PEPTIDES FOR VACCINE AGAINST BIRCH ALLERGY
Field of the Invention
The present invention relates to compositions for preventing or treating allergy
to birch.
Background of the Invention
T-cell antigen recognition requires antigen presenting cells (APCs) to present
antigen fragments (peptides) on their cell surface in association with molecules of the
major histocompatibility complex (MHC). T cells use their antigen specific T-cell
receptors (TCRs) to recognise the antigen fragments presented by the APC. Such
recognition acts as a trigger to the immune system to generate a range of responses to
eradicate the antigen which has been recognised.
Recognition of external antigens by the immune system of an organism,
such as man, can in some cases result in diseases, known as atopic
conditions. Examples of the latter are the allergic diseases including asthma, atopic
dermatitis and allergic rhinitis. In this group of diseases, B lymphocytes generate
antibodies of the IgE class (in humans) which bind externally derived antigens, which
are referred to in this context as allergens since these molecules elicit an allergic
response. Production of allergen-specific IgE is dependent upon T lymphocytes which
are also activated by (are specific for) the allergen. Allergen-specific IgE antibodies
bind to the surface of cells such as basophils and mast cells by virtue of the expression
by these cells of surface receptors for IgE.
Crosslinking of surface bound IgE molecules by allergen results in
degranulation of these effector cells causing release of inflammatory mediators such as
histamine, 5-hydroxtryptamine and lipid mediators such as the sulphidoleukotrienes. In
addition to IgE-dependent events, certain allergic diseases such as asthma are
characterised by IgE-independent events.
Allergic IgE-mediated diseases are currently treated with agents which provide
symptomatic relief or prevention. Examples of such agents are anti-histamines, b2
agonists, and glucocorticosteroids. In addition, some IgE-mediated diseases are treated
by desensitisation procedures that involve the periodic injection of allergen
components or extracts. Desensitisation treatments may induce an IgG response that
competes with IgE for allergen, or they may induce specific suppressor T cells that
block the synthesis of IgE directed against allergen. This form of treatment is not
always effective and poses the risk of provoking serious side effects, particularly
general anaphylactic shock. This can be fatal unless recognised immediately and
treated with adrenaline. A therapeutic treatment that would decrease or eliminate the
unwanted allergic-immune response to a particular allergen, without altering the
immune reactivity to other foreign antigens or triggering an allergic response itself
would be of great benefit to allergic individuals.
Pollen allergens are recognised as a major cause of allergic diseases in humans
and animals, including asthma, allergic rhinitis and allergic dermatitis. At least 10% of
the population of the USA suffers from pollen allergies at various times and to varying
extents. Proteins present in tree pollen, in particular from trees of the order Fagales,
for example birch, alder, hazel, hornbeam and oak, are particularly important. Of these
species, birch pollen allergens are the most frequent initiators of allergic responses to
tree pollen (Jarolim et al: Allergy 1989; 44(6):385-95). For example, approximately
25% of hayfever sufferers are responsive to birch pollen. Hayfever is the common term
for a form of seasonal allergy characterised by sneezing, runny nose and itching eyes.
Allergy to tree pollen is most problematic during the spring months, with the birch
pollen season typically occurring around April (in the northern hemisphere). However,
some related types of tree such as alder and hazel can release airborne pollen as early
as January (northern hemisphere). These are followed by elm, willow and ash in
March, with oak in late April and early May.
It has been calculated that for adults in the United States, hayfever is the 5th
leading chronic disease and a major cause of work absenteeism, resulting in nearly 4
million missed or lost workdays each year, resulting in a total cost of more than $700
million in total lost productivity. Allergies are also the most frequently reported
chronic condition in children, limiting activities for more than 40% of them. Each
year, allergies account for more than 17 million outpatient office visits in the United
States; seasonal allergies such as hayfever account for more than half of these allergy
visits.
A therapeutic or preventative treatment would therefore be of great benefit to
humans that suffer or are at risk of suffering from tree allergy.
Summary of the Invention
The present inventors have discovered that certain peptide fragments derived
from the major allergens in the pollens of birch species are useful in desensitising
individuals to these allergens. Peptide fragments derived from Bet v2, Bet vl, Bet v3,
Bet v4, Bet v6 and Bet v7 of birch (family name: Betulucaea) are particularly useful.
The peptides of the invention were selected as MHC class P-binding T cell
epitopes through use of in silico analysis to predict peptide-MHC interactions and
MHC class Pbinding assays. Additional epitopes were identified by homology.
A difficulty associated with approaches to desensitisation based on peptide
immunisation lies in how to select an appropriate size and region of the allergen as the
basis for the peptide to be used for immunisation. The size of the peptide of choice is
crucial. If the peptide is too small, the vaccine would not be effective in inducing an
immunological response. If the peptides are too large, or if the whole antigen is
introduced into an individual, there is the risk of inducing adverse reactions, such as
anaphylaxis, which may be fatal.
The polypeptides of the invention have been selected to retain T cell specificity
whilst being small enough in size to not possess significant tertiary structure that would
enable them to retain the conformation of an IgE-binding epitope of the whole
molecule. The polypeptides of the invention therefore do not induce significant
crosslinking of adjacent specific IgE molecules on cells such as mast cells and
basophils and consequently do not cause significant histamine release.
An advantage of the invention is the ability of the peptides to broadly target
Major Histocompatibility Complex (MHC) molecules. T cell receptors (TCRs) are
highly variable in their specificity. Variability is generated, as with antibody molecules,
through gene recombination events within the cell. TCRs recognise antigen in the form
of short peptides bound to molecules encoded by the genes of the Major
Histocompatibility Complex (MHC). These gene products are the same molecules that
give rise to "tissue types" used in transplantation and are also referred to as Human
Leukocyte Antigen molecules (HLAs) which terms may be used interchangeably.
Individual MHC molecules possess peptide binding grooves which, due to their shape
and charge are only capable of binding a limited group of peptides. The peptides bound
by one MHC molecule may not necessarily be bound by other MHC molecules.
When a protein molecule such as an antigen or allergen is taken up by antigen
presenting cells such as B lymphocytes, dendritic cells, monocytes and macrophages,
the molecule is enzymatically degraded within the cell. The process of degradation
gives rise to peptide fragments of the molecule which, if they are of the appropriate
size, charge and shape, may then bind within the peptide binding groove of certain
MHC molecules and be subsequently displayed upon the surface of antigen presenting
cells. If the peptide/MHC complexes are present upon the antigen presenting cell
surface in sufficient numbers they may then activate T cells which bear the appropriate
peptide/MHC-specific T cell receptors.
Due to the polymorphic nature of the MHC, individuals in an outbred
population such as man will express different combinations of MHC molecules on
their cell surfaces. Since different MHC molecules can bind different peptides from the
same molecule based on the size, charge and shape of the peptide, different individuals
will display a different repertoire of peptides bound to their MHC molecules.
Identification of universal MHC-binding peptide epitopes in an outbred population
such as man is more difficult than in inbred animals (such as certain strains of
laboratory mice). On the basis of differential MHC expression between individuals and
the inherent differences in peptide binding and presentation which this brings, it is
unlikely that a single peptide can be identified which will be of use for desensitisation
therapy in man.
The peptides of the invention, however, provide a broad coverage of efficacy
over the human population by targeting multiple different MHC molecules. A vaccine
formulated with a peptide of the invention would therefore have broad utility.
Accordingly, the present invention provides a composition suitable for use in
preventing or treating allergy to birch pollen by tolerisation comprising:
i) at least one of the polypeptides of SEQ ID NO: 74 (BIR12B;
AKYMVIQGEPGRVIRGK), SEQ ID NO: 72 (BIRl 1; FPQFKPQEITGIMK), SEQ ID
NO: 7 1 (BIR10; GSVWAQSSSFPQFK), SEQ ID NO: 73 (BIR12A;
PTGMFVAGAKYMVIQGR), SEQ ID NO: 75 (BIRl 3; IKYMVIQGEAGAVIRGK
and SEQ ID NO: 76 (BIRl ; EAGAVIRGKKGSGGIT), or a variant of any thereof,
and
ii) at least one of the polypeptides of SEQ ED NO: 53 (Bir02J;
PAARMFKAFILEGDKLVPK), SEQ ED NO: 48 (Bi I; FNYETETTSVIPAARK),
SEQ ED NO: 54 (Bir04; PGTEKKISFPEGFPFKYV), SEQ ED NO: 67 (Bir09;
ETLLRAVESYLLAHSDAY), SEQ ED NO: 60 (BER07; SNErKTVATPDGGSELK),
and SEQ ID NO: 63 (Bir07C; SNEDOVATPEGGSILK), or a variant of any thereof,
wherein said variant is:
I) a longer polypeptide of up to 30 amino acids in length which comprises
the sequence of the corresponding polypeptide specified in (i) or (ii), or
II) a polypeptide of 9 to 30 arnino acids in length which comprises a
sequence that has at least 65% homology to the sequence of the corresponding
polypeptide specified in (i) or (ii), which sequence is capable of tolerising to
said corresponding polypeptide; or
PI) a polypeptide of length 9 to 30 amino acids which comprises a
sequence of, or a sequence that has at least 65% homology to, at least 9
contiguous amino acids of the sequence of the corresponding polypeptide
specified in (i) or (ii), which sequence of at least 9 contiguous amino acids or
homologous sequence is capable of tolerising to said corresponding
polypeptide.
Also provided is a composition suitable for use in preventing or treating allergy to
birch pollen by tolerisation comprising at least three different polypeptides, selected
from:
(a) Bir 1 B (AKYMVIQGEPGRVERGK), or a variant thereof;
(b) Bir02J (PAARMFKAFILEGDKLVPK), or a variant thereof;
(c) BirO 11(FNYETETTSVIPAARK) or a variant thereof;
(d) Bir04 (PGTEKKISFPEGFPFKYV) or a variant thereof;
(e) Bir09 (ETLLRAVESYLLAHSDAY) or a variant thereof;
(f) Birl6A (AEREREFKRFDANGEGK) or a variant thereof;
(g) Bir07 (SNEEKTVATPDGGSILK) or a variant thereof;
(h) Bir07C (SNEEKEVATPEGGSILK) or a variant thereof;
(i) BirO 11 (FPQFKPQEITGIMK) or a variant thereof;
0' ) Bir 5 (SLNTLRLRREFDLFDK) or a variant thereof;
wherein said variant is:
I) a longer polypeptide of up to 30 amino acids in length which comprises
the sequence of the corresponding polypeptide specified in (a) to (j), or
P) a polypeptide of 9 to 30 amino acids in length which comprises a
sequence that has at least 65% homology to the sequence of the corresponding
polypeptide specified in (a) to (j), which sequence is capable of tolerising to
said corresponding polypeptide; or
PG) a polypeptide of length 9 to 30 amino acids which comprises a
sequence of, or a sequence that has at least 65% homology to, at least 9
contiguous amino acids of the sequence of the coiresponding polypeptide
specified in (a) to (j), which sequence of at least 9 contiguous amino acids or
homologous sequence is capable of tolerising to said corresponding
polypeptide.
Description of the sequences mentioned herein
SEQ ID NOS: 1 to 80 provide the polypeptide sequences of the invention as set
out in Tables 1 to 8. SEQ ID NOS: 1 to 34 and 45 to 70 correspond to peptides
derived from Bet vl. SEQ ID NOS: 7 1 to 76 correspond to peptides derived from Bet
v2. SEQ ID NOS: 35, 36 and 77 correspond to peptides derived from Bet v3. SEQ ID
NOS: 37 to 39, 78 and 79 correspond to peptides derived from Bet v4. SEQ ID NOS:
40 to 43 and 80 correspond to peptides derived from Bet v6. SEQ ID NO: 44
corresponds to a peptide derived from Bet v7.
Detailed description of the invention
The invention concerns peptides which can be used in tolerisation. Such
peptides may comprise, consist of, or consist essentially of the sequences shown in any
of SEQ D NOS: 1 to 80. Variants of these specific peptides may also be used. The
variants may comprise, consist of, or consist essentially of sequences which are
fragments of either any of SEQ ID NOS: 1 to 80 or homologues of any of SEQ ID
NOS: 1 to 80.
The invention also provides products and formulations comprising the
polypeptides of the invention and compositions, products and vectors comprising
polynucleotides capable of expressing the polypeptides of the invention for use in
preventing or treating birch allergy by tolerisation. Such tolerisation will typically be
to an epitope (for example a MHC class P-binding T cell epitope) present in any of
SEQ ID NOS: 1 to 80.
Tree species
Species of tree from the family Betulaceae, commonly known as birch, are
responsible for a high proportion of tree allergy worldwide, particularly allergies
associated with tree pollen, such as hayfever. Other important tree species include
alder, hazel, hornbeam and oak.
Birch trees, for example Silver Birch (Betula penduld), tolerate a wide range of
habitats, with soil pH from approximately 3.5 to approximately 7. They are native to
most of Europe and parts of Asia, but are common throughout the world, being found
in the temperate, boreal, and arctic zones of the Northern Hemisphere, especially in
Canada and other parts of North America. Birch trees typically flower between April
and May (Northern Hemisphere).
Peptidefragments of birchpollen allergens
The present inventors have identified the regions in certain birch pollen
allergen proteins which comprise MHC Class P-binding T cell epitopes. The present
inventors have also shown that regions corresponding to MHC Class P-binding T cell
epitopes within the major birch pollen allergens are highly conserved between different
isoforms of said allergens. Based on this information, peptides derived from the
relevant regions of each protein are suitable for preventing or treating birch allergy by
tolerisation to all isoforms of that protein.
The peptides of the invention are derived directly or by homology from the
protein allergens Bet v2 (SEQ ID NOS: 7 1 to 76), Bet vl (SEQ ID NOS: 1to 34 and
45 to 70), Bet v3 (SEQ ID NOS: 35, 36 and 77), Bet v4 (SEQ ID NOS: 37 to 39, 78
and 79), Bet v6 (SEQ ID NOS: 40 to 43 and 80) and Bet v7 (SEQ ID NO: 44). The
terms "peptide" and "polypeptide" are used interchangeably herein. The above
proteins are also referred to herein as "the allergens". Tables 1 to 7 set out the
sequences of the peptides of the invention (SEQ IDNOS: 1to 80), indicating the
parent protein from which each peptide derives. The composition of the invention
comprises at least one polypeptide selected from SEQ ID NOS: 1 to 80 or a variant of
any thereof.
