The present invention relates to polypeptides and pharmaceutical formulations
5 which may be used for preventing or treating allergy to moulds of the Cladosporium
and/or Alternaria genus.
Background to the Invention
Mould allergens are recognised as a major cause of allergic diseases in humans
10 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
15 are dependent on moisture levels in the home, indoor mould levels are higher when
outdoor mould 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
20 disease based on the types of mould spores collected in the air: Alternaria,
Cladosporium, Aspergillus, Penicillium, Helminthosporz~mE, picoccum, Fusarium,
Aureohasidium, 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.
25 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
30 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
5 North America and Europe. It is effectively impossible to avoid Alternaria allergens.
Summarv of the Invention
The invention provides a polypeptide, or a pharmaceutically acceptable salt *.
thereof, suitable for use in preventing or treating allergy to Cladosporium and/or
10 Alternaria, which is up to 30 amino acids in length and comprises:
(I) the amino acid sequence:
(a) GGYKAAVRPTMLE (SEQ ID NO: 35; Cla35),
(b) AEVYQKLKSLTKK (SEQ ID NO: 3 1; Cla16),
(c) VAITYASRAQGAE (SEQ ID NO: 32; Cla25),
15 (d) GHHFKERGTGSLVIT (SEQ ID NO: 33; Cla26), or
(e) ANYTQTKTVSIRL (SEQ ID NO: 34; Cla29); or
(11) a T cell epitope-containing variant sequence which is a said amino acid
sequence (I) having up to six amino acid modifications, each of which is independently
; a deletion, substitution or insertion. -
20 The invention further provides a pharmaceutical formulation which comprises a
pharmaceutically acceptable carrier or diluent and a polypeptide of the invention, or a
pharmaceutically acceptable salt thereof.
The invention additionally provides a polypeptide, salt or pharmaceutical
formulation of the invention for use in a method of treating or preventing allergy to
25 Cladosporium and/or Alternaria.
The invention further provides a method of treating an individual for allergy to
Cladosporium and/or Alternaria or of preventing in an individual allergy to
Cladosporium and/or Alternaria, which method comprises administering to said
individual a therapeutically or prophylactically effective amount of a polypeptide, salt
30 ; or pharmaceutical formulation of the invention.
The invention also provides use of a polypeptide or salt of the invention for the
manufacture of a medicament for the prevention or treatment of allergy to
Cladosporium and/or Alternaria.
The invention additionally provides an in vitro method of determining whether T
5 cells recognize a polypeptide or salt of the invention, which method comprises
contacting said T cells with said polypeptide or salt and detecting whether said T cells
are stimulated by said polypeptide or salt.
The invention further provides a method of preparing a pharmaceutical
formulation of the invention, comprising combining one or more polypeptides or salts
10 described herein with a pharmaceutically acceptable carrier or diluent.
Description of the sequences
SEQ ID NOS: 1 to 57 provide amino acid sequences as set out in Examples 1 to
7. In more detail:
15 SEQ ID NOS: 1 to 9 and 3 1 correspond to amino acid sequences derived from
the protein Cla h6.
SEQ ID NO: 37 corresponds to an amino acid sequence derived from the protein
Cla h7.
SEQ ID NOS: 10 to 20,30,32,33 and 5 1 correspond to amino acid sequences
20 derived from the protein Cla h8.
SEQ ID NOS. 21 to 29 and 34 correspond to amino acid sequences derived from
the protein Cla h10.
SEQ ID NOS: 35, 36 and 53 correspond to amino acid sequences derived from
the protein Cla c9.
25 SEQ ID NOS: 38 to 42 and 52 correspond to amino acid sequences derived from
'the protein Alt a6.
SEQ ID NOS: 43 to 49 and 54 correspond to amino acid sequences derived from
the protein Alt a8.
SEQ ID NO: 50 corresponds to an amino acid sequence derived from the protein
30 Alt a10.
SEQ ID NOS: 55 to 57 correspond to the amino acid sequences of control
polypeptides used in Example 6.
NCBI or Uniprot accession numbers for the proteins referred to above are
provided in Examples 1 to 7.
Detailed Descri~tiono f the Invention
5 The present invention is concerned with preventing or treating allergy to
Cladosporiunz and/or Alternaria and provides polypeptides, and pharmaceutically
acceptable salts thereof, suitable for this use. Said polypeptides or salts may be provided
in pharmaceutical formulations.
10 Amino acid sequences and variant amino acid sequences
A polypeptide of the invention may comprise, consist or consist essentially of an
amino acid sequence as shown in any one of SEQ ID NOS 3 1 to 35.
Alternatively, a polypeptide of the invention may comprise, consist or consist
essentially of a T cell epitope-containing variant sequence which is an amino acid
15 sequence as shown in any one of SEQ ID NOS 3 1 to 35 having up to six 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 fbnctional properties of T cell epitopes are discussed further below.
20 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.
25 A variant sequence may have fewer than six amino acid modifications. For
example, said variant sequence may have 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.
30 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
modification that is a deletion, the deleted amino acid is preferably removed from the
5 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.
10 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
15 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
20 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
25 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
30 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 SEQ ID NOS 3 1 to 35 having one, two, three, four, five, or six amino acids
5 removed from the N-terminus of said sequence of SEQ ID NOS 3 1 to 35.
In another embodiment, a variant sequence is an amino acid sequence of any one
of SEQ ID NOS 3 1 to 35 having one, two, three, four, five, or six amino acids removed
from the C-terminus of said sequence of SEQ ID NOS 3 1 to 35.
In another embodiment, a variant amino acid sequence is an amino acid
10 sequence of any one of SEQ ID NOS 3 1 to 35 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 SEQ ID NOS 3 1 to 35 having one,
two or three amino acids removed from the N- andlor C-terminus of said sequence of
15 SEQ ID NOS 3 1 to 35, and optionally no other modifications.
Specific examples of variant amino acid sequences which have at least one
deletion include:
the variant sequence YQKLKSLTK (SEQ ID NO. 6), which is the amino
acid sequence of AEVYQKLKSLTKK (SEQ ID NO. 3 1) having three amino acids
20 removed from the N-terminus and one amino acid removed from the C terminus;
- the variant sequence ITYASRAQG (SEQ ID NO: 13). which is the
amino acid sequence of VAITYASRAQGAE (SEQ ID NO. 32) having two amino acids
removed from both the N-terminus and the C terminus;
- the variant sequence ERGTGSLVI (SEQ ID NO: 14). which is the amino .
25 acid sequence of GHHFKERGTGSLVIT (SEQ ID NO: 33) having five amino acids
removed from the N-terminus and one amino acid removed from the C terminus; and
the variant sequence YTQTKTVSI (SEQ ID NO: 29), which is the
amino acid sequence of ANYTQTKTVSIRL (SEQ ID NO: 34) having two amino acids
removed from the N-terminus and two amino acids removed from the C terminus.
30
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
5 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:
10 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
15 with pyroglutamate. A variant sequence having pyroglutamate susbstituted 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.
20 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.
