5. For determination of anti-human 



35 



• • * 



WO 99/67293 PCT/US99/13959 



- 47 - 

° IgE crossreactivity, human IgE ELISAs were conducted in 

human IgE-coated 96-well microtiter plates coated in a 
likewise fashion with a human IgE myeloma protein 
(American Biosystems, Inc. cat. no. A113) at 5 jxg/mL. The 

5 peptide or human IgE-coated wells were incubated with 250 

|iL of 3% by weight of gelatin in PBS, at 37°C for 1 hr to 
block non-specific protein binding sites, washed three 
times with PBS containing 0.05% by volume TWEEN 20 and 
then dried. Test samples were serially diluted with PBS 

10 

containing 20% by volume normal goat serum, 1% by weight 
gelatin and 0.05% by volume TWEEN 20. 100 nL of the 
diluted sample was added to each of the wells and allowed 
to react for 1 hr at 37°C. The wells were then washed six 

15 times with 0.05% by volume TWEEN 20 in PBS to remove 

unbound labeled antibodies. 100 \xL of horseradish 
peroxidase labeled anti-rat IgG goat antibody or anti- 
guinea pig IgG goat antibody at predetermined optimal 

20 dilution in 1% by volume normal goat serum, 0.05% by 

volume TWEEN 20 in PBS were added to each well and 
incubated at 37°C for 30 minutes. The wells were washed 
six times with 0.05% by volume TWEEN 20 in PBS to remove 

25 unbound labeled antibody conjugate and reacted with 100 (iL 

of the substrate mixture containing 0.04% by weight 
orthophenylenediamine (OPD) and 0.12% by volume hydrogen 
peroxide in sodium citrate buffer pH 5.0, for 15 minutes. 
Reactions were stopped by the addition of 100 [iL of 1.0 M 

30 

H 2 S0 4 and the absorbance at A492 was measured. ELISA 
titers, expressed as logio of reciprocal dilution, were 
calculated based on linear regression analysis of the 
absorbances, with cutoff A492 set at 0.5. This cutoff 



35 



WO 99/67293 PCT/US99/13959 



10 



15 



- 48 - 

value was rigorous as the values for diluted normal guinea 
pig control samples run with each assay were less than 
0.15. 

Results. 

Candidate target antigen sites are described in 
Table 2. They are shown either as "a" peptides attached 
to KLH carrier or as "b" peptides attached to synthetic Th 
sites or as "c" peptides attached to synthetic Th and Inv. 
Either rats or guinea pigs were immunized as described in 
Specific Procedures above and hyperimmune antisera 
collected at week 8 were analyzed by anti-peptide ELISA 
and anti-human IgE ELISA as described in Specific 
Procedures. 

Many of the CH2/3 and CH3 peptide immunogens were 
immunogenic, as they evoked anti-peptide antibodies with 
titers in the range of logio 2-5. The CH2/3 antigenic 
20 target sites comprising long segments of the human e chain 

from 301-376 (numbering scheme of Table 1) were all 
strongly crossreactive with human IgE, as shown by log 10 
titers on the anti-human IgE ELISA of greater than 3. 
Crossreactivity was lost for some CH3 peptides which 
initiated at position 342 and beyond (e.g., entries 21 and 
22). However, for CH3 peptides which included a 
relatively short region comprising 354-372, 
crossreactivity was largely restored (e.g, entries 27, 28, 
30 and 29) with the exception of entry 31 (354-368). Another 

short region of crossreactivity is seen in entry 20 
(cyclic peptide spanning positions 374-385) . 

As evidenced by the lack of crossreactivity of 



25 



35 



WO 99/67293 PCT/US99/13959 



10 



15 



- 49 - 

entries 14, 17, 23, 24, 25, and 26, a stretch of sequence 
that extends from 365 to 413 is devoid of crossreactivity, 
despite overlap with the 354-372 region of crossreactivity 
and a crossreactive region represented by entry 20 (374- 
385). Interestingly, the short crossreactivities 
exemplified by entries 27, 28, 29 (354-372) and 20 (374- 
385) are lost in the conformation of the long cyclized 
peptide entry 17 (365-396), despite their overlap in those 
crossreactive regions. Crossreactive sites which overlap 
non-crossreactive sites are again to be found beyond a 
region that starts around position 399 and extends to 
position 445, as shown by the crossreactivities of entries 
15 and 30, and the weak crossreactivities of entries 19 
(432-445) and 23 (404-413) . It is significant that of two 
similarly cyclized peptides which include position 418, 15 
(413-435) and 18 (404-434), only entry 15 (SEQ ID NO:5), 
in which the cysteine at position 418 has been substituted 
by serine, is crossreactive with human IgE. A CH4 site 
that corresponds to an IgE effector site described by 
Stanworth (Stanworth et al., Lancet, 1990; 336:1279-1281) 
failed to show crossreactivity (entry 34). 

These results demonstrate that crossreactivity for 
25 igE peptides is a complex phenomenon influenced by 

conformational features, and cannot be predicted from a 
straightforward analysis of overlapping linear peptides. 
Candidate IgE-CH3 domain antigens were selected from among 
the conjugates shown to be crossreactive with human IgE in 
Table 2 and used for further analyses. 

EXAMPLE 2 



20 



30 



35 



WO 99/67293 



PCT/US99/13959 



10 



- 50 - 

IDENTIFICATION OF EFFECTOR SITE 
ON THE HUMAN IqE MOLECULE 

IgE-CH3 domain antigen peptides were selected for 
further analysis from among those peptide conjugates of 
Table 2 that exhibited high affinity crossreactivities to 
human IgE, as evidenced by anti-IgE titers for their 
respective antisera of greater than logi 0 =3. Guinea pig 
hyperimmune sera were produced as described above. Guinea 
pig IgG antibodies were purified from the hyperimmune sera 
by protein A affinity chromatography and analyzed by a 
functional assay for determination of ability of anti-IgE 
to inhibit the sensitization of human basophils by 
allergen-specific IgE. The endpoint of the assay is 
15 expressed as per cent inhibition of IgE-mediated histamine 

release. 

Guinea pig IgG antibodies were purified from serum 
by Protein A affinity chromatography (ImmunoPure® 
Immobilized Recomb® Protein A, Pierce) and the eluted 
antibodies were prepared at a standard concentration of 8 
mg/ml in 25 mM PIPES buffer, 0.15 M NaCl, pH 7.2. A 
control antibody preparation was prepared from the pooled 
serum of guinea pigs immunized with an irrelevant peptide 
25 immunogen. These antibodies were used in assays that 

measure the reduction in IgE-mediated sensitization of 
human basophils. Human basophils were prepared from the 
venous blood of volunteers using centrif ugation through 
Percoll density gradients (MacGlashan. J Allergy Clin 
Immunol, 1993; 91:605-615). The banded leukocytes were 
collected, washed, and resuspended in 0.1 ml of PAGCM 
buffer as described (MacGlashan, 1993) except that the 
PAGCM buffer used to suspend the cells was made up with 



20 



30 



WO 99/67293 PCT/US99/13959 



- 51 - 

water containing 44% D 2 0. The IgE used for the assay was 
allergen-specific, either human BPO-specific IgE or 
chimeric human IgE specific for HIV glycoprotein gpl20. 
The allergen-specific IgE used for sensitization at 0.25 
5 fxg/ml was preincubated with an equal volume of purified 

guinea pig antibody at 8 mg/ml, total volume 0.1 ml, for 
15 minutes at 37°C, prior to being added to the basophils. 
The antibody mixture was added to the cells and incubated 
for 20 minutes to allow for sensitization of the cells by 
uncomplexed IgE . The sensitized cells were then 
stimulated by addition of the allergen, either BP0 2 i-HSA 
or a gpl20 polypeptide as described (MacGlashan, 1993) . 

After an appropriate incubation period (usually 45 

15 minutes), the cells were separated from the supernatant 

and the supernatant assayed for histamine content by an 
automated fluorimetric technique (Siraganian, Anal 
Biochem r 1974; 57:' 383-394). All reactions were performed 

20 in duplicate. The percentage of histamine release was 

calculated from the ratio of sample to total histamine 
after spontaneous release was subtracted from both. 
Results are expressed as per cent inhibition of histamine 
release, as determined from the ratio of histamine release 

25 by experimental antibody to histamine release by the 

control antibody of irrelevant specificity. Histamine 
release assays on human basophils were kindly performed 
under coded conditions by Dr. Donald W. MacGlashan, The 

30 Johns Hopkins University School of Medicine, Johns Hopkins 

Asthma and Allergy Center, Baltimore. 
Results 

The results for inhibition of histamine release 



35 



WO 99/67293 PCT/US99/13959 



- 52 - 

° assays are shown in Table 3 for guinea pig anti-peptide 

antibodies that displayed crossreactivities for human IgE 
of logio >3. Determinations were made from antibodies 
purified from 8 week bleeds, except for antibodies against 
5 peptide entries 15b and 15c which were also characterized 

from serum collected on week 12. The inhibition results 
shown for anti-15b and anti-15c antibodies, of 61% and 
71%, were made on the antibodies purified from bleeds 
taken on weeks 8 and 12, respectively. Separate animals 

10 had been immunized with 15b and 15c, but antibodies from 

both sets of animals had been pooled for the 8 and 12 week 
results shown in Table 3. (The guinea pigs of these 
groups had received an additional dose of peptide 

15 conjugate on week 10 and so had retained high antibody 

levels for the 12 week bleed) . The significant inhibitory 
reactivity of the anti-15 antibodies was unexpected in 
comparison to the reactivities of the IgE crossreactive 
antibodies evoked by the remainder of the peptides shown 

20 in Table 3. These other IgE-CH3 domain antigenic peptides 

failed to provide inhibition, or presented levels of 
inhibition for histamine release that were negligible and 
non-reproducible . 

25 Histamine release inhibition results and IgE 

crossreactivities for antibodies elicited by IgE-CH3 
domain antigen peptides that overlap with the antigenic 
site (SEQ ID NO:5) of peptide entries 15b (SEQ ID NO:14) 
and 15c (SEQ ID NO: 15) may be compared. The IgE antigens 

30 

represented by peptide entries 19, 23, 24, and 33 comprise 
short overlaps with the entry 15 antigen sequence (SEQ ID 
NO: 5). They compare unfavorably to entry 15 for 
crossreactivity to IgE, and are devoid of inhibitory 



35 



WO 99/67293 PCT/US99/1 39S9 



- 53 - 

° activity. The IgE antigen sequence (SEQ ID NO: 44) of 

entry 18 comprises the entire antigen sequence of entry 
15, except that (1) the carboxyl terminal lysine, is 
deleted, (2) the naturally occurring cysteine at position 

5 418 is retained, and (3) there are nine additional N- 

terminal amino acids. It is non-crossreactive with IgE 
and fails to inhibit histamine release. In contrast, the 
immunogens of entry 15, having antigen SEQ ID NO: 5, 
provide unexpected reactivities. The IgE-CH3 domain 

10 antigen sequence of entry 15, with a cyclic structure 

specified by introduced terminal cysteines, and with no 
contribution from the cysteine at position 418 (which has 
been replaced) , provides an antigen that is crossreactive 

15 with IgE and elicits antibodies which inhibit IgE 

sensitization. 

Antibodies elicited by entry 15b (SEQ ID NO: 14) 
and 15c (SEQ ID NO: 15) were prepared from 13 week bleeds 
and tested individually. By week 13, both crossreactivity 
for IgE, as determined by IgE ELISA, and per cent 
inhibition of histamine release had diminished from the 
values of week 12. Nevertheless, antibodies from both 
preparations were found to be individually effective in 

25 reducing histamine release: anti-15b inhibited 28% and 

anti-15c inhibited 20%. 

The extent by which histamine release was 
inhibited by either of these antibodies was dose 

3Q dependent, as evidenced by the effect of dilution on the 

antibodies. When a preparation of anti-15b from week 13 
was assayed at full concentration (8 mg/ml), then at 1:3 
and 1:9 dilutions, per cent inhibition of histamine 
release was 28%, 21%, and 14% respectively. 



35 



WO 99/67293 PCT/US99/13959 



10 



15 



- 54 - 

A preparation of guinea pig anti-15b was tested by 
direct challenge of IgE-sensitized basophils, in the 
absence of allergen, as an evaluation of its ability to 
crosslink receptor-bound IgE and induce degranulation. 
Histamine release by anti-15b was equivalent to the level 
of spontaneous histamine release by the donor cells. This 
indicates that antibody of specificity for the SEQ ID NO: 5 
IgE antigen is non-anaphylactogenic. Thus, active 
immunization with peptide conjugate immunogens comprising, 
the IgE-CH3 domain antigen SEQ ID NO: 5 (SEQ ID NOS:14 and 
15) elicits non-anaphylactogenic anti-IgE antibodies that 
inhibit IgE-mediated sensitization without themselves 
causing histamine release. These actively evoked 
polyclonal antibodies display specificity for an IgE 
effector site that has not been described by previous 
studies, including prior studies of therapeutic and non- 
anaphylactogenic anti-IgE monoclonal antibodies intended 
for treatment of allergy by passive immunization (U.S. 
20 4,940,782, U.S. 5,420,251, and Presta et al., 1993). 

EXAMPLE 3 

ISOTYPE SPECIFICITY AND 
25 POTENTIAL FOR IMMUNOSUPPRESSION 

The polyclonal antibodies elicited by active 
immune response to SEQ ID N0S:14 and 15 were examined for 
specificity to IgE in comparison to IgG. Anti-15b guinea 
30 pig antibodies described in Example 2 that were prepared 

from the 12 week bleed were subjected to a parallel 
comparison of crossreactivities to IgE and IgG, by the IgE 
ELISA described in Example 1 and by a similar IgG ELISA. 



35 



WO 99/67293 



PCT/US99/13959 



10 



15 



- 55 - 

For the IgE ELISA, plates were coated with the human IgE 
myeloma at 5 ug/ml. For the IgG ELISA, the plates were 
coated with human purified IgG (Sigma reagent grade human 
IgG), also at 5 ug/ml. The purified guinea pig anti-15b 
was tested for reactivities in both ELISAs at 
concentrations of 0.5 and 0.1 ug/ml. Results were 
compared to antibodies purified from control guinea pig 
serum and to a "no antibody" control. The A 490 values for 
anti-15b antibody on IgE were 1.126 at 0.5 ug/ml and 0.344 
at 0.1 ug/ml. The A 490 values for anti-15b antibody on IgG 
were equal to control antibody and background values. 
There was no crossreactivity of the guinea pig anti-15b to 
human IgG. The peptide composition of the invention did 
not evoke antibodies that recognize IgG antibodies, and 
therefore are isotype specific for IgE. They will 
suppress IgE-mediated allergic reactions and not result in 
undesirable immunosuppression of IgG protective antibody 
20 responses . 

EXAMPLE 4 

REPRESENTATIVE PEPTIDE CONJUGATES OF THE INVENTION 

The immunogenic peptide conjugates of the 
invention shown in Table 4A, which are wholly synthetic 
peptides, were synthesized by the solid-phase method 
outlined in Example 1. Each peptide in the Table can be 
represented by the formula (A)„-(Th)„-(B) 0 -(IgE-CH3 domain 
antigen) -X, but peptides of the other formulas disclosed 
above are understood to be encompassed within the peptides 
of this invention. The IgE-CH3 domain antigen sequence is 
SEQ ID NO: 5, 6, or 8 in the peptides of Table 4A, but it 



25 



30 



WO 99/67293 PCT/US99/13959 



- 56 - 

° is understood that homologous IgE-CH3 domain antigen 

sequences from other mammalian species are encompassed 
within the peptides of this invention. The immunogenic 
peptides comprise Th sites derived from foreign pathogens ' 
5 (e.g., SEQ ID NO:20, 87), and also artificial Th (e.g., 

SEQ ID NOS:14, 18, 21 and 90). In addition to the 
examples shown in Table 4A, other pathogen-related Th may 
be selected from among the promiscuous Th sites 
exemplified in Table 5, and artificial Th may be selected 
10 from among the Th sites exemplified in Table 6. Each 

peptide of this example has Gly-Gly or (d-N)Lys spacers 
between immunogenic elements, but peptides of the 
invention may have other spacers (e.g., SEQ ID NO: 16) or 
J5 no spacers. 

Peptides of these examples also comprise an 
optional Inv immunostimulatory site (e.g., SEQ ID NOS:15- 
19 and 22) . It is understood however that the invention 
is not limited to Inv as an additional immunostimulatory 
element. As shown by the KLH conjugate, peptide 
conjugates of the invention also include an IgE-CH3 domain 
antigen coupled to a carrier protein. 
Materials and methods 

Representative peptide constructs of the invention 
as listed in Table 4A (SEQ ID NOS: 18, 85, 87, 88, 90 and 
91) were synthesized, cleaved, cyclized and purified as 
described in Example 1. The peptide constructs were 
formulated for immunization into small animals such as 
guinea pigs, or into larger animals such as pigs or 
baboons for evaluation of their immunogenicities . 
Peptides were suspended in a volume of 0.5 mL containing 



20 



25 



30 



35 



WO 99/67293 PCT/US99/13959 



- 57 - 

° representative emulsifiers or adjuvants such as ISA51, 

ISA720, DDA or monophosphoryl lipid A (MPL) . The dose was 
100 |ig of peptide for guinea pigs or 300 \xq of peptide for 
swine or baboons and the animals were immunized 
5 intramuscularly. 