In other words, the invention provides a composition for use in the prevention
or treatment of birch allergy by tolerisation comprising at least three, preferably at least
four different polypeptides selected from any of SEQ ID NOS: 1to 80, or a variant of
any thereof. It is preferred that none of the selected polypeptides are variants of the
same original sequence defined by any one of SEQ ID NOS: 1 to 80. In other words, it
is preferred that each of the three or four polypeptides are different original baseline
sequences defined by any one of SEQ ID NOS: 1 to 80, or are variants of different
original baseline sequences defined by any one of SEQ ID NOS: 1 to 80.
Preferably, the composition will comprise polypeptides which derive from
more than one allergen. For example, the composition may comprise one or more
polypeptides or variants thereof derived from Bet v 2 and one or more polypeptides or
variants thereof derived from Bet v 1. Additional polypeptides may optionally be
included which derive from Bet v 3, Bet v 4, Bet v 6 and/or Bet v 7.Accordingly, in
some embodiments, the composition comprises
i) at least one of the polypeptides of SEQ ID NO: 74, 72, 71, 73, 75 and 76 (which are
derived from Bet v 2), or a variant of any thereof as defined herein; and
ii) at least one of the polypeptides of SEQ ID NOS: 1to 34 and 45 to 70 (which are
derived from Bet v 1), or a variant of any thereof; and optionally
iii) at least one of the polypeptides of:
(a) SEQ ID NOs: 35, 36 and 77 (which are derived from Bet v 3), or a variant of
any thereof as defined herein; and/or
(b) SEQ ID NOs: 37 to 39, 78 and 79 (which derived from Bet v 4), or a variant of
any thereof as defined herein; and/or
(c) SEQ ID NO:s 40 to 43 and 80 (which are derived from Bet v 6), or a variant of
any thereof as defined herein; and/or
(d) SEQ D NO: 44 (which is derived from Bet v 7), or a variant thereof as defined
herein.
The composition may thus comprise any combination of one or more
polypeptides selected from group (i), one or more polypeptides selected from group (ii)
and optionally one or more polypeptides from group (iii) (a) to (d) as defined above.
Groups (i), (ii) and (iii) (a) to (d) correspond to peptides derived from different Bet
allergens, as described above. Combining polypeptides derived from different Bet
allergens may allow for broad coverage of birch pollen allergy observed in the general
population by providing tolerising epitopes from more than one birch pollen allergen.
Non-limiting examples of compositions selected as defined above include:
One, two or more polypeptides selected from SEQ ID NO: 74, 72, 71, 73, 75 and 76 or
variants of any thereof, at least one polypeptide selected from group (ii) or variant of
any thereof, and optionally at least one polypeptide or variant thereof selected from
groups (iii) (a) and/or (b); or
One, two or more polypeptides selected from SEQ D NO: 74, 72, 71, 73, 75 and 76 or
variants of any thereof and two, three, four or five polypeptides selected from group (ii)
or variants of any thereof, and optionally at least one polypeptide or variant thereof
selected from groups (iii) (a) and/or (b); or
One, two or more polypeptides selected from SEQ ID NO: 74, 72, 71, 73, 75 and 76 or
variants of any thereof and two, three, four or five polypeptides selected from group (ii)
or variants of any thereof, and at least one polypeptide from group (iii) b).
In one embodiment, the composition comprises:
(i) at least one of the polypeptides of SEQ ID NO: 74 (BIR12B;
AKYMVIQGEPGRVIRGK), SEQ ID NO: 72 (BIRl 1; FPQFKPQEITGIMK), SEQ ID
NO: 7 1 (BIR10; GSVWAQSSSFPQFK), SEQ ID NO: 73 (BIR12A;
PTGMFVAGAKYMVIQGR), SEQ ID NO: 75 (BIRl 3; IKYMVIQGEAGAVIRGK
and SEQ ID NO: 76 (BIR14; EAGAVTRGKKGSGGIT),or a variant of any thereof,
and
ii) at least one of the polypeptides of SEQ ID NO: 53 (Bir02J;
PAARMFKAFILEGDKLVPK), SEQ ID NO: 48 (BirOlI; FNYETETTSVIPAARK),
SEQ ID NO: 54 (Bir04; PGTDCKISFPEGFPFKYV), SEQ ID NO: 67 (Bir09;
ETLLRAVESYLLAHSDAY), SEQ ID NO: 60 (BIR07; SNEIKTVATPDGGSILK),
and SEQ ID NO: 63 (Bir07C; SNEKJVATPEGGSILK), or a variant of any thereof.
In another embodiment, the composition further comprises at least one additional
polypeptide of (i) or (ii) or variant thereof not selected above. In another embodiment,
the composition further comprises at least one additional polypeptide of SEQ ID NO:
77 (BIR15; SLNTLRLRPJFDLFDK) or SEQ ID NO: 78 (BIR16A;
AERERTFKRFDANGEGK), or a variant of any thereof. In a preferred embodiment,
the composition comprises:
(a) the polypeptide Bir 12B (AKYMVIQGEPGRVIRGK), or a variant thereof;
(b) the polypeptide Bir02J (PAARMFKAFILEGDKLVPK), or a variant thereof;
and
(c) the polypeptide BirO 1(FNYETETTS VIPAARK) or a variant thereof;
In a particularly preferred embodiment, the composition comprises the
polypeptide Birl2B (AKYMVIQGEPGRVIRGK) or a variant thereof, the polypeptide
Bir02J (PAARMFKAFILEGDKLVPK) or a variant thereof, the polypeptide BirO II
(FNYETETTSVrPAARK) or a variant thereof, the polypeptide Bir04
(PGTIKKISFPEGFPFKYV) or a variant thereof, the polypeptide Bir09
(ETLLRAVESYLLAHSDAY) or a variant thereof, the polypeptide Bir07C
(SNETKTVATPEGGSTLK)or a variant thereof, and the polypeptide Birl6A
(AERERTFKRFDANGEGK) or a variant thereof, and optionally no further
polypeptides.
In a further particularly preferred embodiment, the composition comprises the
polypeptide Birl2B (AKYMVIQGEPGRVIRGK) or a variant thereof, the polypeptide
Bir02J (PAARMFKAFILEGDKLVPK) or a variant thereof, the polypeptide BirOlI
(FNYETETTSVrPAARK) or a variant thereof, the polypeptide Bir04
(PGTIKKISFPEGFPFKYV) or a variant thereof, the polypeptide Bir07C
(SNEDGVATPEGGSILK) or a variant thereof, the polypeptide Birl6A
(AERERTFKRFDANGEGK) or a variant thereof, and the polypeptide Bir09B
(KEMGETLLRAVESYLLAHS) or a variant thereof, and optionally no further
polypeptides.
In a further particularly preferred embodiment, the composition comprises the
polypeptide Birl2B (AKYMVIQGEPGRVIRGK) or a variant thereof, the polypeptide
Bir02J (PAARMFKAFILEGDKLVPK) or a variant thereof, the polypeptide BirO I I
(FNYETETTSVTPAARK) or a variant thereof, the polypeptide Bir04
(PGTIKKISFPEGFPFKYV) or a variant thereof, the polypeptide Bir07C
(SNEIKTVATPEGGSTLK) or a variant thereof, and the polypeptide Bir 16A
(AERERTFKRFDANGEGK) or a variant thereof, and optionally no further
polypeptides.
The invention also provides a product comprising a peptide, variant or
composition according to the invention. The invention provides a product comprising:
i) at least one of the polypeptides of SEQ ID NO: 74 (BIR12B;
AKYMVIQGEPGRVIRGK), SEQ ID NO: 72 (BIR1 1; FPQFKPQEITGIMK), SEQ ID
NO: 7 1 (BIR10; GSVWAQSSSFPQFK), SEQ ID NO: 73 (BIR12A;
PTGMFVAGAKYMVIQGR), SEQ ID NO: 75 (BIR13; IKYMVIQGEAGAVIRGK
and SEQ ID NO: 76 (BIR14; EAGAVIRGKKGSGGIT), or a variant of any thereof as
defined in (I) to (III), and
ii) at least one of the polypeptides of SEQ ID NO: 53 (Bir02J;
PAARMFKAFILEGDKLVPK), SEQ ID NO: 48 (BirOlI; FNYETETTSVIPAARK),
SEQ ID NO: 54 (Bir04; PGTIKKISFPEGFPFKYV), SEQ ID NO: 67 (Bir09;
ETLLRAVESYLLAHSDAY), SEQ ID NO: 60 (BIR07; SNEIKJVATPDGGSILK),
and SEQ ID NO: 63 (Bir07C; SNEIKIVATPEGGSILK), or a variant of any thereof as
defined in (I) to (III),wherein each different polypeptide is for simultaneous, separate or
sequential use in preventing or treating allergy to birch pollen by tolerisation.
Variants of the polypeptides of SEQ ID NOS: 1to 80 are mentioned herein. A
variant of any of SEQ ID NOS: 1 to 80 will typically be functional. By functional it is
meant that the variant is one which:
(a) comprises or consists of a sequence which binds to the same MHC class II
molecule as the corresponding polypeptide of SEQ ID NOS: 1 to 80; and/or
(b) comprises or consists of a sequence which is recognised by a T cell which
recognises the corresponding polypeptide of SEQ ID NOS: 1 to 80; and/or
(c) is capable of inducing a late phase response in an individual with birch allergy;
and/or
(d) is capable of tolerising an individual to the corresponding polypeptide.
Recognition by a T cell may be tested by measuring the ability of a peptide or
variant to induce T cell proliferation in a sample of T cells. The induction of a late
phase response may also be tested in this way when the sample of T cells is taken from
an individual with birch allergy. Methods of testing the induction of T cell proliferation
are well known in the art and one such method is exemplified in Example 8.
Variants of SEQ ID NOS: 1 to 80 may be fragments derived by truncation, e.g.
by removal of one or more amino acids from the N and/or C-tenninal ends of a
polypeptide. Fragments may also be generated by one or more internal deletions,
provided that the core 9 amino acids that makes up the T cell epitope is not
substantially disrupted.
For example, a variant of SEQ ID NO: 1may comprise a fragment of SEQ ID
NO: 1, i.e. a shorter sequence. This may include a deletion of one, two, three or four
amino acids from the N-terminal end of SEQ ID NO: 1 or from the C-terminal end of
SEQ ID NO: 1. Such deletions may be made from both ends of SEQ ID NO: 1.
A variant of SEQ ID NO: 1 may include additional amino acids (for example
from the sequence of the parent protein from which the peptide derives) extending
beyond the end(s) of SEQ ID NO: 1. A variant of a polypeptide may typically be a
longer polypeptide of up to 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in
length which comprises the sequence of the corresponding polypeptide of SEQ ID
NOS: 1 to 80.
A variant may include a combination of the deletions and additions discussed
above. For example, amino acids may be deleted from one end of SEQ ID NO: 1, but
additional amino acids from the full length parent protein sequence may be added at
the other end of SEQ ID NO: 1. The same discussion of variants above also applies to
SEQ ID NOS: 2 to 80.
A variant may alternatively be a polypeptide of 9 to 30, 11 to 20 or 13 to 17
amino acids in length which comprises a sequence that has at least 65% sequence
identity to the sequence of the corresponding polypeptide of SEQ D NOS: 1 to 80.
More preferably a suitable variant may comprise at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, at least 95%, or at least 98% amino acid identity to the
corresponding polypeptide of SEQ ID NOS: 1 to 80.
A variant may be a polypeptide of length 9 to 30, 1 to 20 or 13 to 17 amino
acids which comprises a sequence of, or a sequence that has, at least 65% sequence
identity to at least 9 (for example at least 10, 11, 12 or 13) or more contiguous amino
acids of the sequence of the corresponding polypeptide of SEQ ID NOS: 1 to 80. These
contiguous amino acids may typically comprise a MHC class Pepitope, for example
which binds to any of the MHC molecules mentioned herein.
A variant peptide may include one or more amino acid substitutions from the
amino acid sequence of any of SEQ ID NOS: 1 to 80 or a fragment thereof. A variant
peptide may comprise sequence having at least 65% sequence identity to at least 9 or
more contiguous arrrino acids in any of SEQ ID NOS: 1 to 80. More preferably a
suitable variant may comprise at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, or at least 98% amino acid identity to at least 9 contiguous
amino acids of any of SEQ ID NOS: 1 to 80. This level of amino acid identity may be
seen at any section of the peptide, although it is preferably the core region. The level
of amino acid identity is over at least 9 contiguous amino acids but it may be at least
10, 11, 12, 13, 14, 15 or at least 16 or 17 amino acids, depending on the size of the
peptides of comparison. Accordingly, any of the above-specified levels of identity may
be across the entire length of sequence.
In connection with amino acid sequences, "sequence identity" refers to
sequences which have the stated value when assessed using ClustalW (Thompson et
al., 1994, supra) with the following parameters:
Pairwise alignment parameters -Method: accurate, Matrix: PAM, Gap open penalty:
10.00, Gap extension penalty: 0.10; Multiple alignment parameters -Matrix: PAM, Gap
open penalty: 10.00, % identity for delay: 30, Penalize end gaps: on, Gap separation
distance: 0, Negative matrix: no, Gap extension penalty: 0.20, Residue-specific gap
penalties: on, Hydrophilic gap penalties: on, Hydrophilic residues: GPSNDQEKR.
Sequence identity at a particular residue is intended to include identical residues which
have simply been derivatized.
A variant peptide may comprise 1, 2, 3, 4, 5 or more, or up to 10 amino acid
substitutions from any of SEQ ID NOS: 1 to 80. Substitution variants preferably
involve the replacement of one or more amino acids with the same number of amino
acids and making conservative amino acid substitutions. For example, an amino acid
may be substituted with an alternative amino acid having similar properties, for
example, another basic amino acid, another acidic amino acid, another neutral amino
acid, another charged amino acid, another hydrophilic amino acid, another hydrophobic
amino acid, another polar amino acid, another aromatic amino acid or another aliphatic
amino acid. Some properties of the 20 main amino acids which can be used to select
suitable substituents are as follows:
Further variants include those in which instead of the naturally occurring amino
acid the amino acid which appears in the sequence is a structural analog thereof.
Amino acids used in the sequences may also be modified, e.g. labelled, providing the
function of the peptide is not significantly adversely affected.