25 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
30 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 L.
properties of the 20 main amino acids which can be used to select suitable substituents
are as follows:
kla laliphatic, hydrophobic, neutral l hydrophobic, neutral
phe laromatic, hydrophobic, neutral l ~ rIp~olar , hydrophilic, charged (+) I
; Cys
Asp
Glu
Gly
His
Ile
Lys
5 ' Specific examples of variant amino acid sequences which have at least one
substitution include:
- the variant sequence AEVYQKLKALAKK (SEQ ID NO: 52) is the
amino acid sequence of AEVYQKLKSLTKK (SEQ ID NO: 3 1) having two
substitutions. The amino acid S at position 9 of SEQ ID NO: 3 1 is substituted with A,
10 and the T at position 11 of SEQ ID NO: 3 1 is substituted with A. Other preferred
variant sequences of SEQ ID NO: 3 1 include sequences with alternative , preferably
similar, substitutions at positions 9 and 11. For example, instead of substituting with A,
both the S and T amino acids at positions 9 and 11 could be independently replaced
with G or V;
15 - the variant sequence LAITYNSRAEGAE (SEQ ID NO: 54) is the amino
acid sequence of VAITYASRAQGAE (SEQ ID NO: 32) having three substitutions. .
The amino acid V at position 1 of SEQ ID NO: 32 is substituted with L, the A at
position 6 of SEQ ID NO: 32 is substituted with N, and the Q at position 10 of SEQ ID
NO: 32 is substituted with E. Other preferred variant sequences of SEQ ID NO: 32
20 include sequences with alternative, preferably similar, substitutions at positions 1, 6 and
polar, hydrophobic, neutral
polar, hydrophilic, charged (-)
polar, hydrophilic, charged (-)
aliphatic, neutral
aromatic, polar, hydrophilic,
I I I
charged (+)
aliphatic, hydrophobic, neutral
polar, hydrophilic, charged(+)
Asn
Pro
Gln
Ser
Thr
eu
polar, hydrophilic, neutral
hydrophobic, neutral
polar, hydrophilic, neutral
polar, hydrophilic, neutral
polar, hydrophilic, neutral
Val
Trp
aliphatic, hydrophobic, neutral Tyr
aliphatic, hydrophobic, neutral
aromatic, hydrophobic, neutral
aromatic, polar, hydrophobic
10. For example, instead of substituting L with V, the amino acid at position 1 could.be
replaced with G or A; instead of substituting A with N, the amino acid at position 6
could be replaced with Q, E or D; and instead of substituting Q with E, the amino acid
at position 10 could be replaced with N or D;
5 - the variant sequence GLHFRERKTGSLVIT (SEQ ID NO: 44) is the
amino acid sequence of GHHFKERGTGSLVIT (SEQ ID NO: 33) having three
substitutions The amino acid H at position 2 of SEQ ID NO: 33 is substituted with L,
the K at position 5 of SEQ ID NO: 33 is substituted with R, and the G at position 8 of
SEQ ID NO: 33 is substituted with K. Other preferred variant sequences of SEQ ID
10 NO: 33 include sequences with alternative, preferably similar, substitutions at positions
2, 5 and 8. For example, instead of substituting H with L, the amino acid at position 1
. could be replaced with V, G or A; instead of substituting K with R, the amino acid at,
position 6 could be replaced with H; and instead of substituting G with K, the amino
acid at position 10 could be replaced with H or R; and
15 - the variant sequence GGYKAAVRPT-Nle-LE (SEQ ID NO: 36) is the
amino acid sequence of GGYKAAVRPTMLE (SEQ ID NO: 35) having one
substitution. The amino acid M at position 11 of SEQ ID NO: 35 is substituted with
Norleucine.
20 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
25 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
30 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
5 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
10 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 SEQ ID NOS 3 1 to 35 having one, two, three, four, five, or six amino acids
15 inserted at the N-terminus of said sequence of SEQ ID NOS 3 1 to 35.
In another embodiment, a variant sequence is an amino acid sequence of any one
of SEQ ID NOS 3 1 to 35 having one, two, three, four, five, or six amino acids inserted
at the C-terminus of said sequence of SEQ ID NOS 3 1 to 35.
In another embodiment, a variant sequence is an amino acid sequence of any qne
20 of SEQ ID NOS 3 1 to 35 having a number of amino acids inserted at both the N- and Cterminus
of said sequence of SEQ ID NOS 3 1 to 35, 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 SEQ ID NOS 3 1 to 35 having one,
two or three amino acids inserted at the N- and/or C-terminus of said sequence of SEQ
25 ID NOS 3 1 to 35, 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 C-
30 terminus 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 SEQ ID NOS 3 1 to 35 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
SEQ ID NOS 3 1 to 3 5.
A specific example of a variant amino acid sequence which has at least one .
5 insertion is:
- the variant sequence VAITYASRAQGAEK (SEQ ID NO: 30), which is
the amino acid sequence of VAITYASRAQGAIZ (SEQ ID NO: 32) having a lysine
inserted at the C terminus.
In some variant sequences there may be substitutions and insertions. For
10 example:
- the variant sequence of LAITYNSRAEGAEK (SEQ ID NO: 43) is the
sequence of SEQ ID NO: 32 having three substitutions as described above for SEQ ID
NO: 54 and in addition having a lysine inserted at the C-terminus. Other variant
sequences based on SEQ ID NO: 43 may have alternative, preferably similar
15 , substitutions as described above for SEQ ID NO: 54, as well as a lysine inserted at the
C-terminus.
Polypeptides
A polypeptide of the invention is up to 30 amino acids in length and comprises,
20 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
25 maximum length of 30, 25,20, 17 or 15 amino acids.
A polypeptide 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.
30 A polypeptide 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.
5 A polypeptide 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
10 and/or C-terminus of said amino acid sequence or variant sequence.
Put another way, a polypeptide of the invention may have a sequence consisting
of said amino acid sequence or variant sequence having an N-terminal and/or Cterminal
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
15 the polypeptide, as defined above.
The amino acids in an N-terminal extension of a said amino acids 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.
20 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,
25 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
30 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
5 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 t a
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.
10 Specific examples of polypeptides of the invention which include an N-terminal
and/or C-terminal extension to an amino acid sequence or variant sequence include the
following:
- AEVYQKLKSLTKK (SEQ ID NO: 3 1) may have a C-terminal
extension of one, two, three, four or five amino acids corresponding to the one, two,
15 three, four or five contiguous amino acids immediately C-terminal to
AEVYQKLKSLTKK in the native sequence of Cla h 6, that is the amino acids R, Y, G,
Q and S. For example, where all five said contiguous amino acids are present, the
polypeptide of the invention has the amino acid sequence of
AEVYQKLKSLTKKRYGOS (SEQ ID NO: 1; C-terminal extension is underlined).
20 - AEVYQKLKALAKK (SEQ ID NO: 52) may have a C-terminal
extension of one, two, three or four amino acids corresponding to the one, two, three or
four contiguous amino acids immediately C-terminal to AEVYQKLKALAKK in the
native sequence of Alt a 6, that is the amino acids T, Y, G and Q. For example, where
all four said contiguous amino acids are present, the polypeptide of the invention has the
25 amino acid sequence of AEVYQKLKALAKKTYGQ (SEQ ID NO:38; C-terminal
extension is underlined).
