CA2186595A1 - Synthetic peptide based immunogens for the treatment of allergy - Google Patents

Abstract

The present invention relates to a method for eliciting the production in healthy mammals, including humans, of high titer antibodies to an effector site in human IgE heavy chain, i.e. a site in the CH4 domain of the -chain, by the use of compositions of synthetic peptide immunogens in either a radially branching multimeric form (such as branching octameric or hexadecameric peptides) or a linearly arranged monomeric form, to inhibit mast cell activation and reduce allergen-induced IgE production. It also relates to the use of such "optimally" designer, carrier protein free, IgE -chain related immunogens as key components in a synthetic vaccine to provide an immunotherapy for the treatment of allergy. The subject peptides contain immune stimulator sequences, including a built-in helper T cell epitope tandemly linked in a specific orientation, to aid in stimulating the immune response towards the IgE CH4 domain.

Description

Wo 95/26365 2 1 ~3 6 5 ~ 5 P~ 4l C PEPTIDE BASED IMMUNOGENS
FOR THE TREATMENT OF AI.I ERGY
CROSS ~ ;N~ TO RT'T ~TEn APPLICATION
This i5 a rrnt;nllAt;on-in-part application of pending Application Serial No. 08/218,461 filed March 28, 1994 which i9 a c~nt i nllAtion of pending application Serial No. 08/060,798 filed May 10, 1993 which is a crnt;nllAt;r-n-in-part of pendlng application Serial No. 07/847, 745, filed March 6, 1992, now ~hAn~r)nPd~ which was a continuation-in-part of application Serial No. 07/637,364, filed January 4, 1991, now AhAn~ n~d.
FIELD OF THE INVENTION
The present invention relates to the use of a composition of a synthetic peptide, in a linear or radially branching multimeric form, as an immunogen for eliciting the production in healthy mammals, including humans, of high titer ~nt;hr~rl;es to the effector site on the CH4 domain of the ~-chain of the human IgE heavy chain, and to the use of the composition as a vaccine to provide an immunotherapy for the treatment of allergy.
BACKGROUND OF TH~ INVENTION
Immunotherapy for the prevention of IgE-mediated allergic responses, such as asthma and hay fever, as known and practiced since early in this century, has been by desensitization or hyposensitization, wherein a gradually increasing amount of an allergen is given to a patient to reduce the effects of subsequent exposure to that allergen~1) Limitations to such an allergen-based immunotherapy include difficulties in identifying the allergen involved and, if an allergen is identified, the adverse reactions f requently caused by the use of the nt; ~; erl allergen~2~ .
Other trf~;~ A for the relief of allergies employ drugs to block the cascade of cellular events that W095l26365 2186595 - 2 - r~ /41 --is responsible for allergic reactions. These drugs include anti-histamines, decongestants, ,t'~ agonists, and corticosteroids. Anti-histamines, decongestants, and 1;~2 agonists act on events downstream of IgE in the allergic cascade, making them F~ll ;i~t;ve remedies which address 5 only the allergy symptoms. Preventative trP~tr tR must act on cellular events closer to the initiation of IgE-mediated allergic reactions. These palliatives provide relief~that is short term and partial. Moreover, the relief .of symptoms is frerluently Arl ~~ 1ed by adverse 10 side effects, e.g. anti-histamines may cause restlessness or drowsiness, and ~/;72 agonists have sometimes been associated with increased morbidity in asthmatic patients.
Corticosteroids are powerful immunosuppressants and are highly efficacious for the treatment of allergic 15 symptoms. However, they stimulate adverse h~
activities and may cause an undesirably broad immunosuppression.
To avoid the shortcomings of the known therapeutic drugs, it would be more desirable to prevent 20 allergic responses by selective suppression targeted to IgE. This may be accomplished either by suppressing IgE
synthesis, such as is achieved by the inconvenient desensiti7~t;~n method; or by blocking the process by which IgE-allergen complexes stimulate the degranulation 25 of mast cells and basophils with the co~comitant release of the chemical mediators of hypersensitivity.
At a more fl1n~ l level, Stanworth et al. (3-7 and others~a~13) have used synthetic IgE ~-chain peptides and the corr~Rp~m~;nr antibodies to study the role of 30 cytophilic peptides in cell signaling processes, in an attempt to elucidate the molecular basis for the immunological triggering of mast cells a~d basophils.
Among the many IgE peptides st~ldied over the past two decades ~Table l), a potential effector site 35 within the Fc CH4 domain of the human ~-chain (~ysj97-, .. . , . . ,,, , . ,,,,, .. . ,, .. ,, . . _ _ _ _ _ . _ _ _ _ _ _ _ _ w09s/2636s Z 1 ~365q5 r~ C~74 Phes~6, shown in Table 2 by double underlining) was thedecapeptide. It was synthesized and used for structure/activity studies (3~ . This IgE CH4 domain-derived decapeptide was f ound to be capable of activating dose-dependent histamine release from isolated rat peritoneal 5 mast cells in a non-cytolytic manner resembling the IgE-mediated mast cell triggering process (4) . Precise structural requirements for this peptide effector site were deduced through structure-activity studies using multiple synthetic analogue~ of the ~-chain 10 decapeptide(3 4 5).
Anti - IgE CH4 peptide antibodies derived f rom ln; 7~tiong with ~ chain-related "peptide-carrier protein conjugates" were also used for structure action studies on the degranulation of IgE-sensitized cells, by 15 observing inhibitory activities (5~ 12) .
The f~C;h;l;ty of using a peptide based vaccine to provide immunotherapy to patients with IgE-mediated sensitivities has been suggested by Stanworth et al. (14 15) He used the previously identified ~-chain decapeptide with 20 a se~uence of Lys-Thr-~ys-Gly-Ser-Gly-Phe-Phe-Val-Phe-NH2(3) ~SEQ ID N~:1) conjugated to a "carrier protein", such as keyhole limpet hemocyanin (KLH) or the purified protein derivative (PPD) of tuberculin, and found that the "peptide-carrier protein~ conjugates elicited decapeptide-25 specific antibodies. For example, a rabbit anti-peptide serum, selected on the basis of its better-than-average anti-peptide titer, reduced the decapeptide-induced histamine release from rat peritoneal mast cells in a titer-dependent fashion. This inhibitory activity was 30 further confirmed by in vivo tests in a rat passive cutaneous anaphylaxis (PCA) model system. The effect of this rabbit anti-peptide serum on anaphylaxis was assessed, by meaYul~ t of the area of blueing and by an estimate of color intensity when given to rats which had 35 been previously sensitized by multiple allergen Wo95/26365 21 ~65~95 r~ .. Ir~74l --application prior to anaphylactic ~ n~e with the allergen . _ In the same study, results o~tained in rats using immunogens containing such "decapeptide-protein carrier con~ugates" gave pr~l ;m1ni~ry indications of S feasibility for their use as a vaccine for the treatment of allergy.
However, this strategy has met with considerable difficulties. The major deficiencies of this prototype "decapeptide-protein carrier conjugate~ vacci~e include a 10 less-than-optimal immune stimulatory capability and manufacturing difficulties stemming from the poorly def ined composition of the carrier protein a~d the non-uniformity of the con~ugation reaction It has also been found that the resultant antisera raised by such 15 peptide-protein co~jugates frequently contain more antibodies directed ~t the epitopes on the protein ~;
carrier, e.g. Keyhole ~impet ~lemocyanin (KLH), than to the target -peptide ~S) .

WO 95/2636S 2 t ~ 6 5 9 5 r~"~ . ~ /41 M
_.
o ~ 3 Ow ; $
_ ~ r~ 1 N
O~
C
5 ~_ I _ -~i ~ . ~ 5 ~ r~ ~i ~ a c 10 u3 u ~, v _ O ~ c E v r_ ~ ~æ D ' y ,, ~æ
C~ C o o 0 C
, . 8 ~ c~ C E CJ 8 ~a o '0 ~ a~r ., .E = ._ ~
20 ~ v m . C~ 3 E ~ I n 0 9~ ~ ~ c a ~ r~
-- E N r~ r~ ' r~ r7 u C C
a j~
C u u 3 0 0 ~ ~
~, K o r~ r~ r '~
y O~ ~ O rO r~
C W ~ W r W W ~ ~ ;
E ~ K ~ ~ ~ ", Ui K ~ 8 ~ L

.

WO95126365 21 8 6 5 9 5 6 T~l/~ /Q~741 U~ _ o~ 0 o , 0o -- ~A
~ _ o a s ~ O
0 '1 -~ ,O
V

0 C ~ 80 ol 0 0 0 O
U . O O O C O O
_ ._ O ~ . 0 . ._ ._ _ ~
0 cV U ~ . Ll ~ V O O 01 0 ._~ =O o ~ O ~ _ , j Vo Vo 01~ ._ 15~3 _ u o ~ n ~ V V O o o ~c ~
C _ F C V ~ C C -- 0 ~ E E '0 ~ V
O U ~ ~ 0 - O '- V V O ~
_ _ ~ 2 ~ ~ ." N~ ~ _ ~ O
20 . ù 8 ~ ; 'C 0 0 C C C
.C V
E ~t N N N ~ ~ y O UU ' U U U U U U

C ' ' o~ o. ._ , c j~ c t~
1~ ~ .~ ~ 10 _ IA o u -- ~ ~ ~ I~ r-- -- ~ N
~ -- N N ~
_ . _ _ . _ . _ . _ _ . . . _ . . _ _ _ .

WOgs/26365 2 1 ~ 65 95 P~ 374l It is known to those of akill in the art, small peptides are poor immunogenE. To make small peptides immunogenic, they are usually joined to large carrier proteins by chemical coniugation or by gene fusion. These processes, however, generally produce unpredictable conformational changes in a peptide. Further, the immune response i8 frequently misdirected to the immuno~l, n:lnt carrier. Consequently, the development of a potent vaccine to provide long-lasting relief from allergies awaits further immunogen design.
In Table 2, the amino acid 3equences for the CH2 to CX4 domains of rat IgE ~-chain(l6) and mouse ~-chain~l7 are aligned with the amino acid sequence for human ~-chain(lS~ (SEQ ID NOS:2-4) to provide a guide for IgE-related peptide fragments previously reported. It is to be noted that in human IgE ~ heavy chain, :~ next to Q at position 2~2 is not present in the original IgE myeloma ND
sequence. Gaps, indicated by dashes, have been introduced to maximize homology. Matches of homologous residue positions are boxed. The positions on the ~ sequences which have been studied for structural activity (Table 1) are underlined in Table 2. The structurally active IgE
CH4 decapeptide sequence in the human IgE CH4 domain is double underlined (SEQ ID NO:1). The amino acid code used in the Table is: A, alanine; R, arginine; N, asparagine; D, aspartic acid; C, cysteine; Q, g1~1t~m;n~;
E, glutamic acid; G, glycine; H, histidine; I, isoleucine;
L, leucine; K, lysine; M, methionine; F, phenyl~l~n;n~; P, proline; S, serine; T, threonine; W, tryptophan; Y, tyrosine; V, valine.

W0 95126365 2 1 8 6 5 9 5 r~ . /41 Y 1~
O O I ~ N ~ O
O l_ Y O ~ ~ ~
U U U
~ ~ Y I ~ ~ I Cl O O
< ~ C~ I S Z l ~-- _ _ al ~D
O _ I O O ~ ~ O ~
~ ~ ~. ~ ~ _ 1:1 ~ lU
O O ~ ~ ~L . _ _ ~
S ~ I_ <n u) (.7 ~ 1 3 ~ _ c n. ~ o o ~
N ~ CC ~ _ _ ~ C C
C~ _ _ _ U O ~
I O Cl I ~ ~ ~ ~ ~ ~
111 S .C ~ ~ C C~
_ I~
I ~ I
0 s O A Cl = Cl L~ --.1~ 0 ~ ~-- In O~
s~~n o = o. I ~ ~ ~_ ~ -- N ¦ I ~ ¦ ~
=' I U U U I _ S S
¦ Cl O ¦ V Y ¦ K Iy ~y ¦ = =
l J a~ I U ~ o 3 _ y I ~
I- - I I - cl ~ In N ~ S ~ ~ ~ L~l L~
201 ~ ~ o Y K C.
z¦ u u u L~ K Y
~. 1 1. 1. C 1 1~1 111 1 I o ~ I I l_ ~ r n o~ r UU I C~
o ~ 3 3 ~ o~ ~
O~ r / ~ 3 2S _ u~ ~ ~- ~
Y , L~ o ~ I ~ ~ I

1. -- ` Z y ~ "., ~
o ._ U o C~ o U K ~ U U U
~U O ~ O ~ ~1 0 K ~1: = K

~ WO 95/26365 2 1 8 6 5 9 5 PCIIUS95103741 O G S ¦ ¦
~ Z
G W
~ , ~
C~ ~
r_ ~ Cl ~r Z Q ~-~:1 G
- - z O r ly . o V
~ ~ Ul .~ ~ ~ (O G
0 Z ~ Z G y o ~ a o7 o C
C ~
U L ~ ~ ~.
I r- r_ S
. . ~ ~ _ ~: ~
1~ K G G' Cl ~o Y Y Y I ~
~CU C- Z ~ -- --C\l 1 07 Ll I ~n IL
~~ ~ ~ .
I_ G ~
.Q ~ . .
G Ul r-- ¦ G C ¦
~ ~ G
20 ~ ~ ~ G' Y
,.
~r ~ >
~~IL ~ 1:1 AGG G ~ ~
r_. C .C G G
~ W ~ ~O vl r~r G' ,. ..
v , ~ u.
r-~o G
Y
r-r-r~
I_ _ _ I Cl Cl I
U> O~ U~ Y
GG G O~
~ ~ I Y I
"~ G G or ~
rG-- G G
O ~ ~ 0 ~0 = IY ~: z ~, W0 95l26365 2 1 8 6 5 9 5 r~ 741 ~
o ~ 10 --~IST OF R~ ;N~
1. Noon B. "Prophylactic inoculation against hay fever. "
~ancet, i:1572-1573 (1911).

2. World Health Organization and Tnt~rn;~ion~l U~ion of Immunological Societies Working Group Report: Current status of allergen immunotherapy. Lancet, i :259-261 (1989) .

3. Stanworth, Kings, Roy, -et al. Biochem. ~., 180:665-668 (1979).
10 4. Stanworth. Mol. Immunol, 21:1183-1190 (1984).
5 . Stanworth, and Burt. Mol . Tmmllnnl ., 23 :1231-1235 (1986) .
6. Burt, and Stanworth. ~llr ,J. Immunol., 17:437-440 (1987) .
1~ 7. Stanworth. Mol. Immunol., 25:1213-1215 ~1988) .
8. Ha,lJ-u~ . Science, 189:389-390 (1975) .
9 . Kenten, Helm, T~hi ~;lk~3, et al. Proc. Natl . Acad Sci ., USA, 81:2955-2956 (1984) .
10. Coleman, E3elm, Stanworth, and Gould. Eur. J.
Immunol ., 15: 966-969 (1985) .
11. Chretien, Helm, Marsh, et al. J. Immunol., 141-3128-3134 (1988).
12. Robertson, and Lin. Mol. Immunol., 25:103-113 (1988).
13. Helm, Kebo, Vercelli, et al. Proc. Natl. Acad. Sci., USA 86: 9465-9469 (1989) .
14. Stanworth, Jones, ~ewin, and Nayyar. Allergy treatment with a peptide vaccine. ~ancet, 336:1279-1281 (1990).
15. Stanworth, I.ewin, Nayyar, and Jones. Immunoreactive peptides and antibodies and their use in anti-allergy treatment. EPO 403 312 A1 (1990).
16. Kindsroger et al., DNA 1:335_343 (1982) .
17. Ishida et al., EMBO 1:1117-1123 (1982).

18 . Dorrington and Bennlch, Immunol . Rev., 41 : 3 -25 ( 1978 ) .

