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TITLE: GROUP 1 MITE POLYPEPTIDE VARIANTS
FIELD OF THE INVENTION
The present invention relates to variants of the group 1 mite polypeptide
antigens aller-
gens having an altered antigenic profile, compared to the parent group 1
polypeptide allergens,
processes for making such variants, compositions comprising the variants and
use of the vari-
ants in immuno-therapy such as allergy vaccination and/or desensitisation.
BACKGROUND OF THE INVENTION
Antigenic polypeptides heterologeous to humans and animals, such as the group
1
mite polypeptide allergens, present e.g., in excrements of dust mites
Dermatophagoides
pteronyssinus (Der p 1 ) or Dermatophagoides farinae (Der f 1 ), can induce
immunological re-
sponses in susceptible individuals, such as an atopic allergic response, in
humans and ani-
mals. Allergic responses may range from hay fever, rhinoconjunctivitis,
rhinitis, and asthma,
and in cases when the sensitised individual is exposed, e.g., to bee sting or
insect bites, even
to systemic anaphylaxis and death.
An individual may become sensitised to such polypeptides, termed allergens, by
inhala-
tion, direct contact with skin or eyes, ingestion or injection. The general
mechanism behind an
allergic response is divided into a sensitisation phase and a symptomatic
phase. The sensiti-
sation phase involves a first exposure of an individual to an allergen. This
event activates spe-
cific T- and B-lymphocytes, and leads to the production of allergen specific
antibodies, such as
immunoglobulin E (IgE). The specific IgE antibodies bind to IgE receptors on
mast cells and
basophils, among others, and the symptomatic phase is initiated upon a second
exposure to
the same or a homologous allergen. The allergen will bind to the cell-bound
IgE, and the pofy-
clonal nature of the antibodies results in bridging and clustering of the IgE
receptors, and sub-
sequently in the activation of mast cells and basophils. This activation
results in the release of
various chemical mediators, such as histamine, heparin, proteases,
prostaglandin D2 and leu-
kotrienes, involved in the early as well as late phase reactions of the
symptomatic phase of
allergy.
For certain forms of IgE-mediated allergies, a therapy exists, called specific
allergy
vaccination (SAV) or immuno therapy (IT), which comprises repeated parenteral
or mucosal
(e.g., s ublingual) a dministration o f a Ilergen p reparations formulated a s
a v accine ( Int. A rch.
Allergy Immunol., 1999, vol., 119, pp1-5). T his leads to reduction of the
allergic symptoms,
most likely due to induction of a protective, non lgE-based immune response,
possibly by
modulation of the existing Th2 response and/or a redirection of the immune
response towards
the immunoprotective (Th1 ) pathway (Int. Arch. Allergy Immunol., 1999, vol.
119, pp1-5).
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Compared to other types of vaccination, allergy vaccination is complicated by
the pres-
ence of an existing and ongoing immune response in the allergic patients. The
presence of
allergen specific IgE antibodies on effector cells, such as mast cells and
basophils, in affected
tissues, may result in allergic symptoms upon exposure to antigens. Thus, the
inherent risk of
adverse events or side effects limits the antigen dose, which can be
administered, and has ne-
cessitated prolonged (12-36 months) and cumbersome treatment regimes where the
delivered
dose slowly is increased over time.
There is thus a need to provide modified allergens, with a lower inherent risk
of induc-
ing adverse events, which can be used for specific allergy vaccination. These
modified aller-
gens should have a reduced capacity for binding, and especially cross-linking,
antigen-specific
IgE molecules. At the same time it is important that they retain the tertiary
structure, and to
some degree the immunogenicity, of the parent allergen, in order to be able to
elicit the protec-
tive (IgG-based) immune response in the patient.
In order to produce such modified proteins it is desirable to first identify
the minimal B
cell epitopes on the molecule. An epitope is the structural area on a complex
antigen that can
combine with an antibody, while the minimal epitope contains the amino acids
involved directly
in antibody binding.
B-cell epitopes can in nature be continuous, discontinuous or a combination
thereof,
but must contain around 10 amino acids in order to elicit an antibody
response. One may iden-
tify larger regions or areas of the molecule comprising an epitope and a
minimal epitope, but
when desiring to alter immunogenic properties of a polypeptide by introducing
mutations in the
molecule one will realize the importance of firstly identifying the minimal
epitope because it is
far less feasible to prepare modified polypeptides by mutating amino acids if
the number of
amino acids, which potentially is to be mutated, exceeds 5-10 amino acids.
This is because the
number of possible variants increases steeply with the number of amino acids
involved in the
mutation strategy (many more permutations possible) and because it may be less
useful to
modify amino acids which are not part of an epitope.
Several studies have been aimed at identifying epitopes in group 1 dust mite
allergens:
Green et al., Int. Arch. Allergy Appl. Immunol., vol. 92, pp 30-38, 1990;
Green et al. J.
Immunol., vol. 147 pp. 3768-3773, 1991 and Green & Thomas, Mol. Immunol. Vol.
29(2), pp
257-262, 1992, disclose antibody-binding fragments of Der p 1. The antibody
binding regions
disclosed are very large (from 11-56 amino acid residues) and together they
cover almost all
amino acids of the molecule.
Lombardero et al., J. Immunol., vol. 144(4), pp 1353-1360, 1990, disclose that
B cell
epitopes on Der p 1 are conformational, i.e., the epitope is made up of non-
contiguous parts of
the molecule and thus highly dependent on correct tertiary structure, and that
antibody binding
is sensitive to denaturation of the protein.
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Collins et al., Clin. Exp. Allergy, vol. 26(1 ), pp 36-42, 1996, conclude that
IgE binding
epitopes of Der p 1 and Der f 1 are discontinuous in nature.
Jeannin et al., Mol. Immunol. Vol. 29(6), pp 739-749, 1992, used predictions
of hydro-
phobicity and solvent accessibility to amino acid residues on a three-
dimensional model of Der
p 1 to identify 4 putative antibody binding peptides: N52-C71, C117-Q133, 6176-
1187 and
V188-Y199. The four peptides could induce low levels of histamine release in
basophils from
40-60% of a panel of dust mite allergic patients. Histamine release requires
cross-linking of at
least two IgE molecules and the authors speculate that the peptides must have
bound non-
specifically to serum components, and thus acted as haptens.
Furmonaviciene et al., Clin. Exp. Allergy, 29, pp 1563-1571, 1999, suggest
L147 to
Q160 of Der p 1 to be the potential epitope recognised by a monoclonal mouse
anti-Der p 1
antibody.
Pierson-Mullany et al., M o1. Immunol., 37, pp 613-620, 2000, r eported t hat
peptides
representing residues T1 to T21, E59 to Y93, Y155 to W187 and 1209 to 1221 of
Der p 1 can
parially inhibit human serum binding to Der p 1.
WO 99/47680 (ALK-ABELLO) discloses that allergens may be modified to render
these
polypeptides less allergenic. This disclosure concerns mainly modification of
the birch pollen
protein, Bet v 1 and Venom allergen Ves V 5.
WO 02/40676 (ALK-ABELLO) discloses modified allergens, said modifications
alleg-
edly causing the allergenicity of the allergen to be reduced. In this
disclosure amino acids suit-
able for modification are selected by virtue of their solvent accessibility,
i.e. if they are present
on the surface of the allergen or they are selected if they are conserved vis
a vis homologeous
allergens of the same taxonomic group.
WO 01/29078 ( HESKA) d escribes recombinant expression of group 1 mite p
roteins,
nucleotide sequences encoding these proteins and nucleotide sequences modified
to enable
expression of the proteins in certain microorganisms. The group 1 mite
polypeptides of this dis-
closure are said to bind to IgE which also bind to native group 1 mite
polypeptides.
EP-A-1 219 300 describes a method for administering an allergy vaccine.
In the art various suggestions are made as to the antibody binding epitopes of
group 1
mite p olypeptides s uch a s D er p 1 . H owever, t he a rt a ither c oncerns
( 1 ) I inear a pitopes o f
which most have low relevance in allergy, (2) regions which are too large to
contain informa
tion of specific amino acids involved in antibody recognition, (3) epitopes
selected by choosing
those areas which have a higher solvent accessibility without considering if
the epitope is de
facto involved in antibody binding, (4) non human epitopes or (5) a
combination of one or more
of (1 )-(4).
The inadequacy of the epitope identification in for example Der p 1 may be the
reason
why very different potential epitopes on for example Der p 1 have been
reported in different
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documents. For example in WO 02/40676 residues E13, P24, R20, Y50, S67, R78,
R99,
Q109, 8128, 8156, 8161, P167 and W192 are selected as being important for the
allergenicity
of Der p 1, while in Pierson-Mullany et al. the epitopes are contemplated to
be T1 to T21, E59
to Y93, Y155 to W 187 and 1209 to 1221, in Furmonaviciene et al. the major
epitope is deter-
s mined to be L147 to Q160, and in Jeannin et al, (where an almost identical
approach as in WO
02/40676 is utilised), N52-C71, C117-Q133, 6176-1187 and V188-Y199 are
identified.
Further, two studies have aimed to reduce activity or 'allergenic activity' by
mutation in
residues of the active site, maturation site, or cysteine-bridge formation
sites:
WO 99/25823 (Smith Kline Beecham) discloses variants of Der p 1 in which a)
C34 is
mutated, b) the pro-peptide site is modified, e.g. by deletion of NAET
sequence or c) H170 is
mutated.
WO 03/016340 (Smith Kline Beecham) discloses variants of Der p 1 in which
either of
six cysteinses (C4, C31, C65, C71, C103, or C117) are mutated.
The ambiguity of the art concerning epitopes, of e.g., Der p 1, means that
presently no
conclusive and reliable data is available on epitopes of Group 1 mite
polypeptides and even
less on amino acids comprised in said epitopes suitable for mutation with the
purpose of re-
ducing the antigenicity of these polypeptides.
SUMMARY OF THE INVENTION
Attempts to reduce the allergenicity of polypeptide allergens have been
conducted. It
2o was found that small changes in the epitope, may affect the binding to an
antibody. This may
change the properties of such an epitope, e.g., by converting it from a high
affinity to a low af-
finity epitope towards antibodies, or even result in epitope loss, i.e. that
the epitope cannot suf
ficiently bind an antibody to elicit an antigenic response.
In order to produce modified group 1 mite polypeptides with improved
properties as a
vaccine agent, it is an advantage to first identify the minimal B cell
epitopes on the molecule.
An epitope is the smallest structural area on a complex antigen that can bind
an antibody. B-
cell epitopes can be continuous or discontinuous in nature. The minimal
epitope consists of the
specific amino acids directly involved in antibody binding.
The present invention relates to variants of group 1 mite polypeptide
antigens, including
Der p 1, comprising a mutation in a minimal epitope and thus having an altered
immmunogenic
profile in exposed animals, including humans.
The applicant has identified amino acids in group 1 mite polypeptides which
are involved in
antibody binding epitopes and for which a mutation have an altering,
preferably reducing, ef-
fect on the antibody binding, particularly IgE binding of the polypeptide.
Hence, the present in-
vention provide in a first aspect a variant of a group 1 mite polypeptide,
wherein the mature
polypeptide of the variant comprises one or more mutations in the positions or
corresponding
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to the positions consisting of A10, A12, E13, G29, G30, G32, A46, Y47, S54,
L55, D64, A66,
S67, G73, T75, 180, Q84, N86, G87, S92, Y93, Y96, A98-Q101, 8104-P106, Q109-
1113, A132,
1144-D146, D148, 8151, 1158-Q166, N179, A180, 6182-D184, A205, 1208 of SEQ ID
NO: 1 or
alternatively 10, 12, 13, 29, 30, 32, 46, 47, 54, 55, 64, 66, 67, 73, 75, 80,
84, 86, 87, 92, 93,
96, 98-101, 104-106, 109-113, 132, 144-146, 148, 151, 158-166, 179-180, 182-
184, 205, 208
of the mature Der p 1 polypeptide.
Particularly, said variants have reduced IgE-binding, more particularly
combined with pre-
served immunogenicity for inducing protective responses (vide supra). Still
more preferably,
the variants have an altered immmunogenic profile in exposed animals,
including humans, as
compared to the native group 1 mite polypeptide.
In further aspects the invention provides a nucleotide sequence encoding the
variant of
the invention; a nucleotide construct comprising the nucleotide sequence
encoding the variant,
operably linked to one or more control sequences that direct the production of
the variant in a
host cell; a recombinant expression vector comprising the nucleotide construct
of the invention
and to a recombinant host cell comprising the nucleotide construct of the
invention.
In a still further aspect the invention provides a method of preparing a
variant of the in-
vention comprising:
(a) cultivating a recombinant host cell of the invention under conditions
conducive for pro-
duction of the variant of the invention and
(b) recovering the variant.
In still further aspects the invention provides a composition comprising a
variant of the
invention and a pharmaceutically acceptable carrier and a method for preparing
such a phar-
maceutical composition comprising admixing the variant of the invention with
an acceptable
pharmaceutical carrier.
In still further aspect the invention provides a variant or a composition of
the invention
for use as a medicament.
In still further aspect the invention provides use of a variant or the
composition of the
invention for the preparation of a medicament for the treatment of an
immunological disorder.
In still further aspect the invention provides use of the variant or the
composition of the
invention for the treatment of a disease.
In still further aspect the invention provides use of the variant or the
composition of the
invention for the treatment of an immunological disorder.
In a still further aspect the invention provides a kit comprising the variant
of the inven-
tion immobilized on a solid support.
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BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows histamine release in one representative donor, donor 1, in
response to stimula-
tion with group 1 mite polypeptide (nDerp1 ) and the group 1 mite polypeptide
variants, rec-
proDer p 1, rec-Der p 1 and DP070.
Figure 2 shows normalized histamine release, ECSO was calculated for the group
1 mite poly-
peptide variants. The dose response curves in nDer p 1-specific IgE serum
isolated from 14
patients with dust-mite allergy were plotted and fitted to a sigmoid curve,
and the ECSO was
calculated for group 1 mite polypeptide variants and normalized to group 1
mite polypeptide
(nDer p 1 ).
SEQUENCE LISTING
The present application contains information in the form of a sequence
listing, which is ap-
pended to the application and also submitted on a data carrier accompanying
this application.
The contents of the data carrier are fully incorporated herein by reference.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
The term "native polypeptide" as used herein is to be understood as a
polypeptide
essentially in its naturally occurring form. A native polypeptide may be for
example be a wild
type polypeptide, i.e. a polypeptide isolated from the natural source, a
polypeptide in its natu-
rally occurring form obtained via genetic engineering by expression in host
organism different
from the natural source or by polypeptide synthesis.
An "epitope" or a "B-cell epitope", as used in this context, is an antigenic
determinant
and the structural area on a complex antigen that can combine with or bind an
antibody. It can
be discontinuous in nature, but will in general have a size of 1 kD or less
(about 10 amino ac-
ids or less). The size may be 3 to 10 amino acids or 5 to 10 amino acids or
even 7 to 10 amino
acids, depending on the epitope and the polypeptide.
The term "epitope pattern" as used herein is to be understood as a consensus
se-
quence of antibody binding peptides. An example is the epitope pattern A R R *
R. The sign "*'a
in this notation indicates that the aligned antibody binding peptides included
a non-consensus
moiety between the second and the third arginine. That moiety may be any amino
acid or a
few amino acids or no amino acid. Epitope patterns are used to identify
epitopes and minimal
epitopes on complex antigens.
The term "Anchor amino acid" as used herein is to be understood as conserved
indi-
vidual amino acids of an epitope pattern recurring in all peptides bound by
monospecific anti-
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bodies used to define that pattern. Anchor amino acid will usually also be the
amino acid of a
minimal epitope on the full polypeptide.
The "antigenicity" of a polypeptide indicates, in this context, its ability to
bind antibod-
ies e.g., of IgE and/or IgG and/or other immunoglobulin classes. The 'IgE-
antigenicity' of a
polypeptide as used herein, indicates its ability to bind IgE antibodies.
The "immunogenicity" of a polypeptide indicates its ability to stimulate
antibody pro-
duction and immunological reactions in exposed animals, including humans.
The "allergenicity "of a polypeptide indicates its ability to stimulate IgE
antibody pro-
duction and allergic sensitization in exposed animals, including humans.
The term "parent" or "parent group 1 mite polypeptide" is to be understood as
a
group 1 mite polypeptide (also refered to as group 1 mite allergen) before
introducing the mu-
tations according to the invention. In particular the parent group 1 mite
polypeptide is the na-
tive group 1 mite polypeptide.
Group 1 mite polypeptides
As described in the art such as WO 01/29078, mites produce several classes or
groups
of allergens, one of which is known as Group 1 allergens. Group 1 allergens,
displaying con-
siderable cross-reactivity, have been found in Dermatophagoides pteronyssinus,
Dermato-
phagoides farinae, Dermatophagoides siboney, Dermatophagoides microceaus,
Blomia tropi-
calis and Euroglyphus maynei, see for example, Thomas et al, 1998, Allergy 53,
821-832.
Group 1 mite allergens share significant homology with a family of cysteine
proteases
including actinidin, papain, cathepsin H and cathepsin B. which is why they
often are referred
to as Group 1 mite cysteine proteases. The Group 1 mite allergens are commonly
found in the
feces of mites and are thought to function as digestive enzymes in the mite
intestine.