Where the peptide has a sequence that varies from the sequence of any of SEQ ID
NOS: 1 to 80 or a fragment thereof, the substitutions may occur across the full length
of the sequence, within the sequence of any of SEQ ED NOS: 1 to 80 or outside the
sequence of any of SEQ ED NOS: 1 to 80. For example, the variations described
herein, such as additions, deletions, substitutions and modifications, may occur within
the sequence of any of SEQ ED NOS: 1 to 80. A variant peptide may comprise or
consist essentially of the amino acid sequence of any of SEQ ED NOS: 1 to 80 in which
one, two, three, four or more amino acid substitutions have been made. A variant
peptide may comprise a fragment of the parent protein that is larger than any of SEQ
ED NOS: 1 to 80. In this embodiment, the variations described herein, such as
substitutions and modifications, may occur within and/or outside the sequence of any
of SEQ ED NOS: 1 to 80. For example, one or more positively charged residues may
be added at the N and/or C terminus of the native sequence of the peptide of any of
SEQ ED NOS: 1 to 80.
The variant peptides of the invention are 9 to 30 amino acids in length
inclusive. Preferably, they may be from 9 to 20 or more preferably 3 to 17 amino
acids in length. The peptides may be the same length as the peptide sequences in any
one of SEQ ID NOS: 1 to 80.
The peptides may be chemically derived from the polypeptide allergen, for
example by proteolytic cleavage or can be derived in an intellectual sense from the
polypeptide allergen, for example by making use of the amino acid sequence of the
polypeptide allergen and synthesising peptides based on the sequence. Peptides may be
synthesised using methods well known in the art.
The term "peptide" includes not only molecules in which amino acid residues
are joined by peptide (-CO-NH-) linkages but also molecules in which the peptide
bond is reversed. Such retro-inverso peptidomimetics may be made using methods
known in the art, for example such as those described in Meziere et al (1997) J .
Immunol. 159, 3230-3237. This approach involves making pseudopeptides containing
changes involving the backbone, and not the orientation of side chains. Meziere et al
( 997) show that, at least for MHC class II and T helper cell responses, these
pseudopeptides are useful. Retro-inverse peptides, which contain NH-CO bonds
instead of CO-NH peptide bonds, are much more resistant to proteolysis.
Similarly, the peptide bond may be dispensed with altogether provided that an
appropriate linker moiety which retains the spacing between the carbon atoms of the
amino acid residues is used; it is particularly preferred if the linker moiety has
substantially the same charge distribution and substantially the same planarity as a
peptide bond. It will also be appreciated that the peptide may conveniently be blocked
at its N-or C-terminus so as to help reduce susceptibility to exoproteolytic digestion.
For example, the N-terminal amino group of the peptides may be protected by reacting
with a carboxylic acid and the C-terminal carboxyl group of the peptide may be
protected by reacting with an amine. Other examples of modifications include
glycosylation and phosphorylation. Another potential modification is that hydrogens
on the side chain amines of R or K may be replaced with methylene groups (-NH - » -
NH(Me) or -N(Me)2) .
Analogues of peptides according to the invention may also include peptide
variants that increase or decrease the peptide's half-life in vivo. Examples of analogues
capable of increasing the half-life of peptides used according to the invention include
peptoid analogues of the peptides, D-arnino acid derivatives of the peptides, and
peptide-peptoid hybrids. A further embodiment of the variant polypeptides used
according to the invention comprises D-amino acid forms of the polypeptide. The
preparation of polypeptides using D-arnino acids rather than L-amino acids greatly
decreases any unwanted breakdown of such an agent by normal metabolic processes,
decreasing the amounts of agent which needs to be administered, along with the
frequency of its administration.
The peptides provided by the present invention may be derived from splice
variants of the parent proteins encoded by R A generated by alternative splicing of
the primary transcripts encoding the parent protein chains. The peptides may also be
derived from amino acid mutants, glycosylation variants and other covalent derivatives
of the parent proteins which retain at least an MHC-binding property of the allergens.
Exemplary derivatives include molecules wherein the peptides of the invention are
covalently modified by substitution, chemical, enzymatic, or other appropriate means
with a moiety other than a naturally occurring amino acid. Further included are
naturally occurring variants of the parent proteins found in different mites. Such a
variant may be encoded by an allelic variant or represent an alternative splicing variant.
Variants as described above may be prepared during synthesis of the peptide or
by post- production modification, or when the peptide is in recombinant form using the
known techniques of site- directed mutagenesis, random mutagenesis, or enzymatic
cleavage and/or ligation of nucleic acids.
In any of the embodiments of the invention, typical examples of variants as
described herein may be as follows:
- a variant of BirOlI is BirOlF (FNYETEATSVIPAARK), BirOlG
(FNYEIEATSVIPAARK) or BirO 1H (FNYEIETTSVIPAARK); and/or
- a variant of Bir02J is Bir02E (PAARLFKAFILEGDTLIPK), Bir02G
(PAAJRLFKAFILEGDNLIPK), Bir02I (PAARMFKAFILD) or Bir02D
(PAARMFKAFILDGDKLVPK); and/or
- a variant of Bir09 is selected from Bir09A (GETLLRAVESYLLAHS), Bir09B
(KEMGETLLRAVESYLLAHS) or Bir09C (KEKGETLLRAVESYLLAHS); and/or
- a variant of Birl6B is Birl6A (AEREPJFKRFD ANGEGK) .
It will be understood that SEQ ID NOS: 1 to 80 are polypeptide sequences
which comprise a T cell epitope that consists of a core of typically 9 amino acids,
which are the minimal essential sequence required for MHC class II binding.
However, the polypeptides of SEQ ID NOS: 1 to 80 may also comprise additional
residues flanking the core. The peptides may therefore comprise a region containing a
T cell epitope, in which some residues may be modified without affecting the function
of the epitope. Thus, for example, the sequences of any of SEQ ID NOS: 1 to 80 may
be altered to improve their solubility, and accordingly a variant of any of SEQ ID NOS:
1 to 80 will preferably be more soluble than the corresponding polypeptide of SEQ ID
NOS: 1 to 80 under equivalent conditions. Methods for evaluating the solubility of
peptides are well known in the art and one such method is exemplified in Example 9.
Improved solubility is advantageous for the tolerisation of subjects to allergens
from which the peptides of the invention derive, since administration of poorly soluble
agents to subjects causes undesirable, non-tolerising inflammatory responses. The
solubility of the peptides may be improved by altering the residues which flank the
region containing a T cell epitope. A peptide of the invention may be engineered to be
more soluble such that it comprises:
i) N terminal to the residues of the peptide which flank a T cell epitope: one to six
contiguous amino acids corresponding to the one to six contiguous amino acids
immediately N terminal to said residues in the sequence of the protein from which
the peptide derives; and/or
ii) C terminal to the residues of the peptide which flank a T cell epitope: one to six
contiguous amino acids corresponding to the one to six contiguous amino acids
immediately C terminal to the said residues in the sequence of the protein from
which the peptide derives; or
iii)both N and C terminal to the residues of the peptide which flank a T cell epitope, at
least one amino acid selected from arginine, lysine, histidine, glutamate and
aspartate.
Optionally, the peptides may additionally be engineered to be more soluble
such that:
i) any cysteine residues in the native sequence of the peptide are replaced with serine
'
18
or 2-aminobutyric acid; and /or
ii) any hydrophobic residues in the upto three amino acids at the N o C terminus of the
native sequence of the peptide, which are not comprised in a T cell epitope, are
deleted; and/or
5 iii) any two consecutive amino acids comprising the sequence Asp-Gly in the upto four
amino acids at the N or C terminus of the native sequence of the peptide, which are not
comprised in a T cell epitope, are deleted; and/or
iv) one or more positively charged residues are added at the N and/or C terminus of the
native sequence of the peptide.
10 Preferably the peptides and variants of the invention are capable of causing T
cell proliferation in at least 20 % of samples of T cells, wherein each sample is
obtained from different birch allergic individuals in the population. The compositions
of the invention are preferably capable of inducing T cell proliferation in 30 % or more
samples of T cells obtained from of a panel of birch allergic individuals. More
15 preferably, the compositions are capable of inducing T cell proliferation in 35% or
more, 40 % or more, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, or 90 %
or more of samples obtained from sensitized individuals in a panel. The number of
individuals in a panel of birch allergic individuals may be any number greater than one,
for example at least 2, 3, 5, 10, 15, 20, 30, 50, 80, or at least 100 individuals.
20 It is preferred if the peptides, variants and compositions of the invention cause
T cell proliferation, but do not lead to the release of histamine from enriched basophils
or mast cell preparations from a sensitised individual. There may be some Mstarnine
release, but preferably the peptides, variants and compositions do not cause significant
amounts of Mstarnine to be released. Significant histamine release may be considered
25 to be the release of 20% or more of the total available leukocyte histamine when a
sample of leukocytes from an individual is stimulated with a composition in vitro. A
peptide, variant or composition of the invention preferably causes the release of less
than 5%, less than 4%, less than 3%, less than 2% or less than 1% of the total available
leukocyte Mstarnine when a sample of leukocytes from an individual is stimulated with
30 a composition in vitro. A normal individual typically has an approximate leukocyte
Mstarnine content of 150ng/10 cells.
Suitable peptides or variants capable of binding to TCRs may be derived
empirically or selected according to known criteria. Within a single peptide there are
certain residues which contribute to binding within the MHC antigen binding groove
and other residues which interact with hypervariable regions of the T cell receptor
(Allen et al (1987) Nature 327: 713-5).
Within the residues contributing to T cell receptor interaction, a hierarchy has
been demonstrated which pertains to dependency of T cell activation upon substitution
of a given peptide residue. Using peptides which have had one or more T cell receptor
contact residues substituted with a different amino acid, several groups have
demonstrated profound effects upon the process of T cell activation. Evavold & Allen
(1991) Nature 252: 1308-10) demonstrated the dissociation of T cell proliferation and
cytokine production. In this in vitro model, a T cell clone specific for residues 64-76 of
haemoglobin (in the context of I-Ek), was challenged with a peptide analogue in which
a conservative substitution of aspartic acid for glutamic acid had been made. This
substitution did not significantly interfere with the capacity of the analogue to bind to
I-Ek.
Following in vitro challenge of a T cell clone with this analogue, no
proliferation was detected although IL-4 secretion was maintained, as was the capacity
of the clone to help B cell responses. In a subsequent study the same group
demonstrated the separation of T cell-mediated cytolysis from cytokine production. In
this instance, the former remained unaltered while the latter was impaired. The efficacy
of altered peptide ligands in vivo was initially demonstrated in a murine model of EAE
(experimental allergic encephalomyelitis) by McDevitt and colleagues (Smilek et al
(1991) Proc Natl Acad Sci USA 88 : 9633-9637). In this model EAE is induced by
immunisation with the encephalitogenic peptide Acl-1 1 of MBP (myelin basic
protein). Substitution at position four (lysine) with an alanine residue generated a
peptide which bound well to its restricting element (Aa"Ab ), but which was nonimmunogenic
in the susceptible PL/JxSJLFl strain and which, furthermore prevented
the onset of EAE when administered either before or after immunisation with the
encephalitogenic peptide. Thus, residues can be identified in peptides which affect the
ability of the peptides to induce various functions of T-cells.
Advantageously, peptides may be designed to favour T-cell proliferation and
induction of desensitisation. Metzler and Wraith have demonstrated improved
tolerogenic capacity of peptides in which substitutions increasing peptide-MHC
affinity have been made (Metzler & Wraith(1993) Int Immunol ~ : 1 159-65). That an
altered peptide ligand can cause long-term and profound anergy in cloned T cells was
demonstrated by Sloan-Lancaster et al (1993) Nature 363: 156-9.
The compositions of the invention are capable of inducing a late phase response
in an individual that is sensitised to the allergens. The term "late phase response"
includes the meaning as set forth in Allergy and Allergic Diseases (1997) A. B. Kay
(Ed.), Blackwell Science, pp 1113-1 130. The late phase response may be any late
phase response (LPR). Preferably, the peptides are capable of inducing a late asthmatic
response (LAR) or a late rhinitic response, or a late phase skin response or a late phase
ocular response. Whether or not a particular peptide can give rise to a LPR can be
determined using methods well known in the art; a particularly preferred method is that
described in Cromwell O, Durham SR, Shaw RJ, Mackay J and Kay AB. Provocation
tests and measurements of mediators from mast cells and basophils in asthma and
allergic rhinitis. In: Handbook of Experimental Immunology (4) Chapter 127, Editor:
Weir DM, Blackwell Scientific Publications, 1986.
Thus, preferably, the individual peptides and variants of the invention are able
to induce a LPR in an individual who has been sensitised to the allergens. Whether or
not an individual has been sensitised to the allergens may be determined by well
known procedures such as skin prick testing with solutions of allergen extracts,
induction of cutaneous LPRs, clinical history, allergen challenge and
radioallergosorbent test (RAST) for measurement of allergen specific IgE. Whether or
not a particular individual is expected to benefit from treatment may be determined by
the physician based, for example, on such tests.
Desensitising or tolerising an individual to the allergens means inhibition or
dampening of allergic tissue reactions induced by the allergens in appropriately
sensitised individuals. It has been shown that T cells can be selectively activated, and
then rendered unresponsive. Moreover the anergising or elimination of these T-cells
leads to desensitisation of the patient for a particular allergen. The desensitisation
manifests itself as a reduction in response to an allergen or allergen-derived peptide, or
preferably an elimination of such a response, on second and further administrations of ·
the allergen or allergen-derived peptide. The second administration may be made after
a suitable period of time has elapsed to allow desensitisation to occur; this is preferably
any period between one day and several weeks. An interval of around two weeks is
preferred.
Although the compositions of the invention are able to induce a LPR in a birch
allergic individual, it should be appreciated that when a composition is used to treat a
patient it is preferable that a sufficiently low concentration of the composition is used
such that no observable LPR will occur but the response will be sufficient to partially
desensitise the T cells such that the next (preferably higher) dose may be given, and so
on. In this way the dose is built up to give full desensitisation but often without ever
inducing a LPR in the patient. Although, the composition or peptide is able to do so at
a higher concentration than is adrninistered.
The compositions of the invention preferably are capable of inducing a late
phase response in 50 % or more of a panel of birch allergic individuals from the
population. More preferably, the compositions are capable of inducing a LPR in 55%
or more, 60 % or more, 65 % or more, 70% or more, 75% or more, 80% or more, 85%
or more, or 90 % or more of sensitized individuals in a panel. Whether or not the
compositions are able to induce a LPR in a certain percentage of a panel of subjects
can be determined by methods which are well known in the art.