WO95/26365 21 86595 ~",J /41 19 . Brett et al Eur. J. Immunol . . 23 :1608-1614 (1993) .
20. Weismuller et al., Int. J. PePtide Re~., 40:255-260 ( 1992 ) .
21. Celiq et al., J. Immunol., 140:1808-1815 (1988) .
22. Demotz et al., J. Immunol., 142:394-402 (1989) .
23 . Chong et al, Infect . Immun., 60 :4640-4647 (1992) .
24 . Grant ed., Svnthetic PePtide~: A User' s Guide, W.H.Freeman & Co., New York, NY, (1992) pp. 382.
25. O'Hagan et al., Vaccine, 9:768-771 (1991) .
0 26. Eldridge et al., Molec. Immunol., 28:287-294 (1991).
27. Mar(~ s~n et al., J. Immunol136:2231-2239 (1986).

wo ss/2636s 2 1 ~ 6 5 ~ 5 r~ 41 OB~ECTS OF THE INVENTIQN
It is an obj ective of the present invention to employ a group of IgE ~-chain based E2eptide immunogens chemically synthesized in either a radially branching form or a linear T helper epitope c~)nt~;n;n~ form, to elicit high titer antibodies to the decapeptide effector site of the CH4 domain of the human ~-chain, when introduced to mammals, including humans.
Another obj ective is to desig~ optimal peptide immunogens, with specif ic amino acid sequences taken f rom the human IgE heavy chain CH4 domai~ ( IgE CH4 ) attached to peptides ~ nt~;n;ng ~JL~ CC~ U8 human helper T cell epitopes in a specific orientation which, when introduced into mammals, including humans, will stimulate production of high titers of ~ff;r~ us antibodies to the effector site on human IgE CH4. These antibodies should inhibit mast cell activation, reduce the release of chemical mediators such as histamines that are responsible for allergy symptoms, depress IgE-mediated passive cutaneous anaphylaxis (PCA) reaction, and suppress allergen-induced IgE production by B lymphocytes.
A further objective is to develop an effective IgE ~-chain peptide-based vaccine, employing compositions .,,nti~;n;n~ guch branching multimeric or linear immunogens, to provide immunotherapy for the treatment of allergic reactions .
SI~RY OF THE INVENTION
According to the present invention, peptide immunogens are made by solid phase synthesis. The peptide immunogens comprise a series of radially branched multimeric peptides containing a ten amino acid IgE CH4 peptide (SEQ ID NO:1), or an immunogenic analog thereof; a series of multimeric branched peptides ~nt~;n;n~ the IgE
CH4 peptide (SEQ ID NO:l) or an immunogenic analog thereof together with a helper T-cell epitope (Th epitope); and a W0 95~26365 2 1 ~ ~ 5 ~ 5 ~ 41 series of linear monomeric peptides ~nti~;n;ng the IgE CH4 peptide (SEQ ID NO:1) or an immunogenic analog thereof together with a portion of a helper T-cell epitope (Th epitope) . The IgE CH4 peptide is taken from the Fc region 5 of the IgE heavy chain, i.e. ~-chain CH4 domain (IgE CH4) .
of the three series of peptide immunogens, the linear peptides are preferred. Compositions containing these peptides are used to immunize healthy mammals, e.g. guinea pigs, rats, and humans, to elicit the production of high 10 titer antisera specific for the IgE CH4 effector site (SEQ
ID NO:1) and free of irrelevant ~nt;hn~l;es According to the present invention, vaccines ~nntA;n;n~ the synthetic peptides as the key immunogen may also be prepared with an effective amount of a multimeric-15 branching peptide or a linear peptide in the presence of aproper adjuvant and/or delivery vehicle. It is expected that such vaccine compositions will elicit a more focused anti- IgE peptide response than those of the peptide-carrier protein conjugates currently used by Stanworth et 20 al (1~), thus providing a better immunotherapy for the treatment of allergy.
DETAI~ED ~ 'RTPTION OF THE INVENTION
This invention is directed to the use of a novel group of peptide-based immunogens for the generation of high titer ~nt;ht~ to an effector site on the CH4 domain of human IgE ~ heavy chain (SEQ ID NO:1) in healthy mammals, including humans, for the treatment of IgE-mediated allergic diseases.
It is generally accepted that allergy symptoms, the; ~ te result of IgE-dependent hypersensitivities, are caused by chemical mediators released by mast cells and basophils. The release is triggered when a mast cell or basophil that has been sensitized with surface-bound IgE binds to an allergen for which the surface-bound IgE
is specific. The triggering is actuated by the binding of w0 9s/~636s ` 2 1 8 6 5 q 5 ~ . /41 --0 - 14 _ the allergen to the Fab' portion of the surface-bound IgE
in an antigen-antibody type interaction. The ~
allergen/antibody bindi~g crosslinks the bivalent surface-bound IgE and ind~ces a conformational change in the 5 distal Fc region o~ IgE, the region of IgE in direct contact with a high af f inity Fc receptor on the cell surface. By a ~ Anif~m as yet not precisely understood, the con~ormational change activates the cell-IgE-allergen complex with the r~sultant release of mediators, including 10 histamine, by the cell. Effector site (8) on IgE are believed to participate in the triggering event. The presence o~ specific anti-IgE antibodies directed against such ~effector sites", through either active or passive ; 7ation, may lead to inhibition of ~the cell 15 activation process in hosts suffering from allergic reactions by interf ering with the interaction between the gE "effector sites" and the cell surface.
Such interventions through the use of specif ic anti-IgE Ant;ho~l;es~ i.e a kind of immunotherapy, can be 20 achieved either passively, through prophylactic treatment with specific "site-directed" Ant;ho~ to IgE, or, more preferably, actively, by providing the host with a vaccine comprised o_ site-directed peptide immunogens, to elicit the production by the host of its own site-directed anti-25 IgE antibodies . It is believed that active ; ; zat; ~ nwill provide a more ef~e~ive and 1 onger lasting protection .
Among the sites f rom the Fc region of circulating IgE that have been studied for flln~t;~nAl 30 activity, a region on the CH4 domain of the IgE molecule (7Iys~97-Phe~06) has been i~ont;f;P~l as a conformational e__ector involved in::the trlggering of mast cells and basophils~3-8~l~). See Table 1 and the areas underlined in Table 2. A decapeptide~derived _rom this site with the 35 sequence ~ys-Thr-l.ys-Gly-Ser-G~y-Phe-Phe-Val-Phe-NEI2 (SEQ

woss/2636s 21 8659~ r~ /41 ID NO:1) waE found to approximate the conformation of this effector site. This is evidenced by the ability of the decapeptide to elicit dose-~lPp~on~lpnt histamine release from rat mast cells in a manner resembling the 5 immunological triggering process(~).
Stanworth et al. (14~15) demonstrated the feasibility of providing immunotherapy to patients with IgE-mediated allergic reactions through the use of experimental vaccines by using the IgE CH4 decapeptide (SEQ ID NO:1) coupled to a carrier protein, keyhole limpet hemocyanin (KLH) as an immunogen. Animal immune sera obtained from such; i z~tions were found by Stanworth et al. (14~15) to moderately reduce the decapeptide-induced histamine release from rat peritoneal mast cells in a 15 titer-flPrpn~pnt fashion. Inhibitory activity by the immune sera generated was further confirmed by in vivo passive cutaneouE anaphylaxis (PCA) tests under conditions of multiple allergen application.
A maj or def iciency of the prototype ~ IgE CH4 20 peptide" vaccine developed by Stanworth et al is its weak immunogenicity, a problem inherently associated with almost all self-antigens.
In the present invention, specif ic immunogens are provided wherein synthetic immune stimulatory elements are linked to the CH4 decapeptide of IgE (SEQ ID NO:1) in a specif ic orientation such that potent antibodies directed to this effector site on IgE can be broadly generated in a genetically diverse host population. In turn, these antibodies block the stimulatory action of IgE
on mast cells and basophils, thus resulting in an effective treatment to prevent IgE-~ tP~ allergic diseases .
The peptide immunogens of the present invention are capable of eliciting antibodies with serological croEs-reactivity with the target amino acid Eequence of wo 95~26365 2 1 8 6 5 9 5 P~ /41 the Fc region of IgE (SEQ ID NO:1) while being subst ~nt; ;3 1 1 y ; nr:lr~hl P of mediating non- cytolytic histamine release.
The initial do3e, e.g. C.2-2.5 mg; preferably 1 5 mg, of immunogen is to be administered by in~ection, preferably intramuscular, followed by repeat (booster~
doses. Dosage will depend on the age, weight and general health of the patient a5 i5 well krlown in the therapeutic arts .
While there i9 no particular limitation to the species of mammals suitable for the production of antibodies, it is generally preferred to use mice, rabbits, guinea pigs, pigs, goats, rats or sheep, etc. as the hosts.
For active; ; z~t; on, the term ~immunogen~
referred to herein relates to synthetic peptides which are capable of ; n~ r1 nr, antibodies against the IgE CH4 decapeptide (SEQ ID NO:1), which antibodies lead to the suppression of IgE-mediated basophil and mast cell 20 degranulation. The; ~ of this invention included multimeric peptides or its analogs with a branching lysyl core matrix structure.
These branched multimeric peptides have the r~;lh; l; ty of independently eliciting an immune response 25 in a host animal. The analogs of IgE CE4 decapeptide (SEQ
ID NO:1) include the synthetic peptide analogs described by Stanworth et al. (3'4'5), which are incorporated herein by reference. To be suitable, the r~lec~ r weight of the immunogen should be higher than 5, 000 and preferably be higher than 10,000. The repeating branch unit for the peptide should be equal to or higher than 4.
Bifunctional amino acids such as lysine followed by attachment to an amino acid with a pref erably non -charged side chain, such a5 Gly or Ala, are useful in the 35 making of the core matrix structure. By inserting an wo gs/26365 2 1 ~ 6 5 9 5 r~".J.. ~ /41 amino acid in one additional coupling cycle in between two di-Boc-Lysine coupling cycles, the amino acid acts as a spacer in between the peptide branches to allow maximum freedom to attain the conformation necessary for optimal 5 presentation.
The immunogen ref erred to in the present invention also included linear peptides which contain promiscuous helper T cell epitopes (Th epitopes). These Th epitopes were covalently attached in a defined fashion 10 to the decapeptide effector sequence (SEQ ID NO:1), with or without a spacer, so as to be adj acent to the N
terminus of the decapeptide, in order to evoke efficient antibody responses. The immunogen may also be comprised of an immune stimulatory sequence corresponding, for 15 example, to a domain of an invasin protein f rom the bacteria Yersinia spp~l9). The invasin domain may also be attached through a spacer to a Th epitope.
The '~immunogen" of the present invention minimizes the generation of irrelevant antibodies, thus 20 eliciting a more focused immune response to the ~target sequence", i.e., the desired IgE CH4 cross-reactivity (SEQ
ID NO:1), without producing undesirable side effects which may complicate the immunotherapy process for the treatment of allergy.
However, when a short target sequence, such as the 10 amino acid IgE CE4 segment ~ys-Thr-~ys-Gly-Ser-Gly-Phe-Phe-Val-Phe (SEQ ID NO:1), is used to design a carrier protein-free immunogen, one faces serious challenges. A
short peptide antigen is usually a T cell-dependent antigen, i . e . the presence of a T helper epitope is required to render a short " target " peptide immunogenic .
The short IgE CH4 decapeptide (SEQ ID NO :1) or an immunogenic analog thereof doe8 not contain a T helper cell epitope. The branched multimeric and linear immunogens comprising the short IgE CH4 decapeptide are . _ . _ .. _ . .. _ . . . . . , . , . . . _ _ _ W0 9s~26365 2 1 ~3 6 5 ~ 5 P~ /41 designed h~rP;n ~n provide for artificially built-in functional helper T-cell epitopes.
The peptides immunogens of this invention are represented by the formula (A) n~ (Th) ~n~ (B) O- ( IgE CH4 peptide) p wherein is an amino acid, CY-N~2, a fatty acid, a derivative oi a fatty acid, or an invasin domain;
B is an amino acid;
Th is a helper T cell epitope or an immune Pnhi~nr;ng analog or segment~ thereof;
IgE CH4 peptide i5 Lys-Thr-Lys-Gly-Ser-Gly-Phe-Phe-Val-Phe (S~Q ID NO:1) or an immunogenic analog thereof;
n is from 1 to 10;
m is from 1 to 4;
o is from 0 to 10; and p is from 1 to 3.
The peptide; r~Pn~ of the present invention comprise from about 20 to about 100 amino acid residues, preferably from about 20 to about 50 amino acid residues and more preferably from about 20 to about 35 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, ~-alanine, ornithi~e, norleucine, norvaline, hydroxyproline, thyroxine, ~-amino butyric acid, homoserine, citrulline and the like. Naturally-occurriAg amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, me~h;nn;n~, phe~yl~l~n;np, proline, serine, threonine, 35 tryptophan, tyrosine and valine. Moreover, when n is ~ Wo ss/2636s 2 1 dr 6 s q ~ P~ /41 greater than one, and two or more of the A groups are amino acids, then each amino acid is independently the same or dif f erent .
When A is a fatty acid, such as stearic acid or S palmitic acid or a fatty acid derivative, such as a tripalmitoyl cysteine (Pam3Cys) group, it acts as an adjuvant by f~nh~n~; n~ the immunostimulating properties of the Th epitope(20). When A is a fatty acid or its derivative it i9 usually located at the amino t~l; nllc of 10 the peptide. Furthermore, when one of A is a fatty acid, there are 2 or 3 additional amino acid A moieties. The fatty acids useful in the invention have a hydrocarbon chain of 8 to 24 carbon atoms which may be saturated or unsaturated .
When A is an invasin domain, it is an immune St;r~ tr1ry epitope from the invasin protein of a Yersinia species. This immune stimulatory property results from the ~r~h; l; ty of this invasin domain to interact with the ~l integrin molecules present on T cells, particularly 20 activated immune or memory T cells . The specif ic sequence for an invasin domain found to interact with the ,~l integrins has been described by Brett et al (19) In a preferred embodiment, the invasin domain (Inv) for linkage to a promiscuous Th epitope has the sequence:
Thr-Ala-Lys-Ser-I,ys-~ys-Phe-Pro-Ser-Tyr-Thr-Ala-Thr-Tyr-Gln-Phe (SEQ ID NO: 25) or is an immune stimulatory analog thereof from the corresponding region in another Yersinia species invasin protein. Such analogs may contain substitutions, deletions or insertions to accommodate strain to strain variation, provided that the analogs retain immune St; r~ t~ properties .
In one embodiment, n is 4 and A is ~Y-NH2, lysine, lysine and lysine in that order. In another 35 embodiment n is 1 and A is ~Y-NH2. In yet another woss/z636s 2 1 8 6 5 9 5 ~ '0~741 embodiment, n is 4 and A is ~-NH2, an invasin domain (Inv), glycine,and glycinc in that order.
B comprises naturally occurring or the non-naturally occurring amino acids as described above. Each B may be ;n~pPn~ntly the same or different. When B is lysine, a branched polymer can be formed. For example, if o is 7 and all seven B groups are lysine then a branching K core (K~K2K) is formed when the peptide synthesis is conducted without protecting the lysyl side chain ~-amino group. Peptides with a K core have eight branch arms, with each branch arm being i~nt~ ~1 and represented as "(A) n~ (Th)=-" or "(IgE CH4 peptide with built-in-Th)-".
In addition, the amino acids of B can form a flexible hinge, or spacer, to enhance the immune response to the Th epitope and IgE CH4 decapeptide or an analog thereof.
Examples of sequences encoding flexible hinges can be found in the i -globulin 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:24), where Xaa is any amino acid, preferably aspartic acid.
Immunogenicity can also be improved through the addition of spacer residues (e.g. Gly-Gly) between the promiscuous Th epitope and the IgE CH4 decapeptide or an analog thereof. In addition to physically separating the Th epitope from the B cell epitope (i.e., the IgE CH4 decapeptide site or an analog thereof), the glycine residues can disrupt any artifactual secondary structures created by the joining of the Th epitope with the IgE CH4 decapeptide (SEQ ID NO:l) or an analog thereof and thereby ~l;m'n~te interference between the T and/or ~ cell responses. Thus, the.conformational separation between the helper cell and the antibody eliciting domains permits more efficient interactions between the presented 3S immunogen and the appropriate Th and B cells.