Group 1 allergens from different mites are highly homologous, approximately 25
kilo-
dalton (kD) secretory glycoproteins, that are synthesized by the cell as a pre-
pro-protein that is
processed to a mature form. D, farinae, D. pteronyssinus, and E. maynei Group
1 proteins, for
example, share about 80% identity. In particular, Group 1 allergens from D.
farinae and D.
pteronyssinus, also referred to as Der f 1 and Der p 1 proteins, respectively,
show extensive
cross-reactivity in binding IgE and IgG. In patients that are mite allergic,
approximately 80% to
90% of the individuals have IgE that is reactive to Group 1 allergens (Thomas,
Adv. Exp. Med.
Biol., 409, pp. 85-93, 1996).
Group 1 mite allergens thus include native polypeptides known in the art as
Der p 1 ob-
tainable from Dermatophagoides pteronyssinus (NCBI accession number: P08176,
SEQ ID
N0:1), Der f 1 obtainable from Dermatophagoides farinae (NCBI accession
number: P16311,
SEQ ID N0:2), Eur m 1 obtainable from Euroglyphus maynei (NCBI accession
number:
P25780, SEQ ID NO: 3), Der m 1 obtainable from Dermatophagoides microceaus
(NCBI ac-
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cession number: P16312, SEQ ID NO: 4), and Blo t 1 obtainable from Blomia
tropicalis (NCBI
accession number: Q95PJ4, SEQ ID NO: 5). Thus, in the context of this patent,
the term group
1 mite allergens includes in particular native group 1 mite allergens, but
also includes ho-
mologs to the native group 1 allergens, such as recombinant variants with
disrupted N-
glycosylation motifs, and hybrids of the above mentioned mite allergens, e.g.
as created by
family shuffling as described in the art (J.E. Ness, et al, Nature
Biotechnology, vol. 17, pp. 893-
896, 1999).
In silico identification of epitope patterns and epitopes in group 1 mite
polypeptides
Group 1 mite polypeptides may be epitope mapped using the proprietary in
silico epi-
tope mapping tool disclosed in detail in WO 00/26230 and WO 01/83559. In
brief, this tool
comprises a database of epitope patterns (determined from an input of peptide
sequences,
known to bind specifically to anti-protein antibodies) and an algorithm to
analyse 3-D structure
of a given protein against the epitope pattern database. This will determine
the possible epi
topes on that protein, and the preference of each amino acid in the protein
sequence to be part
of epitopes.
Identifyingi antibody-binding peptides:
Antibody-binding peptides can be identified by many different ways. One is to
synthe-
size a number of peptides of known sequence, and test for their ability to
bind antibodies of
interest, e.g., in ELISA or other immunochemical assays. Such data are
available in great
abundance in the literature.
A particularly effective way is to prepare a library of many different random
peptide se-
quences and select experimentally only the ones that bind antibodies well and
specific (i.e.,
can be outcompeted by the protein towards which the antibodies were raised).
Phage display
techniques are well suited for this way of finding antibody bidning peptides:
In a phage display system, a sequence encoding a desired amino acid sequence
is in-
corporated into a phage gene coding for a protein displayed on the surface of
the phage. Thus,
the phage will make and display the hybrid protein on its surface, where it
can interact with
specific target agents. Given that each phage contains codons for one specific
sequence of a
determined length, an average phage display library can express 108 - 1 O'2
different random
sequences. If the displayed sequence resembles an epitope, the phage can be
selected by an
epitope-specific antibody. Thus, it is possible to select specific phages from
the bulk of a large
number of phages, each expressing their one hybrid protein.
It is important that the amino acid sequence of the (oligo)peptides presented
by the
phage display system have a sufficient length to present a significant part of
an epitope to be
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identified. The oligopeptides may have from 5 to 25 amino acids, preferably at
least 8 amino
acids, such as 9 amino acids.
The antibodies used for reacting with the oligopeptides can be polyclonal or
mono-
clonal. In particular, they may be IgE antibodies to ensure that the epitopes
identified are IgE
epitopes, i.e., epitopes inducing and binding IgE. The antibodies may also be
monospecific,
meaning they are isolated according to their specificity for a certain
protein. Polyclonal antibod-
ies are preferred for building up data on antibody-binding peptides to be used
in the in silico
mapping tool in order to obtain a broader knowledge about the epitopes of a
polypeptide.
These reactive peptides, by virtue of their reactivity against antibodies, to
some degree
1 o resemble the appearance of an epitope on a full polypeptide.
Identifyina epitope patterns from reactive petides
The reactive (oligo)peptides identified e.g. by phage display are compared and
aligned
in order to identify common epitope patterns, which then can be used for
identification of anti-
body binding epitopes on a 3-dimensional polypeptide.
In the alignment conservative alternatives to an amino acid such as aspartate
and glu-
tamate, lysine and arginine, serine and threonine are considered as one or
equal.
Thus, the alignment results in a number of patterns, which depend on the
chosen number of
residues of the peptides. Using for example a 7-mer peptide, the pattern may
have the form:
XX**XXX,
where "*" in this notation indicates a non-consensus moiety which may be any
amino acid or
group of amino acids or no amino acid, while X is one of the following 13
residue types: AG,
C, DE, FY, H, IL, KR, M, NQ, P, ST, V, and W, where the pairs AG, DE, FY, IL,
KR, NQ, ST
are conservative alternatives and considered equal. Accordingly, 3 peptides
such as
AKSNNKR
AKSMNKR
AKTPN KK
would create a pattern of [AG] [KR] [ST] * [NQ] [KR] [KR], where the residues
AG KR ST and
NQ KR KR are consensus residues shared by all 3 peptides and thus the epitope
pattern
would be AG KR ST * NQ KR KR. The patterns are chosen to describe a complete
set of reac-
tive (oligo)peptides (obtained e.g., by a phage display and antibody reaction)
by the fewest
possible patterns.
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The epitope patterns may be determined directly from the reactive peptides; if
for ex-
ample a library of 7-mer reactive peptides is made, one can use each different
reactive 7 mer
peptide, taking conservative alternatives into account, as an epitope pattern
in the epitope
mapping approach as described below.
It is also possible to reduce the number of epitope patterns to be examined in
the epi-
tope mapping by removing redundant patterns and/or by employing experimental
designs as
known in the art (See example 1 ).
Within the identified epitope patterns some amino acids are conservative,
called anchor
amino acids. The anchor amino acids recur in all or a majority of the reactive
peptides.
Epitope mapping algorithm
When a pitope p atterns have b een i dentified t hey a re s ubsequently c
ompared t o t he
three-dimensional coordinates of the amino acid sequence of the polypeptide of
interest, in or-
der to identify combinations of residues on the polypeptide surface
corresponding to the con-
sensus sequences) or epitope pattern(s). In this way, amino acids residues,
which are impor-
tant for antibody binding, can be identified.
Once one or more epitope patterns have been identified, any polypeptide for
which a
three-dimensional structure is known may be analysed for epitopes matching the
epitope pat-
terns. Finding an epitope on a polypeptide is achieved by searching the
surface of the polypep-
tide in the following way:
(1) For all amino acids in the polypeptide it is examined if (a) the amino
acid type match
the first amino acid of an epitope pattern and (b) the surface accessibility
greater than
or equal to a chosen threshold allowing the amino acid to be immunological
interactive.
Those amino acid satisfying 1 (a) and 1 (b) are selected.
(2) For all amino acids within a selected distance (e.g. 10 Angstroms) of the
amino acids
selected in step 1 it is examined if (a) the amino acid type matches the
second amino
acid of the pattern and (b) the surface accessibility greater than or equal to
a chosen
threshold allowing the amino acid to be immunological interactive. Those amino
acid
satisfying 2(a) and 2(b) are selected
(3) For all amino acids within a selected distance (e.g., 10 Angstroms) of the
amino acids
selected in step 2 it is examined if (a) the amino acid type matches the third
amino acid
of the pattern and (b) the surface accessibility greater than or equal to a
chosen
threshold allowing the amino acid to be immunological interactive. Those amino
acid
satisfying 3(a) and 3(b) are selected.
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This procedure (step 3) is repeated for all amino acids in the epitope pattern
consensus
sequence. The coordinates of its C-alpha atom define the spatial positioning
of an amino acid.
The surface solvent accessibility threshold is given in percent of an average
for the particular
residue type (see example 2).
If matching amino acids for all amino acids in the epitope pattern can be
found in the
structure of the polypeptide it is a very strong indication that an epitope
has been found.
However, it is also checked that the size of the epitope is satisfactory,
i.e., the distance
between any two residues is below a given threshold, usually 25 A.
The a pitopes found may b a ranked a nd weighted a ccording to their total a
ccessible
1 o surface area, in order to improve further the predictability of the tool.
Finally, when all possible epitopes have been mapped for the protein of
interest, one
can provide a score for each amino acid of the protein by adding up the number
of times it ap-
pears in an epitope pattern. This score will be an indication of the
likelyhood that modification
(substitution, insertion, deletion, glycosylation or chemical conjugation) of
that amino acid will,
result in a variant with a lower antigenicity. All amino acids of the protein
can then be ranked
according to this score and those with highest scores can be selected for
mutagenesis.
The epitope mapping tool can be adjusted, such that only a subset of the known
reac-
tive peptides are included as data set for building epitope patterns, and thus
for conducting
epitope mapping. For instance, one may choose only to include peptides
reactive to IgE anti-
bodies (rather than to IgG or other antibodies), or one may include only
peptides reactive to
human antibodies etc. One may choose to involve only peptides reactive against
the target
protein in order to get a more specific response; however, in general,
peptides reactive to anti-
bodies that in turn were raised against any protein are included.
If no three-dimensional structure coordinates are available for the protein of
interest,
one can map the epitope patterns directly to the primary sequence of the
protein of interest.
From all the above information, it is obvious that the epitopes are
conveniently deter-
mined using this epitope mapping tool.
Further, the in silico epitope mapping tool can be used to predict if mutating
one amino
acid residue will result in that the new variant overall will have fewer
epitopes. Thus, some or
all 19 possible substitutions can be tested in a given position, the epitope
mapping procedure
repeated for a model structure of each of these proposed variants, and the
best variants) can
be constructed by mutation and tested experimentally.
Identified eaitopes of group 1 mite polypeptides
Using the epitope mapping tool the present inventors have surprisingly found
that
amino acids corresponding to positions A10, A12, E13, G29, G30, G32, A46, Y47,
L55, D64,
A66, S67, G73, T75, 180, Q84, N86, G87, S92, Y93, Y96, A98-Q101, 8104-P106,
Q109-1113,
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A132, I 144-D 146, D 148, 8151, I 158-Q 166, N 179, A180, G 182-D 184, A205,
1208 of SEQ I D
NO: 1 or alternatively 10, 12, 13, 29, 30, 32, 46, 47, 55, 64, 66, 67, 73, 75,
80, 84, 86, 87, 92,
93, 96, 98-101, 104-106, 109-113, 132, 144-146, 148, 151, 158-166, 179-180,
182-184, 205,
208 of the mature Der p 1 polypeptide in particularly that of SEQ ID N0:1, are
comprised in the
epitopes of native group 1 mite polypeptides. Group 1 mite polypeptides are as
stated above
highly homologeous and the corresponding positions in Group 1 mite
polypeptides of various
sources may easily be found by aligning such polypeptides with SEQ ID N0:1.
Group 1 mite polypeptide variants
Selection of positions for mutation
Once the epitopes of a polypeptide have been determined, variants of the
polypeptide
with modified antigenic properties can be made by mutating one or more of the
amino acid
residues comprised in the epitope. In this context mutation encompasses
deletion and/or sub-
stitution of an amino acid residue and/or insertion of one or more amino acids
before or after-
that residue.
When providing a polypeptide variant suitable as a vaccine agent, for
treatment of al-
lergies, it is particularly desirable to alter IgE epitopes to reduce the
binding of IgE, while at the
same time maintaining the ability of the variant to invoke an immune response
in humans and
animals, hence it is desirable that the polypeptides retain the three-
dimensional conformation
of the parent polypeptide. Further, it is particularly desirable that the
variant is capable of
stimulating T-cells sufficiently, preferable at the level of the parent
polypeptide or better. Still
further it is desirable that the variant is capable of invoking an IgG
response in human and ani-
mals.
The epitope identified may be mutated by substituting at least one amino acid
of the
epitope. In a particular embodiment at least one anchor amino acid is mutated.
The mutation
will often be a substitution with an amino acid of different size,
hydrophilicity, polarity and/or
acidity, such as a small amino acid in exchange of a large amino acid, a
hydrophilic amino acid
in exchange of a hydrophobic amino acid, a polar amino acid in exchange of a
non-polar
amino acid and a basic in exchange of an acidic amino acid.
3o Other mutations may be the insertion or deletion of at least one amino acid
of the epi-
tope, particularly deleting an anchor amino acid. Furthermore, an epitope may
be mutated by
substituting some amino acids, and deleting and/or inserting others.
The mutations) performed may be performed by standard techniques well known to
a
person skilled in the art, such as site-directed mutagenesis (see, e.g.,
Sambrook et al. (1989),
Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, NY).
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The mutagenesis may be spiked mutagenesis which is a form of site-directed
mutagenesis, in which the primers used have been synthesized using mixtures of
oligonucleo-
tides at one or more positions.
A general description of nucleotide substitution can be found in e.g., Ford et
al., 1991,
Protein Expression and Purification 2, pp. 95-107.
The polypeptide variant of the invention concerns variants of parent group 1
mite poly-
peptides comprising one or more mutations in the parent polypeptide in the
positions or corre-
sponding to the positions consisting of A10, A12, E13, G29, G30, G32, A46,
Y47, 554, L55,
D64, A66, S67, G73, T75, 180, Q84, N86, G87, S92, Y93, Y96, A98, R99, E100,
Q101, 8104,
8105, P 106, Q 109, 8110, F 111, 6112, 1113, A 132, 1144, K145, D 146, D 148,
8151, 1158, 1159,
Q 160, 8161, D 162, N 163, G 164, Y165, Q 166, N 179, A180, G 182, V183, D
184, A205, 1208 of
SEQ ID NO: 1 or 10, 12, 13, 29, 30, 32, 46, 47, 54, 55, 64, 66, 67, 73, 75,
180, 84, 86, 87, 92,
Y93, 96, 98, 99, 100, 101, 104, 105, 106, 109, 110, 111, 112, 113, 132, 144,
145, 146, 148,
151, 158, 159, 160, 161, 162, 163, 164, 165, 166, 179, 180, 182, 183, 184,
205, 208 of the
mature Der p 1 polypeptide,
In another particular embodiment of the above the variant polypeptide of the
invention
comprises one or more mutations in the parent polypeptide in the positions or
corresponding to
the positions consisting of A10, A12, G29, G30, G32, A46, Y47, S54, L55, D64,
A66, G73,
T75, 180, Q84, N86, G87, S92, Y93, Y96, A98, E100, Q101, 8104, 8105, P106,
8110, F111,
6112, 1113, A132, 1144, K145, D146, 1158, 1159, Q160, D162, N163, 6164, Y165,
Q166,
N179, A180, 6182, V183, D184, A205, 1208 of SEQ ID NO: 1 or 10, 12, 29, 30,
32, 46, 47, 55,
64, 66, 73, 75, 180, 84, 86, 87, 92, Y93, 96, 98, 100, 101, 104, 105, 106,
110, 111, 112, 113,
132, 144, 145, 146, 158, 159, 160, 162, 163, 164, 165, 166, 179, 180, 182,
183, 184, 205, 208
of the mature Der p 1 polypeptide,
In still another particular embodiment the variant polypeptide of the
invention comprises
one or more mutations in the parent polypeptide in the positions or
corresponding to the posi-
tions consisting of A10, A12, G29, G30, G32, A46, S54, L55, D64, A66, G73,
T75, Q84, N86,
G87, S92, Y96, E100, Q101, 8104, 8105, P106, 8110, 1113, A132, 1144, K145,
D146, D162,
N163, 6164, Y165, N179, A180, 6182, V183, D184 and A205 of SEQ ID NO: 1 or 10,
12, 29,
30, 32, 46, 55, 64, 66, 73, 75, 84, 86, 87, 92, 96, 100, 101, 104, 105, 106,
110, 113, 132, 144,
145, 146, 162, 163, 164, 165, 179, 180, 182, 183, 184 and 205 of the mature
Der p 1 polypep-
tide,
Particularly the variant has an altered antibody binding profile as compared
the parent
group 1 mite polypeptide, more particularly the variant has a reduced IgE-
binding, more par-
ticularly combined with preserved immunogenicity for inducing protective
responses (vide su-
pra). Still more preferably, the variant has an altered immmunogenic profile
in exposed ani-
mals, including humans, as compared to the parent group 1 mite polypeptide.
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Further the variant has in particular an altered IgE-antigenicity as compared
to the
parent group 1 mite polypeptide.
Still further the variant has in particular at least the same T-cell
stimulatory effect
compared to the parent group 1 mite polypeptide as measured by the procedure
in example 6.
Still further the variant induces an altered immunogenic response in exposed
animals,
including humans, as compared to the parent group 1 mite polypeptide.
Still further the variant induces in particular an altered immunogenic
response in
humans, as compared to the parent group 1 mite polypeptide.