Nucleic acids and vectors
The individual peptides that make up the compositions and products of the
invention may be administered directly, or may be administered indirectly by
expression from an encoding sequence. For example, a polynucleotide may be
provided that encodes a peptide of the invention, such as any of the peptides described
above. A peptide of the invention may thus be produced from or delivered in the form
of a polynucleotide which encodes, and is capable of expressing, it. Any reference
herein to the use, delivery or administration of a peptide of the invention is intended to
include the indirect use, delivery or ackriinistration of such a peptide via expression
from a polynucleotide that encodes it.
Accordingly, the invention provides a composition for use in preventing or
treating allergy to birch by tolerisation comprising at least one polynucleotide sequence
which when expressed causes the production of a composition according to the
invention for use in preventing or treating allergy to birch by tolerisation.
The terms "nucleic acid molecule" and "polynucleotide" are used
interchangeably herein and refer to a polymeric form of nucleotides of any length,
either deoxyribonucleotides or ribonucleotides, or analogs thereof. Non-limiting
examples of polynucleotides include a gene, a gene fragment, messenger RNA
(mRNA), cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any
sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A
polynucleotide of the invention may be provided in isolated or purified form.
A nucleic acid sequence which "encodes" a selected polypeptide is a nucleic acid
molecule which is transcribed (in the case of DNA) and translated (in the case of
mRNA) into a polypeptide in vivo when placed under the control of appropriate
regulatory sequences. The boundaries of the coding sequence are determined by a start
codon at the 5 (amino) terrninus and a translation stop codon at the 3' (carboxy)
terminus. For the purposes of the invention, such nucleic acid sequences can include,
but are not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic
sequences from viral or prokaryotic DNA or RNA, and even synthetic DNA sequences.
A transcription termination sequence may be located 3' to the coding sequence.
Polynucleotides of the invention can be synthesised according to methods well
known in the art, as described by way of example in Sambrook et al (1989, Molecular
Cloning - a laboratory manual; Cold Spring Harbor Press).
The polynucleotide molecules of the present invention may be provided in the
form of an expression cassette which includes control sequences operably linked to the
inserted sequence, thus allowing for expression of the peptide of the invention in vivo
in a targeted subject. These expression cassettes, in turn, are typically provided within
vectors (e.g., plasmids or recombinant viral vectors) which are suitable for use as
reagents for nucleic acid immunization. Such an expression cassette may be
administered directly to a host subject. Alternatively, a vector comprising a
polynucleotide of the invention may be administered to a host subject. Preferably the
polynucleotide is prepared and/or administered using a genetic vector. A suitable
vector may be any vector which is capable of carrying a sufficient amount of genetic
information, and allowing expression of a peptide of the invention.
Expression vectors are routinely constructed in the art of molecular biology and
may for example involve the use of plasmid DNA and appropriate initiators,
promoters, enhancers and other elements, such as for example polyadenylation signals
which may be necessary, and which are positioned in the correct orientation, in order to
allow for expression of a peptide of the invention. Other suitable vectors would be
apparent to persons skilled in the art. By way of further example in this regard we refer
to Sambrook et al.
Thus, a polypeptide of the invention may be provided by delivering such a
vector to a cell and allowing transcription from the vector to occur. Thus, the
invention also provides a vector for use in preventing or treating allergy to birch pollen
by tolerisation comprising four or more polynucleotide sequences which encode a
different polypeptide of the invention. Preferably, a polynucleotide of the invention or
for use in the invention in a vector is operably linked to a control sequence which is
capable of providing for the expression of the coding sequence by the host cell, i.e. the
vector is an expression vector.
"Operably linked" refers to an arrangement of elements wherein the
components so described are configured so as to perform their usual function. Thus, a
given regulatory sequence, such as a promoter, operably linked to a nucleic acid
sequence is capable of effecting the expression of that sequence when the proper
enzymes are present. The promoter need not be contiguous with the sequence, so long
as it functions to direct the expression thereof. Thus, for example, mtervening
untranslated yet transcribed sequences can be present between the promoter sequence
and the nucleic acid sequence and the promoter sequence can still be considered
"operably linked" to the coding sequence.
A number of expression systems have been described in the art, each of which
typically consists of a vector containing a gene or nucleotide sequence of interest
operably linked to expression control sequences. These control sequences include
transcriptional promoter sequences and transcriptional start and termination sequences.
The vectors of the invention may be for example, plasmid, virus or phage vectors
provided with an origin of replication, optionally a promoter for the expression of the
said polynucleotide and optionally a regulator of the promoter. A "plasmid" is a vector
in the form of an extrachromosomal genetic element. The vectors may contain one or
more selectable marker genes, for example an ampicillin resistence gene in the case of
a bacterial plasmid or a resistance gene for a fungal vector. Vectors may be used in
vitro, for example for the production of DNA or RNA or used to transfect or transform
a host cell, for example, a mammalian host cell. The vectors may also be adapted to be
used in vivo, for example to allow in vivo expression of the polypeptide.
A "promoter" is a nucleotide sequence which initiates and regulates
transcription of a polypeptide-encoding polynucleotide. Promoters can include
inducible promoters (where expression of a polynucleotide sequence operably linked to
the promoter is induced by an analyte, cofactor, regulatory protein, etc.), repressible
promoters (where expression of a polynucleotide sequence operably linked to the
promoter is repressed by an analyte, cofactor, regulatory protein, etc.), and constitutive
promoters. It is intended that the term "promoter" or "control element" includes fulllength
promoter regions and functional (e.g., controls transcription or translation)
segments of these regions.
A polynucleotide, expression cassette or vector according to the present
invention may additionally comprise a signal peptide sequence. The signal peptide
sequence is generally inserted in operable linkage with the promoter such that the
signal peptide is expressed and facilitates secretion of a polypeptide encoded by coding
sequence also in operable linkage with the promoter.
Typically a signal peptide sequence encodes a peptide of 10 to 30 amino acids
for example 15 to 20 amino acids. Often the amino acids are predominantly
hydrophobic. In a typical situation, a signal peptide targets a growing polypeptide
chain bearing the signal peptide to the endoplasmic reticulum of the expressing cell.
The signal peptide is cleaved off in the endoplasmic reticulum, allowing for secretion
of the polypeptide via the Golgi apparatus. Thus, a peptide of the invention may be
provided to an individual by expression from cells within the individual, and secretion
from those cells.
Alternatively, polynucleotides of the invention may be expressed in a suitable
manner to allow presentation of a peptide of the invention by an MHC class P
molecule at the surface of an antigen presenting cell. For example, a polynucleotide,
expression cassette or vector of the invention may be targeted to antigen presenting
cells, or the expression of encoded peptide may be preferentially stimulated or induced
in such cells.
In some embodiments, the polynucleotide, expression cassette or vector will
encode an adjuvant, or an adjuvant will otherwise be provided. As used herein, the
term "adjuvant" refers to any material or composition capable of specifically or nonspecifically
altering, enhancing, directing, redirecting, potentiating or initiating an
antigen-specific immune response.
Polynucleotides of interest may be used in vitro, ex vivo or in vivo in the
production of a peptide of the invention. Such polynucleotides may be administered or
used in the prevention or treatment of allergy by tolerisation.
Methods for gene delivery are known in the art. See, e.g., U.S. Patent Nos.
5,399,346, 5,580,859 and 5,589,466. The nucleic acid molecule can be introduced
directly into the recipient subject, such as by standard intramuscular or intradermal
injection; transdermal particle delivery; inhalation; topically, or by oral, intranasal or
mucosal modes of administration. The molecule alternatively can be introduced ex
vivo into cells that have been removed from a subject. For example, a polynucleotide,
expression cassette or vector of the invention may be introduced into APCs of an
individual ex vivo. Cells containing the nucleic acid molecule of interest are re¬
introduced into the subject such that an immune response can be mounted against the
peptide encoded by the nucleic acid molecule. The nucleic acid molecules used in
such immunization are generally referred to herein as "nucleic acid vaccines."
The polypeptides, polynucleotides, vectors or cells of the invention may be
present in a substantially isolated form. They may be mixed with carriers or diluents
which will not interfere with their intended use and still be regarded as substantially
isolated. They may also be in a substantially purified form, in which case they will
generally comprise at least 90%, e.g. at least 95%, 98% or 99%, of the proteins,
polynucleotides, cells or dry mass of the preparation.
Antigen presenting cells (APCs)
The invention encompasses the use in vitro of a method of producing a
population of APCs that present the peptides of the invention on their surface, that may
be subsequently used in therapy. Such a method may be carried out ex vivo on a
sample of cells that have been obtained from a patient. The APCs produced in this
way therefore form a pharmaceutical agent that can be used in the treatment or
prevention of birch allergy by tolerisation. The cells should be accepted by the
immune system of the individual because they derive from that individual. Delivery of
cells that have been produced in this way to the individual from whom they were
originally obtained, thus forms a therapeutic embodiment of the invention.
Formulations and compositions
The peptides, polynucleotides, vectors and cells of the invention may be
provided to an individual either singly or in combination. Each molecule or cell of the
invention may be provided to an individual in an isolated, substantially isolated,
purified or substantially purified form. For example, a peptide of the invention may be
provided to an individual substantially free from the other peptides. Alternatively, four
or more peptides in the composition may be coupled chemically together, using
standard peptide coupling reagents, to provide a single peptide containing the preferred
epitopes. Such peptides would be screened for basophil histamine release to confirm
lack of Mstamine release as per the individual peptides. In a further embodiment, four
or more peptides in the composition may be provided as part of a single polypeptide
chain i.e by recombinant means from an encoding polynucleotide. The four or more
peptides may be fused contiguously, or may alternatively be separated by appropriate
linkers.
Whilst it may be possible for the peptides, polynucleotides or compositions
according to the invention to be presented in raw form, it is preferable to present them
as a pharmaceutical formulation. Thus, according to a further aspect of the invention,
the present invention provides a pharmaceutical formulation for use in preventing or
treating allergy to birch by tolerisation comprising a composition, vector or product
according to the invention together with one or more pharmaceutically acceptable
carriers or diluents and optionally one or more other therapeutic ingredients. The
carrier (s) must be 'acceptable' in the sense of being compatible with the other
ingredients of the formulation and not deleterious to the recipient thereof. Typically,
carriers for injection, and the final formulation, are sterile and pyrogen free.
Preferably, the carrier or diluent is thioglycerol or thioanisole.
Formulation of a composition comprising the peptide, polynucleotides or cells
of the invention can be carried out using standard pharmaceutical formulation
chemistries and methodologies all of which are readily available to the reasonably
skilled artisan.
For example, compositions containing one or more molecules or cells of the
invention can be combined with one or more pharmaceutically acceptable excipients or
vehicles. Auxiliary substances, such as wetting or emulsifying agents, pH buffering
substances and the like, may be present in the excipient or vehicle. These excipients,
vehicles and auxiliary substances are generally pharmaceutical agents that do not
induce an immune response in the individual receiving the composition, and which
may be administered without undue toxicity. Pharmaceutically acceptable excipients
include, but are not limited to, liquids such as water, saline, polyethyleneglycol,
hyaluronic acid, glycerol, thioglycerol and ethanol. Pharmaceutically acceptable salts
can also be included therein, for example, mineral acid salts such as hydrochlorides,
hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as
acetates, propionates, malonates, benzoates, and the like. A thorough discussion of
pharmaceutically acceptable excipients, vehicles and auxiliary substances is available
in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).
Such compositions may be prepared, packaged, or sold in a form suitable for
bolus aclministration or for continuous administration. Injectable compositions may be
prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi-dose
containers containing a preservative. Compositions include, but are not limited to,
suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable
sustained-release or biodegradable formulations. Such compositions may further
comprise one or more additional ingredients including, but not limited to, suspending,
stabilizing, or dispersing agents. In one embodiment of a composition for parenteral
administration, the active ingredient is provided in dry (for e.g., a powder or granules)
form for reconstitution with a suitable vehicle (e. g., sterile pyrogen-free water) prior to
parenteral adrninistration of the reconstituted composition. The pharmaceutical
compositions may be prepared, packaged, or sold in the form of a sterile injectable
aqueous or oily suspension or solution. This suspension or solution may be formulated
according to the known art, and may comprise, in addition to the active ingredient,
additional ingredients such as the dispersing agents, wetting agents, or suspending
agents described herein. Such sterile injectable formulations may be prepared using a
non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol,
for example. Other acceptable diluents and solvents include, but are not limited to,
Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic
mono-or di-glycerides.
Other parentally-administrable compositions which are useful include those which
comprise the active ingredient in macrocrystalline form, in a liposomal preparation, or
as a component of a biodegradable polymer systems. Compositions for sustained
release or implantation may comprise pharmaceutically acceptable polymeric or
hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble
polymer, or a sparingly soluble salt.
Alternatively, the peptides or polynucleotides of the present invention may be
encapsulated, adsorbed to, or associated with, particulate carriers. Suitable particulate
carriers include those derived from polymethyl methacrylate polymers, as well as PLG
microparticles derived from poly(lactides) and poly(lactide-co-glycolides). See, e.g.,
Jeffery et al. (1993) Pharm. Res. 10:362-368. Other particulate systems and polymers
can also be used, for example, polymers such as polylysine, polyarginine,
polyonuthine, spermine, spermidine, as well as conjugates of these molecules.
The formulation of any of the peptides, polynucleotides or cells mentioned
herein will depend upon factors such as the nature of the substance and the method of
delivery. Any such substance may be administered in a variety of dosage forms. It may
be administered orally (e.g. as tablets, troches, lozenges, aqueous or oily suspensions,
dispersible powders or granules), topically, parenterally, subcutaneously, by inhalation,
intravenously, intramuscularly, intrasternally, transdermally, intradermally,
sublingually, intranasally, buccally or by infusion techniques. The substance may also
be administered as suppositories. A physician will be able to determine the required
route of administration for each particular individual.
The compositions of formulations of the invention will comprise a suitable
concentration of each peptide/polynucleotide/cell to be effective without causing
adverse reaction. Typically, the concentration of each peptide in the composition will
be in the range of 0.03 to 200 nmol/ml. More preferably in the range of 0.3 to 200
nmol/ml, 3 to 180 nmol/ml, 10 to 150 nmol7ml, 50 to 200nmol/ml or 30 to 120
nmol/ml. The composition or formulations should have a purity of greater than 95% or
98% or a purity of at least 99%.