- VAITYASRAQGAE (SEQ ID NO: 32) may have a N-terminal extension
of one, two, three, four, five, six or seven amino acids corresponding to the one, two,
three, four, five, six or seven contiguous amino acids immediately N-terminal to
30 ' VAITYASRAQGAE in the native sequence of Cla h 8, that is the amino acids C, A, E,
M, G, A 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 Cterminal
to VAITYASRAQGAE in the native sequence of Cla h 8. That is the amino
acids E, N and V. For example, where all seven 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
5 CAEMGAAVAITYASRAQGAEW (SEQ ID NO: 10; N and C-terminal extensions
are underlined).
- GHHFKERGTGSLVIT (SEQ ID NO: 33) may have a C-terminal
extension of one, two, three, four, five, six, seven, eight, nine or ten amino acids
corresponding to the one, two, three, four, five, six, seven, eight, nine or ten contiguous
10 amino acids immediately C-terminal to GHHFKERGTGSLVIT in the native sequence
of Cla h 8, that is the amino acids A, S, M, S, G, H, I, A, N and F. For example, where
all ten said contiguous amino acids are present, the polypeptide of the invention has the
amino acid GHHFKERGTGSLVITASMSGHIANF (SEQ ID NO: 1 1, C-terminal
, extension underlined).
15 - ANYTQTKTVSIRL (SEQ ID NO: 34) may have a N-terminal extension
of one amino acid corresponding to the one amino acid immediately N-terminal to
ANYTQTKTVSIRL in the native sequence of Cla h 10, that is the amino acid L. It may
also have a C-terminal extension of one, two, three, four, five or six amino acids
corresponding to the one, two, three, four, five or six contiguous amino acids
20 immediately C-terminal to ANYTQTKTVSIRL in the native sequence of Cla h 10.
That is the amino acids G, D, A, L, F and G. For example, where the one amino acid is
present in the N-terminal extension and all six contiguous amino acids are present in the
C-terminal extension the polypeptide of the invention has the amino acid sequence of
I&VYTQTKTVSIRLGDALFG (SEQ ID NO:27, N- and C-terminal extensions
25 underlined).
- GGYKAAVRPTMLE (SEQ ID NO: 35) may have a N-terminal
extension of one, two, three, four, five, six, seven, eight, nine or ten amino acids
corresponding to the one, two, three, four, five, six, seven, eight, nine or ten contiguous
amino acids immediately N-terminal to GGYKAAVRPTMLE in the native sequence of
30 Cla c 9, that is the amino acids E, S, N, Y, S, A, I, V, E and K. It may also have a Cterminal
extension of one, two, three, four, five, six, seven, eight, nine or ten amino
acids corresponding to the one, two, three, four, five, six, seven, eight, nine or ten
contiguous amino acids immediately C-terminal to GGYKAAVRPTMLE in the native
sequence of Cla c 9, that is the amino acids E, I, E, S, E, A, K, V, A and S. For
example, where all ten contiguous amino acids are present in the N-terminal extension
and all ten contiguous amino acids are present in the C-terminal extension the
5 polypeptide of the invention has the amino acid sequence of
ESNYSAIVEKGGYKAAVRPTMLEEIESEAKVAS (SEQ ID NO: 53; N- and Cterminal
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
10 - 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
15 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
20 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 SEQ ID NOS: 3 1 to 35. Preferably positively charged residues
such as arginine and lysine are added. Amino acids selected from histidine, glutamate
25 and aspartate may be added.
Additional polypeptides disclosed herein include, for example, polypeptides
comprising, consisting or consisting essentially of an amino acid sequence of any one of
SEQ ID NOS 2 to 5,7 to 9, 12, 15 to 26,28,37, 39 to 42 and 45 to 5 1, or a variant
sequence derived therefrom. The variant sequences referred to in this paragraph are
30 derived from each corresponding original amino acid sequence in the same manner as is
described above with respect to variant sequences derived from SEQ ID NOS: 3 1 to 35.
T cell epitopes
A polypeptide 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 sequences contains a T cell
5 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 hnctional .
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.
10 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
15 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, andlor the ability of a polypeptide comprising the
epitope to activate a T cell, preferably when bound to an MHC Class I1 molecule.
The ability of a polypeptide to bind to an MHC molecule may be evaluated
20 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.
25 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).
3 0 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 I1 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.
5 Typically, a polypeptide has substantially the same MHC Class I1 binding
characteristics as another polypeptide if both polypeptides are capable of binding
specifically to one or more MHC Class I1 molecules belonging to the same MHC Class
I1 allele supertype family. Examples of MHC Class I1 allele supertype families include
HLA-DRl , HLA-DR3, HLA-DR4, HLA-DR7, HLA-DR8, HLA-DRl 1 , HLA-DRI 3,
10 HLA-DRl5 and HLA-DR51. Most preferably, both polypeptides will bind specifically
to the same MHC Class I1 molecule, that is to an MHC Class I1 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
15 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
20 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 a1 (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
25 in which substitutions increasing polypeptide-MHC affinity have been made (Metzler &
Wraith(1993) Int Immunol: 11 59-65). That an altered polypeptide ligand can cause
long-term and profound anergy in cloned T cells was demonstrated by Sloan-Lancaster
et a1 (1993) Nature 363: 156-9.
30 Sequence identity
T cell epitope-containing variant sequences 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 SEQ ID NOS: 3 1 to 35. 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 SEQ ID
5 NOS: 31 to 35.
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, 1 1, 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 .
10 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 a].,
15 1994, supra) with the following parameters:
Pairwise alignment parameters - Method: accurate, Matrix: PAM, Gap open
penalty: 10.00, Gap extension penalty: 0.10; Multiple alignment parameters -Matrix:
PAM, Gap open penalty: 10.00, % identity for delay: 30, Penalize end gaps: on, Gap
separation distance: 0, Negative matrix: no, Gap extension penalty: 0.20, Residue-
20 , specific 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
25 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
30 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.
5 : 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
10 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.
15 Pharmaceutically acceptable salts of polypeptides can be prepared by any
, suitable technique. Typically, salification involves reaction of the polypeptide or a sqlt
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
20 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
25 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
30 methods known in the art, for example such as those described in Meziere et a1 (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 a1 (1997) show that, at least for MHC class 11 and T helper cell responses;
these pseudopolypeptides are usekl. 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
5 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.
10 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 (-NH;! + -
1 5 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
20 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 metaboFc
processes, decreasing the amounts of agent which needs to be administered, along with
25 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
30 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
5 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
10 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
15 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 .
20 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
25 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
30 prevent dimer formation such that any cysteine residues are replaced with serine or 2-
aminobutyric acid.