In still another particular embodiment the variant polypeptide of the
invention comprises
one or more mutations in the parent polypeptide in the positions or
corresponding to the posi
tions consisting of A10, A12, G30, G32, A46, Y47, S54, L55, D64, A66, S67,
G87, S92, A98,
R99, E 100, Q 101, 8105, 8110, F 111, 6112, 1113, 1144, K 145, D 146, D 148,
8151, 1159, Q 160,
8161, D162, N163, 6164, Y165, Q166, N179, A180, 6182, V183, D184, A205, 1208
of SEQ
ID N0:1 or 10, 12, 30, 32, 46, 47, 54, 55, 64, 66, 67, 87, 92, 98, 99, 100,
101, 105, 110, 111,
112, 113, 144, 145, 146, 148, 151, 159, 160, 161, 162, 163, 164, 165, 166,
179, 180, 182, 183,
184, 205, 208 of the mature Der p 1 polypeptide. .
In still another particular embodiment of the above the variant polypeptide of
the invention
comprises one or more mutations in the parent polypeptide in the positions or
corresponding to
the positions consisting of A10, A12, G32, S54, L55, A66, S67, G87, A98, R99,
F111, 6112,
I 113, I 144, D 146, D 148, I 159, R 161, G 164, Q 166, A 180, D 184, A 205
and I 208 o f S EQ I D
N0:1 or 10, 12, 32, 54, 55, 66, 67, 87, 98, 99, 111, 112, 113, 144, 146, 148,
159, 161, 164,
166, 180, 184, 205 and 208 of the mature Der p 1 polypeptide.
Mutations directly providing for reduced antigenicity
When providing mutants having a reduced antigenicity it may be particularly
interesting to sub-
stitute an amino acid in an epitope of a parent group 1 mite polypeptide with
an amino acid
having different properties. Hence, in a particular embodiment, the variant
polypeptide of the
invention comprises a mutation selected from the group consisting of
10 substituted by a residue selected from the group consisting of V, Y, Q, N,
E and D;
12 substituted by a residue selected from the group consisting of V, Y, Q, N
and F;
32 substituted by a residue selected from the group consisting of V, Y, E, D,
N and Q;
54 substituted by a residue selected from the group consisting of N, A, T, V,
and Q.
55 substituted by a residue selected from the group consisting of V, N, and Q;
66 substituted by a residue selected from the group consisting of V, H, Y, D,
E, N, and Q;
67 substituted by a residue selected from the group consisting of V, H, Y, D,
E, N and Q;
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87 substituted by a residue selected from the group consisting of V, Y, D and
E;
98 substituted by a residue selected from the group consisting of V, N, Q, D
or E
99 substituted by a residue selected from the group consisting of H, Y, V, N,
Q, E and D;
111 substituted by a residue selected from the group consisting of V, H and W;
112 substituted by a residue selected from the group consisting of V, H, N, Q,
E, D and Y;
113 substituted by a residue selected from the group consisting of V, H, N, Q,
E, D and Y;
144 substituted by a residue selected from the group consisting of A, G, Y, N,
Q or V
146 substituted by a residue selected from the group consisting of Y, H, V, I,
L, N and Q:
148 substituted by a residue selected from the group consisting of Y, V, I and
L;
159 substituted by a residue selected from the group consisting of V, Y, A and
G;
161 substituted by a residue selected from the group consisting of A, G, V and
Y;
164 substituted by a residue selected from the group consisting of V, H and W;
166 substituted by a residue selected from the group consisting of S, W, Y and
F;
180 substituted by a residue selected from the group consisting of V, N, Q and
Y;
184 substituted by a residue selected from the group consisting of V, M and Y;
205 substituted by a residue selected from the group consisting of V, W and H;
208 substituted by a residue selected from the group consisting of A, G, V, W
and H;
said numbering being positions of or corresponding to positions of the mature
Der p1
polypeptide.
In still another particular embodiment the variant polypeptide of the
invention comprises a mu-
tation selected from the group consisting of
A10 substituted by a residue selected from the group consisting of V, Y, Q, N,
E and D;
A12 substituted by a residue selected from the group consisting of V, Y, Q, N
and F;
G32 substituted by a residue selected from the group consisting of V, Y, E, D,
N and Q;
S54 substituted by a residue selected from the group consisting of N, A, T, V,
and Q;
L55 substituted by a residue selected from the group consisting of V, N and Q;
A66 substituted by a residue selected from the group consisting of V, H, Y, D,
E, N and Q;
S67 substituted by a residue selected from the group consisting of V, H, Y, D,
E, N and Q;
G87 substituted by a residue selected from the group consisting of V, Y, D and
E;
A98 substituted by a residue selected from the group consisting of V, N, Q, D
or E
R99 substituted by a residue selected from the group consisting of H, Y, V, N,
Q, E and D;
F111 substituted by a residue selected from the group consisting of V, H and
W;
6112 substituted by a residue selected from the group consisting of V, H, N,
Q, E, D and Y;
1113 substituted by a residue selected from the group consisting of V, H, N,
Q, E, D and Y;
CA 02523402 2005-10-24
WO 2004/096844 PCT/DK2004/000280
1144 substituted by a residue selected from the group consisting of A, G, Y,
N, Q or V
D146 substituted by a residue selected from the group consisting of Y, H, V,
I, L, N and Q:
D148 substituted by a residue selected from the group consisting of Y, V, I
and L;
1159 substituted by a residue selected from the group consisting of V, Y, A
and G;
8161 substituted by a residue selected from the group consisting of A, G, V
and Y;
6164 substituted by a residue selected from the group consisting of V, H and
W;
Q166 substituted by a residue selected from the group consisting of S, W, Y
and F;
A180 substituted by a residue selected from the group consisting of V, N, Q
and Y;
D184 substituted by a residue selected from the group consisting of V, M and
Y;
A205 substituted by a residue selected from the group consisting of V, W and
H;
1208 substituted by a residue selected from the group consisting of A, G, V, W
and H;
said numbering being positions of or corresponding to positions of SEQ ID
N0:1.
In a further embodiment the parent group 1 mite polypeptide has in its mature
form a
sequence which displays at least 80% identity to SEQ ID N0:1; in particular at
least 90 % iden-
tity; in particular at least 95 % identity, more particularly 98 % identity,
more particularly 100
identity to SEQ ID NO:1 or 100 % identity to Der p 1.
In a further embodiment the variant group 1 mite polypeptide has in its mature
form a
sequence which displays at least 80% identity to SEQ ID N0:1; in particular at
least 90 % iden-
tity; in particular at least 95 % identity, more particularly 98 % identity,
more particularly 100
identity to SEQ ID N0:1 or 100 % identity to Der p 1.
In a further embodiment the parent group 1 mite polypeptide has in its mature
form a
sequence which displays at least 80% identity to SEQ ID N0:2; in particular at
least 90 % iden-
tity; in particular at least 95 % identity, more particularly 98 % identity,
more particularly 100
identity to SEQ ID N0:2 or 100 % identity to Eur m1.
In a further embodiment the variant group 1 mite polypeptide has in its mature
form a
sequence which displays at least 80% identity to SEQ ID N0:2; in particular at
least 90 % iden-
tity; in particular at least 95 % identity, more particularly 98 % identity,
more particularly 100
3o identity to SEQ ID N0:2 or 100 % identity to Eur m1.
In a further embodiment the parent group 1 mite polypeptide has in its mature
form a.
sequence which displays at least 80% identity to SEQ ID N0:3; in particular at
least 90 % iden-
tity; in particular at least 95 % identity, more particularly 98 % identity,
more particularly 100
identity to SEQ ID N0:3 or 100 % identity to Der f1.
In a further embodiment the variant group 1 mite polypeptide has in its mature
form a
sequence which displays at least 80% identity to SEQ ID N0:3; in particular at
least 90 % iden-
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tity; in particular at least 95 % identity, more particularly 98 % identity,
more particularly 100
identity to SEQ ID N0:3 or 100 % identity to Der f1.
In a further embodiment the parent group 1 mite polypeptide has in its mature
form a
sequence which displays at least 80% identity to Der m1; in particular at
least 90 % identity; in
particular at least 95 % identity, more particularly 98 % identity; more
particularly 100 % iden-
tity to Der m1.
In a further embodiment the variant group 1 mite polypeptide has in its mature
form a
sequence which displays at least 80% identity to Der m1; in particular at
least 90 % identity; in
particular at least 95 % identity, more particularly 98 % identity; more
particularly 100 % iden
tity to Der m1.
In a further embodiment the parent group 1 mite polypeptide has in its mature
form a
sequence which displays at least 80% identity to SEQ ID N0:5; in particular at
least 90 % iden-
tity; in particular at least 95 % identity, more particularly 98 % identity,
more particularly 100
identity to SEQ ID N0:5 or 100 % identity to Blo t 1.
In a further embodiment the variant group 1 mite polypeptide has in its mature
form a
sequence which displays at least 80% identity to SEQ ID N0:5; in particular at
least 90 % iden-
tity; in particular at least 95 % identity, more particularly 98 % identity,
more particularly 100
identity to SEQ ID N0:5 or 100 % identity to Blo t 1.
The risk linked to protein engineering in order to eliminate epitopes that new
epitopes
are made, or existing epitopes are duplicated is reduced by testing the
planned mutations at a
given position in the 3-dimensional structure of the protein of interest
against the found epitope
patterns thereby identifying the mutations for each position that are feasible
for obtaining the
desired properties of the polypeptide.
In a particular embodiment of the invention, it will be an advantage, to
establish a 1i
brary of diversified mutants each having one or more changed amino acids
introduced and se
lecting those variants, which show the best effect as vaccine agent while the
fewest side ef
fects. A diversified library can be established by a range of techniques known
to the person
skilled in the art (Reetz MT; Jaeger KE, in Biocatalysis - from Discovery to
Application edited
by F essner W D, V o1. 200, p p. 31-57 ( 1999); S temmer, N ature, v o1. 3 70,
p .389-391, 1994;
Zhao and Arnold, Proc. Natl. Acad. Sci., USA, vol. 94, pp. 7997-8000, 1997; or
Yano et al.,
Proc. Natl. Acad. Sci., USA, vol. 95, pp 5511-5515, 1998). In a more
preferable embodiment,
substitutions are found by a method comprising the following steps: 1 ) a
range of substitutions,
additions, and/or deletions are listed encompassing several epitopes, 2) a
library is designed
which introduces a randomized subset of these changes in the amino acid
sequence into the
target gene, e.g., by spiked mutagenesis, 3) the library is expressed, and
preferred variants
are selected. In a m ost preferred embodiment, this m ethod is s upplemented
with additonal
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rounds of screening and/or family shuffling of hits from the first round of
screening (J.E. Ness,
et al, Nature Biotechnology, vol. 17, pp. 893-896, 1999).
Mutations providing for increased alycosylation to amino acids in the epitope
area
In another approach, the mutations are designed, such that recognition sites
for post-
translational modifications are introduced in the epitope areas, and the
protein variant is ex-
pressed in a suitable host organism capable of the corresponding post-
translational modifica-
tion. These post-translational modifications may serve to shield the epitope
and hence lower
the immunogenicity of the protein variant relative to the protein backbone.
Post-translational
modifications include glycosylation, phosphorylation, N-terminal processing,
acylation, ribosy-
lation and sulfatation. A good example is N-glycosylation. N-glycosylation is
found at sites of
the sequence Asn-Xaa-Ser, Asn-Xaa-Thr, or Asn-Xaa-Cys, in which neither the
Xaa residue
nor the amino acid following the tri-peptide consensus sequence is a proline
(T. E. Creighton,
'Proteins - Structures and Molecular Properties, 2nd edition, W.H. Freeman and
Co., New
York, 1993, pp. 91-93). It is thus desirable to introduce such recognition
sites in the sequence
of the backbone protein. The specific nature of the glycosyl chain of the
glycosylated protein
variant may be linear or branched depending on the protein and the host cells.
Another exam-
ple is phosphorylation: The protein sequence can be modified so as to
introduce serine pho-
phorylation sites with the recognition sequence arg-arg-(xaa)n ser (where n =
0, 1, or 2), which
can be phosphorylated by the cAMP-dependent kinase or tyrosine phosphorylation
sites with
the recognition sequence -lys/arg - (xaa)3 - asp/glu- (xaa)3 - tyr, which can
usually be pho-
phorylated b y t yrosine-specific kinases (T.E. C reighton, " Proteins- S
tructures a nd molecular
properties", 2nd ed., Freeman, NY, 1993).
Mutations arovidina for covalent coniuaation of polymers to amino acids in the
epitope area
Another way of making mutations that will change the antigenic properties of a
polypep-
tide is to react or conjugate polymers to amino acids in or near the epitope,
thus blocking or
shielding the access to the anchor amino acids and thus the binding of
antibodies and/or recp-
tors to those amino acids. If no amino acid suitable for conjugation with a
polymer exists in the
parent polypeptide a suitable mutation is the insertion of one or more amino
acids being at-
tachment sites and/or groups and/or amino acids for polymer conjugation.
Which amino acids to substitute and/or insert depends in principle on the
coupling
chemistry to be applied. The chemistry for preparation of covalent
bioconjugates can be found
in "Bioconjugate Techniques", Hermanson, G.T. (1996), Academic Press Inc.,
which is hereby
incorporated as reference.
It a particular embodiment activated polymers are conjugated to amino acids in
or near
the epitope area.
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It is preferred to make conservative substitutions in the polypeptide when the
polypep-
tide has to be conjugated, as conservative substitutions secure that the
impact of the substitu-
tion on the polypeptide structure is limited.
In the case of providing additional amino groups, this may be done by
substitution of
Arginine to Lysine, both residues being positively charged, but only the
Lysine having a free
amino group suitable as an attachment groups.
In the case of providing additional carboxylic acid groups, the conservative
substitution
may for instance be an Asparagine to Aspartic acid or Glutamine to Glutamic
acid substitution.
These residues resemble each other in size and shape, except from the
carboxylic groups be-
1 o ing present on the acidic residues.
In the case of providing SH-groups the conservative substitution may be done
by sub-
stitution of Threonine or Serine to Cysteine.
Verification of variants having altered antigenic properties
The mutation of amino acids, comprised in an epitope, will cause the antigenic
proper-
ties of the polypeptide to change, as predicted by the in silico determination
of the epitopes.
However, the quantitative effect of the mutation on the antigenicity, i.e.,
the antibodybinding,
and the immunogenicity of the variant, is suitably determined using various in
vivo or in vitro
model systems. For that use, the polypeptide variant of interest can be
expressed in larger
scale and purified by conventional techniques. Then the functionality and
specific activity may
2o be tested by cysteine protease activity assays, in order to assure that the
variant has retained
three-dimensional structure.
In vitro systems include assays measuring binding to IgE in serum from dust
mite aller-
gic patients or exposed animals, cytokine expression profiles or proliferation
responses of pri-
mary T-cells from dust mite allergic patients or T cell clones or T cell line
generated from dust
mite allergic patients (Current protocols in Immunology, chapter 7 and 9), or
exposed animals,
and histamine release from basophils from dust mite allergic patients.
The IgE antibody binding can be examined in detail using, e.g., direct or
competitive ELISA (C-
ELISA), histamine release assays on basophil cells from allergic patients, or
IgE -stripped ba-
sophils from whole blood incubated with IgE-containing serum from allergic
patients, or by
other or other solid phase immunoassays or cellular assays (see Example 9).
The use of
stripped basophils from whole blood is described in: Knol EF, Kuijpers TW, Mul
FP, Roos D.
Stimulation of human basophils results in homotypic aggregation. A response
independent of
degranulation. J Immunol. 1993 Nov 1;151(9):4926-33; and in: Budde IK, Aalbers
M, Aalberse
RC, van der Zee JS, Knol EF. Reactivity to IgE-dependent histamine-releasing
factor is due to
monomeric IgE. Allergy. 2000 Ju1;55(7):653-7.
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In a particular embodiment the ability of the polypeptide variant to bind IgE
is reduced
at least 3 times as compared to the binding ability of the orginal or parent
group 1 mite poly-
peptide, preferably 5 times reduced, more preferably 10 times reduced, or more
preferably 50
times reduced.
In a further particular embodiment the ability of the polypeptide variant to
induce hista-
mine release in basophil cells from subjects allergic to dust mites is reduced
least 3 times, as
compared to that of the parent group 1 mite polypeptide, preferably 10 times
reduced, more
preferably 50 times.
In a further embodiment, the ability of the polypeptide variant to invoke a
recall T-cell
response in lymphocytes from animals, including humans, previously exposed to
the orginal or
parent group 1 mite allergen is measured, preferably the strength of the
response is compara-
ble to or higher than that to the parent group 1 mite allergen.
In a particular embodiment the in vivo verification comprises skin prick
testing (SPT), in
which a dust mite allergic subject/indvidual is exposed to intradermal or
subcutaneous injection
of group 1 mite polypeptides and the IgE reactivity, measured as the diameter
of the wheat and
flare reaction, in response to a polypeptide variant of the invention is
compared to that to the
parent group 1 mite polypeptide (Kronquist et al., Clin. Exp. Allergy, 2000,
vol. 30, pp. 670-
676).
Animal Models of allergy and SIT - Experimental Immunization of animals with
each of
the compositions
The in vivo immunogenic properties of the polypeptide variant of the invention
may
suitably be measured in an animal test, wherein test animals are exposed to a
vaccination al-
lergen polypeptide and the responses are measured and compared to those of the
target aller-
gen or other appropriate references. The immune response measurements may
include com-
paring reactivity of serum IgG, IgE or T-cells from a test animal with target
polypeptide and the
polypeptide variant. Animal immunization can be conducted in at least two
distinct manners: on
naive animals and on pre-sensitized animals (to better simulate the vaccine
situation). In the
context of this invention affinity of immunoglobulins towards the target
antigen is tested.
