The present invention relates to polypeptides and pharmaceutical formulations
which may be used for preventing or treating allergy to moulds of the Alternaria and/or
Cladosporium genus.
Background to the Invention
Mould allergens are recognised as a major cause of allergic diseases in humans
and animals, including asthma, allergic rhinitis, allergic conjunctivitis and allergic
dermatitis. In colder climates, moulds can be found in the outdoor air starting in the late
winter, and peaking in the late summer to early fall months (July to October). In warmer
climates, mould spores may be found throughout the year, with the highest levels found
in the late summer to early fall months. While indoor moulds can occur year round and
are dependent on moisture levels in the home, indoor mould levels are higher when
outdoor mold levels are higher. Therefore, a common source of indoor mould is from
the outside environment, although can also be from indoor mould contamination.
There are thousands of types of mould; however, only a few of these are
commonly associated with allergy. The following are the most likely causes of allergic
disease based on the types of mould spores collected in the air: Alternaria,
Cladosporium, Aspergillus, Penicillium, Helminthosporum, Epicoccum, Fusarium,
Aureobasidium, Phoma, Rhizopus, Mucor, Smuts and Yeasts. Moulds in the genus
Alternaria, in particular Alternaria Alternata, and the genus Cladosporium are
considered to be among the most important allergenic fungi.
Cladosporium is the most common airborne outdoor mould. Alternaria is one of
the main allergens affecting children. In temperate climates, airborne Alternaria spores
are detectable from for most of the year (typically May to November in the northern
hemisphere), with peaks in late summer and autumn. Dispersion of Alternaria spores
occurs during dry periods. These feature higher wind velocity and lower relative
humidity, which result in peak dispersion during sunny afternoon periods
Although considered to be an outdoor mould, Alternaria will grow anywhere
that provides sufficient moisture and a suitable growth substrate. Accordingly,
Alternaria is commonly found indoors, in particular in damp areas such as basements,
kitchens or bathrooms. Alternaria is commonly found in refrigerator drip trays, air
conditioners, waste containers, mattresses, foam rubber pillows, or even in condensation
on windows. It is one of the most common mould spores found in house dust in both
North America and Europe. It is effectively impossible to avoid Alternaria allergens.
Summary of the Invention
The invention provides a pharmaceutical formulation comprising a
pharmaceutically acceptable carrier or diluent and a polypeptide or a pharmaceutically
acceptable salt thereof selected from at least three of:
(a) a polypeptide comprising the amino acid sequence of
WSWKIGPAIATGNT (Alt28; SEQ ID NO: 101) or a T cell epitope-containing variant
sequence derived from said amino acid sequence, or a said salt thereof;
(b) a polypeptide comprising the amino acid sequence of
KYRRVVRAGVKVAQTAR (Alt34A; SEQ ID NO: 107) or a T cell epitope-containing
variant sequence derived from said amino acid sequence, or a said salt thereof;
(c) a polypeptide comprising the amino acid sequence of
KYAGVFVSTGTLGGG (SEQ ID NO: 112) or a T cell epitope-containing variant
sequence derived from said amino acid sequence, or a said salt thereof ;
(d) a polypeptide comprising the amino acid sequence of
AEVYQKLKALAKKTYGQ (Alt 13A; SEQ ID NO: 83) or a T cell epitope-containing
variant sequence derived from said amino acid sequence, or a said salt thereof;
(e) a polypeptide comprising the amino acid sequence of
SLGFNIKATNGGTLD (AltOlA; SEQ ID NO: 60) or a T cell epitope-containing
variant sequence derived from said amino acid sequence, or a said salt thereof;
(f) a polypeptide comprising the amino acid sequence of
SAKRMKVAFKLDIEK (Alt06; SEQ ID NO: 72) or a T cell epitope-containing variant
sequence derived from said amino acid sequence, or a said salt thereof;
(g) a polypeptide comprising the amino acid sequence of
DITYVATATLPNYCR (SEQ ID NO: 111) or a T cell epitope-containing variant
sequence derived from said amino acid sequence, or a said salt thereof; and
(h) a polypeptide comprising the amino acid sequence of
GWGVMVSHRSGET (Alt 14; SEQ ID NO: 84) or a T cell epitope-containing variant
sequence derived from said amino acid sequence, or a said salt thereof;
wherein a T cell epitope-containing variant sequence of a said amino acid
sequence is said amino acid sequence having up to seven amino acid modifications,
each of which is independently a deletion, substitution or insertion, and each
polypeptide is up to 30 amino acids in length.
The invention further provides a pharmaceutical formulation of the invention for
use in a method of treating or preventing allergy to Cladosporium and/'or Alternaria.
The invention also provides a method of treating an individual for allergy to
Alternaria and/or Cladosporium or of preventing in an individual allergy to Alternaria
and/or Cladosporium, which method comprises administering to said individual a
therapeutically or prophylactically effective amount of a pharmaceutical formulation of
the invention.
The invention further provides use of the at least three polypeptides or salts
described above for the manufacture of a medicament for the prevention or treatment of
allergy to Alternaria and/or Cladosporium.
The invention additionally provides an in vitro method of determining whether T
cells recognise a polypeptide of a pharmaceutical formulation of the invention, which
method comprises contacting said T cells with said pharmaceutical formulation and
detecting whether said T cells are stimulated by a said polypeptide.
The invention also provides a method of preparing a pharmaceutical
formulation of the invention, comprising combining at least three polypeptides or salts
described above with a pharmaceutically acceptable carrier or diluent.
The invention further provides a polypeptide, or a pharmaceutically acceptable
salt thereof, which is up to 30 amino acids in length and comprises:
(I) the amino acid sequence:
(a) WSWKIGPAIATGNT (Alt28; SEQ ID NO: 101),
(b) KYRRVVRAGVKVAQTAR (Alt34A; SEQ ID NO: 107), or
(c) KYAGVFVSTGTLGGG (SEQ ID NO: 112); or
(II) a T cell epitope-containing variant sequence which is a said amino acid
sequence (I) having up to seven amino acid modifications, each of which is
independently a deletion, substitution or insertion.
The invention also provides a polypeptide or salt of the invention for use in a
method of treating or preventing allergy to Alternaria and/or Cladosporium, and use of
a polypeptide or salt of the invention for the manufacture of a medicament for the
prevention or treatment of allergy to Alternaria and/or Cladosporium. The invention
additionally provides method of treating an individual for allergy to Alternaria and/or
Cladosporium or of preventing in an individual allergy to Alternaria and/or
Cladosporium, which method comprises administering to said individual a
therapeutically or prophylactically effective amount of a polypeptide or salt of the
invention.
Description of the sequences
SEQ ID NOS: 1 to 118 provide amino acid sequences as set out in Examples 1
to 10. In more detail:
SEQ ID NOS: 1 to 6, 59 to 65 and 111 correspond to amino acid sequences
derived from protein Alt al .
SEQ ID NOS: 7 to 11, 66 to 78 and 113 correspond to amino acid sequences
derived from protein Alt a2.
SEQ ID NOS: 12 to 22 and 79 to 87 correspond to amino acid sequences
derived from protein Alt a6.
SEQ ID NOS: 23 to 29, 88, 89, 112 and 114 correspond to amino acid sequences
derived from protein Alt a7.
SEQ ID NOS: 30 to 35 and 90 to 98 correspond to amino acid sequences derived
from protein Alt a8.
SEQ ID NOS: 36 to 48 and 99 to 101 correspond to amino acid sequences
derived from protein Alt a10.
SEQ ID NOS: 49 to 58, 102 to 107 and 115 correspond to amino acid sequences
derived from protein Alt a13.
SEQ ID NOS: 108 to 110 correspond to amino acid sequences derived from
homologues of the above Alternaria proteins.
SEQ ID NOS: 116 to 118 correspond to amino acid sequences of control
polypeptides used in Example 9 .
NCBI accession numbers for the proteins referred to above are provided in
Examples 1 to 10.
Detailed Description of the Invention
The present invention is concerned with preventing or treating allergy to
Cladosporium and/or Alternaria and provides combinations of polypeptides, and
pharmaceutically acceptable salts thereof, suitable for this use. The combinations of
polypeptides or salts may be provided in pharmaceutical formulations.
Amino acid sequences and variant amino acid sequences
A polypeptide of the invention may preferably comprise, consist or consist
essentially of an amino acid sequence as shown in any one of:
(a) WSWKIGPAIATGNT (Alt28; SEQ ID NO: 101),
(b) KYRRVVRAGVKVAQTAR (Alt34A; SEQ ID NO: 107),
(c) KYAGVFVSTGTLGGG (Altl 8; SEQ ID NO: 112),
(d) AEVYQKLKALAKKTYGQ (Alt 13A; SEQ ID NO: 83),
(e) SLGFNIKATNGGTLD (AltOlA; SEQ ID NO: 60),
(f) SAKRMKVAFKLDIEK (Alt06; SEQ ID NO: 72),
(g) DITYVATATLPNYCR (SEQ ID NO: 111), or
(h) GWGVMVSHRSGET (Alt 14; SEQ ID NO: 84);
Other polypeptides of the invention comprise, consist or consist essentially of
an amino acid sequence as shown in:
(i) IEKLRSNITVQYDI (Alt33; SEQ ID NO: 105),
0) SAFRSIEPELTVY (Alt 10; SEQ ID NO: 77),
(k) GYTGKIKIAMD VASSE (Alt15; SEQ ID NO: 86),
Alternatively, a polypeptide included in a pharmaceutical formulation of the
invention may comprise, consist or consist essentially of a T cell epitope-containing
variant sequence which is an amino acid sequence as shown in any one of (a) to (k)
having up to seven amino acid modifications, each of which is independently a deletion,
substitution or insertion.
It is preferred that the modifications in a variant sequence do not alter the
functional properties of a T cell epitope present in the corresponding original amino
acid sequence. The functional properties of T cell epitopes are discussed further below.
In preferred variant sequences, sufficient contiguous amino acids of the
corresponding original amino acid sequence are retained to contain a T cell epitope.
Typically, such a variant sequence retains at least 8, preferably at least 9, contiguous
amino acids of the original amino acid sequence. The variant sequence may retain from
8 to 12 amino acids or from 9 to 12 amino acids of the original amino acid sequence.
A variant sequence may have fewer than seven amino acid modifications. For
example, said variant sequence may have up to six or up to five amino acid
modifications, preferably up to four said amino modifications, more preferably up to
three amino acid modifications, and most preferably only one or two amino acid
modifications. All said modifications are independently a deletion, substitution or
insertion.
In a particularly preferred embodiment, the variant sequence has one or two
amino acid modifications, the or each of which independently is a deletion or
substitution.
Deletions
Where a T cell epitope-containing variant sequence has an amino acid
modifications that is a deletion, the deleted amino acid is preferably removed from the
N- or C-terminus of the corresponding original amino acid sequence. That is, the
variant sequence is a truncation of the original amino acid sequence formed by
removing one or more contiguous amino acids from the N- and/or C-terminus of the
original sequence. Such a variant sequence may optionally have no other deletions or
no other modifications.
A deleted amino acid may less preferably be removed from an internal position
in the corresponding original amino acid sequence. By removal from an internal
position it is meant that a deleted amino acid is not itself at the N- or C-terminus of the
original amino acid sequence and nor is it removed as part of a sequence of contiguous
amino acids including the N- or C-terminus of the original amino acid sequence. That
is, to be considered to be deletion from an internal position, said deletion must occur
independently of deletion from the N- or C-terminus of the original amino acid
sequence.
For example, given an original sequence ABCDEFGH, an example variant
sequence having an internal deletion of two amino acids could be ADEFGH, that is B
and C are removed from internal positions and the original terminal residues A and H
are retained. By contrast, a deletion of two contiguous amino acids from the Nterminus
of the same original sequence would result in the variant sequence CDEFGH,
in which A and B are removed and C is now at the N-terminus. The deletion of B in
this case is not a removal from an internal position, because it is removed as one of the
two contiguous amino acids including the N-terminus of the original sequence.
Where more than one deletion occurs in a variant sequence, the deleted amino
acids may be removed from any combination of the N-terminus and/or the C-terminus
and/or an internal position. Preferred variant sequences have no more than one deletion
from an internal position. In particularly preferred variant sequences there is no deletion
from an internal position, and the deleted amino acids are removed from any
combination of the N- and/or C-terminus of the original sequence. That is, the deleted
amino acids may all be removed from the N-terminus of the original sequence, or they
may all be removed from the C-terminus of the original sequence, or some amino acids
may be removed from each end of the original sequence.
Thus, in one embodiment, a variant sequence is an amino acid sequence of any
one of (a) to (k) having one, two, three, four, five, six or seven amino acids removed
from the N-terminus of said sequence of (a) to (k).
In another embodiment, a variant sequence is an amino acid sequence of any one
of (a) to (k) having one, two, three, four, five, six or seven amino acids removed from
the C-terminus of said sequence of (a) to (k).
In another embodiment, a variant amino acid sequence is an amino acid
sequence of any one of (a) to (k) having a number of amino acids removed from both
the N- and C-terminus of said sequence, provided that said sequence has no more than
six modifications in total. A preferred embodiment of such a variant sequence is an
amino acid sequence of any one of (a) to (k) having one, two or three amino acids
removed from the N- and/or C-terminus of said sequence, and optionally no other
modifications.