WO 95/26365 2 1 ~ ~ 5 9 5 I~ 9!"'~741 Th is a Th epitope comprising natural or non-natural amino acids. A Th epitope may con3ist of a rnnt;nllnus or discnnt;nllnus epitope; not every amino acid of Th is necessarily part of the epitope. Th epitopes, including analog3 and segments thereof, to be suitable for the present invention are capable of ~nh~n~l ns or stimulating an immune response to the IgE CH4 decapeptide (SEQ ID N0:1) or an analog thereof. Th epitopes that are n~l n~nt and promiscuous are highly and broadly reactive in animal and human populations with widely divergent MEIC types(2l~231. The Th domain suitable for the present invention 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 2 ), then each Th epitope may be independently the same or dif f erent .
Th epitope analogs include substitutions, additions, deletions and insertions of from one to about 10 amino acid residues in the Th epitope. Th ~e_ ~ are contiguous portions of a Th epitope that are suf f icient to enhance or stimulate an immune response to the IgE CII4 decapeptide (SEQ ID N0:1) or an analog thereof.
Th epitopes of the present invention include hepatitis B surface and core antigen helper T cell epitopes (B~Th and HBCTh), 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~
Th), Chlamydia trachomati6 major outer membrane protein helper T cell epitopes (CT Th), ~l;rhth~ria toxin helper T
cell epitopes (DT Th), Pla6modium falciparum circumsporozoite helper T cell epitopes (PF Th), Schisto60ma mansoni triose rhnsph~te isomerase helper T
cell epitopes (SM Th), Escherichia coli TraT helper T cell epitopes (TraT Th) and immune-~nh~n-~;n~ analogs and segments of any of these Th epitopes. Examples of Th _ _ _ _ _ _ _ _ . . .. .. , ...... _ .. . _ .. . .. , . , _ _ _ _ wo g5n6365 2 1 8 6 5 9 5 ~ . /41 epitope se~uences are provided below:
B. Th: Phe-Phe-Leu-Leu=Thr-Arg-Ile-Leu-Thr-Ile-Pro-Gln-Ser-Leu-Asp (SEQ ID NO:5) S PTl Th: Lys-Lys-Leu-l~rg-Arg-Leu-Leu-Tyr-Met-Ile-Tyr-Met-Ser-Gly-Leu-Ala-Val -Arg-Val-~is-VaI -Ser-Lys-Glu-Glu-GlI:L-Tyr-Tyr-Asp-Tyr (SEQ ID NO:6) TTI Th: Lys-Lys-Gln-Tyr-Ile-Lys-Ala-Asn-ser-Lys-phe-Ile 0 Gly-Ile-Thr-Glu-Leu (SEQ ID NO:7) TT, Th: Lys - Lys - Phe -Asn -Asn - Phe - Thr -Val - Ser - Phe - Trp - Leu -Arg-Val-Pro-Lys-Val-Ser-Ala-Ser-His-Leu (SEQ ID NO:8) PT~, Th: Tyr-Met-ser-Gly-Leu-Ala-val-Arg-val-His-val-ser Lys-Glu-Glu (SEQ ID NO: 9 ) TT, Th: Tyr-Asp-Pro-Asn-Tyr-Leu-Arg-Thr-Asp-Ser-Asp-Lys-Asp-Arg-Phe-Leu-Gln-Thr-Met-Val-Lys-Leu-Phe-Asn-Arg-Ile-Lys (SEQ ID NO:10) PT2 Th: Gly-Ala-Tyr-Ala-Arg-Cys-Pro-Asn-Gly-Thr-Arg-Ala-Leu-Thr-Val -Ala-Glu-Leu-Arg-Gly-Asn-Ala -Glu-Leu 2S (SEQ ID NO:11) MV" Th: Ser-Glu-Ile-Lys-Gly-Val-Ile-Val-~is-Arg-Leu-Glu-Gly (SEQ ID NO:12) and Leu-Ser-Glu-Ile-Lys-Gly-Val-Ile-Val-EIis-Arg-Leu-Glu-Gly-Val (SEQ ID NO:61) Bc Th: Val-Ser-Phe-Gly-Val-Trp-Ile-Arg-Thr-Pro-Pro-Ala-Tyr-Arg-Pro-Pro-Asn-Ala-Pro-Ile-Leu 3S (SEQ ID NO:1~) ~ WO 95/26365 2 18 6 5 9 5 r~ . /41 MVF2 Th: Gly-Ile-Leu-Glu-Ser-Arg-Gly-Ile-Lys-Ala-Arg-Ile-Thr-Xis-Val-Asp-Thr-Glu-Ser-Tyr (SEQ ID NO:26) TT4 Th: Trp-Val-Arg-Asp-Ile-Ile-Asp-Asp-Phe-T~r-Asn-Glu-Ser-Ser-Gln-Lys-Thr (SEQ ID NO:27) TTs Th: Asp-Val-Ser-Thr-Ile-Val-Pro-Tyr-Ile-Gly-Pro-Ala-Leu-Asn-E~is-Val (SEQ ID NO:28) CT Th: Ala-Leu-Asn-Ile-Trp-Asp-Arg-Phe-Asp-Val-Phe-Cys-Thr-Leu-Gly-Ala-Thr-Thr-Gly-Tyr-Leu-Lys-Gly-Asn-Ser ( SEQ ID NO: 2 9 ) DTl Th: Asp-Ser-Glu-Thr-Ala-Asp-Asn-Leu-Glu-Lys-Thr-Val-Ala-Ala-Leu-Ser-Ile-Leu-Pro-Gly-Ile-Gly-Cys (SEQ ID NO:30) DT2 Th: Glu-Glu-Ile-Val-Ala-Gln-Ser-Ile-Ala-Leu-Ser-Ser-Leu-Met-Val-Ala-Gln-Ala-Ile-Pro-Leu-Val-Gly-Glu-Leu-Val-Asp- Ile-Gly-Phe-Ala-Ala-Thr-Asn-Phe-Val-Glu - Ser - Cys ( SEQ ID NO: 3 l ) PF Th: Asp-Ile-Glu-Lys-Lys-Ile-Ala-Lys-Met-Glu-Lys-Ala-Ser-Ser-Val-Phe-Asn-Val-Val-Asn-Ser (SEQ ID NO:32) SM Th: Lys-Trp-Phe-Lys-Thr-Asn-Ala-Pro-Asn-Gly-Val-Asp-Glu-Lys-Ile-Arg-Ile (SEQ ID NO:33) TraTl Th: Gly-Leu-Gln-Gly-Lys-Ile-Ala-Asp-Ala-Val-Lys-Ala-Lys-Gly (SEQ ID NO:34) TraT2 Th: Gly-Leu-Ala-Ala-Gly-Leu-Val-Gly-Met-Ala-Ala-Asp-Ala-Met-val-Glu-Agp-val-A8n (SEQ ID NO:35) _ _ _ _ _ _ _ _ _ , . ,, . , ,, .. .. _ . _ . _ _ _ . _ _,, ... _ Wo 9s/26365 21 8 6 5 9 5 PCrNS95/03741 ~

o TraT3 Th: Ser-Thr-Glu-Thr-Gly-Asn-Gln-His-His-Tyr-Gln-Thr-Arg-Val-Val-Ser-Asn-Ala-Asn-Lys (SEQ ID NO:36) Preferably, the Th epitope is HB" Th, PTl Th, PT2 Th, TTl Th, TT3 Th, or MV~l Th.
In the monomeric linear peptides of this inventio~, as described by the Formula (A)n~ (Th)m- (B)~,- (IgE
CH4 peptide), the Th epitope is covalently attached through spacer B to the N terminus of the IgE CH4 decapeptide (SEQ ID NO:1) . The IgE CH4 peptide is Lys-Thr-Lys-Gly-Ser-Gly-Phe-Phe-Val-Phe (SEQ ID NO:1), a decapeptide. The IgE CH4 peptide may be replaced by an immunogenic analog. The immunogenic analogs thereof= may contain a substitution, addition, deletion, or insertion of from one to about four amino acid residues provided that the analog is capable of eliciting an immune response crossreactive with the IgE CH4 decapeptide (SEQ ID NO:1).
The substitutions, additio~s, and insertions may be made with natural or non-natural amino acids as def ined herein .
Immunogenic ana~ogs of the IgE C~4 peptide (SEQ NO:1) have been identified by Stanworth et al. (3'4'5) and are incorporated herein by ref erence .
Accordingly, preferred peptide immunogens of this invention are monomeric peptides cnnt~;n~n~ IgE CH4 decapeptide (SEQ ID NO:1) or an immunogenic analog thereof and Th. More specifically, preferred peptide immunogens are those linear constructs ~ nt~inin~ IgE CH4 (SEQ ID
NO:1) or an immunogenic analog thereof; a spacer (e.g Gly-Gly); a Th epitope selected from the group consisting HB, Th, PTl Th, PT2 Th, TTl Th, TT3 Th, and MV~l Th (SEQ ID
NOS:5,6,11,7,10,61, respectively) i and optionally the Inv domain (SEQ ID NO:25). Preferred peptide immunogen compositions include, for example, Peptide Nos. 19-23 and 28 (Table8 5 and 6, SEQ ID-Nos:5l-55~62) wo 95/26365 2 ~ 8 ~ 5 ~ ~ r~ 'IQ3741 The peptide; AgF-n~ of this inveIItion may be made by chemical 9ynthesi5 well known to the ordinarily skilled artisan. See, for example, Grant, ed. SYnthetic Pe1?tides~24~. Hence, peptide immunogens may be synthesized using the automated Merrif ield techni~ues of solid phase synthesis with the ~-NH, protected by either t-Boc or F-moc chemistry using side chain protected amino acids on, for example, an Applied Biosystems Peptide Synthesizer Model 430A or 431. To synthesize a K core moiety on which to synthesize peptide branches, Di-~, ~ (t-Boc) lysine residues are used in place of t-Boc lysine with a 2, ~ -dichlorobenzyl protecting ~ - amino group .
When A is a fatty acid, it may be added easily to the N-terminus of the resin bound peptide by the well known carbodiimide method. To add PamlCys, the lipoamino acid S- [2, 3-Bis (palmitoyloxy) - (2R) -propyl-N-palmitoyl- (R) -cysteine (Pam3Cys) is chemically synthesized. Pam3Cys may then be coupled to the N terminus of a peptide by solid-phase synthesis using Pam3Cys-OH in the final coupling 20 step to link the 1; ro~m; n~ acid to a resin-bound peptide chain .
To improve the solubility of the final coupled lipopeptide product, the solid-phase peptide can be elongated with additional serine and ly3ine residues at the N-terminus.
Af ter 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 by HP~C and characterized biochemically, for example, by amino acid analysis or by seguencing. Purification and characterization methods for peptides are well known to one of ordinary skill in the art.
Other chemical means to generate linear Th- Ig~
. .. . .. . . . _, . . . .... .

WO 95t26365 2 1 8 6 5 9 5 P~l/L /41 CH4 decapeptide constructs of the invention include the ligation of the haloacetylated and the cysteinyl peptide through the formation of a "thloether" linkage. For example, cysteine can be added to the C terminus of a Th- -containing peptide and the thiol group of cysteine iB used to form a covalent bond to :ar electrophilic group such as an N'Y chloroacetyl-modified or a --lP;m;~f~-derivatized ~-or e-NH2 group of a lysine residue that is attached to the N-terminus of the IgE CH4 decapeptide ~ID SEQ NO:l) or an 0 immunogenic analog thereof.
The subject peptides can also be polymerized.
Polymerization can be accomplished for example by reaction between glutaraldehyde and the -~I, groups of the lysine residues using routine methodology. The linear "A-Th-spacer-IgECH4" peptide constructs (e.g., Peptide Nos. 19-23 and 28, SEQ ID NOS:51-55 and 62) may also be polymerized or co-polymerized by utilization of an additional cysteine added to the N-t~rm;nllC of the linear "A-Th-spacer-Igl3CH4" construct. The thiol group of the N-terminal cysteine may be used for the formation of a nthioether" bond with a halochloroacetyl-modified or a maleimide-derivatized ~- or e-NH2 group of a lysine residue that is ~tt~ h~d to the N-terminus of a branched poly-lysyl core molecule (e.g., K2K, K~K2K, K8K~K2K).
Alternatively, the longer linear peptide -, ~ may be synthesized by well kno~n recombinant DNA tech~iques. Any standard manual on DNA technology provides detailed protocols to produce the peptides of the invention. To construct a gene encoding a peptide of this 30 invention, the amino acid sequence i8 reverse translated into a nucleic acid sequence,- and preferably using ~
optimized codon usage for the organism in which the gene will be expressed. Next, a synthetic gene is made, typically by synthe8izing overlapping oligonucleotides which encode the peptide and any regulatory elements, if - -wo ss/263 r~
65 2 1 ~6595 ., '~ 741 necessary. The synthetic gene is inserted in a euitable cloning vector and recombinants are obtained and characterized. The peptide is then expressed under suitable conditions d~y~ r iate for the selected expression syatem and ho5t. The peptide is purified and characterized by ~tandard methods.
The efficacy of the peptide immunogen of the present invention may be esti~hl; ~hPd by injecting the immunogen into an animal, and then monitoring the humoral immune response to IgE CH4 decapeptide (SEQ ID NO:1) or an immunogenic analog thereof, as detailed in the Examples.
Suitable animals include mice, rats, rilbbits, guinea pigs, pigs, goats, sheep, or the like.
Another aspect of this invention provides a vaccine composition comprising an effective amount of one or more of the peptide; -3~is of this invention in a pharmaceutically acceptable delivery system. Such vaccine compositions are used for prevention of atopic allergic reactions ;n~ lng allergic rhinitis, those of food allergies, asthma, anaphylaxis, and other IgE ~ ted hypersensitive reactions such as virally-induced asthma.
Accordingly, the subject peptide immunogens can be formulated as a vaccine composition using adjuvants, pharmaceutically-acceptable carriers or other ingredients routinely provided in vaccine compositions. Such formulations are readily f~t~rmln~d by one of ordinary skill in the art and include formulations for immediate release and/or for 3ustained release, and for induction of systemic immunity and/or induction of localized mucosal immunity, which may be accomplished by, ~or example, immunogen entrapment by microparticles. The formulation may also include adjuvants or emulsifiers such as alum, incomplete Freund's adjuvant, liposyn, saponin, squalene, ~121, emulsigen and ISA 720 and the like.
The vaccine of the preeent invention may be , .. . , . ... . . . . _ wo g5,26365 2 1 8 6 5 9 5 - }~ 4l --administered by any convenient route i nr~ ; nr subcutaneous, orall intramuscular, or other parenteral or enteral route. It may be administered as a single dose or in multiple dogeg. T ~ 7~tion schedules are readily determined by the ordinarily skilled artisan.
The vaccine composltions of the instant invention contain an effective amount of one or more of the synthetic peptide immuno3ens cnnt~;n;nr the IgE CH4 decapeptide or its immunogenic analog and a pharmaceutically acceptable carrier. The dosage unit form may be ~u- lAt.'f3~ to contain about 0.5 f~g to about 1 mg of each peptide per kg body weight. When delivered in multiple doses, the effective dose may be conveniently divided to contain the appropriate amounts per unit dosage form.
The vaccine compositions of the present invention may be formulated to contain a cocktail of two or more of the subject peptide immunogens to enhance immunoefficacy in a broader population and thus provide a better immune response against IgE CH4 decapeptide. For example, a cocktail of Peptide Nos. 19, 20, 21, 23, and 4 is u3eful. The composition may also be formulated to comprise lipopeptides to provide a built-in adjuvant. The immune response to synthetic IgE CH4 decapeptide-rnn~A;n;ng immunogens may also be improved by delivery through entrapment in or on biodegradable microparticles of the type described by O'Hagan et al~25~. The; ,_lls can be encapsulated with or without adjuvant, including covalently attached Pam3Cys, and such microparticles may carry an immune 5t; lAl~nry adjuvant such as Freund's Incomplete Adjuvant or alum. The microparticles function to pot.on~; Ate immune responses to the immunogen, including localized mucosal immunity. Such localized immunity is P~pec;A-ly desirable, for example, for I rosAl1y locali~ed 35 allergic reactions. Vaccine compoSitionS in Wogs/26365 21 ~5q~ P~ /41 microparticular form may al90 be formulated to provide time-controlled release for sustained or periodic responses, for oral administration, and for topical administration (25-20, Examples of specific peptide immunogens are provided herebelow to illustrate the present invention and are to be used to limit the scope of the invention.
~X~MP~ R 1 syNr~ cIs OF OCTAMERIC PEPTIDE IMMUNO~
The following multimeric peptides were synthesized:
Pe~tide No. 1 [LysThrLys&lySerGlyPhePheValPheGlyProGlyLysThrLysGlySerGly PhePheValPheGlyLysMet] cLys~Lys2Lys, (SEQ ID NO:23) Pe~tide No. 2 [LysThrLysGlySerGlyPhePheValPheGlyProGlyLysThrLysGlySerGly PhePheValPheGlyProGly3~ysThrLysGlySerGlyPhePheValPheGlyLys Met]8Lys~Lys2Lys, (S~Q ID NO:13) The synthesis of the multimeric peptides proceeds by the limited sequential propagation of a trifunctional amino acid to serve as a low molecular weight matrix core i8 the basis for the f~ t; r~n of a branching multimeric peptide antigen system. The trifunctional amino acid, Boc-Lys (Boc), or di- (Boc) -Lys is most suitable since both Ns- and N~- amino acid groups are available as reactive ends. Thus, sequential propagation of di- (Boc) -Lys will generate 2n reactive ends.
For example, the first coupling of di- (Boc) -Lys onto a solid phase resin will produce two reactive amino ends to bind two peptide chains. Sequential generations of a second, third, and fourth step with di- (Boc) -Lys will therefore generate respectively tetravalent, octavalent, and hpy~Apcpvalent ends for binding multimeric peptide 3S chains antigens. Such multimeric peptides are useful as ... . .. . _ . _ . . _ . . ... .. ... .. . . _ _ _ _ _ _ _ _ .

wo 95/26365 2 1 8 6 5 9 5 r~ 741 ~

O
immunogens. Branched octameric Peptide Nos. 1 and 2 as described above were synthesized f or use as lmmunogens .
The branched antigens contain a small heptalysyl core surrounded by a layer of high density of uniform peptide-5 antigens around the core matrix. This design differs fromthe conv~nt;r,nAl peptide-carrier conjugate antigen which r~nt;:l;nc a large protein carrier such as PPD or KLEI and a small peptide antigen randomly distributed o~ the surface of the protein carrier in many undefined forms.
The synthesis of the octameric peptide immunogens employs a ;n~t;on of Boc-amino acid resin-bound benzhydrylamide and tBoc-chemistry. For example, an 8-branched heptalysyl core resin was prepared by coupling di-t-Boc ~ys onto an extra low loading of 0.14 mmole/g MBHA (4-methylbenzhydrylamine) resin on a Biosearch 9500 instrument . Two coupling cycles of di- (Boc) -Lys for each was followed by two capping reactions using 0.3 M
acetylimidazole in DMF dimethylformamide.
Another two di- (Boc) -I,ys couplings were added onto the first di- (NH2) Lys-resin. The substitution level of synthetic octameric resin was then determined by the ninhydrin test and found to have an appropriate level of free -~I2 groups, based on the theoretical coupling yield, and was used thereafter for the synthesis of octameric peptide immunogen according to the standard t-Boc procedure.
Acid=labile tert-butyloxycarbonyl (t-Boc) was used for the protection of N-~ amino acid. The following functional si~f--rh~;n protecting groups were used: O-benzyl f or Thr, Ser, Glu and Tyr; N~ - tosyl f or Arg; BOM, i e BOC-Nim-Benzyloxymethyl for ~iis, N6-dichlorobenzyloxycarbonyl for Lys; S-4-methylbenzyl- for Cys; O-cyclohexyl for Asp and C~iO for Trp.
The successive amino acids of Peptides No. 1 and 3S No. 2 were added from the C- to N- t~orTn;nllc as dictated by Wo gs/2636s 2 1 8 6 5 9 5 r~ 74l the sequences of Peptide Nos . 1 and 2 (SEQ ID NOS: 23 ,13 ) .
The resultant octameric peptidyl re9ins for Peptide No. 1 and Peptide No. 2 were cleaved by anhydrous HF at OoC for 1 hr in the presence of 10~ v/v anisole. The released multimeric antigens were extracted with acetic acid, washed twice with ether and lyophilized to dryness. The lyophilized multimeric peptides were used as immunogens.
EXAMPT,~ 2 ACTI~7E IMMUNIZATION WITH BRANCHED OCTA~R~T~
PEPTIDE IMMUNOGENS USING CFA A~D IFA AS ADJYVANTS
(a) Immunization Procedure Groups of Guinea Pigs (N=3 per group) were immunized with each of the tWQ IgE CH4-related multimeric peptide immunogens (Peptide Nos. l and 2) and with Peptide No. 3 (SEQ ID NO:l) conjugated to KLH, according to the following protocol: Each animal was injected subcutaneously with a mixture (200 ILL) of the peptide-based immunogen or conjugate (100 ~Lg/mL) emulsified with an equal volume of complete Freund' s adjuvant (CFA) .
Subcutaneous inj ections of the peptide-based immunogen mixed with incomplete Freund' s adjuvant (IFA) were repeated at days 21, 42, and 63.
(b) AssaY of G1~1n~-~ Piqs lmml]n~ sera bY meas~ inc~ their Anti-IcrE CH4 related PePtide res~onse Anti-peptide antibody activity i9 determined by ELISA (enzyme-linked immunosorbentassay) using 96-well f lat bottom microtiter plates which were coated with the corrf~pnn-~;ng; n~Pn, Aliquotg (100 ~L) of a peptide immunogen solution at a c~n~nt~^~tion of 5 llg/mL were incubated for 1 hour at 37C. The plates were blocked by another ;nc~llhz~t;nn at 37C for 1 hour with a 3~
gelatin/PBS solution. The blocked plates were then dried and used for the assay. Aliquots (100 IlL) of the test guinea pig sera, starting with a i:10 dilution in a sample dilution buf~er and ten-fold serial dilutiong thereafter, _ _ _ _ _ _ _ _ _ _ _ _ . _ . . . . . . . . . ... . .. ...... .

