Note: Descriptions are shown in the official language in which they were submitted.
Wo 95/26365 2 1 ~3 6 5 ~ 5 P~ 4l
-- 1 --
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
-- 3
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 --
-- 4
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) .
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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.
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~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
O - 11 --
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
O -- 12 -
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
O -- 13 --
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
O -- 15 -
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
O -- 16 --
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
O -- 17 -
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
O -- 18 --
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
- 19 -
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
- 20 -
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
- 21 -
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
O - 22 --
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
- 23 -
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 ~
-- 24 --
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
- 25 -
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
- 26 --
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
- 27 -
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 --
- 28 -
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
O - 29 -
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 ~
-- 30 -
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
- 31 -
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 --
- 32 -
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
- 33 -
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
- 34 --
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.
3S
WO 9S/26365 PCT/US9S/03741
2 1 86595
-- 35 -
o
TABLE 3
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)
3S
wo 95/Z6365 P~,ll~., _.'^~741 ~
2~ 86595
-- 36 -
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
?~
-- 37 --
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.
TA3LE 4
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
- 38 --
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
-- 39 -
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
-- 40 -
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
21 86595
- 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
O -- 42 --
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
21 86595
-- 43 --
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 ~
-- 44 --
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
-- 45 -
O
TA3LE 6
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
-- 46 --
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
-- 47 -
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 ~
-- 48 --
(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
O -- 50 --
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
- 51 -
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
-- 52 -
O
TA13LE 7
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
- 53 --
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
65 70 75
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
95 100 105
Cys Ala Asp Ser Asn Pro Arg Gly Val Ser Ala Tyr Leu Ser Arg
110 115 120
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
140 145 150
Thr Trp Ser Arg Ala Ser Gly Lys Pro Val Asn Asn Ser Thr Arg
155 160 165
Lys Glu Glu Lys Gln Arg Asn Gly Thr Leu Thr Val Thr Ser Thr
170 175 180
20 Leu Pro Val Gly Thr Arg Asp Trp Ile Glu Gly Glu Thr Tyr Gln
185 190 195
Cys Arg Val Thr His Pro His Leu Pro Arg Ala Leu Met Arg Ser
200 205 210
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
230 235 240
25 Ala Cys Leu Ile Gln Asn Phe Met Pro Glu Asp Ile Ser Val Gln
245 250 255
Trp Leu His Asn Glu Val Gln Leu Pro Asp Ala Arg His Ser Thr
260 265 270
Thr Gln Pro Arg Lys Thr Lys Gly Ser Gly Phe Phe Val Phe Ser
275 280 285
Arg Leu Glu Val Thr Arg Ala Glu Trp Gln Glu Lys Asp Glu Phe
290 295 300
Ile Cys Arg Ala Val l~is Glu Ala Ala Ser Pro Ser Gln Thr Val
305 310 315
Gln Arg Ala Val Ser Val Asn Pro Gly Lys
320 325
(4 ) INFORMATION FOR SEQ ID NO : 3:
WO95l26365 2 1 8 6595 ~ 741
-- 56 -
(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
25 30
15 Arg Arg Cys Ser Asp A~p Glu Pro: Arg Gly Val Ile Thr Tyr Leu
35 40 45
Ile Pro Pro Ser Pro Leu Asp Leu Tyr Glu Asn Gly Thr Pro Lys
50 55 60
Leu Thr Cys Leu Val Leu Asp Leu Glu Ser Glu Glu Asn Ile Thr
65 70 75
Val Thr Trp Val Arg Glu Arg Ly8 Lys Ser Ile Gly Ser Ala Ser
80 85 90
Gln Arg Ser Thr Lys Xis His Asn Ala Thr Thr Ser Ile Thr Ser
95 100 105
Ile Leu Pro Val Asp Ala Lys Asp Trp Ile Glu Gly Glu Gly Tyr
110 115 120
Gln Cys Arg Val Asp His Pro His Phe Pro Lys Pro Ile Val Arg
125 130 135
Ser Ile Thr Lys Ala Leu Gly Leu Arg Ser Ala Pro Glu Val Tyr
140 145 150
Val Phe Leu Pro Pro Glu Glu Glu Glu Lys Asn Lys Arg Thr Leu
155 160 165
Thr Cys Leu Ile Gln Asn Phe Phe Pro Glu
170 175
(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
-- 57 -
(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
5 10 15
Ile His Arg Cys Asp Pro Asn Ala Phe l~is Ser Thr Ile Gln Leu
20 25 30
Tyr Cys Phe Ile Tyr Gly His Ile Leu Asn Asp Val Ser Val Ser
35 40 45
Trp Leu Met Asp Asp Arg Glu Ile Thr Asp Thr Leu Ala Gln Thr
50 55 60
Val Leu Ile Lys Glu Glu Gly Lys Leu Ala Ser Thr Cys Ser Lys
65 70 75
Leu Asn Ile Thr Glu Gln Gln Trp Met Ser Glu Ser Thr Phe Thr
80 85 90
Cys Arg Val Thr Ser Gln Gly Cys Asp Tyr Leu Ala His Thr Arg
95 100 105
Arg Cys Pro Asp His Glu Pro Arg Gly Ala Ile Thr Tyr Leu Ile