Specific examples of variant amino acid sequences which have at least one
deletion include:
the variant sequence QKLKALAKKTYGQ (SEQ ID NO: 18), which is
the amino acid sequence of AEVYQKLKALAKKT YGQ (SEQ ID NO: 83) having four
amino acids removed from the N-terminus;
the variant sequence DITYVATATLPNY (SEQ ID NO: 5) which is the
amino acid sequence of DITYVATATLPNYCR (SEQ ID NO: 111) having two amino
acids removed from the C terminus;
the variant sequence RVVRAGVKVAQTA (SEQ ID NO: 58). which is
the amino acid sequence of KYRRVVRAGVKVAQTAR (SEQ ID NO: 107) having
three amino acids removed from the N-terminus and one amino acid removed from the
C terminus; and
the variant sequence YEKYRRVVRAGVKV (SEQ ID NO: 106) which
is the amino acid sequence of KYRRVVRAGVKVAQTAR (SEQ ID NO: 107) having
five amino acid residues removed from the C-terminus, and an N-terminal extension of
two amino acids corresponding to the two contiguous amino acids immediately Nterminal
to KYRRVVRAGVKVAQTAR in the native sequence of Alt a 13.
Substitutions
Where a T cell epitope-containing variant sequence has an amino acid
modification that is a substitution, the substitution may occur at any position in the
original amino acid sequence. It is preferred that said substitution does not introduce a
proline or a cysteine. It is also preferred that said substitution is a conservative
substitution.
By conservative substitution, it is meant that an amino acid may be substituted
with any alternative amino acid having similar properties. The following is a nonexhaustive
list of examples:
The amino acids with basic side chains, such as lysine, arginine or histidine,
may each be independently substituted for each other.
The amino acids with acidic side chains, such as aspartate and glutamate, may
each be independently substituted for each other, or for their amide derivatives,
asparagine and glutamine. A glutamate or glutamine may also preferably be replaced
with pyroglutamate. A variant sequence having pyroglutamate substituted for glutamate
or glutamine is particularly preferred where said pyroglutamate will correspond to the
N-terminus of a polypeptide of the invention which comprises, consists or consists
essentially of the variant sequence. Polypeptides with pyroglutamate at the N-terminus
typically have improved stability during manufacture.
The amino acids with aliphatic side chains, such as glycine, alanine, valine,
leucine and isoleucine, may each be independently substituted for each other.
Particularly preferred substitutions in this category are limited to the amino acids with
smaller aliphatic side chains, that is glycine, alanine, valine, which may preferably each
be independently substituted for each other.
Other preferred substitutions include the substitution of methionine with
norleucine (Nle).
Additionally, in more general terms, a neutral amino acid may be substituted
with another neutral amino acid, a charged amino acid may be substituted with another
charged amino acid, a hydrophilic amino acid may be substituted with another
hydrophilic amino acid, a hydrophobic may be substituted with another hydrophobic
amino acid, a polar amino acid may be substituted with another polar amino acid, and
an aromatic amino acid may be substituted with another aromatic amino acid. Some
properties of the 20 main amino acids which can be used to select suitable substituents
are as follows:
Specific examples of variant amino acid sequences which have at least one
substitution are:
The variant sequence SAKR-Nle-KVAFKLDIEK (SEQ ID NO: 73) is
the amino acid sequence of SAKRMKVAFKLDIEK (SEQ ID NO: 72) having one
substitution. The amino acid M at position 5 of SEQ ID NO: 72 is substituted with
norleucine; and
The variant sequence DITYVATATLPNYSR (SEQ ID NO: 62) is the
amino acid sequence of DITYVATATLPNYCR (SEQ ID NO: 111) having one
substitution. The amino acid C at position 14 of SEQ ID NO: 111 is substituted with
Serine. Other preferred variant sequences of SEQ ID NO: 111 include sequences with
alternative, preferably similar, substitutions at position 14. For example, instead of
substituting with S, the C amino acid at position 14 could be replaced with T, G, A or
V.
In some variant sequences there may be substitutions and deletions. Specific
examples are:
the variant sequence AEVYQKLKSLTK (SEQ ID NO: 108) is the
amino acid sequence of AEVYQKLKALAKKTYGQ (SEQ ID NO: 83) having five
deletions at the C-terminus and two substitutions made at positions 9 (Ser for Ala) and
11 (Thr for Ala). Other preferred variant sequences of SEQ ID NO: 83 include
sequences with alternative, preferably similar, substitutions at positions 9 and 11. For
example, instead of substituting with S and T, both the A amino acids at positions 9 and
11 could be independently replaced with G or V; and
the variant sequence of SAKR-Nle-KVAFK (SEQ ID NO: 113) is the
amino acid sequence of SAKRMKVAFKLDIEK (Alt06; SEQ ID NO: 72) having five
deletions at the C-terminus and one substitution made at position 5 (Nle for Met).
Insertions
Where a variant sequence has an amino acid modification that is an insertion,
the added amino acid may be inserted at any position in the original amino acid
sequence. It is preferred that the insertion does not introduce a proline or a cysteine.
Preferably, an amino acid may be inserted at the N-terminus and/or C-terminus
of the original sequence. That is, the variant sequence is an extension of the original
amino acid sequence formed by adding amino acids to the N- and/or C-terminus of the
original sequence. Such a variant sequence may optionally have no other insertions or
no other modifications.
Less preferably, an amino acid may be inserted at an internal position. By
insertion at an internal position it is meant that an amino acid is inserted at any position
which is C-terminal to the amino acid at the N-terminus of the original sequence, or that
an amino acid is inserted at any position which is N-terminal to the amino acid at the Cterminus
of the original sequence.
Where more than one insertion occurs in a variant sequence, the added amino
acids may be inserted at any combination of the N-terminus and/or the C-terminus
and/or an internal position. Preferred variant sequences have no more than one insertion
at an internal position. In particularly preferred variant sequences there is no insertion at
an internal position, and the added amino acids are inserted at any combination of the
N- and/or C-terminus of the original sequence. That is, the added amino acids may all
be inserted at the N-terminus of the original sequence, or they may all be inserted at the
C-terminus of the original sequence, or some amino acids may be inserted at each end
of the original sequence. That is, the added amino acids may be considered to extend
the original sequence at the N- and/or C-terminus.
Thus, in one embodiment, a variant sequence is an amino acid sequence of any
one of (a) to (k) having one, two, three, four, five, six or seven amino acids inserted at
the N-terminus of said sequence of (a) to (k).
In another embodiment, a variant sequence is an amino acid sequence of any one
of (a) to (k) having one, two, three, four, five, six or seven amino acids inserted at the
C-terminus of said sequence of (a) to (k).
In another embodiment, a variant sequence is an amino acid sequence of any one
of (a) to (k) having a number of amino acids inserted at both the N- and C-terminus of
said sequence of (a) to (k), provided that said sequence has no more than seven
modifications in total. A preferred embodiment of such a variant sequence is an amino
acid sequence of any one of (a) to (k) having one, two or three amino acids inserted at
the N- and/or C-terminus of said sequence of (a) to (k), and optionally no other
modifications.
A variant sequence having a charged amino acid inserted at the N- and/or Cterminus
is particularly preferred where said charged amino acid will correspond to the
N- and/or C- terminus of the polypeptide of the invention which comprises, consists or
consists essentially of the variant sequence. Charged residues at the N- and/or Cterminus
of a polypeptide can improve the solubility of a polypeptide. Preferred
charged amino acids include lysine, arginine and histidine. Lysine is particularly
preferred. Thus, a particularly preferred variant sequence is an amino acid sequence of
any one of (a) to (k) having one or more charged amino acids, preferably one or more
lysine residues, inserted at the N- and/or C-terminus of said sequence of (a) to (k).
Specific examples of variant amino acid sequences which have at least one
insertion include:
the variant sequence KSAFRSIEPELTVYK (SEQ ID NO: 78), which is
the amino acid sequence of SAFRSIEPELTVY (SEQ ID NO: 77) having a lysine
inserted at the N-terminus and at the C terminus; and
the variant sequence KKYAGVFVSTGTLGGGK (SEQ ID NO: 89),
which is the amino acid sequence of KYAGVFVSTGTLGGG (SEQ ID NO: 112)
having a lysine inserted at the N-terminus and at the C terminus.
Polypeptides
A polypeptide included in a pharmaceutical formulation of the invention is up to
30 amino acids in length and comprises, consists or consists essentially of an amino acid
sequence or variant sequence as defined above.
Said polypeptide may preferably be up to 25 amino acids in length, more
preferably up to 20 amino acids in length or up to 17 amino acids in length, and most
preferably up to 15 amino acids in length. Put another way, the polypeptide may have a
maximum length of 30, 25, 20, 17 or 15 amino acids.
A polypeptide included in a pharmaceutical formulation of the invention is
preferably at least 8 amino acids in length, more preferably at least 9 amino acids in
length, most preferably at least 12 amino acids in length. Put another way, the
polypeptide may have a minimum length of 8, 9, or 12 amino acids.
A polypeptide included in a pharmaceutical formulation of the invention may be
of a length defined by any combination of a said minimum and a said maximum length.
For example, the polypeptide may be 8 to 30, 8 to 25, 8 to 20, 8 to 17 or 8 to 15 amino
acids in length. The polypeptide may be 9 to 30, 9 to 25, 9 to 20, 9 to 17 or 9 to 15
amino acids in length. The polypeptide may be 12 to 30, 12 to 25, 12 to 20, 12 to 17 or
12 to 15 amino acids in length. A preferred polypeptide is of 9 to 30 amino acids in
length, more preferably 9 to 20 amino acids in length. A particularly preferred
polypeptide is of 12 to 17 amino acids in length.
A polypeptide included in a pharmaceutical formulation of the invention may
comprise an amino acid sequence or variant sequence as defined above. Therefore, said
polypeptide may include additional amino acids which are not defined by said amino
acid sequence or variant sequence. The additional amino acids may be described as
flanking said amino acid sequence or variant sequence. That is, the additional amino
acids are included at the N-terminus and/or C-terminus of said amino acid sequence or
variant sequence.
Put another way, a polypeptide included in a pharmaceutical formulation of the
invention may have a sequence consisting of said amino acid sequence or variant
sequence having an N-terminal and/or C-terminal extension of a number of amino acids.
The maximum number of amino acids in the N-terminal and/or C-terminal extension is
determined by the maximum length of the polypeptide, as defined above.
The amino acids in an N-terminal extension of a said amino acid sequence or
variant sequence preferably correspond to the amino acids immediately N-terminal to
the said amino acid sequence in the native sequence of the protein from which it
derives.
The amino acids in a C-terminal extension of a said amino acid sequence or
variant sequence preferably correspond to the amino acids immediately C-terminal to
the said amino acid sequence in the native sequence of the protein from which it
derives.
The N-terminal and/or C-terminal extension may be the one, two, three, four,
five, six, seven, eight, nine or ten amino acids corresponding respectively to the one,
two, three, four, five, six, seven, eight, nine or ten contiguous amino acids immediately
N- terminal or C-terminal to said amino acid sequence in the sequence of the protein
from which it derives.
That is, the N-terminal and/or C-terminal extension is of from one to ten amino
acids corresponding respectively to the one to ten contiguous amino acids immediately
N-terminal or C-terminal to the said amino acid sequence in the native sequence of the
protein from which it derives.
Preferably, the N-terminal and/or C-terminal extension is of from one to six
amino acids corresponding respectively to the one to six contiguous amino acids
immediately N-terminal or C-terminal to the said amino.
More preferably, the N-terminal and/or C-terminal extension is of from one to
four amino acids corresponding respectively to the one to four contiguous amino acids
immediately N-terminal or C-terminal to the said amino.
Most preferably, the N-terminal and/or C-terminal extension is of from one to
two amino acids corresponding respectively to the one to two contiguous amino acids
immediately N-terminal or C-terminal to the said amino acid sequence.
Specific examples of polypeptides which include an N-terminal and/or Cterminal
extension to an amino acid sequence or variant sequence include the following:
AEVYQKLKALAKKTYGQ (SEQ ID NO: 83) may have an N-terminal and/or
a C-terminal extension of one, two, three, four, five or six amino acids corresponding
respectively to the one, two, three, four, five or six contiguous amino acids immediately
N- or C-terminal to AEVYQKLKALAKKTYGQ in the native sequence of Alt a 6, that
is the amino acids E, A, M, R, Q and G at the N-terminus and S, A,G, N, V and G at the
C-terminus. For example, where a one amino acid extension is present both at the Nand
C-terminus, the polypeptide of the invention has the amino acid sequence of
GAEVYQKLKALAKKTYGQS (SEQ ID NO: 12; N- and C-terminal extensions are
underlined).