In a particular embodiment the affinity of animal IgG and/or IgG1 and/or IgG4
following
administration of the variant molecule is tested.
In the method according to the invention the test animals can either be naive
animals or
pre-sensitized animals.
A number of model systems are based on the use of naive animals:
In a particular embodiment the in vivo verification comprises exposing a mouse
to a
parent target allergen by the intranasal route. Useful in vivo animal models
include the mouse
intranasal test (MINT) model (Robinson et al., Fund. Appl. Toxicol. 34, pp. 15-
24, 1996).
CA 02523402 2005-10-24
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In a further p articular a mbodiment t he i n v ivo verification c omprises a
xposing a t est
animal to a polypeptide variant by the intratracheal route. Useful in vivo
animal models include
the guinea pig intratracheal (GPIT) model (Ritz, et al. Fund. App!. Toxicol.,
21, pp. 31-37,
1993) and the rat intratracheal (rat-IT) model (VllO 96/17929, Novo Nordisk).
In a further particular embodiment, the in vivo verification comprises
exposing a test
animal subcutaneously to the target allergen and the vaccination allergen
variant. A suitable
model is the mouse subcutaneous (mouse-SC) model (WO 98/30682, Novo Nordisk).
In a further particular embodiment, the method comprises exposing the test
animal in-
traperitoneally. ALK-Abello disclose (W002/40676) a method to assess the
ability of allergen
variants (of the birch pollen allergen bet v 1 ) to induce IgG antibodies upon
immunization of
mice: BALB/C mice were immunized intraperitoneally with the relevant allergy
variant or con-
trols, four times at dose intervals of 14 days. The proteins were conjugated
to 1.25 mg/mL al-
hydogel (AIOH gel, 1,3%, pH8-8.4, Superfos Biosector). The mice were immunized
with either
1 or 10 ug protein/dose. Blood samples were drawn at day 0, 14, 21, 35, 49,
and 63 and ana-
lysed by direct ELISA using rBet v 1 coated microtiterplates and biotinylated
rabbit anti mouse
IgG antibodies as detecting antibodies.
In yet a further embodiment, the method comprise using transgenic mice capable
of fa-
cilitating production of donor-specific immunity as test animals. Such mice
are disclosed by
Genencor International (WO 01/15521).
Also, a number of studies have assessed the effect of allergy vaccination
compositions
in animal models, in which the animals were sensitized to the relevant
allergen prior to expo-
sure to the vaccination composition:
Mice: Li et al. (J. Allergy Clin. Immunol. vol. 112, pp159-167, 2003) disclose
a mice-
based system to assess efficacy of allergy vaccines. The mice are sensitized
intra-gastrically
with a food allergen, and the treatment is introduced as an intra-rectum
injection. In a separate
allegy vaccination system, Hardy et al. (AM J. Respir. Crit Care Med, vol 167,
pp. 1393-1399,
2003) show that mice can be sensitized by i ntraperitonal injection, and that
allergy vaccine
compounds can be administered intrtracheally withthe animals anaestethized.
Sudowe et al.,
(Gene T herapy, vol. 9, 147-156, 2002) show that intraperitoneal injection in
mice could be
made to produce either TH1 or TH2 responses.
Rats: Wheeler et al., (Int. Arch. Allergy Immunol, vol. 126, pp. 135-139,
2001) disclose
a rat allergy model in which rats are injected subcutaneously along with
adjuvant. These 'aller-
gic' rats can then be made to conduct an allergy-vaccine like response, when
subjected to
subsequent injections with trial vaccine compositions.
Guinea Pigs: Nakamoto et al., (Clin Exp. Allergy, vol. 27, pp 1103-1108 1997)
demon-
strate the use of guinea pigs as model system for SIT. Guinea pigs were
injected intraperito-
neally and boosted twice, and then they were exposed to the vaccine compound
to register
21
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WO 2004/096844 PCT/DK2004/000280
decreases in allergenicity by measuring antibody titers as a function of the
compound, formula-
tion, or mode of application.
Preparation of nucleotide constructs, vectors, host cells, protein variants
and polymers
for conjugation
In accordance with the present invention there may be employed conventional
molecu-
lar biology, microbiology, and recombinant DNA techniques well known to a
person skilled in
the art. Such techniques are explained fully in the literature. See, e.g.,
Sambrook, Fritsch &
Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold
Spring Harbor
Laboratory Press, Cold Spring Harbor, New York (herein "Sambrook et al.,
1989") DNA Clon-
ing: A Practical Approach, Volumes I and II ID.N. Glover ed. 1985);
Oligonucleotide Synthesis
(M.J. Gait ed. 1984); Nucleic Acid Hybridization (B.D. Names & S.J. Higgins
eds (1985));
Transcription And Translation (B.D. Names & S.J. Higgins, eds. (1984)); Animal
Cell Culture
(R.1. Freshney, ed. (1986)); Immobilized Cells And Enzymes (IRL Press,
(1986)); B. Perbal, A
Practical Guide To Molecular Cloning (1984).
The method may in a particular embodiment be carried out to express group 1
dust
mite proteins as inclusion bodies in E.coli or in soluble form in
methylotrophic yeasts such as
Pichia pastoris, as described in WO 01/29078 (HESKA) describing recombinant
expression of
group 1 m ite p roteins i ncluding n ucleotide s equences m odified t o a
nable a xpression o f t he
polypeptides in microorganisms.
A preferred method is to express the group 1 dust mite proteins in
S.cerevisiae cells, as
described by Chua et al. (J. Allergy Clin Immunol. 1992, vol. 89, pp 95-102).
Another preferred method is to express group 1 dust mite proteins in insect
cells such
as Drosophila (Jacquet et al, Clin Exp. Allergy, 2000, vol. 30 pp. 677-84) or
Spodoptera
frugiperda Sf9 cells infected with a bacullovirus system (Shoji, et al.,
Biosci. Biotech. Biochem.
1996, vol. 60, pp. 621-25).
Nucleotide sequences
The present invention also encompasses a nucleotide sequence encoding a
polypep-
tide variant of the invention. As described, a description of standard
mutation of nucleotide se-
quences to encode polypeptide variants by nucleotide substitution can be found
in e.g., Ford
et al., 1991, Protein Expression and Purification 2, p. 95-107. Other standard
methods, such
as site-directed mutagenesis is described in e.g., Sambrook et al. (1989),
Molecular Cloning. A
Laboratory Manual, Cold Spring Harbor, NY.
A "nucleotide sequence" is a single- or double-stranded polymer of
deoxyribonucleo-
tide or ribonucleotide bases read from the 5' to the 3' end. Nucleotide
sequences include RNA
22
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WO 2004/096844 PCT/DK2004/000280
and DNA, and may be isolated from natural sources, synthesized in vitro, or
prepared from a
combination of natural and synthetic molecules.
The techniques used to isolate or clone a nucleotide sequence alias a
nucleotide se-
quence encoding a polypeptide are known in the art and include isolation from
genomic DNA,
preparation from cDNA, or a combination thereof. The cloning of the nucleotide
sequences of
the present invention from such genomic DNA can be effected, e.g., by using
the well known
polymerase chain reaction (PCR) or antibody screening of expression libraries
to detect cloned
DNA fragments with shared structural features. See, e.g. Innis et al., 1990, A
Guide to Meth-
ods and Application, Academic Press, New York. Other nucleotide amplification
procedures
such as ligase chain reaction (LCR), ligated activated transcription (LAT) and
nuceic acid se-
quence-based amplification (NASBA) may be used. The nucleotide sequence may be
cloned
from a strain producing the polypeptide, or from another related organism and
thus, for exam-
ple, may be an allelic or species variant of the polypeptide encoding region
of the nucleotide
sequence.
The term "isolated" nucleotide sequence as used herein refers to a nucleotide
se-
quence which is essentially free of other nucleotide sequences, e.g., at least
about 20% pure,
preferably at least about 40% pure, more preferably about 60% pure, even more
preferably
about 80% pure, most preferably about 90% pure, and even most preferably about
95% pure,
as determined by agarose gel electorphoresis. For example, an isolated
nucleotide sequence
can be obtained by standard cloning procedures used in genetic engineering to
relocate the
nucleotide sequence from its natural location to a different site where it
will be reproduced.
The cloning procedures may involve excision and isolation of a desired
nucleotide fragment
comprising the nucleotide sequence encoding the polypeptide, insertion of the
fragment into a
vector molecule, and incorporation of the recombinant vector into a host cell
where multiple
copies or clones of the nucleotide sequence will be replicated. The nucleotide
sequence may
be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations
thereof. Such
isolated molecules are those that are separated from their natural environment
and include
cDNA and genomic clones. Isolated DNA molecules of the present invention are
free of other
genes with which they are ordinarily associated, and may include naturally
occurring 5' and 3'
untranslated regions such as promoters and terminators. The identification of
associated re-
gions will be evident to one of ordinary skill in the art (see for example,
Dynan and Tijan, Na-
ture 316: 774-78, 1985).
Nucleotide construct
As used herein the term "nucleotide construct" is intended to indicate any
nucleotide
molecule of cDNA, genomic DNA, synthetic DNA or RNA origin. The term
"construct" is in-
tended to indicate a nucleotide segment which may be single- or double-
stranded, and which
23
CA 02523402 2005-10-24
WO 2004/096844 PCT/DK2004/000280
may be based on a complete or partial naturally occurring nucleotide sequence
encoding a
polypeptide of interest. The construct may optionally contain other nucleotide
segments.
The DNA of interest may suitably be of genomic or cDNA origin, for instance
obtained
by preparing a genomic or cDNA library and screening for DNA sequences coding
for all or
part of the polypeptide by hybridization using synthetic oligonucleotide
probes in accordance
with standard techniques (cf. Sambrook et al., supra).
The nucleotide construct may also be prepared synthetically by established
standard
methods, e.g., the phosphoamidite method described by Beaucage and Caruthers,
Tetrahe-
dron Letters 22 (1981 ), 1859 - 1869, or the method described by Matthes et
al., EMBO Journal
3 (1984), 801 - 805. According to the phosphoamidite method, oligonucleotides
are synthe-
sized, e.g., in an automatic DNA synthesizer, purified, annealed, ligated and
cloned in suitable
vectors.
Furthermore, the nucleotide construct may be of mixed synthetic and genomic,
mixed
synthetic and cDNA or mixed genomic and cDNA origin prepared by ligating
fragments of syn-
thetic, genomic or cDNA origin (as appropriate), the fragments corresponding
to various parts
of the entire nucleotide construct, in accordance with standard techniques.
The nucleotide construct m ay also be prepared by polymerise chain reaction
using
specific primers, for instance as described in US 4,683,202 or Saiki et al.,
Science 239 (1988),
487 - 491.
The term nucleotide construct may be synonymous with the term expression
cassette '
when the nucleotide construct contains all the control sequences required for
expression of a
coding sequence of the present invention.
The term "coding sequence" as defined herein is a sequence which is
transcribed
into mRNA and translated into a polypeptide of the present invention when
placed under the
control of the above mentioned control sequences. The boundaries of the coding
sequence
are generally determined by a translation start codon ATG at the 5'-terminus
and a translation
stop codon at the 3'-terminus. A coding sequence can include, but is n of
limited to, DNA,
cDNA, and recombinant nucleotide sequences.
The term "control sequences" is defined herein to include all components which
are
necessary or advantageous for expression of the coding sequence of the
nucleotide sequence.
Each control sequence may be native or foreign to the nucleotide sequence
encoding the
polypeptide. Such control sequences include, but are not limited to, a leader,
a polyadenyla
tion sequence, a propeptide sequence, a promoter, a signal sequence, and a
transcription
terminator. At a minimum, the control sequences include a promoter, and
transcriptional and
translational stop signals. The control sequences may be provided with linkers
for the purpose
of introducing specific restriction sites facilitating ligation of the control
sequences with the cod-
ing region of the nucleotide sequence encoding a polypeptide.
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WO 2004/096844 PCT/DK2004/000280
Transcriptional and translational control sequences are DNA regulatory
sequences,
such as promoters, enhancers, terminators, and the like, that provide for the
expression of a
coding sequence in a host cell. In eukaryotic cells, polyadenylation signals
are control se-
quences.
The nucleotide constructs of the present invention may also comprise one or
more nu-
cleotide sequences which encode one or more factors that are advantageous in
the expression
of the polypeptide, e.g., an activator (e.g., a trans-acting factor), a
chaperone, and a process-
ing protease. Any factor that is functional in the host cell of choice may be
used in the present
invention. The nucleotides encoding one or more of these factors are not
necessarily in tan-
dem with the nucleotide sequence encoding the polypeptide.
Proaeptides
The control sequence may also be a propeptide coding region, which codes for
an
amino acid sequence positioned at the amino terminus of a polypeptide. The
resultant poly-
peptide is~known as a proenzyme or propolypeptide (or a zymogen in some
cases).
A propolypeptide is generally inactive and can be converted to mature active
polypep-
tide by catalytic or autocatalytic cleavage of the propeptide from the
propolypeptide. In the pre-
sent invention protease of the family of subtilisin-like serine proteases have
proven useful for
activation of propeptides. Subtilisin could be added to the crude cell
supernatant, to filtron con-
centrated supernatant, or to material that had been purified by column
chromatography. The
2o subtilisin could be removed by an extra chromatography step or inactivated
with barley chy-
motrypsin inhibitor (Ci-2A). The time of incubation could range from 1 to 21
or 24 hours. The
pro-der p 1 or corresponding propeptide of the variants were cleaved at the
native processing
site (as verified by Edman degradation and N-terminal sequencing) to give the
N-terminal se-
quence TNACSIN.
The preferred Subtilisins are SavinaseTM (Subtilisin from Bacillus clausii.
Novozymes
commercial product.), BPN' (Subtilisin Novo from Bacillus amyloliquefaciens,
Swis-
sProt:SUBT_BACAM see Siezen et al., Protein Engng. 4 (1991) 719-737), PD498
(Subtilisin
from a Bacillus sp., GeneSeqP:AAW24071; W09324623A1) and B34 (Subtilisin from
Bacillus
alcalophilus, patent WO 0158275). The most preferred is BPN' (BASBPN) (Siezen
et al., Pro
tein Engng. 4 (1991 ) 719-737)) dosed to a final concentration ranging from
0,25 to 165 mi
crog/ml, preferably 16,5 to 165 microgram/ml.
The propeptide coding region may be obtained from the Bacillus subtilis
alkaline prote-
ase gene (aprE), the Bacillus subtilis neutral protease gene (nprT), the
Saccharomyces cere-
visiae alpha-factor gene, or the Myceliophthora thermophilum laccase gene (WO
95/33836).
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Activators
An activator is a protein which activates transcription of a nucleotide
sequence encod-
ing a polypeptide (Kudla et al., 1990, EMBO Journal 9:1355-1364; Jarai and
Buxton, 1994,
Current Genetics 26:2238-244; Verdier, 1990, Yeast 6:271-297). T he nucleotide
sequence
encoding an activator may be obtained from the genes encoding Bacillus
stearothermophilus
NprA (nprA), Saccharomyces cerevisiae heme activator protein 1 (hap1 ),
Saccharomyces cer-
evisiae galactose metabolizing protein 4 (gal4), and Aspergillus nidulans
ammonia regulation
protein (areA). For further examples, see Verdier, 1990, supra and MacKenzie
et al., 1993,
Journal of General Microbiology 139:2295-2307.
Chaperones
A chaperone is a protein which assists another polypeptide in folding properly
(Hartl et
al., 1994, TIBS 19:20-25; Bergeron et al., 1994, TIBS 19:124-128; Demolder et
al., 1994,
Journal of Biotechnology 32:179-189; Craig, 1993, Science 260:1902-1903;
Gething and
Sambrook, 1992, Nature 355:33-45; Puig and Gilbert, 1994, Journal of
Biological Chemistry
269:7764-7771; Wang and Tsou, 1993, The FASEB Journal 7:1515-11157; Robinson
et al.,
1994, Bio/Technology 1:381-384). The nucleotide sequence encoding a chaperone
may be
obtained from the genes encoding Bacillus subtilis GroE proteins, Aspergillus
oryzae protein
disulphide isomerase, Saccharomyces cerevisiae calnexin, Saccharomyces
cerevisiae
BiP/GRP78, and Saccharomyces cerevisiae Hsp70. For further examples, see
Gething and
Sambrook, 1992, supra, and Hartl et al., 1994, supra.
Processing protease
A processing protease is a protease that cleaves a propeptide to generate a
mature
biochemically active polypeptide (Enderlin and Ogrydziak, 1994, Yeast 10:67-
79; Fuller et al.,
1989, Proceedings of the National Academy of Sciences USA 86:1434-1438; Julius
et al.,
1984, Cell 37:1075-1089; Julius et al., 1983, Cell 32:839-852). The nucleotide
sequence en-
coding a processing protease may be obtained from the genes encoding
Aspergillus niger
Kex2, Saccharomyces cerevisiae dipeptidylaminopeptidase, Saccharomyces
cerevisiae Kex2,
and Yarrowia lipolytica dibasic processing endoprotease (xpr6).
Promoters
3o The control sequence may be an appropriate promoter sequence, a nucleotide
se
quence which is recognized by a host cell for expression of the nucleotide
sequence. The
promoter sequence contains transcription and translation control sequences
which mediate the
expression of the polypeptide. The promoter may be any nucleotide sequence
which shows
transcriptional activity in the host cell of choice and may be obtained from
genes encoding ex
tracellular or intracellular polypeptides either homologous or heterologous to
the host cell.