Polypeptide combinations
A polypeptide of the invention, or salt thereof, may be provided in combination
with one or more fbrther polypeptides, or salts thereof, of the invention. Accordingly,
' two, three, four, or five polypeptides or salts thereof may be provided together in
5 combination. For example, a suitable combination may comprise two, three, four or five
different polypeptides selected from the following:
i) a polypeptide comprising, consisting or consisting essentially of the
amino acid sequence of SEQ ID NO: 35 or a said variant sequence derived therefrom,
or a said salt thereof;
10 ii) a polypeptide comprising, consisting or consisting essentially of the
amino acid sequence of SEQ ID NO: 3 1 or a said variant sequence derived therefrom,
or a said salt thereof;
iii) a polypeptide comprising, consisting or consisting essentially of the
amino acid sequence of SEQ ID NO: 32 or a said variant sequence derived therefrom,
15 or a said salt thereoc .
iv) a polypeptide comprising, consisting or consisting essentially of the
amino acid sequence of SEQ ID NO: 33 or a said variant sequence derived therefrom,
or a said salt thereof; and
v) a polypeptide comprising, consisting or consisting essentially of the
20 amino acid sequence of SEQ ID NO: 34 or a said variant sequence derived therefrom,
or a said salt thereof.
It is preferred that the combination should include no more than one
polypeptide, or salt thereof, selected from each of i) to v). It is particularly preferred
that the combination includes at least one polypeptide, or salt thereof, selected from i).
25 A combination as defined above may further include one or more additional
. polypeptides. Additional polypeptides disclosed herein include, for example,
polypeptides comprising, consisting or consisting essentially of an amino acid sequence
ofanyoneofSEQIDNOs2to5,7to9, 12, 15to26,28,37,39to42and45to51,or
a variant sequence derived therefrom. In some embodiments, the further polypeptides
30 in a combination are selected only from polypeptides which comprise, consist or consist
essentially of an amino acid sequence of any one of SEQ ID NOS: 1 to 29, 37, 5 1 and 53
(which are derived directly from Cladosporzum allergens), or a variant sequence derived
therefrom. In other embodiments, the further polypeptides may comprise, consist or
consist essentially of an amino acid sequence of any one of SEQ ID NOS: 1 to 29 and
37 to 54, or a variant sequence derived therefrom. The variant sequences referred to in
this paragraph are derived from each corresponding original amino acid sequence in the
5 same manner as is described above with respect to variant sequences derived from SEQ
ID NOS: 3 1 to 35.
Preferably, a combination will include a polypeptide comprising, consisting or
consisting essentially of a first amino acid sequence (or variant derived therefrom) and
at least one further polypeptide comprising, consisting or consisting essentially of a
10 second amino acid sequence (or variant derived therefrom), wherein said second amino
acid sequence derives from a different allergen to said first amino acid sequence. The
allergens from which each of the amino acid sequences disclosed herein derive are set
out in the section entitled "Description of Sequences" above. By including sequences
which derive from more than one Cla (andlor Alt) allergen, a combination may allow
15 for broad coverage of mould allergy observed in the general population by providing T
cell epitopes from more than one mould allergen.
Particularly preferred combinations include at one, two or three further
polypeptides selected from:
(f) GWGVMVSHRSGET (SEQ ID NO: 39; Alt14);
20 (g) GYTGKIKIAMDVASSE (SEQ ID NO: 4 1 ; Alt 15), and
(h) WSWKIGPAIATGNT (SEQ ID NO. 50; Alt28).
Any of the polypeptide combinations described above may optionally comprise
no further polypeptides, or no further polypeptides derived from Alternaria andlor
Cladosporium allergens. Any of the polypeptide combinations described above may be
25 incorporated in a pharmaceutical formulation of the invention as described in more
detail below.
Medical uses and methods
A preferred aspect of the invention is the prevention or treatment of allergy. In
30 this aspect, the invention provides a polypeptide or salt of the invention or a
pharmaceutical formulation of the invention for use in a method of treating or
preventing allergy to Cladosporium and/or Alternaria. The polypeptide, salt or
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
Cladosporium and/or Alternaria genus.
The invention further provides a use of a polypeptide or salt of the invention for
5 the manufacture of a medicament for the prevention or treatment of allergy to
Cladosporium and/or Alternaria.
The invention further provides a method of treating an individual for allergy to
Cladosporiurn and/or Alternaria or of preventing in an individual allergy to
Cladosporiurn and/or Alternaria, which method comprises administering to said
10 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
15 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
20 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 invention thus provides a polypeptide or salt of the invention for use in a
method of preventing or treating allergy to Cladosporiurn and/or Ahernaria as described
25 above, wherein said method further comprises administering at least one, preferably two
or more additional polypeptides of the invention. The multiple peptides of this method
may each be administered simultaneously, sequentially or concurrently.
The polypeptide, salt or pharmaceutical formulation of the invention may treat
or prevent the allergy by desensitising or tolerising to Cladosporium and/or Alternaria
30 allergens. A polypeptide of the invention 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
5 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 anergising or elimination of these T-cells leads to
desensitisation of the patient for a particular allergen The desensitisation manifests
10 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. .
15 The individual to whom the polypeptide, salt or pharmaceutical formulation is
administered may be asymptomatic. A prophylactically effective amount of the
polypeptide or 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.
20 Alternatively, the individual to whom the polypeptide, salt or 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
polypeptide or pharmaceutical formulation is administered to such an individual. A
therapeutically effective amount is an amount which is effective to ameliorate one or
25 , more symptoms of allergy.
The individual to whom the polypeptide, salt or 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
30 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
5 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.
10 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 1 1 common DRB 1 allele families are shown
in Table 1 (Data from HLA Facts Book, Parham and Barber).
15 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
20 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 DRBI alleles is:
25 4 - at least 9%
7 - at least 10%
DRB 1
%
Reference
population
Yo
1
6.4
9.4
3
14.7
11.1
4
15.7
12.8
7
8.8
13.2
8
3.4
3.7
11
8.3
13.4
12
3.9
2.3
13
14.7
10.2
14
2.9
3.2
15
17.6
10.7
16
2.5
3.6
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
5 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.
10 The allergic individual is typically allergic to moulds of the Cladosporium
genus, particularly Cladosporium herbarium and/or Cladosporium cladosporoides. The
allergic individual may be allergic to moulds of the Alternaria genus, particularly
Alternaria altemata. The allergic individual may be allergic both to moulds of the
Cladosporium and Alternata genus.
15 The polypeptides, salts or 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 tebe
allergic to moulds of the Alternaria and Cladosporiunz genus. In particular where
multiple polypeptides are provided in combination in a pharmaceutical formulation,
20 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
25 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, salts and pharmaceutical formulations
30 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
5 stimulated with a pharmaceutical formulation in vitro. A polypeptide, salt or
pharmaceutical formulation of the invention preferably causes the release of less than
596, less than 494, 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
10 histamine content of 1 50ng/1 o7 cells.
Pharmaceutical formulations
Each polypeptide or salt of the invention may be provided to an individual in in
isolated, substantially isolated, purified or substantially purified form. For example, a
15 polypeptide or salt of the invention 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.
Thus, according to a further aspect of the invention, the invention further
20 provides a pharmaceutical formulation which comprises a pharmaceutically acceptable
carrier or diluent and a polypeptide, or a pharmaceutically acceptable salt thereof, of the
invention. The pharmaceutical formulation may comprise any combination of
polypeptides or salts of the invention as described above.
The carrier(s) or diluent(s) present in the pharmaceutical formulation must be-
25 "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 thioglycero1,thioanisole or methionine.