Animals received injection on weeks 0, 3 and 6, or 
0, 2 and 4 weeks as specified in Table 4B. Test bleeds 
from 8 weeks post initial immunization were evaluated for 
crossreactivities to IgE by human IgE or dog IgE ELISA as 

10 described in Example 1, except that for the dog IgE ELISA 

a dog IgE myeloma protein (Bethyl Laboratories Inc., 
Montgomery TX) was used for plate coating at 1 ng/mL, and 
horseradish peroxidase labeled protein A/G reagent (Pierce 

15 Chemical Co., Rockford IL) at a predetermined optimal 

dilution was used as the tracer for detection of dog IgE. 
The peptide-induced crossreactivities were also evaluated 
for capacity to inhibit IgE-mediated histamine release. 

20 Guinea pig, pig, or baboon IgG were purified from 

representative immune sera by protein A affinity 
chromatography and analyzed by functional assay for 
determination of ability to inhibit the sensitization of 
human basophils by allergen-specific IgE, as described in 

25 details in Example 2. The endpoint of the assay is 

expressed as per cent inhibition of IgE-mediated histamine 
release in comparison to control antibody of the same 
species that was raised with specificity for an irrelevant 

30 antigen, as shown in Table 4B. 

Results 

The representative peptide constructs were of 
relevant immunogenicity, as all peptides tested elicited 



35 



WO 99/67293 



PCT/US99/13959 



- 58 - 

° strong site-directed cross reactivities to the 

corresponding human IgE or dog IgE, as shown by Log i0 
titers on the anti-human IgE or anti-dog IgE ELISAs of 
greater than 3 (Table 4B) . Inhibition of IgE-mediated 
5 sensitization was observed for guinea pig, pig, and baboon 
antibodies as evaluated by the ability of the anti-IgE 
peptide antibodies to inhibit histamine release by 
basophils- This functional crossreactivity by the baboon 
antibodies is noteworthy insomuch as the neutralization of 

10 human IgE by the baboon IgG is nearly a human system. 
Thus, the efficacy of a peptide construct of the 
invention, as an agent for the immunotherapy of allergy by 
active immunization, is indicated in a model that is 

15 nearly homologous for species of peptide and target 
species . 

EXAMPLE 5 

IMMUNIZATION OF MICE AND 
20 EVALUATION OF IN VIVO EFFICACY 

Efficacy of peptides of SEQ ID NOS:24 and 25 (37b 
and 38b) is evaluated with five groups of 16 mice by the 
immunization and sensitization protocol outlined below. 

25 

Groups of 16 mice (Balb/c) , female, 8-10 weeks 
old, are immunized subcutaneously with the indicated 
peptide composition of the invention. The mice are given 
20 fig/0.2ml doses on weeks 0, 3, 6, and 11. The first 
dose is prepared with Complete Freunds Adjuvant, 
subsequent doses with Incomplete Freunds Adjuvant. The 
mice are sensitized to a hapten conjugate, diphenylated 
KLH (DNP-KLH), on weeks 7 and 10. Sensitization is 



35 



WO 99/67293 PCT/US99/U959 



- 59 - 

0 accomplished by intraperitoneal administration of DNP-KLH 

in 0.4% Alum, 5 ng/0 . 2ml/dose . Mock immunizations and 
sensitizations are accomplished in control groups by 
administration of adjuvant with phosphate-buff ered-saline. 

5 The groups are as follows: 

1: Immunize/mock sensitize, with peptide 37b and 0.4% 
Alum 

2: Immunize/sensitize, with peptide 37b and DNP-KLH 
1Q 3: Mock immunize/sensitize, with Freunds and DNP-KLH 

4: Immunize/mock sensitize, with peptide 38b and 0.4% 
Alum 

5: Immunize/sensitize, with peptide 38b and DNP-KLH 

Serum is collected on weeks 0, 5, 7, 9, 10, 11, 

13, 16, and 20. Splenocytes are prepared from pairs of 

mice from each group on weeks 10 and 11. 

IgG response to the peptide antigens and to DNP is 

monitored by conventional ELISA assays, using an anti- 

20 

mouse IgG horseradish peroxidase conjugate, and microtiter 
plates whose wells are coated with unconjugated peptide 37 
(mouse IgE-CH3 domain antigen peptide, SEQ ID NO: 8) for 
peptide ELISA, and plates coated with DNP-BSA conjugate 

25 for DNP ELISA. Cross-reactivity of anti-37b antibodies 

with mouse IgE are monitored by a conventional IgG ELISA 
on plates coated with mouse monoclonal IgE SPE 7 (Sigma). 
IgG response to peptide immunogens is compared to mouse 

30 IgE crossreactivity among the groups throughout the 20 

week course, to determine 1) primary and secondary 
responses, 2) the presence of undesirable 
immunosuppression of IgG responsiveness, and, 3) the 
occurrence of a desirable reduction in anti-IgE reactivity 



35 



WO 99/67293 PCT/US99/13959 



- 60 - 

° during weeks 10-20 as evidence of reversibility and safety 
of the antibody response to the peptide composition of the 
invention. 

On weeks 7, 9, 10, 11, 13, and 16, IgE response is 
5 monitored by whole IgE EL ISA and by DNP-specific ELISA. 
On weeks 10 and 11 splenocyte B cells that secrete IgE 
with specificity for DNP are enumerated by DNP-specific 
ELISPOT assay. Also, because serum IgE levels may not be 
completely predictive of anaphylaxis, i.e., IgE 

10 determinations may miss significant effects on in vivo 
sensitivity, sensitization of the mice is measured by 
Passive Percutaneous Anaphylaxis assay of mouse serum in 
rats (heterologous PCA) . Heterologous PCA is preferred to 

15 autologous PCA assay in mice because rat skin mast cells 
are selectively cross-sensitized by mouse IgE as opposed 
to mouse IgG. Therefore, the heterologous mouse/rat PCA 
reaction is IgE-specific and is not confounded by IgG- 
mediated anaphylaxis which may occur in autologous mouse 

20 

PCA assay (Maekawa and Ovary, J Immunol Methods, 1984; 
71:229-239) . 

ELISA, ELISPOT, and PCA results are compared 
between groups for immunosuppression of IgE responsiveness 

25 and for isotypic specificity of the immunosuppression. 

Experimental methods are described below. 
Whole IgE ELISA 

For an ELISA to measure total mouse IgE in serum, 

30 microtiter plates are coated with monoclonal rat anti- 

mouse IgE, R35-72 ( Pharmingen) , at 1 ^ig/ml. The plates 
are coated, washed and blocked as described. Serially 
diluted mouse sera are added to the plates and incubated. 



35 



WO 99/67293 



PCT/US99/13959 



10 



15 



- 61 - 

Captured IgE is detected by reaction with biotinylated 
monoclonal rat anti-mouse IgE, R35-118 ( Pharmingen) , 
followed by sequential additions of streptavidin- 
horseradish peroxidase (Pierce) and OPD. A 492 values are 
determined. 

DNP-specific IgE ELISA 

For an ELISA to determine DNP hapten-specif ic 
mouse IgE in serum from mice that have been sensitized 
with DNP-KLH, microtiter wells are coated with DNP-BSA 
conjugate (Molecular Probes, Inc.) at 5 |ag/ml. Captured 
IgE with specificity for DNP hapten is detected as 
described above. 
DNP-specific ELISPOT 

For an ELISPOT assay to determine B cells that 
secrete DNP hapten-specif ic mouse IgE, DNP-BSA conjugate 
at 5 ^ig/ml is used to coat the wells of sterile microtiter 
plates whose wells are lined with 0.45 ^im nitrocellulose 
filters, for example a MULTISCREEN HA Plate (Millipore 
Inc., cat. no. MAHAS4510) . Serially diluted splenocytes, 
prepared from sensitized and control mice, are added to 
the wells and incubated overnight at 37° C under 5% C0 2 . 
25 The cells are washed from the plates and IgE-secreting 

cells with specificity for DNP hapten are counted as 
localized spots on the filters following staining by 
alkaline phosphatase con jugated-rat monoclonal antibody 
R35-118 with 5-bromo-4-chloro-3-indoyl phosphate (Sigma) 

30 

as colored substrate. 
Heterologous PCA 

Serial dilutions of sera from immunized/ 
sensitized and control mice are injected intradermally 

35 



20 



WO 99/67293 



PCT/US99/13959 



- 62 - 

into the shaved backs of adult male Sprague-Dawley rats. 
Anesthetized animals receive 10-12 injections of diluted 
serum into each of three parallel rows on the dorsal skin 
(50 pl/site) . Each pattern of injections is replicated in 
duplicate animals. After a 24 hour latent period, for 
effective sensitization of skin mast cells, rats are 
challenged by intravenous injection of 1 mg of DNP-BSA in 
1% Evans blue dye in PBS. In 30 minutes to 1 hour, rats 
are asphyxiated and skinned so that blueing reactions can 
be observed on the inside of the dorsal skin. A PCA titer 
is determined from the highest serum dilution which 
results in a readily definable spot. 



15 EXAMPLE 6 

IMMUNIZATION OF MICE AND INHIBITION OF 
PASSIVE CUTANEOUS ANAPHYLAXIS 

To study the effect of immunization by an 

20 

immunogenic peptide of the invention on an IgE-mediated 
inflammatory reaction, an antibody response was elicited 
to the mouse IgE-CH3 target antigenic site, SEQ ID NO: 8, 
by immunizing mice with a peptide of the invention. The 

25 resulting mouse antiserum was then used to suppress the 

passive cutaneous anaphylaxis (PCA) triggered by the 
crosslinking of mouse IgE bound to rat mast cells. 
Materials and methods 

30 Balb/c mice were immunized with a peptide 

composition of the invention, SEQ ID NO: 25, as described 
in Example 5, except that the subcutaneous injections were 
given on weeks 0, 3, and 6 only and the mice were not 



35 



WO 99/67293 PCT/US99/139S9 



- 63 - 

O 

sensitized. On week 8, mouse sera were collected and 
evaluated for crossreactivity to IgE by mouse IgE ELISA. 
The mouse IgE ELISA was as described for the human IgE 
ELISA in Example 1 except that microtiter wells were 
5 coated with 1 jig/ml of mouse anti-DNP IgE monoclonal 
antibody SPE7 (Sigma Chemical Co., St. Louis MO), and 
horseradish peroxidase (HRP) -labeled goat anti-mouse IgG 
(Kirkegaard and Perry Laboratories, Gaithersburg MD) was 
used for detection of captured mouse IgG. Thirteen out of 

10 

20 immunized mice had crossreactive antibodies for mouse 
IgE. Sera was pooled from seven mice showing ELISA titers 
against mouse IgE of >logio 2.3 for use as the site- 
specific anti-IgE. 

^ Another group of 10 balb/c mice was used to 

produce murine IgE. This group was sensitized by a single 
intraperitoneal administration of ovalbumin (Oa) on 0.4% 
Alum, 1.0 jig/0.2 ml. IgE content of the mouse sera was 

20 measured at day 20 by the whole IgE ELISA described in 

Example 5, except that captured IgE was detected by HRP- 
labeled sheep anti-mouse IgE supplied by The Binding Site 
Inc. (San Diego, CA) . Out of the 10 mice, 7 had 

2 5 appreciable IgE responses of titer >logi 0 1.6. These sera 

were pooled for use as the anti-Oa IgE working stock. 

The IgE serum pool was serially diluted 1:62, 
1:124 and 1:248 into PBS and then further diluted with an 
equal volume of the site-specific anti-IgE serum. Thus, 

30 

final dilutions for mouse IgE were 1:124, 1:248, and 1:496 
while mouse anti-IgE was diluted 1:2. Control dilutions 
of IgE were prepared having only PBS as diluent. 

The IgE dilutions, with and without anti-IgE 



35 



WO 99/67293 PCT/US99/13959 



- 64 - 

serum, were incubated for 1 hour at 37° and 50 nl of each 
was taken for evaluation by heterologous PCA reaction. 

Results 

5 The 50 |al samples of diluted mouse IgE were 

injected intradermally into the shaved back of rats in a 
pattern that was a set of two rows of four injections. 
The rows were a row of three controls of IgE diluted 
10 1:124, 1:248, and 1:496 in PBS only, in parallel with a 

row of the serially diluted IgE incubated with the site- 
specific anti-IgE. The fourth injection of each row was 
PBS only, as a control for the tissue trauma. The 
pattern was duplicated on two rats. 

15 

After 24 hours, PCA reactions were induced by 
intravenous injection of 1 mg of DNP-Oa conjugate in 1% 
Evans blue dye. One hour later, the rats were euthanized 
and skinned. The DNP-Oa allergen had crosslinked 

20 receptor-bound mouse anti-Oa IgE on the rat mast cells. 

The crosslinking triggered degranulation, increased 
permeability of the Evans blue dye, and the appearance of 
blue zones on the underside of the rat skins proportional 

25 to the extent of degranulation. However, wherever free 

IgE had been depleted by the site-specific murine anti- 
IgE, less was available to sensitize the rat mast cells 
and PCA reactions were suppressed. PCA reactions were 
evaluated by measuring the diameters of the blue zones on 

30 the undersides of the rat skins in two directions at right 

angles and taking the average. Results are shown in Table 
7 for the duplicate inhibition of PCA determinations on 
two rats. 



35 



WO 99/67293 



PCT/US99/13959 



- 65 - 

The rats differed by their inherent sensitivities 
to the mouse IgE so that control and anti-IgE inhibited 
PCA reactions should be compared only on the same rat. 
Mouse IgE-mediated PCA reactions were inhibited in both 
rats by the murine antiserum with specificity for the 
target antigenic site on mouse IgE. Thus, the antibody 
response that results from immunization by a peptide 
composition specific for the target antigenic site of a 
non-human IgE resulted in suppression of the inflammatory 
response mediated by the selfsame non-human IgE. 