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The term "promoter" is used herein for its art-recognized meaning to denote a
portion
of a gene containing DNA sequences that provide for the binding of RNA
polymerase and ini-
tiation of transcription. Promoter sequences are commonly, but not always,
found in the 5' non-
coding regions of genes.
Examples of suitable promoters f or directing t he transcription of the
nucleotide con-
structs of the present invention, especially in a bacterial host cell, are the
promoters obtained
from t he E . coli I ac o peron, the S treptomyces c oelicolor a garase gene
(dagA), the B acillus
subtilis levansucrase gene (sacB), the Bacillus subtilis alkaline protease
gene, the Bacillus
licheniformis alpha-amylase gene (amyL), the Bacillus stearothermophilus
maltogenic amylase
gene (amyM), the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), the
Bacillus amylo-
liquefaciens BAN amylase gene, the Bacillus licheniformis penicillinase gene
(penP), the Bacil-
lus subtilis xylA and xylB genes, and the prokaryotic beta-lactamase gene
(Villa-Kamaroff et
al., 1978, Proceedings of the National Academy of Sciences USA 75:3727-3731 ),
as well as
the tac promoter (DeBoer et al., 1983, Proceedings of the National Academy of
Sciences USA
80:21-25) , or the Bacillus pumilus xylosidase gene, or by the phage Lambda PR
or PL pro-
moters or the E. coli lac, trp or tac promoters. Further promoters are
described in "Useful pro-
teins from recombinant bacteria" in Scientific American, 1980, 242:74-94; and
in Sambrook et
al., 1989, supra.
Examples of suitable promoters f or directing t he transcription of the
nucleotide con-
structs of the present invention in a filamentous fungal host cell are
promoters obtained from
the genes encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic
pro-
teinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid
stable alpha-amylase,
Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor
miehei lipase, As-
pergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate
isomerase, Aspergillus
nidulans acetamidase, Fusarium oxysporum trypsin-like protease (as described
in U.S. Patent
No. 4,288,627, which is incorporated herein by reference), and hybrids
thereof. Particularly
preferred promoters for use in filamentous fungal host cells are the TAKA
amylase, NA2-tpi (a
hybrid of the promoters from the genes encoding Aspergillus niger neutral (-
amylase and As-
pergillus oryzae triose phosphate isomerase), and glaA promoters. Further
suitable promoters
for use in filamentous fungus host cells are the ADH3 promoter (McKnight et
al., The EMBO J.
4 (1985), 2093 - 2099) or the tpiA promoter.
Examples of suitable promoters for use in yeast host cells include promoters
from yeast
glycolytic genes (Hitzeman et al., J. Biol. Chem. 255 (1980), 12073 - 12080;
Alber and Kawa-
saki, J. Mol. Appl. Gen. 1 (1982), 419 - 434) or alcohol dehydrogenase genes
(Young et al., in
Genetic Engineering of Microorganisms for Chemicals (Hollaender et al, eds.),
Plenum Press,
New York, 1982), or the TP11 (US 4,599,311 ) or ADH2-4c (Russell et al.,
Nature 304 (1983),
652 - 654) promoters.
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Further useful promoters are obtained from the Saccharomyces cerevisiae
enolase
(ENO-1 ) gene, the Saccharomyces cerevisiae galactokinase gene (GAL1 ), the
Saccharomy-
ces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase
genes
(ADH2/GAP), and the Saccharomyces cerevisiae 3-phosphoglycerate kinase gene.
Other
useful promoters for yeast host cells are described by Romanos et al., 1992,
Yeast 8:423-488.
In a mammalian host cell, useful promoters include viral promoters such as
those from Simian
Virus 40 (SV40), Rous sarcoma virus (RSV), adenovirus, and bovine papilloma
virus (BPV).
Examples of suitable promoters for directing the transcription of the DNA
encoding the
polypeptide of the invention in mammalian cells are the SV40 promoter
(Subramani et al., Mol.
Cell Biol. 1 (1981 ), 854 -864), the MT-1 (metallothionein gene) promoter
(Palmiter et al., Sci
ence 222 (1983), 809 - 814) or the adenovirus 2 major late promoter.
An example of a suitable promoter for use in insect cells is the polyhedrin
promoter (US
4,745,051; Vasuvedan et al., FEBS Lett. 311, (1992) 7 - 11), the P10 promoter
(J.M. Vlak et
al., J. Gen. Virology 69, 1988, pp. 765-776), the Autographa californica
polyhedrosis virus ba-
sic protein promoter (EP 397 485), the baculovirus immediate early gene 1
promoter (US
5,155,037; US 5,162,222), or the baculovirus 39K delayed-early gene promoter
(US
5,155,037; US 5,162,222).
Terminators
The control sequence may also be a suitable transcription terminator sequence,
a se-
2o quence recognized by a host cell to terminate transcription. The terminator
sequence is oper-
ably linked to the 3' terminus of the nucleotide sequence encoding the
polypeptide. Any termi-
nator which is functional in the host cell of choice may be used in the
present invention.
Preferred terminators for filamentous fungal host cells are obtained from the
genes en-
coding Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase,
Aspergillus nidu
lans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium
oxysporum
trypsin-like protease for fungal hosts) the TP11 (Alber and Kawasaki, op.
cit.) or ADH3
(McKnight et al., op. cit.) terminators.
Preferred terminators for yeast host cells are obtained from the genes
encoding Sac-
charomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1 ),
or Sac-
3o charomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other
useful terminators
for yeast host cells are described by Romanos et al., 1992, supra.
Polyadenylation signals
The control sequence may also be a polyadenylation sequence, a sequence which
is
operably linked to the 3' terminus of the nucleotide sequence and which, when
transcribed, is
recognized by the host cell as a signal to add polyadenosine residues to
transcribed mRNA.
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WO 2004/096844 PCT/DK2004/000280
Any polyadenylation sequence which is functional in the host cell of choice
may be used in the
present invention.
Preferred polyadenylation sequences for filamentous fungal host cells are
obtained
from the genes encoding Aspergillus oryzae TAKA amylase, Aspergillus niger
glucoamylase,
Aspergillus nidulans anthranilate synthase, and Aspergillus niger alpha-
glucosidase.
Useful polyadenylation sequences for yeast host cells are described by Guo and
Sherman,
1995, Molecular Cellular Biology 15:5983-5990.
Polyadenylation sequences are well known in the art for mammalian host cells
such as SV40
or the adenovirus 5 Elb region.
1o Signal seauences
The control sequence may also be a signal peptide coding region, which codes
for an
amino acid sequence linked to the amino terminus of the polypeptide which can
direct the ex-
pressed polypeptide into the cell's secretory pathway of the host cell. The 5'
end of the coding
sequence of the nucleotide sequence may inherently contain a signal peptide
coding region
naturally linked in translation reading frame with the segment of the coding
region which en-
codes the secreted polypeptide. Alternatively, the 5' end of the coding
sequence may contain
a signal peptide coding region which is foreign to that portion of the coding
sequence which
encodes the secreted polypeptide. A foreign signal peptide coding region may
be required
where the coding sequence does not normally contain a signal peptide coding
region. Alterna-
2o tively, the foreign signal peptide coding region may simply replace the
natural signal peptide
coding region in order to obtain enhanced secretion relative to the natural
signal peptide cod-
ing region normally associated with the coding sequence. The signal peptide
coding region
may be obtained from a glucoamylase or an amylase gene from an Aspergillus
species, a li-
pase or proteinase gene from a Rhizomucor species, the 'gene for the alpha-
factor from Sac-
charomyces cerevisiae, an amylase or a protease gene from a Bacillus species,
or the calf
preprochymosin gene. However, any signal peptide coding region capable of
directing the ex-
pressed polypeptide into the secretory pathway of a host cell of choice may be
used in the
present invention.
A " secretory signal sequence" i s a D NA s equence t hat a ncodes a p
olypeptide ( a
"secretory peptide" that, as a component of a larger polypeptide, directs the
larger polypeptide
through a secretory pathway of a cell in which it is synthesized. The larger
polypeptide is
commonly cleaved to remove the secretory peptide during transit through the
secretory path-
way.
An effective signal peptide coding region for bacterial host cells is the
signal peptide
coding region obtained from the maltogenic amylase gene from Bacillus NCIB
11837, the Ba-
cillus stearothermophilus alpha-amylase gene, t he Bacillus licheniformis
subtilisin gene, the
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Bacillus licheniformis beta-lactamase gene, the Bacillus stearothermophilus
neutral proteases
genes (nprT, nprS, nprM), and the Bacillus subtilis PrsA gene. Further signal
peptides are de-
scribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.
An effective signal peptide coding region for filamentous fungal host cells is
the signal
peptide coding region obtained from Aspergillus oryzae TAKA amylase gene,
Aspergillus niger
neutral amylase gene, the Rhizomucor miehei aspartic proteinase gene, the
Humicola lanugi-
nosa cellulase or lipase gene, or the Rhizomucor miehei lipase or protease
gene, Aspergillus
sp. amylase or glucoamylase, a gene encoding a Rhizomucor miehei lipase or
protease. The
signal peptide is preferably derived from a gene encoding A. oryzae TAI<A
amylase, A. niger
neutral (-amylase, A. niger acid-stable amylase, or A. niger glucoamylase.
Useful signal peptides for yeast host cells are obtained from the genes for
Saccharo-
myces cerevisiae a-factor and Saccharomyces cerevisiae invertase. Other useful
signal pep-
tide coding regions are described by Romanos et al., 1992, supra.
For secretion from yeast cells, the secretory signal sequence may encode any
signal peptide
which ensures efficient direction of the expressed polypeptide into the
secretory pathway of the
cell. The signal peptide may be a naturally occurring signal peptide, or a
functional part
thereof, or it may be a synthetic peptide. Suitable signal peptides have been
found to be the a
factor signal peptide (cf. US 4,870,008), the signal peptide of mouse salivary
amylase (cf. O.
Hagenbuchle et al., Nature 289, 1981, pp. 643-646), a modified
carboxypeptidase signal pep
2o tide (cf. L.A. Valls et al., Cell 48, 1987, pp. 887-897), the yeast BAR1
signal peptide (cf. WO
87/02670), or the yeast aspartic protease 3 (YAP3) signal peptide (cf. M. Egel-
Mitani et al.,
Yeast 6, 1990, pp. 127-137).
For efficient secretion in yeast, a sequence encoding a leader peptide may
also be in-
serted downstream of the signal sequence and uptream of the DNA sequence
encoding the
polypeptide. The function of the leader peptide is to allow the expressed
polypeptide to be di-
rected from the endoplasmic reticulum to the Golgi apparatus and further to a
secretory vesicle
for secretion into the culture medium (i.e. exportation of the polypeptide
across the cell wall or
at least through the cellular membrane into the periplasmic space of the yeast
cell). The leader
peptide may be the yeast a-factor leader (the use of which is described in
e.g., US 4,546,082,
EP 16 201, EP 123 294, EP 123 544 and EP 163 529). Alternatively, the leader
peptide may
be a synthetic leader peptide, which is to say a leader peptide not found in
nature. Synthetic
leader peptides may, for instance, be constructed as described in WO 89/02463
or WO
92/11378.
For use in insect cells, the signal peptide may conveniently be derived from
an insect
gene (cf. WO 90/05783), such as the lepidopteran Manduca sexta adipokinetic
hormone pre-
cursor signal peptide (cf. US 5,023,328).
CA 02523402 2005-10-24
WO 2004/096844 PCT/DK2004/000280
For use in insect cells, the signal peptide may conveniently be derived from
an insect
gene (cf. WO 90/05783), such as the lepidopteran Manduca sexta adipokinetic
hormone pre-
cursor signal peptide (cf. US 5,023,328).
Other regulator seauences
It may also be desirable to add regulatory sequences which allow the
regulation of the
expression of the polypeptide relative to the growth of the host cell.
Examples of regulatory
systems are those which cause the expression of the gene to be turned on or
off in response
to a chemical or physical stimulus, including the presence of a regulatory
compound. Regula-
tory systems in prokaryotic systems would include the lac, tac, and trp
operator systems. In
1o yeast, the ADH2 system or GAL1 system may be used. In filamentous fungi,
the TAKA alpha-
amylase promoter, Aspergillus niger glucoamylase promoter, and the Aspergillus
oryzae glu-
coamylase promoter may be used as regulatory sequences. Other examples of
regulatory se-
quences are those which allow for gene amplification. In eukaryotic systems,
these include the
dihydrofolate reductase gene which is amplified in the presence of
methotrexate, and the met-
allothionein genes which are amplified with heavy metals. In these cases, the
nucleotide se
quence encoding the polypeptide would be placed in tandem with the regulatory
sequence.
Recombinant expression vector comprising nucleotide construct
The present invention also relates to a recombinant expression vector
comprising a nu-
cleotide sequence of the present invention, a promoter, and transcriptional
and translational
stop signals. The various nucleotide and control sequences described above may
be joined
together to produce a recombinant expression vector which may include one or
more conven-
ient restriction sites to allow for insertion or substitution of the
nucleotide sequence encoding
the polypeptide at such sites. Alternatively, the nucleotide sequence of the
present invention
may be expressed by inserting the nucleotide sequence or a nucleotide
construct comprising
the sequence into an appropriate vector for expression. In creating the
expression vector, the
coding sequence is located in the vector so that the coding sequence is
operably linked with
the appropriate control sequences for expression, and possibly secretion.
"Operably linked", when referring to DNA segments, indicates that the segments
are
arranged so that they function in concert for their intended purposes, e.g.,
transcription initiates
in the promoter and proceeds through the coding segment to the terminator.
An "Expression vector" is a DNA molecule, linear or circular, that comprises a
seg-
ment encoding a polypeptide of interest operably linked to additional segments
that provide for
its transcription. Such additional segments may include promoter and
terminator sequences,
and optionally one or more origins of replication, one or more selectable
markers, an enhan-
31
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WO 2004/096844 PCT/DK2004/000280
cer, a polyadenylation signal, and the like. Expression vectors are generally
derived from
plasmid or viral DNA, or may contain elements of both.
The recombinant expression vector may be any vector (e.g., a plasmid or
virus), which
can be conveniently subjected to recombinant DNA procedures and can bring
about the ex-
pression of the nucleotide sequence. The choice of the vector will typically
depend on the
compatibility of the vector with the host cell into which the vector is to be
introduced. The vec-
tors may be linear or closed circular plasmids. The vector may be an
autonomously replicating
vector, i.e., a vector which exists as an extrachromosomal entity, the
replication of which is in-
dependent of chromosomal replication, e.g., a plasmid, an extrachromosomal
element, a
minichromosome, or an artificial chromosome. The vector may contain any means
for assur
ing self-replication. Alternatively, the vector may be one which, when
introduced into the host
cell, is integrated into the genome and replicated together with the
chromosomes) into which it
has been integrated. The vector system may be a single vector or plasmid or
two or more vec
tors or plasmids which together contain the total DNA to be introduced into
the genome of the
host cell, or a transposon. ,
The vectors of the present invention preferably contain one or more selectable
markers
which permit easy selection of transformed cells. A selectable marker is a
gene the product of
which provides for biocide or viral resistance, resistance to heavy metals,
prototrophy to
auxotrophs, and the like. Examples of bacterial selectable markers are the dal
genes from Ba-
2o cillus subtilis or Bacillus licheniformis, or markers which confer
antibiotic resistance such as
ampicillin, kanamycin, chloramphenicol, tetracycline, neomycin, hygromycin or
methotrexate
resistance. A frequently used mammalian marker is the dihydrofolate reductase
gene (DHFR).
Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1,
and URA3.
A selectable marker for use in a filamentous fungal host cell may be selected
from the group
including, but not limited to, amdS (acetamidase), argB (ornithine
carbamoyltransferase), bar
(phosphinothricin acetyltransferase), hygB (hygromycin phosphotransferase),
niaD (nitrate re-
ductase), pyre (orotidine-5'-phosphate decarboxylase), sC (sulfate
adenyltransferase), trpC
(anthranilate synthase), and glufosinate resistance markers, as well as
equivalents from other
species. Preferred for use in an Aspergillus cell are the amdS and pyre
markers of Aspergillus
nidulans or Aspergillus oryzae and the bar marker of Streptomyces
hygroscopicus. Further-
more, selection may be accomplished by co-transformation, e.g., as described
in WO
91/17243, where the selectable marker is on a separate vector.
The vectors of the present invention preferably contain an elements) that
permits sta-
ble integration of the vector into the host cell genome or autonomous
replication of the vector
in the cell independent of the genome of the cell.
The vectors of the present invention may be integrated into the host cell
genome when
introduced into a host cell. For integration, the vector may rely on the
nucleotide sequence
32
CA 02523402 2005-10-24
WO 2004/096844 PCT/DK2004/000280
encoding the polypeptide or any other element of the vector for stable
integration of the vector
into the genome by homologous or nonhomologous recombination. Alternatively,
the vector
may contain additional nucleotide sequences for directing integration by
homologous recombi-
nation into the genome of the host cell. The additional nucleotide sequences
enable the vector
to be integrated into the host cell genome at a precise locations) in the
chromosome(s). To
increase the likelihood of integration at a precise location, the
integrational elements should
preferably contain a sufficient number of nucleotides, such as 100 to 1,500
base pairs, pref-
erably 400 to 1,500 base pairs, and most preferably 800 to 1,500 base pairs,
which are highly
homologous with the corresponding target sequence to enhance the probability
of homologous
recombination. The integrational elements may be any sequence that is
homologous with the
target sequence in the genome of the host cell. Furthermore, the integrational
elements may
be non-encoding or encoding nucleotide sequences. On the other hand, the
vector may be
integrated into the genome of the host cell by non-homologous recombination.