SLGFNIKATNGGTLD (SEQ ID NO: 60) may have an N-terminal and/or a Cterminal
extension of one, two, three, four, five or six amino acids corresponding to the
one, two, three, four, five or six contiguous amino acids immediately N-and/or Cterminal
to SLGFNIKATNGGTLD in the native sequence of Alt a 1. For example,
where six contiguous amino acids are present in the N-terminal extension and two
contiguous amino acids are present in the C-terminal extension, the polypeptide of the
invention has the amino acid sequence of EGTYYNSLGFN ATNGGTLDFT (SEQ
ID NO: 2; N- and C-terminal extensions are underlined).
lEKLRSNITVQYDI (SEQ ID NO: 105) may have a N-terminal extension of
one, two or three amino acids corresponding to the one, two, or three contiguous amino
acids immediately N-terminal to lEKLRSNITVQYDI in the native sequence of Alt a
13, that is the amino acids P, K and T. It may also have a C-terminal extension of one,
two or three amino acids corresponding to the one, two or three contiguous amino acids
immediately C-terminal to lEKLRSNITVQYDI in the native sequence of Alt a 13. That
is the amino acids L, E and R. For example, where all three contiguous amino acids are
present in the N-terminal extension and all three contiguous amino acids are present in
the C-terminal extension the polypeptide of the invention has the amino acid sequence
of PKTIEKLRSNIT V YDILER (SEQ ID NO: 115; N and C-terminal extensions are
underlined).
GWGVMVSHRSGET (Alt 18; SEQ ID NO: 84) may have a N-terminal
extension of one, two, three, four, five or six amino acids corresponding to the one, two,
three, four, five or six amino acids immediately N-terminal to GWGVMVSHRSGET in
the native sequence of Alt a 7, that is the amino acids K, D, A, F, G and A. It may also
have a C-terminal extension of one, two or three amino acids corresponding to the one,
two, or three contiguous amino acids immediately C-terminal to GWGVMVSHRSGET
in the native sequence of Alt a 7 . That is the amino acids E, D and V. For example,
where all six amino acids are present in the N-terminal extension and all three
contiguous amino acids are present in the C-terminal extension the polypeptide of the
invention has the amino acid sequence of KDA GAGWGVMVSHRSGETEDV (SEQ
15, N- and C-terminal extensions underlined).
The amino acids in the N-terminal and/or C-terminal extension may not
correspond exactly to amino acids in the native sequence of the protein from which an
amino acid sequence or variant sequence derives. The N-terminal and/or C-terminal
extension may include a sequence derived from said native sequence which has been
modified, for example to improve stability, solubility or manufacturability of the
polypeptide. For example, a methionine in the native sequence may be substituted with
nor-leucine, and/or one or more charged residues may be added at the N-terminus of a
N-terminal extension and/or the C-terminus of a C-terminal extension. Preferably
positively charged residues such as arginine and lysine are added. Amino acids selected
from histidine, glutamate and aspartate may be added.
Alternatively, the amino acids of an N-terminal and/or C-terminal extension may
not correspond to amino acids in native sequence of the protein from which an amino
acid sequence or variant sequence derives. They may instead be any suitable amino
acids, preferably selected to improve stability, solubility or manufacturability of the
polypeptide. For example, one or more charged residues may be added at the N and/or
C terminus of any of the amino acid sequences or variant sequences of the invention.
Preferably positively charged residues such as arginine and lysine are added. Amino
acids selected from histidine, glutamate and aspartate may be added.
T cell epitopes
A polypeptide included in a pharmaceutical formulation of the invention is up to
30 amino acids in length and comprises, consists or consists essentially of an amino acid
sequence or variant sequence as defined above. Each said amino acid sequence and said
variant sequence contains a T cell epitope. The T cell epitope is preferably an MHC
Class II-binding T cell epitope. It is preferred that the modifications in a variant
sequence do not alter the functional properties of a T cell epitope present in the
corresponding original amino acid sequence.
In preferred variant sequences, sufficient contiguous amino acids of the
corresponding original amino acid sequence are retained to contain a T cell epitope.
Typically, such a variant sequence retains at least 8, preferably at least 9, contiguous
amino acids of the original amino acid sequence.
The presence of a T cell epitope may preferably be confirmed by analysis
performed in silico, for example using bioinformatic software as described in Examples
1 to 5 . Alternatively, the presence of a T cell epitope may be confirmed by direct
evaluation of its functional properties. Particular functional properties of T cell epitopes
include the ability of a polypeptide comprising the epitope to bind to an MHC molecule,
preferably an MHC Class II molecule, and/or the ability of a polypeptide comprising the
epitope to activate a T cell, preferably when bound to an MHC Class II molecule.
The ability of a polypeptide to bind to an MHC molecule may be evaluated
using any suitable method, such as a competition assay. A preferred in vitro assay is
described in Example 6 .
The ability of a polypeptide to activate a T cell may also be evaluated using any
suitable method. Preferred methods include the measurement of one or more
parameters associated with T cell activation, such as proliferation or cytokine release.
Preferred assays for these parameters are described in Example 7 . Relevant cytokines
include IFN-gamma, IL-13 and IL-10. In the context of the present invention, a
polypeptide is typically considered to have activated a T cell if it induces release of one,
two, or all of IFN-gamma, IL-13 and IL-10. The polypeptide preferably induces a
release of greater than 50 pg/ml of the given cytokine(s).
As mentioned above, it is preferred that the modifications in a variant sequence
do not alter the functional properties of a T cell epitope present in the corresponding
original amino acid sequence. Thus, a polypeptide comprising, consisting or consisting
essentially of a variant amino acid sequence should have substantially the same MHC
class II binding properties and substantially the same T cell activation properties as a
polypeptide comprising, consisting or consisting essentially of the corresponding
original amino acid sequence.
Typically, a polypeptide has substantially the same MHC Class II binding
characteristics as another polypeptide if both polypeptides are capable of binding
specifically to one or more MHC Class II molecules belonging to the same MHC Class
II allele supertype family. Examples of MHC Class II allele supertype families include
HLA-DR1, HLA-DR3, HLA-DR4, HLA-DR7, HLA-DR8, HLA-DR1 1, HLA-DR13,
HLA-DR15 and HLA-DR51. Most preferably, both polypeptides will bind specifically
to the same MHC Class II molecule, that is to an MHC Class II molecule encoded by
the same allele.
Typically, a polypeptide has substantially the same T cell activation properties
as another polypeptide if both polypeptides specifically activate a T cell expressing the
same T cell receptor. Preferably, there should be no significant difference in the level
of activation induced by each polypeptide. The level of activation may be assessed by
monitoring proliferation and/or cytokine release, as described above.
Suitable polypeptides comprising, consisting or consisting essentially a variant
sequence may be derived empirically or selected according to known criteria. Within a
single polypeptide 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). Advantageously,
peptides may be designed to favour T-cell proliferation and induction of desensitisation.
Metzler and Wraith have demonstrated improved tolerogenic capacity of polypeptides
in which substitutions increasing polypeptide-MHC affinity have been made (Metzler &
Wraith(1993) Int Immunol: 1159-65). That an altered polypeptide ligand can cause
long-term and profound energy in cloned T cells was demonstrated by Sloan-Lancaster
et al (1993) Nature 363: 156-9.
Sequence identity
T cell epitope-containing variant sequences of polypeptides included in
pharmaceutical formulations of the invention may alternatively be described in terms of
their sequence identity to a corresponding original amino acid sequence. For example, a
variant sequence may have at least 65% identity to an amino acid sequence of any one
of (a) to (k). More preferably, a variant sequence may have at least 70%, at least 75%, at
least 80% , at least 85%, at least 90%, or at least 95% amino acid identity to an amino
acid sequence of any one of (a) to (k).
Sequence identity is typically evaluated over a number of contiguous amino
acids in the original amino acid sequence. For example, sequence identity may be
measured over at least 9, 10, 11, 12, 13, 14, or 15 contiguous amino acids in the original
amino acid sequence, depending on the size of the peptides of comparison. It is
preferred that sequence identity be measured over at least 9 contiguous amino acids in
the original amino acid sequence. It is particularly preferred that sequence identity is
measured over the entire length of the corresponding original amino acid 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, Residuespecific
gap penalties: on, Hydrophilic gap penalties: on, Hydrophilic residues: G, P, S,
N, D, Q, E, K, and R. Sequence identity at a particular residue is intended to include
identical residues which have simply been derivatized.
Salts
The invention encompasses any pharmaceutically acceptable salt of a
polypeptide of the invention. Pharmaceutically acceptable salts of a polypeptide of the
invention include, for example, mineral acid salts such as chlorides, hydrochlorides,
hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as
acetates, propionates, malonates, benzoates, and the like. A hydrochloride salt or an
acetate salt is preferred.
Synthesis
A polypeptide of the invention can be prepared by any suitable technique.
Solid-phase peptide synthesis (SPPS) is a preferred technique. This involves formation
of the peptide on small solid beads.
Using SPPS, the peptide remains covalently attached to a bead during synthesis.
The peptide is synthesised using repeated cycles of coupling-washing-deprotectionwashing.
In particular, the free N-terminal amine of a solid-phase attached peptide is
coupled to a single N-protected amino acid unit. This unit is then deprotected, revealing
a new N-terminal amine to which a further protected amino acid is attached. These
steps are repeated until the peptide is complete. The peptide is then cleaved from the
beads using a suitable reagent.
Suitable protecting groups, reagents, solvents and reaction conditions for SPPS
are well known to those skilled in the art and as such conditions can be determined by
one skilled in the art by routine optimization procedures.
Pharmaceutically acceptable salts of polypeptides can be prepared by any
suitable technique. Typically, salification involves reaction of the polypeptide or a salt
thereof with a suitable reagent, typically acid, to obtain the pharmaceutically acceptable
salt selected.
For example, a hydrochloride salt of a polypeptide can be prepared by initially
cleaving the polypeptide from the solid phase using trifluoroacetic acid. The
polypeptide will thus initially be a trifluoroacetate salt. The trifluoroacetate salt can
then be converted into a hydrochloride salt by any known technique, such as ion
exchange on a suitable column using hydrochloric acid as an eluent.
The polypeptide or polypeptide salt products can be purified, where required, by
any suitable technique. High pressure liquid chromatography (HPLC) can be used, for
example.
The term "polypeptide" 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 pseudopolypeptides
containing changes involving the backbone, and not the orientation of side chains.
Meziere et al (1997) show that, at least for MHC class II and T helper cell responses,
these pseudopolypeptides are useful. Retro-inverse polypeptides, 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 polypeptides 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 (-NH2— -
NH(Me) or -N(Me)2) .
Analogues of polypeptides according to the invention may also include peptide
variants that increase or decrease the polypeptide's half-life in vivo. Examples of
analogues capable of increasing the half-life of polypeptides used according to the
invention include peptoid analogues of the peptides, D-amino 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-amino 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 polypeptides provided by the present invention may be derived from splice
variants of the parent proteins encoded by mRNA generated by alternative splicing of
the primary transcripts encoding the parent protein chains. The polypeptides may also
be derived from amino acid mutants, glycosylation variants and other covalent
derivatives of the parent allergen proteins. Exemplary derivatives include molecules
wherein the polypeptides 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 variant amino acid
sequences of the parent proteins. Such a variant amino acid sequence may be encoded
by an allelic variant or represent an alternative splicing variant.
Modifications as described above may be prepared during synthesis of the
peptide or by post-production modification, or when the polypeptide is in recombinant
form using the known techniques of site-directed mutagenesis, random mutagenesis, or
enzymatic cleavage and/or ligation of nucleic acids.
The polypeptides described herein may also be modified to improve
physicochemical characteristics. Thus, for example, original amino acid sequences may
be altered to improve their solubility, and accordingly a polypeptide of the invention
having a variant sequence will preferably be more soluble than a polypeptide having the
corresponding original amino acid sequence under equivalent conditions. Methods for
evaluating the solubility of polypeptides are well known in the art.
Improved solubility is advantageous for the tolerisation of subjects to allergens
from which the polypeptides of the invention derive, since administration of poorly
soluble agents to subjects causes undesirable, non-tolerising inflammatory responses.
The solubility of the polypeptides may be improved by altering the residues which flank
the region containing a T cell epitope. For example, N and C terminal to the residues of
the polypeptide which flank a T cell epitope, at least one amino acid may be added
selected from arginine, lysine, histidine, glutamate and aspartate. In other examples:
i) any hydrophobic residues in the up to three amino acids at the N or C terminus
of the native sequence of the polypeptide, which are not comprised in a T cell epitope,
are deleted; and/or
ii) any two consecutive amino acids comprising the sequence Asp-Gly in the up
to four amino acids at the N or C terminus of the native sequence of the polypeptide,
which are not comprised in a T cell epitope, are deleted; and/or
iii) one or more positively charged residues are added at the N and/or C terminus
of the native sequence of the polypeptide.
Optionally, any polypeptides containing cysteine residues may be engineered to
prevent dimer formation such that any cysteine residues are replaced with serine or 2-
aminobutyric acid.
Polypeptide combinations
The invention provides combinations of polypeptides including at least three
polypeptides or salts thereof. Each polypeptide is up to 30 amino acids in length and is
preferably independently selected from the polypeptides of (a) to (h) as set out above.
The combination of polypeptides may also be independently selected from the
polypeptides of (a) to (k) as set out above. Such a combination of peptides is preferably
provided in a pharmaceutical formulation as described below.
Three, four, five, six, seven, eight or more polypeptides selected from the
polypeptides of (a) to (k), or said salts thereof, may be provided together in
combination. It is preferred that only one polypeptide, or salt thereof, is selected from
each of (a) to (k).
More preferably, three, four, five, six, seven, or eight polypeptides selected from
the polypeptides of (a) to (h) or said salts thereof, are provided together in combination.
It is preferred that only one polypeptide, or salt thereof is selected from each of (a) to
(h).