A composition containing one or more polypeptides or salts of the invention can
30 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
5 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 *
10 auxiliary substances is available in Remington's Pharmaceutical Sciences (Mack Pub.
Co., N. J. 1991).
The polypeptide or salt is typically present at 0.1% to 50% by weight in the
pharmaceutical formulation, more preferably at 0.1% to 5% by weight. The polypeptide
or salt may be present at less than 0.1% by weight in the pharmaceutical formulation.
15 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
20 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
25 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
30 formulation of the invention, comprising combining a polypeptide or salt as described
, above, or a combination of polypeptide of polypeptides and 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
polypeptide(s), or salt(s) in dry form and reconstituting said polypeptide(s), or salt(s)
with a said pharmaceutically acceptable carrier or diluent.
The pharmaceutical formulations may be prepared, packaged, or sold in the form
5 ' 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
10 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
15 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 of the present invention may be encapsulated,
20 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(1actides) and poly(1actide-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,
25 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,
30 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.
5 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,
10 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
15 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 nmollml. The concentration of each polypeptide may
be more preferably in the range of 0.3 to 200 nmollml, 3 to 180 nmollml, 10 to 150
nmol/ml, 50 to 200nmol/ml or 30 to 120 nmol/ml. The pharmaceutical formulation
20 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 one or
more polypeptides of the invention. An adjuvant is preferably administered in an
, amount which is sufficient to augment the effect of the polypeptide(s) of the inventian
or vice versa. The adjuvant or further therapeutic agent may be an agent that
25 potentiates the effects of a polypeptide of the invention. 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,
30 prednisone, prednisolone, methylprednisolone, betamethasone and triamcinolone. A
preferred glucocorticoid is dexamethasone.
In an embodiment where one or more polypeptides of the invention 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
5 formulations. A pharmaceutical formulation may therefore be prepared which
comprises a polypeptide of the invention 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
10 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 of the invention is to be administered to an
15 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
20 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
25 liquid jet injection system. The administration may be made using a patch, such as a
microtine patch. Compositions 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,
30 , suppositories, sublingual administration, and active or passive transdermal delivery .
techniques.
Llosages
Administration of the polypeptides, salts or pharmaceutical formulations of the
' invention may be by any suitable method as described above. Suitable amounts of the
5 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
10 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
15 ' and timing of the administration regime. Suitable doses of a polypeptide or salt of the
invention may be in the order of up to 10 pg, up to 15pg, up to 20pg, up to 25pg, up to
30pg, up to 50pg, up to 100pg, up to 500 pg or more per administration. Suitable doses
may be less than 15pg, but at least lng, or at least 2ng, or at least 5ng, or at least 50ng,
or least 100ng, or at least 500ng, or at least lpg, or at least 10pg. For some
20 polypeptides of the invention, the dose used may be higher, for example, up to 1 mg, up
to 2 mg, up to 3 mg, up to 4 mg, up to 5 mg or higher. Such doses may be provided in a
liquid formulation, at a concentration suitable to allow an appropriate volume for
administration by the selected route. 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 3 . 5 ~ "
25 refers to a total peptide or salt concentration of up to 35pg in a composition comprising
a combination or more than one peptide or salt.
Nucleic acids and vectors
The polypeptides of the invention may be administered directly, or may be
30 administered indirectly by expression from an encoding sequence. For example, a
polynucleotide may be provided that encodes a polypeptide of the invention. A
polypeptide of the invention may thus be produced from or delivered in the form of a
polynucleotide which encodes, and is capable of expressing, it. Any reference herein to
the use, delivery or administration of a peptide of the invention is intended to include
the indirect use, delivery or administration of such a peptide via expression from a
polynucleotide that encodes it.
5 In this aspect, the invention provides a polynucleotide which encodes a
polypeptide comprising, consisting or consisting essentially of the amino acid sequence
of any one of SEQ ID NOS 3 1 to 35 or a variant derived therefrom.
The terms "nucleic acid molecule" and "polynucleotide" are used
interchangeably herein and refer to a polymeric form of nucleotides of any length, either
10 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.
15 Polynucleotides can be synthesised according to methods well known in the art,
as described by way of example in Sambrook et a1 (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 of the invention. Such polynucleotides may be
20 administered or used in the prevention or treatment of allergy to Cladosporium and/or
Alternaria.
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 intradennal
25 injection; transdermal particle delivery; inhalation; topically, or by oral, intranasal or
mucosal modes of administration. The molecule alternatively can be introduced ex vivo
into cells that have been removed from a subject. For example, a polynucleotide,
expression cassette or vector of the invention may be introduced into APCs of an
individual ex vivo. Cells containing the nucleic acid molecule of interest are re-
30 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 (AYCs)
The invention encompasses the use in vitro of a method of producing a
population of APCs that present the peptides of the invention on their surface. Said
5 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 andlor Alternaria. The cells
should be accepted by the immune system of the individual because they derive from
10 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
15 the individual.
In vitro method
The invention further provides an in vitro method of determining whether T '
cells recognize a polypeptide or salt of the invention, which method comprises
20 contacting said T cells with said polypeptide or salt and detecting whether said T cells
are stimulated by said polypeptide or salt. Preferably, the method comprises use of a
polypeptide, or salt thereof, comprising, consisting or consisting essentially of the
amino acid sequence of any one of SEQ ID NOS 3 1 to 35 or a variant derived therefrom
The above method may be carried out to determine whether an individual has, or
25 is at risk of having, an allergy to Cladosporium and/or Alternaria.
The invention is illustrated by the following Examples:
' Exam~les
3 0
Example 1
MHC Class 11 binding search
The aim of this study is to identify a distinct panel of polypeptides having
sequences with strong affinities for the seven most common human MHC Class I1
HLA-DRBl* allotypes (covering in total around 63% of the allotypes found in the
average Caucasian population). In order to identify said polypeptides in the major
5 Cladosporium allergens Cla h 6, Cla h 8 and Cla h 10 from Cladosporium herbarium
and in Cla c 9 from Cladosporium cladosporoides, an in silico approach known as
"peptide threading" was performed using the commercially available EpiMatrix
algorithm (EpiVax Inc.) This is a bioinformatic method of analysing a polypeptide
having a given sequence for the potential to be accommodated within the binding
10 groove of MHC class I1 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. @e Groot et a1 ., AIDS Research and
' Human Retroviruses 13 : 539-4 1 (1 997)). The procedure for developing matrix motifs '
15 was published by Schafer et al, 16 Vaccine 1998 (1998). In this Example, binding
potential for HLA DR1, DR2, DR3, DR4, DR7, DR8, DR11, DR13 and DR15 is
assessed. Putative MHC ligands are selected by scoring each 10-mer frame in a
polypeptide 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.
20 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
25 , 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,
30 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 the known isoform
of Cla h 6 (NCBI accession no: P42040). These analyses identified core polypeptides
5 (and their flanking sequences) derived from the above sequences which are predicted to
have good MHC class-I1 binding. The 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
10 ' stabilizing flanks (N-terminal and C-terminal, not bold) were included for use with thk
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
15 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
20
Based on a fbrther analysis of Epimatrix data, and based on homology with
MHC-binding polypeptides from Alt a 6, the following additional sequences from Cla h
6 were also identified as having suitable MHC-binding properties: MAVAKAAAA
(SEQ ID NO: 5), YQKLKSLTK (SEQ ID NO: 6), WGVMVSHRS (SEQ ID NO: 7),
YTGQIKIAM (SEQ ID NO: 8), IKIAMDVAS (SEQ ID NO: 9).