WO 99/67293 



PCT/US99/13959 



-66- 





M 


> 


M 


> 




o 

H 




>* 


>* 


fa 


fa 






Eh 










Z 


w 


CO 


to 




m 




H 


H 


Eh 


Eh 






CM 


CM 


CM 


CM 




H 


s 


> 


> 








p; 




CO 


CO 






CO 


< 


Eh 






H 


p 


Q 


Q 








Q 


a 


w 


W 






u: 




o 


o 




U 


o 


Z 


Z 








CO 


CO 


CO 


CO 


o 
in 

CO 






X 


2; 


z 




cm 


a 


a 










iJ 


> 




2 




M 


> 


w 


a 


o 

VD 


Eh 


> 


w 


H 


















fa 


>H 


>* 


>* 


CN 






X 










x 


u 


a 


O 
















w 


rl 




o 










u 


a 


a 






a 


Q 


Q 


Q 




CO 


CO 


>* 


>* 








a 


a 


a 


w 






fa 


^ 








> 


M 


> 


H 




o 
o 




CO 


£h 


H 


H 






CM 


CM 


Cm 


CM 






h3 


fa 


fa 




ro 






M 


W 


H 






CO 


CO 


CO 


CO 






CJ 


CJ 


CJ 










Z 




z 






CM 


CM 


CM 


CM 


ro 
id 






i* 


>* 






















CM 


Cm 


CM 


CN 
















w 




Oi 




o 




CO 


CO 


H 


H 




o 


a 


a 


a 








CO 


CO 


CO 


CO 


ro 




























Oi 


X 


>i 


CJ 




>H 


>* 




>H 


o 
in 






H 










£-4 


H 


Eh 


Eh 






«t 


CO 


H 


H 


CN 


CM 




CO 


CO 








CO 


CO 


CO 


CO 






CO 


Eh 


H 


H 




04 


X 


X 


x 








ft 




< 


< 






> 


> 


> 


< 




1 


H 


i 


I 




o 




►4 


> 














o 


o 




fa 


Q 


fa 


fa 




CN 




w 




S*S 










oi 


p: 


oi 




X 


O 


< 










o 




O 








CM 


CM 


CM 


CM 




U 


> 












w 


u 


w 


w 








Q 




W 




CD 


CM 


CM 


CM 








o 




w 


w 


o 




CO 


CO 


o 


X 




O 


i 


I 


i 








H 




« 




ro 
ro 




Q 


W 


Q 


Q 




a 


2; 


Q 


Q 










Eh 


H 


M 




< 


CO 


CO 


CM 




u 


CJ 
i 


CJ 
I 


o 








CO 


O 
CM 


> 


l 






CJ 


U 
i 


CJ 
Oi 


U 

pi 


o 


CO 


CO 


CO 










< 


< 


> 


> 








i 


Oi 


p: 


CN 


CO 


CO 


CO 


M 








H 


Eh 


z 


Eh 






Eh 


i 


H 


Eh 




a 


X 


X 


CO 




o 

00 




CO 


>h 


a 


a 






CO 


• 


X 


X 






fa 


►J 


>* 




CN 






CM 


< 


< 






Q 


i 


< 


< 




M 


■J 


►J 


CO 








Q 


fa 


X 








W 


US 




•J 








Q 










> 


M 


H 


Eh 


O 




fa 


CtJ 




J* 




> 


> 


> 


CM 








D 


z 


Q 


Q 


CN 

ro 




Eh 




Z 


Q 




Eh 






CM 








£ 


H 


>H 


H 




X 






U 




CM 


CM 


Cm 


CO 








*^ 




)— ( 


M 






u 


o 


o 


O 




CM 


CM 




tJ 








i 






pa 






Of 




0 


o 


O 
CO 


H 


M 


Eh 


CO 








a 




Pi 


a: 








fa 


CO 


CO 


CN 


fa 


fa 


H 


X 








o 




o 


i— i 






tr* 


f_i 


£-4 






a 


Z 












Q 


a 


Q 


Q 






t 


fa 


i 


i 








> 


> 




O 

r- 




W 


> 


2 


S 






> 


o 


> 


> 




CO 


< 


Cm 


CM 




CN 










t-i 






a 






PJ 




u 


CJ 
















S 








u 


CJ 


o 


CJ 


CN 
CN 


> 




< 


> 








Eh 


M 


X 


CO 






Eh 


Eh 


Eh 


Eh 




H 




CN 


ro 








H 


CN 


















<D 
U 


1 




2 




1 






1 


i 


i 








i 


| 


i 


1 


w Q 

H 




Q 

H 


Q w 

M 


8 






e 


9 


Q 

H 


u> Q 
M 






a 


Q 

M 


Q 

M 


u Q 
H 


Sequ© 


Human 
(Seq 


Dog e 


o- " 

O 4-> 

s 3 


CD 

« £ 


(Seq 




1 Human 


(Seq 


Dog e 
(Seq 


aj a) 

S 2 


a) ^ 

3 CD 

2 W 




i 


(Seq 


w 

O 1 

m cu 


D 1 

S a 


CJ) 

D CD 

| 2 



WO 99/67293 



PCT/US99/13959 



-67- 















CU 


CU 


CU 




CU 






Q 








Z 






H 


CO 


CO 




< 


CU 


< 




< 






CU 


a 




cu 


CO 






H 


H 


H 




< 




CO 




CO 






•4 


H 




M 


H 




H 


H 


E- 


H 


Oi 


p; 


oi 




Oi 






O 


CU 










o 


O 


< 


< 




CU 








a 






> 


CO 










Z 


Z 


Z 


Z 




o 


o 


o 




u 






w 


Q 




CO 


a 




t 


fa 


i 


Z 




CO 


CU 






cu 


o 










P 


a 




OJ 


X 


X 






1 


i 


1 




a 


CO 




X 


Oi 




o 


o 




o 


Q 




X 




H 








H 












►4 






^ 


















« 


















o 
o\ 


w 


i 


1 








< 


E-» 




H 








o 


a 




o 




ro 


w 




H 






H 


H 


H 




H 








> 


> 




> 


> 




u: 


Z 


CO 


>* 




CO 


CO 


CO 




CO 








CO 


CO 




CO 


CO 




o: 




as 






Pi 


oi 


Oi 




Oi 








w 


H 




H 


H 




H 




a 


o» 


o 
ro 


s 


> 


> 




> 








Q 


Q 




Q 






CO 


a 


CO 


CO 






M 


M 




M 








ca 


< 






Ed 






Cu 


<c 






< 


Q 


CU 




CU 




o 




cu 


cu 




cu 


Cu 




Z 


z 


CO 


CO 




p: 








« 








£ 








{14 




> 


> 




> 




Cu 


CU 


CU 




CU 










En 






b 




i 


i 


w 


CO 












Pu 










Z 




z 


Z 




CU 


(X 


CO 


Eh 




w 




w 




Q 








a 


a 




a 


a 


o 

00 






« 








CU 


CU 




CU 








M 


M 




M 




ro 












s 








PJ 










(J 




•4 






CO 


CO 


Pi 


1 




H 




Q 




a 








CJ 


u 




U 








w 




W 


o 


> 


> 


> 




> 










Eh 




H 


H 






Pi 




O 


^ 


Pi 


o; 


oi 




M 










►J 










CO 


>* 


> 


Z 




U 


u 


u 




u 










Eh 






Eh 














O 


>* 


o 




o 








» 


> 




i 


l 




H 






H 






>< 










^ 




a; 


o: 




Oi 


Oi 








> 


> 




H 


E- 


o 




a 










Q 




i 


i 




z 


Z 








H 


W 


w 




















Ui 


o 


> 


1 


M 


> 




0 


o 


CD 












Q 


Eh 




z 


a 


ro 


H 
CD 


S 

o 


z 


Z 

>i 




W 

M 


w 

M 


Ex] 

H 




M 






Oi 

CO 


a 






CO 






w 


W 


i 


O 
H 


s 














o 


w 




Ex] 


Da 




CO 


s 


W 


W 


Tl* 


Q 


Q 


Q 




Q 






cu 


Da 






w 




o* 


E-* 


CO 


CO 




Pi 
















i 




1 






< 


< 


W 


W 




H 


H 


*< 




<< 






w 


w 




w 


CO 














O 




Q 




> 


o 




cu 


cu 




cu 


cu 




Q 


Q 


Q 


Q 




> 


> 


> 




> 


m 




H 


cu 




cu 


cu 




> 


> 




> 




04 


CU 


CU 




CU 












a 


cu 


O 
vo 


> 


> 


> 


> 




a 
















Pu 






b 


ro 


.4 




J 










H 




H 












> 


> 




U 


U 


U 


U 




CO 


CO 


CO 




CO 








>* 






>< 






H 


H 


JH 


O 
O 


Eh 


H 


H 




H 






> 


> 




> 


> 




H 


H 


■J 






> 


> 


M 




M 






w 


a 




Ei] 


Da 




H 




ro 






H (N ro 
















ro 








i 


1 






0 0 

? s : 


0 










£ 


s 




i 


i 




to Q 

1-4 


Q 

M 


Q 

M 


co Q 

H 




to Q Q 1 

H M » 


ij 
-« 


CO 


Q 

H 




to 


Q 

M 


Q 

M 




a 


to Q 

M 




Human 
(Seq ! 


Dog e 
(Seq : 


Rat e 
(Seq 


Mouse 
(Seq 




Human 
(Seq 
Dog e 
(Seq 
Rat 8 
(Seq 


Mouse 


(Seq 




i 


(Seq 


Dog e 
(Seq 


CO 

U 

m 

Oi 


(Seq 


g 8^ 

S 2 



WO 99/67293 



PCT/US99/13959 



-68- 



o 

CN 

in 



o 

H 

m 



o 
o 
in 



o 



ts 


u 


W 


w 








X 


> 


> 


H 


H 


< 


> 


> 


> 


OS 


O 


os 


a 


u 


u 


u 


u 


H 


H 


H 




fa 


fa 


fa 


fa 


W 


S*J 


i 


i 


Q 




o 


a 






^ 




w 




E-« 


OS 


o» 


O 


o 


o» 


1 


w 


ft 


H 










w 


D 








> 




H 


OS 


OS 








CO 






*> 


> 


> 


> 


w 


w 


Pa 


W 






►q 




os 


OS 


OS 


os 


CO 


CO 


CO 


CO 


fa 


fa 


fa 


fa 


> 


M 


M 


H 


fa 


fa 


fa 


fa 


fa 


fa 


fa 


fa 


O 


< 


OS 


o 






a 


a 


i 


OS 


S3 




co 


CO 


CO 


i 


o 


o 


o 


o 


NS 


CO 




z 


E-» 


> 




CO 


*S 






*S 


OS 






►J 






04 




t 


o 


1 




o 


H 


£h 






H 


H 


E-* 




H 


£-* 




CO 


i 


CO 


CO 


a 




X 




OS 


O 


a 


a 




Q 


CO 


CO 



cu 
> 

CO 

> 
< 

OS 

a 
> 

a 

CO 
CO 

< 



OS 


pS 


os 


CO 


04 


04 


o 


w 




CO 


OS 


o» 








< 




< 



tN 


CO 
















i 






0 


Q 


CO 


a 




M 


0) 


H 






cr 


cr 






4J 0) 


i 





8 



<D tn a> jj a> 3 a> 
w o w ^ co g co 



8 9 u 8 

« co a) 

o* tr w tr 

$ m 3» 4-> <u 2 <D 

co 0 co nj co o co 



WO 99/67293 



PCT/US99/13959 



-69- 




< 

co to 

M > y 



o 



o 
ro 





0) 

4J 
-H 
CO 



•H 



•H 

s 

w 



I 



-H 



s 



00 
CM 

£ Q 



CO 



a 2 



o 
ro 

S 



a: 



a h 



ro 



ro A 



ro 



n 
t 

GO 
(N 
ro 



CN 



CO 



s 1 



I 



s 

CO 



CN 



rH 

ro 



CO 

ro t 

5 $ 



CO 

r 



CO 

T 



CO 

t 



CO 

t 



g i 



WO 99/67293 



PCT/US99/13959 



-70- 



T3 
0) 



CM 



Cross-reactivity with 
hunan IgE 


? 

u 
u 

*H 

H W 

^ 01 

< M 

CO 3 

M •£ 

o 

1 


3.92 


r- 
ro 

ro 


3.49 


4.71 


3.76 


2.94 


4.31 


2.79 


3.77 


1.47 


0.77 


Immunostimulatory 
sequence attached 
to Target Antigenic 
Site 


KLH 




KLH 




8 
i 

o 

H 

CO 

a 


OK 

CQ 

S8 
8S 

H 


KLH 




KLH 


8 
i 

to 

s 


8 

, e 

8 2 

i £ 
S S 












O 






(0 


XI 


o 


IgE Derived Target Antigenic Site 


Amino Acid Sequence 


m 

9 2 

1 a 

501 o 

M 

1 

a* 

CO 

1 

O 


1 

CO 

i 

CO) ro 

1 s 

Q Q Q 


CADSNPRGVSAYLSRPSPFDLFIRKSPTI 

(SEQ ID NO:36) 


ro 

§ 

Q 

H 

8f 

co 

H 
H 

CO 

! 


CO 

ro 

8 

9 
g 
S ~ 

H 

a 

i 

n 


S Q 

i 8 

§ = 

i 

i 

i 

O H 


1 a 

1 s 

£ 8 

I 
1 

s 

II 

l! 


(C) KQRNGTLT (C) 

(SEQ ID NO:41) 


Entry No . ; 
Description t 


VD 

ro 
i 

ro 

iH 

ro ^ 
w CO 
m t 

6 H 


CM 
VO 

ro 
i 

H 
O 

ro _ 
w CO 
ro t 

n ~ 


CH2/3 (328-356) 


CH2/3 (317-356) 


CH2/3 (313-356) 


VD 

in 
ro 
1 

iH 

o 

ro ^ 

~ CO 

ro t 

5 ~ 


CH2/3 (301-376) 


(C)CH3 (391-398) 




00 


<n 


o 

rH 


rH 
rH 


H 


ro 

rH 





WO 99/67293 



PCT/US99/13959 



-71- 



•H 

« 1 
81 

i 

CO .£ 

CO 4J 



13 

XJ 

*H 



5 




4J O 4J " 

0) u <u 1 

M 01 O 
4-> 



•rt 
CO 

o 

•H 

tn 

-H 

a 



I 



ft 

tn 



co 

8 



3 

8 S 



"8 

CO 



£8 

01 I 



g 

Q 

M 

Of 

CO 



CO 

t 

i £ 
ro U 

H — ' 
— + 

5 r 

^ in 
O ro 



a 



U CO 



5i 



in 

CM 



cn 



3 



8 

i 



8 



8 



8 
8g 



8 

i 



8 

8£ 



8 



0 

CO 



s 

9 

CO 



§ 
s 

CO 



CO 

g 

Q 

IH 

& 

CO 



8 

CO 



o 



1 



e 

& 

CO 



in 
ro 



ro 

5 



i 

CM 

CO * 

ro — . 
in 

CO 

r> ro 
ro i 
ro 
ro co 

B? 



in 
ro 
i 

in 



ro 

5 



O 



ro 



ro 

5 



CN 



WO 99/67293 



PCT/US99/13959 



-72- 



t 

s 1 

85 



5 

0) 
■U 
•H 

H 

CO 

a 

o 
ft 

5 



u fi e 

O XS 0) 

u u O) 

(d to -h 

H iJ ii 



a> 

n o 



is 



ss 



98 
8 4 



Is 

H 

r 



01 fc 

98 
86 



o 



8 
& 



(0 

9 



CD 
4_> 
•rt 
CO 



(D 

S 

T? 

5 

-H 
J* 

ft 



0 

Of 

to 



O 

m 



a 
s 

CO 



g 
s 

8 

CO 



s 

CO 



(1) 

CO 
-H 



a 2 



O 

~ u 

5 S 



5i 



6 



i 

n 
r- 

U oo 











u 












m 


o\ 


r- 


vo 


r> 


m 


i 


i 


in 


in 


m 





in 
in 

8 

8 
8 

CO 



5 



WO 99/67293 



PCT/US99/13959 



-73- 



u 

0) 

Si? 



o 




CO 
U 

s 

d) 

1 

•H 

ft 



& -a 



S ? 



O 

ro 



^ O 



4 * ! 

ro i 

« i s 

■-• » 3 

to r " 

a & 



-8 



Q i 

a o 

CO 



•a * 

S o 



*H tQ 

ft Si 

St tj 

•a "u 

3 g 



■H 

00 



. 8 

U -H 
P J_> ^ 



£ " 



8 S 
8 s 



o 
c 

■rH 

8 



CO 

■9 



5 g 



CO 

5 I 



WO 99/67293 



PCT/US99/13959 



- 74 - 
Table 3 



Evaluation of Anti-IgE Antibodies 
for Inhibition of Histamine Release 



5 


IgE 

Antigen 
Entry 


IgE Antigen 
Description 


Immunogenic Elements 
Attached to IgE Antigen 


% 

Inhibition 
of 




No. 


{SEQ ID NO) 






Histamine 
Releaset 




1 


CH2/3 (328-376) (G, 58 -+S) 

(SEQ ID NO:28) 


a 


KLH 


0 • 


10 


2 


CH2/3 (317-376) (Q 58 -*S) 


a 


KLH 


14% 






/CFO ID NO'29) 


b 


1,4,9 PALINDROMIC Tn-GG- 


17% and 0 




5 


CH2/3 (328-362) (Q 59 ->S) 

(SEQ ID NO:32) 


a 


KLH 


0 




6 


CH2/3 (317-362) (C, 5 a->S) 

(SEQ ID NO:33) 


a 


KLH 


0 


15 


7 


CH2/3 (313-362) (G, 5 8-+S) 

(SEQ ID NO:34) 


a 


KLH 


6% 




8 


CH2/3 (301-362) (Q 58 -»S) 

(SEQ ID NO:35) 


a 


KLH 


6% 




11 


CH2/3 (313-356) 

(SEQ ID NO:38) 


a 


KLH 


6% 


20 


15 


(C)CH3 (413-435) (C) * 

(C<18->S) 


b 


Syn Th(l,2,4)-GG 


58%* and 








c 


Inv-GG-Syn Th ( 1 , 2 , 4 ) -GG- 


71%-e 






(SEQ ID N0:5) 










20 


(C)CH3 (374-382-(C)-383- 
385)* 


b 


HBs 19 _32Tn-GG 


0 


25 




(SEQ ID NO:46) 








30 


CH3 (399-424) 

(SEQ ID NO:55) 


b 


HBs x9 -32Th-GG- 


9% and 0 




32 


(OCH3 (370-390) (C) * 

(SEQ ID NO:57) 


b 


HBs ig - 32 Th-GG- 


0 



* Cyclized peptide 
30 (C) Cysteine introduced into native sequence for cyclization 
(C->S) Serine substituted for cysteine ssidue 
% Results are shown for pooled anti-15b and anti-15c IgG's. 

t Histamine release inhibition by antibodies to peptides, purified from serum 
collected at week 8, except as otherwise noted by© 

© Histamine release inhibition by antibodies to peptides, azllected at week 

35 n - 



WO 99/67293 



PCT/US99/13959 



§ 4 

o oi 



CO 



a 

8 

CO 



-75- 



CO H 



2 H 



crv 

H 

8 

a 

8 

CO 



o 



M 

B 



w 

CO 



CO 




S S S 

S 1 s s 



3 8 
ti 

in 

g 

Q 

H 

8 

CO 



8 
8 

co ^ s 



CO » 

i w n 

8? * 

I rH CO 

si 8 

eg S 



as 

S £ ° 

(X T3 • • 
<D D 

- m-i _ 

^•H Q 

- rH M 

dip 

co -h ra 

H 01 M 



p s 

h *h 55 

CO -H Q 

HH H 
I 

& .. 