It is particularly preferred that the above combinations of at least three
polypeptides comprise a polypeptide of (a) or a said salt thereof. That is a polypeptide,
or a salt thereof, comprising, consisting essentially or consisting of the amino acid
sequence of (Alt28; SEQ ID NO: 101), or a corresponding variant sequence.
The above combinations of at least three polypeptides may also preferably
comprise at least one polypeptide of (b), (c) or (d), or a said salt thereof. That is a
polypeptide, or a salt thereof, comprising, consisting essentially or consisting of the
amino acid sequence of any one of KYRRVVRAGVKVAQTAR (Alt34A; SEQ ID NO:
107), KYAGVFVSTGTLGGG (SEQ ID NO: 112), or AEVYQKLKALAKKTYGQ
(Altl3A;SEQ ID NO: 83), or a corresponding variant sequence of any thereof.
A preferred variant sequence of SEQ ID NO: 107 is YEKYRRVVRAGVKV
(Alt34, SEQ ID NO: 106). Alt34 may be used in place of SEQ ID: 107 in any
polypeptide combination and pharmaceutical formulation described herein. A preferred
variant sequence of SEQ ID NO: 112 is KKYAGVFVSTGTLGGGK (Alt 18; SEQ ID
NO: 89). Altl8 may be used in place of SEQ ID: 112 in any polypeptide combination
and pharmaceutical formulation described herein.
It is particularly preferred that the above combinations of at least three
polypeptides comprise a polypeptide or salt of (a), (b) and (c). It is also particularly
preferred that the above combinations of at least three polypeptides comprise a
polypeptide of (d) or a said salt thereof. That is a polypeptide, or a salt thereof,
comprising, consisting essentially or consisting of the amino acid sequence of
AEVYQKLKALAKKTYGQ (Altl3A;SEQ ID NO: 83) or a corresponding variant
sequence. Another preferred combination of at least three polypeptides comprises a
polypeptide or salt of (a), (b) and (d).
The above combinations of at least three polypeptides may also preferably
comprise at least one polypeptide of (e) or (f), or a said salt thereof. That is a
polypeptide, or a salt thereof, comprising, consisting essentially or consisting of the
amino acid sequence of SLGFNIKATNGGTLD (AltOlA; SEQ ID NO: 60), or
SAKRMKVAFKLDIEK (Alt06; SEQ ID NO: 72, or a corresponding variant sequence
of either thereof. A preferred variant sequence of Alt06 is SAKR-Nle-KVAFKLDIEK
(Alt06A, SEQ ID NO: 73). Alt06A may be used in place of Alt06 in any polypeptide
combination and pharmaceutical formulation described herein.
The above combinations of at least three polypeptides may also preferably
comprise at least one polypeptide of (e) or (f), or a said salt thereof. That is a
polypeptide, or a salt thereof, comprising, consisting essentially or consisting of the
amino acid sequence of DITYVATATLPNYCR (SEQ ID NO: 111), or
GWGVMVSHRSGET (Alt 14;SEQ ID NO: 84), or a corresponding variant sequence of
any thereof. Preferred variant sequences of SEQ ID NO: 111 are KDITYVATATLPNY
(Alt02; SEQ ID NO: 61) and DITYVATATLPNYSR (Alt02A; SEQ ID NO: 62). Alt02
or Alt02A may be used in place of SEQ ID: 111 in any polypeptide combination and
pharmaceutical formulation described herein. A preferred variant sequence of Altl4 is
GWGV-Nle-VSHRSGET (Alt14A, SEQ ID NO: 85). Altl4A may be used in place of
Alt 14 in any polypeptide combination and pharmaceutical formulation described herein.
The invention also provides a combination of at least three polypeptides
comprising a polypeptide of (d), or a said salt thereof, and at least two further
polypeptides selected from the polypeptides of (a) to (c) and (e) to (h) or said salts
thereof. For example, a combination of the polypeptides of (b), (c) and (d) or salts
thereof may be provided. The invention further provides a combination of at least three
polypeptides comprising a polypeptide of (b), or a said salt thereof, and at least two
further polypeptides selected from the polypeptides of (a), (c) and (d) to (h) or said salts
thereof. The invention additionally provides a combination of at least three polypeptides
comprising a polypeptide of (c), or a said salt thereof, and at least two further
polypeptides selected from the polypeptides of (a), (b) and (d) to (h) or said salts
thereof.
The above combinations of at least three polypeptides may further comprise
at least one additional polypeptide selected from:
(1) a polypeptide comprising the amino acid sequence of
KKVSMAIAKAAAAEK (Altl 1: SEQ ID NO: 79) or a T cell epitope-containing
variant sequence derived from said amino acid sequence;
(m) polypeptide comprising the amino acid sequence of
SYNVAKAGCIHLAK (Alt22; SEQ ID NO: 92) or a T cell epitope-containing variant
sequence derived from said amino acid sequence;
(n) polypeptide comprising the amino acid sequence of
KLWHSMIPMGRDAK (Alt24; SEQ ID NO: 95) or a T cell epitope-containing variant
sequence derived from said amino acid sequence;
(o) polypeptide comprising the amino acid sequence of
KRSLLVFAVRSSMELRK (Alt27; SEQ ID NO: 99) or a T cell epitope-containing
variant sequence derived from said amino acid sequence; and
(p) polypeptide comprising the amino acid sequence of
NWLTLHTAALGPTAK (Alt3 1; SEQ ID NO: 103) or a T cell epitope-containing
variant sequence derived from said amino acid sequence;
wherein a T cell epitope-containing variant sequence of a said amino acid
sequence is as defined above and each polypeptide is up to 30 amino acids in length.
A preferred variant sequence of Altl 1 is Altl 1A (SEQ ID NO:81). A preferred
variant sequence of Alt22 is Alt22A (SEQ ID NO: 93). A preferred variant sequence of
Alt24 is Alt24A (SEQ ID NO: 96). A preferred variant sequence of Alt27 is Alt27A
(SEQ ID NO: 100).
The above combinations of at least three polypeptides may also further comprise
a polypeptide, or a salt thereof, comprising, consisting essentially or consisting of the
amino acid sequence of any one of IEKLRSNITVQYDI (Alt33; SEQ ID NO: 105),
GYTGKIKIAMDVASSE (Altl 5; SEQ ID NO: 86), or SAFRSIEPELTVY (AltlO; SEQ
ID NO: 77), or a corresponding variant thereof, A preferred variant sequence of Altl 5
(SEQ ID NO: 86) is GYTGKIKI A-Nle-DVASSE (Altl 5A, SEQ ID NO: 87).
All of the above selections are subject to the combination preferably comprising
twelve polypeptides in total or less, more typically, ten polypeptides or less, preferably
less than nine, more preferably less than eight polypeptides, such as seven or six
polypeptides or less. The combination may comprise four, five, six or seven
polypeptides. The combination of the invention most preferably comprises six or seven
polypeptides.
A preferred core polypeptide combination is (1):
a polypeptide having the amino acid sequence WSWKIGPAIATGNT
(Alt28; SEQ ID NO: 101), or a corresponding variant sequence, or a said salt
thereof;
a polypeptide having the amino acid sequence KYAGVFVSTGTLGGG
(SEQ ID NO: 112) or a corresponding variant sequence (particularly
preferably Altl 8), or a said salt thereof;
a polypeptide having the amino acid sequence KYRRVVRAGVKVAQTAR
(Alt34A; SEQ ID NO: 107) or a corresponding variant sequence, or a said
salt thereof; and
a polypeptide having the amino acid sequence AEVYQKLKALAKKTYGQ
(Altl3A; SEQ ID NO: 83) or a corresponding variant sequence, or a said salt
thereof.
Preferably, one or both of the following additional polypeptides may be added to
the core polypeptide combination of (1) to create a combination of five or six
polypeptides:
a polypeptide having the amino acid sequence SLGFNIKATNGGTLD
(AltO 1A; SEQ ID NO: 60) or a corresponding variant sequence, or a said salt
thereof; and
- a polypeptide having the amino acid sequence SAKRMKVAFKLDIEK
(Alt06; SEQ ID NO: 72) or a corresponding variant sequence (such as
Alt06A), or a said salt thereof.
Optionally, one or two of the following further additional polypeptides may be
added to the combination of (1) or to the above combination of five or six
polypeptides to create a combination of six, seven or eight polypeptides:
a polypeptide having the amino acid sequence DITYVATATLPNYCR (SEQ
ID NO: 111) or a corresponding variant sequence (such as Alt02 or Alt02A),
or a said salt thereof; and
a polypeptide having the amino acid sequence GWGVMVSHRSGET
(Alt 14; SEQ ID NO: 84) or a corresponding variant sequence (such as
Alt 14A), or a said salt thereof.
Another preferred core polypeptide combination is (2):
a polypeptide having the amino acid sequence WSWKIGPAIATGNT
(Alt28; SEQ ID NO: 101), or a corresponding variant sequence, or a said salt
thereof;
a polypeptide having the amino acid sequence KYAGVFVSTGTLGGG
(SEQ ID NO: 112) (particularly preferably Altl8) or a corresponding
variant sequence, or a said salt thereof;
a polypeptide having the amino acid sequence KYRRVVRAGVKVAQTAR
(Alt34A; SEQ ID NO: 107) or a corresponding variant sequence, or a said
salt thereof;
a polypeptide having the amino acid sequence AEVYQKLKALAKKTYGQ
(Altl3A; SEQ ID NO: 83) or a corresponding variant sequence, or a said salt
thereof; and
a polypeptide having the amino acid sequence SLGFNIKATNGGTLD
(AltO 1A; SEQ ID NO: 60) or a corresponding variant sequence, or a said salt
thereof.
Optionally, one or two of the following further additional polypeptides may be
added to the core combination of (2) to create a combination of six or seven
polypeptides:
- a polypeptide having the amino acid sequence SAKRMKVAFKLDIEK
(Alt06; SEQ ID NO: 72) or a corresponding variant sequence (such as
Alt06A), or a said salt thereof;
a polypeptide having the amino acid sequence DITYVATATLPNYCR (SEQ
ID NO: 111) or a corresponding variant sequence (such as Alt02 or Alt02A),
or a said salt thereof; and
a polypeptide having the amino acid sequence GWGVMVSHRSGET
(Alt 14; SEQ ID NO: 84) or a corresponding variant sequence (such as
Alt 14A), or a said salt thereof.
An especially preferred polypeptide combination comprises Alt28 (SEQ ID NO:
101), or a said salt thereof; Altl8, or a said salt thereof; Alt34A (SEQ ID NO: 107) or a
corresponding variant sequence (such as Alt34), or a said salt thereof; Altl3A (SEQ ID
NO: 83), or a said salt thereof; and AltO 1A (SEQ ID NO: 60), or a said salt thereof.
One, two or three peptides, each selected from one of the following groups of peptides,
may also preferably be added to the especially preferred polypeptide combination: (I)
Alt06 or Alt06A or a said salt of either thereof, (II) Alt02 or Alt02A, or a said salt of
either thereof, and (III) Altl4 or Altl4A, or a said salt of either thereof. The especially
preferred polypeptide combination may comprise a polypeptide from group (I) and a
polypeptide from group (II), a polypeptide from group (I) and a polypeptide from group
(III), or a polypeptide from group (I) and a polypeptide from group (II).
The above polypeptide combinations may be suitable for use in preventing or
treating allergy to Alternaria and/or Cladosporium. The invention provides polypeptide
combinations which are suitable for tolerisation against both Alternaria and
Cladosporium allergens by virtue of representing epitope regions which are
homologous between these moulds. In this aspect, the following polypeptides, or salts
thereof, may preferably be used.
SEQ ID NOs 83 to 87 represent sequences derived from Alt a 6 which contain
epitope regions which are homologous with Cla h 6 . SEQ ID NO: 108 is a sequence
derived from Cla h 6 which contains an epitope region which is homologous with Alt a
6 . SEQ ID NO: 108 may be used in place of, or in addition to any Alt a 6 peptide in a
combination described herein.
SEQ ID NOs 90, 91, and 94 to 98 represent sequences derived from Alt a 8
which contain epitope regions which are homologous with Cla h 8 . SEQ ID NOs: 109
and 110 are sequences derived from Cla h 8 which contains epitope regions which are
homologous with Alt a 8 . One or more of SEQ ID NOs: 109 and 110 may be used in
place of, or in addition to any Alt a 8 peptide in a combination described herein.
SEQ ID NO 101 represents a sequence derived from Alt a 10 which contains an
epitope region which is homologous with Cla h 10. SEQ ID NO: 101 may be used in
place of, or in addition to any Alt a 10 peptide in a combination described herein.
In a broader aspect, the invention provides combinations of polypeptides
including at least three polypeptides each of which is up to 30 amino acids in length and
comprise the amino acid sequence of any of SEQ ID NOs 1 to 110 or a corresponding
variant sequence. Such combinations typically include i) at least one polypeptide
comprising the amino acid sequence of any of SEQ ID NOS: 1 to 6 and 59 to 65 (which
are derived from Alt al), or a corresponding variant sequence; ii) at least one
polypeptide comprising the amino acid sequence of SEQ ID NOS: 7 to 11 and 66 to 78
(which are derived from Alt a2), or a corresponding variant sequence; and iii) at least
one polypeptide comprising the amino acid sequence of SEQ ID NOS: 12 to 22 and 79
to 87 (which are derived from Alt a6), or a corresponding variant sequence, preferably
where in said polypeptide comprises AEVYQKLKALAKKTYGQ (Alt 13A; SEQ ID
NO: 83) or a corresponding variant sequence.