These nucleo
tide sequences may be any sequence that is homologous with a target sequence
in the ge
nome of the host cell, and, furthermore, may be non-encoding or encoding
sequences.
For autonomous replication, the vector m ay f urther comprise an origin of
replication
enabling the vector to replicate autonomously in the host cell in question.
Examples of bacte-
rial origins of replication are the origins of replication of plasmids pBR322,
pUC19, pACYC177,
pACYC184, pUB110, pE194, pTA1060, and pAMf31. Examples of origin of
replications for use
in a yeast host cell are the 2 micron origin of replication, the combination
of CEN6 and ARS4,
and the combination of CEN3 and ARS1. The origin of replication may be one
having a muta-
tion which makes its functioning temperature-sensitive in the host cell (see,
e.g., Ehrlich, 1978,
Proceedings of the National Academy of Sciences USA 75:1433).
More than one copy of a nucleotide sequence encoding a polypeptide of the
present
invention may be inserted into the host cell to amplify expression of the
nucleotide sequence.
Stable amplification of the nucleotide sequence can be obtained by integrating
at least one ad-
ditional copy of the sequence into the host cell genome using methods well
known in the art
and selecting for transformants.
The procedures used to ligate the elements described above to construct the
recombi-
nant expression vectors of the present invention are well known to one skilled
in the art (see,
e.g., Sambrook et al., 1989, supra).
Host cell comprising nucleotide constructs
The present invention also relates to recombinant host cells, comprising a
nucleotide se-
quence or nucleotide construct or recombinant expression vector of the
invention, which are
advantageously used in the recombinant production of the polypeptide variants
of the inven-
33
CA 02523402 2005-10-24
WO 2004/096844 PCT/DK2004/000280
tion. The term "host cell" encompasses a parent host cell and any progeny
thereof, which is
not identical to the parent host cell due to mutations that occur during
replication.
The cell is preferably transformed with a vector comprising a nucleotide
sequence of
the invention followed by integration of the vector into the host chromosome.
"Transforma
tion" means introducing a vector comprising a nucleotide sequence of the
present invention
into a host cell so that the vector is maintained as a chromosomal integrant
or as a self
replicating extra-chromosomal vector. Integration is generally considered to
be an advantage
as the nucleotide sequence is more likely to be stably maintained in the cell.
Integration of the
vector into the host chromosome may occur by homologous or non-homologous
recombination
as described above.
The choice of a host cell will to a large extent depend upon the gene encoding
the
polypeptide and its source. The host cell may be a unicellular microorganism,
e.g., a prokary-
ote, or a non-unicellular microorganism, e.g., a eukaryote. Useful unicellular
cells are bacterial
cells such as gram positive bacteria including, but not limited to, a Bacillus
cell, e.g., Bacillus
alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,
Bacillus coagulans,
Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium,
Bacillus
stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis; or a
Streptomyces cell, e.g.,
Streptomyces lividans or Streptomyces murinus, or gram negative bacteria such
as E. coli and
Pseudomonas sp. In a preferred embodiment, the bacterial host cell is a
Bacillus lentus, Bacil-
lus licheniformis, Bacillus stearothermophilus or Bacillus subtilis cell. The
transformation of a
bacterial host cell may, for instance, be effected by protoplast
transformation (see, e.g., Chang
and Cohen, 1979, Molecular General Genetics 168:111-115), by using competent
cells (see,
e.g., Young and Spizizin, 1961, Journal of Bacteriology 81:823-829, or Dubnar
and Davidoff-
Abelson, 1971, Journal of Molecular Biology 56:209-221), by electroporation
(see, e.g., Shi-
gekawa and Dower, 1988, Biotechniques 6:742-751 ), or by conjugation (see,
e.g., Koehler and
Thorne, 1987, Journal of Bacteriology 169:5771-5278).
The host cell may be a eukaryote, such as a mammalian cell, an insect cell, a
plant cell
or a fungal cell.
Useful mammalian cells include Chinese hamster ovary (CHO) cells, HeLa cells,
baby
hamster kidney (BHK) cells, COS cells, or any number of other immortalized
cell lines avail
able, e.g., from the American Type Culture Collection.
Examples of suitable mammalian cell lines are the COS (ATCC CRL 1650 and 1651
),
BHK (ATCC CRL 1632, 10314 and 1573, ATCC CCL 10), CHL (ATCC CCL39) or CHO
(ATCC
CCL 61 ) cell lines. Methods of transfecting mammalian cells and expressing
DNA sequences
introduced in the cells are described in e.g., Kaufman and Sharp, J. Mol.
Biol. 159 (1982), 601
- 621; Southern and Berg, J. Mol. Appl. Genet. 1 (1982), 327 - 341; Loyter et
al., Proc. Natl.
Acad. Sci. USA 79 (1982), 422 - 426; Wigler et al., Cell 14 (1978), 725;
Corsaro and Pearson,
34
CA 02523402 2005-10-24
WO 2004/096844 PCT/DK2004/000280
Somatic Cell Genetics 7 (1981 ), 603, Ausubel et al., Current Protocols in
Molecular Biology,
John Wiley and Sons, Inc., N.Y., 1987, Hawley-Nelson et al., Focus 15 (1993),
73; Ciccarone
et al., Focus 15 (1993), 80; Graham and van der Eb, Virology 52 (1973), 456;
and Neumann et
al., EMBO J. 1 (1982), 841 - 845.
In a preferred embodiment, the host cell is a fungal cell. "Fungi" as used
herein in-
cludes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota
(as defined
by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th
edition, 1995,
CAB International, University Press, Cambridge, UK) as well as the Oomycota
(as cited in
Hawksworth et al., 1995, supra, page 171 ) and all mitosporic fungi
(Hawksworth et al., 1995,
supra). Representative groups of Ascomycota include, e.g., Neurospora,
Eupenicillium
(=Penicillium), Emericella (=Aspergillus), Eurotium (=Aspergillus), and the
true yeasts listed
above. Examples of Basidiomycota include mushrooms, rusts, and smuts.
Representative
groups of Chytridiomycota include, e.g., Allomyces, Blastocladiella,
Coelomomyces, and
aquatic fungi. Representative groups of Oomycota include, e.g.,
Saprolegniomycetous aquatic
fungi (water molds) such as Achlya. Examples of mitosporic fungi include
Aspergillus, Penicil-
lium, Candida, and Alternaria. Representative groups of Zygomycota include,
e.g., Rhizopus
and Mucor.
In a preferred embodiment, the fungal host cell is a yeast cell. "Yeast" as
used herein
includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and
yeast be-
longing to the Fungi Imperfecti (Blastomycetes). The ascosporogenous yeasts
are divided into
the families Spermophthoraceae and Saccharomycetaceae. The latter is comprised
of four
subfamilies, Schizosaccharomycoideae (e.g., genus Schizosaccharomyces),
Nadsonioideae,
Lipomycoideae, and Saccharomycoideae (e.g., genera Pichia, Kluyveromyces and
Saccharo-
myces). The basidiosporogenous yeasts include the genera Leucosporidim,
Rhodosporidium,
Sporidiobolus, Filobasidium, and Filobasidiella. Yeast belonging to the Fungi
Imperfecti are
divided into two families, Sporobolomycetaceae (e.g., genera Sorobolomyces and
Bullera) and
Cryptococcaceae (e.g., genus Candida). Since the classification of yeast may
change in the
future, for the purposes of this invention, yeast shall be defined as
described in Biology and
Activities of Yeast (Skinner, F.A., Passmore, S.M., and Davenport, R.R., eds,
Soc. App. Bacte-
riot. Symposium Series No. 9, 1980. The biology of yeast and manipulation of
yeast genetics
are well known in the art (see, e.g., Biochemistry and Genetics of Yeast,
Bacil, M., Horecker,
B.J., and Stopani, A.O.M., editors, 2nd edition, 1987; The Yeasts, Rose, A.H.,
and Harrison,
J.S., editors, 2nd edition, 1987; and The Molecular Biology of the Yeast
Saccharomyces,
Strathern et al., editors, 1981 ).
The yeast host cell may be selected from a cell of a species of Candida,
Kluyveromy-
ces, Saccharomyces, Schizosaccharomyces, Candida, Pichia, Hansehula, , or
Yarrowia. In a
preferred embodiment, the yeast host cell is a Saccharomyces carlsbergensis,
Saccharomy-
CA 02523402 2005-10-24
WO 2004/096844 PCT/DK2004/000280
ces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,
Saccharomyces kluy-
veri, Saccharomyces norbensis or Saccharomyces oviformis cell. Other useful
yeast host cells
are a Kluyveromyces lactis Kluyveromyces fragilis Hansehula polymorpha, Pichia
pastoris Yar-
rowia lipolytica, Schizosaccharomyces pombe, Ustilgo maylis, Candida maltose,
Pichia guiller-
mondii and Pichia methanolio cell (cf. Gleeson et al., J. Gen. Microbiol. 132,
1986, pp. 3459-
3465; US 4,882,279 and US 4,879,231 ).
In a preferred embodiment, the fungal host cell is a filamentous fungal cell.
"Filamen-
tous fungi" include all filamentous forms of the subdivision Eumycota and
Oomycota (as de-
fined by Hawksworth et al., 1995, supra). The filamentous fungi are
characterized by a vege-
tative mycelium composed of chitin, cellulose, glucan, chitosan, mannan, and
other complex
polysaccharides. Vegetative growth is by hyphal elongation and carbon
catabolism is obliga-
tely aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces
cerevisiae is by
budding of a unicellular thallus and carbon catabolism may be fermentative. In
a more pre-
ferred embodiment, the filamentous fungal host cell is a cell of a species of,
but not limited to,
~ 5 Acremonium, A spergillus, F usarium, H umicola, M ucor, Myceliophthora, N
eurospora, P enicil-
lium, Thielavia, Tolypocladium, and Trichoderma or a teleomorph or synonym
thereof. In an
even more preferred embodiment, the filamentous fungal host cell is an
Aspergillus cell. In
another even more preferred embodiment, the filamentous fungal host cell is an
Acremonium
cell. In another even more preferred embodiment, the filamentous fungal host
cell is a Fusa-
2o rium cell. In another even more preferred embodiment, the filamentous
fungal host cell is a
Humicola cell. In another even more preferred embodiment, the filamentous
fungal host cell is
a Mucor cell. In another even more preferred embodiment, the filamentous
fungal host cell is a
Myceliophthora cell. In another even more preferred embodiment, the
filamentous fungal host
cell is a Neurospora cell. In another even more preferred embodiment, the
filamentous fungal
25 host cell is a Penicillium cell. In another even more preferred embodiment,
the filamentous
fungal host cell is a Thielavia cell. In another even more preferred
embodiment, the filamen-
tous fungal host cell is a Tolypocladium cell. In another even more preferred
embodiment, the
filamentous fungal host cell is a Trichoderma cell. In a most preferred
embodiment, the fila-
mentous fungal host cell is an Aspergillus awamori, Aspergillus foetidus,
Aspergillus japonicus,
30 Aspergillus niger, Aspergillus nidulans or Aspergillus oryzae cell. In
another most preferred
embodiment, the filamentous fungal host cell is a Fusarium cell of the section
Discolor (also
known as the section Fusarium). For example, the filamentous fungal parent
cell may be a
Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium
culmorum,
Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium
negundi,
35 Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium
sarcochroum,
Fusarium sulphureum, or Fusarium trichothecioides cell. In another prefered
embodiment, the
filamentous fungal parent cell is a Fusarium strain of the section Elegans,
e.g., Fusarium ox-
36
CA 02523402 2005-10-24
WO 2004/096844 PCT/DK2004/000280
ysporum. In another most preferred embodiment, the filamentous fungal host
cell is a Humi-
cola insolens or Humicola lanuginosa cell. In another most preferred
embodiment, the filamen-
tous fungal host cell is a Mucor miehei cell. In another most preferred
embodiment, the fila-
mentous fungal host cell is a Myceliophthora thermophilum cell. In another
most preferred em-
bodiment, the filamentous fungal host cell is a Neurospora crassa cell. In
another most pre-
ferred embodiment, the filamentous fungal host cell is a Penicillium
purpurogenum cell. In an-
other most preferred embodiment, the filamentous fungal host cell is a
Thielavia terrestris cell
or an Acremonium chrysogenum cell. In another most preferred embodiment, the
Trichoderma
cell is a Trichoderma harzianum, Trichoderma koningii, Trichoderma
longibrachiatum, Tricho-
derma reesei or Trichoderma viride cell. The use of Aspergillus spp. for the
expression of pro-
teins is described in, e.g., EP 272 277, EP 230 023.
The nucleotide sequences, DNA, of the invention may be modified such as to
optimize
the codon usage for a preferred particular host organism in which it will be
expressed. Exam-
ples of this are published for yeast (Woo JH, et al, Protein Expression and
Purification, Vol. 25
(2), pp. 270-282, 2002), fungi (Te'o et al, FEMS Microbiology Letters, Vol.
190 (1 ) pp. 13-19
(2000)), and other microorganisms, as well as for Der p 1 expressed in
mammalian cells (Mas-
saer M, et al, International Archives of Allergy and Immunology, Vol. 125 (1
), pp. 32-43, 2001 ).
In a particular embodiment the host cell is an insect cell and/or insect cell
line. The in-
sect cell line used as the host may suitably be a Lepidoptera cell line, such
as Spodoptera
frugiperda cells or Trichoplusia ni cells (cf. US 5,077,214). Culture
conditions may suitably be
as described in, for instance, WO 89/01029 or WO 89/01028, or any of the
aforementioned
references.
Plants
The present invention also relates to a transgenic plant, plant part, or plant
cell which
has been transformed with a nucleic acid sequence encoding a polypeptide (i.e.
variant) of the
present invention so as to express and produce the polypeptide in recoverable
quantities. The
polypeptide may be recovered from the plant or plant part. Alternatively, the
plant or plant part
containing the recombinant polypeptide may be used as such for vaccine
purposes.
In a particular embodiment, the polypeptide is targeted to the endosperm
storage
vacuoles in seeds. This can be obtained by synthesizing it as a precursor with
a suitable signal
peptide, see Horvath et al in PNAS, Feb. 15, 2000, vol. 97, no. 4, p. 1914-
1919.
The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous (a
monocot)
or engineered variants thereof. Examples of monocot plants are grasses, such
as meadow
grass (blue grass, Poa), forage grass such as festuca, lolium, temperate
grass, such as
Agrostis, and cereals, e.g., wheat, oats, rye, barley, rice, sorghum, and
maize (corn). Exam-
ples of dicot plants are tobacco, legumes, such as lupins, potato, sugar beet,
pea, bean and
37
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WO 2004/096844 PCT/DK2004/000280
soybean, and cruciferous plants (family Brassicaceae), such as cauliflower,
rape seed, and the
closely related model organism Arabidopsis thaliana. Low-phytate plants as
described e.g. in
US patent no. 5,689,054 and US patent no. 6,111,168 are examples of engineered
plants.
Examples of plant parts are stem, callus, leaves, root, fruits, seeds, and
tubers. Also
specific plant tissues, such as chloroplast, apoplast, mitochondria, vacuole,
peroxisomes, and
cytoplasm are considered to be a plant part. Furthermore, any plant cell,
whatever the tissue
origin, is considered to be a plant part.
Also included within the scope of the present invention are the progeny of
such plants,
plant parts and plant cells.
The transgenic plant or plant cell expressing a polypeptide of the present
invention may
be constructed in accordance with methods known in the art. Briefly, the plant
or plant cell is
constructed by incorporating one or more expression constructs encoding a
polypeptide of the
present invention into the plant host genome and propagating the resulting
modified plant or
plant cell into a transgenic plant or plant cell.
Conveniently, the expression construct is a nucleic acid construct which
comprises a
nucleic acid sequence encoding a polypeptide of the present invention operably
linked with
appropriate regulatory sequences required for expression of the nucleic acid
sequence in the
plant or plant part of choice. Furthermore, the expression construct may
comprise a selectable
marker useful for identifying host cells into which the expression construct
has been integrated
and DNA sequences necessary for introduction of the construct into the plant
in question (the
latter depends on the DNA introduction method to be used).
The choice of regulatory sequences, such as promoter and terminator sequences
and
optionally signal or transit sequences are determined, for example, on the
basis of when,
where, and how the polypeptide is desired to be expressed. For instance, the
expression of
the gene encoding a polypeptide of the present invention may be constitutive
or inducible, or
may be developmental, stage or tissue specific, and the gene product may be
targeted to a
specific tissue or plant part such as seeds or leaves. Regulatory sequences
are, for example,
described by Tague et al., 1988, Plant Physiology 86: 506.
For constitutive expression, the 35S-CaMV promoter may be used (Franck et al.,
1980,
Cell 21: 285-294). Organ-specific promoters may be, for example, a promoter
from storage
sink tissues such as seeds, potato tubers, and fruits (Edwards & Coruzzi,
1990, Ann. Rev.