M 03 CO 



8 



8 

8 
8 

CO 



. 58 * 

i-l ^ -h Q 

°* H Q, 

" — M CD CO 



in 



8 

co 



WO 99/67293 



PCT/US99/13959 



-76- 



<D 
3 

4J 

o 
u 



Eh 



8 £ 



8 

CO 



co H 

H 
CO 



s 

CO 



H 



8 

o 

•»- H 

o> a 

* H 
— CO 



J? CO 

- Q 

H 



S8 



l CO 

£8S 

i 

H h m 

CU - — • H 

sis 



H M-l 
-— -H 
CO f-H 

St 

ea . 



as 




8 

1 




O 

VD 






g 


2 8 


- IM 
tJ» *rt 
* »-t 


8 


I Q 


H gi 

CO -H 
H DQ 


& 

CO 


& Of 

CO CO 



8 



CO CO 



8 

e 
s 

CO 



a 



a 

CO 



WO 99/67293 



PCT/US99/13959 



-77- 



B 



Bis 



w 
to 



ii * 



e 

— H 

|§ 

is 

to 

to CD 
M O 



g 

CO 



M Si 
Q w 

X 



to CO 

t I 

I I 1 
j5 1 



H * CO 

§=*g 



a 



CO 



00 
CO 

8 

o e 

CO 



c to 

H 

£ * - 

H N (N 
H H " 



CO 



§ 

a 




"5 



WO 99/67293 



PCT/US99/13959 



- 78 - 
Table 4B 



Immunogenicity of Representative Peptide Constructs 
of the Invention 



SEQ ID NO 
of peptide contructs 


Species 
immunized 


Site-directed 
crossreactivity to IgE 
(Loq 10 titer) 


% HR C 


%HR d 
inhibition 


Human IgE 
Target 


SEQ ID NO: 18 


GP* 


4.4 e 


1 


96 


SEQ ID NO:85 


GP a 


4.2 e 


3 


87 


SEQ ID NO: 18 


Pig a 


4.1 e 


3 


84 


SEQ ID NO: 18 


Baboon* 


4.8 e 


8 


53 


Dog IgE 
Target 


SEQ ID NO: 87 


GP* 


3.4 £ 


NT 


NT 


SEQ ID NO: 88 


GP 6 


3.2 £ 


NT 


NT 


SEQ ID NO: 90 


GP 6 


3.2 ( 


NT 


NT 


SEQ ID NO: 91 


GP* 


3.2 f 


NT 


NT 



a Guinea pigs, pigs and baboon were immunized with human IgE 
15 peptide constructs at 0, 3 and 6 weeks, with sera collected at 8 wpi 
for testing by human IgE ELISA and inhibition of HR. 
b Guinea pigs were immunized with dog IgE peptide constructs at 
0, 2 and 4 weeks with sera collected at 6 wpi for dog IgE 

ELISA. 

c Average % HR. 

d % HR inhibition = control - %HR/control x 100 

20 

GP: Guinea pig 
NT: Not tested 



25 



30 



35 



WO 99/67293 



PCT/US99/13959 



- 79 - 
Table 5 

o Amino Acid Sequences of 

Foreign Pathoqen^Derived Th Epitopes 



5 



10 



20 



Description of Th 


SEQ ID NO 


Amino Acid Sequences 


MVF 2 88-302 Th 


01 


T ^FTfCCTTVHRLEGV 
jjO Hi it\u v x vni\jju<jv 


urn r* TV* 

MVF 25 8-277 Tn 






i 1 830-644 ^n 


DO 


I ilVrtlN Dive X Ui 1 CtAJ 


941-966 An 


C A 
OH 


rviM. IN in r Ivor v»,ur\ vriw ori«jxi u 


PT 149 -i76 Th 


c c 
DO 


WT DDT T VMTVMCPT &\7R VHV9 KTFriYYnY 


TT73-99 Th 


66 


vriDMVT DTncn^nurTOTMUTfT "PMPTfc* 


PTis-ii Th 


67 


/"■7VV7V OPDMrTD TV T TVZVTTT D/^MZiPT 

bAYAKLrNo 1 tinh I VttilxjtvjiMMCiii 


HBs 19 -32 Th 


68 


FFLLTRILT1 r\Jo.LU 


HBc 120 -ho Th 


69 


VSFGVWIRTPPAYRPPNAPIL 


HBc 2 i-g 0 Th 


70 


SDFFPSVRDLLDTASALYRE 


HBc 50 -69 Th 


71 


PHHTALRQAI LCWGELMTLA 


TT 615 . 63 i Th 


72 


WVRDI I DDFTNESSQKT 


MTTT — — A 1 rpU / M _ \ 

MIV Cfpdi Irlg IN J 


7^ 


RAGRAILHI PTRIROGLER 


HIV gp41 Th 6 (C-) 


74 


AVAEGTDRV I EVLQRAGRAI L 


CT A8106-130 Th 


75 


ALNIWDRFDVFTLGATSGYLKGNS 


CT Pll Th 


12 


TINKPKGYVGKE 


DTI Th 


76 


DSET ADNLEKTVAALS I LPGHG 


DT4 Th 


77 


EEIVAQSIALSSLMVAQAIPLVGELVDIGFAATNFVESC 


PF Th 


78 


DIEKKIAKMEKASSVFNVVNS 


SM Th 


79 


KWFKTNAPNGVDEKIRI 


TraTl Th 


80 


GLQGK I ADAVKAKG 


TraT4 Th 


81 


GLAAGLVGMAADAMVEDVN 


TraT6 Th 


82 


STETGNQHHYQTRWSNANK 


SMTPITh 


86 


KWFKTNAPNGVDEKIRI 



25 



30 



35 



WO 99/67293 



PCT/US99/13959 



- 80 - 



Table 6 

Amino Acid Sequences of 
Representative Artificial Th Epitopes and SSAL 



5 



Description of Th 


SEQ ID NO: 


Amino Acid Sequence 


(1,4,9 PALINDROMIC) Th 


10 


ISEIKGVIVHKIEGI 
MT RT TRM TM 
L L V 


Syn Th(l f 2 f 4) 


9 


KKKIITITRIITIITTID 


IS (1,4, 9 PALINDROMIC) LF 
simplified Th 


60 


ISISEIKGVIVHKIEGILF 
T RT TR T 


IS (1,4, 9 PALINDROMIC) LF Th 


11 


ISISEIKGVIVHKIEGILF 
MT RT TRM TM 
L L V 


ArtMVF Th 


89 


ISLTEIRTVIVTRLETVLF 



15 



20 



25 



30 



35 



WO 99/67293 



PCT/US99/13959 



Table 7 
Inhibition of PCA Reaction 





Rat #5 


Rat #6 


IgE 


No Anti-IgE 


Anti-IgE 


No Anti-IgE 


Anti-IgE 


Dilution 


(mm) 


1:2 (mm) 


(mm) 


1:2 (mm) 


0 


0 


0 


0 


0 


1:496 


0 


0 


4.3 


0 


1:248 


0 


0 


7.0 


6.0 


1:124 


11 


4* 


13.0 


12.7 



* very pale blue 

10 



15 



20 



25 



35 



WO 99/67293 82 PCT/US99/13959 



10 



25 



30 



CLAIMS 

We claim: 

1. An IgE-CH3 domain antigen peptide between about 
25 and about 29 amino acids in length containing two 
cysteine residues separated by about 23 amino acid 
residues, selected from the group consisting of SEQ ID 
NO:5, SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:84, 
homologous sequences from the epsilon heavy chain of 
mammalian IgE-CH3, and crossreactive and immunologically 
functional analogs thereof. 



2. An IgE-CH3 domain antigen peptide of claim 1 
selected from the group consisting of SEQ ID NO: 5, SEQ ID 

15 NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 84. 

3. A synthetic peptide of about 50 to about 90 amino 
acids, which comprises 

20 (a) a helper T cell (Th) epitope, 

(b) an IgE-CH3 domain antigen peptide according to 
claim l;and 

(c) an immunostimulatory invasin domain. 



4. A peptide conjugate comprising a helper T cell 
epitope sequence (Th) covalently attached to an IgE-CH3 
domain antigen peptide according to claim 1. 

5. A peptide conjugate represented by the formula 

(A) n - (IgE-CH3 domain antigen) - (B) 0 - (Th) m -X 



35 



WO 99/67293 PCT/US99/13959 

83 



15 



o or 

(A) n -(Th) m -{B)o-(IgE-CH3 domain antigen)-X 

wherein 

each A is independently an amino acid or a general 
5 immunostimulatory sequence; 

each B is chosen from the group consisting of amino 
acids, -NHCH(X)CH 2 SCH 2 CO-, -NHCH (X) CH 2 SCH 2 CO (e-N) Lys-, 

10 -NHCH (X)CH 2 S-succinimidyl (e-N) Lys-, and -NHCH (X) CH 2 S- 

(succinimidyl) -; 

each Th is independently a sequence of amino acids 
that constitutes a helper T cell epitope, or an immune 
enhancing analog or segment thereof; 

IgE-CH3 domain antigen represents the sequence of an 
IgE-CH3 domain antigen peptide according to claim 1; 
X is an amino acid a-COOH or a-CONH 2 ; 
n is from 0 to about 10; 
m is from 1 to about 4; and 
o is from 0 to about 10. 

25 6. A peptide conjugate represented by the formula 

(IgE-CH3 domain antigen) - (B.) 0 ~ (Th) m - (A) n -X 

or 

(Th) m - (B) 0 - (IgE-CH3 domain antigen) - (A) n -X 

30 wherein 

each A is independently an amino acid or a general 
immunostimulatory sequence; 



20 



35 



WO 99/67293 ^ M PCT/US99/13959 

84 



o each B is chosen from the group consisting of amino 

acids, -NHCH<X)CH 2 SCH 2 CO-, -NHCH (X) CH 2 SCH 2 CO (e-N) Lys-, 
-NHCH (X) CH 2 S-succinimidyl (e-N) Lys-, and -NHCH (X) CH 2 S- 
( succinimidyl ) - ; 

5 each Th is independently a sequence of amino acids 

that constitutes a helper T cell epitope, or an immune 
enhancing analog or segment thereof; 

IgE-CH3 domain antigen represents the sequence of an 
IqE-CH3 domain antigen peptide according to claim 1; 

10 

X is an amino acid a-COOH or a-CONH 2 ; 

n is from 0 to about 10; 

m is from 1 to about 4; and 

o is from 0 to about 10. 

7. A peptide conjugate of any one of claims 4-6 
wherein said Th is an SSAL. 

20 

8. A peptide conjugate of any one of claims 4-6 
wherein said IgE-CH3 domain antigen peptide has an amino 
acid sequence selected from the group consisting of SEQ ID 
N0:5, SEQ ID N0:6, SEQ ID N0:7, SEQ ID NO:8, and SEQ ID 

25 

NO:84 . 

9. A peptide conjugate of claim 7 wherein said IgE- 
CH3 domain antigen peptide has an amino acid sequence 

30 selected from the group consisting of SEQ ID NO: 5, SEQ ID 

N0:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:84. 



35 



WO 99/67293 _ c PCT/US99/13959 

ob 



10 



25 



30 



10. A peptide conjugate of any one of claims 4-6 
wherein said Th has an amino acid sequence selected from 
the group consisting of SEQ ID NOS: 9-12 and SEQ ID NOS: 
61-82 and 84. 

11. A peptide conjugate of claim 7 wherein said Th 
has an amino acid sequence selected from the group 
consisting of SEQ ID NOS: 9-12 and SEQ ID NOS: 61-82 and 
84. 



12. A peptide conjugate of claim 8 wherein said Th 
has an amino acid sequence selected from the group 
consisting of SEQ ID NOS: 9-12 and SEQ ID NOS: 61-82 and 

15 84. 

13. A peptide conjugate of claim 9 wherein said Th 
has an amino acid sequence selected from the group 

20 consisting of SEQ ID NOS: 9-12 and SEQ ID NOS: 61-82 and 

84. 



14. A peptide comprising an amino acid sequence 
selected from the group consisting of SEQ ID NOS: 14, 15, 
17-27, 85, 87, 88, 90, 91. 

15. A peptide conjugate of claim 5 or 6, wherein at 
least one A is an invasin domain. 

16. A peptide conjugate of claim 5 or 6 wherein n is 
3, and (A) 3 is (invasin domain) rGly-Gly. 



35 



WO 99/67293 PCT/US99/13959 

86 



10 



15 



20 



25 



30 



17. A peptide conjugate of . claim 15 wherein said 
invasin domain has the amino acid sequence of SEQ ID 
NO:13. 

18. A peptide conjugate of claim 16 wherein said 
invasin domain has the amino acid sequence of SEQ ID 
NO:13. 

19. A peptide conjugate comprising a carrier protein 
covalently attached to one or more IgE-CH3 domain antigen 
peptides according to claim 1. 

20. The peptide conjugate of claim 19 wherein the 
carrier protein is keyhole limpet hemocyanin. 

21. A peptide comprising an amino acid sequence 
selected from the group consisting of SEQ ID NOS:14, 15, 
26, 90. 

22. A branched polymer comprising a lysine, 
trilysine, or heptalysine core, covalently attached to 
two, four, or eight peptide conjugates, respectively, of 
any one of claims 4-6 or 14. 

23. A polymer comprising one or more peptide 
conjugates of any one of claims 4-6 or 14, cross-linked by 
a bifunctional crosslinking agent. 



35 



WO 99/67293 



87 



PCT/US99/13959 



10 



20 



24. A pharmaceutical composition comprising an 
immunologically effective amount of a peptide or peptide 
conjugate of any one of claims 4-6 or 14, and a 
pharmaceutical^ acceptable carrier. 

25. A pharmaceutical composition of claim 23, 
wherein said immunologically effective amount of said 
peptide or peptide conjugate is between about 0.5 ^g and 
about 1 mg per kilogram body weight per dose. 



26. A method for inducing anti-IgE antibody 
production in a mammal which comprises administering to 
15 said mammal a pharmaceutical composition of claim 23. 



27. A method for inducing anti-IgE antibody 
production in a mammal which comprises administering to 
said mammal a pharmaceutical composition of claim 24. 



28. A nucleic acid comprising a sequence which 
encodes a peptide of any one of claims 1-6. 



25 



30 



35 



WO 99/67293 PCT/US99/139S9 

SEQUENCE LISTING 

o 

(1) GENERAL INFORMATION: 

(i) APPLICANT: UNITED BIOMEDICAL INC., et al. 

(ii) TITLE OF INVENTION: PEPTIDE COMPOSITION AS 
5 IMMUNOGEN FOR THE TREATMENT OF ALLERGY 

(iii) NUMBER OF SEQUENCES: 91 

(iv) CORRESPONDENCE ADDRESS: 

10 (A) ADDRESSEE: Morgan & Finnegan 

(B) STREET: 345 Park Avenue 

(C) CITY: New York 

(D) STATE: NY 

(E) COUNTRY: USA 

(F) ZIP : 10154-0053 

15 

(v) COMPUTER READABLE FORM: 

(A) MEDIUM TYPE: Floppy disk 

(B) COMPUTER: IBM PC compatible 

(C) OPERATING SYSTEM: PC-DOS/MS-DOS 
20 (D) SOFTWARE: WORD 8.0 



(vi) CURRENT APPLICATION DATA: 

(A) APPLICATION NUMBER: To be assigned 

(B) FILING DATE: 21-JUNE-1999 

(C) CLASSIFICATION: 

25 

(vii) PRIOR APPLICATION DATA: 

(A) APPLICATION NUMBER: US 09/100,287 

(B) FILING DATE: 20-JUN-1998 

(C) CLASSIFICATION: 514 

30 

(viii) ATTORNEY/AGENT INFORMATION: 

(A) NAME: MARIA C.H.LIN 

(B) REGISTRATION NUMBER: 29,323 

(C) REFERENCE/ DOCKET NUMBER: 1151-4153PC1 



35 



WO 99/67293 PCT/US99/13959 



10 



15 



20 



25 



(ix) TELECOMMUNICATION INFORMATION: 

(A) TELEPHONE: 212-758-4800 

(B) TELEFAX: 212-751-6849 

(2) INFORMATION FOR SEQ ID NO:l: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 325 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

(ix) FEATURE: 

(A) NAME/KEY: i chain of human IgE 

(x) REFERENCE: Dorrington and Bennich, Immunol Rev, 

1978, 41:3-25. 