In one aspect of the invention an adjuvant may be used in combination with the
polypeptide/polynucleotides/cells of the invention. The adjuvant is preferably
administered in an amount which is sufficient to augment the effect of the
polypeptide/polynucleotides/cells of the invention or vice versa. The adjuvant or
other therapeutic agent may be an agent that potentiates the effects of the molecule of
the invention. For example, the other agent may be an immunomodulatory molecule or
an adjuvant which enhances the response to the peptide or cell of the invention.
In one embodiment therefore, the peptides, polynucleotides, cells or
compositions of the invention are used for therapy in combination with one or more
other therapeutic agents. The agents may be administered separately, simultaneously
or sequentially. They may be administered in the same or different compositions.
Accordingly, in a method of the invention, the subject may also be treated with a
further therapeutic agent.
A composition may therefore be formulated which comprises a molecule and/or
cell of the invention and also one or more other therapeutic molecules. A composition
of the invention may alternatively be used simultaneously, sequentially or separately
with one or more other therapeutic compositions as part of a combined treatment.
Non-limiting examples of adjuvants include vitamin D, rapamycin and
glucocorticoid steroids such as dexamethasone, fluticasone, budesonide, mometasone,
beclomethasone, hydrocortisone, cortisone acetate, prednisone, prednisolone,
methylprednisolone, betamethasone and triamcinolone. A preferred glucocorticoid is
dexamethasone.
Therapeutic methods and individual to be treated
The present invention relates to peptides, polynucleotides, vectors and cells that
are capable of desensitising or tolerising human individuals to the allergens described
above and are therefore useful in the prevention or treatment of birch allergy. The
invention provides compositions, products, vectors and formulations for use in
preventing or treating allergy to birch by tolerisation. The invention also provides a
method of tolerising or desensitizing a birch allergic individual comprising
adrrririistering, either singly or in combination the polypeptides/polynucleotides/cells of
the invention as described above.
The individual to be treated or provided with the composition or formulation
of the invention is preferably human. It will be appreciated that the individual to be
treated may be known to be sensitised to the allergens, at risk of being sensitised or
suspected of being sensitised. The individual can be tested for sensitisation using
techniques well known in the art and as described herein. Alternatively, the individual
may have a family history of allergy to birch. It may not be necessary to test an
individual for sensitisation to birch because the individual may display symptoms of
allergy when exposed to birch. By exposure is meant proximity to, for example, a
birch plant, or a substance or product derived from a birch plant, or a substance or
product containing or comprising either of the above. The substance or product
derived from a birch plant is typically birch pollen. By proximity is meant 10 metres
or less, 5 metres or less, 2 metres or less, 1 metre or less, or 0 metres from the items
described above. Symptoms of allergy can include itchy eyes, runny nose, breathing
difficulties, red itchy skin or rash.
The individual to be treated may be of any age. However, preferably, the
individual may be in the age group of 1 to 90, 5 to 60, 10 to 40, or more preferably 18
to 35.
Preferably, the individual to be treated is from a population that has MHC allele
frequencies within the range of frequencies that are representative of the Caucasian
population. Reference population allele frequencies for 11 common DRB1 allele
families are shown in Table 1 (Data from HLA Facts Book, Parham and Barber).
Reference frequencies were obtained by analysis of multiple studies reporting
frequencies and the figures shown are mean values. Preferably therefore, the
individual to be treated is from a population that has equivalent MHC allele
frequencies as the reference population for the alleles referred to Table 1 (such as for at
least 1, 2, 3, 4, 5 or all of the alleles), for example within the ranges of those figures
plus or minus 1, 2, 3, 5, 10, 15 or 20%.
Preferably the individual is from a population where the allele frequencies of
the following DRB1 alleles is:
4 - at least 9%
7 - at least 10%
11 - at least 8%.
The individual may have had allergy to birch for at least 2 weeks, 1 month, 6
months, 1 year or 5 years. The individual may suffer from a rash, nasal congestion,
nasal discharge and/or coughing caused by the allergy. The individual may or may not
have been administered with other compositions/compounds which treat birch allergy.
The individual may live in a geographical region which has:
- a temperate, boreal or arctic climate, and/or:
- a typical soil pH in the range of about 3.5, to about 7.5.
The individual typically suffers from allergy to birch pollen in a particular
season. The season typically corresponds to the flowering season of birch, which is
typically spring, preferably early spring (for example from April to May in the
Northern hemisphere). The allergic individual is typically allergic to birch pollen from
any tree in the subgenus Betula, for example Betula pendula or Betula pubescens.
Combination immunotherapy
Since many individuals are allergic, or may require desensitizing to several
polypeptide antigens, the current invention also provides means of desensitizing
individuals that are allergic to multiple antigens. "Tolerance" induced in an individual
to a first polypeptide antigen or allergen can create in the individual a "tolergeneic
environment" wherein inappropriate immune responses to other antigens can be
downregulated in order to provide tolerance to other antigens.
This finding means that individuals allergic to multiple allergens can be treated
in a greatly reduced time period, and that individuals seriously allergic to some
allergens (e.g., peanuts) but more mildly allergic to other allergens (e.g., cat dander)
can benefit from a therapy wherein tolerance to the milder allergen is established and
then this tolergeneic environment is used to provide tolerance to the other, more
extreme allergen. In addition, individuals suffering from an autoimmune disorder who
are additionally sensitised (or otherwise immune) to an unrelated antigen or allergen
can benefit from a treatment regime wherein tolerance to the unrelated antigen or
allergen is first established and then this tolergeneic environment is used to provide
tolerance to the autoantigen associated with the autoimmune disorder.
A method is therefore provided for desensitising a birch allergic individual to
birch allergen as described above and one or more further different polypeptide
antigens. The method entails, in a first step, administering to the individual a
composition/product/formulation (primary composition) according to the invention as
described herein and wherein the administration is carried out in a manner sufficient to
generate a hyporesponsive state against birch allergen. Once a hyporesponsive state
has been established toward birch allergen, or at least a shift toward desensitisation has
occurred, the method entails administration of a secondary composition comprising a
second, different polypeptide antigen to which the individual is to be sensitised.
Aclministration of the secondary composition is carried out in such a way as to take
advantage of the tolerogenic environment established by use of the primary
composition, where it is now possible to establish tolerance to the second, different
polypeptide antigen. The secondary composition is coadministered with either the first
primary composition or a larger fragment of the birch allergen. By "coadministered" it
is meant either the simultaneous or concurrent administration, e.g., when the two are
present in the same composition or administered in separate compositions at nearly the
same time but at different sites, as well as the delivery of polypeptide antigens in
separate compositions at different times. For example, the secondary composition may
be delivered prior to or subsequent to delivery of the first composition at the same or a
different site. The timing between deliveries can range from about several seconds
apart to about several minutes apart, several hours apart, or even several days apart.
Furthermore, different delivery methods can be employed.
The second polypeptide antigen is preferably an allergen different to the birch
allergen. Suitable allergens for use in the methods of the invention can of course be
obtained and/or produced using known methods. Classes of suitable allergens include,
but are not limited to, dust mite allergens, pollens, animal dander (especially cat
dander), grass allergens, molds, dusts, antibiotics, stinging insect venoms, and a variety
of environmental (including chemicals and metals), drug and food allergens. Common
tree allergens include pollens from cottonwood, popular, ash, birch, maple, oak, elm,
hickory, and pecan trees; common plant allergens include those from mugwort,
ragweed, English plantain, sorrel-dock and pigweed; plant contact allergens include
those from poison oak, poison ivy and nettles; common grass allergens include rye
grass, Timothy, Johnson, Bermuda, fescue and bluegrass allergens; common allergens
can also be obtained from molds or fungi such as Alternaria, Fusarium,
Hormodendrum, Aspergillus, Micropolyspora, Mucor and thermophilic actinomycetes;
epidermal allergens can be obtained from house or organic dusts (typically fungal in
origin), or from animal sources such as feathers, and dog dander; common food
allergens include milk and cheese (diary), egg, wheat, nut (e.g., peanut), seafood (e.g.,
shellfish), pea, bean and gluten allergens; common environmental allergens include
metals (nickel and gold), chemicals (formaldehyde, trinitrophenol and turpentine),
Latex, rubber, fiber (cotton or wool), burlap, hair dye, cosmetic, detergent and perfume
allergens; common drug allergens include local anesthetic and salicylate allergens;
antibiotic allergens include penicillin, tetracycline and sulfonamide allergens; and
common insect allergens include bee, wasp and ant venom, and cockroach calyx
allergens. Particularly well characterized allergens include, but are not limited to, the
major cat allergen Fel dl, bee venom phospholipase A2 (PLA) (Akdis et al. (1996) J.
Clin. Invest. 98:1676-1683) and the multi-epitopic recombinant grass allergen
rKBG8.3 (Cao et al. (1997) Immunology 90:46-51). These and other suitable allergens
are commercially available and/or can be readily prepared as extracts following known
techniques.
Preferably, the second polypeptide allergen is a whole tree pollen allergen or
allergen fragment selected from the list of allergen sequences and database accession
numbers (NCBI Entrez accession numbers) below. NCBI is the National Center for
Biotechnology information and is a division of the US National Institutes of Health.
The NCBI web site, from which access to the database may be sought, is
www.ncbi.nlm.nih.gov/. Allergen sequences and database accession numbers (NCBI
Entrez accession numbers):
Olive tree
Olive sequences
416610 Ole e 1
EDEPQPPVSQFfflQGQVYCDTCRAGFITELSEFIPGASLRLQCKDKENGDVTFTE
VGYTRAEGLYSMLVERDHKNEFCEITLISSGRKDCNEIPTEGWAKPSLKFKLNT
VNGTTRTVNPLGFFKKEALPKCAQVYNKLGMYPPNM
Tree allergen sequences (mainly birch) sequences:
130975 Bet v 2
MSWQTYVDEHLMCDIDGQASNSLASArVGHDGSVWAQSSSFPQFKPQEITGIM
KDFEEPGHLAPTGLHLGGIKYMVIQGEAGAVIRGKKGSGGITIKKTGQALVFGI
YEEPVTPGQCNMWERLGDYLIDQGL
1942360 Bet v 2
MSWQTYVDEHLMCDIDGQGEELAASAIVGHDGSVWAQSSSFPQFKPQEITGIM
KDFEEPGHLAPTGLHLGGIKYMVIQGEAGAVrRGKKGSGGITIKKTGQALVFGI
YEEPVTPGQCNMWERLGDYLIDQGL
166953 Bet v 2
MSWQTYVDEHLMCDIDGQASNSLASAIVGHDGSVWAQSSSFPQFKPQEITGIM
KDFEEPGHLAPTGLHLGGIKYMVIQGEAGAVIRGKKGSGGITIKKTGQALVFGI
YEEPVTPGQCNMWERLGDYLIDQGL
541814 Bet v 2
MSWQTWDEHLMCDIDGQASNSLASAIVGHDGSVWAQSSSFPQFKPQEITGIM
KDFEEPGHLAPTGLHLGGIKYMVIQGEAGAVIRGKKGSGGITIKKTGQALVFGI
YEEPVTPGQCNMWERLGDYLIDQGL
2488678 Bet v 2
MSWQTYVDEHLMCDIDGQASNSLASAIVGHDGSVWAQSSSFPQFKPQEITGIM
KDFEEPGHLAPTGLHLGGIKYMVIQGEAGAVIRGKKGSGGITIKKTGQALVFGI
YEEPVTPGQCNMWERLGDYLIDQGL
1829894 Bet v 2
MSWQTYVDEHLMCDIDGQASNSLASArVGHDGSVWAQSSSFPQFKPQEITGIM
KDFEEPGHLAPTGLHLGGIKYMVIQGEAGAVIRGKKGSGGITIKKTGQALVFGI
YEEPVTPGQCNMWERLGDYLIDQGL
1168696 Bet v 3
MPCSTEAMEKAGHGHASTPRKRSLSNSSFRLRSESLNTLRLRRIFDLFDKNSDG
nTVDELSRALNLLGLETDLSELESTVKSFTREGNIGLQFEDFISLHQSLNDSYFA
YGGEDEDDNEEDMRKSILSQEEADSFGGFKVFDEDGDGYISARELQMVLGKL
GFSEGSEIDRVEKMIVSVDSNRDGRVDFFEFKDMMRSVLVRSS
809536 Bet v 4
MADDHPQDKAERERIFKRFDANGDGKISAAELGEALKTLGSITPDEVKHMMA
EIDTDGDGFISFQEFTDFGRANRGLLKDVAKIF
543675 Que a I - Quercus alba=oak trees (fragment)
GVFTXESQETSVIAPAXLFKALFL
543509 Car b I - Carpinus betulus=hornbeam trees (fragment)
GVFNYEAETPSVIPAARLFKSYVLDGDKLIPKVAPQAIXK
543491 Aln g I - Alnus glutinosa=alder trees (fragment)
GVFNYEAETPSVIPAARLFKAFILDGDKLLPKVAPEAVSSVENI
1204056 Rubisco
VQCMQVWPPLGLKKFETLSYLPPLSSEQLAKEVDYLLRKNLIPCLEFELEHGFV
YREHNRSPGYYDGRYWTMWKLPMFGCNDSSQVLKELEECKKAYPSAFIRIIGF
DDK
Additional tree allergen sequences (NCBI entrez accession number):
131919; 128193; 585564; 1942360; 2554672; 2392209; 2414158; 1321728; 1321726;
1321724; 1321722; 1321720; 1321718; 1321716; 1321714; 1321712; 3015520;
2935416; 464576; 1705843; 1168701; 1168710; 1168709; 1168708; 1168707;
1168706; 1168705; 1168704; 1168703; 1168702; 1842188; 2564228; 2564226;
2564224; 2564222; 2564220; 2051993; 1813891; 1536889; 534910; 534900; 534898;
1340000; 1339998; 2149808; 66207; 2129477; 1076249; 1076247; 629480; 481805;
81443; 1361968; 1361967; 1361966; 1361965; 1361964; 1361963; 1361962;
1361961; 1361960; 1361959; 320546; 629483 ; 629482; 629481; 541804; 320545;
81444; 541814:; 629484; 47491 1; 452742; 1834387; 298737; 298736; 1584322;
1584321; 584320; 1542873; 1542871; 1542869; 1542867; 1542865; 1542863;
1542861; 1542859; 1542857; 1483232; 1483230; 1483228; 558561; 551640; 488605;
452746; 452744; 452740; 452738; 452736; 452734; 452732; 452730; 452728;
450885; 17938; 17927; 17925; 17921; 297538; 510951; 289331; 289329; 166953 .