Genet. 24: 275-303), or from metabolic sink tissues such as meristems (Ito et
al., 1994, Plant
Mol. Biol. 24: 863-878), a seed specific promoter such as the glutelin,
prolamin, globulin, or
albumin promoter from rice (Vllu et al., 1998, Plant and Cell Physiology 39:
885-889), a Vicia
faba promoter from the legumin B4 and the unknown seed protein gene from Vicia
faba (Con-
rad et al., 1998, Journal of Plant Physiology 152: 708-711 ), a promoter from
a seed oil body
protein (Chen et al., 1998, Plant and Cell Physiology 39: 935-941 ), the
storage protein napA
38
CA 02523402 2005-10-24
WO 2004/096844 PCT/DK2004/000280
promoter from Brassica napus, or any other seed specific promoter known in the
art, e.g., as
described in WO 91/14772. Furthermore, the promoter may be a leaf specific
promoter such
as the rbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant
Physiology 102: 991-
1000, the chlorella virus adenine methyltransferase gene promoter (Mitra and
Higgins, 1994,
Plant Molecular Biology 26: 85-93), or the aldP gene promoter from rice
(Kagaya et al., 1995,
Molecular and General Genetics 248: 668-674), or a wound inducible promoter
such as the
potato pint promoter (Xu et al., 1993, Plant Molecular Biology 22: 573-588).
A promoter enhancer element may also be used to achieve higher expression of
the
variant of the present invention in the plant. For instance, the promoter
enhancer element may
be an intron which is placed between the promoter and the nucleotide sequence
encoding a
polypeptide of the present invention. For instance, Xu et al., 1993, supra
disclose the use of
the first intron of the rice actin 1 gene to enhance expression.
Still further, the codon usage may be optimized for the plant species in
question to im-
prove expression (see Horvath et al referred to above).
The selectable marker gene and any other parts of the expression construct may
be
chosen from those available in the art.
The nucleic acid construct is incorporated into the plant genome according to
conven-
tional techniques known in the art, including Agrobacterium-mediated
transformation, virus-
mediated transformation, microinjection, particle bombardment, biolistic
transformation, and
electroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,
Bio/Technology 8:
535; Shimamoto et al., 1989, Nature 338: 274).
Presently, Agrobacterium tumefaciens-mediated gene transfer is the method of
choice
for generating transgenic dicots (for a review, see Hooykas and Schilperoort,
1992, Plant Mo-
lecular Biology 19: 15-38). However it can also be used for transforming
monocots, although
other transformation methods are generally preferred for these plants.
Presently, the method
of c hoice for generating t ransgenic m onocots i s p article b ombardment (
microscopic gold o r
tungsten particles coated with the transforming DNA) of embryonic calli or
developing embryos
(Christou, 1992, Plant Journal 2: 275-281; Shimamoto, 1994, Current Opinion
Biotechnology 5:
158-162; Vasil et al., 1992, Bio/Technology 10: 667-674). An alternative
method for transfor-
mation of monocots is based on protoplast transformation as described by
Omirulleh et al.,
1993, Plant Molecular Biology 21: 415-428.
Following transformation, the transformants having incorporated therein the
expression
construct are selected and regenerated into whole plants according to methods
well-known in
the art.
The present invention also relates to methods for producing a polypeptide of
the pre-
sent invention comprising (a) cultivating a transgenic plant or a plant cell
comprising a nucleic
39
CA 02523402 2005-10-24
WO 2004/096844 PCT/DK2004/000280
acid sequence encoding a variant of the present invention under conditions
conducive for pro-
duction of the variant; and (b) recovering the variant.
Methods of preparing group 1 mite polypeptide variants
The polypetide variants of the invention may be prepared by (a) transforming a
suitable
host cell with a nucleotide construct of the invention, (b) cultivating the
recombinant host cell of
the invention comprising a nucleotide construct of the invention under
conditions conducive for
production of the variant of the invention and (c) recovering the variant. The
method may in a
particular embodiment be carried out as described in WO 01/29078 (HESI<,4)
describing re-
combinant expression of group 1 mite proteins including nucleotide sequences
modified to en-
able expression of the polypeptides in microorganisms.
Transformation
Fungal cells may be transformed by a process involving protoplast formation,
transfor-
mation of the protoplasts, and regeneration of the cell wall in a manner known
per se. Suitable
procedures for transformation of Aspergillus host cells are described in EP
238 023 and Yelton
et al., 1984, Proceedings of the National Academy of Sciences USA 81:1470-
1474. A suitable
method of transforming Fusarium species is described by Malardier et al.,
1989, Gene 78:147-
156 or in copending US Serial No. 08/269,449. Examples of other fungal cells
are cells of fila-
mentous fungi, e.g., Aspergillus spp., Neurospora spp., Fusarium spp. or
Trichoderma spp., in
particular strains of A. oryzae, A. nidulans or A. niger. The use of
Aspergillus spp. for the ex
pression of proteins is described in, e.g., EP 272 277 and EP 230 023. The
transformation of
F. oxysporum may, for instance, be carried out as described by Malardier et
al., 1989, Gene
78: 147-156.
Yeast may be transformed using the procedures described by Becker and
Guarente, In
Abelson, J.N. and Simon, M.I., editors, Guide to Yeast Genetics and Molecular
Biology, Meth-
ods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito
et al., 1983,
Journal of Bacteriology 153:163; and Hinnen et al., 1978, Proceedings of the
National Acad-
emy of Sciences USA 75:1920. Mammalian cells may be transformed by direct
uptake using
the calcium phosphate precipitation method of Graham and Van der Eb (1978,
Virology
52:546).
Transformation of insect cells and production of heterologous polypeptides
therein may
be performed as described in US 4,745,051; US 4, 775, 624; US 4,879,236; US
5,155,037; US
5,162,222; EP 397,485) all of which are incorporated herein by reference.
CA 02523402 2005-10-24
WO 2004/096844 PCT/DK2004/000280
Cultivation
The transformed or transfected host cells described above are cultured in a
suitable nu-
trient medium under conditions permitting the production of the desired
molecules, after which
these are recovered from the cells, or the culture broth.
The medium used to culture the cells may be any conventional medium suitable
for
growing the host cells, such as minimal or complex media containing
appropriate supplements.
Suitable media are available from commercial suppliers or may be prepared
according to pub-
lished recipes (e.g., in catalogues of the American Type Culture Collection).
The media are
prepared using procedures known in the art (see, e.g., references for bacteria
and yeast; Ben-
nett, J.W. and LaSure, L., editors, More Gene Manipulations in Fungi, Academic
Press, CA,
1991 ).
Recovery
In a particular embodiment the polypeptide variant of the invention is in an
isolated and
purified form. Thus the polypeptide variant of the invention is provided in a
preparation which
more than 20 %w/w pure, particularly more than 50% w/w pure, more particularly
more than
75% w/w pure, more particularly more than 90% wlw pure and even more
particularly more
than 95% w/w pure. The purity in this context is to be understood as the
amount of polypeptide
variant of the invention present in the preparation of the total polypeptide
material in the prepa-
ration.
2o When applied to a polypeptide, the term "isolated" indicates that the
polypeptide is
found in a condition other than its native environment, such as apart from
blood and animal
tissue. In a preferred form, the isolated polypeptide is substantially free of
other proteins, par-
ticularly other proteins of animal origin. It is preferred to provide the
polypeptides in a highly
purified form, i.e., greater than 95% pure, more preferably greater than 99%
pure.
If the molecules are secreted into the nutrient medium, they can be recovered
directly
from the medium. If they are not secreted, they can be recovered from cell
lysates. The mole-
cules are recovered from the culture medium by conventional procedures
including separating
the host cells from the medium by centrifugation or filtration, precipitating
the proteinaceous
components of the supernatant or filtrate by means of a salt, e.g., ammonium
sulphate. The
proteins may be matured in vitro e.g., by acidification to induce
autocatalytic processing (Jac
quet et al., Clin Exp Allergy, 2002, vol. 32 pp 1048-53), and they may be
purified by a variety of
chromatographic procedures, e.g., ion exchange chromatography, gelfiltration
chromatogra
phy, affinity chromatography, or the like, dependent on the type of molecule
in question (see,
e.g., Protein Purification, J-C Janson and Lars Ryden, editors, VCH
Publishers, New York,
1989).
41
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WO 2004/096844 PCT/DK2004/000280
Activation of polymers
In case the variant of the invention is to be conjugated to one or more
polymers and if
the p olymeric molecules t o b a c onjugated w ith t he p olypeptide i n q
uestion a re n of a ctive i t
must be activated by the use of a suitable technique. It is also contemplated
according to the
invention to couple the polymeric molecules to the polypeptide through a
linker. Suitable link-
ers are well-known to the skilled person.
Methods and chemistry for activation of polymeric molecules as well as for
conjugation
of polypeptides are intensively described in the literature. Commonly used
methods for activa-
tion of insoluble polymers include activation of functional groups with
cyanogen bromide, pe-
riodate, glutaraldehyde, biepoxides, epichlorohydrin, divinylsulfone,
carbodiimide, sulfonyl hal-
ides, trichlorotriazine etc. (see R.F. Taylor, (1991), "Protein
immobilisation. Fundamental and
applications", Marcel Dekker, N.Y.; S.S. Wong, (1992), "Chemistry of Protein
Conjugation and
Crosslinking", CRC Press, Boca Raton; G.T. Hermanson et al., (1993),
"Immobilized Affinity
Ligand Techniques", Academic Press, N.Y.). Some of the methods concern
activation of in-
soluble polymers but are also applicable to activation of soluble polymers
e.g., periodate, tri
chlorotriazine, sulfonylhalides, divinylsulfone, carbodiimide etc. The
functional groups being
amino, hydroxyl, thiol, carboxyl, aldehyde or sulfydryl on the polymer and the
chosen attach
ment group on the protein must be considered in choosing the activation and
conjugation
chemistry which normally consist of i) activation of polymer, ii) conjugation,
and iii) blocking of
residual active groups.
In the following a number of suitable polymer activation methods will be
described
shortly. However, it is to be understood that also other methods may be used.
Coupling polymeric molecules to the free acid groups of polypeptides may be
per-
formed with the aid of diimide and for example amino-PEG or hydrazino-PEG
(Pollak et al.,
(1976), J. Amr. Chem. Soc., 98, 289291) or diazoacetatelamide (Wong et al.,
(1992), "Chem-
istry of Protein Conjugation and Crosslinking", CRC Press).
Coupling polymeric molecules to hydroxy groups are generally very difficult as
it must
be performed in water. Usually hydrolysis predominates over reaction with
hydroxyl groups.
Coupling p olymeric m olecules t o free s ulfhydryl g roups c an b a r eached
w ith s pecial
groups like maleimido or the ortho-pyridyl disulfide. Also vinylsulfone (US
patent no. 5,414,135,
(1995), Snow et al.) has a preference for sulfhydryl groups but is not as
selective as the other
mentioned.
Accessible Arginine residues in the polypeptide chain may be targeted by
groups com-
prising two vicinal carbonyl groups.
Techniques involving coupling electrophilically activated PEGs to the amino
groups of
Lysines may also be useful. Many of the usual leaving groups f or alcohols
give rise to an
amine linkage. For instance, alkyl sulfonates, such as tresylates (Nilsson et
al., (1984), Meth-
42
CA 02523402 2005-10-24
WO 2004/096844 PCT/DK2004/000280
ods in Enzymology vol. 104, Jacoby, W. B., Ed., Academic Press: Orlando, p. 56-
66; Nilsson
et al., (1987), Methods in Enzymology vol. 135; Mosbach, K., Ed.; Academic
Press: Orlando,
pp. 65-79; Scouten et al., (1987), Methods in Enzymology vol. 135, Mosbach,
K., Ed., Aca-
demic Press: Orlando, 1987; pp 79-84; Crossland et al., (1971), J. Amr. Chem.
Soc. 1971, 93,
pp. 4217-4219), mesylates (Harris, (1985), supra; Harris et al., (1984), J.
Polym. Sci. Polym.
Chem. Ed. 22, pp 341-352), aryl sulfonates like tosylates, and para-
nitrobenzene sulfonates
can be used.
Organic sulfonyl chlorides, e.g., Tresyl chloride, effectively converts
hydroxy groups in
a number of polymers, e.g., PEG, into good leaving groups (sulfonates) that,
when reacted
with n ucleophiles I ike a mino g roups i n p olypeptides a Ilow s table I
inkages t o b a formed b e-
tween polymer and polypeptide. In addition to high conjugation yields, the
reaction conditions
are in general mild (neutral or slightly alkaline pH, to avoid denaturation
and little or no disrup-
tion of activity), and satisfy the non-destructive requirements to the
polypeptide.
Tosylate is more reactive than the mesylate but also more unstable decomposing
into
PEG, dioxane, and sulfonic acid (Zalipsky, (1995), Bioconjugate Chem., 6, 150-
165). Epoxides
may also been used for creating amine bonds but are much less reactive than
the above men-
tinned groups.
Converting PEG into a chloroformate with phosgene gives rise to carbamate
linkages
to Lysines. This theme can be played in many variants substituting the
chlorine with N-hydroxy
2o succinimide (US patent no. 5,122,614, (1992); Zalipsky et al., (1992),
Biotechnol. Appl. Bio-
chem., 15, p. 100-114; Monfardini et al., (1995), Bioconjugate Chem., 6, 62-
69, with imidazole
(Allen et al., (1991 ), Carbohydr. Res., 213, pp 309-319), with para-
nitrophenol, DMAP (EP 632
082 A1, (1993), Looze, Y.) etc. The derivatives are usually made by reacting
the chloroformate
with the desired leaving group. All these groups give rise to carbamate
linkages to the peptide.
Furthermore, isocyanates and isothiocyanates may be employed yielding ureas
and
thioureas, respectively.
Amides may be obtained from PEG acids using the same leaving groups as
mentioned
above and cyclic imid thrones (US patent no. 5,349,001, (1994), Greenwald et
al.). The reac-
tivity of these compounds is very high but may make the hydrolysis to fast.
PEG succinate made from reaction with succinic anhydride can also be used. The
hereby comprised ester group make the conjugate much more susceptible to
hydrolysis (US
patent no. 5,122,614, (1992), Zalipsky). This group may be activated with N-
hydroxy succinim-
ide.
Furthermore, a special linker can be introduced. The oldest being cyanuric
chloride
(Abuchowski et al., (1977), J. Biol. Chem., 252, 3578-3581; US patent no.
4,179,337, (1979),
Davis et al.; Shafer et al., (1986), J. Polym. Sci. Polym. Chem. Ed., 24,
375378.
43
CA 02523402 2005-10-24
WO 2004/096844 PCT/DK2004/000280
Coupling of PEG to an aromatic amine followed by diazotation yields a very
reactive
diazonium salt which in situ can be reacted with a peptide. An amide linkage
may also be ob-
tained by reacting an azlactone derivative of PEG (US patent no. 5,321,095,
(1994),
Greenwald, R. B.) thus introducing an additional amide linkage.
As some polypeptides do not comprise many Lysines it may be advantageous to
attach
more than one PEG to the same Lysine. This can be done e.g., by the use of 1,3-
diamino-2-
propanol.
PEGs may also be attached to the amino-groups of the polypeptide w ith
carbamate
linkages (WO 95/11924, Greenwald et al.). Lysine residues may also be used as
the back-
1 o bone.
The coupling technique used in the examples is the N-succinimidyl carbonate
conju-
gaion technique descried in WO 90/13590 (Enzon).
Compositions
The present invention also relates to a composition comprising a variant of
the inven-
tion a nd o ptionally a p harmaceutically a cceptable c arrier a nd/or a
djuvant a nd a m ethod f or
preparing such a composition comprising admixing the variant of the invention
with an accept-
able pharmaceutical carrier and/or adjuvant. In a particular embodiment the
composition is
suitable for treating an immunological disorder, such as allergy in animals or
humans, such as
a vaccine.
2o Pharmaceutical carriers and/or adjuvants includes saline, glycerol,
aluminium hydrox
ide, aluminium phosphate, calcium phosphate, saponins (e.g., Q21 and Quill A),
squalene
based emulsions (e.g., MF59), monophosphoryl lipid A (and synthetic mimics),
polylactide co
glycolid (PLG) particles, ISCOMS, liposomes, chitosan, bacterial DNA (e.g.,
unmethylated
CpG c ontaining s equences). S uitable c arriers a Iso i nclude p
harmaceutically a cceptable s o1
vents and/or tabletting aids/auxilliaries.
Use of vaccination antigen polypeptide variants and compositions containing
them
In a further aspect the invention provide use of the variant or the
composition of the in-
vention as a medicament, particularly for the treatment of an immunological
disorder, such as
allergy in animals and humans and/or for the preparation of a medicament for
the treatment of
3o such immunological disorder.
Traditionally, a Ilergy v accination i s p erformed b y p arenteral, i
ntranasal, or s ublingual
administration in increasing doses over a fairly long period of time, and
results in, so called,
desensitisation of the patient. The exact immunological mechanism is not
known, but induced
differences in the phenotype of allergen specific T and B cells are thought to
be of particular
importance.
44
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WO 2004/096844 PCT/DK2004/000280
Compared to conventional types of vaccination, allergy vaccination is
complicated by
the existence of an ongoing immune response in allergic patients. This immune
response is
characterised by the presence of allergen specific IgE, that will mediate the
release of allergic
mediators, thereby inducing allergic symptoms upon exposure to allergens.
Thus, allergy vac-
s cination using native and/or naturally occurring allergens has an inherent
risk of side effects
being in the utmost consequence life threatening to the patient.