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



Val Cys Ser Arg Asp Phe Thr Pro Pro Thr Val Lys 

1 5 10 

He Leu Gin Ser Ser Cys Asp Gly Gly Gly His Phe 

15 20 
Pro Pro Thr He Gin Leu Leu Cys Leu Val Ser Gly 

25 30 35 

Tyr Thr Pro Gly Thr He Asn He Thr Trp Leu Glu 

40 45 
Asp Gly Gin Val Met Asp Val Asp Leu Ser Thr Ala 
50 55 60 

Ser Thr Thr Gin Glu Gly Glu Leu Ala Ser Thr Gin 

65 70 
Ser Glu Leu Thr Leu Ser Gin Lys His Trp Leu Ser 
30 75 80 

Asp Arg Thr Tyr Thr Cys Gin Val Thr Tyr Gin Gly 

85 90 95. 

His Thr Phe Glu Asp Ser Thr Lys Lys Cys Ala Asp 
100 105 



35 



WO 99/67293 



PCT/US99/13959 



10 



15 



20 



25 



30 



Ser Asn 
110 
Pro Ser 



Pro Arg Gly Val 



Pro Phe Asp Leu 
125 

Thr lie Thr Cys Leu Val 
135 

Lys Gly Thr Val Asn Leu 
145 150 
Gly Lys Pro Val Asn His 
160 

Lys Gin Arg Asn Gly Thr 
170 

Leu Pro Val Gly Thr Arg 
185 

Thr Tyr Gin Cys Arg Val 
195 

Arg Ala Leu Met Arg Ser 
205 210 
Pro Arg Ala Ala Pro Glu 
220 

Pro Glu Trp Pro Gly Ser 
230 

Ala Cys Leu lie Gin Asn 
245 

Ser Val Gin Trp Leu His 
255 

Asp Ala Arg His Ser Thr 
265 270 
Lys Gly Ser Gly Phe Phe 
280 

Val Thr Arg Ala Glu Trp 
290 

lie Cys Arg Ala Val His 
305 

Gin Thr Val Gin Arg Ala 
315 

Lys 
325 



Ser Ala Tyr Leu Ser Arg 
115 120 
Phe lie Arg Lys Ser Pro 
130 

Val Asp Leu Ala Pro Ser 
140 

Thr Trp Ser Arg Ala Ser 
155 

Ser Thr Arg Lys Glu Glu 
165 

Leu Thr Val Thr Ser Thr 
175 180 
Asp Trp He Glu Gly Glu 
190 

Thr His Pro His Leu Pro 
200 

Thr Thr Lys Thr Ser Gly 
215 

Val Tyr Ala Phe Ala Thr 
225 

Arg Asp Lys Arg Thr Leu 
235 240 
Phe Met Pro Glu Asp He 
250 

Asn Glu Val Gin Leu Pro 
260 

Thr Gin Pro Arg Lys Thr 
275 

Val Phe Ser Arg Leu Glu 
285 

Gin Glu Lys Asp Glu Phe 
295 300 
Glu Ala Ala Ser Pro Ser 
310 

Val Ser Val Asn Pro Gly 
320 



35 



(2) INFORMATION FOR SEQ ID NO: 2: 



WO 99/67293 



4 



PCT/US99/13959 



10 



20 



(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 312 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

(ix) FEATURE: 

(A) NAME /KEY : i chain of dog IgE 

(x) REFERENCE: Patel et al., Immunogenetics, 

1995; 41:282-286. 

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



Ala Cys Ala Leu Asn Phe He Pro Pro Thr Val Lys 
15 10 
15 Leu Phe His Ser Ser Cys Asn Pro Val Gly Asp Thr 

15 20 
His Thr Thr He Gin Leu Leu Cys Leu He Ser Gly 

25 30 35 

Tyr Val Pro Gly Asp Met Glu Val He Trp Leu Val 

40 45 
Asp Gly Gin Lys Ala Thr Asn lie Phe Pro Tyr Thr 
50 55 60 

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

65 70 
His Ser Glu Leu Asn He Thr Gin Gly Glu Trp Val 
25 75 80 

Ser Gin Lys Thr Tyr Thr Cys Gin Gly Phe Thr Phe 

85 90 95 

Lys Asp Glu Ala Arg Lys Cys Ser Glu Ser Asp Pro 
100 105 
30 Arg Gly Val Thr Ser Tyr Leu Ser Pro Pro Ser Pro 

110 115 120 

Leu Asp Leu Tyr Val His Lys Ala Pro Lys lie Thr 

125 130 
Cys Leu Val Val Asp Leu Ala Thr Met Glu Gly Met 
135 140 

35 



WO 99/67293 PCT/US99/13959 



10 



15 



20 



25 



30 



Asn Leu Thr Trp Tyr Arg Glu Ser Lys Glu Pro Val 
145 150 155 

Asn Pro Gly Pro Leu Asn Lys Lys Asp His Phe Asn 

160 165 
Gly Thr He Thr Val Thr Ser Thr Leu Pro Val Asn 
170 175 180 

Thr Asn Asp Trp He Glu Gly Glu Thr Tyr Tyr Cys 

185 190 
Arg Val Thr His Pro His Leu Pro Lys Asp He Val 

195 200 
Arg Ser He Ala Lys Ala Pro Gly Lys Arg Ala Pro 
205 210 215 

Pro Asp Val Tyr Leu Phe Leu Pro Pro Glu Glu Glu 

220 225 
Gin Gly Thr Lys Asp Arg Val Thr Leu Thr Cys Leu 
230 235 240 

lie Gin Asn Phe Phe Pro Ala Asp He Ser Val Gin 
245 250 

Trp Leu Arg Asn Asp Ser Pro He Gin Thr Asp Gin 

255 260 
Tyr Thr Thr Thr Gly Pro His Lys Val Ser Gly Ser 
265 270 275 

Arg Pro Ala Phe Phe lie Phe Ser Arg Leu Glu Val 

280 285 
Ser Arg Val Asp Trp Glu Gin Lys Asn Lys Phe Thr 
290 295 300 

Cys Gin Val Val His Glu Ala Leu Ser Gly Ser Arg 
305 310 



(2) INFORMATION FOR SEQ ID NO: 3: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 313 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 



35 



WO 99/67293 



6 



PCT/US99/13959 



10 



15 



(ix) FEATURE: 
(A) NAME /KEY : i chain of rat IgE 

(x) REFERENCE: Steen et al., J Mol Biol, 1984; 
177:19-32. 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: 

Ala Arg Pro Val Asn He Thr Lys Pro Thr Val Asp 

1 5 10 

Leu Leu His Ser Ser Cys Asp Pro Asn Ala Phe His 

15 20 
Ser Thr He Gin Leu Tyr Cys Phe Val Tyr Gly His 

25 30 35 

He Gin Asn Asp Val Ser He His Trp Leu Met Asp 

40 45 
Asp Arg Lys He Tyr Asp Thr His Ala Gin Asn Val 
50 55 60 

Leu He Lys Glu Glu Gly Lys Leu Ala Ser Thr Tyr 

65 70 
Ser Arg Leu Asn He Thr Gin Gin Gin Trp Met Ser 

75 80 
Glu Ser Thr Phe Thr Cys Lys Val Thr Ser Gin Gly 

85 90 95 

Glu Asn Tyr Trp Ala His Thr Arg Arg Cys Ser Asp 

100 105 
Asp Glu Pro Arg Gly Val He Thr Tyr Leu He Pro 
110 H5 120 

Pro Ser Pro Leu Asp Leu Tyr Glu Asn Gly Thr Pro 
25 125 130 

Lys Leu Thr Cys Leu Val Leu Asp Leu Glu Ser Glu 

135 140 
Glu Asn He Thr Val Thr Trp Val Arg Glu Arg Lys 
145 150 155 

30 Lys Ser He Gly Ser Ala Ser Gin Arg Ser Thr Lys 

160 165 
His His Asn Ala Thr Thr Ser He Thr Ser He. Leu 
170 175 . 180 

Pro Val Asp Ala Lys Asp Trp He Glu Gly Glu Gly 
185 190 



20 



WO 99/67293 PCT/US99/13959 



Tyr Gin Cys Arg Val Asp His Pro His Phe Pro Lys 

195 200 
Pro He Val Arg Ser He Thr Lys Ala Leu Gly Leu 
205 210 215 

Arg Ser Ala Pro Glu Val Tyr Val Phe Leu Pro Pro 

220 225 
Glu Glu Glu Glu Lys Asn Lys Arg Thr Leu Thr Cys 
230 235 240 

Leu He Gin Asn Phe Phe Pro Glu Asp He Ser Val 

245 250 
Gin Trp Leu Gin Asp Ser Lys Leu He Pro Lys Ser 

255 260 
Gin His Ser Thr Thr Thr Pro Leu Lys Thr Asn Gly 
265 270 275 

Ser Asn Gin Arg Phe Phe lie Phe Ser Arg Leu Glu 

280 285 
Val Thr Lys Ala Leu Trp Thr Gin Thr Lys Gin Phe 
290 295 300 

15 Thr Cys Arg Val He His Glu Ala Leu Arg Glu Pro 

305 310 

Arg 



10 



20 



(2) INFORMATION FOR SEQ ID NO: 4: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 313 amino acids 
<B) TYPE: amino acid 
(D) TOPOLOGY: linear 

25 (ii) MOLECULE TYPE: protein 

(ix) FEATURE: 

(A) NAME/KEY: i chain of mouse IgE 



30 



(x) REFERENCE: Ishida et al., EMBO, 1982 j 

1:1117-1123 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: 



35 



WO 99/67293 



8 



PCT/US99/13959 



10 



15 



20 



25 



30 



Val Arg Pro Val Thr His Ser Leu Ser Pro Pro Trp 

1 5 10 

Ser Tyr Ser He His Arg Cys Asp Pro Asn Ala Phe 

15 20 
His Ser Thr He Gin Leu Tyr Cys Phe He Tyr Gly 

25 30 35 

His He Leu Asn Asp Val Ser Val Ser Trp Leu Met 

40 45 
Asp Asp Arg Glu He Thr Asp Thr Leu Ala Gin Thr 
50 55 60 

Val Leu He Lys Glu Glu Gly Lys Leu Ala Ser Thr 

65 70 
Cys Ser Lys Leu Asn He Thr Glu Gin Gin Trp Met 

75 80 
Ser Glu Ser Thr Phe Thr Cys Arg Val Thr Ser Gin 

85 90 95 

Gly Cys Asp Tyr Leu Ala His Thr Arg Arg Cys Pro 

100 105 
Asp His Glu Pro Arg Gly Ala He Thr Tyr Leu He 
110 115 120 

Pro Pro Ser Pro Leu Asp Leu Tyr Gin Asn Gly Ala 

125 130 
Pro Lys Leu Thr Cys Leu Val Val Asp Leu Glu Ser 

135 140 
Glu Lys Asn Val Asn Val Thr Trp Asn Gin Glu Lys 
145 150 155 

Lys Thr Ser Val Ser Ala Ser Gin Trp Tyr Thr Lys 

160 165 
His His Asn Asn Ala Thr Thr Ser He Thr Ser He 
170 175 180 

Leu Pro Val Val Ala Lys Asp Trp He Glu Gly Tyr 

185 190 
Gly Tyr Gin Cys He Val Asp Arg Pro Asp Phe Pro 

195 200 
Lys Pro He Val Arg Ser He Thr Lys Thr Pro Gly 
205 210 215 

Gin Arg Ser Ala Pro Glu Val Tyr Val Phe Pro Pro 

220 225 
Pro Glu Glu Glu Ser Glu Asp Lys Arg Thr Leu Thr 
230 235 240 



35 



WO 99/67293 



9 



PCT/US99/13959 



10 



20 



Cys Leu He Gin Asn Phe Phe Pro Glu Asp He Ser 

245 250 
Val Gin Trp Leu Gly Asp Gly Lys Leu He Ser Asn 

255 260 
Ser Gin His Ser Thr Thr Thr Pro Leu Lys Ser Asn 
265 270 275 

Gly Asn Gin Gly Phe Phe He Phe Ser Arg Leu Glu 

280 285 
Val Ala Lys Thr Leu Trp Thr Gin Arg Lys Gin Phe 
290 295 300 

Thr Cys Gin Val He His Glu Ala Leu Gin Lys Pro 
305 310 

Arg 



(2) INFORMATION FOR SEQ ID NO: 5: 



(i) SEQUENCE CHARACTERISTICS: 
15 (A) LENGTH: 25 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 



(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: 



Cys Gly Glu Thr Tyr Gin Ser Arg Val Thr His Pro 

1 5 10 

His Leu Pro Arg Ala Leu Met Arg Ser Thr Thr Lys 
25 15 20 

Cys 
25 



(2) INFORMATION FOR SEQ ID NO: 6: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 25 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 



10 

WO 99/67293 PCT/US99/13959 



10 



15 



20 



25 



30 



<ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: 

Cys Gly Glu Thr Tyr Tyr Ser Arg Val Thr His Pro 

1 5 10 

His Leu Pro Lys Asp He Val Arg Ser He Ala Lys 
15 20 

Cys 
25 

(2) INFORMATION FOR SEQ ID NO: 7: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 25 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

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

Cys Gly Glu Gly Tyr Gin Ser Arg Val Asp His Pro 

1 5 10 

His Phe Pro Lys Pro He Val Arg Ser He Thr Lys 
15 20 

Cys 
25 

(2) INFORMATION FOR SEQ ID NO: 8: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 25 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

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



35 



11 

WO 99/67293 PCT/US99/13959 



Cys Gly Tyr Gly Tyr Gin Ser He Val Asp Arg Pro 

1 5 10 

Asp Phe Pro Lys Pro He Val Arg Ser He Thr Leu 
15 20 

Cys 
25 



(2) INFORMATION FOR SEQ ID NO: 9: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 18 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

15 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: 

Lys Lys Lys He He Thr He Thr Arg He He Thr 

15 10 
He He Thr Thr He Asp 



(2) INFORMATION FOR SEQ ID NO: 10: 

(i) SEQUENCE CHARACTERISTICS: 
25 (A) LENGTH: 15 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 



30 



(ii) MOLECULE TYPE: peptide 

(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 1 



35 



WO 99/67293 



12 



PCT/US99/13959 



(D) OTHER INFORMATION: /note= "lie, Met or 

Leu" 

(ix) FEATURE: 

(A) NAME /KEY: Modif ied-site 

(B) LOCATION: 2 
(D) OTHER INFORMATION: /note= "Ser or Thr" 

(ix) FEATURE: 

(A) NAME /KEY : Modif ied-site 

(B) LOCATION: 5 

(D) OTHER INFORMATION: /note= "Lys or Arg" 

(ix) FEATURE: 

(A) NAME /KEY : Modif ied-site 

(B) LOCATION: 6 

(D) OTHER INFORMATION: /note= "Gly or Thr" 

15 (ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 10 

(D) OTHER INFORMATION: /note= "His or Thr" 



10 



20 



(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 11 

(D) OTHER INFORMATION: /note= "Lys or Arg" 

(ix) FEATURE: 
25 (A) NAME/KEY: Modif ied-site 

(B) LOCATION: 12 

(D) OTHER INFORMATION: /note= "lie, Met or Leu" 



30 



35 



(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 14 

(D) OTHER INFORMATION: /note= "Gly or Thr' 

(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 



13 

WO 99/67293 PCT/US99/13959 



(B) LOCATION: 15 

(D) OTHER INFORMATION: /note- "lie, Met or Val" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: 



Xaa Xaa Glu He Xaa Xaa Val He Val Xaa Xaa Xaa 

1 5 10 

Glu Xaa Xaa 
15 



(2) INFORMATION FOR SEQ ID NO: 11: 

10 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 19 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

15 (ii) MOLECULE TYPE: peptide 



(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 3 

2Q (D) OTHER INFORMATION: /note= "He, Met or Leu" 

(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 4 

(D) OTHER INFORMATION: /note= "Ser or Thr" 

25 

(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 7 

(D) OTHER INFORMATION: /note= "Lys or Arg" 

30 

(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 8 

(D) OTHER INFORMATION: /note= "Gly or Thr" 



35 



WO 99/67293 



PCT/US99/13959 



(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 12 

(D) OTHER INFORMATION : /note= "His or Thr" 



(ix) FEATURE: 

(A) NAME /KEY: Modif ied-site 

(B) LOCATION: 13 

(D) OTHER INFORMATION: /note= "Lys or Arg" 



(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 14 

(D) OTHER INFORMATION: /note= "lie, Met or Leu" 



(ix) FEATURE: 

(A) NAME/KEY : Modif ied-site 

(B) LOCATION: 16 

(D) OTHER INFORMATION: /note= "Gly Or Thr" 

(ix) FEATURE.: 

(A) NAME /KEY : Modif ied-site 

(B) LOCATION: 17. 

