Note: Descriptions are shown in the official language in which they were submitted.
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EPITOPES OR MIMOTOPES DERIVED FROM THE C-EPSILON-3 OR C-EPSILON-4 DOMAINS OF
IGE, ANTAGO-
NISTS THEREOF, AND THEIR THERAPEUTIC USES
The present invention relates to the provision of novel medicaments for the
treatment, prevention or amelioration of allergic disease. In particular, the
novel
s medicaments are epitopes or mimotopes derived from the Cs3 or Cs4 domains of
IgE.
These novel regions may be the target for both passive and active
immunoprophylaxis
or immunotherapy. The invention further relates to methods for production of
the
medicaments, pharmaceutical compositions containing them and their use in
medicine. Also forming an aspect of the present invention are ligands,
especially
1o monoclonal antibodies, which are capable of binding the IgE regions of the
present
invention, and their use in medicine as passive immunotherapy or
immunoprophylaxis.
In an allergic response, the symptoms commonly associated with allergy are
brought about by the release of allergic mediators, such as histamine, from
immune
15 cells into the surrounding tissues and vascular structures. Histamine is
normally stored
in mast cells and basophils, until such time as the release is triggered by
interaction
with allergen specific IgE. The role of IgE in the mediation of allergic
responses, such
as asthma, food allergies, atopic dermatitis, type-I hypersensitivity and
allergic
rhinitis, is well known. On encountering an antigen, such as pollen or dust
mite
2o allergens, B-cells commence the synthesis of allergen specific IgE. The
allergen
specific IgE then binds to the FcsRI receptor (the high affinity IgE receptor)
on
basophils and mast cells. Any subsequent encounterwith allergen leads to the
triggering of histamine release from the mast cells or basophils, by cross-
linking of
neighbouring IgE/ FcsRI complexes (Sutton and Gould, Nature, 1993, 366: 421-
428;
2s EP 0 477 231 B 1 ).
IgE, like all immunoglobulins, comprises two heavy and two light chains. The
E heavy chain consists of five domains: one variable domain (VH) and four
constant
domains (CE1 to Cs4). The molecular weight of IgE is about 190,000 Da, the
heavy
chain being approximately 550 amino acids in length. The structure of IgE is
3o discussed in Padlan and Davis (Mol. Immunol., 23, 1063-75, 1986) and Helm
et al.,
(2IgE model structure deposited 2/10/90 with PDB (Protein Data Bank, Research
Collabarotory for Structural Bioinformatics; http:\pdb-browsers.ebi.ac.uk)).
Each of
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the IgE domains consists of a squashed barrel of seven anti-parallel strands
of
extended ((3-) polypeptide segments, labelled a to f, grouped into two (3-
sheets. Four
~3-strands (a,b,d & e) form one sheet that is stacked against the second sheet
of three
strands (c f & g) (see FIG.8). The shape of each (3-sheet is maintained by
lateral
packing of amino acid residue side-chains from neighbouring anti-parallel
strands
within each sheet (and is further stabilised by main-chain hydrogen-bonding
between
these strands). Loops of residues, forming non-extended (non-~3-)
conformations,
connect the anti-parallel ~i-strands, either within a sheet or between the
opposing
sheets. The connection from strand a to strand b is labelled as the A-B loop,
and so
on. The A-B and d-a loops belong topologically to the four-stranded sheet, and
loop f
g to the three-stranded sheet. The interface between the pair of opposing
sheets
provides the hydrophobic interior of the globular domain. This water-
inaccessible,
mainly hydrophobic core results from the close packing of residue side-chains
that
face each other from opposing ~i-sheets.
In the past, a number of passive or active immunotherapeutic approaches
designed to interfere with IgE-mediated histamine release mechanism have been
investigated. These approaches include interfering with IgE or allergen/IgE
complexes
binding to the FcsRI or FcERII (the low affinity IgE receptor) receptors, with
either
passively administered antibodies, or with passive administration of IgE
derived
2o peptides to competitively bind to the receptors. In addition, some authors
have
described the use of specific peptides derived from IgE in active immunisation
to
stimulate histamine release inhibiting immune responses.
In the course of their investigations, previous workers in this field have
encountered a number of considerations, and problems, which have to be taken
into
account when designing new anti-allergy therapies. One of the most dangerous
problems revolves around the involvement of IgE cross-linking in the histamine
release signal. It is most often the case that the generation of anti-IgE
antibodies
during active vaccination, are capable of triggering histamine release per se,
by the
cross-linking of neighbouring IgE-receptor complexes in. the absence of
allergen. This
3o phenomenon is termed anaphylactogenicity. Indeed many commercially
available
anti-IgE monoclonal antibodies which are normally used for IgE detection
assays, are
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anaphylactogenic, and consequently useless and potentially dangerous if
administered
to a patient.
Whether or not an antibody is anaphylactogenic, depends on the location of
the target epitope on the IgE molecule. However, based on the present state of
knowledge in this area, and despite enormous scientific interest and
endeavour, there
is little or no predictability of what characteristics any antibody or epitope
may have
and whether or not it might have a positive or negative clinical effect on a
patient.
Therefore, in order to be safe and effective, the passively administered, or
vaccine induced, antibodies must bind in a region of IgE which is capable of
interfering with the histamine triggering pathway, without being anaphylactic
per se.
The present invention achieves all of these aims and provides medicaments
which are
capable of raising non-anaphylactic antibodies which inhibit histamine
release. These
medicaments may form the basis of an active vaccine or be used to raise
appropriate
antibodies for passive immunotherapy, or may be passively administered
themselves
for a therapeutic effect.
Much work has been carried out by those skilled in the art to identify
specific
anti-IgE antibodies which do have some beneficial effects against IgE-mediated
allergic reaction (WO 90/15878, WO 89/04834, WO 93/05810). Attempts have also
been made to identify epitopes recognised by these useful antibodies, to
create peptide
mimotopes of such epitopes and to use those as immunogens to produce anti-IgE
antibodies.
WO 97/31948 describes an example of this type of work, and further describes
IgE peptides from the C~3 and CE4 domains conjugated to carrier molecules for
active
vaccination purposes. These immunogens may be used in vaccination studies and
are
said to be capable of generating antibodies which subsequently inhibit
histamine
release in vivo . In this work, a monoclonal antibody (BSW 17) was described
which
was said to be capable of binding to IgE peptides contained within the CE3
domain
which are useful for active vaccination purposes.
EP 0 477 231 B 1 describes immunogens derived from the CE4 domain of IgE
(residues 497-506, also known as the Stanworth decapeptide), conjugated to
Keyhole
Limpet Haemocyanin (KLH) used in active vaccination immunoprophylaxis. WO
96/14333 is a continuation of the work described in EP 0 477 231 B1.
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Other approaches are based on the identification of peptides derived from Cs3
or Cs4, which themselves compete for IgE binding to the high or low affinity
receptors on basophils or mast cells (WO 93/04173, WO 98/24808, EP 0 303 625 B
1,
EP 0 341 290).
The present invention is the identification of novel sequences of IgE which
are
used in active or passive immunoprophylaxis or therapy. These sequences have
not
previously been associated with anti-allergy treatments. The present invention
provides peptides, per se, that incorporate specific isolated epitopes from
continuous
portions of IgE which have been identified as being surface exposed, and
further
1o provides mimotopes of these newly identified epitopes. These peptides or
mimotopes
may be used alone in the treatment of allergy, or may be used vaccines to
induce auto
anti-IgE antibodies during active immunoprophylaxis or immunotherapy of
allergy to
limit, reduce, or eliminate allergic symptoms in vaccinated subjects.
Surprisingly, the anti-IgE antibodies induced by the peptides of the present
15 invention are non-anaphylactogenic and are capable of blocking IgE-mediated
histamine release from mast cells and basophils.
The regions of human IgE which are peptides of the present invention, and
which may serve to provide the basis for peptide modification are:
20 Table 1
PeptideSequence Location sequenceSEQ
and IgE Domain ID NO.
PS RASGKPVNHSTRKEEKQRNGTL Cs3 1
P6 GTRDWIEGE CE3 2
P7 PHLPRALMRSTTKTSGPRA Cs3/Cs4 3
P8 PEWPGSRDKRT CE4 (Pro451-Thr461)4
P9 EQKDE Cs4
P200 LSRPSPFDLFIRKSPTITC CE3 6
P210 WLHNEVQLPDARHSTTQPRKT Cs4 7
1-90N LFIRKS CE3 g 1
2-90N PSKGTVN Cs3 g2
3-90N LHNEVQLPDARHSTTQPRKTKGS Cs4 g3
4-90N SVNPGK CE4 g4
Peptides that incorporate these epitopes form a preferred aspect of the
present
invention. Mimotopes which have the same characteristics as these epitopes,
and
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immunogens comprising such mimotopes which generate an immune response which
cross-react with the IgE epitope in the context of the IgE molecule, also form
part of
the present invention.
The present invention, therefore, includes isolated peptides encompassing
these IgE epitopes themselves, and any mimotope thereof. The meaning of
mimotope
is defined as an entity which is sufficiently similar to the native IgE
epitope so as to
be capable of being recognised by antibodies which recognise the native IgE
epitope;
(Gheysen, H.M., et al., 1986, Synthetic peptides as antigens. Wiley,
Chichester, Ciba
foundation symposium 119, p130-149; Gheysen, H.M., 1986, Molecular
to Immunology, 23,7, 709-715); or are capable of raising antibodies, when
coupled to a
suitable carrier, which antibodies cross-react with the native IgE epitope.
The mimotopes of the present invention may be peptidic or non-peptidic. A
peptidic mimotope of the surface exposed IgE epitopes identified above, may
also be
of exactly the same sequence as the native epitope: Such a molecule is
described as a
15 mimotope of the epitope, because although the two molecules share the same
sequence, the mimotope will not be presented in the context of the whole IgE
domain
structure, and as such the mimotope may take a slightly different conformation
to that
of the native IgE epitope. It will also be clear to the man skilled in the art
that the
above identified linear sequences (P1 to P7), when in the tertiary structure
of IgE, lie
2o adjacent to other regions that may be distant in the primary sequence of
IgE. As such,
for example, a mimotope of P1 may be continuous or discontinuous, in that it
comprises or mimics segments of P 1 and segments made up of these distant
amino
acid residues.
The mimotopes of the present invention mimic the surface exposed regions of
25 the IgE structure, however, within those regions the dominant aspect is
thought by the
present inventors to be those regions within the surface exposed area which
correlate
to a loop structure. The structure of the domains of IgE are described in
"Introduction
to protein Structure" (page 304, 2"d Edition, Branden and Tooze, Garland
Publishing,
New York, ISBN 0 8153 2305-0) and take the form a (3-barrel made up of two
30 opposing anti-parallel ~3-sheets (see FIG. 8). The mimotopes may comprise,
therefore,
a loop with N or C terminal extensions which may be the natural amino acid
residues
from neighbouring sheets. As examples of this, P 100 contains the A-B loop of
Cs3,
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P8 contains the A-B loop of CE4, PS contains the C-D loop of Cs3 and P 110
contains
the C-D loop of Cs4. Accordingly, mimotopes of these loops form an aspect of
the
present invention. Particularly preferred loops are the C-D loops of Cs3 or
Cs4, and
the A-B loop of CE4.