Wo gs/26365 2 ~ 8 6 5 9 5 P~ 741 --were added to the peptide coated plates. The plates were incubated for 1 hour at 37C. Normal guinea pig serum was used as a control.
The plates were washed six times with O . 059 PBS/Tween~D buffer. 100 ~L of horseradish peroxidase labelled goat-anti-guinea piy antibody was added at a dilution of 1:1,000 in conjugate dilution buffer (Phosphate buffer rnnt~;n;ng 0.5M NaCl, and normal goat serum). The plates were incubated for 1 hour at 37C
IO before being washed as above_ Aliquots (100 ILL) of o-phçnyl.on~ m; n~ substrate solution were then added. The color was allowed to develop for 5-15 minutes before the enzymatic color reaction was stopped by the addition of 50 ~L 2N H2SO~. The A~92"", of the ~m~nt~ntC of each well was read in a plate reader.
The immunogens, Peptide No. 1 and its closely related derivative Peptide No. 2, both in branching multimeric form, were found to be ef~ective in P1;~-it;n~
antibodie8 specific to the IgE CH4 target se~uence ~SEQ ID
NO:l) through an ELIS~ inhibition assay. The results, whçn compared to a control; ~ , the KLH conjugate of monomeric Peptide No. 3 (IgE~CH4 deca~eptide SEQ ID NO:1) showed that these two multimeric peptide antigens g~n~ 3tf~-1 a higher level of antibody titers than the KLH
2S conjugate.
The succçssful results of these; ; 7~tion experiments indicated the generation of a Th~epitope in the multimeric 8ystem as a result of insertion of Gly-Lys-Met at the C-t~rm;nll~ of the peptide sç~ence (see SEQ ID
NOS: 23 and 13, Peptide Nos . 1 and 2 ) and indicated the importance of certain orientations for effective presentation to the immune system. Other experiments showed that merely making 8- or even 16-branched IgE
peptide immunogens c-~rt~;n;n~ the IgE CH4 decapeptide (SEQ
3S ID NO:1) or multiple repeats thereof, in other I Wo gs/2636S 2 1 ~ 6 5 9 5 ~1111 . ~741 orientations, were not effective in the induction of anti-IgE CH4 responses. In fact, out of a total of 19 branched multimeric con3truct3, Peptide Nos. 1 and 2 were the only ones to diEplay enhanced immunogenicity. In this respect, the high immunogenicity observed with multimeric Peptide Nos. 1 and 2 required exper; t~tion and was not predictable by one skilled in the art.
In addition, the results obtained suggest that a spacer sequence, i . e ., Gly-Pro-Gly, incorporated between the 3hort IgE CH4 AP~ tA, i3 nece3sary to allow free pre3entation of the epitopes conf erred by the 3ubunit 3equence The insertion of a 3pacer, i.e., Gly-I,y3-Met, at the C-terminu3 prior to linkage to the branched ly3ine core re3in was al30 found to be nece33ary for the immunogenicity of multimeric branched IgE CH4 decapeptide ~SEQ ID NO:1) 3ynthetic con3truct3.
EX~MpT ~ 3 IMMUNIZATION OF R~TS WITH I~INEAR
IMMUNOGENS (SEO ID NOS:15-22) A . Immunocen pre~arat ion: Peptide immunogen3 A-H (Table 3) are 3ynthf~A; 7~rl by 301id pha3e 3ynthe3i3 u3ing F-moc chemi3try on an Applied Bio3y3tems Peptide Synthe3izer Model 430A or 431 according to manu~acturer~ 3 in3tructions. After complete assembly of the peptide, the resin is treated according to standard ~,uc~-lu-e3 to cleave the peptide f rom the re3in and deprotect the functional groups on amino acid 3ide chain3. The 3tructure of the peptide immunogen3 from the amino terminu3 to the carboxyl t~ ; nllA i3 as follows: Peptide immunogen A i9 a linear peptide with t_ree domains: 3 lysine residues (3K), the hepatitis B surface antigen helper T cell epitope (H;3,Th epitope) and IgE CH4 peptide.
Peptide immunogen A is thus represented as 3K-HB,Th-IgE
CH4 peptide . The actual 3equence3 f or Peptide ; - j A
and fûr Peptide immunogen3 B-H are 3hown in Table 5 (SEQ
. _ .. . .. . , . . .. _ . . .. . _ _ . .. _ _ . . _ ..... ... . ..

Wo 95/26365 PCr/US95/03741 21 865q5 O
OS :15 -22 ) .
For i ; 7Ations at weeks 0, 2 and 5, each peptide immunogen is dissolved and combined with an adjuvant solution ~Complete Freund' 9 Adjuvant, Incomplete Freund'3 Adjuvant, or 0.29~ Alum) to result in a final concentration of 0.5 mg/ml. The 601ution i8 stored at 4C
until use and vortexed for 3 ~to 5 min prior to injection.
Each rat receives 100 ,~g per~injection.
B. T ; 7Ation schedule and serum collection:
Sprague-Dawley rats (n=5) are immunized subcutaneously ( s . c . ) . Booster inj ection3 are given B . C . at weeks 2 and 5. Blood is collected at weeks 3, 6, 7 and 11.
Blood collection from the middle caudal artery is perf ormed f ollowing anesthesia of the rats by intraperitoneal injection of 1 mL of sodium pp~t~hArhital (64 . 8 mg/mL; Anthony Products Co., Accadia, CA) diluted 1 to 10 in 0.996 NaCl. The tails are kept in 4~C i 0.5C
water for 2 min and rapidly massaged with paper towels (i.e_, milked). Blood is collected immediately into a 5 mL syringe outfitted with a 23 gauge needle. Typically, 2 to 2 . 5 mL of blood is obtained . The serum is collected by centrifugation for 25 min at 3000 rpm. The serum is aliquoted in 300 ILL volumes and stored frozen until used for ELISA assays.

o Sequences of Peptide Immunogens A-H
Peptide Immunoge~ Amino Acid Sequence A 3K-HB~Th-IgECH4 Lys-Lys-Lys-Phe-Phe-Leu-Leu-Thr-Arg-Ile-Leu-Thr-Ile-Pro-Gln-Ser-Leu-Asp-Lys-Thr-Lys-Gly-Ser-Gly-Phe-Phe-Val -Phe ( SEQ ID NO: 15 ) B PT~Th- IgECH4 Lys-Lys-Leu-Arg-Arg-Leu-Leu-Tyr-Met-Ile-Tyr-Met-Ser-Gly-Leu-Ala-Val -Arg-Val -His-Val -Ser-Lys-Glu-Glu-Gln-Tyr-Tyr-Asp-Tyr-Lys-Thr-Lys-Gly-Ser-Gly-Phe-Phe-Val-Phe (SEQ ID NO:16) C PT,"Th-IgECH4 Tyr-Met-Ser-Gly-Leu-Ala-Val-Arg-Val-His-Val -Ser-Lys-Glu-Glu-Lys -Thr-Lys-Gly-Ser-Gly-Phe-Phe-Val -Phe ( SEQ ID NO :17 ) D TT1Th-IgECH4 Lys-Lys-Gln-Tyr-Ile-Lys-Ala-Asn-Ser-Lys-Phe-Ile-Gly-Ile-Thr-Glu-Leu-Lys-Thr-Lys-Gly-Ser-Gly-Phe-Phe-Val-Phe (SEQ ID NO:18) E TT2Th-IgECH4 Lys-Lys-Phe-Asn-Asn-Phe-Thr-Val-Ser-Phe-Trp-Leu-Arg-Val-Pro-Lys-Val -Ser-Ala-Ser-His-Leu-Lys-Thr-Lys-Gly-Ser-Gly-Phe-Phe-Val-Phe (S-Q ID NO:19) wo 95/Z6365 P~,ll~., _.'^~741 ~
2~ 86595 F TT3Th-IgECH4 Tyr-Asp-Pro-Asn-Tyr-~eu-Arg-Thr-Asp-Ser-Asp-Lys-Asp-Arg-Phe-l,eu-Gln-Thr-Met-Val-Ly~-Leu-Phe-Asn-Arg-Ile-Lys-Lys-Thr-Lys -Gly-Ser-Gly-phe-phe-val -Phe (SEQ ID NO:20) G PT2Th-IgECH4 ~ Gly-Ala-Tyr-Ala-Arg-Cys-Pro-Asn-Gly-Thr-Arg-Ala -Leu-Thr-Val -Ala-Glu-Leu-Arg-Gly-Asn-Ala-Glu-Leu-Lys -Thr-Lys -0 Gly-Ser-Gly-Phe-Phe-Val-Phe (SEQ ID NO:21) X MV"lTh-IgECH4 Ser-Glu-Ile-Lys-Gly-Val-Ile-Val-His-Arg-Leu-Glu-Gly-val-Leu-Lys-Thr-Lys -Gly-Ser-Gly-Phe-Phe-Val-Phe (SEQ ID NO:22) qpT ,~: 4 T~NIZATION OF RATS WITX T TNEZ~R
I I~NOGENS ( SEO ID NOS: 3 7 - 5 0 ) Linear peptide immunogens represented as A-Th-GG-IgE CX~L , where A may be either NH2- , Lys-Lys (2K), Lys-Lys-Lys ~3K), or an lnv~in ~' ~1n (Inv) (SEQ ID NO:2~), Th is a T helper peptide, GG is a Gly-Gly spacer, and IgE
CEI4 is the target decapeptide (SEQ ID NO:1), are synthesized as described in Example 3. These peptide immunogens are shown in Table 4 as Peptide Immunogens Nos.

4-17 (SEQ ID NOS:37-SC). The synthesized and cleaved peptides are used to immunize rats to test for efficacy.
Efficacy is evaluated on groups of five rats by the experimental immunization protocol outlined below.
Experimental Def3ign:
Immunogen: Peptide Nos. 4-17 (1 per trial) Dose: 100 ~g per; i 7~tion Route: intramuscular ~ WOgsl2636~ P~ ... '/Q~741 ?~

o Ad~uvant: Freund' s Complete/Incomplete Dose .Schedule: week 0 (FCA), 3 and 6 weeks ( IFA) Bleed Schedule: weeks 0, 3, 6, 8, 10

5 Species: Sprague-Dawley rats Group size: 5 Assay: ELISA f or anti -peptide activity, solid-phase immunosorbent ig rn~n~ ic Peptide No. 3 of the IgE CH4 decapeptide se~uence (SEQ ID
N0:1) .
Blood is collected, processed into serum, and stored prior to titering by ELISA as described in Example 2, with the exception of using horseradish peroxidase-labelled goat anti-rat IgG antibody instead of goat anti-guinea pig IgG as the tracer.

SeSIuences of Peptide Immunogens Nos. 4-17 Peptide Immunogen Amino Acid Se~uence 4 TTlTh-GG-IgECH, Lys-Lys-Gln-Tyr-Ile-Lys-Ala-Asn-Ser-Lys-phe-Ile-Gly-Ile-Thr-Glu-Leu-Gly-Gly-Lys-Thr-Lys -Gly-Ser-Gly- Phe - Phe -Val - Phe (SEQ ID N0.37) TT~Th-GG-IgECH~ Lys-Lys-Phe-Asn-Asn-P~e-Thr-Val-Ser-Phe-Trp-Leu-Arg-Val-Pro-Lys-Val-Ser-Ala-Ser-~is-Leu-Gly-Gly-Lys-Thr-Lys-Gly-Ser-Gly-Phe-Phe-Val-Phe (SEQ ID N0:38)

6 PT,~Th-GG-IgECH~ Tyr-Met-Ser-Gly-Leu-Ala-Val-Arg-Val -lIis-Val-Ser-Lys-Glu-Glu-Gly-Gly-Lys-Thr-Lys -Gly-Ser-Gly-Phe-Phe - Val - Phe (SEQ ID N0:39) WO 9~26365 2 1 ~ 6 5 9 5 r.~ '74l

7 MVF2Th-GG-IgECH; Gly-Ile-Leu-Glu-Ser-Arg-Gly-Ile-Lys-Ala-Arg- Ile-Thr-His -VaI-Asp-Thr-Glu-Ser-Tyr-Gly-Gly-Lys-Thr-Lys-Gly-Ser-Gly-Phe-Phe-Val-Phe ~SEQ ID N0:40)

8 TT4Th-GG-IgECH~ Trp-Val-Arg-Asp-Ile-Ile-Asp-Asp-S Phe-Thr-As~-Glu-Ser-Ser-Gl~-Lys-Thr-Gly-Gly-Lys-Thr-Lys-Gly-Ser-Gly- Phe - Phe -Val - Phe (SEQ ID N0:41)

9 TT~Th-GG-IgECH; Asp-Val-Ser-Thr-Ile-Val-Pro-Tyr-Ile-Gly-Pro-Ala-Leu-Asn-His -Val-Gly - Gly - Lys - Thr - Lys - Gly - Ser - Gly -Phe-Phe-Val-Phe (SEQ ID N0:42)

10 CTTh-GG-IgECH. Ala-Leu-Asn-Ile-Trp-Asp-Arg-Phe-Asp-Val-Phe-Cys-Thr-Leu-Gly-Ala-Thr-Thr-~ly-Tyr-Leu-Lys-Gly-Asn-Ser-Gly-Gly-Lys-Thr-Lys -Gly-Ser-Gly-Phe-Phe-Val-Phe (SEQ ID ~O: 43 )

11 DTlTh-GG- IgECH~ Asp-Ser-Glu-Thr-Ala-Asp-Asn-Leu-Glu-Lys-Thr-Val-Ala-Ala-Leu-Ser-Ile-Leu-Pro-Gly-Ile-Gly-Cys-Gly-Gly-Lys -Thr-Lys -Gly-Ser-Gly-Phe-Phe-Val-Phe (SEQ ID N0:44)

12 DT2Th-Gg-IgECH4 Glu-Glu-Ile-Val-Ala-Gln-Ser-Ile-Ala-Leu-Ser-Ser-Leu-Met-VaL-Ala-Gln-Ala-Ile-Pro-Leu-Val-Gly-Glu-Leu-Val-Asp- Ile-Gly-Phe-Ala-Ala-Thr-Asn-Phe-Val-Glu-Ser-Cys-Gly-Gly-Lys -Thr-Lys-Gly-Ser-Gly-Phe-Phe-Val- (SEQ ID N0:4~)

13 PFTh-GG-IgECH~ ~sp-Ile-Glu-Lys-Lys-Ile-Ala-Lys-Met-Glu-Lys -Ala-Ser-Ser-Val-Phe-Asn-Val -Val -Asn-Ser-Gly-Gly-Lys-Thr-Lys -Gly-Ser-Gly-Phe-Phe-Val-Phe ( SEQ ID ~O: 4 6 )

14 SMTh-GG-IgECH4 Lys-Trp-Phe-Lys-Thr-Asn-Ala-Pro-Asn-Gly-Val -Asp-Glu-Lys - Ile-Arg-Ile-Gly-Gly-Lys-Thr-Lys-Gly-Ser-Gly-Phe-Phe-Val-Phe ( SEQ ID NO: 4 ` ) -~ W0 95/26365 2 1 P~ 6 5 `9 5 ~ 741