110 115 120
Pro Pro Ser Pro Leu Asp Leu Tyr Gln Asn Gly Ala Pro Lys Leu
125 130 135
Thr Cys Leu Val Leu Asp Leu Glu Ser Glu Lys Asn Val Asn Val
140 145 150
Thr Trp Asn Gln Glu Lys Lys Thr Ser Val Ser Ala Ser Gln Trp
155 160 165
Tyr Thr Lys His His Asn Asn Ala Thr Thr Ser Ile Thr Ser Ile
170 175 180
Leu Pro Val Val Ala Lys Asp Trp Ile Glu Gly Tyr Gly Tyr Gln
185 190 195
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
215 220 225
Val Phe Pro Pro Pro Glu Glu Glu Ser Glu Asp Lys Arg Thr Leu
230 235 240
Thr Cys Leu Ile Gln Asn Phe Phe Pro Glu Asp Ile Ser Val Gln
245 250 255
Trp Leu Gly Asp Gly Lys Leu Ile Ser Asn Ser Gln His Ser Thr
260 265 270
Thr Thr Pro Leu Lys Ser Asn Gly Ser Asn Gln Gly Phe Phe Ile
275 280 285
Phe Ser Arg Leu Glu Val Ala Lys Thr Leu Trp Thr Gln Arg Lys
295 300
WO 95/26365 2 1 8 6 5 9 5 r~~ ?/41 --
- 58 --
Gln Phe Thr Cys Glrl Val Ile His Glu Ala Leu Gln l~ys Pro Arg
305 310 315
( 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
10 15
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
- 59 -
(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
25 1 5 10 15
(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 --
-- 60 -
o
Tyr Asp Pro Asn Tyr Leu Arg Thr Asp Ser Asp Lys Asp Arg Phe
1 0 1 5
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
- 61 -
(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
5 10 15
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
5 10 15
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 ~ -
O -- 62 --
( 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
5 10 15
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
1 5 10 15
Glu Leu Lys Thr Lys Gly Ser Gly Phe Phe Val Phe
20 25
(20) INFORMATIO~ FOR SEQ ID NO:19:
U~N~ CHil~RACTERISTICS:
WO 95/263~5 2 1 ~ 6 ~ 9 ~ P~ 4l
-- 63 -
(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
5 10 15
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
5 10 15
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~
-- 65 -
(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
5 10 15
(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 --
- 66 -
;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
5 - 10 15
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
20 25
(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
- 67 -
(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
1 5 10 15
Gln Ala Ile Pro Leu Val Gly Glu Leu Val Asp Ile Gly Phe Ala Ala
20 25 30
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
5 10 15
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
5 10 15
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
3S
W095/26365 2 1 ~ 6595 F~ 741 --
. .
-- 68 --
(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
5 10 15
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
5 10 15
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
10 15
. .
WO95/26365 l_l/~J.. 'n'~741
~ 1 8~
- 69 -
Leu Gly Gly Lys Thr Lys Gly Ser Gly Phe Phe Val Phe
20 25
( 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
1 5 10 15
Val Ser Ala Ser His Leu Gly Gly Lys Thr Lys Gly Ser Gly Phe Phe
20 25 30
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
5 10 15
Gly Lys Thr Lys Gly Ser Gly Phe Phe Val Phe
20 25
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
-- 70 --
Thr Glu Ser Tyr Gly Gly Lys Thr Lys Gly Ser Gly Phe Phe Val Phe
20 25 30
(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
20 25
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
5 10 15
Gly Gly ~ys Thr Lys Gly Ser Gly Phe Phe Val Phe
20 25
(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
21 86595
O - 71 -
Thr Thr Gly Tyr Leu Lys Gly Asn Ser Gly Gly Lys Thr Lys Gly Ser
20 25 30
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
20 25 30
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
1 5 10 15
Gln Ala Ile Pro Leu Val Gly Glu Leu Val Asp Ile Gly Phe Ala Ala
25 30
Thr Asn Phe Val Glu Ser Cys Gly Gly Lys Thr Lys Gly Ser Gly Phe
35 40 45
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
-- 72 --
(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
20 25 30
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
5 10 15
Ile Gly Gly Lys Thr Lys Gly Ser Gly Phe Phe Val Phe =~
20 25
(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
5 10 15
Lys Thr Lys Gly Ser Gly Phe Phe Val Phe
20 25
( 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
- 73 -
(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
20 25 30
( 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
1 5 10 15
Asn Ala Asn Lys Gly Gly ~ys Thr Lys Gly Ser Gly Phe Phe Val Phe
20 25 30
(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
5 10 15
Gly Lys Thr Lys Gly Ser Gly Phe Phe Val Phe
20 25
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 --
-- 74 --
O
(xi) SEQUENOE DESCRIPTIO~:: SEQ ID NO:52:
Leu Ser Glu Ile Ly5 Gly Val Ile Val His Arg Leu Glu Gly Val Gly
5 10 15
Gly Lys Thr Lys Gly Ser Gly Phe Phe Val Phe
20 25
~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
10 15
15 Val Arg Val Eis Val Ser Lys Glu Glu Gln Tyr Tyr Asp Tyr Gly Gly
20 25 30
Lys Thr Lys Gly Ser Gly Phe Phe Val Phe
35 40
(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
10 15
Glu Leu Arg Gly Asn Ala Glu Leu Gly Gly Lys Thr Lys Gly Ser Gly
20 25 30
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
-- 75 --
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
-- 76 -
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
20 25 30
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
20 25 30
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~
2 1 8 6 5 9 5 PCT/US95/03741
-- 77 --
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
15 1 5 10 15
( 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
35 40