Peptide mimotopes of the above-identified IgE epitopes may be designed for a
particular purpose by addition, deletion or substitution of elected amino
acids. Thus,
the peptides of the present invention may be modified for the purposes of ease
of
conjugation to a protein carrier. For example, it may be desirable for some
chemical
conjugation methods to include a terminal cysteine to the IgE epitope. In
addition it
may be desirable for peptides conjugated to a protein carrier to include a
hydrophobic
terminus distal from the conjugated terminus of the peptide, such that the
free
unconjugated end of the peptide remains associated with the surface of the
carrier
protein. This reduces the conformational degrees of freedom of the peptide,
and thus
increases the probability that the peptide is presented in a conformation
which most
closely resembles that of the IgE peptide as found in the context of the whole
IgE
molecule. For example, the peptides may be altered to have an N-terminal
cysteine
and a C-terminal hydrophobic amidated tail. Alternatively, the addition or
substitution
of a D-stereoisomer form of one or more of the amino acids may be performed to
create a beneficial derivative, for example to enhance stability of the
peptide. Those
2o skilled in the art will realise that such modified peptides, or mimotopes,
could be a
wholly or partly non-peptide mimotope wherein the constituent residues are not
necessarily confined to the 20 naturally occurring amino acids. In addition,
these may
be cyclised by techniques known in the art to constrain the peptide into a
conformation that closely resembles its shape when the peptide sequence is in
the
context of the whole IgE molecule. A preferred method of cyclising a peptide
comprises the addition of a pair of cysteine residues to allow the formation
of a
disulphide bridge.
Further, those skilled in the art will realise that mimotopes or immunogens of
the present invention may be larger than the above-identified epitopes, and as
such
may comprise the sequences disclosed herein. Accordingly, the mimotopes of the
present invention may consist of addition of N and/or C terminal extensions of
a
number of other natural residues at one or both ends. The peptide mimotopes
may also
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be retro sequences of the natural IgE sequences, in that the sequence
orientation is
reversed; or alternatively the sequences may be entirely or at least in part
comprised
of D-stereo isomer amino acids (inverso sequences). Also, the peptide
sequences may
be retro-inverso in character, in that the sequence orientation is reversed
and the
amino acids are of the D-stereoisomer form. Such retro or retro-inverso
peptides have
the advantage of being non-self, and as such may overcome problems of self
tolerance
in the immune system (for example Pl4c).
Alternatively, peptide mimotopes may be identified using antibodies which are
capable themselves of binding to the IgE epitopes of the present invention
using
techniques such as phage display technology (EP 0 552 267 BI). This technique,
generates a large number of peptide sequences which mimic the structure of the
native
peptides and are, therefore, capable of binding to anti-native peptide
antibodies, but
may not necessarily themselves share significant sequence homology to the
native IgE
peptide. This approach may have significant advantages by allowing the
possibility of
identifying a peptide with enhanced immunogenic properties (such as higher
affinity
binding characteristics to the IgE receptors or anti-IgE antibodies, or being
capable of
inducing polyclonal immune response which binds to IgE with higher affinity),
or
may overcome any potential self antigen tolerance problems which may be
associated
with the use of the native peptide sequence. Additionally this technique
allows the
2o identification of a recognition pattern for each native-peptide in terms of
its shared
chemical properties amongst recognised mimotope sequences.
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Examples of such mimotopes are:
Table 2
Peptide Sequence Description SEQ ID
NO.
PI1 CRASGKPVNHSTRKEEKQRNGLL PS mimotope 8
Pl la (Ac) GKPV~1HSTGGC PS mimotope 9
PI Ib (Ac) GKPVNHSTRKEEKQRNGC PS mimotope 10
P1 lc CGKPVNHSTRKEEKQRNGLL (NH,) PS mimotope I I
PI Id (Ac) RASGKPVNHSTGGC PS mimotope 12
P 12 CGTRDWIEGLL P6 mimotope 13
P 12a CGTRDWIEGETL (NHS) P6 mimotope 14
P 12b (Ac) GTRDWIEGETGC P6 mimotope 15
P 13 CHPHLPRALMLL P7 mimotope I 6
Pl3a CGTHPHLPRALM (NHZ) P7 mimotope 17
Pl3b (Ac) THPHLPRALMRSC P7 mimotope 18
Pl3c (Ac) GPHLPRALMRSSSC P7 mimotope 19
P14 APEWPGSRDKRTC P8 mimotope 20
Pl4a (Ac) APEWPGSRDKRTLAGGC P8 mimotope 21
Pl4b CGGATPEWPGSRDKRTL (NHz) P8 mimotope 22
Pl4c CTRKDRSGPWEPA (NHz) P8 retro 23
Pl4d* (Ac) APCWPGSRDCRTLAG P8 mimotope 24
(cyclic)
Pl4d (Ac) ACPEWPGSRDRCTLAG P8 mimotope 25
(cyclic)
C-I C CATPEWPGSRDKRTLCG P8 mimotope 26
14
C-1 C CATPEWPGSRDKRTCG P8 mimotope 27
13
C3C12 TPCWPGSRDKRCG P8 mimotope 28
P9a CGAEWEQKDEL (NHz) P9 mimotope 29
P9b (Ac) AEWEQKDEFIC P9 mimotope 30
P9b* (Ac) GEQKDEFIC P9 mimotope 31
P9a* CAEGEQKDEL (NHZ) P9 mimotope 32
Carll CPEWPGCRDKRTG P8 mimotope 85
Carl2 TPEWPGCRDKRCG P8 mimotope 86
Alternatively, peptide mimotopes may be generated with the objective of
increasing the immunogenicity of the peptide by increasing its affinity to the
anti-IgE
peptide polyclonal antibody, the effect of which may be measured by techniques
known in the art such as (Biocore experiments) . In order to achieve this the
peptide
sequence may be electively changed following the general rules:
* To maintain the structural constraints, prolines and glycines should not be
1o replaced
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* Other positions can be substituted by an amino acid that has similar
physicochemical properties.
As such, each amino acid residue can be replaced by the amino acid that most
closely resembles that amino acid. For example, A may be substituted by V, L
or I, as
described in the following table.
Original residueExemplary Preferred
substitutions substitution
A V, L, I V
R K,Q,N K
N Q, H,K,R Q
D E E
C S S
Q N N
E D D
G A A
H N, Q,K,R N
I L, V, M, A, L
F
L I, V, M, A, I
F
K R, Q, N R
M L, F, I L
F L, V, I, A, W
Y,W
P A A
S T T
T I S S
W Y~ F Y
Y W, F,T,S F
V I, L, M, F, L
A
Particularly preferred IgE peptides are P8 and variants thereof (such as P14
or
P 14a). These peptides, when coupled to a carrier are potent in inducing anti-
IgE
immune responses, which responses are capable of inhibiting histamine release
from
1o human basophils. Variants, or mimotopes, of P8 are described primarily as
any
peptide based immunogen which is capable of inducing an immune response, which
response is capable of recognising P8. Without being limiting to the scope of
the
present invention, some variants of P8 may be described by a general formula
in
which certain amino acids may be replaced by their closest counterparts. Using
this
technique, P8 peptide mimotopes may be described by the general formula:
P~ Xu Xz~ P~ xs~ X4~ Xs~ X6~ xs~ Xs
or,
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P, X~, Xz, P, G, X4, R, D, Xs, Xs
wherein; X, is an amino acid selected from E, D, N, or Q; X, is an amino acid
selected from W, Y, or F; X3 is an amino acid selected from G or A, X4 is an
amino
acid selected from S, T or M; Xs is an amino acid selected from R or K; and X6
is an
amino acid selected from D or E.
P8 mimotopes may also be identified using antibodies which are capable
themselves of binding to P8, using techniques such as phage display technology
(EP 0
552 267 B 1 ). Monoclonal antibodies such as P 14/23, P 14/31 and P 14/33 are
particularly suitable in this regard.
1o The present invention, therefore, provides novel epitopes, and mimotopes
thereof, and their use in the manufacture of pharmaceutical compositions for
the
prophylaxis or therapy of allergies. Immunogens comprising at least one of the
epitopes or mimotopes of the present invention and carrier molecules are also
provided for use in vaccines for the immunoprophylaxis or therapy of
allergies.
15 Accordingly, the epitopes, mimotopes, or immunogens of the present
invention are
provided for use in medicine, and in the medical treatment or prophylaxis of
allergic
disease. Preferred immunogens and vaccines of the present invention comprise
the
IgE epitope P8, or mimotopes thereof, including P14.
The present inventors have shown that different methods by which the epitope
20 or mimotope is presented has significant effects upon binding to monoclonal
antibodies and to the immune response after vaccination. For example, when
using
cyclised peptides, altering the length and phase of the loop may have
significant
effects on the binding activity of the cyclised mimotopes to the P14
monoclonal
antibodies (P14/23, P14/31 or P14/33). As such the present inventors have
developed
25 a novel system which selects the sites of cyclisation, thereby increasing
the
probability that the cyclised peptides are held in the correct loop structure,
which
comprises the correct amino acid residues. In this way, the peptide is likely
to be
constrained in a conformation that most closely resembles that which the
peptides
would normally adopt if they were in the context of the whole IgE domain.
Hence,
3o without limiting the present invention the cyclised mimotopes which follow
these new
rules form one preferred aspect of the present invention.
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Putative mimotope sequences that are not consistent with these rules may still
raise useful antisera (for example P 14 and P 11 ), as such the following
examples are
only a sub-set of the types of mimotopes of the present invention.
Examples of preferred peptides that follow these newly defined structural
rules
are:
Table 3
Peptide sequence Mimotope of SEQ ID NO.
CSRPSPFDLFIRKSPTITC A-B loop of 33
CE3
CSRPSPFDLFIRKSPTC A-B loop of 35
CE3
CPSPFDLFIRKSPTITC A-B loop of 41
Cs3
CPSPFDLFIRKSPC A-B loop of 43
CE3
CTWSRASGKPVNHSTC C-D loop of 58
Cs3
CTWSRASGKPVNHC C-D loop of 60
CE3
CSR.ASGKPVNHSTC C-D loop of 66
Cs3
CSRASGKPVNHC C-D loop of 68
Cs3
CYAFATPEWPGSRDKRTLAC A-B loop of 45
Cs4
CYAFATPEWPGSRDKRTC A-B loop of 47
Cs4
CFATPEWPGSR.DKRTLAC A-B loop of 53
Ce4
CFATPEWPGSRDKRTC A-B loop of 55
C~4
CQWLHNEVQLPDARHC C-D loop of 70
CE4
CQWLHNEVQLPDAC C-D loop of 72
Cs4
CLHNEVQLPDARHC C-D loop of 78
Cs4
CLHNEVQLPDAC C-D loop of 80
C4
It is envisaged that the mimotopes of the present invention will be of a small
size, such that they mimic a region selected from the whole IgE domain in
which the
1o native epitope is found. Peptidic mimotopes, therefore, should be less than
100 amino
acids in length, preferably shorter than 75 amino acids, more preferably less
than 50
amino acids, and most preferable within the range of 4 to 25 amino acids long.
Specific examples of preferred peptide mimotopes are P 14 and P 1 l, which are
respectively 13 and 23 amino acids long. Non-peptidic mimotopes are envisaged
to be
15 of a similar size, in terms of molecular volume, to their peptidic
counterparts.
It will be apparent to the man skilled in the art which techniques may be used
to confirm the status of a specific construct as a mimotope which falls within
the
scope of the present invention. Such techniques include, but are not
restricted to, the
following. The putative mimotope can be assayed to ascertain the
immunogenicity of
20 the construct, in that antisera raised by the putative mimotope cross-react
with the
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native IgE molecule, and are also functional in blocking allergic mediator
release
from allergic effector cells. The specificity of these responses can be
confirmed by
competition experiments by blocking the activity of the antiserum with the
mimotope
itself or the native IgE, and/or specific monoclonal antibodies that are known
to bind
the epitope within IgE. Specific examples of such monoclonal antibodies for
use in
the competition assays include P 14/23, P 14/31 or P 14/33, which would
confirm the
status of the putative mimotope as a mimotope of P8.