(D) OTHER INFORMATION: /note= "lie, Met or Val" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll: 



25 



He Ser Xaa Xaa Glu He Xaa Xaa Val He Val Xaa 



1 5 
Xaa Xaa Glu Xaa Xaa Leu Phe 
15 



10 



(2) INFORMATION FOR SEQ ID NO: 12: 

30 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 12 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 



35 



WO 99/67293 



15 



PCT/US99/13959 



(ii) MOLECULE TYPE: peptide 

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

Thr lie Asn Lys Pro Lys Gly Tyr Val Gly Lys Glu 
15 10 



(2) INFORMATION FOR SEQ ID NO: 13: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 16 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: 

Thr Ala Lys Ser Lys Lys Phe Pro Ser Tyr Thr Ala 

1 5 10 

Thr Tyr Gin Phe 
15 



(2) INFORMATION FOR SEQ ID NO: 14: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 4 5 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: 

Lys Lys Lys He He Thr He Thr Arg He He Thr 

1 5 10 

He He Thr Thr He Asp Gly Gly Cys Gly Glu Thr 
15 20 



16 

WO 99/67293 PCT/US99/13959 



Tyr Gin Ser Arg Val Thr His Pro His Leu Pro Arg 

25 30 35 

Ala Leu Met Arg Ser Thr Thr Lys Cys 
40 45 



(2) INFORMATION FOR SEQ ID NO: 15: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 63 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: 

Thr Ala Lys Ser Lys Lys Phe Pro Ser Tyr Thr Ala 
15 i 5 10 

Thr Tyr Gin Phe Gly Gly Lys Lys Lys He He Thr 
15 .20 



10 



20 



25 



He Thr Arg He He Thr He He Thr Thr He Asp 

25 30 35 

Gly Gly Cys Gly Glu Thr Tyr Gin Ser Arg Val Thr 

40 45 
His Pro His Leu Pro Arg Ala Leu Met Arg Ser Thr 
50 55 60 

Thr Lys Cys 



(2) INFORMATION FOR SEQ ID NO: 16: 



(i) SEQUENCE CHARACTERISTICS: 
30 (A) LENGTH: 6 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 



35 



17 

WO 99/67293 PCT/US 99/1 3959 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: 



10 



Pro Pro Xaa Pro Xaa Pro 
1 5 



(2) INFORMATION FOR SEQ ID NO: 17: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 59 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: 

Thr He Asn Lys Pro Lys Gly Tyr Val Gly Lys Glu 
15 i 5 10 

Gly Gly Lys Lys Lys He He Thr He Thr Arg He 

15 20 
He Thr He He Thr Thr He Asp Gly Gly Cys Gly 

25 30 35 

Glu Thr Tyr Gin Ser Arg Val Thr His Pro His Leu 

40 45 
Pro Arg Ala Leu Met Arg Ser Thr Thr Lys Cys 
50 55 



20 



(2) INFORMATION FOR SEQ ID NO: 18: 

25 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 4 6 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

30 

(ii) MOLECULE TYPE: peptide 



35 



(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: .4 



WO 99/67293 



18 



PCT/US99/13959 



(D) OTHER INFORMATION: /note= "Ser or Thr" 

(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 7 

(D) OTHER INFORMATION: /note= "Lys or Arg" 

(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 8 

(D) OTHER INFORMATION: /note= "Gly Or Thr" 



(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 12 

(D) OTHER INFORMATION: /note= "His Or Thr" 

(ix) FEATURE: 

(A) NAME /KEY: Modif ied-site 

(B) LOCATION: 13 

(D) OTHER INFORMATION: /note= "Lys or Arg" 



(ix) FEATURE: 
20 (A) NAME/KEY: Modif ied-site 

(B) LOCATION: 16 

(D) OTHER INFORMATION: /note- "Gly Or Thr" 



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

25 He Ser He Xaa Glu He Xaa Xaa Val He Val Xaa 

1 5 10 

Xaa He Glu Xaa He Leu Phe Gly Gly Cys Gly Glu 

15 20 

30 Thr Tyr Gin Ser Arg Val Thr His Pro His Leu Pro 

25 30 35 

Arg Ala Leu Met Arg Ser Thr Thr Lys Cys 

40 45 



35 



(2) INFORMATION FOR SEQ ID NO: 19: 



WO 99/67293 



PCT/US99/13959 



o (i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 63 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

5 

(ix) FEATURE: 

(A) NAME /KEY: Modif ied-site 

(B) LOCATION: 21 

(D) OTHER INFORMATION: /note= "Ser or Thr" 

10 

(ix) FEATURE: 

(A) NAME /KEY : Modif ied-site 

(B) LOCATION: 24 

(D) OTHER INFORMATION: /note= "Lys or Arg" 

15 (ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 25 

(D) OTHER INFORMATION: /note= "Gly Or Thr" 



(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 2 9 

(D) OTHER INFORMATION: /note= "His Or Thr" 



(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 30 

(D) OTHER INFORMATION: /note= "Lys or Arg" 



(ix) FEATURE: 
30 (A) NAME/KEY: Modif ied-site 

(B) LOCATION: 33 

(D) OTHER INFORMATION : /note= "Gly. Or Thr" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: 



35 



WO 99/67293 



20 



PCT/US99/13959 



10 



Thr Ala Lys Ser Lys Lys Phe Pro Ser Tyr Thr Ala 

1 5 10 

Thr Gin Phe Gly Gly He Ser He Xaa Glu He Xaa 

15 20 
Xaa Val He Val Xaa Xaa He Glu Xaa He Leu Phe 

25 30 35 

Gly Gly Cys Gly Glu Thr Tyr Gin Ser Arg Val Thr 

40 45 
His Pro His Leu Pro Arg Ala Leu Met Arg Ser Thr 
50 55 60 

Thr Lys Cys 



(2) INFORMATION FOR SEQ ID NO: 20: 



(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 60 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 



(ii) MOLECULE TYPE: peptide 

(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 18 

(D) OTHER INFORMATION: /note= "Ser or Thr" 



(ix) FEATURE: 

(A) NAME /KEY : Modif ied-site 
25 (B) LOCATION: 21 

(D) OTHER INFORMATION: /note= "Lys or Arg" 

(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 
30 (B) LOCATION: 22 

(D) OTHER INFORMATION: /note= "Gly or Thr" 

(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 
35 (B) LOCATION: 2 6 



WO 99/67293 



21 



PCT/US99/13959 



10 



15 



20 



(D) OTHER INFORMATION : /note- "His or Thr" 

(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 27 

(D) OTHER INFORMATION: /note- "Lys or Arg" 

(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 30 

(D) OTHER INFORMATION: /note= "Gly or Thr" 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 

Thr He Asn Lys Pro Lys Gly Tyr Val Gly Lys Glu 

1 5 10 

Gly Gly He Ser He Xaa Glu He Xaa Xaa Val He 

15 20 
Val Xaa Xaa He Glu Xaa He Leu Phe Gly Gly Cys 

25 30 35 

Gly Glu Thr Tyr Gin Ser Arg Val Thr His Pro His 

40 45 
Leu Pro Arg Ala Leu Met Arg Ser Thr Thr Lys Cys 
50 55 60 



(2) INFORMATION FOR SEQ ID NO:21: 

(i) SEQUENCE CHARACTERISTICS: 
25 (A) LENGTH: 42 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

30 

(ix) FEATURE: 

(A) NAME /KEY : Modif ied-site 

(B) LOCATION: 1 

(D) OTHER INFORMATION: /note- "He, Met or Leu" 



35 



WO 99/67293 



PCT/US99/13959 



(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 2 

(D) OTHER INFORMATION: /note= "Ser or Thr" 



(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 
(B) LOCATION: 5 

(D) OTHER INFORMATION: /note= "Lys or Arg" 



(ix) FEATURE: 

(A) NAME /KEY: Modif ied-site 

(B) LOCATION: 6 

(D) OTHER INFORMATION: /note= "Gly or Thr" 



(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 10 

15 (D) OTHER INFORMATION: /note= "His or Thr" 

(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 11 

0Ci (D) OTHER INFORMATION: /note= "Lys or Arg" 



(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 12 

(D) OTHER INFORMATION: /note= "He, Met or Leu" 

25 

(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 14 

(D) OTHER INFORMATION: /note= "Gly or Thr" 

30 

(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 15 

(D) OTHER INFORMATION:- /note= "lie, Met or Val" 



35 



WO 99/67293 



PCT/US99/13959 



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

o 

Xaa Xaa Glu He Xaa Xaa Val He Val Xaa Xaa Xaa 

1 5 10 

Glu Xaa Xaa Gly Gly Cys Gly Glu Thr Tyr Gin Ser 

15 20 
Arg Val Thr His Pro His Leu Pro Arg Ala Leu Met 
5 25 30 35 

Arg Ser Thr Thr Lys Cys 
40 



10 (2) INFORMATION FOR SEQ ID NO: 22: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 60 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

15 

(ii) MOLECULE TYPE: peptide 

(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 
20 (B) LOCATION: 19 

(D) OTHER INFORMATION: /note= "He, Met or Leu" 



(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 20 

(D) OTHER INFORMATION: /note= "Ser or Thr" 



(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 23 

(D) OTHER INFORMATION: /note= "Lys or Arg" 

(ix) FEATURE: 

(A) NAME /KEY : Modif ied-site 

(B) LOCATION: 24 

(D) OTHER INFORMATION: /note= "Gly or Thr" 



24 

WO 99/67293 PCT/US99/13959 



10 



20 



25 



(ix) FEATURE: 

(A) NAME /KEY: Modif ied-site 

(B) LOCATION: 28 

(D) OTHER INFORMATION: /note= "His or Thr" 

(ix) FEATURE: 

(A) NAME /KEY : Modif ied-site 

(B) LOCATION: 29 

(D) OTHER INFORMATION: /note= "Lys or Arg" 

(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 30 

(D) OTHER INFORMATION: /note= "He, Met or Leu* 



(ix) FEATURE: 
15 (A) NAME /KEY: Modif ied-site 

(B) LOCATION: 32 

(D) OTHER INFORMATION: /note= "Gly or Thr" 



(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 33 

(D) OTHER INFORMATION: /note= "lie, Met or Val" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 



Thr Ala Lys Ser Lys Lys Phe Pro Ser Tyr Thr Ala 

1 5 10 

Thr Tyr Gin Phe Gly Gly Xaa Xaa Glu lie Xaa Xaa 

15 20 

Val He Val Xaa Xaa Xaa Glu Xaa Xaa Gly Gly Cys 

30 25 30 35 

Gly Glu Thr Tyr Gin Ser Arg Val Thr His Pro His 

40 45 

Leu Pro Arg Ala Leu Met Arg Ser Thr Thr Lys Cys 

50 55 60 



35 



WO 99/67293 



PCT/US99/13959 



o (2) INFORMATION FOR SEQ ID NO: 23: 

<i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 56 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

5 

(ii) MOLECULE TYPE: peptide 

(ix) FEATURE: 

(A) NAME /KEY: Modif ied-site 
10 (B) LOCATION: 15 

D) OTHER INFORMATION: /note- "lie, Met or Leu" 

(ix) FEATURE: 

(A) NAME /KEY: Modif ied-site 

(B) LOCATION: 16 

15 (D) OTHER INFORMATION: /note- "Ser or Thr" 

(ix) FEATURE: 

(A) NAME /KEY: Modif ied-site 

(B) LOCATION: 19 

20 (D) OTHER INFORMATION: /note- "Lys or Arg" 

(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 20 

(D) OTHER INFORMATION: /note- "Gly or Thr" 

25 

(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 24 

(D) OTHER INFORMATION: /note= "His or Thr" 

30 

(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 25 

(D) OTHER INFORMATION: 7note= "Lys or Arg" 



35 



WO 99/67293 



26 



PCT/US99/13959 



{ix) FEATURE: 

(A) NAME /KEY : Modif ied-site 

(B) LOCATION: 26 

(D) OTHER INFORMATION: /note= "lie, Met or Leu" 



(ix) FEATURE: 

(A) NAME /KEY: Modif ied-site 

(B) LOCATION: 28 

(D) OTHER INFORMATION: /note= "Gly or Thr" 

(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 2 9 

(D) OTHER INFORMATION: /note- "He, Met, or Val" 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: 

Thr He Asn Lys Pro Lys Gly Tyr Val Gly Lys Glu 

1 5 10 

Gly Gly Xaa Xaa Glu He Xaa Xaa Val He Val Xaa 

15 20 
Xaa Xaa Glu Xaa Xaa Gly Gly Cys Gly Glu Thr Tyr 

25 30 35 

Gin Ser Arg Val Thr His Pro His Leu Pro Arg Ala 

40 45 
Leu Met Arg Ser Thr Thr Lys Cys 
50 55 



(2) INFORMATION FOR SEQ ID NO: 24: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 4 6 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 4 



27 

WO 99/67293 PCT/US99/13959 



(D) OTHER INFORMATION: /note= "Ser or Thr" 

o 

(ix) FEATURE: 

(A) NAME /KEY : Modif ied-site 

(B) LOCATION: 7 

(D) OTHER INFORMATION: /note= "Lys or Arg" 

5 (ix) FEATURE: 

(A) NAME /KEY : Modif ied-site 

(B) LOCATION: 8 

(D) OTHER INFORMATION: /note= "Gly or Thr" 



(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 12 

(D) OTHER INFORMATION: - /note= "His or Thr" 



(ix) FEATURE: 
15 (A) NAME /KEY: Modif ied-site 

(B) LOCATION: 13 

(D) OTHER INFORMATION: /note= "Lys or Arg" 



20 



(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 16 

(D) OTHER INFORMATION: /note= "Gly or Thr" 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: 

25 Ile ser lie Xaa Glu He Xaa Xaa Val He Val Xaa 

1 5 10 

Xaa Ile Glu Xaa Ile Leu Phe Gly Gly Cys Gly Tyr 

15 20 

3 q Gly Tyr Gin Ser Ile Val Asp His Pro Asp Phe Pro 

25 30 35 

Lys Pro Ile Val Arg Ser Ile Thr Lys Cys 

40 45 



35 



WO 99/67293 



28 



PCT/US99/I3959 



(2) INFORMATION FOR SEQ ID NO: 25: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 4 5 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

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

Lys Lys Lys He He Thr He Thr Arg He lie Thr 

15 10 
He He Thr Thr He Asp Gly Gly Cys Gly Tyr Gly 

15 20 
Tyr Gin Ser He Val Asp His Pro Asp Phe Pro Lys 

25 30 35 

Pro He Val Arg Ser He Thr Lys Cys 
15 40 45 

(2) INFORMATION FOR SEQ ID NO: 26: 



10 



20 



25 



(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 4 5 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

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



Lys Lys Lys He lie Thr lie Thr Arg lie lie Thr 
15 10 
3Q He lie Thr Thr lie Asp Gly Gly Cys Gly Glu Thr 

15 20 

Tyr Tyr Ser Arg Val Thr His Pro His Leu Pro Lys 
25 30 35 



35 



29 

WO 99/67293 PCT/US99/13959 



Asp lie Val Arg Ser lie Ala Lys Cys 
40 45 



(2) INFORMATION FOR SEQ ID NO: 27: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 4 6 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(ix) FEATURE: 

(A) NAME /KEY : Modif ied-site 

(B) LOCATION: 1 

(D) OTHER INFORMATION: /note- "Met or Leu" 

15 (ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 2 

(D) OTHER INFORMATION: /note= "Thr" 



10 



20 



(ix) FEATURE: 

(A) NAME/KEY : Modif ied-site 

(B) LOCATION: 7 

(D) OTHER INFORMATION: /note= "Arg 1 



(ix) FEATURE: 
25 (A) NAME/KEY: Modif ied-site 

(B) LOCATION: 8 

(D) OTHER INFORMATION: /note= "Thr" 

(ix) FEATURE: 
30 (A) NAME/KEY: Modif ied-site 

(B) LOCATION: 12 

(D) OTHER INFORMATION: /note= "Thr" 



35 



(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 



30 

WO 99/67293 PCT/US99/13959 



10 



(B) LOCATION: 13 

(D) OTHER INFORMATION: /note= "Arg" 

(ix) FEATURE: 

(A) NAME /KEY: Modif ied-site 

(B) LOCATION: 14 

(D) OTHER INFORMATION: /note= "Met or Leu" 

(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 16 

(D) OTHER INFORMATION: /note= "Thr" 

(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 17 

(D) OTHER INFORMATION: /note= "Met or Val" 



15 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: 



lie Ser lie Ser Glu lie Lys Gly Val lie Val His 

15 10 
Lys He Glu Gly He Leu Phe Gly Gly Cys Gly Glu 

15 20 
Thr Tyr Tyr Ser Arg Val Thr His Pro His Leu Pro 

25 30 35 

Lys Asp He Val Arg Ser lie Ala Lys Cys 
40 45 



20 



25 



30 



(2) INFORMATION FOR SEQ ID NO: 28: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 49 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 