Cedar sequences
493634 Cry j IB precursor
MDSPCLVALLVFSFVIGSCFSDNPIDSCWRGDSNWAQNRMKLADCAVGFGSST
MGGKGGDLYTVTNSDDDPVNPPGTLRYGATRDRPLWIIFSGNMNIKLKMPMY
IAGYKTFDGRGAQVYIGNGGPCVFIKRVSNVIIHGLYLYGCSTSVLGNVLINESF
GVEPVHPQDGDALTLRTATNIWIDHNSFSNSSDGLVDVTLTSTGVTISNNLFFN
HHKVMSLGHDDAYSDDKSMKVTVAFNQFGPNCGQRMPRARYGLVHVANNN
YDPWTIYAIGGSSNPTILSEGNSFTAPNESYKKQVTIRIGCKTSSSCSNWVWQST
QDVFYNGAYFVSSGKYEGGNIYTKKEAFNVENGNATPHLTQNAGVLTCSLSK
RC
493632 Cry j IA precursor
MDSPCLVALLVLSFVIGSCFSDNPIDSCWRGDSNWAQNRMKLADCAVGFGSS
TMGGKGGDLYTVTNSDDDPVNPAPGTLRYGATRDRPLWlIFSGNMNIKLKMP
MYIAGYKTFDGRGAQVYIGNGGPCVFIKRVSNVIIHGLHLYGCSTSVLGNVLIN
ESFGVEPVHPQDGDALTLRTATNIWIDHNSFSNSSDGLVDVTLSSTGVTISNNLF
FNFiHKVMLLGHDDAYSDDKSMKVTVAFNQFGPNCGQRMPRARYGLVHVAN
NNYDPWTIYAIGGSSNPTILSEGNSFTAPNESYKKQVTIRIGCKTSSSCSNWVW
QSTQDVFYNGAYFVSSGKYEGGNIYTKKEAFNVENGNATPQLTKNAGVLTCS
LSKRC
1076242 Cry j II precursor - Japanese cedar
MAMKLIAPMAFLAMQLIIMAAAEDQSAQIMLDSVVEKYLRSNRSLRKVEHSR
HDAINIFNVEKYGAVGDGKHDCTEAFSTAWQAACKNPSAMLLVPGSKKFVVN
NLFFNGPCQPHFTFKVDGIIAAYQNPASWKNNRIWLQFAKLTGFTLMGKGVID
GQGKQWWAGQCKWVNGREICNDRDRPTAIKFDFSTGLIIQGLKLMNSPEFHL
VFGNCEGVKIIGISITAPRDSPNTDGIDIFASKNFHLQKNTIGTGDDCVAIGTGSS
NIVffiDLICGPGHGISIGSLGRENSRAEVSWHVNGAKFIDTQNGLRIKTWQGGS
GMASHIIYENVEMNSENPILINQFYCTSASACQNQRSAVQIQDVTYKNIRGTSA
TAAAIQLKCSDSMPCKDIKLSDISLKLTSGKIASCLNDNANGYFSGHVIPACKNL
SPSAKRKESKSHKHPKTVMVENMRAYDKGNRTRILLGSRPPNCTNKCHGCSP
CKAKLVIVHRIMPQEYYPQRWICSCHGKIYHP
1076241 Cry j Pprotein - Japanese cedar
MAMKFIAPMAFVAMQLnMAAAEDQSAQIMLDSDIEQYLRSNRSLRKVEHSRH
DAINIFNVEKYGAVGDGKHDCTEAFSTAWQAACKKPSAMLLVPGNKKFVVN
NLFFNGPCQPHFTFKVDGIIAAYQNPASWKNNRJWLQFAKLTGFTLMGKGVID
GQGKQWlVAGQCKWWGREICNDRDRPTAIKFDFSTGLnQGLKLMNSPEFHL
VFGNCEGVIOIGISITAPRDSPNTDGIDIFASKNFHLQKNTIGTGDDCVAIGTGSS
NIVIEDLICGPGHGISIGSLGRENSRAEVSYVHVNGAKFIDTQNGLRIKTWQGGS
GMASHnYENVEMINSENPILINQFYCTSASACQNQRSAVQIQDVTYKNIRGTSA
TAAAIQLKCSDSMPCKDIKLSDISLKLTSGKIASCLNDNANGYFSGHVIPACKNL
SPSAKRKESKSHKHPKTVMVKNMGAYDKGNRTRILLGSRPPNCTNKCHGCSP
CKAKLVIVHRIMPQEYYPQRWMCSRHGKIYHP
541803 Cry j I precursor - Japanese cedar
MDSPCLVALLVLSFVIGSCFSDNPIDSCWRGDSNWAQNRMKLADCAVGFGSS
TMGGKGGDLYTVTNSDDDPVNPPGTLRYGATRDRPLWIIFSGNMNIKLKMPM
YIAGYKTFDGRGAQVYIGNGGPCVFKRVSNVIIHGLHLYGCSTSVLGNVLINES
FGVEPVHPQDGDALTLRTATNIWIDHNSFSNSSDGLVDVTLSSTGVTISNNLFF
NHHKVMLLGHDDAYSDDKSMKVTVAFNQFGPNCGQRMPRARYGLVHVANN
NYDPWTIYAIGGSSNPTILSEGNSFTAPNESYKKQVTIRIGCKTSSSCSNWVWQS
TQDVFYNGAYFVSSGKYEGGNIYTKKEAFNVENGNATPQLTKNAGVLTCSLS
KRC
541802 Cryj I precursor- Japanese cedar
MDSPCLVALLVFSFVIGSCFSDNPIDSCWRGDSNWAQNRMKLADCAVGFGSST
MGGKGGDLYTVTNSDDDPVNPAPGTLRYGATRDRPLWIIFSGNMNIKLKMPM
YIAGYKTFDGRGAQVYIGNGGPCVFIKRVSNVIIHGLYLYGCSTSVLGNVLINES
FGVEPVHPQDGDALTLRTATNIWIDHNSFSNSSDGLVDVTLTSTGVTISNNLFF
NHKVMSLGHDDAYSDDKSMKVTVAFNQFGPNCGQRMPRARYGLVHVANN
NYDPWTIYAIGGSSNPTELSEGNSFTAPNESYKKQVTIRIGCKTSSSCSNWVWQS
TQDVFYNGAYFVSSGKYEGGNIYTKKEAFNVENGNATPHLTQNAGVLTCSLS
KRC
Delivery methods
Once formulated the compositions of the invention can be delivered to a subject
in vivo using a variety of known routes and techniques. For example, a composition
can be provided as an injectable solution, suspension or emulsion and administered via
parenteral, subcutaneous, epidermal, intradermal, intramuscular, intraarterial,
intraperitoneal, intravenous injection using a conventional needle and syringe, or using
a liquid jet injection system, or using a patch. Compositions can also be administered
topically to skin or mucosal tissue, such as nasally, intratracheally, intestinal, rectally
or vaginally, or provided as a finely divided spray suitable for respiratory or pulmonary
administration. Other modes of administration include oral administration,
suppositories, sublingual administration, and active or passive transdermal delivery
techniques.
Where a peptide of the invention is to be administered, it is preferred to
administer the peptide to a site in the body where it will have the ability to contact
suitable antigen presenting cells, and where it, or they, will have the opportunity to
contact T cells of the individual. Where an APC is to be administered, it is preferred to
administer the APC to a site in the body where it will have the ability to contact, and
activate, suitable T cells of the individual.
Delivery regimes
Administration of the peptides/polynucleotides/cells (such as the composition
containing a plurality of peptides) may be by any suitable method as described above.
Suitable amounts of the peptide may be determined empirically, but typically are in the
range given below. A single administration of each peptide may be sufficient to have a
beneficial effect for the patient, but it will be appreciated that it may be beneficial if the
peptide is adrninistered more than once, in which case typical administration regimes
may be, for example, once or twice a week for 2-4 weeks every 6 months, or once a
day for a week every four to six months. As will be appreciated, each peptide or
polynucleotide, or combination of peptides and/or polynucleotides may be
administered to a patient singly or in combination.
Dosages for administration will depend upon a number of factors including the
nature of the composition, the route of administration and the schedule and timing of
the administration regime. Suitable doses of a molecule of the invention may be in the
order of up to 15mg, up to 0mg, up to 5 g, up to 30mg, up to 50mg, up to 100mg, up to
500 g or more per administration. Suitable doses may be less than 5 g, but at least
lng, or at least 2ng, or at least 5ng, or at least 50ng, or least lOOng, or at least 500ng, or
at least 1mg, or at least 10mg. For some molecules of the invention, the dose used may
be higher, for example, up to 1 mg, up to 2 mg, up to 3 mg, up to 4 mg, up to 5 mg or
higher. Such doses may be provided in a liquid formulation, at a concentration suitable
to allow an appropriate volume for administration by the selected route.
Kits
The invention also relates to a combination of components described herein
suitable for use in a treatment of the invention which are packaged in the form of a kit
in a container. Such kits may comprise a series of components to allow for a treatment
of the invention. For example, a kit may comprise one or more different peptides,
polynucleotides and/or cells of the invention, or one or more peptides, polynucleotides
or cells of the invention and one or more additional therapeutic agents suitable for
simultaneous administration, or for sequential or separate administration. The kit may
optionally contain other suitable reagent(s) or instructions and the like.
The invention is illustrated by the following Examples:
Example 1
MHC Class II binding search
The aim of this study is to identify a distinct panel of peptides with strong
affinities for the eight most common human MHC Class PHLA-DRB1 * allotypes. In
order to identify binding peptides in the major birch allergens Bet vl, Bet v2, Bet v3,
Bet v4, Bet v6, an in silico approach was carried out using the commercially available
EpiMatrix algorithm (EpiVax Inc.) This is a bioinformatic analysis of peptides from a
sequence for the potential to be accommodated within the binding groove of MHC
class PHLA-DR molecules.
EpiMatrix is a matrix-based algorithm that ranks 9 amino acid residue
sequences, overlapping by 8 amino acids, from any polypeptide sequence by estimated
probability of binding to each of the selected MHC molecules. (De Groot et al., AIDS
Research and Human Retroviruses 13:539-41 (1997). The procedure for developing
matrix motifs was published by Schafer et al, Vaccine 16:1880-4 (1998). In this
Example, binding potential for HLA DR1, DR3, DR4, DR7, DR8, DR11, DR13 and
DR15 is assessed. Putative MHC ligands are selected by scoring each 9-mer frame in a
protein sequence. This score is derived by comparing the sequence of the 9-mer to the
matrix of amino acid sequences known to bind to each MHC allele. Retrospective
studies have demonstrated that EpiMatrix accurately predicts published MHC ligands
(Jesdale et al., in Vaccines '97 (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.,
1997)). Successful Prediction of peptides which bind to multiple MHC molecules has
also been confirmed.
The EpiMatrix data for each allergen is shown either:
As overlapping 9 mer peptide data with the binding (Z) score for each allele
and the number of 'hits' for the eight alleles (Z scores of equal to or greater than the
top 5% of predicted binders): or
As a cluster report where the data from analysing multiple sequences from the
database is 'clustered' to give an overview of binding for all variants of the protein.
The "EpiMatrix hits' refers to the number of high predicted Z binding scores for the
eight alleles within that sequence whilst the "EpiMatrix Cluster Score" is derived from
the number of hits normalized for the length of the cluster. Cluster Score is thus the
excess or shortfall in predicted aggregate MHC binding properties relative to a random
peptide standard. A cluster score above 10 is considered to indicate broad MHC
binding properties.
EpiMatrix analyses were performed on the entire sequences of known isoforms
of Bet vl, listed below with their corresponding NCBI accession numbers:
Bet vl L P43185; Bet vl E P43178;
Bet vl M P43186; Bet vl D/H P43177;
Betvl P43184; Bet vl C P43176;
Bet vl J P43183; Bet vl B P45431;
Bet vl G P43180; Bet vl A PI 5494;
Bet vl F/I P43179;
Epimatrix analyses were also performed on additional known Bet vl sequences
indexed by accession number in Table 2.
These analyses identified core peptides (and their flanking sequences) derived
from the above sequences which are predicted to have good MHC class-Pbinding.
These sequences are shown below in Tables 1 and 2. As shown, many of the peptides
identified are highly conserved between different Bet vl isoforms.
In Tables 1 and 2:
"Residues in main sequence" gives the location of the peptide within the sequences
that were analysed. The core peptide (underscored middle amino acids in bold) defines
the actual binding sequence that was identified during the analysis. The stabilizing
flanks (N-terminal and C-terminal, not bold) were included for use with the core
sequence and are typically required to aid manufacture of the peptides. "Number of
hits" refers to the number of high predicted binding affinities for all MHC types tested
within the sequence. The "EpiMatrix Cluster Score" is derived from the number of hits
normalized for the length of the cluster. Cluster Score is thus the excess or shortfall in
predicted aggregate MHC binding properties relative to a random peptide standard. A
score above 10 is considered to indicate broad MHC binding properties.

Example 2
EpiMatrix analyses as above were performed on the entire sequence of a known
isoform of Bet v 3 (NCBI accession no: P43187). These analyses identified a core
peptide (and its flanking sequence) derived from the above sequence which is predicted
to have good MHC class-Pbinding. The sequence is shown below in Table 3.
Headings and notes for Table 3 are as with Table 1 above.
A sequence at residues 80 to 94 of P43 187, TVKSFTREGNIGLQF (Peptide ID NO.
P55, SEQ ID NO: 36), was also predicted to have good MHC-Class Pbinding.
Additional in silico analysis of other birch allergen sequences from Bet v 3 is shown
here:
Example 3
EpiMatrix analyses as above were performed on the entire sequence of known
isoforms of Bet v 4 (NCBI accession nos: Q39419, CAA73 147). These analyses
identified core peptides (and their flanking sequences) derived from the above
sequences which are predicted to have good MHC class-II binding. These sequences
are shown below in Table 4. Headings and notes for Table 4 are as with Table 1
above.
Example 4
EpiMatrix analyses as above were performed on the entire sequence of a known
isoform of Bet v 6 (NCBI accession no: 065002). These analyses identified core
peptides (and their flanking sequences) derived from the above sequences which are
predicted to have good MHC class-Pbinding. These sequences are shown below in
Table 5. Headings and notes for Table 5 are as with Table 1 above.
Example 5
EpiMatrix analyses as above were performed on the entire sequence of a known
isoform of Bet v 7 (NCBI accession no: CAC841 16). These analyses identified a core
peptide (and its flanking sequence) derived from the above sequence which is predicted
to have good MHC class-U binding. This sequence is shown below in Table 6.