In one embodiment of the present invention at least one IgE epitope or
mimotope are linked to Garner molecules to form immunogens for vaccination
1o protocols, preferably wherein the Garner molecules are not related to the
native IgE
molecule. The mimotopes may be linked via chemical covalent conjugation or by
expression of genetically engineered fusion partners, optionally via a linker
sequence.
As one embodiment, the peptides of the present invention are expressed in a
fusion
molecule with the fusion partner, wherein the peptide sequence is found within
the
15 primary sequence of the fusion partner.
The covalent coupling of the peptide to the immunogenic carrier can be carried
out in a manner well known in the art. Thus, for example, for direct covalent
coupling
it is possible to utilise a carbodiimide, glutaraldehyde or (N-[y-
maleimidobutyryloxy]
succinimide ester, utilising common commercially available heterobifunctional
20 linkers such as CDAP and SPDP (using manufacturers instructions). After the
coupling reaction, the immunogen can easily be isolated and purified by means
of a
dialysis method, a gel filtration method, a fractionation method etc.
In a preferred embodiment the present inventors have found that peptides,
particularly cyclised peptides may be conjugated to the carrier by preparing
25 Acylhydrazine peptide derivatives.
The peptides/protein carrier constructs can be produced as follows.
Acylhydrazine peptide derivatives can be prepared on the solid phase as shown
in the
following scheme 1 Solid Phase Peptide Synthesis:
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Scheme 1
Rink-Resin
Solid Phase Peptide Synthesis
X-AAL..C(Trt)....C(Trt)....AAn-Lys(Dde)-Rink-Resin
Hydrazine hydrate
X-AAi...C(Trt)....C(Trt)....AAr,-Lys(NH~-Rink-Resin
(i) Succinic anhydride
(ii) HBTU/HOBt/NnRVI/NZH4
O
X AAI...C(Trt)....C(Trt)....AA"-Lys-Rink-Resin Z= ~N ~ ~
t
N I-
TFA
O
~ N t-~t ~
X-AAA...C(-SH)....C(-SH)....AA~,-Lys-CONHZ Z = O
t
N I-a
H2O2 oxidation
n
X-AA~...C....C....AA,~-Lys-CONH2
t
N I-a
These peptide derivatives can be readily prepared using the well-known
'Fmoc' procedure, utilising either polyamide or polyethyleneglycol-polystyrene
(PEG-PS) supports in a fully automated apparatus, through techniques well
known in
the art [techniques and procedures for solid phase synthesis are described in
'Solid
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Phase Peptide Synthesis: A Practical Approach' by E. Atherton and R.C.
Sheppard,
published by IRL at Oxford University Press (1989)]. Acid mediated cleavage
afforded the linear, deprotected, modified peptide. This could be readily
oxidised and
purified to yield the disulphide-bridged modified epitope using methodology
outlined
in 'Methods in Molecular Biology, Vol. 35: Peptide Synthesis Protocols (ed.
M.W.
Pennington and B.M. Dunn), chapter 7, pp91-171 by D. Andreau et al.
The peptides thus synthesised can then be conjugated to protein carriers using
the following technique:
Introduction of the aryl aldehyde functionality utilised the succinimido
active
1o ester (BAL-OSu) prepared as shown in scheme 2 (see WO 98/17628 for further
details). Substitution of the amino functions of a carrier eg BSA (bovine
serum
albumin) to ~SO% routinely give soluble modified protein. Greater substitution
of the
BSA leads to insoluble constructs. BSA and BAL-OSu were mixed in equimolar
concentration in DMSO/buffer (see scheme) for 2 hrs. This experimentally
derived
protocol gives ~50% substitution of BSA as judged by the Fluorescamine test
for free
amino groups in the following Scheme 2/3 - Modified Carrier Preparation:
14
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Scheme 2
HO O
H /~
O O-(CHZ)a-COZH + ~N=C=N--( ) + HO-N
O
Dioxan
HO
H / \ O O
O OWC~)a
O-N BAL-OSu
O
Scheme 3
BSA~(NI-i~)m + mBAL-OSu
40% DMSO/buffer
(pH 7.25,0.2 M NaOAc)
2 hr.
OH
O
BSA~MNH~CO-(CHZ)a-O ~ ~ H
BSA-BAL
Simple combination of modified peptide and derivatised carrier affords
peptide carrier constructs readily isolated by dialysis - Scheme 4 -
Peptide/carrier
s conjugate:
IS
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Scheme 4
OH
O
BSA O . \ / H + 3 m ~ X-AA1...C....C....AA°-Li s-CONHZ
NH-Z
m
BSA-BAL
50% DMSO/buffer
(pH 3.5, 0.1 M NaHC02)
~8-16 hr.
H
BSA H O
N-N
'' NH
O
H2N~ Lys...AA~...C.........C...AA~-X
O m
The types of carriers used in the immunogens of the present invention will be
readily known to the man skilled in the art. The function of the Garner is to
provide
cytokine help in order to help induce an immune response against the IgE
peptide. A
non-exhaustive list of carriers which may be used in the present invention
include:
Keyhole limpet Haemocyanin (KLH), serum albumins such as bovine serum albumin
(BSA), inactivated bacterial toxins such as tetanus or diptheria toxins (TT
and DT), or
recombinant fragments thereof (for example, Domain 1 of Fragment C of TT, or
the
1o translocation domain of DT), or the purified protein derivative of
tuberculin (PPD).
Alternatively the mimotopes or epitopes may be directly conjugated to liposome
Garners, which may additionally comprise immunogens capable of providing T-
cell
help. Preferably the ratio of mimotopes to carrier is in the order of 1:1 to
20: l, and
preferably each carrier should carry between 3-15 peptides.
In an embodiment of the invention a preferred carrier is Protein D from
Haemophilus influenzae (EP 0 594 610 B 1). Protein D is an IgD-binding protein
from
Haemophilus influenzae and has been patented by Forsgren (WO 91/18926, granted
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EP 0 594 610 B 1 ). In some circumstances, for example in recombinant
immunogen
expression systems it may be desirable to use fragments of protein D, for
example
Protein D 1/3~d (comprising the N-terminal 100-110 amino acids of protein D
(GB
9717953.5)).
Another preferred method of presenting the IgE peptides of the present
invention is in the context of a recombinant fusion molecule. For example, EP
0 421
635 B describes the use of chimaeric hepadnavirus core antigen particles to
present
foreign peptide sequences in a virus-like particle. As such, immunogens of the
present
invention may comprise IgE peptides presented in chimaeric particles
consisting of
1o hepatitis B core antigen. Additionally, the recombinant fusion proteins may
comprise
the mimotopes of the present invention and a carrier protein, such as NS 1 of
the
influenza virus. For any recombinantly expressed protein which forms part of
the
present invention, the nucleic acid which encodes said immunogen also forms an
aspect of the present invention.
15 Peptides used in the present invention can be readily synthesised by solid
phase procedures well known in the art. Suitable syntheses may be performed by
utilising "T-boc" or "F-moc" procedures. Cyclic peptides can be synthesised by
the
solid phase procedure employing the well-known "F-moc" procedure and polyamide
resin in the fully automated apparatus. Alternatively, those skilled in the
art will know
2o the necessary laboratory procedures to perform the process manually.
Techniques and
procedures for solid phase synthesis are described in 'Solid Phase Peptide
Synthesis:
A Practical Approach' by E. Atherton and R.C. Sheppard, published by IRL at
Oxford
University Press (1989). Alternatively, the peptides may be produced by
recombinant
methods, including expressing nucleic acid molecules encoding the mimotopes in
a
25 bacterial or mammalian cell line, followed by purification of the expressed
mimotope.
Techniques for recombinant expression of peptides and proteins are known in
the art,
and are described in Maniatis, T., Fritsch, E.F. and Sambrook et al.,
Molecular
cloning, a laboratory manual, 2nd Ed.; Cold Spring Harbor Laboratory Press,
Cold
Spring Harbor, New York ( 1989).
3o The immunogens of the present invention may comprise the peptides as
previously described, including mimotopes or analogues thereof, or may be
immunologically cross-reactive derivatives or fragments thereof. Also forming
part of
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the present invention are portions of nucleic acid which encode the immunogens
of
the present invention or peptides, mimotopes or derivatives thereof.
The present invention, therefore, provides the use of novel epitopes or
mimotopes (as defined above) in the manufacture of pharmaceutical compositions
for
the prophylaxis or therapy of allergies. Immunogens comprising the mimotopes
or
peptides of the present invention, and carrier molecules are also provided for
use in
vaccines for the immunoprophylaxis or therapy of allergies. Accordingly, the
mimotopes, peptides or immunogens of the present invention are provided for
use in
medicine, and in the medical treatment or prophylaxis of allergic disease.
1o Vaccines of the present invention, may advantageously also include an
adjuvant. Suitable adjuvants for vaccines of the present invention comprise
those
adjuvants that are capable of enhancing the antibody responses against the IgE
peptide
immunogen. Adjuvants are well known in the art (Vaccine Design - The Subunit
and
Adjuvant Approach, 1995, Pharmaceutical Biotechnology, Volume 6, Eds. Powell,
M.F., and Newman, M.J., Plenum Press, New York and London, ISBN 0-306-44867
X). Preferred adjuvants for use with immunogens of the present invention
include
aluminium or calcium salts (hydroxide or phosphate).
The vaccines of the present invention will be generally administered for both
priming and boosting doses. It is expected that the boosting doses will be
adequately
2o spaced, or preferably given yearly or at such times where the levels of
circulating
antibody fall below a desired level. Boosting doses may consist of the peptide
in the
absence of the original Garner molecule. Such booster constructs may comprise
an
alternative carrier or may be in the absence of any carrier.
In a further aspect of the present invention there is provided an immunogen or
vaccine as herein described for use in medicine.
The vaccine preparation of the present invention may be used to protect or
treat a mammal susceptible to, or suffering from allergies, by means of
administering
said vaccine via systemic or mucosal route. These administrations may include
injection via the intramuscular, intraperitoneal, intradermal or subcutaneous
routes; or
3o via mucosal administration to the oral/alimentary, respiratory,
genitourinary tracts. A
preferred route of administration is via the transdermal route, for example by
skin
patches. Accordingly, there is provided a method for the treatment of allergy,
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comprising the administration of a peptide, immunogen, or ligand of the
present
invention to a patient who is suffering from or is susceptible to allergy.
The amount of protein in each vaccine dose is selected as an amount which
induces an immunoprotective response without significant adverse side effects
in
typical vaccinees. Such amount will vary depending upon which specific
immunogen
is employed and how it is presented. Generally, it is expected that each dose
will
comprise 1-1000 pg of protein, preferably 1-500 fig, more preferably 1-100 pg,
of
which 1 to SO~g is the most preferable range. An optimal amount for a
particular
vaccine can be ascertained by standard studies involving observation of
appropriate
immune responses in subjects. Following an initial vaccination, subjects may
receive
one or several booster immunisations adequately spaced.