35 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: 



WO 99/67293 



31 



PCT/US99/13959 



Cys Ala Asp Ser 
1 

Leu Ser Arg Pro 
15 

Lys Ser Pro Thr 
25 

Ala Pro Ser Lys 
40 

Arg 



Asn Pro Arg Gly 
5 

Ser Pro Phe Asp 
20 

lie Thr Ser Leu 
30 

Gly Thr Val Asn 



Val Ser Ala Tyr 
10 

Leu Phe lie Arg 

Val Val Asp Leu 
35 

Leu Thr Trp Ser 
45 



(2) INFORMATION FOR SEQ ID NO: 29: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 60 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 



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

Gin Gly His Thr Phe Glu Asp Ser Thr Lys Lys Cys 

1 5 10 

Ala Asp Ser Asn Pro Arg Gly Val Ser Ala Tyr Leu 

15 20 
Ser Arg Pro Ser Pro Phe Asp Leu Phe lie Arg Lys 

25 30 35 

Ser Pro Thr lie Thr Ser Leu Val Val Asp Leu Ala 

40 45 
Pro Ser Lys Gly Thr Val Asn Leu Thr Trp Ser Arg 
50 55 60 



(2) INFORMATION FOR SEQ ID NO: 30: 

(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 64 amino acids 



32 

WO 99/67293 PCT/US99/13959 



10 



15 



20 



25 



(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30: 

Gin Val Thr Tyr Gin Gly His Thr Phe Glu Asp Ser 

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

15 20 
Ser Ala Tyr Leu Ser Arg Pro Ser Pro Phe Asp Leu 

25 30 35 

Phe lie Arg Lys Ser Pro Thr lie Thr Ser Leu Val 

40 45 
Val Asp Leu Ala Pro Ser Lys Gly Thr Val Asn Leu 
50 55 60 

Thr Trp Ser Arg 

(2) INFORMATION FOR SEQ ID NO: 31: 

<i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 7 6 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

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



Gin Lys His Trp Leu Ser Asp Arg Thr Tyr Thr Ser 

1 5 10 

Gin Val Thr Tyr Gin Gly His Thr Phe Glu Asp Ser 
15 20 
30 Thr Lys Lys Cys Ala Asp Ser Asn Pro Arg Gly Val 

25 30 35 

Ser Ala Tyr Leu Ser Arg Pro Ser Pro Phe Asp Leu 

40 45 
Phe lie Arg Lys Ser Pro Thr He Thr Ser Leu Val 
50 55 60 

35 



WO 99/67293 



33 



PCT/US99/13959 



Val Asp Leu Ala Pro Ser Lys Gly Thr Val Asn Leu 

65 70 

Thr Trp Ser Arg 
75 

(2) INFORMATION FOR SEQ ID NO: 32: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 35 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 



(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: 



Cys Ala Asp Ser 
1 

Leu Ser Arg Pro 
15 

Lys Ser Pro Thr 
25 



Asn Pro Arg Gly 
5 

Ser Pro Phe Asp 
20 

lie Thr Ser' Leu 
30 



Val Ser Ala Tyr 
10 

Leu Phe lie Arg 

Val Val Asp 
35 



(2) INFORMATION FOR SEQ ID NO: 33: 



(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 4 6 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 



(ii) MOLECULE TYPE: peptide 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33: 

Gin Gly His Thr Phe Glu Asp Ser Thr Lys Lys Cys 

1 5 10 

Ala Asp Ser Asn Pro Arg Gly Val Ser Ala Tyr Leu 
15 20 



34 

WO 99/67293 PCT/US99/I3959 



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

25 30 35 

Ser Pro Thr He Thr Ser Leu Val Val Asp 
40 45 



10 



(2) INFORMATION FOR SEQ ID NO: 34: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 50 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34: 

Gin Val Thr Tyr Gin Gly His Thr Phe Glu Asp Ser 
15 i 5 10 

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

15 20 
Ser Ala Tyr Leu Ser Arg Pro Ser Pro Phe Asp Leu 

25 30 35 

Phe He Arg Lys Ser Pro Thr He Thr Ser Leu Val 
40 45 

Val Asp 
50 



20 



25 



(2) INFORMATION FOR SEQ ID NO: 35: 



(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 62 amino acids 

30 (B) TYPE: amino acid 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 



35 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35: 



WO 99/67293 



35 



PCT/US99/13959 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35: 



Gin Lys His Trp 
1 

Gin Val Thr Tyr 
15 

Thr Lys Lys Cys 
25 

Ser Ala Tyr Leu 
40 

Phe lie Arg Lys 
50 

Val Asp 



Leu Ser Asp Arg 
5 

Gin Gly His Thr 
20 

Ala Asp Ser Asn 
30 

Ser Arg Pro Ser 

Ser Pro Thr lie 
55 



Thr Tyr Thr Ser 
10 

Phe Glu Asp Ser 

Pro Arg Gly Val 
35 

Pro Phe Asp Leu 
45 

Thr Ser Leu Val 
60 



(2) INFORMATION FOR SEQ ID NO: 36: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 29 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36: 

Cys Ala Asp Ser Asn Pro Arg Gly Val Ser Ala Tyr 

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

15 20 
Lys Ser Pro Thr lie 
25 



(2) INFORMATION FOR SEQ ID NO: 37: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 40 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 



36 

WO 99/67293 PCT/US99/13959 



(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37: 

Gin Gly His Thr Phe Glu Asp Ser Thr Lys Lys Cys 

1 5 10 

Ala Asp Ser Asn Pro Arg Gly Val Ser Ala Tyr Leu 

15 20 
Ser Arg Pro Ser Pro Phe Asp Leu Phe lie Arg Lys 

25 30 35 

Ser Pro Thr lie 
40 

(2) INFORMATION FOR SEQ ID NO: 38: 

(i) SEQUENCE CHARACTERISTICS: 
15 (A) LENGTH: 44 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 



10 



20 



25 



30 



(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: 

Gin Val Thr Tyr Gin Gly His Thr Phe Glu Asp Ser 

1 5 10 

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

15 20 
Ser Ala Tyr Leu Ser Arg Pro Ser Pro Phe Asp Leu 

25 30 35 

Phe lie Arg Lys Ser Pro Thr lie 
40 

(2) INFORMATION FOR SEQ ID NO: 39: 



35 



(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 56 amino acids 



37 

WO 99/67293 PCT/US99/1 3959 



10 



15 



(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39: 

Gin Lys His Trp Leu Ser Asp Arg Thr Tyr Thr Ser 

15 10 
Gin Val Thr Tyr Gin Gly His Thr Phe Glu Asp Ser 

15 20 
Thr Lys Lys Cys Ala Asp Ser Asn Pro Arg Gly Val 

25 30 35 

Ser Ala Tyr Leu Ser Arg Pro Ser Pro Phe Asp Leu 

40 45 
Phe He Arg Lys Ser Pro Thr He 
50 55 

(2) INFORMATION FOR SEQ ID NO: 40: 



<i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 76 amino acids 
20 (B) TYPE: amino acid 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40: 

25 

Gin Lys His Trp Leu Ser Asp Arg Thr Tyr Thr Cys 

15 10 
Gin Val Thr Tyr Gin Gly His Thr Phe Glu Asp Ser 
15 20 
30 Thr Lys Lys Cys Ala Asp Ser Asn Pro Arg Gly Val 

25 30 35 

Ser Ala Tyr Leu Ser Arg Pro Ser Pro Phe Asp Leu 

40 45 
Phe He Arg Lys Ser Pro Thr He Thr Cys Leu Val 
50 55 60 

35 



38 

WO 99/67293 PCT/US99/13959 



Val Asp Leu Ala Pro Ser Lys Gly Thr Val Asn Leu 

65 70 

Thr Trp Ser Arg 
75 



(2) INFORMATION FOR SEQ ID NO: 41: 

5 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 10 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

10 

(ii) MOLECULE TYPE: peptide 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41: 

Cys Lys Gin Arg Asn Gly Thr Leu Thr Cys 
15 i 5 10 



(2) INFORMATION FOR SEQ ID NO: 42: 



(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 45 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 



(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42: 



Gin Lys His Trp 
1 

Gin Val Thr Tyr 

30 15 

Thr Lys Lys Cys 
25 

Ser Ala Tyr Leu 
40 



Leu Ser Asp Arg 
5 

Gin Gly His Thr 
20 

Ala Asp Ser Asn 
30 

Ser Arg Pro Ser 



Thr Tyr Thr Cys 
10 

Phe Glu Asp Ser 

Pro Arg Gly Val 
35 

Pro 
45 



35 



WO 99/67293 



39 



PCT7US99/13959 



10 



20 



25 



30 



(2) INFORMATION FOR SEQ ID NO: 43: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 34 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43: 



Cys Pro Ser Lys Gly Thr Val Asn Leu Thr Trp Ser 

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

15 20 
Lys Glu Glu Lys Gin Arg Asn Gly Thr Cys 
15 25 30 



(2) INFORMATION FOR SEQ ID NO: 44: 



(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 33 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44: 

Cys Pro Val Gly Thr Arg Asp Trp lie Glu Gly Glu 

1 -5 10 

Thr Tyr Gin Cys Arg Val Thr His Pro His Leu Pro 

15 20 
Arg Ala Leu Met Arg Ser Thr Thr Cys 
25 30 



35 



WO 99/67293 



40 



PCTAJS99/13959 



(2) INFORMATION FOR SEQ ID NO: 45: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 14 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45: 

Ser Thr Thr Lys Thr Ser Gly Pro Arg Ala Ala Pro Glu Val 
15 10 

(2) INFORMATION FOR SEQ ID NO: 46: 

(i) SEQUENCE CHARACTERISTICS: 
15 (A) LENGTH: 14 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 



10 



20 



25 



30 



35 



(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: 

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

(2) INFORMATION FOR SEQ ID NO: 47: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 19 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47: 



41 

WO 99/67293 PCT/US99/13959 



10 



15 



20 



30 



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

15 10 
Lys Ser Pro Thr lie Thr Cys 
15 



(2) INFORMATION FOR SEQ ID NO: 48; 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 13 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: 

Cys Val Gly Thr Arg Asp Trp He Glu Gly Glu Pro Cys 
1 5 10 



(2) INFORMATION FOR SEQ ID NO: 49: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 15 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

25 (ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49; 



Cys Pro Pro Val Gly Thr Arg Asp Trp He Glu Gly 

1 5 10 

Glu Pro Cys 
15 



35 



(2) INFORMATION FOR SEQ ID NO: 50: 



42 

WO 99/67293 PCT/US99/13959 



10 



(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 16 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50: 

Cys Lys Glu Glu Lys Gin Arg Asn Gly Thr Leu Thr 
1 5 10 

Val Thr Ser Cys 
15 



15 



20 



30 



(2) INFORMATION FOR SEQ ID NO: 51: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 8 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 



(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51; 

Lys Glu Glu Lys Gin Arg Asn Gly 
25 i 5 

(2) INFORMATION FOR SEQ ID NO: 52: 



(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 11 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 



35 



(ii) MOLECULE TYPE: peptide 



43 



WO 99/67293 



PCT/US99/13959 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: 

Cys Trp Ser Arg Ala Ser Gly Lys Pro- Val Cys 
1 5 10 



(2) INFORMATION FOR SEQ ID NO: 53: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 21 amino acids 

(B) TYPE: amino acid 
<D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53: 

15 Pro Thr lie Thr Cys Leu Val Leu Asp Leu Ala Pro 

1 5 10 

Ser Lys Gly Thr Val Asn Leu Thr Cys 
15 20 



10 



20 



30 



(2) INFORMATION FOR SEQ ID NO: 54: 



(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 16 amino acids 

(B) TYPE: amino acid 
25 (D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54: 



Pro Thr lie Thr Cys Leu Val Leu Asp Leu Ala Pro 

1 5 10 

Ser Lys Gly Thr 
15 



35 



44 

WO 99/67293 PCT/US99/13959 



10 



15 



20 



25 



30 



(2) INFORMATION FOR SEQ ID NO: 55: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 25 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55: 

Thr Ser Thr Leu Pro Val Gly Thr Arg Asp Trp lie 

1 5 10 

Glu Gly Glu Thr Tyr Gin Cys Arg Val Thr His Pro 
15 20 

His 
25 

(2) INFORMATION FOR SEQ ID NO: 56: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 16 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56: 

Pro Thr He Thr Ser Leu Val Leu Cys Leu Ala Pro 

1 5 10 

Ser Lys Gly Cys 
15 



(2) INFORMATION FOR SEQ ID NO: 57: 



35 



(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 amino acids 



45 



WO 99/67293 



PCT/US99/13959 



10 



20 



(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57: 

Cys Val Asn Leu Thr Trp Ser Arg Ala Ser Gly Lys 

1 5 10 

Pro Val Asn His Ser Thr Arg Lys Glu Glu Cys 
15 20 

(2) INFORMATION FOR SEQ ID NO: 58: 



(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 53 amino acids 

(B) TYPE: amino acid 
15 (D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58: 



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



His Ser Thr Arg Lys Glu Glu Lys Gin Arg Asn Gly 
15 20 
25 Thr Leu Thr Val Thr Ser Thr Leu Pro Val Gly Thr 

25 30 35 

Arg Asp Trp He Glu Gly Glu Thr Tyr Gin Cys Arg 

40 45 
Val Thr His Pro His 

30 50 



35 



(2) INFORMATION FOR SEQ ID NO: 59: 
(i) SEQUENCE CHARACTERISTICS: 



WO 99/67293 



46 



PCT/US99/13959 



10 



15 



20 



(A) LENGTH : 10 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59: 

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

(2) INFORMATION FOR SEQ ID NO: 60: 

<i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 19 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 4 

(D) OTHER INFORMATION: /note= "Ser or Thr 1 



(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 7 

25 ( d) OTHER INFORMATION: /note- "Lys or Arg" 



(ix) FEATURE: 

(A) NAME /KEY : Modif ied-site 

30 (B) LOCATION: 8 

(D) OTHER INFORMATION: /note= "Gly or Thr" 

(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 12 



10 



15 



20 



25 



30 



47 

WO 99/67293 PCT/US99/13959 



(D) OTHER INFORMATION: /note- "His or Thr" 

(ix) FEATURE: 

(A) NAME /KEY: Modif ied-site 

(B) LOCATION: 13 

(D) OTHER INFORMATION: /note= "Lys or Arg" 

(ix) FEATURE: 

(A) NAME /KEY: Modif ied-site 

(B) LOCATION: 16 

(D) OTHER INFORMATION: /note= "Gly or Thr" 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60: 

He Ser He Xaa Glu He Xaa Xaa Val He Val Xaa 

1 5 10 

Xaa He Glu Xaa He Leu Phe 
15 

(2) INFORMATION FOR SEQ ID NO: 61: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 15 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61: 

Leu Ser Glu He Lys Gly Val He Val His Arg Leu 

1 5 10 

Glu Gly Val 
15 

(2) INFORMATION FOR SEQ ID NO: 62: 



35 



(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 20 amino acids 

(B) TYPE: amino acid 



10 



48 

WO 99/67293 PCT/US99/13959 



(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 

{xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62: 

Gly He Leu Glu Ser Arg Gly He Lys Ala Arg He 

1 5 10 

Thr His Val Asp Thr Glu Ser Tyr 
15 20 

(2) INFORMATION FOR SEQ ID NO: 63: 



(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 17 amino acids 

(B) TYPE: amino acid 
15 (D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63: 



20 



25 



30 



Lys Lys Gin Tyr He Lys Ala Asn Ser Lys Phe He 

1 5 10 

Gly He Thr Glu Leu 
15 



(2) INFORMATION FOR SEQ ID NO: 64: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 22 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 



35 



49 

WO 99/67293 PCT/US99/13959 



10 



15 



20 



30 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64: 

Lys Lys Phe Asn Asn Phe Thr Val Ser Phe Trp Leu 

1 5 10 

Arg Val Pro Lys Val Ser Ala Ser His Leu 
15 20 



(2) INFORMATION FOR SEQ ID NO: 65: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 30 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65: 

Lys Lys Leu Arg Arg Leu Leu Tyr Met He Tyr Met 

1 5 10 

Ser Gly Leu Ala Val Arg Val His Val Ser Lys Glu 

15 20 
Glu Gin Tyr Tyr Asp Tyr 
25 30 



(2) INFORMATION FOR SEQ ID NO: 6.6: 

25 (i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 27 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 



(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66: 



35 



Tyr Asp Pro Asn Tyr Leu Arg Thr Asp Ser Asp Lys 
1 5 10 



50 



WO 99/67293 



PCT/US99/13959 



10 



Asp Arg Phe Leu Gin Thr Met Val Lys Leu Phe Asn 

15 20 
Arg lie Lys 
25 

(2) INFORMATION FOR SEQ ID NO: 67: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 24 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67: 