Combining polypeptides, or salts thereof, derived from different Alt allergens
may allow for broad coverage of mould allergy observed in the general population by
providing tolerising epitopes from more than one mould allergen. The above
combinations comprising polypeptides, or salts thereof, from Alt al, Alt a2, and Alt a6
may further comprise:
(iv) at least one polypeptide comprising the amino acid sequence of any of SEQ
ID NOS: 23 to 29 and 88 to 89 (which are derived from Alt a7) or a corresponding
variant sequence, or a said salt thereof; and/or
(v) at least one polypeptide comprising the amino acid sequence of any of SEQ
ID NOS: 30 to 35 and 90 to 98 (which are derived from Alt a8) or a corresponding
variant sequence, or a said salt thereof; and/or
(vi) at least one polypeptide comprising the amino acid sequence of any of SEQ
ID NOS: 36 to 48 and 99 to 101 (which derived from Alt a 10) or a corresponding
variant sequence, or a said salt thereof; and/or
(vii) at least one polypeptide comprising the amino acid sequence of any of SEQ
ID NOS: 49 to 58 and 102 to 107 (which are derived from Alt a 13) or a corresponding
variant sequence, or a said salt thereof.
Non-limiting examples of such combinations include:
(1) a polypeptide or salt comprising AEVYQKLKALAKKTYGQ (Altl3A;SEQ
ID NO: 83) or a corresponding variant sequence, at least two polypeptides or salts
selected from group (ii), and at least two polypeptides or salts selected from group (iii);
(2) a polypeptide or salt comprising AEVYQKLKALAKKTYGQ (Altl3A; SEQ
ID NO: 83) or a corresponding variant sequence, at least one, preferably two
polypeptides or salts selected from group (ii), at least two, preferably two polypeptides
or salts selected from group (iii), and at least one polypeptide or salt selected from
group (vi); and
(3) a polypeptide or salt comprising AEVYQKLKALAKKTYGQ (Altl3A; SEQ
ID NO: 83) or a corresponding variant sequence, at least one, preferably two
polypeptides or salts selected from group (ii), at least two, preferably two polypeptides
or salts selected from group (iii), and at least one, preferably two polypeptides or salts
selected from group (vii).
Any of the polypeptide combinations described above may optionally comprise
no further polypeptides or no further peptides derived from Alternaria and/or
Cladosporium allergens. Any of the polypeptide combinations described above may be
incorporated in a pharmaceutical formulation of the invention as described in more
detail below.
In a broader aspect, the invention provides a polypeptide, or a pharmaceutically
acceptable salt thereof which is up to 30 amino acids in length and comprises, consists
essentially or consists of the amino acid sequence of any one of SEQ ID NOs 1 to 110
or a corresponding variant sequence.
The polypeptide, or pharmaceutically acceptable salt thereof, preferably
comprises:
(I) the amino acid sequence:
(a) WSWKIGPAIATGNT (Alt28; SEQ ID NO: 101),
(b) KYRRVVRAGVKVAQTAR (Alt34A; SEQ ID NO: 107), or
(c) KYAGVFVSTGTLGGG (SEQ ID NO: 112); or
(II) a T cell epitope-containing variant sequence which is a said amino acid
sequence (I) having up to seven amino acid modifications, each of which is
independently a deletion, substitution or insertion.
Other polypeptides of the invention or pharmaceutically acceptable salts thereof,
comprise the amino acid sequence (d) AEVYQKLKALAKKTYGQ (Alt 13A; SEQ ID
NO: 83), (e) SLGFNIKATNGGTLD (AltOlA; SEQ ID NO: 60), (f)
SAKRMKVAFKLDIEK (Alt06; SEQ ID NO: 72); (g) DITYVATATLPNYCR (SEQ
ID NO: 111); and (h) GWGVMVSHRSGET (Alt 14; SEQ ID NO: 84), or a
corresponding variant sequence as described above.
Preferably, a polypeptide of the invention comprises, consists or consists essentially of
the amino acid sequence of any one of SEQ ID NOs 83, 60, 61, 62, 72, 73, 106, 107,
101, 84, 85, or 89.
The invention also provides a pharmaceutical formulation comprising a
polypeptide of the invention, and a pharmaceutically acceptable carrier or diluent. The
invention further provides a polypeptide of the invention for use in a method of treating
or preventing allergy to Alternaria and/or Cladosporium. The invention additionally
provides use of a polypeptide of the invention for the manufacture of a medicament for
the prevention or treatment of allergy to Alternaria and/or Cladosporium.
Medical uses and methods
A preferred aspect of the invention is the prevention or treatment of allergy. In
this aspect, the invention provides a pharmaceutical formulation of the invention (which
comprises at least three polypeptides or salts) for use in a method of treating or
preventing allergy to Alternaria and/or Cladosporium. The pharmaceutical formulation
of the invention may prevent or treat the allergy by tolerisation. The tolerisation may be
to one or more protein allergens of the Alternaria and/or Cladosporium genus.
The invention further provides a use of the at least three polypeptides or salts
described above for the manufacture of a medicament for the prevention or treatment of
allergy to Alternaria and/or Cladosporium.
The invention further provides a method of treating an individual for allergy to
Alternaria and/or Cladosporium or of preventing in an individual allergy to Alternaria
and/or Cladosporium, which method comprises administering to said individual a
therapeutically or prophylactically effective amount of a polypeptide or salt of the
invention or of a pharmaceutical formulation of the invention. The method may thus
reduce or ameliorate the symptoms of allergy in the individual suffering from the
allergy. The method may improve the condition of the individual suffering from the
allergy. The method may prevent or delay the appearance of symptoms of allergy in the
individual. Symptoms of allergy to mould are discussed below.
In each of the methods and uses mentioned in this section, a polypeptide or salt
may be replaced with a combination of polypeptides or salts as was defined in the
previous section. As such, the invention encompasses a scenario in which a
combination of polypeptides or salts is used in a method of treating or preventing
allergy to Cladosporium and/'or Alternaria. In said scenario, the polypeptides in a
combination need not be administered together, and/or need not be not part of the same
pharmaceutical formulation. The multiple peptides of this method may each be
administered simultaneously, sequentially or concurrently.
The pharmaceutical formulation of the invention may treat or prevent the allergy
by desensitising or tolerising to Cladosporium and/'or Alternaria allergens. A
polypeptide comprised in the pharmaceutical formulation may be used to tolerise or
desensitise an individual to the allergen from which it is derived. Desensitising an
individual to the allergens means inhibition or dampening of allergic tissue reactions
induced by the allergens in appropriately sensitised individuals. The term "tolerisation"
refers to an ability to suppress, or abolish a response to an antigen, such as an allergic
response to a protein allergen. Tolerisation is also an ability to diminish or abolish an
unwanted immune response, or to desensitise a subject to a protein allergen.
Tolerisation may be determined by in vitro analysis of T cell responses or by
observation of a reduction in the symptoms in an individual.
In more detail, T cells can be selectively activated, and then rendered
unresponsive. Moreover the energising 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. This 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 four weeks is preferred.
The individual to whom the pharmaceutical formulation is administered may be
asymptomatic. A prophylactically effective amount of the pharmaceutical formulation
is administered to such an individual. A prophylactically effective amount is an amount
which prevents the onset of one or more symptoms of allergy.
Alternatively, the individual to whom the pharmaceutical formulation is
administered may be in need thereof. That is, the individual may exhibit one or more
symptoms of allergy. A therapeutically effective amount of the r pharmaceutical
formulation is administered to such an individual. A therapeutically effective amount is
an amount which is effective to ameliorate one or more symptoms of allergy.
The individual to whom the pharmaceutical formulation is administered is
preferably human. The individual may be known to be sensitised to mould 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 mould.
It may not be necessary to test an individual for sensitisation to mould because
the individual may display symptoms of allergy when exposed to mould. By exposure
is meant proximity to, for example, a mould or a substance or product derived from a
mould. 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 an itching nose, sneezing, ocular tearing, an itchy throat, itchy palate, itchy
eyes, runny nose, breathing difficulties, bronchospasm, asthma, red itchy skin or rash.
The individual 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 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 DRBl allele families are shown
in Table 1 (Data from HLA Facts Book, Parham and Barber).
Table 1
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 DRBl alleles is:
4 - at least 9%
7 - at least 10%
11 - at least 8%.
The individual may have had allergy to mould for at least 2 weeks, 1 month, 6
months, 1 year, 5 years or more than 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 mould allergy. The individual may live in a geographical region which has a
temperate, semi-tropical, tropical, or arctic climate. The individual typically suffers
from allergy to mould in a particular season but the allergy may be perennial. Seasonal
allergy to mould may commonly occur in autumn in the Northern hemisphere.
The allergic individual is typically allergic to moulds of the Alternaria genus,
particularly Alternaria alternata. The allergic individual may be allergic to moulds of
the Cladosporium genus, particularly Cladosporium herbarium and/or Cladosporium
cladosporoides. The allergic individual may be allergic both to moulds of the Alternata
and Cladosporium.
The polypeptides or salts described herein and the pharmaceutical formulations
of the invention may be screened in panels of mould allergic individuals to confirm
their suitability for use. The panel of mould allergic individuals may comprise
individuals known or not known to be allergic to moulds of the Alternaria and
Cladosporium genus. In particular where multiple polypeptides are provided in
combination in a pharmaceutical formulation, they may be screened for their ability to
cause T cell proliferation in at least 20 % of samples of T cells, wherein each sample is
obtained from different mould allergic individuals in the population. Preferably, the
pharmaceutical formulation will induce T cell proliferation in at least 30 % of samples
of T cells obtained from of a panel of mould allergic individuals. More preferably, the
pharmaceutical formulation will induce T cell proliferation in 35% or more, 40 % or
more, 50 % or more, 60 % or more, 70% or more, 80 % or more, or 90 % or more of
samples in the panel. The number of individuals in a panel of mould 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.
It is also preferred that the polypeptides or salts described herein and the
pharmaceutical formulations of the invention cause T cell proliferation, but do not lead
to the release of histamine from leucocyte samples from a sensitised individual. The
histamine release profile of a polypeptide, salt or pharmaceutical formulation may thus
be confirmed. Suitable leucocyte samples include enriched basophils or mast cell
preparations. There may be some histamine release, but preferably the amounts
released are not significant. Significant histamine release may be considered 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 pharmaceutical formulation in vitro.
A polypeptide or salt described herein or a pharmaceutical formulation 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 histamine when a sample of leukocytes
from an individual is stimulated with a composition in vitro. A normal individual
typically has an approximate leukocyte histamine content of 150ng/10 cells.
Pharmaceuticalformulations
Each polypeptide or salt described herein may be provided to an individual in an
isolated, substantially isolated, purified or substantially purified form. For example,
where polypeptides or salts of a combination described herein are not administered
together, a polypeptide or salt described herein may be provided to an individual
substantially free from other polypeptides or salts thereof. Whilst it may be possible for
the polypeptides or salts to be presented in raw form, it is preferable to present them as
a pharmaceutical formulation.
A pharmaceutical formulation of the invention preferably comprises at least
three polypeptides or salts as described above and a pharmaceutically acceptable carrier
or diluent. The pharmaceutical formulation may comprise any combination of
polypeptides or salts described above.
The carrier(s) or diluent(s) present in the pharmaceutical formulation 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 water. The carrier or diluent may comprise thioglycerol, methionine or thioanisole.
A composition containing at least three polypeptides or salts as described above
can be combined with one or more pharmaceutically acceptable excipients or vehicles
to produce a pharmaceutical formulation. 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).
The polypeptides or salts are typically present at 0.1% to 50% by weight in the
pharmaceutical formulation, more preferably at 0 .1% to 5% by weight. The
polypeptides or salts may be present at less than 0 .1% by weight in the pharmaceutical
formulation.
The pharmaceutically acceptable carrier or diluent is typically present at 50% to
99.9 % by weight in the pharmaceutical formulation, more preferably at 95% to 99.9 %
by weight. The pharmaceutically acceptable carrier or diluents may be present at more
than 99. 9% by weight in the pharmaceutical formulation.
Pharmaceutical formulations include, but are not limited to pharmaceutically
acceptable solutions, lyophilisates, suspensions, emulsions in oily or aqueous vehicles,
pastes, and implantable sustained-release or biodegradable formulations. Such
pharmaceutical formulations may further comprise one or more additional ingredients
including, but not limited to, suspending, stabilizing, or dispersing agents. A
lyophilisate may comprise one or more of trehalose, thioglycerol, methionine and
thioanisole. In one embodiment of a pharmaceutical formulation for parenteral
administration, the active ingredient is provided in dry form (e.g., a lyophilisate, powder
or granules) for reconstitution with a suitable vehicle (e. g., sterile pyrogen-free water)
prior to parenteral administration of the reconstituted pharmaceutical formulation.
The invention further provides a method of preparing a pharmaceutical
formulation of the invention, comprising combining at least three polypeptides or salts
as described above with a pharmaceutically acceptable carrier or diluent. Preferably,
said method prepares a pharmaceutical formulation for parenteral administration, and
comprises providing said polypeptides, or salts in dry form and reconstituting said
polypeptides, or salts with a said pharmaceutically acceptable carrier or diluent.