In a related aspect of the present invention are ligands capable of binding to
the peptides of the present invention. Example of such ligands are antibodies
(or Fab
fragments). Also provided are the use of the ligands in medicine, and in the
manufacture of medicaments for the treatment of allergies. The term "antibody"
herein is used to refer to a molecule having a useful antigen binding
specificity. Those
skilled in the art will readily appreciate that this teen may also cover
polypeptides
which are fragments of or derivatives of antibodies yet which can show the
same or a
closely similar functionality. Such antibody fragments or derivatives are
intended to
2o be encompassed by the term antibody as used herein.
Particularly preferred ligands are monoclonal antibodies. For example, P14/23,
P 14/31 or P 14/33 are monoclonal antibodies which recognise P8 (which were
raised
by vaccination with a P 14 immunogen). The hybridomas of these antibodies were
deposited as Budapest Treaty patent deposit at ECACC (European Collection of
Cell
Cultures, Vaccine Research and Production Laboratory, Public Health Laboratory
Service, Centre for Applied Microbiology Research, Porton Down, Salisbury,
Wiltshire, SP4 OJG, UK) on 26 January 2000 under Accession No.s 00012610,
00012611, 00012612 respectively. Also forming an important aspect of the
present
invention is the use of these monoclonal antibodies in the identification of
novel
3o mimotopes of IgE, for subsequent use in allergy therapy, and the use of the
antibodies
in the manufacture of a medicament for the treatment or prophylaxis of
allergy. All of
these monoclonal antibodies function in vitro in inhibiting histamine release
from
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human basophils, and also P 14/23 and P 14/31 have been shown to inhibit
passive
cutaneous anaphylaxis in vivo.
Therefore, mimotopes of IgE CE4 that are capable of binding to P 14/23,
P 14/31 or P 14/33, and immunogens comprising these mimotopes, form an
important
aspect of the present invention. Vaccines comprising mimotopes that are
capable of
binding to P 14/23, P 14/31 or P 14/33 are useful in the treatment of allergy.
Additionally, antibodies induced in one animal by vaccination with the
peptides or immunogens of the present invention, may be purified and passively
administered to another animal for the prophylaxis or therapy of allergy. The
peptides
to of the present invention may also be used for the generation of monoclonal
antibody
hybridomas (using know techniques e.g. Kohler and Milstein, Nature, 1975, 256,
p495), humanised monoclonal antibodies or CDR grafted monoclonals, by
techniques
known in the art. Such antibodies may be used in passive immunoprophylaxis or
immunotherapy, or be used in the identification of IgE peptide mimotopes.
15 As the ligands of the present invention may be used for the prophylaxis or
treatment of allergy, there is provided pharmaceutical compositions comprising
the
ligands of the present invention. Preferred pharmaceutical compositions for
the
treatment or prophylaxis of allergy comprise the monoclonal antibodies P
14/23,
P 14/31 or P 14/33.
20 Aspects of the present invention may also be used in diagnostic assays. For
example, panels of ligands which recognise the different peptides of the
present
invention may be used in assaying titres of anti-IgE present in serum taken
from
patients. Moreover, the peptides may themselves be used to type the
circulating anti-
IgE. It may in some circumstances be appropriate to assay circulating anti-IgE
levels,
25 for example in atopic patients, and as such the peptides and poly/mono-
clonal
antibodies of the present invention may be used in the diagnosis of atopy. In
addition,
the peptides may be used to affinity remove circulating anti-IgE from the
blood of
patients before re-infusion of the blood back into the patient.
Also forming part of the present invention is a method of identifying peptide
30 immunogens for the immunoprophylaxis or therapy of allergy comprising using
a
computer model of the structure of IgE, and identifying those peptides of the
IgE
which are surface exposed. These regions may then be formulated into
immunogens
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and used in medicine. Accordingly, the use of P 14/23, P 14/31 or P 14/33 in
the
identification of peptides for use in allergy immunoprophylaxis or therapy
forms part
of the present invention.
Vaccine preparation is generally described in New Trends and Developments
in Vaccines, edited by Voller et al., University Park Press, Baltimore,
Maryland,
U.S.A. 1978. Conjugation of proteins to macromolecules is disclosed by
Likhite, U.S.
Patent 4,372,945 and by Armor et al., U.S. Patent 4,474,757.
Description of drawings
1o FIG 1, Surface exposure of Cs3 an Ce4 of human IgE as calculated from the
Padlan
and Davis model 1986.
FIG 2, Histamine release inhibition and anaphylactogenicity of P14 antiserum.
Monoclonal Antibodies, PTmAb0005 and PTmAb001 l, which were used as positive
controls, were added at 1 pg/ml to anti-BSA sera diluted 1/100 and 1/500
(final). The
anti-P14 antisera were added at 1/100 and 1/500 final dilution. Cells were
taken from
an allergic patient sensitive to grass pollen, histamine release was triggered
by
incubation with this grass pollen allergen.
FIG 3, Histamine release inhibition and anaphylactogenicity of anti-P 14
antiserum.
The P 14 antiserum from different mice, was added at different dilutions (80X
or 40X)
2o to contain approximately 1 pg/ml of anti-IgE antibody as measured by IgE
receptor-
bound ELISA. Three negative controls were used: Anti-BSA antiserum, non-
specific
IgGl and a mixture of non-specific IgGI diluted in anti-BSA antiserum. mAbl 1
is a
monoclonal antibody known to inhibit histamine release and was used as a
positive
control (added at 2pg/ml).
FIG 4, Histamine release inhibition and anaphylactogenicity of anti-P 14
antiserum.
Anti-P14 Antisera from different mice were added at a 1/50 final dilution.
Monoclonal Abs were added at 2 pg/ml either in assay buffer or in anti-BSA
sera
dilution 1/50. Three negative controls were used: Anti-BSA antiserum, non-
specific
IgGI and a mixture of non-specific IgGl diluted in anti-BSA antiserum. mAbl l
is a
3o monoclonal antibody known to inhibit histamine release and was used as a
positive
control (added at 2~g/ml).
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FIG 5, Antibody response anti-P 11. Peptide P 11 is coated at 1 pg/ml in
carbonate
buffer at +4°C overnight. After saturation of plates, two-fold serial
dilution of sera are
added and incubated for lh at 37°C. Bound IgG is detected with a
biotinylated anti-
mouse Ab followed by streptavidin-POD and TMB substrate. Time points measured
A. days 14 post vaccination 1, and day 14 post v2; B, Day 14 post v3.
FIG 6, Anti-P11 IgG anti-human IgE titres. Human IgE was coated at 1 pg/ml.
Two-
fold serial dilutions of sera ("BSA pool" is a pool of the control group) or
PTmAb0005 (a positive control monoclonal antibody) were incubated for lh at
37°C.
Bound IgG is detected with a biotinylated anti-mouse Ab.
1 o FIG 7, Histamine release inhibition studies with anti-P 14 monoclonal
antibodies, on
allergic basophils donated by dustmite allergic patients (A 10 and A 11 ) and
from grass
pollen allergic patients (G8 and G4). PT11 (PTmAb0011) was used as a positive
control, and non-specific IgG2a was used as an isotype control for the P14/23,
P14/31
and P14/33.
FIG 8, IgE domain structure. (A) Each domain is composed of two facing (3-
sheets,
shown in outline, one of 4 anti-parallel (3-strands (labelled 4) and the other
of 3 anti-
parallel (3-strands (labelled 3). (B) The seven strands are shown
topographically as
block arrows labelled a to f, partitioned between the two sheets as shown. The
loop-
connectivity of the strands is shown topologically with curved arrows: solid
arrows
2o are intra-sheet loops and dashed arrows are inter-sheet loops. In the IgGl
Fc domain
structures a short c' strand forms part of the C-D loop, as is predicted for
IgE Fc.
FIG 9, (A) Predicted structural alignment of the A-B loop sequences of human
IgE
domains Cs2, 3 & 4 with the equivalent segments from the crystallographically
determined structure of human IgGI Fc (domains C~y2 & Cy3). [3-strands in the
IgGl
structure are underlined and labelled a and b; amino acid residues at the ends
of each
sequence segment are numbered. Vertical arrows below the block of sequences
point
to predicted optimal cyclisation positions, labelled and connected by dashed
or solid
lines as shown in FIG l Ob. (B) Predicted structural alignment of the c d
loops of
human IgE Cs2,3 & 4 with human IgGI Fc. (3-strands in the IgGI structure are
3o underlined and labelled c, c ~ and d; amino acid residues at the ends of
each sequence
segment are numbered. Residues highlighted by the shaded boxes form (Cy2 &
Cy3)
or are predicted to form (Cs2, by homology model refinement and experiment,
Cs3,
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CE4, by homology-modelling) a protected core within the loop. Residues within
the
plain bold boxes are predicted to be involved in recognition by receptors
and/or
antibodies. Vertical arrows below the block of sequences point to predicted
optimal
cyclisation positions, labelled and connected by dashed or solid lines as
shown in FIG
l lb.
FIG 10, (A) The schematic structure of the A-B hairpin at the sheet-sheet
interface of
Ig constant domains. Adjacent anti-parallel ~i-strands are shown as solid
arrows,
labelled a and b. Residues along strand a are labelled i, those along strand b
are
labelled j. Residues i+n & j+m, where both n and m are zero or even, form part
of the
1o sheet-sheet interface within a domain. Residues i+n & j+m, where both n and
m are
odd, form part of the solvent-exposed surface of a domain. The A-B loop is
shown as
a black arrow. (B) The schematic structure of the A-B hairpin as in figure 3a,
with
residue positions optimal for cyclisation connected by dashed or solid
dumbbells.
FIG 11, (A) The schematic structure of the C-D hairpin (loop plus supporting
(3-
strands) at the edge of the sheet-sheet interface of Ig constant domains.
Opposing anti-
parallel ~3-strands are shown as solid arrows, labelled c and d. Residues
along strand c
are labelled i, those along strand d are labelled j. Residues i+n & j+m, where
n is odd
but m is even, forni part of the sheet-sheet interface within a domain.
Residues i+n &
j+m, where n is zero or even but m is odd, form part of the solvent-exposed
surface of
2o a domain. The c d loop, containing the short c' strand, is shown as a black
arrow. (B)
The schematic structure of the c d hairpin, with residue positions optimal for
cyclisation connected by dashed or solid dumbbells.
The present invention is illustrated by but not limited to the following
examples.
Part 1, Active vaccination studies
Exam les
1.1 Peptide identification
The peptides were identified by the following technique.
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The modelled structure of human IgE has been described Padlan and Davies (Mol.
Immunol., 23, 1063-75, 1986). Peptides were identified which were both
continuous
and solvent exposed. This was achieved by using Molecular Simulations software
(MSI) to calculate the accessibility for each IgE amino acid, the accessible
surface
was averaged over a sliding window of five residues, and thereby identifying
regions
of the IgE peptides which had an average over that 5-mer of greater than 802.
The results of the test are shown in FIG 1.
Results
From figure 1 there are a number of native peptides which may be used as
immunogens for raising antibodies against IgE.
Table 4, Native surface exposed and continuous IgE peptides using the 1986
Padlan
and Davies model.
PeptideSequence Location sequenceSEQ
and IgE Domain ID NO.
PS RASGKPVNHSTRKEEKQRNGTL CE3 1
P6 GTRDWIEGE Cs3 2
P7 PHLPRALMRSTTKTSGPRA CE3/Cs4 3
P8 PEWPGSRDKRT CE4 (Pro451-Thr461)4
P9 EQKDE CE4 5
P200 LSRPSPFDLFIRKSPTITC Cs3 6
P210 WLHNEVQLPDARHSTTQPRKT Cs4 7
In addition to those peptides identified above, the following peptides have
been
identified using the same selection criteria with the Helm et al. IgE model
(2IgE
model structure deposited 2/10/90 with PDB (Protein Data Bank, Research
2o Collabarotory for Structural Bioinformatics; http:\pdb-
browsers.ebi.ac.uk)).