Based on homology with Cla h 6 and further design and screening, the following
sequences derived from Alt a 6 were also identified as having suitable MHC-binding
5 properties:
SEQ ID NO: 38 (Altl3A; AEVYQKLKALAKKTYGQ), SEQ ID NO: 39
(Alt14; GWGVMVSHRSGET), SEQ ID NO: 40 (Alt14A; GWGV-Nle-VSHRSGET),
SEQ ID NO: 41 (Alt15; GYTGKIKIAMDVASSE), SEQ ID NO: 42 (Alt15A;
GYTGKIKIA-Nle-DVAS SE). Nle: Norleucine.
10
Examule 2
EpiMatrix analyses as above were performed on the entire sequence of the
known isoform of Cla h 8 (NCBI accession no: POCOYS). This analysis identified core
sequences (with their flanking sequences) derived from said Cla h 8 isoform which are
15 predicted to have good MHC class-I1 binding properties. These sequences are shown
below in Table 3. Headings and notes for Table 3 are as with Table 2 above.
20
Based on a firther analysis of Epimatrix data, and based on homology with
MHC binding polypeptides from Alt a 8, the following additional sequences from Cla h
8 were also identified as having suitable MHC-binding properties: ITYASRAQG (SEQ
ID NO: 13), ERGTGSLVI (SEQ ID NO: 14), YNVAKAGCI (SEQ ID NO: 15),
WRDFARVNS (SEQ ID NO: 16), FVPKETQQL (SEQ ID NO: 17), WHSMIPMGR
(SEQ ID NO: 18), LKGAYVYFA (SEQ ID NO: 19), YVYFASDAS (SEQ ID NO: 20).
SEQ ID NO: s 19 and 20 are variants of SEQ ID NO: 12.
Based on homology with Cla h 8 and further design and screening, the following
5 sequences derived from Alt a 8 were also identified as having suitable MHC-binding
properties:
SEQ ID NO: 43 (Alt20; LAITYNSRAEGAEK), SEQ ID NO: 44 (Alt21;
GLHFRERKTGSLVIT), SEQ ID NO: 45 (Alt23; NEWRDFARVNSISP), SEQ ID NO:
46 (Alt 24; KLWHSMIPMGRDAK), SEQ ID NO: 47 (Alt24A; KLWHS-Nle-IP-Nle-
10 GRDAK), SEQ ID NO: 48 (Alt24B KLWHS-Nle-IPMGRDAK), SEQ ID NO: 49 (Alt
24C; KLWHSMIP-Nle-GRDAK).
Examole 3
EpiMatrix analyses as above were performed on the entire sequence of known
15 isoforms of Cla h 10 (NCBI accession nos: P42041.2; P40108). This analysis identified
core sequences (with flanking sequences) derived from the said Cla h 10 isoform which
are predicted to have good MHC class-I1 binding. These sequences are shown below in
Table 4. Headings and notes for Table 4 are as with Table 2 above.
20 Table 4
P40108
P4 0 10 8
P4010 8
P4 0 10 8
P4 0 10 8
P 4 0 10 8
: .
. .
. ... ...........-.-.. ....'...I.. :
?:.!:? .... ~:...~~.':~ ~~~~
.: ...
: i ~ ~ ~ ~ ; ~ ~ ~ , ~ ~ ~ < ~ ~ ~ ~ ~
, ..: .., +
PDTFNnrKKEPIGVCRS
GSTWGRTILKAAASSNLKKV
A1 SWVNFGIFFNHGQCCCAG
YDKE'VQKE'KERAQKNWGDPF
EDAIKLGNASTYGLAAAVHTKNL
LANYTQTKTVS IRLGDALFG
-0.65
0 . 2 8
0. 8 1
-1.03
- 0 . 0 1
0 . 13
7
11
9
l1
10
9
.:...i."......3. ..0.
.j.,. .6.. ..... :.:..,'. .., .c. .
. .-, ..I .... ............ .
........... ......?..
...... .,.. ......
............ .....,. ..
P58
P59
P60
P 6 1
P62
P 6 3
22
23
24
2 5
26
2 7
Based on a further analysis of Epimatrix data, and based on homology with Alt a
10 MHC binding peptides, the following additional sequence from Cla h 10 was also
identified as having suitable MHC-binding properties: WKIGPAIAT (SEQ ID NO: 28),
YTQTKTVSI (SEQ ID NO: 29).
5 Based on homology with Cla h 10 and further design and screening, the
following sequence derived from Alt a 10 was also identified as having suitable MHCbinding
properties:
SEQ ID NO: 50 (Alt28; WSWKIGPAIATGNT).
10 Examule 4
EpiMatrix analyses as above were performed on the entire sequence of a known
isoform of Cladosporium cladospovoides vacuolar serine protease (Cla c 9; Uniprot
accession no: BOL807). This analysis identified a core sequence (with flanking
sequences) derived from the said vacuolar serine protease which is predicted to have
15 good MHC class-I1 binding. This sequence is shown below in Table 5. Headings and
notes for Table 5 are as with Table 2 above.
Table 5
20 In addition, the sequence GLFISTGTQGGGQ (SEQ ID NO: 37) derived from
Cla h 7 was identified based on homology with an MHC-binding polypeptide from Alt a
7.
Examule 5
25 The sequences set out in Table 6 were selected by the inventors as having
desirable characteristics, based on the analyses performed in Examples 1 to 4 and a
consideration of solubility and other physicochemical characteristics. The sequences of
40
SEQ ID NOS: 30 and 36 are preferred variant sequences derived from SEQ ID NOS: 32
and 35, 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 6 were produced and were particularly preferred
5 for screening in subsequent assays. The polypeptide of SEQ ID NO: 30 (Cla25A) is
typically used in place of the polypeptide of SEQ ID NO: 32 (Cla25) in said assays.
Table 6
10 Example 6
In vitro binding analysis
Polypeptides having the sequences identified in Examples 1 to 5 are prescreened
for solubility in an aqueous, acidic milieu and the peptides are tested in an in
vitro MHC Class I1 binding assay.
15
Methods
The assay employed is a competitive MHC class I1 binding assay, wherein each
polypeptide is analysed for its ability to displace a known control binder from each of
' the human MHC class 11 allotypes investigated. The allotypes and control polypeptidis
20 used in this study are those shown below:
Peptide
Cla 16
Cla25
Cla25A
Cla26
Cla29
Cla3 5
Cla35A
Residues
in parent
187-199 (Cla h 6)
49-61 (Cla h 8)
49-6 1 +K (Cla h 8)
144-158 (Cla h 8)
478-490 (Cla h 10)
337-349 (Cla c 9)
337-349
Sequence
AE WQKLKSLTKK
VAITYASRAQGAE
VAITYASRAQGAEK_
GHHFKERGTGSLVIT
ANYTQTKTVSIRL
GGYKAAVRPTMLE
GGYKAAVRPT-Nle-LE
SEQ
ID NO.