The pharmaceutical formulations 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 parenterally-administrable pharmaceutical formulations which are useful
include those which comprise the active ingredient in microcrystalline form, in a
liposomal preparation, or as a component of a biodegradable polymer systems
pharmaceutical formulations 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 polypeptides described herein 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, polyornithine, spermine,
spermidine, as well as conjugates of these molecules.
The formulation of any of the polypeptides mentioned herein will depend upon
factors such as the nature of the polypeptide and the method of delivery. The
pharmaceutical formulation 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, intralymphatically (such as to lymph nodes in the groin),
intrasternally, transdermally, intradermally, epidermally, sublingually, intranasally,
buccally or by infusion techniques. The administration may be intratonsillar. The
administration may be as suppositories. The administration may be made by
iontophoresis. Preferably, the administration is intradermal, epidermal or transdermal.
The administration may be made by a patch, such as a microtine patch.
A physician will be able to determine the required route and means of
administration for each particular individual.
The pharmaceutical formulations of the invention are preferably provided sealed
in a container. Where the pharmaceutical formulation is a pharmaceutically acceptable
solution, the solution may be provided in an ampoule, sealed vial, syringe, cartridge,
flexible bag or glass bottle. Where the pharmaceutical formulation is a lyophilisate, it is
preferably provided in a sealed vial.
The pharmaceutical formulations of the invention will comprise a suitable
concentration of each polypeptide to be effective without causing adverse reaction.
Where the pharmaceutical formulation is for example a lyophilisate, the relevant
concentration will be that of each polypeptide following reconstitution. Typically, the
concentration of each polypeptide in the pharmaceutical formulation when in solution
will be in the range of 0.03 to 200 nmol/ml. The concentration of each polypeptide may
be more preferably in the range of 0.3 to 200 nmol/ml, 3 to 180 nmol/ml, 10 to 150
nmol/ml, 50 to 200nmol/ml or 30 to 120 nmol/ml. The pharmaceutical formulation
should have a purity of greater than 95% or 98% or a purity of at least 99%.
An adjuvant or further therapeutic agent may be used in combination with the
polypeptides described herein. An adjuvant is preferably administered in an amount
which is sufficient to augment the effect of the polypeptide(s) or vice versa. The
adjuvant or further therapeutic agent may be an agent that potentiates the effects of a
polypeptide described herein. For example, the further therapeutic agent may be an
immunomodulatory molecule which enhances the response to the polypeptide of the
invention. 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.
In an embodiment where the polypeptides described herein are used for therapy
in combination with one or more other therapeutic agents or adjuvants, the other
therapeutic agents or adjuvants may be administered separately, simultaneously or
sequentially. They may be administered in the same or different pharmaceutical
formulations. A pharmaceutical formulation may therefore be prepared which
comprises a polypeptide described herein and also one or more other therapeutic agents
or adjuvants. A pharmaceutical formulation of the invention may alternatively be used
simultaneously, sequentially or separately with one or more other therapeutic
compositions as part of a combined treatment. Accordingly, in a method of preventing
or treating allergy according to the invention as described below, the subject may also
be treated with a further therapeutic agent.
Routes of administration
Where a polypeptide or salt described herein is to be administered to an
individual in a pharmaceutical formulation, it is preferred to administer the formulation
to a site in the body of the individual where the polypeptide or salt 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.
Once formulated the pharmaceutical formulations of the invention can be
delivered to a subject in vivo using a variety of known routes and techniques. For
example, a pharmaceutical formulation can be provided as an injectable solution,
suspension or emulsion and administered via parenteral, subcutaneous, epidermal,
intradermal, intramuscular, intralymphatic, intraarterial, intraperitoneal, or intravenous
injection using a conventional needle and syringe, a microneedle and syringe or using a
liquid jet injection system. The administration may be made using a patch, such as a
microtine patch. Pharmaceutical formulations can also be administered topically to skin
or mucosal tissue, such as nasally, intratonsillarly, 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.
Dosages
Administration of the polypeptides or salts described herein or of the
pharmaceutical formulations of the invention may be by any suitable method as
described above. Suitable amounts of the polypeptides or salts to be administered may
be determined empirically, but typically are in the range given below. A single
administration of each polypeptide or salt may be sufficient to have a beneficial effect
for the patient, but it will be appreciated that it may be beneficial if the polypeptide or
salt is administered 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 polypeptide or salt in a
combination of polypeptides or salts 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 pharmaceutical formulation, the route of administration and the schedule
and timing of the administration regime. Suitable doses of a polypeptide or salt
described herein may be in the order of up to 10 g, up to 15 g, up to 20 g, up to 25 g,
up to 30mg, up to 50mg, up to 100mg, up to 500 mg or more per administration. Suitable
doses may be less than 15mg, 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 ^g, or at least 10mg . For some
polypeptides described herein, 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. It will be understood that the above doses refer to
total dose in the case of a combination of peptides or salts. For example, "up to 35m '
refers to a total peptide or salt concentration of up to 35mg in a composition comprising
a combination or more than one peptide or salt.
Nucleic acids and vectors
The polypeptides described herein 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 polypeptide described herein. A
polypeptide described herein 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 described herein is intended to include
the indirect use, delivery or administration of such a peptide via expression from a
polynucleotide that encodes it.
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 may be
provided in isolated or purified form.
Polynucleotides 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 above polynucleotides may be used in vitro, ex vivo or in vivo in the
production of a polypeptide described herein. Such polynucleotides may be
administered or used in the prevention or treatment of allergy to Alternaria and/or
Cladosporium.
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 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."
Antigen presenting cells (APCs)
The invention encompasses the use in vitro of a method of producing a
population of APCs that present the polypeptides described herein, such as a
combination of at least three polypeptides described herein, on their surface. Said
population of APCs may be subsequently used in therapy. Said method of production
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 allergy to Cladosporium and/'or Alternaria. 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.
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.
In vitro method
The invention further provides an in vitro method of determining whether T
cells recognize one or more polypeptides or salts of a pharmaceutical formulation of the
invention, which method comprises contacting said T cells with said pharmaceutical
formulation and detecting whether said T cells are stimulated by said polypeptides.
The above method may be carried out to determine whether an individual has,
or is at risk of having, an allergy to Cladosporium and/'or Alternaria.
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 polypeptides with strong
affinities for the seven most common human MHC Class II HLA-DRB 1* allotypes
(covering in total around 63% of the allotypes found in the average Caucasian
population). In order to identify said polypeptides in the major Alternaria allergens Alt
a 1, Alt a 2, Alt a 6 . Alt a 7, Alt a 8 and Alt a 10 from Alternaria Alternata, an in silico
approach known as "peptide threading" was performed using the commercially
available EpiMatrix algorithm (EpiVax Inc.) This is a bioinformatic analysis of
analysing a polypeptide having a given sequence for the potential to be accommodated
within the binding groove of MHC class II HLA-DR molecules.
EpiMatrix is a matrix-based algorithm that ranks 10 amino acid long segments,
overlapping by 9 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, 16 Vaccine 1998 (1998). In this Example, binding
potential for HLA DR1, DR2, DR3, DR4, DR7, DR8, DR1 1, DR13 and DR15 is
assessed. Putative MHC ligands are selected by scoring each 10-mer frame in a protein
sequence. This score is derived by comparing the sequence of the 10-mer to the matrix
of 10 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 polypeptides which bind to multiple MHC molecules has also
been confirmed.
Estimated probability of binding to a selected MHC molecule is calculated by
EpiMatrix as follows. The polypeptides having a given sequence are scored by
estimating the relative promotion or inhibition of binding for each amino acid,
compared to known MHC binders for a given MHC allele. This information is summed
across the polypeptide and a summary score (EMX score) is assigned to the entire
polypeptide. After comparing the EMX score to the scores of known MHC ligands,
EpiMatrix arrives at an "estimated binding probability" (abbreviated as EBP, but not
strictly a probability). The EBP describes the proportion of polypeptides with EpiMatrix
scores as high or higher that will bind to a given MHC molecule. EBPs range from
100% (highly likely to bind) to less than 1% (very unlikely to bind).
EpiMatrix analyses were performed on the entire sequence of known isoforms of
Alt a 1 (NCBI accession nos: P79085.1; Q6Q128; PQ8NJ79). These analyses identified
core polypeptides (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 2 .
In Table 2 : "Residues in sequence" gives the location of the sequence within the
sequence of the polypeptide that was analysed. The core sequence (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 a polypeptide. "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
polypeptide standard. A score above 10 is considered to indicate broad MHC binding
properties.
Table 2 - Alt a 1
Based on a further analysis of Epimatrix data, the following additional
sequences from Alt a 1 were also identified as having suitable MHC-binding properties:
DITYVATATLPNY (SEQ ID NO: 5), DAYITLVTLPKSS (SEQ ID NO: 6),
DITYVATATLPNYCR (SEQ ID NO: 111).
Example 2
EpiMatrix analyses as above were performed on the entire sequence of the
known isoform of Alt a 2 (NCBI accession no: AAD00097). This analysis identified
core sequences (with their flanking sequences) derived from said Alt a 2 isoform which
are predicted to have good MHC class-II binding properties. These sequences are
shown below in Table 3 . Headings and notes for Table 3 are as with Table 2 above.
Table 3 - Alt a 2
Based on a further analysis of Epimatrix data, the following additional
sequences from Alt a 2 were also identified as having suitable MHC-binding properties:
QLLMLSAKRMKVA (SEQ ID NO: 10), TLYRPRDLLSLLN (SEQ ID NO: 11);
Example 3
EpiMatrix analyses as above were performed on the entire sequence of the
known isoform of Alt a 6 (NCBI accession no: Q9HDT3). This analysis identified core
sequences (with flanking sequences) derived from said Alt a 6 isoform 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 2 above.
Table 4 - Alt
Based on a further analysis of Epimatrix data, the following additional
sequences from Alt a 6 were also identified as having suitable MHC-binding properties:
VSMAIAKAAAAEK (SEQ ID NO: 17), QKLKALAKKTYGQ (SEQ ID NO: 18),
EFIKKAIELKSCN (SEQ ID NO: 19), IELKSCNALLLK (SEQ ID NO: 20),
GYTGKIKIAMDVASSEF (SEQ ID NO: 21), GYTGKIKIAMDVASSEFY (SEQ ID
NO: 22). Based on homology with Alt a 6 and further design and screening, the
following sequence derived from Cla h 6 was also identified as having suitable MHCbinding
properties: SEQ ID NO: 108 (Clal6; AEVYQKLKSLTK).
Example 4
EpiMatrix analyses as above were performed on the entire sequence of the
known isoform of Alt a 7 (NCBI accession no: P42058). This analysis identified core
sequences (with flanking sequences) derived from the said Alt a 7 isoform which are
predicted to have good MHC class-II binding. These sequences are shown below in
Table 5 . Headings and notes for Table 5 are as with Table 2 above.
Table 5 - Alt a 7
P42 0 5 8 7 AIVYYSMYGHIKKMADAELKGI 0 .15 9 .42 P 1 9 2 3
P42 0 5 8 3/ - 4 GDAKLFQVAETLPQEVL 0 .12 8 1 . 1 P 2 0 2 4
P42 0 5 8 GAFWGKYAGVFVSTGTLGGGQE 0 .15 12 19.42 P 2 1 2 5
P42 0 5 8 39 - 162 G I YVPLGYKTAFSMLANLDEVH G 0.45 12 P 2 2 2 6
P42 0 5 8 0 - 19 P SELELNIAQAQGKAFYEAV -0.1 4 8 P 2 3 2 7
Based on a further analysis of Epimatrix data, the following additional
sequences from Alt a 7 were also identified as having suitable MHC-binding properties:
DAKLFQVAETLPQEVLDK (SEQ ID NO: 28), GVFVSTGTLGGGQ (SEQ ID NO:
114), SELELNIAQAQGKAFYE (SEQ ID NO: 29), KYAGVFVSTGTLGGG (SEQ ID
NO: 112).
Example 5
EpiMatrix analyses as above were performed on the entire sequence of the
known isoform of Alt a 8 (NCBI accession no: P0C0Y4). This analysis identified core
sequences (with flanking sequences) derived from the said Alt a 8 isoform which are
predicted to have good MHC class-II binding. These sequences are shown below in
Table 6 . Headings and notes for Table 6 are as with Table 2 above.
Table 6 - Alt a 8
Based on a further analysis of Epimatrix data, the following additional sequence
from Alt a 8 was also identified as having suitable MHC-binding properties:
FVPQDIQKL (SEQ ID NO: 35). Based on homology with Alt a 8 and further design
and screening, the following sequences derived from Cla h 8 were also identified as
having suitable MHC-binding properties: SEQ ID NO: 109 (Alt25,
VAITYASRAQGAEK) and SEQ ID NO: 110 (Alt26, GHHFKERGTGSLVIT).
Example 6
EpiMatrix analyses as above were performed on the entire sequence of the
known isoform of Alt a 10 (NCBI accession no: P42041). This analysis identified core
sequences (with flanking sequences) derived from the said Alt a 10 isoform which are
predicted to have good MHC class-II binding. These sequences are shown below in
Table 7 . Headings and notes for Table 7 are as with Table 2 above.