Table 5, Peptides identified using the Helm et al. 1990 model.
Name Sequence LocationSEQ ID NO.
I-90N LFIRKS Cs3 81
2-90N PSKGTVN Cs3 82
3-90N LHNEVQLPDARHSTTQPRKTKGS Cs4 83
4-90N SVNPGK C~4 84
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These peptides, or mimotopes thereof, were synthesised and conjugated to
carrier
proteins for use in immunogenicity studies.
1.2 Synthesis of IgE peptidelProtein D conjugates using a succinimide-
maleimide
cross-linker
Protein D may be conjugated directly to IgE peptides to form antigens of the
present
invention by using a maleimide-succinimide cross-linker. This chemistry allows
controlled NHz activation of carrier residues by fixing a succinimide group.
to Maleimide groups is a cysteine-binding site. Therefore, for the purpose of
the
following examples, the IgE peptides to be conjugated require the addition of
an N-
terminal cysteine.
The coupling reagent is a selective heterobifunctional cross-linker, one end
of the
15 compound activating amino group of the protein carrier by an succinimidyl
ester and
the other end coupling sulhydryl group of the peptide by a maleimido group.
The
reactional scheme is as the following
a. Activation of the protein by reaction between lysine and succinimidyl ester
3 O
S O '~ - O \
Protein NH2 + ~ N OC-(CH2)3-N
I I
O O ~/
O
O
Protein N~~~ -(CH2)3-
O //
b. Coupling between activated protein and the peptide cysteine by reaction
with
the maleimido group
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0
Protein N~'IC-(CH2)~~~ + gH
O // Pep ide
O O
S ,.wwvw
Protein'' N~~~ -(CH2)3-~~
O //
O
Conjugue
1.3 Preparation of IgE peptide-Protein D conjugate
The protein D is dissolved in a phosphate buffer saline at a pH 7.2 at a
concentration
of 2.5 mg/ml. The coupling reagent (N-[y-maleimidobutyryloxy] succinimide
ester -
GMBS) is dissolved at 102.5 mg/ml in DMSO and added to the protein solution.
1.025 mg of GMBS is used for 1 mg of Protein D. The reaction solution is
incubated
1 hour at room temperature. The by-products are removed by a desalting step
onto a
sephacryl 200HR permeation gel. The eluant used is a phosphate buffer saline
Tween
80 0.1 % pH 6.8. The activated protein is collected and pooled. The peptides
(as
identified in tables 4 or 5, or derivatives or mimotopes thereof] is dissolved
at 4
mg/ml in 0.1 M acetic acid to avoid di-sulfure bond formation. A molar ratio
of
between 2 to 20 peptides per 1 activated Protein D is used for the coupling.
The
peptide solution is slowly added to the protein and the mixture is incubated 1
h at
25°C. The pH is kept at a value of 6.6 during the coupling phase. A
quenching step is
performed by addition of cysteine (0.1 mg cysteine per mg of activated PD
dissolved
at 4 mg/ml in acetic acid 0.1 M), 30 minutes at 25°C and a pH of 6.5.
Two dialysis
against NaCI 150 mM Tween 80 0.1 % are performed to remove the excess of
2o cysteine or peptide.
The last step is sterile filtration through a 0.22 ~m membrane. The final
product is a
clear filtrable solution conserved at 4°C. The final ratio of
peptide/PD may be
determined by amino acid analysis.
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In an analogous fashion the peptides of the present invention may be
conjugated to
other carriers including BSA. A pre-activated BSA may be purchased
commercially
from Pierce Inc.
Mimotopes of P8 (P 14, SEQ ID NO. 20; CLEDGQVMDVDLL) and PS (P 1 l, SEQ
ID NO. 8; CRASGKPVNHSTRKEEKQRNGLL) were synthesised which were
conjugated to both Protein D and BSA using techniques described above.
1.4 ELISA methods
Anti peptide or Anti peptide carrier ELISA
The anti-peptide and anti-Garner immune responses were investigated using an
ELISA
technique outlined below. Microtiterplates (Nunc) are coated with the specific
antigen
in PBS (4° overnight) with either: Streptavidin at 2pg/ml (followed by
incubation
with biotinylated peptide (1pM) for 1 hour at 37°C), Wash 3X PBS-Tween
20 0.1%.
Saturate plates with PBS-BSA 1%-Tween 20 0.1% (Sat buffer) for 1 hr at
37°. Add 1°
antibody = sera in two-step dilution (in Sat buffer), incubate 1 hr 30 minutes
at 37°.
Wash 3X. Add 2° anti-mouse Ig (or anti-mouse isotype specific
monoclonal antibody)
coupled to HRP. Incubate 1 hr at 37°. Wash 5X. Reveal with TMB (BioRad)
for 10
minutes at room temperature in the dark. Block reaction with 0.4N HZS04.
Method for the Detection of Anti-Human IgE Reactivity in Mouse Serum (IgE
plate
bound ELISA)
ELISA plates are coated with human chimaeric IgE at 1 pg/ml in pH 9.6
carbonate/bicarbonate coating buffer for 1 hour at 37°C or overnight at
4°C. Non-
specific binding sites are blocked with PBS/O.OS% Tween-20 containing 5% w/v
Marvel milk powder for 1 hour at 37°C. Serial dilutions of mouse
serum in
PBS/O.OS% Tween-20/1% w/v BSA/4% New Born Calf serum are then added for 1
hour at 37°C. Polyclonal serum binding is detected with goat anti-mouse
IgG-Biotin
(1/2000) followed by Streptavidin-HRP (1/1000). Conjugated antibody is
detected
3o with TMB substrate at 450nm. A standard curve of PTmAb0011 is included on
each
plate so that the anti-IgE reactivity in serum samples can be calculated in
gg/ml.
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Competition of IgE Binding with Mimotope Peptides, Soluble IgE or PTmAb0011
Single dilutions of polyclonal mouse serum are mixed with single
concentrations of
either mimotope peptide or human IgE in a pre-blocked polypropylene 96-well
plate.
Mixtures are incubated for 1 hour at 37°C and then added to IgE-coated
ELISA plates
for 1 hour at 37°C. Polyclonal serum binding is detected with goat anti-
mouse IgG-
Biotin (1/2000) followed by Streptavidin-HRP (1/1000). Conjugated antibody is
detected with TMB substrate at 450nm. For competition between serum and
PTmAb0011 for IgE binding, mixtures of serum and PTmAb0011-biotin are added to
IgE-coated ELISA plates. PTmAb0011 binding is detected with Streptavidin-HRP
(1/1000).
L5 Human Basophil Assays
Two types of assay were performed with human basophils (HBA), one to determine
the anaphylactogenicity of the monoclonal antibodies, consisting of adding the
antibodies to isolated PBMC; and a second to measure the inhibition of Lol P I
(a
strong allergen) triggered histamine release be pre-incubation of the HBA with
the
monoclonal antibodies.
Blood is collected by venepuncture from allergic donors into tubes containing
heparin, and the non-erythrocytic cells were purified. The cells are washed
once in
HBH/HSA, counted, and re-suspended in HBH/HSA at a cell density of 2.0 x 106
per
ml. 100p1 cell suspension are added to wells of a V-bottom 96-well plate
containing
1001 diluted test sample or monoclonal antibody. Each test sample is tested at
a
range of dilutions with 6 wells for each dilution. Well contents are mixed
briefly using
a plate shaker, before incubation at 37°C for 30 minutes.
For each serum dilution 3 wells are triggered by addition of 10.1 Lol p I
extract (final
dilution 1/10000) and 3 wells have lOpl HBH/HSA added for assessment of
anaphylactogenicity. Well contents are again mixed briefly using a plate
shaker,
3o before incubation at 37°C for a further 30 minutes. Incubations are
terminated by
centrifugation at 500g for 5 min. Supernatants are removed for histamine assay
using
a commercially available histamine EIA measuring kit (Immunotech). Control
wells
28
SUBSTITUTE SHEET (RULE 26)
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containing cells without test sample are routinely included to determine
spontaneous
and triggered release. Samples of cells were lysed by 2X freeze/thawing to
assay total
histamine contained in the cells.
The results are expressed as following:
Anaphylactogenesis assay
Histamine release due to test samples =
histamine release from test sample treated cells - % spontaneous histamine
release.
to Blocking assay
The degree of inhibition of histamine release can be calculated using the
formula:
inhibition
= 1 -(histamine release from test sample treated cells*) x 100
(histamine release from antigen stimulated cells*)
Values corrected for spontaneous release.
Example 2, Immunisation of mice with Pl4 conjugates (Pl4-BSA, PI4 -BSA)
induces
production ojanti-human IgE antibodies.
The conjugates comprising the mimotope P14 (25ug protein/dose), described in
example 1, were administered into groups of 10 BalbC mice, adjuvanted with and
oil
in water emulsion containing QS21 and 3D-MPL described in WO 95/17210 .
Boosting was be performed on days 14, 24 and 72, sera was harvested 14 days
after
each immunisation.
The immune responses anti-peptide and anti-plate bound IgE was followed using
ELISA methods described in Example 1. The antiserum was then tested for
anaphylactogenicity and functional activity in the inhibition of histamine
release from
human allergic basophils (methods as described in example 1).
29
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Immunogenicity Results
Both conjugates, PD-P 14 and BSA-P 14, were capable of inducing anti-P 14 and
anti-
IgE immune responses. The results for anti peptide and anti-IgE responses,
induced by
the BSA-P14 conjugates, as measured at day 14 post third and fourth
vaccination, are
shown in table 6. PTmAb0011 is a monoclonal antibody which is known to bind to
the Cs2 domain of IgE, and was used to quantify the anti-IgE responses in
p.g/ml.
Table 6, Immunogenicity results for BSA-P14 conjugates
Anti-peptide Anti-IgE Anti-IgE
responses responses responses
(14 (14
(14 days days
days post post
post 3) 4)
3) (pg/ml (pg/ml
Mid
point (PTmAb0011)) (PTmAb0011))
titre
AV SD GM AV SD GM AV SD GM
25974 22667 15492 9.9 2.18 0.7 22.9 33.5 4.8
fable jootnotes: AV (average), SD (standard deviation), GM (geomean)
Mice vaccinated with BSA alone as controls did not generate any detectable
anti-
peptide or anti-IgE responses.
Functional activity results
The antiserum raised by the P 14 vaccination was found to be functional, in
that it was
potent in the inhibition of histamine release from allergic human basophils
after
triggering with allergen (see FIGS. 2, 3 and 4). Moreover, the antiserum was
not
found to be anaphylactogenic (FIGs. 2, 3 and 4).
Summary
P14 (mimotope of P8) was shown to be capable of raising high titres of anti-
P14 and
anti-IgE antibodies in mice. These antibodies were subsequently shown to be
functional, in that they inhibited histamine release from allergic human
basophils, and
were not anaphylactogenic. P14 and P8, therefore, may be used in the treatment
or
prophylaxis of allergy.
Example 3, Immunisation of mice with Pll conjugates (PII-BSA, Pll -BSA)
induces
production of anti-human IgE antibodies.