15 TraTlTh-GG-IgECH4 Gly-Leu-Gln-Gly-Lys-Ile-Ala-Asp-Ala-Val-Lys -Ala-Lys -Gly-Gly-Gly-Lys -Thr-Lys-Gly-Ser-Gly-Phe-Phe-Val - Phe (SEQ ID N0:48)

16 TraT Th-GG-IgECH~ Gly-Leu-Ala-Ala-Gly-Leu-Val-Gly-2 Met-Ala-Ala-Asp-Ala-Met-Val-Glu-Asp -Val -Asn- Gly- Gly - Lys - Thr - Lys -Gly-Ser-Gly-Phe-Phe-Val-Phe (SEQ ID N0:49)

17 TraT3Th-GG-IgECH~ Ser-Thr-Glu-Thr-Gly-Asn-Gln-His-His-Tyr-Gln-Thr-Arg-Val-Val -Ser-Asn-Ala-Asn-Lys-Gly-Gly-Lys-Thr-Lys -Gly-Ser-Gly-Phe-Phe-Val-Phe (SEQ ID N0:50) WO 95/26365 2 1 8 6 5 9 5 P~ /41 EXAMPT~ 5 ~T7~TIoN OF ~ATS WIT~ T Tr~
T~MUNOGENS (SEO ID NOS:51-56,62~
AND LINEAR IMMUNOGE~NS OF REVERSE POI~RITY
(SE~) ID ~OS:57-60) Peptide immunogens Nos. 18-23 ~ID SEQ ID NOS:51-56) as shown in Table 5, were svnthesized as described in Example 3. The formula for peptide immunogens Nos. 18-23 may be represented as A-Th-GG-IgECH4, wherein A is either the N terminus, Lys-Lys ~2K), Lys-Lys-Lys ~3K), or the invasin domain ~Inv) ~SEQ ID NO:25) separated from the Th sequence by a spacer GG; Th is selected from the group consisting of H3, Th, PTl Th, PT2 Th, MV~l Th, or TT; Th; GG
is a Gly-Gly spacer; and IgECH4 is the IgE CH4 decapeptide ~SEQ ID NO:l) .
Peptide; ~Pn~ with SEQ ID NOS:s7-60, also shown in Table 5, as Peptide Nos. 24-27, were synthesized in an identical fashion to the Peptide Nos. 18-23. These peptides may be represented as IgECH4-GG-Th. These peptide8 are equivalent to Peptide Nos. 19,20,21,23 ~Table 5) in terms of IgECH4 decapeptide, spacer, and Th sequences except that the decapeptide/Th polarity was reversed, i.e., the IgE CH4 decapeptide ~SEQ ID NO:l) was on the N t~nTn;nll~ while Th was located on the C terminus.
These peptide immunogens were used to immunize rats as described in the experimental protocol below, for comparison and demonstration of efficacy.
E~erimental Design:
Immunogen: Peptide Nos. 18-28 ~1 per group) ~SEQ ID NOS:51-60 and 62) Dose: 100 ~Lg per; ; 7~tion Route: intramuscular Adjuvant: Freund' 8 Complete/Incomplete for Peptide Nos. 18-27, 0.4% Alum for . _ .. . . _ .. . _ .. . , _ _ _ _ Wo gs/26365 A ~ . /41 O
Peptide No. 28 Dose Schedule: week O (FCA), 3 and 6 weeks (IFA) for Peptide No9- 8-27, Alum for Peptide No . 2 8 on weeks O, 3, and 6 Bleed Schedule: weeks 0, 3, 6, 8, 10 Species: Sprague-Dawley rats Group size: 5 for Peptide Nos. 27-28, 4 for Peptide No . 2 8 Assay: ELISA for anti-peptide activity, solid-phase substrate is Peptide ~o. 3 ( SEQ ID NO :1 ) .
~3100d was collected, processed into serum, and stored prior to titering by ELISA as described in Example 2 with 15 the exception of substituting horseradish peroxidase-labelled goat anti-rat IgG antibody for anti-guinea pig IgG as the tracer. All sera were assayed by anti-peptide ELISA and those samples which gave A492~m values of 2 0 . 2 at a 1:100 dilution were recorded as seropositive.
The i Innpotencies of Peptide; -/~:ns Nos.

18-28 (SEQ ID NOS:51-60, and 62) were evaluated by the anti-peptide EI,ISA and are shown in Table 6 as the number of rats in each group of 4 or 5 that converted to seropositive reactivity for IgE CH4 Peptide No. 3 on weeks 6 and 8 (i.e. ~ A49~mm 2 0.2 at a 1:100 dilution), in response to the experimental; ; 7~tions .
The peptide; -J~ S of this Example of polarity Th-GG-IgECH4 (Peptide Nos. 18-23 and 28, SEQ ID
NOS:51-56 and 62) showed significant efficacy for the 30 induction of antibodies to the IgE CH4 decapeptide (Peptide No. 3, SEQ ID NO:1). All 6 groups of rats immunized with the peptide immunogens of this polarity (Peptide Nos. 18-23, 28) showed significant conversion to seropositivity compared to the control. Prevalences of 3~i .... , , _ W0 95~26365 2 1 8 6 5 9 5 ~ P~ . /41 seroconYersion for the groups varied from 1/5 to 5/5 by week 6 and seroconversion prevalences c~nt; nll~od to increase between weeks 6 and 8 in response to the third dose of immunogens. Peptide immunogen No. 18 ~nt~;nin~
5 the H~3 Th peptide sequence, Peptide ~ No. 19 with the MVF1 Th peptide and Peptide No. 28 ,-r,nt~;n;n~ the PTlTh peptide sequence were the most effective, with seroconversion prevalences of 4/5, 5/5 and 4/4, respectively, by week 8. Comparison of the immunogenicities of Peptide immunogens Nos. 21 and 22 (SEQ
ID NOS:54,55) demonstrates that the Inv domain peptide provided significant i~ r ~,v~ t by week 8 to~ the immune stimulatory ~-A~h;l;ty of the PT~ Th-ct~ntA;n;n~ peptide ( Table 6 ) .
In contrast, the analogous peptide immunogens with reversed Th polarity (Peptide immunogens Nos. 24-27, SEQ ID NOS:57-60) failed to display significant immunopotency for the seroconversion of rats. This poor immunopotency shows that a Th-GG- IgECH4 amino to carboxyl 20 terminus polarity is critical to the; ,~llicity of the linear peptide immunogens of the invention. A
determination of efficacy for one orientation of target peptide and Th over the other was not predictable by one skilled in the art and i8 unexpected.
TA~3LE 5 Sequences of Peptide=Immunogens Nos. 18-28 Peptide Immunogen Amino Acid Sequence 18 3K-H~3,Th-GG-IgECH~ Phe-Phe-Leu-Leu-Thr-Arg-Ile-Leu-Thr- Ile-Pro-Gln-Ser-Leu-Asp-Gly-Gly-Lys -Thr-Lys-Gly-Ser-Gly-Phe-Phe-Val-Phe (SEQ ID NO:51) 1~220_1 Wo 95/26365 . ~~ '.'Q3~41

19 MVF1Th-GG-IgECH4 Leu-Ser-Glu-Ile-Lys-Gly-Val-Ile-Val-His-Arg-Leu-Glu-Gly-Val-Gly-Gly-Lys-Thr-Lys-Gly-Ser-Gly-Phe-Phe-Val-Phe (S~Q ID NO:52) PT1Th-GG-IgECH4 Lys-Lys-~eu-~rg-Arg-Leu-Leu-Tyr-Met-Ile-Tyr-Met-Ser-Gly-Leu-Ala-S Val-Arg-Val-His-Val-Ser-Lys-GlU-Glu - Gln - Tyr - Tyr -Asp - Tyr - Gly - Gly -Lys -Thr-Lys -Gly-Ser-Gly-Phe-Phe-Val-Phe (SEQ ID N0:53) 21 PT,Th-GG-IgECH~ Gly-Ala-Tyr-Ala-Arg-Cys-Pro-Asn-Gly-Thr-Arg-Ala-Leu-Thr-Val -Ala-Glu-Leu-Arg-Gly-Asn-Ala-Glu-Leu-Gly-Gly-Lys-Thr-Lys-Gly-Ser-Gly-Phe-Phe-Val-Phe (S~Q ID N0:54) 22 Inv-GG-PT2Th-GG-IgECH4 Thr-Ala-Lys-Ser-Lys-Lys-Phe-Pro-Ser-Tyr-Thr-Ala-Thr-Tyr-Gln-Phe-Gly-Gly-Gly-Ala-Tyr-Ala-Arg-Cys-Pro-Asn-Gly-Thr-Arg-Ala-Leu-Thr-Val-Ala-Glu-Leu-Arg-Gly-Asn-Ala-Glu-Leu-Gly-Gly-Lys-Thr-Lys-Gly-Ser-Gly-Phe-Phe-Val-Phe (SEQ ID NO:55) 23 TT3Th-GG-IgECH4 Tyr-Asp-Pro-Asn-Tyr-Leu-Arg-Thr-Asp-Ser-Asp-Lys-Asp-Arg-Phe-Leu-Gln-Thr-Met-Val-Lys-Leu-Phe-Asn-Asp-Arg-Phe-Leu-Gln-Thr-Met-Val-Lys-Leu-Phe-Asn-Arg-Ile-Lys-Gly-Gly-Lys-Thr-Lys-Gly-Ser-Gly-Phe-Phe-Val-Phe (SEQ ID N0:56) 24 IgECH4-GG-MVF1Th Lys-Thr-Lys-Gly-Ser-Gly-Phe-Phe-Val - Phe - Gly - Gly - Leu - Ser - Glu - I le -Lys-Gly-Val- Ile-Val -His-Arg-Leu-Glu-Gly-Val (SEQ ID N0:57) IgECH4-GG-PTlTh Lys-Thr-I,ys-Gly-Ser-Gly-Phe-Phe-Val -Phe-Gly-Gly-Lys-Lys-Leu-Arg-Arg-Leu-Leu-Tyr-Met- Ile-Tyr-Met-Ser-Gly-Leu-Ala-Val -Arg-Val -His -Val-His-Lys-Glu-Glu-Gln-Tyr-Tyr-Asp-Tyr (SEQ ID N0:58) ~$

W0 95/26365 2 1 8 6 5 9 5 ~ S ~741 ~

O
26 IgECH~-GG-PTlTh Lys-Thr-Lys-Gly-Ser-Gly-Phe-Phe-Val-Phe-Gly-Gly-Gly-Ala-Tyr-Ala-Arg-Cys-Pro-Asn-Glu-Thr-Arg-Ala-Leu-Thr-Val-Ala-Glu-Leu-Arg-Gly-Asn-Ala-Glu-Leu (SEQ ID N0:59) 27 IgECH~-GG-TT3Th Lys-Thr-Lys-Gly-Ser-Gly-Phe-Phe-Val -Phe-Gly-Gly-Tyr-Asp-Pro-Asn-Tyr-Leu-Arg-Thr-Asp-Ser-Asp-Lys -Asp-Arg-Phe-Leu-Gln-Thr-Met-Val -Lys - Leu - Phe -Asn -Asp -Arg - Phe - Leu -Gln-Thr-Met -Val -I-ys -Leu-Phe-Asn-Arg-Ile-I,ys (SEQ ID~N0:60) 28 PT~Th-IgECH~ Lys-Lys-I-eu-Arg-Arg-Leu-Leu-Tyr-Met-Ile-Tyr-Met-Ser-Gly-Leu-Ala-Val -Arg-Val-His-Val-Ser-Lys -Glu-Glu - Gln - Tyr - Tyr -Asp - Tyr - ~ys - Thr -Lys -Gly-Ser-Gly-Phe-Phe-Val -Phe (SEQ ID:~0:62) I S _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ .~5 WO95/2636S 2 1 ~6595 ~ u~ ^~741 O

Animals Seroconverted/group*
Peptide Immunogen Week 6 Week 8 18 3~-H3,Th-GG-IgECH~ 4 4 (SEQ ID NO:51) 19 MV"lTh-GG- IgECH4 5 5 (S~Q ID NO:52)

20 PTlTh-GG-IgECH,, 2 3 (SEQ ID NO:53)

21 PT2Th- GG - IgECH~ 1 1 (SEQ ID NO:54) 15 22 Inv-GG-PT2-GG-IgECH, 1 3 (SEQ ID NO:55) 23 TT3Th-GG- IgECHi 3 3 (SEQ ID NO:56) 24 IgECH,~-GG-MV~lTh O o (SEQ ID NO:57) 25 IgECH,,-GG-PTlTh (SEQ ID NO:58) 26 IgECH -GG-PT2Th O o (SEQ ID NO:59) 25 27 IgECHi-GG-TT3Th 0 o (SEQ ID NO:60) 2 8 PTlTh- IgECH~ 4 4 (SEQ ID NO:62) Control T ; 7~tion 0 30(No peptide) *5 animals per group for Peptide Nos. 18-27, 4 animals for Peptide ~o. :28 ,35 WO95l26365 !2 1~3'6 5 9 5 r~ l0~74 EX~MPLE 6 CO~RT1~TT, OF J~TN~ ~R Tl~'WNOGENS
FT~RTT~T R BRO~n~N~ THE RESPONSIVl~ POP~JLATION ~
Est=hl; ~h;n~ the relative- efficacies of the many different linear construct3 rrnt~;nln~ IgE CH4 decapeptide and Th (Examples 3-5) permits selection of useful peptide immunogens to formulate a cocktail vaccine composition. Individual Th-GG-IgEC~I4 constructs carrying r~ n=nt promiscuous Th peptides derived from measles virus F protein, tetanus toxin and pertussis toxin (Peptide Nos. 19-23) were proven by the study of Example 5 to be efficacious in eliciting antibody responses to the IgECH4 decapeptide (SEQ ID NO:1). A formulation rrnt~;n;n~ a mixture of these linear peptides may ~?rovide a desired maximum immunogenicity in a g~n~t; r= 1 1 y diverse population .
The immunopotency of ~uch a composition formulated to contain a mixture of synthetic peptides with the preferred iiA-Th-GG-IgECH4" polarity, Peptide immunogens Nos. 19, 20, 21, 23 (Table 5) and Peptide immunogen No. 4 (Table 4, Example 4) were evaluated in rats by the protocol described in Fxample 5. Each animal in a group o 5 rats were; ; 7'-C~ with 100 /lg doses of an equimolar formulation of the 5 peptides, i.e. 20 ~g of each peptide. The number of rats that converted to seropo~itive reactivity by weeks 5 ~nd 8 were 5 out of 5 (i.e., 100Y6) at both time intervals.
The results demonstrate that a vaccine comprising a cocktail o the~ peptide immunogen~ of the present invention provides improved immunogenicity. It al50 indicates the potential for this mixture, and of like cocktails rl ~ ~8~ of i~dividually efficacious peptides, to induce immunotherapeutic antibody responses in the genetically diverse human population.
3~i .
... , . .. ,, .. ,, , . ,, , . , .. , . ,, . ~

Wo95J2636s 21 ~6595 r ~ 741 EX~MPLE 7 IMMUNIZATIONS WITH cocl~Trq OF
EFFICACIOU,,q T T~R~R IMMr~NOGENS
Est~hl 1 Rh;nr~ the relative efficacies of the many different linear constructs cnnti~;n;ng IgE CH4 decapeptide and Th (Examples 3-5) permits selection of useful peptides for a cocktail of immunogens. Individual constructs carrying a Gly-Gly spacer and promiscuous Th peptides derived from measles virus F protei~, hepatitis B
surface antigen, tetanus toxin and pertussis toxin in the r~Pn cocktail are demonstrated to be ef f icacious (Table 6) . A mixture of these linear peptide immunogens with specif ic polarity with proven ef f icacy may thus provide maximum immunogenicity in a gPnPt;~lly diverse 15 population. The protocol below has been designed to demonstrate efficacy for compositions of the iIlvention f ormulated as mixtures of synthetic peptide immunogens crnt~;n;n~ preferred "A-Th-GG-IgECH4" constructs.
E~g?erimental Design:
T n~PnR (1) ~nrk~; l 1: Peptide Nos. 18, 19, 20 (2) Cocktail 2: Peptide Nos. 18, 19, 22 (3) Positive Control- KLH conjugate of Peptide 3 (One immunogen per group of rats) Do8e: Molar er~uivalents of each synthetic peptide or IgE CH4 equivalent, to equal either 100 ~Lg or 33.3 ~g of peptide per; ; 7~tion Route: iLtramuscular AdjUvants (1) Freund' s Complete/Incomplete (2) 0.4~ Alum (Aluminum hydroxide) (One of either adjuvant per immunogen per group) Dose Schedule: week 0, 2 and 4 weeks WO 95/26365 2 1 8 6 5 9 5 r~".J . ~ ^~74l ~