3 1
3 2
30
3 3
34
3 5
36
Sequence
AKTIAYDEEARRGLE (SEQ ID
NO:%)
PKYVKQNTLKLAT
(SEQ ID NO:56)
ENPVVHFFKNIVTPR
Allotype
DRB 1 *0301
DRB1*1101
D R . 1 * 150 1
Control Polypeptide
Myco. tuberculosisAeprae hsp 65 2-16
Influenza haernagglutinin 307-3 19
Human myelin basic protein 85-99
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
5 control polypeptide. Thus, a polypeptide that has an IC50 value that is parity to the
control polypeptide has an identical binding affinity, while peptides with a ratio less
than one have a higher affinity and those with a ratio greater than one have a lower
affinity.
Solubility in aqueous solution is an essential criterion for a polypeptide to be an
10 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-DRB 1 * binders. Polypeptides which bind to one or more of the
MHC Class I1 allotypes are identified. It would be expected that such polypeptides
15 would have the ability to bind similar allotypes that have not been tested through the
homology of MHC structures.
Examole 7
The following methods are used to evaluate T cell activation characteristics of
20 polypeptides having the sequences identified in Examples 1 to 5.
Cell proliferation assay
The cell proliferation assay is performed on PBMC's (1 40x1 o6 cells required for
all parameters to be tested). Proliferation is measured by the incorporation of the
25 radiolabelled compound 3H-thymidine. In more detail, 100pl 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 37OC for a
maximum of 4 hours. PBMC's isolated as described above are prepared to a
concentration of 2x10~ce lls/ml in complete medium at room temperature. 100pl of cell
30 solution is then distributed into each of the wells of the 96 well plates containing
antigenlpolypeptide. The plates are then incubated for 6 to 8 days. The cultures are
pulsed with tritiated thymidine solution by adding lop1 of tritiated thymidine stock
solution (1.85MBqIml 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
5 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
10 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
15 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) 25nglml and whole Alternaria allergen extract 100 &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 pl containing polypeptides at a concentration of 200 d m l , such that
20 subsequent addition of 100 ~1 of cells would create a final assay concentration of 100
~glml.
Peripheral blood mononuclear cells (PBMCs) were isolated from heparinised
blood by Ficoll density gradient centrifugation. A 100 pl aliquot of a 5x106 celllml
PBMC suspension was then added to each well and the plates placed in a humidified
25 5% C02 incubator at 37OC for 5 days. Following stimulation, culture supernatants (100
yl) 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
30 , 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 pdml 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 6 are summarized in
Table 8.
The number of responders out of the 50 mould allergic subjects tested showing
Polypeptide
Cla16
Cla25A
Cla26
10 an IFNg release of greater than 100pg/ml was also calculated for each polypeptide.
Results for the polypeptides having the sequences of Table 6 are summarized in Table
% responders
36
44
44
Table 9
Polypeptide
Cla29
Cla35
Cla35A
All five polypeptides comprising sequences derived from CIadosporium (the
sequences of Cla16, Cla25A, Cla26, Cla29, and Cla35) showed responses in the
% responders
48
3 4
48
Polypeptide
Cla16
Cla25A
Cla26
population. The polypeptide having the sequence of Cla35A provided for an increased
response compared to the polypeptide having the sequence of Cla35 and was the top-
% responders
16
22
28
20 performing Cladosporium-derived polypeptide, also having the highest proportion of
IFN-g (release >I00 pglml) responders. A polypeptide having the sequence of Cla35 or
Cla35A is preferred for treatment or prevention of Cladosporium allergy. Other top
Polypeptide
Cla29
Cla35
Cla3 5A
performing Cladosporium-derived polypeptides included those having the sequences of
Cla26 and Cla25A. A polypeptide having either of these sequences is therefore also
25 preferred for treatment or prevention of Cladosporium allergy.
A number of polypeptides having sequences derived from Alternaria allergens,
which sequences have homology with corresponding regions of Cladosporium
% responders
28
28
42
allergens, were also tested and showed responses in the population. A positive result
was taken as a cytokine release of greater than 50 pglml 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 are shown in
5 Table 10 below.
Table 10
As shown, the top performing Alternaria-derived polypeptide has the sequence
10 of Alt 13A. Other top performing Alternaria -derived polypeptides include those having
the sequences of Alt14, Altl4A, Alt15, Altl5A and Alt28. These polypeptides induce
positive responses in a high proportion of subjects and are thus advantageous in terms
of population coverage. One or more of these polypeptides may be preferably included
in combination with the Cladosporium-derived peptides described above in a
15 polypeptide combination of the invention for the treatment or prevention of
Cludosporiurn and/or Alternaria allergy.
Example 8 - Preparation of peptides, salts and pharmaceutical formulations
Peptides are prepared as follows. Synthesis is performed in a solid phase peptide
20 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-[(IHBenzotriazol-
1 -yl)(dimethylamino)methylene]-N-methylmethanaminium
25 tetrafluoroborate N-oxide (TBTU) and N,N-diisopropylethylamine (DIPEA) in DMF or
diisopropylcarbodiimide PIC) 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
5 Fmoc-group with 20% (VIV) piperidine in either DMF or 1-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
10 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),
15 triisopropylsilane (TIS), and water. After filtering off and washing the resin with TFA,
the product is precipitated in cold diisopropyl ether (WE). 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%
20 (VN) aqueous acetic acid and loaded onto an ion exchange resin. The elution is
performed with 5% (VIV) aqueous acetic acid. The acetate is filtered through a 0.2 p
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
25 0.01 M HC1 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 HC1. Subsequently, the hydrochloride is filtered through a
' 0.2 pm membrane filter and lyophilized to yield the final product as a white to off-white
30 powders.
An exemplary pharmaceutical formulation of the present invention contains one
or more, such as two, three, four or all of the polypeptides Cla35, Cla16, Cla25, Cla26
and Cla29 in salt form. The polypeptide Cla35A may be used in place of Cla35. The
, polypeptide Cla25A may be used in place of Cla25. The peptide salt is typically an .
acetate or hydrochloride salt. The peptide salt(s) are each typically present at a nominal
concentration of 40 to 220 p . ~T. h e pharmaceutical formulation optionally further
5 comprises one or more of L-methionine as an antioxidant (optionally at a nominal
concentration of 1 to 15mM, typically 5 mM); phosphoric acid, hydrochloric acid or
aqueous ammonia for pH adjustment (as required); and trehalose dihydrate as a tonicity
agent (optionally at a nominal concentration of 260 mM). The pharmaceutical
formulation is prepared in solution prior to being subjected to freeze-drying to produce
10 a lyophilisate.

WE CLAIMS:-
1. A polypeptide, or a pharmaceutically acceptable salt thereof, which is up to 30
amino acids in length and comprises:
5 (I) the amino acid sequence:
(a) GGYKAAVRPTMLE (SEQ ID NO: 35; Cla35),
(b) AEVYQKLKSLTKK (SEQ ID NO: 3 1; Cla16),
(c) VAITYASRAQGAE (SEQ ID NO: 32; Cla25),
(d) GHHFKERGTGSLVIT (SEQ ID NO: 33; Cla26), or
10 (e) ANYTQTKTVSIRL (SEQ ID NO: 34; Cla29); or
(11) a T cell epitope-containing variant sequence which is a said amino acid
sequence (I) having up to six amino acid modifications, each of which is independently
a deletion, substitution or insertion.