Table 7 - Alt a 10
P42 0 4 1 . 2 4 1 0 - 4 3 2 ADVIKI GNNTTYGLAAAVHTSNL 0 . 2 7 1 1 1 , 7 P 4 1 4 4
P42 0 4 1 . 2 T AIEVANALRAGTVWV 0 . 7 8 8 1 2 . 5 P 4 2 -
Based on a further analysis of Epimatrix data, the following additional
sequences from Alt a 10 were also identified as having suitable MHC-binding
properties: SLLVFAVRSSMEL (SEQ ID NO: 45), SLLVFAVRSSMELPIL (SEQ ID
NO: 46), WSWKIGPAIATGN (SEQ ID NO: 47), DNYIQTKTVSIRL (SEQ ID NO:
48).
Example 7
EpiMatrix analyses as above were performed on the entire sequence of the
known isoform of Alt a 13 (NCBI accession no: Q6R4B4). This analysis identified core
sequences (with flanking sequences) derived from the said Alt a 13 isoform which are
predicted to have good MHC class-II binding. These sequences are shown below in
Table 8 . Headings and notes for Table 8 are as with Table 2 above.
Table 8
Based on a further analysis of Epimatrix data, the following additional
sequences from Alt a 13 were also identified as having suitable MHC-binding
properties: NWLTLHTAALGP (SEQ ID NO: 55), EKLRSNITVQYDI (SEQ ID NO:
56), EKYRRVVRAGVKV (SEQ ID NO: 57). RVVRAGVKVAQTA (SEQ ID NO:
58).
Example 8
The sequences set out in Table 9 were selected by the inventors as having
desirable characteristics, based on the analyses performed in Examples 1 to 7 and a
consideration of solubility and other physicochemical characteristics. For example, the
sequences of SEQ ID NOS: 62 and SEQ ID NO: 89 are preferred variant sequences
derived from SEQ ID NOS 111 and 112 respectively. These variant sequences were
selected for improved solubility and/or manufacturability relative to the original amino
acid sequences. Polypeptides consisting of the sequences of Table 9 were produced and
were particularly preferred for screening in subsequent assays.
Table 9
Alt09 KEYQKIFPSIQVI 133-145 (Alt a 2) 76
AltlO SAFRSIEPELTVY 147-159 (Alt a 2) 77
AltlOA KSAFRSIEPELTVYK K147-159K 78
Altll KKVSMAIAKAAAAEK KK1 13-126 (Alt a 6) 79
AltllA KKVS-Nle-AIAKAAAAEK 80
Altl2 AEVYQKLKALAKK 187-199 (Alt a 6) 8 1
Altl3 PyrKLKALAKKTYGQ 191-203 (Alt a 6) 82
A1U3A AEVYQKLKALAKKTYGQ* 187-203 (Alt a 6) 83
Altl4 GWGVMVSHRSGET* 368-380 (Alt a 6) 84
Altl4A GWGV-Nle-VSHRSGET* 368-380 (Alt a 6) 85
Altl5 GYTGKIKIAMDVASSE* 236-251 (Alt a 6) 86
Altl5A GYTGKIKIA-Nle-DVASSE* 236-251 (Alt a 6) 87
Altl7 KLFQVAETLPQEVLDK 35-50 (Alt a 7) 88
Altl8 KKYAGVFVSTGTLGGGK 109-125 (Alt a 7) 89
Alt20 LAITYNSRAEGAEK* 48-61 (Alt a 8) 90
Alt21 GLHFRERKTGSLVIT* 143-157 (Alt a 8) 9 1
Alt22 SYNVAKAGCIHLAK 173-186 (Alt a 8) 92
Alt22A SYNVAKAGSIHLAK 93
Alt23 NEWRDFARVNSISP* 190-203 (Alt a 8) 94
Alt24 KLWHSMIPMGRDAK* 220-233 (Alt a 8) 95
Alt24A KLWHS-Nle-IP-Nle-GRDAK* 96
Alt24B KLWHS-Nle-IPMGRDAK* 97
Alt24C KLWHSMIP-Nle-GRDAK* 98
Alt27 KRSLLVFAVRSSMELRK KR153-166RK(Alta 10) 99
Alt27A KRSLLVFAVRSS-Nle-ELRK 100
Alt28 WSWKIGPAIATGNT* 170-183 (Alt a 10) 101
Alt30 HEIYWDRVSAP 42-53 (Alt a 13) 102
Alt31 NWLTLHTAALGPTAK 112-127 (Alt a 13) 103
Alt32 KYWLYFYKLHPEK 127-139 (Alt a 13) 104
Alt33 IEKLRSNITVQYDI 144-157 (Alt a 13) 105
Alt34 YEKYRRWRAGVKV 193-206 (Alt a 13) 106
Alt34A KYRRWRAGVKVAQTAR 195-211 107
Nle: Norleucine; Pyr: pyroglutamate. Asterisk (*) denotes potentially crossreactive
epitopes between a given Alternaria allergen and its Cladosporium homologue.
Other instances of such epitopes are provided in Examples 1-7.
Example 9
In vitro binding analysis
Polypeptides having the sequences identified in Examples 1 to 8 are prescreened
for solubility in an aqueous, acidic milieu and the polypeptides 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
polypeptide 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 polypeptides
used in this study are typically those shown below:
Each polypeptide is analysed in the competition assay and screened for relative
binding compared to the control polypeptides. Due to the nature of the competitive
assay the data for each polypeptide is determined as a ratio of its own IC50 to that of the
control polypeptide. Thus, a polypeptide that has an IC50 value that is parity to the
control polypeptide has an identical binding affinity, while polypeptides 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 polypeptide to be an
effective therapeutic agent. Therefore, as a consequence of the solubility screen very
hydrophobic polypeptides with a high frequency of large hydrophobic amino acid
residues in multiple binding registers will be eliminated. This is a characteristic of
promiscuous HLA-DRBl* binders. Polypeptides which bind to one or more of the
MHC Class II allotypes are identified. It would be expected that such polypeptides
would have the ability to bind similar allotypes that have not been tested through the
homology of MHC structures.
Example 10
The following methods were used to evaluate T cell activation characteristics of
polypeptides having the sequences identified in Examples 1 to 8 .
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 polypeptide 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 2xl0 6 cells/ml in complete medium at room temperature. IOOmI of cell
solution is then distributed into each of the wells of the 96 well plates containing
antigen/polypeptide. 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 16 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 polypeptide 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 polypeptide-stimulated wells is considered a
positive stimulation of PBMC's by the polypeptide.
Cytokine release assay
Polypeptides for use in this assay were manufactured at small scale
(approximately lOmg batch size, non-GMP). The purity of each polypeptide was at least
95% by HPLC. 96 well culture plates containing polypeptides and controls (the negative
control was culture medium and the positive controls were staphylococcal enterotoxin B
(SEB) 25ng/ml and whole Alternaria allergen extract 100 g/ml) were prepared in
advance and stored at -20°C prior to the day of assay. Polypeptides were added to wells
in a volume of 100 mΐ containing polypeptides at a concentration of 200 g/ml, such that
subsequent addition of 100 mΐ of cells would create a final assay concentration of 100
ΐ .
Peripheral blood mononuclear cells (PBMCs) were isolated from heparinised
blood by Ficoll density gradient centrifugation. A 100 mΐ aliquot of a 5x106 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 supernatants (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 supernatants 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 a cytokine release of greater than 50 pg/ml in one or more of the IL-13, IL-10 and
IFN-g assays. The number of responders out of 50 mould allergic subjects tested was
calculated for each polypeptide for the three cytokines.
Results for the polypeptides having the sequences of Table 9 are summarized in
Table 10.
Table 10
AltlO 42 Alt28 52
AltlOA 34 Alt30 36
Altl l 36 Alt3 1 58
Altl l A 44 Alt32 36
Altl2 38 Alt33 44
Altl3 40 Alt34 38
Alt34A 38
As shown, the top performing peptide was Altl3A. A polypeptide combination
including a polypeptide having the sequence of Altl3A is preferred for treatment or
prevention of Alternaria allergy. Other top performing polypeptides which induce
responses individually in a high proportion of subjects include AltlO, Altl 1A, Altl3,
Alt 14, Altl4A, Altl 5, Altl 5A, Altl 8, Alt22, Alt22A, Alt24A, Alt27, Alt27A, Alt28,
Alt3 1 and Alt33. One or more of these polypeptides may also be preferably included in
any peptide combination of the invention.
An analysis of population coverage was carried out to determine what
polypeptides could advantageously capture additional responses from the population
when included in combination with other peptides. The number of subjects in the
population displaying IL-13 or IFN-g responses to at least one, two or three
polypeptides included in a given combination was analysed. Results are shown below in
Table 11.
Table 11
Altl3A, Altl5, Alt18
AltO 1A, Alt02A, Alt06,
AltlO, A1U3A, Alt33, 45 34 27
Alt34A
AltO 1A, Alt02A, Alt06,
Altl3A, Alt28, Alt33, 44 36 26
Alt34A
AltO 1A, Alt02A, Alt06,
Alt 10, Altl3A, Alt 15, 45 33 25
Alt 18
AltO 1A, Alt02A, Alt06,
Altl3A, Alt 15, Alt 18, 44 35 23
Alt28
The effect of adding different polypeptides can be seen by comparing the
number of additional subjects responding to the different combinations.
Two polypeptides derived from Alt a l (AltO 1A and Alt02A) and one peptide
from Alt a2 (Alt06) were able to provide for an increase in the number of subjects
showing a response to at least one polypeptide, in particular those not showing a
response to top-performing polypeptide Altl3A (86% response, i.e 34/50 individuals).
A combination including any of AltO 1A, Alt02A or Alt06 is thus preferred to increase
population coverage, in particular in combination with polypeptide Alt 13A.
Two polypeptides derived from Alt a 13 (Alt33 and Alt34A) were also
particularly effective in increasing population coverage. When included in a
combination above, they increased the number of subjects responding to at least two
polypeptides from 18 to 32 and the number of subjects responding to at least three
polypeptides from 6 to 24. Thus Alt33 and Alt34A are also preferred to increase
population coverage, in particular in combination with polypeptide Altl3A and/or one
or more polypeptides from AltO 1A, Alt02A or Alt06.
Alternatively polypeptides Alt 15 and Alt 18 could be substituted for
polypeptides Alt 33 and Alt 34. When included in a combination above they achieved
30 subjects responding to at least 2 peptides and 23 subjects responding to at least 3
polypeptides. Thus Alt 15 and Alt 18 are also preferred to increase population coverage,
in particular in combination with polypeptide Altl3A and/or one or more polypeptides
from AltO 1A, Alt02A or Alt06.
Polypeptides AltlO (from Alt a2) and Alt28 (from AltlO) gave further increases
in population coverage when added to a mixture containing AltO 1A, Alt02A, Alt06, Alt
13A, Alt 33 and Alt34A, providing respectively for 34 or 36 subjects responding to at
least 2 peptides and 27 or 26 subjects responding to at least 3 polypeptides. Thus, AltlO
and Alt28 are also preferred in a vaccine to increase population coverage, in particular
in combination with polypeptide Altl3A and/or one or more polypeptides from AltOlA,
Alt02A, Alt06, Alt33 or Alt34A.
Polypeptides AltlO or Alt28 may also be used as an alternative to polypeptides
Alt 33 or Alt 34A. As shown above, a combination including AltlO and Alt28 in place
of polypeptides Alt33 and Alt34A provided respectively for 33 or 35 subjects
responding to at least 2 polypeptides and 25 or 23 subjects responding to at least 3
polypeptides. Thus, AltlO and Alt28 may be preferably provided in combination with
polypeptide Altl3A and/or one or more polypeptides from AltO 1A, Alt02A, Alt06,
Alt 15 or Altl8.
Example 11
The polypeptides identified in Example 10 as providing a basis for combinations
of polypeptides capable of covering responses in a polymorphic population were
analysed for their pharmaceutical development characteristics. An analysis of
population coverage was then carried out for additional combinations of polypeptides
identified as having preferable pharmaceutical development characteristics. _The number
of subjects in the population displaying IL-13 or IFN-g responses to at least one, two or
three polypeptides included in a given combination was analysed. Results are shown
below in Table 12.
Table 12
All polypeptides from Table 9 46 40 36
AltO 1A, Alt28, Alt34A 32 20 3
Altl8, Alt28, Alt34A 28 22 17
AltO 1A, Altl3A,Alt28,Alt34A
42 27 19
Altl3A, Altl8, Alt28, Alt34A 39 26 2 1
Altl3A, Altl8, Alt28, Alt34 39 26 19
Alt01A,Altl3A,Altl8,Alt28,Alt34A 42 28 22
Alt01A,Altl3A,Altl8,Alt28,Alt34 42 28 22
Altl3A, Altl8, Alt28, Alt34A,
43 29 22
AltO 1A, Alt06
Altl3A, Altl8, Alt28, Alt34,
43 29 22
AltO 1A, Alt06
Altl3A, Altl8, Alt28, Alt34A,
43 34 24
AltO 1A, Alt06, Alt02
Altl3A, Altl8, Alt28, Alt34,
43 34 24
AltO 1A, Alt06, Alt02
Altl3A, Altl8, Alt28, Alt34A,
43 29 24
AltO 1A, Alt06, Alt 14
Altl3A, Altl8, Alt28, Alt34,
43 29 24
AltO 1A, Alt06, Alt 14
The effect of adding different polypeptides can be seen by comparing the
number of additional subjects responding to the different combinations.