SUBSTITUTE SHEET (RULE 26)
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Human IgE epitope peptide P11 was coupled to maleimide-activated BSA (Pierce)
(BSA-CRASGKPVNHSTRKEEKQRNGLL). 25 ~g of conjugate formulated in
SBAS2 was injected IM into 8 female BALB/c mice at days 0, 14 and 28. One
control
group of mice was injected with BSA/SBAS2. Blood samples were taken 14 days
after each injection (a fourth bleeding was performed at day 24 post 3 to
increase the
availability of sera). Anti-peptide and anti-IgE antibodies raised by
vaccination were
measured by ELISA, as described in Example 1.
Results
A homogeneous IgG anti-P11 response could be detected already after one
injection,
but increased further after the second and third injection (FIG. Sa and 5b).
All mice
showed an anti-IgE response (ranging from 28 - 244 ~g/ml as expressed in
mAb005
equivalents) after a third injection (FIG. 6).
Part 2, Functional activity of epitope specific monoclonal antibodies
Example 4, Functional activity of monoclonal antibodies raised against P14
Monoclonal antibodies have been generated that recognise specifically P8 and
mimotopes thereof, using techniques known in the art. Briefly, the P14-BSA
conjugate described in part 1 of these examples, was injected into groups of
Balb/C
mice with the o/w adjuvant containing QS21 and 3D-MPL. Spleen cells were taken
and fused with SP2/O B-cell tumour cell line, and supernatants were screened
for
reactivity against both P14 peptide and IgE. Several cell lines were
generated,
amongst which were P 14/23, P 14/31 and P 14/33 which were deposited as
Budapest
Treaty patent deposit at ECACC on 26/1/00 under Accession No.s 00012610,
00012611, 00012612 respectively. All three monoclonal antibodies were
confirmed to
3o bind to IgE, and specifically to P 14, by ELISA binding assays, and P 14
competition
assays against monoclonal antibody binding to IgE.
31
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The functional activity of these monoclonal antibodies was assayed in the
human
basophil histamine release inhibition assay as described in Example 1.
Results
All of the P 14 monoclonal antibodies were tested on basophils taken from four
different allergic patients (A patients were allergic to dust mite antigen, G
patients
were allergic to grass pollen). PT11 (PTmAb0011) was included as a positive
control
antibody which is known to inhibit histamine release in vitro. All of the
three P14
l0 monoclonal antibodies (23, 31, and 33) were potent in inhibiting histamine
release
from allergic basophils (See FIG. 7).
Example 5, Anti-IgE induced in mice after immunisation with conjugate are
capable
of blocking local allergic response in the Monkey Cutaneous Anaphylaxis model.
P 14/23 and P 14/31 have also been tested for in vivo activity. Briefly, the
local skin
mast cells of African green monkeys were shaved and sensitised with
intradermal
administration of 100ng of anti-NP IgE (human IgE anti-nitrophenylacetyl (NP)
.
purchased from Serotech) into both arms. After 24 hours, a dose range of the
2o monoclonal antibodies to be tested were injected at the same injection site
as the
human IgE on one arm. Control sites on the opposite arm of the same animals
received either phosphate buffered saline (PBS) or non-specific human IgE
(specific
for Human Cytomegalovirus (CMV) or Human Immunodeficiency Virus (HN)).
After 5 hours, 10 mg of a BSA-NP conjugate (purchase from Biosearch
Laboratories)
was administered by intravenous injection. After 15-30 minutes, the control
animals
develop a readily observable roughly circular oedema from the anyphylaxis,
which is
measurable in millimeters. Results are expressed in either the mean oedema
diameter
of groups of three monkeys or as a percentage inhibition in comparison to PBS
controls. PTmAb0011, is a monoclonal antibody was used as a positive control.
3o SBmAb0006 was used as a negative control.
32
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Table 7, P 14/23 results
Amount of sampleMean diameter
to be tested of oedema
(,u~ (mm)
P14/23 mAb001 I mAb0006
20 0 ND 12/ 15
0 0 17/19
1 15/13 0 20/20
0.1 15/ 12 ND ND
0.05 15/ 15 ND ND
0 15/ 15 ND 17/ 17
1VL - lVUI 11U11G.
5 Table 8, P 14/31 results
Amount Mean diameter
of of oedema (mm)
sample
to
be tested
(f~~
P14/31 mAb0011 mAb0006
0 ND 15/ 15
10 0 0 15/15
1 22/25 0 20/20
0.1 22/25 ND ND
0.05 25/25 ND ND
0 20/25 ND 20/25
As complete inhibition of anaphylaxis was observed with higher doses of
monoclonal
antibody, these antibodies are not anaphylactogenic per se when administered
in vivo.
10 Example 6, Structural aspects of IgE mimotopes
The present inventors have shown that the conformation in which the epitopes
or
mimotopes of the present invention is important for both anti-mimotope
antibody
recognition, and also for the ability of the peptides to generate a strong
anti-IgE
immune responses. As such the present inventors have developed structural
rules
15 which predict the optimal sites for peptide cyclisation. Peptides that use
these sites of
cyclisation form one prefered aspect of the present invention.
As the full structure of IgE Fc has not been determined, the present inventors
have
refined the currently available models (Helm et al. supra, Padlan and Davis
supra)
33
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using the known structure of Cy2 and Cy3 of IgGI (Deisenhofer J.,
1981.Biochemistry, 20, 2361-2370). In addition, models of the CE2 domain have
been
built by comparison with known Ig folding-unit structures. The present
inventors
have designed these homology models of IgE Fc and thereby predicted the
termini
and the gross structure of infra-sheet (A-B loop, FIG 9A) and inter-sheet
loops in IgE
Fc domains (C-D loop, FIG 9B). Having defined the predicted IgE Fc A-B and C-D
loops together with their supporting (3-strands, mimotopes of the loops may be
derived
from the wild-type (WT) primary sequence of each loop by covalent cyclisation
between chosen specific residues along the adjoining ~i-strands. Cyclisation
is
1o preferably realised by the formation of a disulphide bond between terminal
cysteines
which therefore combine to become a cystine.
Based upon our structural alignments (FIG 9A & 9B) we have derived simple
predictive rules in order to enhance the probability that the conformations
adopted by
~5 a mimotope, after conjugation to a suitable carrier molecule, are similar
to those of the
parent epitope.
Rule 1
The hydrophobic cystine group should replace WT ~3-strand residues that belong
to
2o the water-inaccessible core of the Ig constant domain, formed by the
interface
between the two (3-sheets.
Rule 2i
For infra-sheet loops (e.g. the A-B loop) the cystine group should replace WT
25 residues that are from adjacent anti-parallel (3-strands (see FIG. 8) and
that pack
laterally together on the same side of the sheet. Following rule 1, this will
be on the
domain-interior side of the sheet. The structural derivation of this rule for
the A-B
loops is shown schematically in FIG 10 A and IOB.
3o Rule 2ii
For inter-sheet loops (e.g. the C-D loop) the cystine group should replace WT
residues on anti-parallel ~3-strands, one strand from each sheet. Following
rule 1, the
34
SUBSTITUTE SHEET (RULE 26)
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residues forming the optimal pair pack together from facing (3-sheet surfaces,
so
forning part of the interface between the sheets. The structural derivation of
this rule
for the C-D loops is shown schematically in FIG. 11 A and FIG. 11 B. In the
tables of
putative mimotope sequences that follow, designs predicted to be optimal are
underlined. Below each block of sequences the dotted and solid lines link the
residue
positions chosen for optimal cyclisation, which are also shown in the same way
in
FIG lOB (for A-B loops) and in FIG. 11B (for C-D loops).
Using the sequence alignment as shown in FIG 9A and 9B, together with the
above
to rules, the present inventors have designed the following peptides listed in
tables 9 to
12. The peptides which are underlined (in solid or dotted lines) are the
optimal
peptides according to the above identified rules, the same lines are shown in
FIG 1 OB
and FIG 11B. Non-underlined sequences are mimotopes.
Table 9 , IgE CE3 A-B loop sequences
Peptide SEQ ID NO.
sequence
(solid
and dotted
underlined
are optimal)
341 357
C S R P SP F D L F I R K S P T I T
C
33
C S R P SP F D L F I R K S P T I C 34
_C _S _R_P_S_PF D L F I R K S P T _C 35
C S R P SP F D L F I R K S P C 36
C R P SP F D L F I R K S P C
C R P SP F D L F I R K S P T C
3g
C R P SP F D L F I R K S P T I C
39
C R P SP F D L F I R K S P T I T
C
40
_CP SP F D L F I R K S P T I T
C
_ __ _ _ _ _ _ _ _ _ _ _ _ _ 41
C P SP F D L F I R K S P T I C
C P SP F D L F I R K S P T C 42
C P SP F D L F I R K S P C 43
44
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Table 1 D , IgE Ce4 A-B loop sequences
Peptide SEQ ID NO.
sequence
(solid
and dotted
underlined
are optimal)
446 463
C Y A F A T P E W P G S R D K R T LA C 45
C Y A F A T P E W P G S R D K R T LC 46
_C Y A F A T P E W P G S R D K R T C 4~
C Y A F A T P E W P G S R D K R C 4g
C A F A T P E W P G S R D K R C 49
C A F A T P E W P G S R D K R T C 50
C A F A T P E W P G S R D K R T LC 51
C A F A T P E W P G S R D K R T LA C 52
_C_F_A_T_P_E_W_P_G_S_R_D_K_R_T_LA _C 53
C F A T P E W P G S R D K R T LC 54
C F A T P E W P G S R D K R T C 55
C F A T P E W P G S R D K R C
56
36
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Table ll, IgE CE3 C-D loop sequences
Peptide SEQ ID NO.
sequence
(solid
and dotted
underlined
are optimal)
-
___
373 387
C T W S R A S G K P V N H S TR C 57
C T W S R A S G K P V N H S TC 5g
C T W S R A S G K P V N H S C 59
C T W S R A S G K P V N H C 60
C W S R A S G K P V N H C 61
C W S R A S G K P V N H S C 62
C W S R A S G K P V N H S TC 63
C W S R A S G K P V N H S TR C 64
C S R A S G K P V N H S TR C 65
C _S_RA__S_G_K_P_V_N_H_S_TC 66
C S R A S G K P V N H S C
67
C S R A S G K P V N H C
68
Table 12 , IgE Cs4 C-D loop mimotope sequences
Peptide SEQ ID NO.
sequence
(solid
and dotted
underlined
are optimal)
-
4 4 91
7 7
C Q W L HN E V Q L P D R S C 69
A H
C Q W L HN E V Q L P D R C 70
A H
C Q W L HN E V Q L P D R 71
A C
C Q W L HN E V Q _LP D C 72
A
C W L HN E V Q L P D C 73
A
C W L HN E V Q L P D R 74
A C
C W L HN E V Q L P D R C 75
A H
C W L HN E V Q L P D R S C 76
A H
C L HN E V Q L P D R S C 77
A H
_C_L _H_N_E_VQ _L_P_D _R C 7g
_A _H
C L HN E V Q L P D R
A C
79
C L HN E Q L P D C
V A
37
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IYDICATIO:YS RELATI:'1G TO DEPOSITED t1-tICROORGA:VISVt
OR OTHER BIOLOGICAL :yIATERIAL
(PCT Rule I ibis)
A. ihc indic~tions made below reiate to the depa~ued mtcroor~antsm or other
btolog:cal mate::al ,~te:red ;o w the dcsc::peon
on pace 1 R
:line 7-13
B. IDEVTIFICATION OF DEPOSIT Further deposits ue ideatitied on an additional
sheer
~amc of dc;,ositary institution
European Collection of Cell Cultures
address of dcposituy institution (including postal code errd couan-r~
vaccine Research and Production Laboratory
Public Health Laboratory Service
Centre for Applied Microbiology Research
Porton Down, Salisbury
Wiltshire SP4 OJG, GB
Date of deposit ~ Accession i'lumber 00012610, 00012611
26 January 2000
(26/01/00) and 00012612
C. ADDfTIOi (,aL fYDICATIONS (leave blank ijnor applicable This inlottnation
is continued on an additional shit Q
In respect of those designations where a European Patent is sought, a sample
of the deposited microorganism will be made available until the publication
of the mention of the grant of the European Patent or until the date on which
the application has been refused or withdrawn, only by issue of such a sample
to an expert nominated by the person requesting the sample.