Headings and notes for Table 6 are as with Table 1 above.
Table 6 - Bet v 7
Example 5A
Additional in silico analysis of other birch allergen sequences from Bet v 2 is
shown here:
Example 6
Based on the analyses performed in Examples 1 to 5A, the following peptides
shown in Table 7 were designed for screening in subsequent assays. The design
process involved modification of native sequences to enhance solubility and other
physicochemical characteristics. For example, for Bir12A, residues in parent 62-77R
indicates that the peptide sequence of Birl2A corresponds to residues 62 to 77 of the
parent sequence, with an additional R residue added to the C terminus to improve
solubility. Similarly, for Bir0lF, G, H and I, residues in parent 4-1 8K indicates that
these peptide sequences correspond to residues 4 to 1 of the parent sequence, with an
additional K residue added to the C terminus to improve solubility.
Example 7
In vitro binding analysis
The peptides identified as being potential MHC Class II-binding are prescreened
for solubility in an aqueous, acidic milieu and the peptides are tested in an in
vitro MHC Class II binding assay.
Methods
The assay employed is a competitive MHC class II binding assay, wherein each
peptide is analysed for its ability to displace a known control binder from each of the
human MHC class II allotypes investigated. The allotypes and control peptides used in
this study are typically those shown below:
Each of the peptides from Tables 1 to 7 are analysed in the competition assay and
screened for relative binding compared to the control peptides. Due to the nature of the
competitive assay the data for each peptide is determined as a ratio of its own IC50 to
that of the control peptide. Thus, a peptide that has an IC50 value that is parity to the
control peptide has an identical binding affinity, while peptides with a ratio less than
one have a higher affinity and those with a ratio greater than one have a lower affinity.
Solubility in aqueous solution is an essential criterion for a peptide to be an
effective therapeutic agent. Therefore, as a consequence of the solubility screen very
hydrophobic peptides with a high frequency of large hydrophobic amino acid residues
in multiple binding registers will be eliminated. This is a characteristic of promiscuous
HLA-DRB1* binders. Peptides which bind to one or more of the MHC Class II
allotypes are identified. It would be expected that such peptides would have the ability
to bind similar allotypes that have not been tested through the homology of MHC
structures.
Example 8
The following methods are applied to the same peptides as in Example 7.
Cellproliferation assay
The cell proliferation assay is performed on PBMC's (140xl0 6 cells required
for all parameters to be tested). Proliferation is measured by the incorporation of the
radiolabelled compound 3H-thymidine. In more detail, IOOmI of the appropriate
antigen or peptide concentration is distributed into the appropriate wells of 96 well
plates. The plates are then placed into a humidified 5% C02 incubator set at 37°C for a
maximum of 4 hours. PBMC's isolated as described above are prepared to a
concentration of 2x 106 cells/ml in complete medium at room temperature. 1OOmI of
cell solution is then distributed into each of the wells of the 96 well plates containing
antigen/peptide. The plates are then incubated for 6 to 8 days. The cultures are pulsed
with tritiated thymidine solution by adding IOmI of tritiated thymidine stock solution
( 1.85MBq/ml in serum-free RPMI medium) to each well. The plates are then returned
to the incubator for between 8 and 1 hours. Cultures are then harvested using a
Canberra Packard FilterMate 196 cell harvester. Dried filter mats are counted using an
appropriate beta scintillation counter.
Counts from wells containing peptide are compared statistically to wells
containing media alone (12 wells per group). The non-parametric Mann-Whitney test is
used. The same statistical test is used for all subjects. A statistically significant
difference between media only wells and peptide-stimulated wells is considered a
positive stimulation of PBMC's by the peptide.
Cytokine release assay
The 36 peptides were manufactured at small scale (approximately lOmg batch
size, non-GMP). The purity of each peptide was at least 95% by HPLC. 96 well culture
plates containing peptides and controls (the negative control was culture medium and
the positive controls were staphylococcal enterotoxin B (SEB) 25ng/ml and whole
birch pollen allergen extract 100 mg/ml) were prepared in advance and stored at -20°C
prior to the day of assay. Peptides were added to wells in a volume of 100 mΐ
containing peptides at a concentration of 200 ύ, such that subsequent addition of
100 mΐ of cells would create a final assay concentration of 100 m
Peripheral blood mononuclear cells (PBMCs) were isolated from heparinised
blood by Ficoll density gradient centrifugation. A 100 mΐ aliquot of a 5x1 06 cell/ml
PBMC suspension was then added to each well and the plates placed in a humidified
5% C02 incubator at 37°C for 5 days. Following stimulation, culture supematants
(100 mΐ) were harvested for testing by multiplex bead assay.
Multiplex cytokine bead assays (IL-10, IL-13, Interferon gamma (IFN-g)) were
performed on thawed culture supematants according to the manufacturer's instructions.
Single measurements were performed for each culture supernatant sample. After
completion of the multiplex assay, individual cytokine levels were determined by
interpolation from the standard curve generated in the assay. A positive result was
taken as being greater than 100 pg/ml for the EL-13 and EFN-g assays or >4 times the
background for the EL- 10 assays. The number of responders out of 47 birch allergic
subjects tested was calculated for each peptide for the three cytokines. Results for EL-
13 or EFN-g are summarized in Table 9.
Peptides which induce positive response in a high proportion of subjects are
desirable for inclusion in a vaccine. As shown, the top performing peptides were
Bir02J (top of the 02 series), Bir Oil (top of the 0 1 series) and Birl2B. The core of any
vaccine should ideally contain these peptides. The second best performing peptides
were Bir04 and Bir09 (top of the 09 series) which may be added to the core mixture of
Bir02J, BirOlI and Birl2B. The third best performing peptides were Bir07, Bir07C,
Birl 1 and Bir16A. Additional peptides from this group may be added to the vaccine
mixture to further increase coverage. Birl 5 was the fourth best performing peptide and
may also be added to the vaccine mixture. In terms of other peptides in the various
series, BirOlF, 01G or 01H, in that order of preference, are useful variants of BirOlI;
Bir02E, 02G, 021 or lastly 02D are useful variants of Bir02J; Bir09A, 09B or lastly
09C are useful variants of Bir09; and Bir16B is a useful variant of Bir A. A possible
preferred mixture would therefore include Bir02J, BirOlI, Birl2B, Bir04, Bir09,
Bir07C and Birl6A. Birl 1 and/or Birl 5 may also be included, or alternatively
substituted for Bir07C and/or Birl6A.
In terms of IL-10 release, BirOlI, listed above as one of the top 3 peptides for
IL- 13 or IFN-g production, induced IL- 10 responses in 49% of individuals. Bir02I also
induced IL-10 production in a high proportion of individuals (43%). Inclusion of a
strong IL-10 inducing peptide may assist in the induction of tolerance following
vaccination.
Example 9 - Solubility screening
A Introduction
B)
Solubility Testing
A series of matrices containing 260mM trehalose and spanning a pH range of
3.0 to 7.0 plus a solution modified with 2m HCI were prepared as indicated in
Appendix 2. The solubility of each of the nine peptides was evaluated in each of the
matrices in accordance with Appendix 1. Where solubility was achieved initially, but
the peptide precipitated out of solution subsequently then an additional quantity of the
relevant matrix was added to try and achieve solubility of the peptide at ca. 200 mM.
Details of the Birch reference peptides used are indicated in Table 9-2. All
peptides were manufactured by Bachem AG, Bubendorf, Switzerland.
C) Results
The results of the solubility screening are displayed in tables 9-3 to 9-8 below:

Example 9 - Annex 1
PEPTIDE SOLUBILITY STUDIES
Solubility Methodology
The formulation vehicles were prepared and measurement of pH taken.
• Weighing of peptides.
- Approximately 1mg was required for each evaluation.
- Materials were dispensed into containers suitable for subsequent solubility
evaluation , i.e. clear glass HPLC vials (with screw cap) .
· Evaluation of solubility (for each matrix).
- Aliquots of matrix (50 to 100~L) were added as required .
- The peptide solubility was interpreted by visual inspection.
- The description of the sample characteristics following addition of each aliquot
of the solvent was recorded.
- Repeat visual assessment of solubility after 24 hours.
• Where a peptide precipitated out of solution after 24 hours, additional buffer was
added to produce a final concentration of ca. 0.2mM (200nmol per mL should equate
to roughly 0.35mg/mL).
• Calculation of peptide solubilities (initial evaluation).
- Based on absolute amount of powder weighed.
- Determination of molar concentration at which solubility was achieved using
peptide molecular masses and peptide content and purity values.
Calculations
Solubility mg/ml 'as is' = weight(mg) x 1000
dilution( ml)
Solubility mg/ml = weight(mg x 1000 x %Content x %Purity
dilution( ml)
Solubility mmoI/ mI = weight(mg) x 1000 x %Content x %Purity x 1__ x 1000
dilution( ml) MolWt
Example 9 - Annex 2
BUFFERS FOR INITIAL SOLUBILITY AND STABILITY SCREENING
Each matrix was prepared at a concentration of lOmM of the buffering agent. Each
buffer contained 260mM Trehalose dihydrate (FW 378.3).
Preparation of matrix
The procedure indicated is for the preparation of lOOmL of each buffer, but
alternative volumes can be prepared by adjusting the quantities.
· 0.1M stock solutions of sodium citrate and potassium dihydrogen phosphate were
prepared.
• Weight of trehalose dihydrate equivalent to 260mM was transferred to an appropriate
mixing vessel containing 70-80mL of distilled deionised water and allowed to dissolve.
• lOmLof the appropriate stock 0.1M buffer solution was added to the mixing vessel
and stirred .
• The pH of the matrix was adjusted to the desired value by adding 2mM hydrochloric
acid or 0. M sodium hydroxide as required.
• The solutions were finally diluted to lOOg weight and the pH re-assessed.
Buffers for initial solubility and stability screening, shown as Buffer salt or pH
modifier / pH:
2mM HC1 and 260mM trehalose dihydrate/ pH 2.65
lOmM sodium citrate and 260mM trehalose dihydrate/ pH 3.01
lOmM sodium citrate and 260mM trehalose dihydrate/ pH 3.99
lOmM sodium citrate and 260mM trehalose dihydrate/ pH 5.02
1OmM sodium citrate and 260mM trehalose dihydrate/ pH 6.0
lOmM potassium dihydrogen phosphate and 260mM trehalose dihydrate/ pH 6.03
lOmM potassium dihydrogen phosphate and 260mM trehalose dihydrate/ pH 7.03
Example 10 - Histamine release assay
The purpose of this assay was to identify compositions that are capable of
activating blood basophils (as a surrogate for tissue mast cells) resulting in histamine
release that may result in allergic reactions during therapy. A composition comprising
a mixture of peptides that induce histamine release frequently may be considered
unsuitable for use as a vaccine.
Histamine release requires the crosslinking of adjacent specific IgE molecules
on the surface of the basophil. The peptides being evaluated were small ( 11 to 18
amino acids in length) and should not, therefore, possess significant tertiary structure
that would enable them to retain the conformation of an IgE-binding epitope of the
whole molecule. Furthermore, peptide monomers in solution, even if they are bound by
IgE, should not be able to crosslink adjacent IgE molecules.
Histamine release from fresh peripheral whole blood from birch allergic
subjects was evaluated. Peripheral blood basophils were used as a surrogate for tissue
mast cells which were not practical to assay. Blood was incubated in vitro with
mixtures of peptides identified as suitable based on the results of Examples 1 to 9
above. Specifically, the following mixtures were assayed:
Mix 1 -BIR01I, BIR02J, BIR04, BER.12B, BIR16A, BIR07C
Mix 2 - BEROII, BIR02J, BIR04, BIR12B, B 6A, BIR07C, BIR09
Mix 3 - BIR01I, BIR02J, BIR04, BIR12B, BIR16A, BIR07C, BIR09B
Mix 4 - BEROII, BIR02I, BIR04, BIR12B, BIR16A, BI 07C
Mix 5 - BIR01I, BIR02I, BIR04, BIR12B, BIR16A, BIR07C, BIR09
Mix 6 - BIR01I, BIR02I, BIR04, BIR12B, BIR16A, BIR07C, BIR09B
Histamine release in response to whole birch allergen extract was measured in
each subject to confirm basophil sensitisation. A positive control, representing total
Wstamine release, generated by freeze/thawing the cells twice, was included in each
assay. A negative control for spontaneous histamine release was generated by
incubating cells in buffer only.
The assay was performed using the Immunotech Histamine Release
Immunoassay kit according to the manufacturer's instructions. Following the in vitro
challenge of blood basophils with peptide mixtures, whole allergen or buffer in
microtitre plate wells, supematants were removed and the histamine in the samples
converted to acyl Mstamine. Acylated samples were tested by a competitive acyl
histamine ELISA.
Peptide mixtures were assayed for their ability to induce histamine release over
a 5 log 10 range ( 1 to 10,000 ng m ). The concentration range assayed was selected
based on theoretical in vivo doses of peptide that may be achieved during therapy. For
example, a 1m dose (approximately 3 nmol/peptide equivalent) of each peptide
entering a blood volume of 5 litres, would result in a blood concentration of 6ng/ml, at
the lower end of the histamine release assay dose range. The whole birch allergen
extract was used over the same concentration range.
Single measurements were performed for each dilution. After completion of the
ELISA, individual histamine levels were determined by interpolation from the standard
curve generated in the ELISA assay. Results from samples were adjusted to allow for
dilution. Where two or more consecutive dilutions of a peptide/allergen preparation
elicited >15% of the total histamine release seen in the freeze thawed positive control
(>15% of positive control), or where a single value of >15% of positive control was
achieved at the highest concentration tested (10 g/mL for peptides), this was
considered a "positive histamine release".
A total of 40 histamine release assays were completed during the study. Of
these 5 assays were rejected because of failure to meet appropriate QC controls, e.g.
due to unacceptably high levels (>15% of positive control) of spontaneous release in
the medium plus buffer negative control wells.