(CFA/IFA groups reoeive CFA week O, IFA weeks 2 and 4. Alum groups receive Alum formulations for all 3 doses ) Bleed Schedule: weeks 0, 3, 6 and 8 Specie6. Sprague-Dawley rats/group Group size: ~, 6 groups Assay: ELISA f or anti -peptide activity, solid-phase 1 - o~r~ent is Peptide No. 3 (SEQ ID ~O:l) .
Blood is collected, processed into serum, and stored prior to titerirLg by EI-ISA a8 described in Example 5.
This experiment is designed to demonstrate improved perf ormance of the immunogens of the present invention as compared to the: known immunogens of the prior art~ . The results are useful for the evaluation of two mixtures of efficacious peptide immunogens, each ~r~ntA;n;n~ three Th peptides, demonstrate the usefulness 20 of the immune stimulatory Inv domain (cocktail 2 .-nntA;no Inv, cocktail 1 does ~ot), and the efficacy of the:
~djuvant, Alum, in a vaccine~ composition of the invention.
R~AI~PI~E 8 25 CLINICAL TT~TAT~ UsTNG COCRTATT.. C OF IMMUNOf~-~N.~
Establishing the relative ef f icacies of the many different constructs cnntA;n;ng IgE CH4 decapeptide and Th (Examples 3-5) permits selection of representative peptides for a ~ n-ktAl 1 of immunogens . I~dividual constructs carrying a Gly-Gly spacer and Th peptide setluences from measles virus F, hepatiti8 B surface antigen, tetanus toxin and pertussis toxin in the immunogen cocktail are of demonstrated efficacy (Table 6) and are promiscuous for multiple human HIL~ DR antigens, so -Wo 95/26365 2 1 ~ 6 ~ 9 5 P~ . /41 O -- 49 _ as to provide maximum immunogenicity in a genetically diverse human population. Moreover, because these Th peptides are derived from ~-h;l~lrPn's vaccines, childhood vaccinations are a potential source of Th memory in an immunized human population. Thus, children' s vaccines have the potential to afford Pnh~nced immunopotency to anti-allergy vaccines comprised of mixtures of such Th peptides. The clinical protocol below has been designed to demonstrate efficacy for compositions of the invention 0 fULI l:~tP~l as a mixture of: such linear "A-Th-Spacer-Ig~
Ch4 decapeptide" peptide immunogens, in a widely acceptable adjuvant, Alum.
Experimental Design:
Subjects: Hay fever patients Season & Duration: Hay fever seasons, 8 weeks Groups: 4 groups, 1 group/immunogen/dose N=15 per group, 12 receive immunogen, 3 receive placebo Immunogen: Cocktail 1: Peptide Nos. 18, 19, 20, 23 Adjuvant: 0.296 Alum Dose: Molar eguivalents of each synthetic peptide to equal 500 ~g or 125 ~Lg of peptide per dose Route: intramuscular Dose Schedule: week 0, and 4 weeks 25 Evaluation schedule: weeks 0, 4, and 6 Blood is collected, processed into serum, and stored prior to titering by E~ISA as described in Example 5.
Efficacy and safety of the vaccine composition "cocktail 1" are evaluated serologically, by skin reaction tests, the rate of patient usage of hay fever medication, physical examination of patients for allergic symptoms and adverse reactions, and interviewing the patients to obtain their subjective a~sessments of the effect of using the WO9sl2636s 21 P~6595 r~l,~ /0?741 products. Serological evaluations include the af~ iQned ELISA for antipeptide titer, and a standard automated 3pectrofluorimetric assay to determine reduction in histamine levels(1~ as well as to ascertain that the S products do not trigger histamine release. The skin test is an intradermal test in which a standardized solution of allergens is inj ected i~to the upper layers of the skin .
Reactions to the allergens are quantitated by det~r . n i n~
the area of the typical "wheal and f lare" produced in 10 response to the allergens. ~he expected results include significant; ~ ~,v~ ~ in allergic symptoms at the endpoint of the study, and no evidence of histamine release triggered by the vaccine composition of the invention .
This experiment is designed to demonstrate the clinical efficacy of the invention. The results provide an evaluation of a mixture of "A-Th-Spacer-IgB C~I4 decapeptide" immunogens rrn~A;n;n~ four Th peptide sequences f," l ilt~cl with a pharmaceutically acceptable adjuvant, Alum.

W095/26365 21 8 659 J r~ J.; /4l EXAMPB~7 9 IN VITRO PSSAY DEMONSTRATES EFFICACY OF
IqE CH4 DECAPEPTIDE-5PECIFIC ANTIBODIES
Passively-sensitized human basophils are used in a well-known histamine-release assay for an in vitro evaluatio of antibodies induced by immunizations with IgE CH4 decapeptide immunogens. Human basophils are prepared from the venous blood of volunteers and then passively sensitized with IgE specific for benzylpenicilloyl-human serum albumin conjugate (BPO-HSA) that is prepared from the blood of donors hyperimmunogl (~h~ m; C for BPO-HSA-specif ic IgE Xistamine release ~y the sensitized basophils is affected by the addition of either BPO-XSA or IgE CX4 Peptide No. 3 (SEQ ID NO:1). Prior to the addition of the agents to induce histamine release, the basophils are combined with serial dilutions of antise7^um to IgE CH4 decapeptide (SEQ ID- NO: 1) or pre-immune control serum. Samples are analyzed for histamine release by the automated f Luorescence technique . The percentage of histamine reiease is calculated f rom the ratio of sample to total h~Rs~rh; 1 histamine content after spontaneous release is subtracted from both(27~. The capacity of the experimental antiserum to inhibit histamine release is demonstration of in vitro efficacy.
The ability of the IgE CX4 Peptide No. 3 (SEQ ID NO:1) to induce histamine release in a concentration-dependent manner was demonstrated by this assay. The results, presented in Table 7, showed that the IgE CX4 Peptide No.
3 (SEQ ID NO:1) induced histamine release by human basophils and served to validate the relevance of SEQ ID
NO:1 and corresponding antibodies for the human allergic response .

W0 95~26365 2 1 8 6 5 9 5 ~ . /41 O

Inducer ~ Net Histamine Release*
Peptide No. 3 ~ ~ :
150 ~lg/mL (1.3 X 10-iM) 309 60 llg/mL (7 X 10-sM) 13 6 ,~Lg/mL (7 X 10-6M) 2 lOBP0-HSA
0.1 ILg/mL 639~
* Corrected by subtraçtion of 8pontaneous histamine release, 99 3~

WO95/26365 -2 1 86595 1 l,1 /41 o ~ u~;N( :~; LISTING
( 1 ) GENERA~L INFORMATION:
(i) APPLICI~NTS: United Biomedical, Inc. & WANG, Chang Yi (ii) TITLE OF INVENTION: ~YN~ C PEPTIDE BASED
IMMUNOGENS FOR THE TREATMENT OF ALLERGY
(iii) NUMBER OF ~U~N~ S: 62 ( iv) ~ ONL~;NC~; ADDRESS:
(A) AnT~RRc~RR: Maria C.H. Lin (B) STREET: 345 Park Avenue 0 (C) CITY: New York (D) STATE: NY
( E ) COUN TRY: USA
(F) ZIP: 10154 (v) COMPUTER RRAT ART T~ FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: WordPerfect 5 1 (vi) CURRENT APPLICATION- DATA:
(A) APPLICATION NIJMBER:
(B) FILING DATE: 24-MAR-1995 (C) CLASSIFICATION:
(vii) PRIOR APPLIQTION DATA:
(A) APPLICATION NUMBER: US 08/328, 912 (B) FILING DATE: 25-OCT-1994 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/218,461 (B) FILING DATE: 28-MAR-1994 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NI~MBER: US 08/060,798 (B) FILING DATE: 10-MAY-1993 (vii) PRIOR APPLICATION DATA-(A) APPLICATION NUMBER: US 07/847,745 (B) FILING DATE: 06-MAR-1992 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 07/637,364 (B) FILING DATE: 04-JAN-1991 WO 95lZ6365 2 1 8 6 5 9 5 P~ ,. /41 ( vi i i ) ATTORNEY/AGENT INFORMATION:
(A) NAME: Maria C . H . :Lin (B) REGISTRATION NUMBER: 29,323 (C) ~;~ N~:~;/DOCKET N~1MBER: llSl-40611JS4 ( ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 212-758-4800 S (B) TELEFAX: 212-751-6849 (2) INFORMATION- FOR SEQ ID NO:1:
U~;N~; CHARACTERISTICS:
(A) LENGTH: 10 ::
(B) TYPE: amino acid (C) STR~N~ N~ : not applicable ( D ) TOPOLOGY: l inear (ii) MOLECULE TYPE: peptide (x) PUBLICATION :INFORMATION:
(A) AUTHORS: Stanworth et al.
(B) TITLE: The Role Of Non-Antigen Receptors In Mast Cell Signalling Processes (C) JOURNAL: Molecular Immunology (D) VOLUME: 21 (E) ISSUE: 12 (F) PAGES: 1183-1190 (G) DATE: 1984 ~=
(J) PUBLICATION DATE:
20 (K) RELEVANT RESIDUES: 497 to 506 (xi) ~ U~;NC~; DESCRIPTION: SEQ ID NO-I:
Lys Thr Lys Gly Ser Gly Phe Phe Val Phe 5 ~ 10 2S (3) INFORMATION FOR SEQ ID NO:2:
(i) ~;5,~U~;N~:~; r~T~R~t~T~RT~qTIcs (A) LENGTH: 325 (B) TYPE: amino acids (C) sTR~Nn~n-NE~s not applicable (D) TOPOLOGY: Unknown (ii) MOLECULE TYPE: Poylpeptide ~-chain of human IgE
(x) PUBLICATION INFORMATION:
(A) AUTHORS: Dorrington and Bennich (B) TITLE:
(C) JOURNAL: Immunology Review (D) VOLUME: 41 W095/2636S ~l 8~95 I'~_I/U.~.~V. 141 (E) ISSUE:
(F) PAGES: 3-25 (G) DATE: 1978 (Xi ) ~ U~N(~:~ DESCRIPTION SEQ ID NO: 2:
Val Cy8 Ser Arg Asp Phe Thr Pro Pro Thr Val Lys Ile Leu Gln S Ser Ser Cys Asp Gly Gly Gly His Phe Pro Pro Thr Ile Gln Leu Leu Cys Leu Val Ser Gly Tyr Thr Pro Gly Thr Ile Asn Ile Thr Trp Leu Glu Asp Gly Gln Val Met Asp Val Asp Leu Ser Thr Ala Ser Thr Thr Gln Glu Gly Glu Leu Ala Ser Thr Gln Ser Gln Leu Thr Leu Ser Gln Lys His Trp Leu Ser Asp Arg Thr Tyr Thr Cys Gln Val Thr Tyr Gln Gly His Thr Phe Gln Asp Ser Thr Lys Lys Cys Ala Asp Ser Asn Pro Arg Gly Val Ser Ala Tyr Leu Ser Arg 15 Pro Ser Pro Phe Asp Leu Phe Ile Arg Lys Ser Pro Thr Ile Thr Cys Leu Val Leu Asp Leu Ala Pro Ser Lys Gly Thr Val Asn Leu Thr Trp Ser Arg Ala Ser Gly Lys Pro Val Asn Asn Ser Thr Arg Lys Glu Glu Lys Gln Arg Asn Gly Thr Leu Thr Val Thr Ser Thr 20 Leu Pro Val Gly Thr Arg Asp Trp Ile Glu Gly Glu Thr Tyr Gln Cys Arg Val Thr His Pro His Leu Pro Arg Ala Leu Met Arg Ser Thr Thr Lys Thr Ser Gly Pro Arg Ala Ala Pro Glu Val Tyr Ala Phe Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg Thr Leu 25 Ala Cys Leu Ile Gln Asn Phe Met Pro Glu Asp Ile Ser Val Gln Trp Leu His Asn Glu Val Gln Leu Pro Asp Ala Arg His Ser Thr Thr Gln Pro Arg Lys Thr Lys Gly Ser Gly Phe Phe Val Phe Ser Arg Leu Glu Val Thr Arg Ala Glu Trp Gln Glu Lys Asp Glu Phe Ile Cys Arg Ala Val l~is Glu Ala Ala Ser Pro Ser Gln Thr Val Gln Arg Ala Val Ser Val Asn Pro Gly Lys (4 ) INFORMATION FOR SEQ ID NO : 3:

WO95l26365 2 1 8 6595 ~ 741 (i) ~ 52U~ ; r~l~Rz~ RT~TIcs (A) LENGTH: 175 (B) TYPE: amino acids ( C ) STR ~ )NRq~: not appl icab1e ( D ) TOPOLOGY: Unknown (ii) MOLECULE TYPE: Polype~tide .~-chain of rat IgE
(x) PUBLICATION INFORMATION:
(A) AUTHORS: Kindsrogel et al.
(B) TITLE:
( C ) JOUE~L: DNA
( D ) VOLUME:
(E) ISSUE:
(F) PAGES: 335-343 (G) DATE: 1982 (xi) SEQUENCE DESCRIPTION.SEQ ID NO:3:
Asn Leu Asn Ile Thr Gln Gln Gln Trp Met Ser Glu Ser Thr Phe Thr Cys Lys Val Thr Ser Gln Gly Glu Asn Tyr Trp Ala His Thr 15 Arg Arg Cys Ser Asp A~p Glu Pro: Arg Gly Val Ile Thr Tyr Leu Ile Pro Pro Ser Pro Leu Asp Leu Tyr Glu Asn Gly Thr Pro Lys Leu Thr Cys Leu Val Leu Asp Leu Glu Ser Glu Glu Asn Ile Thr Val Thr Trp Val Arg Glu Arg Ly8 Lys Ser Ile Gly Ser Ala Ser Gln Arg Ser Thr Lys Xis His Asn Ala Thr Thr Ser Ile Thr Ser Ile Leu Pro Val Asp Ala Lys Asp Trp Ile Glu Gly Glu Gly Tyr Gln Cys Arg Val Asp His Pro His Phe Pro Lys Pro Ile Val Arg Ser Ile Thr Lys Ala Leu Gly Leu Arg Ser Ala Pro Glu Val Tyr Val Phe Leu Pro Pro Glu Glu Glu Glu Lys Asn Lys Arg Thr Leu Thr Cys Leu Ile Gln Asn Phe Phe Pro Glu (5) INFORMATION FOR SEQ ID NO:4:
(i) ~i~;5~?u~ ; CHARACTERISTICS:
(A) LENGTE~: 315 (B) TYPE: amino acids (C) sTR~ RnNE~ not applicable (D) TOPOLOGY: Unknown (ii) MOLECULE TYPE: Polypeptide ~-chain of mouse IgE