D. DESICi'1ATED STATES FOR WHICH INDICATIONS ARE D2iDE (ijrhe irtdicatiorts
ore nor jor all designated Srctes~
E. SEP.iR.~ITE FURi'lISHL'!C OF INDICATIONS (leave blank ijnor applicable)
The indications listed below will be submitted to the lntemational Bureau
lacer Ispecifvchegenercl ncrueojtheindicertorere.g.. ;~ccrssion
r~umbv ojDeaosir'~
For receiving Office use only For lncernational Bure_u use only
This sheet was received with the inte~ational application ~ This sheet wu
received by the lnte:national Burau on:
Authorized officer
Form pCTIR()/ I 3.t t Julv I 99Q1
Authorized office-.
38
SUBSTfTUTE SHEET (RULE 26)
CA 02363641 2001-08-23
FOR THE PURPOSES OF INFORMATION ONLY
Codes used to identify States party to the PCT on the front pages of pamphlets
publishing international applications under the PCT.
AL Albania ES Spain LS Lesotho SI Slovenia
AM Armenia FI Finland LT Lithuania SK Slovakia
AT Austria FR France LU Luxembourg SN Senegal
AU Australia GA Gabon LV Latvia SZ Swaziland
AZ Azerbaijan GB United KingdomMC Monaco TD Chad
BA Bosnia and GE Georgia MD Republic of TG Togo
Herzegovina Moldova
BB Barbados GH Ghana MG Madagascar TJ Tajikistan
BE Belgium GN Guinea MK The former TM Turkmenistan
Yugoslav
BF Burkina Faso GR Greece Republic of TR Turkey
Macedonia
BG Bulgaria HU Hungary ML Mali TT Trinidad
and Tobago
BJ Benin IE Treland MN Mongolia UA Ukraine
BR Brazil IL Israel MR Mauritania UG Uganda
BY Belarus IS Tceland MW Malawi US United States
of America
CA Canada IT Ttaly MX Mexico UZ Uzbekistan
CF Central African
Re ublic n NE Niger VN Viet Nam
p
CG Con o KE Ken NL Netherlands YU Yugoslavia
g a
Y
CH Switzerland KG Kyrgyzstan NO Norway ZW Zimbabwe
CI C&te d'TvoireKP Democratic NZ New Zealand
People's
CM Cameroon Republic PL Poland
of Korea
CN China KR Republic PT Portugal
of Korea
CU Cuba KZ Kazakstan RO Romania
-
CZ Czech RepublicLC Saint Lucia RU Russian Federation
DE Germany LI LiechtensteinSD Sudan
DK Denmark LK Sri Lanka SE Sweden
EE Estonia LR Liberia SG Singapore
CA 02363641 2001-08-23
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SEQUENCE LISTING
<110> SmithKline Beecham Biolicals s.a.
Peptide Therapeutics Ltd
<120> Vaccine
<130> B45173
<160> 86
<170> FastSEQ for Windows Version 3.0
<210> 1
<211> 22
<212> PRT
<213> Human and artificial sequence
<400> 1
Arg Ala Ser Gly Lys Pro Val Asn His Ser Thr Arg Lys Glu Glu Lys
1 5 ~ 10 15
Gln Arg Asn Gly Thr Leu
2 0
<210> 2
<211> 9
<212> PRT
<213> Human and artificial sequence
<400> 2
Gly Thr Arg Asp Trp Ile Glu Gly Glu
1 5
<210> 3
<211> 19
<212> PRT
<213> Human and artificial sequence
<400> 3
Pio His Leu Pro Asg Ala Leu Met Arg Ser Thr Thr Lys Thr Ser Gly
10 15
Pro Arg Ala
<210> 4
<211> 11
<212> PRT
<213> Human and artificial sequence
<400> 4
Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg Thr
1 5 10
<210> 5
<211> 5
<212> PRT
<213> Human and artificial sequence
<400> 5
Glu Gln Lys Asp Glu
1 5
<210> 6
1
CA 02363641 2001-08-23
WO PCT/EP00/01456
00/50461
<211> 19
<212> PRT
<213> Human and artificial sequence
<400> 6
Leu Ser Pro Ser Pro Phe Asp Leu Arg Ser Thr
Arg Phe Ile Lys Pro
1 5 10 15
Ile Thr
Cys
<210> 7
<211> 21
<212> PRT
<213> Human and artificial sequence
<400> 7
Trp Leu Asn Glu Val Gln Leu Pro Arg Ser Thr
His Asp Ala His Thr
1 5 10 15
Gln Pro Lys Thr
Arg
2 0
<210> 8
<211> 23
<212> PRT
<213> Human and artificial sequence
<400> 8
Cys Arg Ser Gly Lys Pro Val Asn Thr Lys Glu
Ala His Ser Arg Glu
1 5 10 15
Lys Gln Asn Gly Leu Leu
Arg
20
<210> 9
<211> 11
<212> PRT
<213> Human and artificial sequence
<400> 9
Gly Lys Val Asn His Ser Thr Gly
Pro Gly Cys
1 5 10
<210> 10
<211> 18
<212> PRT
<213> Human and artificial sequence
<400> 10
Gly Lys Val Asn His Ser Thr Arg Glu Gln Asn
Pro Lys Glu Lys Arg
1 5 10 15
Gly Cys
<210> 11
<211> 20
<212> PRT
<213> Human and artificial sequence
<400> 11
Cys Gly Pro Val Asn His Ser Thr Glu Lys Arg
Lys Arg Lys Glu G1n
1 5 ~ 10 15
Asn Gly Leu
Leu
20
<210> 12
<211> 14
<212> PRT
2
CA 02363641 2001-08-23
WO PCT/EP00/01456
00/50461
<213> Human and artificial sequence
<400> 12
Arg Ala Gly Lys Pro Val Asn His Gly Gly Cys
Ser Ser Thr
1 5 10
<210> 13
<211> 11
<212> PRT
<213> Human and artificial sequence
<400> 13
Cys Gly Arg Asp Trp Ile Glu Gly
Thr Leu Leu
1 5 10
<210> 14
<211> 12
<212> PRT
<213> Human and artificial sequence
'
<400> 14
Cys Gly Arg Asp Trp Ile Glu Gly Leu
Thr Glu Thr
1 5 10
<210> 15
<211> 12
<212> PRT
<213> Human and artificial sequence
<400> 15
Gly Thr Asp Trp Ile Glu Gly Glu Cys
Arg Thr Gly
1 5 10
<210> 16
<211> 12
<212> PRT
<213> Human and artificial sequence
<400> 16
Cys His His Leu Pro Arg Ala Leu Leu
Pro Met Leu
1 5 10
<210> 17
<211> 12
<212> PRT
<213> Human and artificial sequence
<400> 17
Cys Gly His Pro His Leu Pro Arg Met
Thr Ala Leu
SO 1 5 10
<210> 18
<211> 13
<212> PRT
<213> Human and artificial sequence
<400> 18
Thr His His Leu Pro Arg Ala Leu Ser Cys
Pro Met Arg
1 5 10
<210> 19
<211> 14
<212> PRT
<213> Human and artificial sequence
<400> 19
3
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Gly Pro His Leu Pro Arg Ala Leu Met Arg Ser Ser Ser Cys
1 5 10
<210> 20
<211> 13
<212> PRT
<213> Human and artificial sequence
<400> 20
Ala Pro Trp Pro Gly Ser Arg Asp Thr
Glu Lys Arg Cys
1 5 10
<210> 21
<211> 17
<212> PRT
<213> Human and artificial sequence
<400> 21
Ala Pro Trp Pro Gly Ser Arg Asp Thr Ala Gly
Glu Lys Arg Leu Gly
1 5 10 15
Cys
<210> 22
<211> 17
<212> PRT
<213> Human and artificial sequence
<400> 22
Cys Gly Ala Thr Pro Glu Trp Pro Arg Lys Arg
Gly Gly Ser Asp Thr
1 5 ' 10 15
Leu
<210> 23
<211> 13
<212> PRT
<213> Human and artificial sequence
<400> 23
Cys Thr Lys Asp Arg Ser Gly Pro Pro
Arg Trp Glu Ala
1 5 10
<210> 24
<211> 15
<212> PRT
<213> Human and artificial sequence
<400> 24
Ala Pro Trp Pro Gly Ser Arg Asp Thr Ala Gly
Cys Cys Arg Leu
1 5 10 15
<210> 25
<211> 16
<212> PRT
<213> Human and artificial sequence
<400> 25
Ala Cys Glu Trp Pro Gly Ser Arg Cys Leu Ala
Pro Asp Arg Thr Gly
1 5 10 15
<210> 26
<211> 17
<212> PRT
<213> Human and artificial sequence
4
CA 02363641 2001-08-23
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<400> 26
Cys Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg Thr Leu Cys
1 5 10 15
Gly
<210> 27
<211> 16
<212> PRT
<213> Human and artificialsequence
<400> 27
Cys Ala Pro Glu Trp Pro Ser Arg Asp Lys Arg Thr
Thr Gly Cys Gly
1 5 10 15
<210> 28
<211> 13
<212> PRT
<213> Human and artificialsequence
<400> 28
Thr Pro Trp Pro Gly Ser Asp Lys Arg Cys Gly
Cys Arg
1 5 10
<210> 29
<211> 11
<212> PRT
<213> Human and artificialsequence
<400> 29
Cys Gly Glu Trp Glu Gln Asp Glu Leu
Ala Lys
1 5 10
<210> 30
<211> 11
<212> PRT
<213> Human and artificialsequence
<400> 30
Ala Glu Glu Gln Lys Asp Phe Ile Cys
Trp Glu
1 5 10
<210> 31
<211> 9
<212> PRT
<213> Human and artificialsequence
<400> 31
Gly Glu Lys Asp Glu Phe Cys
Gln Ile
1 5
<210> 32
<211> 10
<212> PRT
<213> Human and artificialsequence
<400> 32
Cys Ala Gly Glu Gln Lys Glu Leu
Glu Asp
1 5 10
-
<210> 33
<211> 19
<212> PRT
<213> Human and artificialsequence
<400> 33
5
CA 02363641 2001-08-23
WO 00/50461 PCT/EP00/01456
Cys Ser Arg Pro Ser Pro Phe Asp Leu Phe Ile Arg Lys Ser Pro Thr
1 5 10 15
Ile Thr Cys
<210> 34
<211> 18
<212> PRT
<213> Human and artificial sequence
<400> 34
Cys Ser Pro Ser Pro Phe Asp Leu Arg Lys Ser Thr
Arg Phe Ile Pro
1 5 10 15
Ile Cys
<210> 35
<211> 17
<212> PRT
<213> Human and artificial sequence
<400> 35
Cys Ser Pro Ser Pro Phe Asp Leu Arg Lys Ser Thr
Arg Phe Ile Pro
1 5 10 15
Cys
<210> 36
<211> 16
<212> PRT
<213> Human and artificial sequence
<400> 36
Cys Ser Pro Ser Pro Phe Rsp Leu Arg Lys Ser Cys