15 2. A polypeptide or salt according to claim 1, wherein the polypeptide comprises a
said variant sequence (11) which has one or two amino acid modifications, the or each of
which independently is a deletion or substitution.
3. A polypeptide or salt according to claim 1 or 2, wherein the or each substitution
20 is a conservative substitution.
4. A polypeptide or salt according to claim 1, wherein the polypeptide comprises a
said variant sequence (11) which has up to two amino acids deleted from the N-terminus
and/or up to two amino acids deleted from the C-terminus.
25
5. A polypeptide or salt according to any one of the preceding claims, wherein the
polypeptide is up to 20 amino acids in length.
6. A polypeptide or salt according to any one of the preceding claims, wherein the
30 polypeptide has an amino acid sequence consisting of a said sequence (I) or (11) having
a N-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 sequence (I) in the native sequence of the protein from which the said sequence (I)
is derived.
7. A polypeptide or salt according to claim 1, wherein the polypeptide has an
5 amino acid sequence selected from said sequences (I), VAITYASRAQGAEK (SEQ ID
NO: 30; Cla25A) and GGYKAAVRPT-Nle-LE (SEQ ID NO: 36; Cla3 5A).
8. A pharmaceutical formulation which comprises a pharmaceutically acceptable
carrier or diluent and a polypeptide, or a pharmaceutically acceptable salt thereof, which
10 is up to 30 amino acids in length and comprises:
(I) the amino acid sequence:
(a) GGYKAAVRPTMLE (SEQ ID NO: 35; Cla3 5),
(b) AEVYQKLKSLTKK (SEQ ID NO: 3 1; Cla16),
(c) VAITYASRAQGAE (SEQ ID NO: 32; Cla25),
15 (d) GHHFKERGTGSLVIT (SEQ ID NO: 33; Cla26), or
(e) ANYTQTKTVSIRL (SEQ ID NO: 34; Cla29); or
(11) a T cell epitope-containing variant sequence which is a said amino acid -
sequence (I) having up to six amino acid modifications, each of which is independently
a deletion, substitution or insertion.
20
9. A pharmaceutical formulation according to claim 8, wherein the polypeptide
comprises a said variant sequence (11) which has one or two amino acid modifications,
the or each of which independently is a deletion or substitution.
25 10. A pharmaceutical formulation according to claim 8 or 9, wherein the or each
substitution is a conservative substitution.
' 1 1. A pharmaceutical formulation according to claim 8, wherein the polypeptide --
comprises a said variant sequence (11) which has up to two amino acids deleted from the
30 N-terminus and/or up to two amino acids deleted from the C-terminus.
12. A pharmaceutical formulation according to any one of claims 8 to 11, wherein
the polypeptide is up to 20 amino acids in length.
13. A pharmaceutical formulation according to any one of claims 8 to 12, wherein
5 the polypeptide has an amino acid sequence consisting of a said sequence (I) or (11)
having a N-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 Cterminal
to the said sequence (I) in the native sequence of the protein from which the
said sequence (I) is derived.
10
14. A pharmaceutical formulation according to claim 8, wherein the polypeptide has
an amino acid sequence selected from said sequences (I), VAITYASRAQGAEK (SEQ
ID NO: 30; Cla25A) and GGYKAAVRPT-Nle-LE (SEQ ID NO: 36; Cla35A).
15 15. A pharmaceutical formulation according to any one of claims 8 to 14, which
comprises two or more said polypeptides or salts thereof.
16. A pharmaceutical formulation according to claim 15, comprising:
- a polypeptide having the amino acid sequence (I)(a) or a said variant
sequence (11) derived therefrom, or a said salt thereof;
- a polypeptide having the amino acid sequence (I)(b) or a said variant
sequence (11) derived therefrom, or a said salt thereof;
- a polypeptide having the amino acid sequence (I)(c) or a said variant
sequence (11) derived therefrom, or a said salt thereof;
- a polypeptide having the amino acid sequence (I)(d) or a said variant
sequence (11) derived therefrom, or a said salt thereof; and
a polypeptide having the amino acid sequence (I)(e) or a said variant
sequence (II) derived therefrom, or a said salt thereof.
30 17. A pharmaceutical formulation according to any one of claims 8 to 16, further
comprising at least one polypeptide, or a pharmaceutically acceptable salt thereof,
which is up to 30 amino acids in length and comprises:
(111) the amino acid sequence:
( f ) GWGVMVSHRSGET (SEQ ID NO: 39; Alt 14);
(g) GYTGKIKIAMDVASSE (SEQ ID NO: 41 ; Alt 15), or
(h) WSWKIGPAIATGNT (SEQ ID NO: 50; Alt28); or
5 (IV) a T cell epitope-containing variant sequence which is a said amino acid
sequence (In) having up to five amino acid modifications, each of which is
independently a deletion, substitution or insertion.
18. A pharmaceutical formulation according to any one of claims 8 to 17, which is
10 sealed in a container.
19. A pharmaceutical formulation according to any one of claims 8 to 18, which is a
pharmaceutically acceptable solution or a lyophilisate.
15 20. A pharmaceutical formulation according to claim 19, wherein the solution is
formulated for intradermal administration, subcutaneous administration, oral
administration, nasal administration, topical administration, sublingual administration,
buccal administration or epidermal administration.
20 ' 2 A pharmaceutical formulation according to claim 19 or 20, wherein the solution
is provided in an ampoule, sealed vial, syringe, cartridge, flexible bag or glass bottle.
22. A pharmaceutical formulation according to claim 19, wherein the lyophilisate is
provided in a sealed vial.
25
23. A polypeptide or salt according to any one of claims 1 to 7 or a pharmaceutical
formulation according to any one of claims 8 to 22 for use in a method of treating or
preventing allergy to Cladosporium and/or Alternaria.
30 24. An in vitro method of determining whether T cells recognize a polypeptide or
' salt as defined in any one of claims 1 to 7, which method comprises contacting said T
cells with said polypeptide or salt and detecting whether said T cells are stimulated by
said polypeptide or salt.
i 25. A method according to claim 24 which is carried out to determine whether an,
5 individual has, or is at risk of having, an allergy to Cludosporium and/or Alternaria.
26. A method of treating an individual for or preventing in an individual allergy to
Cladosporium and/or Alternariu, which method comprises administering to said
individual a therapeutically or prophylactically effective amount of a polypeptide or salt
10 as defined in any one of claims I to 7 or of a pharmaceutical formulation as defined in
any one of claims 8 to 22.
27. Use of a polypeptide or salt as defined in any one of claims 1 to 7 for the
manufacture of a medicament for the prevention or treatment of allergy to
1 5 Cladosporium and/or Alternaria.
28. A method of preparing a pharmaceutical formulation, comprising combining one
or more polypeptides or salts as defined in any one of claims 1 to 17 with a
pharmaceutically acceptable carrier or diluent.