W095/26365 218659rJ P ./~ . /41 (x) PUBLICATION INFORMATION:
~A AUTHORS: Ishida et al.
(B TITLE:
( C JOURNAL: EMBO
( D VOLUME:
(E ISSUE:
(F: PAGES: 1117-1123 5 (G DATE: 1982 (Xi) ~ U~;NI~:~ DESCRIPTION:SEQ ID NO:4:
Val Arg Pro Val Thr His Ser Leu Ser Pro Pro Trp Ser Tyr Ser Ile His Arg Cys Asp Pro Asn Ala Phe l~is Ser Thr Ile Gln Leu Tyr Cys Phe Ile Tyr Gly His Ile Leu Asn Asp Val Ser Val Ser Trp Leu Met Asp Asp Arg Glu Ile Thr Asp Thr Leu Ala Gln Thr Val Leu Ile Lys Glu Glu Gly Lys Leu Ala Ser Thr Cys Ser Lys Leu Asn Ile Thr Glu Gln Gln Trp Met Ser Glu Ser Thr Phe Thr Cys Arg Val Thr Ser Gln Gly Cys Asp Tyr Leu Ala His Thr Arg Arg Cys Pro Asp His Glu Pro Arg Gly Ala Ile Thr Tyr Leu Ile Pro Pro Ser Pro Leu Asp Leu Tyr Gln Asn Gly Ala Pro Lys Leu Thr Cys Leu Val Leu Asp Leu Glu Ser Glu Lys Asn Val Asn Val Thr Trp Asn Gln Glu Lys Lys Thr Ser Val Ser Ala Ser Gln Trp Tyr Thr Lys His His Asn Asn Ala Thr Thr Ser Ile Thr Ser Ile Leu Pro Val Val Ala Lys Asp Trp Ile Glu Gly Tyr Gly Tyr Gln Cys Ile Val Asp Arg Pro Asp Phe Pro Lys Pro Ile Val Arg Ser Ile Thr Leu Pro Gln Val Ser Gln Arg Ser Ala Pro Glu Val Tyr Val Phe Pro Pro Pro Glu Glu Glu Ser Glu Asp Lys Arg Thr Leu Thr Cys Leu Ile Gln Asn Phe Phe Pro Glu Asp Ile Ser Val Gln Trp Leu Gly Asp Gly Lys Leu Ile Ser Asn Ser Gln His Ser Thr Thr Thr Pro Leu Lys Ser Asn Gly Ser Asn Gln Gly Phe Phe Ile Phe Ser Arg Leu Glu Val Ala Lys Thr Leu Trp Thr Gln Arg Lys WO 95/26365 2 1 8 6 5 9 5 r~~ ?/41 --Gln Phe Thr Cys Glrl Val Ile His Glu Ala Leu Gln l~ys Pro Arg ( 6 ) INFORMATION FOR SEQ ID N~: 5:
(i) SEQUENCE f~T~ 'TR~T.~TICS:
(A) LENGTH: 15 (B) TYPE: amino acid (C) ST~N~ l)N~ : not applicable ( D ) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (Xi ) ~ ;yU~;N( :~; DESCRIPTION: SEQ ID NO: 5:
Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile Pro Gln Ser Leu Asp (7) INFORMATION FOR SEQ ID NO:6:
(i) ~:i~;yU~;N~i r~z~ TR~T~TIcs (A) LENGTH: 28 (B) TYPE: amino acid (C) ST~NnRnNR':C: not applicable (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) ~;~;yU~;N~ DESCRIPTION: SEQ ID NO:6:
Lys Lys Leu ~g Arg Leu Leu Tyr Met Ile Tyr Met Ser Gly Leu Ala Val Arg Val His Val Ser Lys Glu Glu G1n Tyr Tyr Asp Tyr 25 ( 8 ) INFORMATION FOR SEQ ID NO: 7:
;QU~;Nt :~; CHARACTERISTICS:
(A) LENGTH: 17 (B) TYPE: amino acid (C) ST~Z~NTlRT)NR~: not applicable ( D ) TOPO~OGY: unknown (ii) MOLECULE TYPE: peptide W095l26365 2 1 86S9~ P~1/lJ,.._.. /41 (xi) ~;uU~ ; DESCRIPTION: SEQ ID NO:7:
Lys Lys Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu 5 ( 9 ) INFORMATION FOR SEQ ID NO: 8:
;UU~N~ R~ RRT~::TIcs:
(A) LENGTH: 22 (B) TYPE: amino acid (C) sTR~NnRnNRq~ not applicable ( D ) TOPOLOGY: unknown lO ( ii ) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Lys Lys Phe Asn Asn Phe Thr Val Ser P~e Trp Leu Arg Val Pro Lys Val Ser Ala Ser His I-eu ( 10 ) INFORMATION FOR SEQ ID NO: 9:
uU~ CHaRACTERISTICS:
(A) LENGTH: 15 (B) TYPE: amino acid (C) sTRANT~RnNRc~ not applicable (b) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) ~ u~;~iu~; DESCRIPTION: SEQ ID NO:9:
Tyr Met Ser Gly Leu Ala Val Arg Val His Val Ser Lys Glu Glu (11) INFORMATION FOR SEQ ID NO:10:
;uu~ CH~RACTERISTICS:
(A) LENGTH: 27 (B) TYPE: amino acid (C) STR~NDRnNRc~ not applicable - (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: pep~ide 90 I N lo WO 95/2636S 2 1 8 6 5 9 5 P~ 4l --o Tyr Asp Pro Asn Tyr Leu Arg Thr Asp Ser Asp Lys Asp Arg Phe Leu Gln Thr Met Val Lys Leu Phe Asn Arg Ile Lys ( 12 ) INFORMATION FOR SEQ ID NO :11:
(i) ~;~;uu~;Nt ~:!; CHAR~CTERISTICS:
(A) LENGTH: 24 (B) TYPE: amino acid~ :
(C) STR~NnRnl~R.~,~: not applicable (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION:~SEQ ID NO:11:
Gly Ala Tyr Ala Arg Cys Pro Asn Gly Thr Arg Ala Leu Thr Val Ala Glu Leu Arg Gly Asn Ala Glu Leu ( 13 ) INFORMATION FOR SEQ I~ NO :12:
;Uu~;N8:~; CHARACTERISTICS:
(A) LENGTH: 15 (B) TYPE: amino acid (C) STR;~l~T)Rn1~s: nb~ applicable (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (Xi) ~ UU~;N~: DESCRIPTION: SEQ ID: NO:12:
25 Ser Glu Ile Lys Gly Val Ile Val His Arg Leu Glu Gly Val Leu (14) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE (~1~Rz~('TRRT~TIcs:
(A) LENGTH: 3 9 (B) TYPE: amino acids (C) STR~l~lnRT)l~R.~: not applicable (D) TOPOLOGY: unknown (ii) MOLECULE TYPE:
3S (A) DESCRIPTION: peptide WO 95/26365 1 ~~ . . /4l ~1 :86~5 (Xi) ~ ;UU~;N~:~; DESCRIPTION:SEQ ID NO:13:
Lys Thr Lys Gly Ser Gly Phe Phe Val Phe Gly Pro Gly Lys Thr Lys Gly Ser Gly Phe Phe Val Phe Gly Pro Gly Lys Thr Lys Gly Ser Gly Phe Phe Val Phe Gly Lys Met (15) ID~FORMATION FOR SEQ ID NO:14:
( i ) ~;~;U U ~ R ~ f'TRR T .~ T I CS:
(A) LENGTH: 21 (E~) TYPE: amino acids (C) STR~NDEDNESS: not applicable ( D ) TOPOLOGY: unknown (ii) MOLECULE TYPE:
(A) DESCRIPTION: peptide (xi) ~;uU~;~ DESCRIPTION:SEQ ID NO:14:
15 Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile heu (16) I~FORM~TION FOR SEQ ID NO:15:

(i) ~:il~UUI~ ; r~ R~t''TRRT.STICS:
(A) LENGTH: 28 (B) TYPE: amino acid (C) STR~l~DRm~RSS: not applicable ( D ) TOPOLOGY: unknown (ii) MOLRCULE TYPE: peptide (xi) ~ ;uu~ DESCRIPTION: SEQ ID NO:15:
Lys Lys Lys Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile Pro Gln Ser Leu Asp Lys Thr ~ys Gly Ser Gly Phe Phe Val Phe (17) INFORM~TION FOR SEQ ID NO:16-;UU~ Rzt~l~RRT~sTIcs (A) LENGTH: 38 (B) TYPE: amino acid (C) sTR~l~nRn~RSS: not applicable Wo 95/26365 r~u.. ~ 741 21 865~5 ~ -( D ) TOPOLOGY: unknown (ii) MOLECULE'TYPE: peptide (Xi) ~ U~N~:~; DESCRIPTION: SEQ ID NO:16:
Lys Lys Leu Arg Arg Leu Leu Tyr Met Iie Tyr Met Ser Gly Leu 1 5 . 10 15 Ala Val Arg Val His Val Ser Lys Glu Glu Gln Tyr Tyr Asp Tyr Lys Thr Lys Gly Ser Gly Phe Phe Val Phe 10 (18) INFORMATION FOR SEQ ID ~0:17:
;uu~;N~:~ ~ThR~ RTqTICS:
~A) LENGTH: 25 (B) TYPE: amino acid (C) sTRANn~nN~q~q not appli abl (D) TOPOLOGY: unknown c e (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID ~0:17:
Tyr Met Ser Gly Leu Ala Val Ar~ Val His Val Ser Ly9 Glu Glu Lys Thr Lys Gly Ser Gly Phe Phe Val Phe (19) INFORMATION FOR SEQ ID NO:18:
U~;N~:~; CH~RACTERISTICS:
(A) LENGTH: 27 (B) TYPE: amino acid (C) Sl'R~N~ N~:.q.~ noE app~icable 25 (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (Xi) ~ U~;N~; DESCRIPTION: SEQ ID NO:18:
Lys Lys Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Lys Thr Lys Gly Ser Gly Phe Phe Val Phe (20) INFORMATIO~ FOR SEQ ID NO:19:
U~N~ CHil~RACTERISTICS:

WO 95/263~5 2 1 ~ 6 ~ 9 ~ P~ 4l (A) LENGTH: 32 (B) TYPE: amino acid (C) STR~ND~nN~ not applicable (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) ~ U~;N~; DESCRIPTION: SEQ ID ~O:l9:
Lys Lys Phe Asn As~ :E~he Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val Ser Ala Ser Xis Leu Lys Thr Lys Gly Ser Gly Phe Phe Val Phe (21) INFORMATION FOR SEQ ID NO:20:
U~;N~:~; CHARACTERISTICS:
(A) LENGTH: 37 (B) TYPE: amino acid (C) sTR~Nn~m~ not applicable (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (Xi) .~ U~:N~:~; DESCRIPTION: SEQ ID NO:20:
Tyr Asp Pro Asn Tyr ~eu Arg Thr Asp Ser Asp Ly3 Asp Arg Phe 20 Leu Gln Thr Met Val Lys Leu Phe Asn Arg Ile Lys Lys Thr Lys Gly Ser Gly Phe Phe Val Phe

(22) INFORMATION FOR SEQ ID NO:21:
25 (i) SEQUENCE CH~RACTERISTICS:
(A) LENGTH: 34 (B) TYPE: amino acid (C) STR~Nn~n~s not applicable ( D ) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (Xi) ~ .!U~;N~-~; DESCRIPTION: SEQ ID NO:21:
Gly Ala Tyr Ala Arg Cys Pro Asn Gly Thr Arg Ala Leu Thr Val 21 8 6595 - 64 - r~ . /41 Ala Glu Leu Arg Gly Asn Ala Glu Leu Lys Thr Lys Gly Ser Gly . 30 Phe Phe Val Phe =~

(23) INFORMATION FOR SEQ ID NO:22:
;UU~;N~: rTT~R1~rT~RT~qTlcs (A) LENGT3~: 25 (B) TYPE: amlno acld ~
(C) STR~NnEnN~qq: not applicable (D) TOPOLOGY_ ~k~own (ii) MOLECULE TYPE: peptide (xi) ~;Uu~:N~: DESCRIPTION: SEQ ID NO:22:
Ser .Glu Ile Lys Gly Val Ile Val His Arg Leu Glu Gly Val Leu Lys Thr Lys Gly Ser Gly Phe Phe Val Phe 15 (24) INFORMATION FOR SEQ ID NO:23:
(i) ~:i~;UU~;N~:~; CHARACTERISTICS:
(A) LENGTH: 2 6 (B) TYPE: amino acids (C) STR~Nn~.nl~.q.q: not applicable (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (Xi) ~;UU~;N-:~; DESCRIPTION:SEQ ID NO:Z3:
Lys Thr Lys Gly Ser Gly Phe Phe Val Phe Gly Pro Gly Lys Thr Lys Gly Ser Gly Phe Phe Val Phe Gly Lys Met (25) INFORMATION FOR SEQ ID NO:24:
(i) ~:i~;UU~;N~ ; rT~R~rT~RT.qTICS:
(A) LENGTH: 6 30 (B) TYPE: amino acids (C) sTR~Nn~nN~..q.q not applicable ( D ) TOPOLOGY: unkr~own (ii) MOLECULE TYPE: linking group WO 95/26365 r~ 5,. /41 21 ~659~

(Xi ) ~ !U~N( '}; DESCRIPTION: SEQ ID NO: 24:
Pro Pro Xaa Pro Xaa Pro '5 (2) INFORMATION FOR SEQ ID NO:25:
2u~;N~ R~ RT.~TICS:
(A) LENGTH: 16 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 10 Thr Ala I-y5 Ser Lys Lys Phe Pro Ser Tyr Thr Ala Thr Tyr Gln Phe (2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE r~T~R~(~T~RT.~TICS
(A) LENGTH: 2 0 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (Xi) ~I:;S.?U~;N~:~; DESCRIPTION: SEQ ID NO:26:
20 Gly Ile Leu Glu Ser Arg Gly Ile Lys Ala Arg Ile Thr ~Iis Val As Thr Glu Ser Tyr (2) INFORMATION FOR SEQ ID NO:27:
25 (i) ~ U~;N~:~ CH~RACTERISTICS:
(A) LENGTH: 17 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) ~I~;(.?U~ DESCRIPTION: SEQ ID NO:27:
Trp Val Arg Asp Ile Ile Asp Asp Phe Thr Asn Glu Ser Ser Gln Lys Thr 35 (2) INFORMATION FOR SEQ ID NO:28:

WO95l26365 2 1 8 6~95 ~ 74l --;52UI~:N~; CHARACTERISTICS:
(A) LENGTH: 16 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear S (ii) MOLECIJIE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
Asp Val Ser Thr Ile Val Pro Tyr Ile Gly Pro Ala ~eu Asn His Val 10 (2) INFOR~ATION FOR SEQ ID NO:29:
( i ) ~i~5.?U l~'N~; CHARACTERISTICS:
(A) ~ENGTH: 25 amino= acids (B) TYPE: amino acid (D) TOPO~OGY: linear (ii) MOLEC~LE TYPE: peptide :~
(Xi) ~ U~;N~ DESCRIPTIO~: SEQ ID NO:29:
Ala Leu Asn Ile Trp Asp Arg Phe Asp Val Phe Cy~ Thr Leu Gly Ala Thr Thr Gly Tyr Leu Lys Gly Asn Ser (2) INFORMATION FOR SEQ ID NO:30:
(i) ~ilSl"~Ul~:N(:~ CHARACTERISTICS:
(A) LENGTH: 23 amino~ acids (B) TYPE: amino acid (D) TOPOLOGY: linear 25 (ii) MOLECULE TYPE: peptide (Xi) ~ 2U~N(:~; DESCRIPTION: SEQ ID NO:30:
Asp Ser Glu Thr Ala Asp A9n Leu Glu l.ys Thr Val Ala Ala I.eu Ser : 15 Ile Leu Pro Gly Ile Gly Cys (2) INFORMATION FOR SEQ ID NO:31: ~ -~
U~;N( ~ R~('TRR T.qTICS -(A) LENGTH: 3 9 amino acld (B) TYPE: amino acid (D) TOPOLOGY: linear WO 95/263G5 ~ 1 ~ G ~ 9 ~ 103741 (ii) MOLECULE TYPE: peptide (Xi) ~I:;~U~;N~ DESCRIPTION: SEQ ID NO:31:
Glu Glu Ile Val Ala Gln Ser Ile Ala Leu Ser Ser Leu Met Val Ala Gln Ala Ile Pro Leu Val Gly Glu Leu Val Asp Ile Gly Phe Ala Ala Thr Asn Phe Val Glu Ser Cy8 (2) INFORM~TION FOR SEQ ID NO:32:
U~:N(~ R~('TRRTqTICS:
(A) LENGTl~: 21 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) ~ u~;N~tl: DESCRIPTION: SEQ ID NO:32:
Asp Ile Glu Lys Lys Ile Ala Lys Met Glu Lys Ala Ser Ser Val Phe Asn Val Val Asn Ser 20 (2) INFORMATION FOR SEQ ID NO:33:
U~;N ~ R ~ t'TRR T ~qT I cs:
(A) LENGTH: 17 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (Xi) ~ UJ:;N~; DESCRIPTION: SEQ ID NO:33:
Lys Trp Phe Lys Thr Asn Ala Pro Asn Gly Val Asp Glu Lys Ile Arg Ile 30 (2) INFORMATION FOR SEQ ID NO:34:
(i) S~;UU~;NCI~ R~f~TRRT~qTIcs (A) LENGTH: 1~ amino acid8 (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide W095/26365 2 1 ~ 6595 F~ 741 --. .

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
Gly Leu Gln Gly Lys Ile Ala Asp Ala Val ~ys Ala Lys Gly 5 _ lO
5 (2) INFORMATION FOR SEQ ID NO:35:
;UU~:N~; CHARACTERISTICS:
(A) LENGTH: 19 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (Xi) ~ ;5,?U~;N~:~; DESCRIPTION:~SEQ ID NO:35:
Gly ~eu Ala Ala Gly ~eu Val Gly Met Ala Ala As~ Ala Met Val Glu Asp Val Asn 15 ( 2 ) INFORMATION FOR SEQ ID: NO: 3 6:
;UU~:N~; CH~RACTERISTICS:
(A) LENGTH: 2 0 amino acids (B) TYPE: amino acld (D) TOPOLOGY: linear (ii) MO~ECU~E TYPE: peptide (xi) ~:UU~;N~; DESCRIPTION: SEQ ID NO:36: : :
Ser Thr Glu Thr Gly Asn Gln His His Tyr Gln Thr Arg Val Val Ser Asn Ala Asn Lys (2) INFORMATION FOR SEQ ID NO:37:
UU~;N~; r~z~ rT~T~c~TIcs (A) LENGTH: 2 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: li~ear (ii) MOLECULE TYPE: peptide (xi) ~;~;UU~;NC:~: DESCRIPTION: SEQ ID NO:37:
ys ~ys Gln Tyr Ile ~ys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu . .

WO95/26365 l_l/~J.. 'n'~741 ~ 1 8~

Leu Gly Gly Lys Thr Lys Gly Ser Gly Phe Phe Val Phe ( 2 ) INFORMATION FOR SEQ ID NO : 3 8:
;UU~;N:~ r~ rTRRT~TIcs (A) LENGTH: 34 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (Xi) ~ UUl~;N~U~ DESCRIPTION: SEQ ID NO:38:
Lys Lys Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val Ser Ala Ser His Leu Gly Gly Lys Thr Lys Gly Ser Gly Phe Phe Val Phe ( 2 ) INFORMATION FOR SEQ ID NO: 3 9:
(i) ~;~;uU~!;N~:~; rT~R~rTRRT~cTIcs (A) LENGTH: 27 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) ~ ;uu~N~; DESCRIPTION: SEQ ID NO:39:
Tyr Met Ser Gly Leu Ala Val Arg Val His Val Ser Lys Glu Glu Gly Gly Lys Thr Lys Gly Ser Gly Phe Phe Val Phe 25 (2) INFORMATION FOR SEQ ID NO:40:
t!;uu~!;N~l~; rT~:~R~rTRRT~TIcs (A) LENGTH: 32 amino aclds (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) ~;UU~;N~'~ DESCRIPTION: SEQ ID NO:40:
Gly Ile Leu Glu Ser Arg Gly Ile Lys Ala Arg Ile Thr His Val As WO 95/26365 2 1 8 6 S 9 5 P.~L~3,. /4l Thr Glu Ser Tyr Gly Gly Lys Thr Lys Gly Ser Gly Phe Phe Val Phe (2) INFORMATION FOR SEQ ID NO:41: :
U~;N~ R~6~T~T~clTIcs (A) LENGTH: 2 9 amino acids (B) TYPE: amino acid (D) TOPO~OGY: linear (ii) MOLECI~E TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:
Trp Val Arg Asp Ile Ile Asp Asp Phe Thr Asn Glu Ser Ser Gln Lys 5 10 . - 15 Thr Gly Gly Lys Thr Lys Gly Ser Gly Phe Phe Val Phe 15 ( 2 ) INFORMATION FOR SEQ ID NO: 4 2:
U~;N(:~ r~R~t~T~ T!::TIcs (A) LENGTH: 2 8 amino acids (B) TYPE: amino acid (D) TOPO~OGY: linear (ii) MOLECULE TYPE: peptide (Xi) ~ U~;N~; DESCRIPTION: SEQ ID NO:42:
Asp Val Ser Thr Ile Val Pro Tyr Ile Gly Pro Ala Leu Asn His Val Gly Gly ~ys Thr Lys Gly Ser Gly Phe Phe Val Phe (2) INFORMATION FOR SEQ ID NO:43:
(i) ~t!;5~U~ R~('T~T~cTIcs (A) LENGTH: 37 amino acids (B) TYPE: amino acid ( D ) TOPOLOGY: l i~ear (ii) MOLECULE TYPE: peptide (Xi) ~ S2U~;N~; DESCRIPTION: SEQ ID NO:43:
Ala Leu Asn Ile Trp Asp Arg Phe Asp Val Phe Cys Thr Leu Gly Ala ~ Wo 95/2636~ ,'Q~74l Thr Thr Gly Tyr Leu Lys Gly Asn Ser Gly Gly Lys Thr Lys Gly Ser Gly Phe Phe Val Phe (2) INFORMATION FOR SEQ ID NO:44:
(i) SEQUENCE t~ rT~T.~TICS:
(A) LENGTH: 3 5 amino acid~
(B) TYPE: amino acid ~ D ) TOPOLOGY: l inear (ii) MOLECULE TYPE: peptide (Xi) ~ U~N~ DESCRIPTION: SEQ ID NO:44:
Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Val Ala Ala Leu Ser Ile Leu Pro Gly Ile Gly Cys Gly Gly Lys Thr Ly~ Gly Ser Gly Phe 15 Phe Val Phe ( 2 ) INFORMATION FOR SEQ ID NO: 4 5:
U~:N(:~; CHARACTERISTICS:
(A) LENGTH: 51 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (Xi) ~ U~ ; DESCRIPTION: SEQ ID NO:45:
Glu Glu Ile Val Ala Gln Ser Ile Ala Leu Ser Ser Leu Met Val Ala Gln Ala Ile Pro Leu Val Gly Glu Leu Val Asp Ile Gly Phe Ala Ala Thr Asn Phe Val Glu Ser Cys Gly Gly Lys Thr Lys Gly Ser Gly Phe Phe Val Phe (2) INFORMATION FOR SEQ ID NO:46:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear WO95/26365 2 1 865 95 I'~_1/U,..~ 741 (ii) MOLECULE TYPE: peptide (xi) ~:uu~;N~; DEscRIpTIoN:-sEQ ID NO:46:
sp Ile Glu Lys Lys Ile Ala Lys Met Glu Lys Ala Ser Ser Val Phe 10 . 15 5 Asn Val Val ~sn Ser Gly Gly Lys Thr Lys Gly Ser Gly Phe Phe Val Phe (2)INFORMATION FOR SEQ ID NO:47:
;Uul:;N~ Rl~'T~RT~cTIcs:
10 (A) LENGTH: 2 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: li~:Lear (ii) MOLECULE TYPE: peptide (Xi) ~ U~;N~; DESCRIPTION: SEQ ID NO:47:
15 Lys Trp Phe Lys Thr ~sn Ala Pro Asn Gly Val Asp Glu Lys Ile Arg Ile Gly Gly Lys Thr Lys Gly Ser Gly Phe Phe Val Phe =~

(2) INFORMATIO~ FOR SEQ ID NO:48:
u~;N~; ~ T~T~TIcs (A) LENGTH: 26 amino acids (B) TYPE: amino ac~d (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) ~ U~;N(:~; DESCRIPTION: SEQ ID NO:48:
ly Leu Gln Gly Lys Ile Ala Asp Ala Val Lys Ala Lys Gly Gly Gl Lys Thr Lys Gly Ser Gly Phe Phe Val Phe ( 2 ) INFORMATION FOR SEQ ID NO: 4 9:
(i) ~i~;UU~;N(~:~; CHARACTERISTICS:
(A) LENGTH: 31 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear .... . . . , . , ... ,, . _ _ _ _ W0 95/26365 2 18 6 5 9 5 r~l" -'/~3741 (ii) MOLECULE TYPE: peptide (Xi) ~ )U~;N~; DESCRIPTION: SEQ ID NO:49:
Gly Leu Ala Ala Gly Leu Val Gly Met Ala Ala Asp Ala Met Val Glu 5 Asp Val Asn Gly Gly Lys Thr Lys Gly Ser Gly Phe Phe Val Phe ( 2 ) INFORMATION FOR SEQ ID NO: 5 0:
;UU~;NO~ CTERISTICS:
(A) LENGTH: 32 amino acids 0 (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:
Ser Thr Glu Thr Gly Asn Gln His His Tyr Gln Thr Arg Val Val Ser Asn Ala Asn Lys Gly Gly ~ys Thr Lys Gly Ser Gly Phe Phe Val Phe (2) INFORMATION FOR SEQ ID NO:51:
(i) ~ iUU~;N~ u~R~t~T~RT~TIcs (A) IENGTH: 27 amino acids (B) TYPE: amino acid ( D ) TOPO~OGY: l inear (ii) MOLECULE TYPE: peptide (xi ) ~;Uu~o~; DESCRIPTION: SEQ ID NO: 51:
Phe Phe Leu Leu Thr Arg Ile ~eu Thr Ile Pro Gln Ser Leu Asp Gly Gly Lys Thr Lys Gly Ser Gly Phe Phe Val Phe 30 (2) INFORMATION FOR SEQ ID NO:52:
(i) ~:;~;UU~;N~:~ CHARACTERISTICS:
(A) LENGTH: 27 amino acids (B) TYPE: amino acid ( D ) TOPOLOGY: l i~ear (ii) MOLECULE TYPE: peptide W095~2636s 2-1 8~59~ 4l --O
(xi) SEQUENOE DESCRIPTIO~:: SEQ ID NO:52:
Leu Ser Glu Ile Ly5 Gly Val Ile Val His Arg Leu Glu Gly Val Gly Gly Lys Thr Lys Gly Ser Gly Phe Phe Val Phe ~2) I~FORM~TION FOR SEQ ID NO:53:
U~;N~; rM~R~rTRRTcTIcs:
(A) LENGTH: . g2 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENOE DESCRIPTION- SEQ ID NO:53:
ye Lys Leu Arg Arg Leu Leu Tyr Met Ile Tyr Met Ser Gly Leu Ala 15 Val Arg Val Eis Val Ser Lys Glu Glu Gln Tyr Tyr Asp Tyr Gly Gly Lys Thr Lys Gly Ser Gly Phe Phe Val Phe (2) INFORMATIO~ FOR SEQ ID NO:54:
)U~;N~:~; CH~R~rTRRT~clTIcs (A) LENGTE: 36 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) ~ U~;N~; DESCRIPTIO~: SEQ ID NO:54:
1y Ala Tyr Ala Arg Cys Pro Asn Gly Thr Arg Ala Leu Thr Val Ala Glu Leu Arg Gly Asn Ala Glu Leu Gly Gly Lys Thr Lys Gly Ser Gly Phe Phe Val Phe (2) INFORMATION FOR SEQ ID NO_55:
U~;N~J~; rM~R~rT~.RT.CTICS:
(A) LENGTH: 54 amino acids (B) TYPE: amino acid WO 95/26365 ~ 4l ?1~ 3 O
(D) TOPOLOGY: linear (ii) MOLECULE'TYPE: pep~cide (xi) ~;UU~;N~; DESCRIPTION: SEU ID NO:55:
5 Thr Ala Lys Ser Lys Lys Phe Pro Ser Tyr Thr Ala Thr Tyr Gln Phe Gly Gly Gly Ala Tyr Ala Arg Cys Pro Asn Gly Thr Arg Ala Leu Thr Val Ala Glu Leu Arg Gly Asn Ala Glu Leu Gly Gly Lys Thr Lys Gly Ser Gly Phe Phe Val Phe (2) INFORMATION FOR SEQ ID NO:56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:
Tyr Asp Pro Asn Tyr Leu Arg Thr Asp Ser Asp Lys Asp Arg Phe Leu 20 Gln Thr Met Val Lys Leu Phe Asn Asp Arg Phe Leu Gln Thr Met Val Lys Leu Phe Asn Arg Ile Lys Gly Gly Lys Thr Lys Gly Ser Gly Phe Phe Val Phe 4 5 25 (2) INFORMATION FOR SEQ ID NO:57:
(i) ~;~(,~U~!;N~; CHARACTERISTICS:
(A) LENGTH: 27 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear 30 (ii) MOLECU~E TYPE: peptide (Xi) ~;S.?U~!;N~; DESCRIPTION: SEQ ID NO:57:
Lys Thr Lys Gly Ser Gly Phe Phe Val Phe Gly Gly Leu Ser Glu Ile Lys Gly Val Ile Val His Arg Leu Glu Gly Val WO 95/26365 21 8 ~ 5 9 5 PCT/US95/03741 O
~2) INFORMATION FOR SEQ ID NO:58:
(i) ~i~;~U~SN~:~; CHARACTER :
(A) LENGTH. 42 ISTICS
amlnO aCl 9 (D) TOPOLOGY: li~ear (ii) MOLECULE TYPE: peptide (Xi) ~ U~;N~; DESCRIPTION: SEQ ID NO:58:
ys Thr Lys Gly Ser Gly Phe Phe Val Phe Gly Gly Lys Lys Leu Arg l0 Arg Leu Leu Tyr Met Ile Tyr Met Ser Gly Leu Ala Val Arg Val His Val His Lys Glu Glu Gln Tyr Tyr Asp Tyr 2) INFORMATION FOR SEQ ID NO:59:
i ~;~ U~NI I~ CHARACTE
( ) (~) LENGTH: 36 amin acids (B) TYPE: amlno acld (D) TOPOLOGY: linear (ii) MO~ECULE TYPE: peptide (xi) ~ ;UU~;N~:~; DESCRIPTIO~: SEQ ID NO:59:
ys Thr Lys Gly Ser Gly Phe Phe Val Phe Gly Gly Gly Ala Tyr Ala Arg Cys Pro Asn Glu Thr Arg Ala Leu Thr Val Ala Glu Leu Arg Gly Asn Ala Glu Leu 2) INFORMATION FOR SEQ ID NO:60:
(i) 8~52UI~;N~ CHARACTERISTICS:
(A) LENGTH: 52 amlno: acids ( B ) TYPE: amlno acid (D) TOPOLOGY: linear (li) MOLECULE TYPE: peptide (Xi) S~ U~;Nt~: DESCRIPTION: SEQ ID NO:60:

WO 95/Z636~

ys Thr Lys Gly Ser Gly Phe Phe Val Phe Gly Gly Tyr Asp Pro Asn Tyr Leu Arg Thr Asp Ser Aap Lys Asp Arg Phe Leu Gln Thr Met Val Lys Leu Phe Asn Asp Arg Phe Leu Gln Thr Met Val Lys Leu Phe Asn Arg Ile Lys 45 2) INFORMATION FOR SEQ ID NO:61:
UI~N~1:5 ~i~RZ~'TRRT~ TIcs (A) LENGTH: 16 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: pep~ide (xi) ~;UUr:N~:~; DESCRIPTION: SEQ ID NO:61:
Ileu Ser Glu Ile Lys Gly Val Ile Val His Arg Leu Glu Gly Val ( 2 ) INFORMATION FOR SEQ ID NO: 62:
U~;N~; rT~AR~ TRRT~cTIcs (A) LENGTH: 4 0 amino acids (B) TYPE: amino acid ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (Xi) ~ l,?U~;N~; D~SCRIPTION: SEQ ID NO:62:
Lys Lys Leu Arg Arg Leu Leu Tyr Met Ile Tyr Met Ser Gly Leu Ala .c 1 5 10 15 ~'J Val Arg Val His Val Ser Lys Glu Glu Gln Tyr Tyr Asp Tyr Lys Thr Lys Gly Ser Gly Phe Phe Val Phe

Claims (44)

I claim:

1. A peptide immunogen represented by the formula:
(A)n-(Th)m-(B)o-(IgE CH4 peptide)p wherein: A is an amino acid, .alpha.-NH2, a fatty acid or a derivative thereof, or an invasin;
B is an amino acid;
Th is a helper T cell epitope, an analog or segment thereof;
IgE CH4 peptide is SEQ ID NO:1 or an immunogenic analog thereof;
n is from 1 to 10;
m is from 1 to 4;
o is from 0 to 10; and p is from 1 to 3.

2. The peptide immunogen of Claim 1 wherein p is 1.

3. The peptide immunogen of Claim 1 wherein Th is selected from the group conaisting SEQ ID NOS:5-12, 14, 26-36, 61 and an immunogenic analog or segment thereof.

4. The peptide immunogen of Claim 2 wherein Th is selected from the group consisting SEQ ID NOS:5-12, 14, 26-36, 61 and an immunogenic analog or segment thereof.

5. The peptide immunogen of Claim 1 selected from the group consisting SEQ ID NOS:13, 15-23, 37-50, 51-56 and 62.

6. The peptide immunogen of Claims 3 selected from the group consisting SEQ ID NOS: 51-56 and 62.

7. The peptide immunogen of Claim 1 wherein A
is a fatty acid.

8. The peptide immunogen of Claim 2 wherein A
is a fatty acid.

9. The peptide immunogen of Claim 1 wherein A
is a fatty acid derivative.

10. The peptide immunogen of Claim 2 wherein A
is a fatty acid derivative.

11. The peptide immunogen of Claim 9 wherein the fatty acid derivative is Pam3Cys.

12. The peptide immunogen of Claim 10 wherein the fatty acid derivative is Pam3Cys.

13. A vaccine composition comprising an effective amount of a peptide immunogen of Claim 1 in a pharmaceutically acceptable delivery system.

14. A vaccine composition comprising an effective amount of a peptide immunogen of Claim 2 in a pharmaceutically acceptable delivery system.

15. A vaccine composition comprising an effective amount of a peptide immunogen of Claim 3 in a pharmaceutically acceptable delivery system.

16. A vaccine composition comprising an effective amount of a peptide immunogen of Claim 4 in a pharmaceutically acceptable delivery system.

17. A vaccine composition comprising an effective amount of a peptide immunogen of Claim 5 in a pharmaceutically acceptable delivery system.

18. A vaccine composition comprising an effective amount of a peptide immunogen of Claim 6 in a pharmaceutically acceptable delivery system.

19. A vaccine composition comprising an effective amount of a peptide immunogen of Claim 7 in a pharmaceutically acceptable delivery system.

20. A vaccine composition comprising an effective amount of a peptide immunogen of Claim 8 in a pharmaceutically acceptable delivery system.

21. A vaccine composition comprising an effective amount of a peptide immunogen of Claim 9 in a pharmaceutically acceptable delivery system.

22. A vaccine composition comprising an effective amount of a peptide immunogen of Claim 10 in a pharmaceutically acceptable delivery system.

23. A vaccine composition comprising an effective amount of a peptide immunogen of Claim 11 in a pharmaceutically acceptable delivery system.

24. A vaccine composition comprising an effective amount of a peptide immunogen of Claim 12 in a pharmaceutically acceptable delivery system.

25. A vaccine composition comprising an effective amount of a mixture of peptide immunogens of Claim 9 in a pharmaceutically acceptable delivery system.

26. A vaccine composition comprising an effective amount of a mixture of peptide immunogens of Claim 10 in a pharmaceutically acceptable delivery system.

27. A vaccine composition comprising an effective amount of a mixture of peptide immunogens of Claim 11 in a pharmaceutically acceptable delivery system.

28. A vaccine composition comprising an effective amount of a mixture of peptide immunogens of Claim 12 in a pharmaceutically acceptable delivery system.

29. A method of treating allergic reactions by administering an effective amount of a vaccine composition according to Claim 13.

30. A method of treating allergic reactions by administering an effective amount of a vaccine composition according to Claim 14.

31. A method of treating allergic reactions by administering an effective amount of a vaccine composition according to Claim 15.

32. A method of treating allergic reactions by administering an effective amount of a vaccine composition according to Claim 16.

33. A method of treating allergic reactions by administering an effective amount of a vaccine composition according to Claim 17.

34. A method of treating allergic reactions by administering an effective amount of a vaccine composition according to Claim 18.

35. A method of treating allergic reactions by administering an effective amount of a vaccine composition according to Claim 19.

36. A method of treating allergic reactions by administering an effective amount of a vaccine composition according to Claim 20.

37. A method of treating allergic reactions by administering an effective amount of a vaccine composition according to Claim 21.

38. A method of treating allergic reactions by administering an effective amount of a vaccine composition according to Claim 22.

39. A method of treating allergic reactions by administering an effective amount of a vaccine composition according to Claim 23.

40. A method of treating allergic reactions by administering an effective amount of a vaccine composition according to Claim 24.

41. A method of treating allergic reactions by administering an effective amount of a vaccine composition according to Claim 25.

42. A method of treating allergic reactions by administering an effective amount of a vaccine composition according to Claim 26.

43. A method of treating allergic reactions by administering an effective amount of a vaccine composition according to Claim 27.

44. A method of treating allergic reactions by administering an effective amount of a vaccine composition according to Claim 28.