Two three peptide combinations: (a) AltOlA, Alt28 and Alt34A and (b) Altl8,
Alt28 and Alt34A, both lacking top performing peptide Altl3A, were compared to
assess differences in population coverage in the absence of Altl3A. The three peptide
combination of (b) provided enhanced coverage in terms of the number of subjects who
respond to all three peptides.
Addition of top performing peptide Altl3A in combinations of four peptides,
such as the combination of Altl8, Alt28, Alt34A and Altl3A significantly improved
coverage in terms of subjects responding to at least one peptide, as expected from the
results in Example 10.
Addition of polypeptides such as AltOlA, Alt06, Alt02 and Alt 14 further
improved population coverage in terms of subjects responding to at least one, two and
three peptides in the various combinations.
Thus, AltO 1A, Alt06, Alt02 and Alt 14 may preferably be provided in
combination with the polypeptides Altl8, Alt28, Alt34A and Altl3A as a basis for
effective coverage of responses in a polymorphic mould allergic population.
Example 12 - Preparation of peptides, salts and pharmaceutical formulations
Peptides are prepared as follows. Synthesis is performed in a solid phase peptide
synthesis (SPPS) reactor and started by suspending the substituted resin in N,Ndimethylformamide
(DMF). After washing of the resin with DMF, each coupling
procedure is performed by addition of the N-a-protected amino acid derivative or the Na-
protected dipeptide to the preceding amino acid in the presence of N-[(1HBenzotriazol-
l-yl)(dimethylamino)methylene]-N-methylmethanaminium
tetrafluorob orate N-oxide (TBTU) and N,N-diisopropylethylamine (DIPEA) in DMF or
diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBt) in a mixture of
methylene chloride (DCM) and DMF. For each single step, the solvents and/or reagents
are added and the reaction mixture is stirred and subsequently filtered to remove
solvents and/or reagents from the resin.
After each successful coupling or capping procedure, an Fmoc-deprotection
procedure is performed. It consists of washing of the resin with DMF, cleaving the
Fmoc-group with 20% (V/V) piperidine in either DMF or l-Methyl-2-pyrrolidone
(NMP), and subsequent washings with DMF and isopropanol (IPA). For each single
step, the solvents and/or reagents are added, and the reaction mixture is stirred and then
filtered to remove the solvents and/or reagents from the resin.
Fmoc-deprotection and coupling procedures are repeated until the resin carries
the complete peptide sequence of the required peptide. The SPPS is completed by a
final Fmoc-deprotection and drying of the peptide resin under reduced pressure.
Acetate or hydrochloride salts of the specified peptides are prepared by the
following methods. The peptide resin is treated with cold trifluoroacetic acid (TFA) at
room temperature for 1.5 to 3 hours in the presence of 1,2-ethanedithiol (EDT),
triisopropylsilane (TIS), and water. After filtering off and washing the resin with TFA,
the product is precipitated in cold diisopropyl ether (TPE). It is then filtered off, washed
with IPE, and dried under reduced pressure. The product is then reconstituted and
purified by high-performance liquid chromatography (HPLC).
For preparation of acetate salts, the trifluoroacetate salt is reconstituted in 5%
(V/V) aqueous acetic acid and loaded onto an ion exchange resin. The elution is
performed with 5% (V/V) aqueous acetic acid. The acetate is filtered through a 0.2 m
membrane filter and lyophilized to yield the final product as a white to off-white
powder.
For preparation of hydrochloride salts, the trifluoroacetate salt is reconstituted in
0.01 M HCl in purified water and filtered where necessary. The solution is loaded onto
a preparative HPLC column for ion exchange into the hydrochloride salt. The ion
exchange is performed by washing the column with a 0.1 M ammonium chloride
solution followed by 0.01 M HCl. Subsequently, the hydrochloride is filtered through a
0.2 m membrane filter and lyophilized to yield the final product as a white to off-white
powders.
An exemplary pharmaceutical formulation of the present invention contains the
components set out in Table 13. The peptide salt is a acetate or hydrochloride salt.
Table 13
Trehalose dihydrate Tonicity agent 260 mM
The formulation is prepared in solution prior to being subjected to freeze-drying
roduce a lyophilisate.

WE CLAIMS:-
1. A pharmaceutical formulation comprising a pharmaceutically acceptable carrier
or diluent and a polypeptide or a pharmaceutically acceptable salt thereof selected from
at least three of:
(a) a polypeptide comprising the amino acid sequence of
WSWKIGPAIATGNT (Alt28; SEQ ID NO: 101) or a T cell epitope-containing variant
sequence derived from said amino acid sequence, or a said salt thereof;
(b) a polypeptide comprising the amino acid sequence of
KYRRVVRAGVKVAQTAR (Alt34A; SEQ ID NO: 107) or a T cell epitope-containing
variant sequence derived from said amino acid sequence, or a said salt thereof;
(c) a polypeptide comprising the amino acid sequence of
KYAGVFVSTGTLGGG (SEQ ID NO: 112) or a T cell epitope-containing variant
sequence derived from said amino acid sequence, or a said salt thereof ;
(d) a polypeptide comprising the amino acid sequence of
AEVYQKLKALAKKTYGQ (Alt 13A; SEQ ID NO: 83) or a T cell epitope-containing
variant sequence derived from said amino acid sequence, or a said salt thereof;
(e) a polypeptide comprising the amino acid sequence of
SLGFNIKATNGGTLD (AltOlA; SEQ ID NO: 60) or a T cell epitope-containing
variant sequence derived from said amino acid sequence, or a said salt thereof;
(f) a polypeptide comprising the amino acid sequence of
SAKRMKVAFKLDIEK (Alt06; SEQ ID NO: 72) or a T cell epitope-containing variant
sequence derived from said amino acid sequence, or a said salt thereof;
(g) a polypeptide comprising the amino acid sequence of
DITYVATATLPNYCR (SEQ ID NO: 111) or a T cell epitope-containing variant
sequence derived from said amino acid sequence, or a said salt thereof; and
(h) a polypeptide comprising the amino acid sequence of
GWGVMVSHRSGET (Alt 14; SEQ ID NO: 84) or a T cell epitope-containing variant
sequence derived from said amino acid sequence, or a said salt thereof;
wherein a T cell epitope-containing variant sequence of a said amino
acid sequence is said amino acid sequence having up to seven amino acid modifications,
each of which is independently a deletion, substitution or insertion, and each
polypeptide is up to 30 amino acids in length.
2 . A pharmaceutical formulation according to claim 1, wherein a polypeptide of (a)
to (h) consists respectively of the amino acid sequence or variant sequence recited in (a)
to (h).
3 . A pharmaceutical formulation according to claim 1 or 2, wherein the variant
sequence of a said amino acid sequence is said amino acid sequence having one or two
amino acid modifications, the or each of which independently is a deletion or
substitution.
4 . A pharmaceutical formulation according to any one of the preceding claims,
wherein the or each substitution is a conservative substitution.
5 . A pharmaceutical formulation according to any one of the preceding claims,
wherein the variant sequence of a said amino acid sequence is said amino acid sequence
having up to two amino acids deleted from the N-terminus and/or up to two amino acids
deleted from the C-terminus.
6 . A pharmaceutical formulation according to any one of the preceding claims,
wherein each polypeptide is up to 20 amino acids in length.
7 . A pharmaceutical formulation according to any one of the preceding claims,
wherein at least one polypeptide has an amino acid sequence or variant sequence as
defined in the preceding claims having aN-terminal and/or C-terminal extension of
from one to six amino acids corresponding respectively to the one to six amino acids
immediately N-terminal or C-terminal to the said amino acid sequence in the native
sequence of the protein from which said amino acid sequence is derived.
8 . A pharmaceutical formulation according to any one of the preceding claims,
which comprises a polypeptide or salt of (a).
9 . A pharmaceutical formulation according to any one of the preceding claims,
which comprises at least one polypeptide or salt of (b), (c), or (d).
10. A pharmaceutical formulation according to any one of the preceding claims,
which comprises a polypeptide or salt of (a), (b) and (c).
11. A pharmaceutical formulation according to claim 9 or 10, which comprises a
polypeptide having the amino acid sequence YEKYRRVVRAGVKV (Alt34, SEQ ID
NO: 106).
12. A pharmaceutical formulation according to any one of claims 9 to 11, which
comprises a polypeptide having the amino acid sequence KKYAGVFVSTGTLGGGK
(Altl8, SEQ ID NO: 89).
13. A pharmaceutical formulation according to any one of the preceding claims,
which comprises a polypeptide or salt of (a), (b) and (d).
14. A pharmaceutical formulation according to any one of the preceding claims,
which comprises a polypeptide or salt of (a), (b), (c) and (d).
15. A pharmaceutical formulation according to any one of the preceding claims,
which comprises at least one polypeptide or salt of (e) or (f).
16. A pharmaceutical formulation according to claim 15, which comprises a
polypeptide having the amino acid sequence SAKR-Nle-KVAFKLDIEK (Alt06A, SEQ
ID NO: 73) , or a salt thereof.
17. A pharmaceutical formulation according to any one of the preceding claims,
which comprises at least one polypeptide or salt of (g) or (h).
18. A pharmaceutical formulation according to claim 17, which comprises a
polypeptide having the amino acid sequence KDITYVATATLPNY (Alt02; SEQ ID
NO: 61), or DITYVATATLPNYSR (Alt02A; SEQ ID NO: 62), or a salt of either
thereof.
19. A pharmaceutical formulation according to claim 17 or 18, which comprises a
polypeptide having the amino acid sequence GWGV-Nle-VSHRSGET (Alt14A, SEQ
ID NO: 85), or a salt thereof.
20. A pharmaceutical formulation according to any one of the preceding claims,
which comprises a polypeptide or salt of (a), (b), (c), (d), and (e).
2 1. A pharmaceutical formulation according to claim 20, which comprises at least
one polypeptide or salt of (f), (g) or (h).
22. A pharmaceutical formulation according to any one of the preceding claims,
which is sealed in a container.
23. A pharmaceutical formulation according to any one of the preceding claims,
which is a pharmaceutically acceptable solution or a lyophilisate.
24. A pharmaceutical formulation according to claim 23, wherein the solution is
formulated for intradermal administration, subcutaneous administration, oral
administration, nasal administration, topical administration, sublingual administration,
buccal administration or epidermal administration.
25. A pharmaceutical formulation according to claim 23 or 24, wherein the solution
is provided in an ampoule, sealed vial, syringe, cartridge, flexible bag or glass bottle.
26. A pharmaceutical formulation according to claim 23, wherein the lyophilisate is
provided in a sealed vial.
27 A pharmaceutical formulation according to any one of the preceding claims for
use in a method of treating or preventing allergy to Alternaria and/or Cladosporium.
28. An in vitro method of determining whether T cells recognize a polypeptide of a
pharmaceutical formulation according to any one of claims 1 to 21, which method
comprises contacting said T cells with said pharmaceutical formulation and detecting
whether said T cells are stimulated by a said polypeptide.
29. A method according to claim 28 which is carried out to determine whether an
individual has, or is at risk of having, an allergy to Alternaria and/or Cladosporium.
30. A method of treating an individual for allergy to Alternaria and/or
Cladosporium or of preventing in an individual allergy to Alternaria and/or
Cladosporium, which method comprises administering to said individual a
therapeutically or prophylactically effective amount of a pharmaceutical formulation as
defined in any one of claims 1 to 21.
31. Use of at least three polypeptides or salts as defined in claims 1 to 2 1 for the
manufacture of a medicament for the prevention or treatment of allergy to Alternaria
and/or Cladosporium.
32. A method of preparing a pharmaceutical formulation of the invention,
comprising combining at least three polypeptides or salts as defined in any one of
claims 1 to 2 1 with a pharmaceutically acceptable carrier or diluent.
33. A polypeptide, or a pharmaceutically acceptable salt thereof, which is up to 30
amino acids in length and comprises:
(I) the amino acid sequence:
(a) WSWKIGPAIATGNT (Alt28; SEQ ID NO: 101),
(b) KYRRVVRAGVKVAQTAR (Alt34A; SEQ ID NO: 107), or
(c) KYAGVFVSTGTLGGG (SEQ ID NO: 112); or
(II) a T cell epitope-containing variant sequence which is a said amino acid
sequence (I) having up to seven amino acid modifications, each of which is
independently a deletion, substitution or insertion.
34. A polypeptide or salt according to claim 33, wherein the polypeptide has an
amino acid sequence selected from KKYAGVFVSTGTLGGGK (Alt 18, SEQ ID NO:
89), and YEKYRRVVRAGVKV (Alt34, SEQ ID NO: 106).
35. A polypeptide or salt according to claim 33 or 34, for use in a method of treating
or preventing allergy to Alternaria and/or Cladosporium.
36. Use of a polypeptide or salt according to claim 33 or 34 for the manufacture of a
medicament for the prevention or treatment of allergy to Alternaria and/or
Cladosporium.
37. A method of treating an individual for allergy to Alternaria and/or
Cladosporium or of preventing in an individual allergy to Alternaria and/or
Cladosporium, which method comprises administering to said individual a
therapeutically or prophylactically effective amount of a polypeptide or salt according
to claim 33 or 34.