Arg Phe Ile Pro
1 5 10 15
<210> 37
<211> 15
<212> PRT
<213> Human and artificial sequence
<400> 37
Cys Arg Ser Pro Phe Asp Leu Phe Lys Ser Pro
Pro Ile Arg Cys
1 5 10 15
<210> 38
<211> 16
<212> PRT
<213> Human and artificial sequence
<400> 38
Cys Arg Ser Pro Phe Asp Leu Phe Lys Ser Pro Cys
Pro Ile Arg Thr
1 5 10 15
<210> 39
<211> 17
<212> PRT
<213> Human and artificial sequence
<400> 39
Cys Arg Ser Pro Phe Asp Leu Phe Lys Ser Pro Ile
Pro Ile Arg Thr
1 5 10 15
Cys
<210> 40
6
CA 02363641 2001-08-23
WO PCT/EP00/01456
00/50461
<211> 18
<212> PRT
<213> Human and artificial sequence
<400> 40
Cys Arg Ser Pro Phe Asp Leu Phe Lys Ser Thr Ile
Pro Ile Arg Pro
1 5 10 15
Thr Cys
<210> 41
<211> 17
<212> PRT
<213> Human and artificial sequence
<400> 41
Cys Pro Pro Phe Asp Leu Phe Ile Ser Pro Ile Thr
Ser Arg Lys Thr
1 5 10 15
Cys
<210> 42
<211> 16
<212> PRT
<213> Human and artificial sequence
<400> 42
Cys Pro Pro Phe Asp Leu Phe Ile Ser Pro Ile Cys
Ser Arg Lys Thr
1 5 10 15
<210> 43
<211> 15
<212> PRT
<213> Human and artificial sequence
<400> 43
Cys Pro Pro Phe Asp Leu Phe Ile Ser Pro Cys
Ser Arg Lys Thr
1 5 10 15
<210> 44
<211> 14
<212> PRT
<213> Human and artificial sequence
<400> 44
Cys Pro Pro Phe Asp Leu Phe Ile Ser Pro
Ser Arg Lys Cys
1 5 10
<210> 45
<211> 20
<212> PRT
<213> Human and artificial sequence
<400> 45
Cys Tyr Phe Ala Thr Pro Glu Trp Ser Arg Lys Arg
Ala Pro Gly Asp
1 5 10 15
Thr Leu Cys
Ala
20
<210> 46
<211> 19
<212> PRT
<213> Human and artificial sequence
<400> 46
Cys Tyr Phe Ala Thr Pro Glu Trp Ser Arg Lys Arg
Ala Pro Gly Asp
CA 02363641 2001-08-23
WO 00/50461 PCT/EP00/01456
1 5 10 15
Thr Leu Cys
<210> 47
<211> 18
<212> PRT
<213> Human and artificial sequence
<400> 47
Cys Tyr Ala Phe Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg
1 5 10 15
Thr Cys
<210> 48
<211> 17
<212> PRT
<213> Human and artificial sequence
<400> 48
Cys Tyr Phe Ala Thr Pro Glu Trp Ser Arg LysArg
Ala Pro Gly Asp
1 5 10 15
Cys
'
<210> 49
<211> 16
<212> PRT
<213> Human and artificial sequence
<400> 49
Cys Ala Ala Thr Pro Glu Trp Pro Arg Asp ArgCys
Phe Gly Ser Lys
1 5 10 15
<210> 50
<211> 17
<212> PRT
<213> Human and artificial sequence
<400> 50
Cys Ala Ala Thr Pro Glu Trp Pro Arg Asp ArgThr
Phe Gly Ser Lys
1 5 10 15
Cys
<210> 51
<211> 18
<212> PRT
<213> Human and artificial sequence
<400> 51
Cys Ala Ala Thr Pro Glu Trp Pro Arg Asp ArgThr
Phe Gly Ser Lys
1 5 10 15
Leu Cys
<210> 52
<211> 19
<212> PRT
<213> Human and artificial sequence
<400> 52
Cys Ala Ala Thr Pro Glu Trp Pro Arg Asp ArgThr
Phe Gly Ser Lys
1 5 10 15
Leu Ala
Cys
g
CA 02363641 2001-08-23
WO 00/50461 PCT/EP00/01456
<210> 53
<211> 18
<212> PRT
<213> Human and artificial sequence
<400> 53
Cys Phe Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg Thr Leu
1 5 ~ 10 15
Ala Cys
<210> 54
<211> 17
<212> PRT
<213> Human and artificial sequence
<400> 54
Cys Phe Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg Thr Leu
1 5 10 15
Cys
<210> 55
<211> 16
<212> PRT
<213> Human and artificial sequence
<400> 55
Cys Phe Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg Thr Cys
1 5 10 15
<210> 56
<211> 15
<212> PRT
<213> Human and artificial sequence
<400> 56
Cys Phe Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg Cys
1 5 10 15
<210> 57
<211> 17
<212> PRT
<213> Human and artificial sequence
<400> 57
Cys Thr Trp Ser Arg Ala Ser Gly Lys Pro Val Asn His Ser Thr Arg
1 5 10 15
Cys
<210> 58
<211> 16
<212> PRT
<213> Human and artificial sequence
<400> 58
Cys Thr Trp Ser Arg Ala Ser Gly Lys Pro Val Asn His Ser Thr Cys
1 5 - 10 15
<210> 59
<211> 15
<212> PRT
<213> Human and artificial sequence
9
CA 02363641 2001-08-23
WO 00/50461 PCT/EP00/01456
<400> 59
Cys Thr Trp Ser Arg Ala Ser Gly Lys Pro Val Asn His Ser Cys
1 5 10 15
<210> 60
<211> 14
<212> PRT
<213> Human and artificial sequence
<400> 60
Cys Thr Ser Arg Ala Ser Gly Lys Asn His Cys
Trp Pro Val
1 5 10
IS <210> 61
<211> 13
<212> PRT
<213> Human and artificial sequence
<400> 61
Cys Trp Arg Ala Ser Gly Lys Pr0 His Cys
Ser Val Asn
1 5 10
<210> 62
<211> 14
<212> PRT
<213> Human and artificial sequence
<400> 62
Cys Trp Arg Ala Ser Gly Lys Pro His Ser Cys
Ser Val Asn
1 5 10
<210> 63
<211> 15
<212> PRT
<213> Human and artificial sequence
<400> 63
Cys Trp Arg Ala Ser Gly Lys Pro His Ser Thr Cys
Ser Val Asn
1 5 10 15
<210> 64
<211> 16
<212> PRT
<213> Human and artificial sequence
<400> 64
Cys Trp Arg Ala Ser Gly Lys Pro His Ser Thr Arg
Ser Val Asn Cys
1 5 10 15
<210> 65
<211> 15
<212> PRT
<213> Human and artificial sequence
<400> 65
Cys Ser Ala Ser Gly Lys Pro Val Ser Thr Arg Cys
Arg Asn His
1 5 10 15
<210> 66
<211> 14
<212> PRT
<213> Human and artificial sequence
<400> 66
Cys Ser Ala Ser Gly Lys Pro Val Ser Thr Cys
Arg Asn His
CA 02363641 2001-08-23
WO PCT/EP00/01456
00/50461
1 5 10
<210> 67
<211> 13
<212> PRT
<213> Human and artificial sequence
<400> 67
Cys Ser Ala Ser Gly Lys Pro Val Ser
Arg Asn His Cys
1 5 10
<210> 68
<211> 12
<212> PRT
<213> Human and artificial sequence
<400> 68
Cys Ser Ala Ser Gly Lys Pro Val Cys
Arg Asn His
1 5 10
<210> 69
<211> 17
<212> PRT
<213> Human and artificial sequence
<400> 69
Cys Gln Leu His Asn Glu Val Gln Asp Ala Arg His Ser
Trp Leu Pro
1 5 10 15
Cys
<210> 70 '
<211> 16
<212> PRT
<213> Human and artificial sequence
<400> 70
Cys Gln Leu His Asn Glu Val Gln Asp Ala Arg His Cys
Trp Leu Pro
1 5 10 15
<210> 71
<211> 15
<212> PRT
<213> Human and artificial sequence
<400> 71
Cys Gln Leu His Asn Glu Val Gln Asp Ala Arg Cys
Trp Leu Pro
1 5 10 15
<210> 72
<211> 14
<212> PRT
<213> Human and artificial sequence
<400> 72
Cys Gln Leu His Asn Glu Val Gln Asp Ala Cys
Trp Leu Pro
1 5 10
<210> 73
<211> 13
<212> PRT
<213> Human and artificial sequence
<400> 73
Cys Trp His Asn Glu Val Gln Leu Ala Cys
Leu Pro Asp
1 5 10
11
CA 02363641 2001-08-23
WO PCT/EP00/01456
00/50461
<210> 74
<211> 14
<212> PRT
<213> Human and artificial sequence
<400> 74
Cys Trp His Asn Glu Val Gln Leu AlaArg Cys
Leu Pro Asp
1 5 10
<210> 75
<211> 15
<212> PRT
<213> Human and artificial sequence
<400> 75
Cys Trp His Asn Glu Val Gln Leu AlaArg His Cys
Leu Pro Asp
1 5 10 15
<210> 76
<211> 16
<212> PRT
<213> Human and artificial sequence
<400> 76
Cys Trp His Asn Glu Val Gln Leu AlaArg His Ser Cys
Leu Pro Asp
1 5 10 15
<210> 77
<211> 15
<212> PRT
<213> Human and artificial sequence
<400> 77
Cys Leu Asn Glu Val Gln Leu Pro ArgHis Ser Cys
His Asp Ala
1 5 10 15
<210> 78
<211> 14
<212> PRT
<213> Human and artificial sequence
<400> 78
Cys Leu Asn Glu Val Gln Leu Pro ArgHis Cys
His Asp Ala
1 5 10
<210> 79
<211> 13
<212> PRT
<213> Human and artificial sequence
<400> 79
Cys Leu Asn Glu Val Gln Leu Pro ArgCys
His Asp Ala
1 5 10
<210> 80
<211> 12
<212> PRT
<213> Human and artificial sequence
<400> 80
Cys Leu Asn Glu Val Gln Leu Pro Cys
His Asp Ala
1 5 10
<210> 81
<211> 6
12
CA 02363641 2001-08-23
WO 00/50461PCT/EP00/01456
<212> PRT
<213> Human and artificial sequence
<400> 81
Leu Phe Arg Lys Ser
Ile
1 5
<210> 82
<211> 7
<212> PRT
<213> Human and artificial sequence
<400> 82
Pro Ser Gly Thr Val Asn
Lys
1 5
<210> 83
<211> 23
<212> PRT
<213> Human and artificial sequence
<400> 83
Leu His Glu Val Gln Leu Pro Asp Ala Arg His Ser Thr Thr
Asn Gln
1 5 10 15
Pro Arg Thr Lys Gly Ser
Lys
20
<210> 84
<211> 6
<212> PRT
<213> Human and artificial sequence
<400> 84
Ser Val Pro Gly Lys
Asn
1 5
<210> 85
<211> 13
<212> PRT
<213> Human and artificial sequence
<400> 85
Cys Pro Trp Pro Gly Cys Arg Asp Lys Arg Thr Gly
Glu
1 5 10
<210> 86
<211> 13
<212> PRT
<213> Human and artificial sequence
<400> B6
Thr Pro Trp Pro Gly Cys Arg Asp Lys Arg Cys Gly
Glu
1 5 10
13