Gly Ala Tyr Ala Arg Cys Pro Asn Gly Thr Arg Ala 
1 5 10 

15 Leu Thr Val Ala Glu Leu Arg Gly Asn Ala Glu Leu 

15 20 



20 



(2) INFORMATION FOR SEQ ID NO: 68: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 15 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 



25 



(ii) MOLECULE TYPE: peptide 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 68: 



30 



Phe Phe Leu Leu Thr Arg He Leu Thr He Pro Gin 

1 5 10 

Ser Leu Asp 
15 



35 



(2) INFORMATION FOR SEQ ID NO: 69: 



51 

WO 99/67293 PCT/US99/13959 



10 



(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 21 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69: 

Val Ser Phe Gly Val Trp He Arg Thr Pro Pro Ala 

1 5 10 

Tyr Arg Pro Pro Asn Ala Pro He Leu 
15 20 



(2) INFORMATION FOR SEQ ID NO: 70: 

15 (i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 20 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 



20 



30 



(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 70; 



Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp 

1 5 10 

25 Thr Ala Ser Ala Leu Tyr Arg Glu 

15 20 



(2) INFORMATION FOR SEQ ID NO: 71: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 20 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 



35 



WO 99/67293 



52 



PCT/US99/13959 



10 



(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:71: 

Pro His His Thr Ala Leu Arg Gin Ala He Leu Cys 

1 5 10 

Trp Gly Glu Leu Met Thr Leu Ala 
15 20 

• (2) INFORMATION FOR SEQ ID NO: 72: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 17 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 72: 

Trp Val Arg Asp He He Asp Asp Phe Thr Asn Glu 

1 5 10 

Ser Ser Gin Lys Thr 
15 

(2) INFORMATION FOR SEQ ID NO: 73: 

25 ( i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 19 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

3 Q (ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73: 



15 



20 



35 



Arg Ala Gly Arg Ala He Leu His He Pro Thr Arg 
1 5 10 



53 



WO 99/67293 



PCT/US99/13959 



He Arg Gin Gly Leu Glu Arg 
15 



10 



(2) INFORMATION FOR SEQ ID NO: 74: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 21 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74: 

Ala Val Ala Glu Gly Thr Asp Arg Val He Glu Val 
15 10 



15 Leu Gin Arg Ala Gly Arg Ala He Leu 

15 20 

(2) INFORMATION FOR SEQ ID NO: 75: 



20 



25 



30 



(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 25 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75: 

Ala Leu Asn He Trp Asp Arg Phe Asp Val Phe Ser 

1 5 10 

Thr Leu Gly Ala Thr Ser Gly Tyr Leu Lys Gly Asn 
15 20 

Ser 
25 



35 



10 



54 

WO 99/67293 PCT/US99/I3959 



(2) INFORMATION FOR SEQ ID NO: 76: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 22 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:76: 

Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Val 

1 5 10 

Ala Ala Leu Ser He Leu Pro Gly His Gly 
15 20 



15 



20 



(2) INFORMATION FOR SEQ ID NO: 77: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 39 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77: 

Glu Glu He Val Ala Gin Ser He Ala Leu Ser Ser 
25 x 5 10 

Leu Met Val Ala. Gin Ala He Pro Leu Val Gly Glu 

15 20 

Leu Val Asp He Gly Phe Ala Ala Thr Asn Phe Val 
25 30 35 

30 Glu Ser Cys 



(2) INFORMATION FOR SEQ ID NO: 78: 



35 



(i) SEQUENCE CHARACTERISTICS: 



5 



10 



55 

WO 99/67293 PCT/US99/13959 



(A) LENGTH: 21 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 78: 

Asp He Glu Lys Lys He Ala Lys Met Glu Lys Ala 

1.5 10 

Ser Ser Val Phe Asn Val Val Asn Ser 
15 20 

(2) INFORMATION FOR SEQ ID NO: 79: 



(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 amino acids 

15 (B) TYPE: amino acid 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

2Q (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 79: 

Lys Trp Phe Lys Thr Asn Ala Pro Asn Gly Val Asp 
1 5 10 

Glu Lys He Arg He 
25 is 



(2) INFORMATION FOR SEQ ID NO: 80: 

3Q (i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 14 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 



35 



(ii) MOLECULE TYPE: peptide 



56 

WO 99/67293 PCT/US99/13959 



10 



15 



20 



30 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 80: 

Gly Leu Gin Gly Lys He Ala Asp Ala Val Lys Ala 

1 5 10 

Lys Gly 



(2) INFORMATION FOR SEQ ID NO: 81: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 19 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:81: 

Gly Leu Ala Ala Gly Leu Val Gly Met Ala Ala Asp 

15 10 
Ala Met Val Glu Asp Val Asn 
15 



(2) INFORMATION FOR SEQ ID NO: 82: 



(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 20 amino acids 

(B) TYPE: amino acid 
25 (D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:82: 



Ser Thr Glu Thr Gly Asn Gin His His Tyr Gin Thr 

1 5 10 

Arg Val Val Ser Asn Ala Asn Lys 
15 2-0 



35 



57 



WO 99/67293 



PCT/US99/13959 



10 



(2) INFORMATION FOR SEQ ID NO: 83: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 15 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:83: 

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

1 5 10 

Pro Thr Cys 
15 



15 (2) INFORMATION FOR SEQ ID NO: 84: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 25 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 84: 

Cys Gly Glu Thr Tyr Lys Ser Thr Val Ser His Pro 

1 5 10 

Asp Leu Pro Arg Glu Val Val Arg Ser He Ala Lys 
15 20 

Cys 
25 



25 



30 



(2) INFORMATION FOR SEQ ID NO:-85: 



35 



58 

WO 99/67293 PCT/US99/I3959 



10 



20 



30 



(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 60 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 

(A) NAME /KEY : Modif ied-site 

(B) LOCATION: 18 

(D) OTHER INFORMATION: /note- "Thr" 

(ix) FEATURE: 

(A) NAME /KEY : Modif ied-site 

(B) LOCATION: 21 

(D) OTHER INFORMATION: /note= "Arg" 



(ix) FEATURE: 

(A) NAME /KEY : Modif ied-site 
15 (B) LOCATION: 22 

(D) OTHER INFORMATION: /note- "Thr" 

(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 26 

(D) OTHER INFORMATION: /note= "Thr" 



(ix) FEATURE: 
- (A) NAME/KEY: Modif ied-site 
(B) LOCATION: 27 
25 (D) OTHER INFORMATION: /note= "Arg" 

(ix) FEATURE: 

(A) NAME/KEY: Modif ied-site 

(B) LOCATION: 30 

(D) OTHER INFORMATION: /note= "Thr" 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 85: 



35 



Thr He Asn Lys Pro Lys Gly Tyr Val Gly Lys Glu 
1 5 10 



WO 99/67293 



PCT/US99/13959 



10 



20 



Gly Gly He Ser He Ser Glu He Lys Gly Val He 

15 20 
Val His Lys He Glu Gly He Leu Phe Gly Gly Cys 

25 30 35 

Gly Gly Thr Tyr Gin Ser Arg Val Thr His Pro His 

40 45 
Leu Pro Arg Ala Leu Met Arg Ser Thr Thr Lys Cys 
50 55 60 

(2) INFORMATION FOR SEQ ID NO: 86: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 17 amino acids 

(B) TYPE: amino acid 
( D ) TOPOLOGY : linear 



(ii) MOLECULE TYPE: peptide 

15 (x i) SEQUENCE DESCRIPTION: SEQ ID NO: 86: 

Lys Trp Phe Lys Thr Asn Ala Pro Asn Gly Val Asp 

1 5 10 

Glu Lys He Arg lie 
15 



(2) INFORMATION FOR SEQ ID NO: 87: 



(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 62 amino acids 

^ 5 (B) TYPE: amino acid 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

30 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 87: 

Lys Trp Phe Lys Thr Asn Ala Pro Asn Gly Val Asp 

1 5 10 

Glu Lys He Arg He Lys Lys Lys Lys lie lie Thr 
15 20 

35 



60 

WO 99/67293 PCT/US99/13959 



lie Thr Arg lie He Thr 
25 30 
Lys Cys Gly Glu Thr Tyr 
40 

Pro His Leu Pro Lys Asp 
50 

Lys Cys 



He He Thr Yhr He Asp 

35 

Tyr Ser Arg Val Thr His 
45 

He Val Arg Ser He Ala 
55 60 



10 



20 



(2) INFORMATION FOR SEQ ID NO: 88: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 57 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 



(ii) MOLECULE TYPE: peptide 

15 ( X i) SEQUENCE DESCRIPTION: SEQ ID NO: 88: 

Thr lie Asn Lys Pro Lys Gly Tyr Val Gly Lys Glu 

1 5 10 

Lys Lys Lys Lys lie He Thr He Thr Arg lie lie 

15 20 
Thr He lie Thr Tyr lie Asp Lys Cys Gly Glu Thr 
25 30 35 

Tyr Tyr Ser Arg Val Thr His Pro His Leu Pro Lys 

40 45 
Asp He Val Arg Ser He Ala Lys Cys 
25 50 55 

(2) INFORMATION FOR SEQ ID NO: 89: 

(i) SEQUENCE CHARACTERISTICS: 
30 (A) LENGTH: 19 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 



35 



(ii) MOLECULE TYPE: peptide 



61 

WO 99/67293 PCT/US9 9/1 3959 



5 



10 



15 



20 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO:89: 

He Ser Leu Thr Glu He Arg Thr Val He Val Thr 

1 5 10 

Arg Leu Glu Thr Val Leu Phe 
15 

(2) INFORMATION FOR SEQ ID NO: 90: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 4 5 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 90: 

He Ser Leu Thr Glu lie Arg Thr Val He Val Thr 

1 5 10 

Arg- Leu Glu Thr Val Leu Phe DLys Cys Gly Glu Thr 

15 20 
Tyr Tyr Ser Arg Val Thr His Pro His Leu Pro Lys 
25 30 35 

Asp He Val Arg Ser He Ala Lys Cys 
40 45 



25 ( 2) INFORMATION FOR SEQ ID NO: 91: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 63 amino acids 

(B) TYPE: amino acid 
3Q (D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 91: 



35 



WO 99/67293 



62 



PCT/US99/13959 



Lys Trp Phe Lys Thr Asn Ala Pro Asn Gly Val Asp 

1 5 10 

Glu Lys He Arg He QLys He Ser Leu Thr Glu He 

15 20 
Arg Thr Val He Val Thr Arg Leu Glu Thr Val Leu 

25 30 35 

Phe DLys Cys Gly Glu Thr Tyr Tyr Ser Arg Val Thr 

40 45 
His Pro His Leu Pro Lys Asp He Val Arg Ser He 
50 55 60 

Ala Lys Cys 



INTERNATIONAL SEARCH REPORT 



Interaational application No. 
PCTAJS99/13959 



A. CLASSIFICATION OF SUBJECT MATTER 

IPC(6) :C07K 16/46; A61K 39/44 

USCL :530/387.I, 403; 424/180.1. 193.1 
According to International Patent Classification (IPC) or to both national classification and IPC 



FIELDS SEARCHED 



Minimum documentation searched (classification system followed by classification symbols) 
U.S. : 530/387.1, 403; 424/180.1, 193.1 



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



Electronic data base consulted during the international search (name of data base and, where practicable, search terms used) 



C DOCUMENTS CONSIDERED TO BE RELEVANT 



Category* 



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



Relevant to claim No. 



BURT, D. S. et ah, Analysis of the interaction between rat 
immunoglobulin E and rat mast cells using anti-peptide antibodies, 
Molecular Immunology, 1987, Vol. 24, No. 4, pages 379-389. 

BURT, D. S. et aL, Inhibition of binding of rat IgE to rat mast cells 
by synthetic IgE peptides, Eur. J. Immunol., 1987, Vol. 17, pages 
437-440, see entire document. 

VERCELLI, D. et ai., The B-cell binding site of human 
immunoglobulin E, LETTERS TO NATURE , 20 April 1989, Vol. 
338, pages 649-651, see entire document. 



1-27 



1-27 



1-27 



px] Further documents are listed in the continuation of Box C. See patent family annex. 



" Special cttegorie* of cited docunienti: 

* A* document defining the general state of the art which i* not considered 

to be of particular relevance 

"E* earlier document published on or after the international filing date 

•L' document which may throw doubts on priority claim (a) or which is 

cited to establish the publication dale of another citation or other 
special reason (as specified) 

'O* document referring to an oral disclosure, use. exhibition or other 

means 

■p* documenl published prior to the international filing date but later than 

the priority date claimed 



later document published after the international filing date or priority 
data and not in conflict with the application but cited to understand 
the principle or theory underlying the invention 

document of particular relevance; the claimed invention cannot be 
considered novel or cannot be considered to involve an inventive step 
when the documenl is taken alone 

document of particular relevance; the cbimed invention cannot be 
considered to involve an inventive step when the document ii 
combined with one or More other such documents, such combination 
being obvious to a per/on skilled in the art 



document memb 




r the same patent family 



Date of the actual completion of the international search 



28 SEPTEMBER 1999 



Date of mailing of lira international search report 

7 OCT 1999, 




Name and mailing address of the ISA/US 
Commissioner of Patents and Trademarks 
Box PCT 

Washington, DC. 20231 
Facsimile No. (703) 305-3230 



GMA 
03) 308-0196 



Form PCT/1SA/210 (second sheetXJuly 1992)* 



INTERNATIONAL SEARCH REPORT 



International application No. 
PCT/US99/13959 



C (Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT 



Category* 



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



HELM, B. et al., The mast cell binding site of human 
immunoglobulin E, Nature, 14 January 1988, vol. 331, pages 180- 
183, see entire document. 

WO 93/04173 Al (GENENTECH INC) 04 March 1993, see entire 
document. 



Relevant to claim No. 



1-27 



1-27 



Form PCT71SA/2I0 (continuation of second sheetXJuly 1992)* 



INTERNATIONAL SEARCH REPORT 



Interoatiooal application No. 
PCT/US99/13959 



Box I Observations where certain claims were found unsearchable (Continuation of item 1 of first sheet) 



This international report has not been established in respect of certain claims under Article l7(2Xa) for the following reasons: 



□ 



Claims Nos.: 

because they relate to subject matter not required to be searched by this Authority, namely: 



□ Claims Nos.: 
because they relate to parts of the international application that do not comply with the prescribed requirements to such 
an extent that no meaningful international search can be carried out, specifically: 



□ 



Claims Nos.: 

becauso they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(a). 



Boa II Observations where unity of invention is lacking (Continuation of Item 2 of first sheet) 



This International Searching Authority found multiple inventions in this international application, as follows: 
Please See Extra Sheet. 



As all required additional search fees were timely paid by the applicant, this international search report covers all searchable 



claims. 



2. Q As all searchable claims could be searched without effort justifying an additional fee. this Authority did not invite payment 

of any additional fee. 

3. | I As only some of the required additional search fees were timely paid by the applicant, this international search report covers 
— only those claims for which fees were paid, specifically claims Nos.: 



nn No required additional search fees were timely paid by the applicant. Consequently, this international search report is 
' — ' restricted to the invention first mentioned in the claims; it is covered by claims Nos.: 
1-27 



Remark on Protest Q The additional search fees were accompanied by the applicant's protest. 

[ [ No protest accompanied the payment of additional search fees. 



Form PCT/1SA/210 (continuation of first sheet(l)XJuty 1992)* 



INTERNATIONAL SEARCH REPORT 



Internationa) Application No. 
PCT/US99/13959 



BOX II. OBSERVATIONS WHERE UNITY OF INVENTION WAS LACKING 
This ISA found multiple inventions as follows: 

This application contains the following inventions or groups of inventions which arc not so linked as to form a single 
inventive concept under PCT Rule 13.1. In order for all inventions to be searched, the appropriate additional search fees 
must be paid. 

I. Group I. claims 1-27. directed to peptides, peptide conjugates, polymeric peptide products and methods of using such 
products to induce antibodies. 

II. Group II, claim 28, directed to nucleic acids encoding the peptide products of Group I. 

In view of conversations with the Applicant's attorney it is presumed that the inventive concept hinges on the identity of 
the lgE-CH3 domain and not on the T helper epitope to which it is attached. The T helper epitopes such as those 
recited by SEQ ID NOS: 9-12. 61-82 and 84 are therefore considered to have the same or corresponding technical 
features. 

The inventions listed as Groups I and II do not relate to a single inventive concept under PCT Rule 13.1 because, under 
PCT Rule 13.2. they lack the same or corresponding special technical features for the following reasons: Group I is 
directed to peptide products which lack the same or corresponding structural and functional features of Group II which is 
directed to nucleic acids. 



Form PCT/ISA/210 (extra shcetXJuly 1992)*