The mixtures tested all showed good histamine release properties. The study
findin s are summarised as follows: WA = whole aller en
CLAIMS
1. A composition suitable for use in preventing or treating allergy to birch pollen
by tolerisation comprising:
i) at least one of the polypeptides of SEQ ID NO: 74 (BIR12B;
AKYMVIQGEPGRVIRGK), SEQ ID NO: 72 (BIR1 1; FPQFKPQEITGMK), SEQ ID
NO: 7 1 (BIR10; GSVWAQSSSFPQFK), SEQ ID NO: 73 (BIR12A;
PTGMFVAGAKYMVIQGR), SEQ ID NO: 75 (BIR13; IKYMVIQGEAGAVTRGK
and SEQ ID NO: 76 (BIR14; EAGAVIRGKKGSGGIT), or a variant of any thereof,
and
ii) at least one of the polypeptides of SEQ ID NO: 53 (Bir02J;
PAARMFKAFILEGDKLVPK), SEQ ID NO: 48 (BirOlI; FNYETETTSVIPAARK),
SEQ ID NO: 54 (Bir04; PGTJKKISFPEGFPFKYV), SEQ ID NO: 67 (Bir09;
ETLLRAVESYLLAHSDAY), SEQ ID NO: 60 (BIR07; SNEJKTVATPDGGSILK),
and SEQ ID NO: 63 (Bir07C; SNEIKIVATPEGGSILK), or a variant of any thereof,
wherein said variant is:
H) a longer polypeptide of up to 30 amino acids in length which comprises
the sequence of the corresponding polypeptide specified in (i) or (ii), or
P) a polypeptide of 9 to 30 amino acids in length which comprises a
sequence that has at least 65% homology to the sequence of the corresponding
polypeptide specified in (i) or (ii), which sequence is capable of tolerising to
said corresponding polypeptide; or
PI) a polypeptide of length 9 to 30 amino acids which comprises a
sequence of at least 9 contiguous amino acids of the sequence of the
corresponding polypeptide specified in (i) or (ii), or a sequence that has at least
65% homology to said at least 9 contiguous amino acids, which sequence of at
least 9 contiguous amino acids or homologous sequence
is capable of tolerising to said corresponding polypeptide.
2. A composition according to claim 1, further comprising at least one additional
polypeptide of (i) or (ii) or variant thereof not selected in claim 1.
3. A composition according to claim 1 or 2, further comprising at least one
additional polypeptide of SEQ ID NO: 77 (BIR15; SLNTLRLRRIFDLFDK ) or SEQ
ID NO: 78 (BIR16A; AERERIFKRFDANGEGK), or a variant of any thereof.
4. A composition according to any one of the preceding claims comprising:
(a) the polypeptide Bir 12B (AKYMVIQGEPGRVIRGK), or a variant thereof;
(b) the polypeptide Bir02J (PAARMFKAFILEGDKLVPK), or a variant thereof;
and
(c) the polypeptide Bir01I (FNYETETTSVIPAARK) or a variant thereof;
5. A composition according to any one of the preceding claims, wherein:
said variant of BirOlI is BirOlF (FNYETEATSVIPAARK), BirOlG
(FNYEIEATSVIPAARK) or BirO 1H (FNYEIETTSVIPAARK); and/or
said variant of Bir02J is Bir02E (PAARLFKAF-LEGDTLIPK), Bir02G
(PAARLFKAFILEGDNLIPK), Bir02I (PAARMFKAFILD) or Bir02D
(PAARMFKAFILDGDKLVPK); and/or
said variant of Bir09 is selected from Bix09A (GETLLRAVES YLLAHS),
Bir09B (KEMGETLLRAVESYLLAHS) or Bir09C (KEKGETLLRAVESYLLAHS);
and/or
said variant of Birl6A is Birl6B (AERERIFKRFDAGGEGK).
6. A composition suitable for use in preventing or treating allergy to birch pollen
by tolerisation comprising at least three different polypeptides, selected from:
(a) Bir 12B (AKYMVIQGEPGRVIRGK), or a variant thereof;
(b) Bir02J (PAARMFKAFILEGDKLVPK), or a variant thereof;
(c) Bir0 11(FNYETETTSVIPAARK) or a variant thereof;
(d) Bir04 (PGTIKKISFPEGFPFKYV) or a variant thereof;
(e) Bir09 (ETLLRAVESYLLAHSDAY) or a variant thereof;
(f) Bir1 6A (AERERIFKRFDANGEGK) or a variant thereof;
(g) Bir07 (SNEJKIVATPDGGSILK) or a variant thereof;
(h) Bir07C (SNEIKIVATPEGGSILK) or a variant thereof;
(i) BirO 11 (FPQFKPQEITGIMK) or a variant thereof;
(j) Birl 5 (SLNTLRLRRIFDLFDK) or a variant thereof;
wherein said variant is:
I) a longer polypeptide of up to 30 amino acids in length which comprises
the sequence of the corresponding polypeptide specified in (a) to (j), or
P) a polypeptide of 9 to 30 amino acids in length which comprises a
sequence that has at least 65% homology to the sequence of the corresponding
polypeptide specified in (a) to (j), which sequence is capable of tolerising to
said corresponding polypeptide; or
PG) a polypeptide of length 9 to 30 amino acids which comprises a
sequence of at least 9 contiguous amino acids of the sequence of the
corresponding polypeptide specified in (a) to (j), or a sequence that has at least
65% homology to said at least 9 contiguous amino acids, which sequence of at
least 9 contiguous amino acids or homologous sequence
is capable of tolerising to said corresponding polypeptide.
7. A composition according to claim 6 comprising:
a) the polypeptide Bir 1 B (AKYMVIQGEPGRVIRGK) or a variant thereof;
b) at least one of the polypeptides Bir02J (PAARMFKAFILEGDKLVPK) and
BirOlI (FNYETETTSVIPAARK), or a variant of any thereof; and
c) at least one additional polypeptide of a) to j ) not selected above.
8. A composition according to claim 7, comprising the polypeptide Birl2B
(AKYMVIQGEPGRVIRGK) or a variant thereof, the polypeptide Bir02J
(PAARMFKAFILEGDKLVPK) or a variant thereof, the polypeptide BirOlI
(FNYETETTSVIPAARK) or a variant thereof, and at least one additional polypeptide
of a) to j ) not selected above.
9. A composition according to any one of claims 6 to 8, comprising the
polypeptide Birl2B (AKYMVIQGEPGRVIRGK) or a variant thereof, the polypeptide
Bir02J (PAARMFKAFILEGDKLVPK) or a variant thereof, the polypeptide BirO 1
(FNYETETTSVIPAARK) or a variant thereof, the polypeptide Bir04
(PGTIKKISFPEGFPFKYV) or a variant thereof, the polypeptide Bir09
(ETLLRAVESYLLAHSDAY) or a variant thereof, the polypeptide Bir07C
(SNEIKIVATPEGGSILK) or a variant thereof, and the polypeptide Birl6A
(AERERIFKRFDANGEGK) or a variant thereof; and optionally no further
polypeptides.
10. A composition according to any one of the claims 6 to 8, comprising the
polypeptide Bir12B (AKYMVIQGEPGRVIRGK) or a variant thereof, the polypeptide
Bir02J (PAARMFKAFILEGDKLVPK) or a variant thereof, the polypeptide BirOlI
(FNYETETTSVrPAARK) or a variant thereof, the polypeptide Bir04
(PGTIKKISFPEGFPFKYV) or a variant thereof, the polypeptide Bir07C
(SNEIKTVATPEGGSILK)or a variant thereof, the polypeptide Birl6A
(AERERIFKRFDANGEGK) or a variant thereof, and the polypeptide Bir09B
(KEMGETLLRAVESYLLAHS) or a variant thereof, and optionally no further
polypeptides.
11. A composition according to any one of the claims 6 to 8, comprising the
polypeptide Birl2B (AKYMVIQGEPGRVIRGK) or a variant thereof, the polypeptide
Bir02J (PAARMFKAFILEGDKLVPK) or a variant thereof, the polypeptide Bir01I
(FNYETETTSVIPAARK) or a variant thereof, the polypeptide Bir04
(PGTIKKISFPEGFPFKYV) or a variant thereof, the polypeptide Bir07C
(SNEIKTVATPEGGSILK)or a variant thereof, and the polypeptide Birl6A
(AERERIFKRFDANGEGK) or a variant thereof, and optionally no further
polypeptides.
1 . A composition according to any one of the claims 6 to 11, wherein:
said variant of BirOlI is BirOlF (FNYETEATSVIPAARK), BirOlG
(FNYEIEATSVIPAARK) or BirOlH (FNYEIETTSVIPAARK); and/or
said variant of Bir02J is Bir02E (PAARLFKAFILEGDTLIPK), Bir02G
(PAARLFKAFILEGDNLIPK), Bir02I (PAARMFKAFILD) or Bir02D
(PAARMFKAFILDGDKLVPK); and/or
said variant of Bir09 is selected from Bir09A (GETLLRAVESYLLAHS),
Bir09B (KEMGETLLRAVESYLLAHS) or Bir09C (KEKGETLLRAVESYLLAHS);
and/or
said variant of Birl 6A is Bir16B (AERERIFKRFDAGGEGK) .
13. The composition according to any one of the preceding claims, wherein the
composition:
- is capable of tolerising at least 50% or at least 60% of a panel of birch pollen allergic
individuals in the population and/or
- comprises at least one further polypeptide up to a total of thirteen unique/different
polypeptides, wherein the further polypeptides:
(a) comprise a sequence having at least 65% sequence identity to at least 9 or more
contiguous amino acids in any of SEQ ID NOs: 1 to 80 not selected above; and
(b) are 9 to 30 amino acids in length.
14. The composition according to any one of the preceding claims, comprising at
least one said polypeptide which is 9 to 20 or 13 to 17 amino acids in length and/or
wherein said polypeptide has at least 70% sequence identity to any of SEQ D NOS: 1
to 80.
15. The composition according to any one of the preceding claims, wherein one or
more of the polypeptides have one or more modifications selected from the following:
(i) N terminal acetylation;
(ii) C terminal amidation;
(iii) one or more hydrogens on the side chain amines of Arginine and/or Lysine
replaced with a methylene group;
(iv) glycosylation; and
(v) phosphorylation.
16. The composition according to any one of the preceding claims wherein at least
one of the peptides has been engineered to be soluble such that it comprises:
i) N terminal to the residues of the peptide which flank a T cell epitope: one to six
contiguous amino acids corresponding to the two to six contiguous amino acids
immediately N terminal to said residues in the sequence of the protein from which the
peptide derives; and/or
ii) C terminal to the residues of the peptide which flank a T cell epitope: one to six
contiguous amino acids corresponding to the one to six contiguous amino acids
immediately C terminal to the said residues in the sequence of the protein from which
the peptide derives; or
iii) both N and C terminal to the residues of the peptide which flank a T cell epitope,
at least one amino acid selected from arginine, lysine, histidine, glutamate and
aspartate,
wherein the polypeptide has a solubility of at least 3.5mg/ml and the T cell epitope has
a solubility of less than 3.5mg/ml.
7. The composition according to any one of the preceding claims wherein at least
one of the peptides has been engineered to be soluble such that additionally:
i) any cysteine residues in the native sequence of the peptide are replaced with
serine or 2-aminobutyric acid; and /or
ii) any hydrophobic residues in the upto three amino acids at the N or C terminus of
the native sequence of the peptide, which are not comprised in a T cell epitope, are
deleted; and/or
iii) any two consecutive amino acids comprising the sequence Asp-Gly in the upto
four amino acids at the N or C terminus of the native sequence of the peptide, which
are not comprised in a T cell epitope, are deleted; and/or
iv) one or more positively charged residues are added at the N and/or C terminus of
the native sequence of the peptide.
18. The composition according to any one of the preceding claims wherein each
polypeptide has a concentration in the range of 0.03 to 200 nmol/ml, 0.3 to 200
nmol/ml, 50 to 200nmol/ml or 30 to 120 nmol/ml.
19. The composition according to any one of the preceding claims further
comprising a pharmaceutically acceptable carrier or diluent and/or optionally one or
more adjuvants selected from a glucocorticoid, vitamin D and/or rapamycin, and/or
comprising no further peptides.
20. A composition for use in preventing or treating allergy to birch pollen by
tolerisation comprising at least one polynucleotide sequence which when expressed
causes the production of a composition as defined in any one of claims 1 to 17.
21. A vector for use in preventing or treating allergy to birch pollen by
tolerisation comprising four or more polynucleotide sequences which encode a
different polypeptide as defined in any one of claims 1 to 17.
22. A product comprising:
i) at least one of the polypeptides of SEQ ID NO: 74 (BIR12B;
AKYMVIQGEPGRVIRGK), SEQ ID NO: 72 (BIRl 1; FPQFKPQEITGIMK), SEQ ID
NO: 7 1 (BIR10; GSVWAQSSSFPQFK), SEQ ID NO: 73 (BIR12A;
PTGMFVAGAKYMVIQGR), SEQ ID NO: 75 (BIRD; EKYMVIQGEAGAVIRGK
and SEQ D NO: 76 (BIR14; EAGAVIRGKKGSGGIT), or a variant of any thereof as
defined in claim 1 (I) to (III), and
ii) at least one of the polypeptides of SEQ ID NO: 53 (Bir02J;
PAARMFKAFILEGDKLVPK), SEQ ID NO: 48 (BirOlI; FNYETETTSVIPAARK),
SEQ ID NO: 54 (Bir04; PGTKKISFPEGFPFKYV), SEQ ID NO: 67 (Bir09;
ETLLRAVESYLLAHSDAY), SEQ ID NO: 60 (BIR07; SNEIKIVATPDGGSILK),
and SEQ ID NO: 63 (Bir07C; SNEIKIVATPEGGSILK), or a variant of any thereof as
defined in claim 1 (I) to (III),
wherein each different polypeptide is for simultaneous, separate or sequential use in
preventing or treating allergy to birch pollen by tolerisation.
23. A pharmaceutical formulation for use in preventing or treating allergy to
birch pollen by tolerisation comprising a composition according to any one of claims 1
to 20; a vector according to claim 2 1; or a product according to claim 22; and a
pharmaceutically acceptable carrier or diluent, and optionally one or more adjuvants
selected from a glucocorticoid, vitamin D and rapamycin.
24. The formulation according to claim 23, formulated for oral administration,
nasal administration, topical administration, subcutaneous administration, sublingual
administration, intradermal administration, buccal administration, epidermal
administration, or for administration by inhalation, by injection, or by a patch.
25. The composition as defined in any one of claims 1to 20 or product as
defined in claim 22, additionally comprising a further polypeptide allergen for use in
tolerising an individual to the further polypeptide allergen.
26. An in vitro method of detemnning whether T cells recognize a composition as
defined in claim 1 comprising contacting said T cells with said composition and
detecting whether said T cells are stimulated by said composition.
27. A method according to claim 26 which is carried out to determine whether an
individual has, or is at risk of having, an allergy to birch