Approaches to circumvent this problem may be divided in three categories. In
practise
measures from more than one category are often combined. First category of
measures in-
cludes the administration of several small and increasing doses over a long
period to reach a
substantial accumulated dose. The theory being, that the protective immune
response slowly is
allowed to be initiated, before potentially anaphylactic doses of allergen is
administrated. Sec-
ond category of measures includes physical modification of the allergen by
incorporation of the
allergen into e.g., a gel formulation such as a aluminium hydroxide. Aluminium
hydroxide has
an adjuvant effect and a depot effect of slow allergen release, thus reducing
the the tissue
concentration of the allergen. Third category of measures include as described
herein modifi-
cation of the allergen for the purpose of reducing allergenicity.
The immunotherapeutic effect of an allergy vaccine can be assessed in a number
of
different ways. One is to measure the specific IgE binding, the reduction of
which indicates a
better safety profile (however not necessarily a better vaccine potential) (WO
99/47680, ALK-
ABELLO). Also, several cellular assays could be employed to show the modified
immunere
sponse indicative of good allergy vaccine potential as shown in several
publications, all of
which are hereby incorporated by reference (van Neerven et al., "T lymphocyte
responses to
allergens: Epitope-specificity and clinical relevance", Immunol Today, 1996,
vol. 17, pp. 526
532; Hoffmann et al., Allergy, 1999, vol. 54, pp. 446-454, WO99/07880).
Basophil histamine
release: e.g., Swoboda et al., Eur. J. Immunol., vol. 32, pp 270-280, 2002.
Also skin prick testing could be employed for example as described in
Kronqvist et al Clin Exp
Allergy 2000 vol 30 pp 670-676
Eventually, clinical trials with allergic patients could be employed using
cellular or clini-
cal end-point measurements. (Ebner et al., Clin. Exp. All., 1997, vol. 27, pp.
107-1015; Int.
3o Arch. Allergy Immunol., 1999, vol. 119, pp 1-5).
EXAMPLES
Methods
Sandwich ELISA
CA 02523402 2005-10-24
WO 2004/096844 PCT/DK2004/000280
Immunoplates (Nunc Maxisorb; Nunc-Nalgene) are coated overnight at 4 degree C
with at suitable dose of polyclonal rabbit anti Der p 1 antibody. The plates
are then washed
thoroughly with 0.15 M Phosphate Buffered Saline (PBS) containing 0.05 % Tween
20 (PBST),
and remaining binding sites are blocked with PBS with 2 % skim milk powder, 1
h at room
temperature. Samples, it can be purified, semi-purified recombinant group 1
mite polypeptide
variant allergen or crude culture broth containing protein of interest, are
added in a suitable
dose or dose-range. The plates are then washed thoroughly with 0.15 M PBST
before the al-
lergens are detected by incubation with biotinylated monoclonal anti Der p 1
antibody (IN-
DOOR) 1 h at room temperature. Wash again in 0.15 M PBST. Conjugate with
complexes of
Streptavidin:Horse Radish Peroxidase (Kierkegaard & Perry) for 1 h at room
temperature. Re
peat washing step and develop by adding 3,3',5,5'-tetramethylbenzidine
hydrogen peroxide
(TMB Plus, ICem-En-Tec) and stop reaction by addition of 0.2 M H2S04. 0D450
will reflex al
lergen binding to the immunoglobin, and it is thus possible to detect and also
determine the
amount of allergen bound if natural Der p 1 (available from Indoor
biotechnologies, product
number: NA-DP1 ) in known concentrations is included in the experiment in a
dose rage.
Example 1: Finding of epitope patterns within oligo peptides with antibody
reactivity
A high diversity library of phages expressing random oligomeric peptides
(hexa, hepta,
octa, nona and/or dodecc peptides) as part of their surface proteins, were
screened for their
capacity to bind antibodies. The phage libraries were obtained from Schafer-N,
Copenhagen,
Denmark.
Antibody samples were raised in animals (Rat, Rabbits or Mice) by parenteral
or muco-
sal administration of each of the proteins listed below. The antibodies were
dissolved in phos-
phate buffered saline (PBS). In some cases, antibodies of specific subclasses
were purified
from the serum of immunised animals by capryilic acid precipitation (for total
IgG) or by affinity
chromatography using paramagnetic immunobeads (Dynal AS) loaded with one of
the follow-
ing antibodies: mouse anti-rat IgG1 or rat anti-mouse IgE.
1 ) amylase AA560 from Bacillus sp. DSM 12649, (Rat IgG)
2) alpha-amylase of Bacillus halmapalus (W096/23873), which is also called
amylase
SP722, (Rat IgG)
3) a variant of SP722 with residues 183 and 184 deleted, called JE-1
(W096/23873),
(Rat IgG and Rabbit IgG)
4) Mycelioptora thermopila laccase (WO 95/33836) (Rabbit IgG),
5) T. lanuginosus lipase (LipolaseT"") (Rat IgG and Rabbit IgG),
46
CA 02523402 2005-10-24
WO 2004/096844 PCT/DK2004/000280
6) family 45 cellulase from Humicola insolens (CarezymeTM) (Rabbit IgG),
7) Bacillus lentus protease (SavinaseT"") (Rat IgG, Mouse IgG, Mouse IgE, and
Rabbit
IgG),
8) Subtilisin Novo (BPN') from B.amyloliquefaciens (Rat IgG),
9) The Y217L variant of Subtilisin Novo (Rat IgG),
10) Subtilisin Carlsberg (AlcalaseT"") (Rat IgG),
11 ) TY145 protease (Rat IgG),
12) CDJ31 protease,
13) Subtilisin 147 (EsperaseT"") (Rat IgG),
14) Bacillolysin from Bacillus amyloliquefaciens (NeutraseT"") (Rat IgG and
Rat IgG1),
and
15) Subtilisin PD498 (WO 93/24623) (Rat IgG and Rabbit IgG).
The phage libraries were incubated with the antibody coated beads. E.g. phages
ex-
pressing oligo-peptides with affinity for mouse IgE antibodies were captured
onto rat anti-
mouse IgE-coated beads and collected by exposing these paramagnetic beads to a
magnetic
field. The collected phages were eluted from the immobilised antibodies by
mild acid treat-
ment, or by elution with intact protein antigen specific for the respective
antibody sample (e.g.,
Savinase for anti-Savinase antibodies). The isolated phages were amplified
using methods
known in the art. Alternatively, immobilised phages were directly incubated
with E.coli for infec-
tion. In short, F-factor positive E. coli (e.g., XL-1 Blue, JM101, TG1) were
infected with M13-
derived vector in the presence of a helper phage (e.g., M13K07), and
incubated, typically in
2xYT containing glucose or IPTG, and appropriate antibiotics for selection.
Finally, cells were
removed by centrifugation. This cycle of events was repeated on the respective
cell super-
natants, minimum 2 times and maximum 5 times. After selection round 2, 3, 4
and 5, a fraction
of the infected E.coli was incubated on selective 2xYT agar plates, and the
specificity of the
emerging phages was assessed immunologically: Phages were transferred to a
nitrocellulase
(NC) membrane. For each plate, 2 NC-replicas were made. One replica was
incubated with
the selection antibodies, the other replica was incubated with the selection
antibodies and the
3o immunogen used to obtain the antibodies as competitor. Those plaques that
were absent in
the presence of immunogen, were considered specific, and were am-plified
according to the
procedure described above.
The specific phage-clones were isolated from the cell supernatant by
centrifugation in
the presence of polyethylenglycol. DNA was isolated, the DNA sequence coding
for the oli-
gopeptide was amplified by PCR, and its DNA sequence was determined, all
according to
47
CA 02523402 2005-10-24
WO 2004/096844 PCT/DK2004/000280
standard procedures known in the art. The amino acid sequence of the
corresponding oli-
gopeptide was deduced from the DNA sequence.
These 256 experimentally determined reactive peptides were supplemented with
infor-
mation on 402 reactive peptides published in the literature:
Allergy 38 (1983) 449-459,
Allergy 56 (2001 ) 118-125;
Allergy 56 s67 (2001 ) 48-51;
Allergy 54 (1999) 1048 -1057;
Arch Biochem Biophys 342 (1997) 244-253
B. B. Res. Com. 219 (1996) 290-293;
Biochem J 293 (1993) 625-632;
Bioinformatics 18 (2002) 1358-1364;
Clin Exp Allergy 24 (1994) 100-108;
Clin Exp Allergy 24 (1994) 250-256;
Clin Exp Allergy 31 (2001) 331-341;
Clin Exp Med 24 (1994) 100-108;
Eur J Biochem 245 (1997) 334-339;
Int Arch Allergy Appl Immunol 89 (1989) 342-348
Int Arch Allergy Appl Immunol 89 (1989) 410-415
Int Arch Appl Immunol 103 (1994) 357-364
Int Arch Appl Immunol 92 (1990) 30-38
J Allergy Clin Immunol 106 (2000) 150-158
J Allergy Clin Immunol 107 (2001 ) 1069-1076
J Allergy Clin Immunol 93 (1994) 34-43
J Biol Chem 271 (1996) 29915-29921
J Clin Invest 103 (1999) 535-542
J Immunol 121 (1989) 275-280
J Immunol 133 (1984) 2668-2673
J Immunol 140 (1988) 611-616
J Immunol 147 (1991 ) 205-211
J Immunol 151 (1993) 5354-5363
J Immunol 151 (1993) 7206-7213
J Immunol Methods 213 (1998) 1-17
Mol Immunol 25 (1988) 355-365
Mol Immunol 28 (1991 ) 1225-1232
Mol Immunol 29 (1992) 1383-1389
48
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WO 2004/096844 PCT/DK2004/000280
Mol Immunol 29 (1992) 257-262
Mol Immunol 30 (1993) 183-189
Mol Immunol 35 (1998) 293-305
Mol Immunol 37 (2000) 789-798
Peptides 21 (2000) 589-599
Protein Scieince 8 (1999) 760-770
Scand J Immunol 27 (1988) 587-591
Science 233 (1986) 747-753
WO 90/11293
WO 99/38978
WO 01/34186
WO 01/39799
WO 01/39799
WO 01/49834
www.csl.gov.uk/allerqen
Thus, in total 658 peptide sequences with specificity for the protein-specific
antibodies,
described above, were obtained. These sequences were collected in a database,
and ana
lysed by sequence alignment to identify epitope patterns observing that
conservative alterna
tives were considered equal (as described above).
Identifyina epitope patterns
In principle, each of the the 658 reactive (oligo)peptide sequences
represented an epi
tope p attern. H owever, i n t he 6 58 r eactive ( oligo)peptide s equences s
ome a pitope p atterns
were redundant and to remove redundency among the epitope patterns, the
reactive
(oligo)peptides sequences were subjected to computerised data analysis.
First all possible dipeptides were generated k orresponding to 132 different
combina-
tions taking conservative alternatives into account. The presence and
frequency of each dipep-
tide among the 658 reactive (oligo)peptide sequences were listed. Next all
possible tripeptides
were generated coresponding to 133 different combinations and again the
presence and fre-
quency of each tripeptide among the 658 reactive (oligo)peptide sequences were
listed. All
possible combinations of the listed dipeptides and tripeptides were then
generated including
those containing 1, 2, 3 or 4 residues inserted bewteen the dipeptides and
tripeptides, these
residues s elected a mong the 13 p ossible r esidue t ypes. T his p rocedure g
enerated a I ist o f
different peptide combinations of 5 to 9 amino acids each containing at least
one dipeptide and
at lest one tripeptide from the initial listings as well as 0 to 4 residues in
between. The
frequency of each peptide combination among the 658 reactive (oligo)peptide
sequences were
then ranked and relevant epitope patterns were selected by a procedure where
reactive
49
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ranked and relevant epitope patterns were selected by a procedure where
reactive peptides
covered by the most frequent combination were first selected and separated
from the group of
the 658 reactive peptides. Then reactive peptides covered by the second most
frequent com-
bination were selected and separated from the remaining group. Then reactive
peptides cov-
ered by the third most frequent combination were selected and separated from
the remaining
group. This procedure was repeated until combinations covering all 658
reactive peptides are
found. This way it was found that 357 combinations (epitope patterns) were
found to cover all
the 658 reactive peptides.
Example 2: Predicting epitopes
The Der p 1 model was built using the following three-dimensional structures
as tem-
plates:
PDB entry Protein
9PAP Papaya papain
2ACT Kiwi actinidin
1 PPO Papaya omega protease
The sequences of the three templates and mature Der p 1 were aligned using
ClustalW
1.7 (as described in Higgins et al., Methods Enzymol., vol. 266, pp 383-402,
1996, and
Thompson, et al., Nucleic Acids Research, vol. 22, pp 4673-4680, 1994. The
alignment is
shown below. As the first 9 residues of mature Der p 1 have no corresponding
residues in any
of the templates, reliable modelling of these residues is not possible, and
hence, the 9 N-
terminal residues are not included in the Der p 1 model.
The "Modeler 5.0" program (Accelrys Software, San Diego, CA, USA) was used to
build
the three-dimensional model of Der p 1. "Modeler 5.0" was started from the
"Insightll" molecu-
lar modelling software (Accelrys Software, San Diego, CA, USA) using the
following parame-
ters: Number of models: 1, Optimize level: None, More options: Yes, Optimize
loop: Yes,
Number of loop models: 2, Loop optimite level: Low, Build hydrogens: None.
Using "Insightll", hydrogens were added and CHARMm potentials and partial
charges
assigned to the model. Using "CHARMm" (Accelrys Software, San Diego, CA, USA),
100 steps
of ABNR mimimization were applied to relax the model.
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Sequence alignment:
>P1;P 1PP0
structureX:P 1PP0:1 . :216 . :unknown:unknown:-1.00:-1.00
---------LPENVDWRKKGAVTPVRHQGSCGSCWAFSAVATVEGINKIRTGKLVELSEQELVDCER--RSHGCK
GGYPPYALEWAKNG-IHLRSKYPYKAKQGTCRAKQVGGPIVKTSGVGRVQPNNEGNLLNAIAKQPVSWVES--
-KGRPFQLYKGGIFEGPCG--TKVDHAVTAVGYGKSGGKGYILIKNSWGTAWGEKGYIRIKRAPGNSPGVCGLYK
SSWPTKN*
>P1;P 2ACT
structureX:P 2ACT:1 . :218 . :unknown:unknown:-1.00:-1.00
---------LPSYVDWRSAGAWDIKSQGECGGCWAFSAIATVEGINKITSGSLISLSEQELIDCGRTQNTRGCD
GGYITDGFQFIINDGGINTEENYPYTAQDGDCDVALQDQKWTIDTYENVPYNNEWALQTAVTYQPVSVALDA--
-AGDAFKQYASGIFTGPCG--TAVDHAIVIVGYGTEGGVDWIVKNSWDTTWGEEGYMRILRNVGG-AGTCGIAT
MPSYPVKY*
>P1;P 9PAP
structureX:P 9PAP:1 . :212 . :unknown:unknown:-1.00:-1.00
---------IPEYVDWRQKGAVTPVKNQGSCGSCWAFSAWTIEGIIKIRTGNLNQYSEQELLDCDR--RSYGCN
GGYPWSALQLVAQYG-IHYRNTYPYEGVQRYCRSREKGPYAAKTDGVRQVQPYNQGALLYSIANQPVSWLQA--
-AGKDFQLYRGGIFVGPCG--NKVDHAVAAVGYGPN----YILIKNSWGTGWGENGYIRIKRGTGNSYGVCGLYT
SSFYPVKN*
>P1;DER P 1 MATURE
sequence:DER P 1 MATURE:1 . :222 . :unknown:unknown:-1.00:-1.00
TNACSINGNAPAEIDLRQMRTVTPIRMQGGCGSCWAFSGVAATESAYLAYRNQSLDLAEQELVDCAS---QHGCH
3O GDTIPRGIEYIQHNG-WQESWRWAREQSCR--RPNAQRFGISNYCQIYPPNVNKIREALAQTHSAIAVIIGI
KDLDAFRHYDGRTIIQRDNGYQPNYHAVNIVGYSNAQGVDWIVRNSWDTNWGDNGYGYFAANIDLMMIEEYPYV
VIL-_-__*
Surface accesibility was measured for each amino amino acid in SEQ ID N0:1
using
the DSSP program (see W.l<absch and C.Sander, Biopolymers 22 (1983) 2577-2637)
in per-
cent of a standard value for that amino acid. The standard values generated
according to es-
tablished methods by analysing average surface accesibility of an amino acid
in a 20-mer ho-
mogeneous peptide in helix formation using the DSSP program. For each of the
20 different
residues the average surface accessibility was as follows:
ResidueAccessiblity
A 62
C 92
D 69
E 156
F 123
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G 50
H 130
I 84
K 174
L 97
M 103
N 85
P 67
Q 127
R 211
S 64
T 80
V 81
W 126
Y 104
Epitopes were predicted by a computer program on a 3-dimensional model of Der
p 1
(SEQ ID N0:1) by using the epitope patterns found in example 1 as follows:
(1 ) For all amino acids in SEQ ID N0:1 it was examined if (a) the amino acid
type match
the first amino acid of an epitope patterns and (b) the solvent surface
accessibility
greater than or equal to a predefined value, e.g., 20 %. Those amino acid
satisfying
1 (a) and 1 (b) are selected.
(2) For all amino acids within a distance of 10A from the amino acids selected
in step 1 it is
examined if (a) the amino acid type matches the second amino acid of the
pattern and
(b) the surface accessibility greater than or equal to the predefined value,
e.g., 20%.
Those amino acid satisfying 2(a) and 2(b) are selected
(3) For all amino acids within a distance 10A from the amino acids selected in
step 2 it is
examined if (a) the amino acid type matches the third amino acid of the
pattern and (b)
the surface accessibility greater than or equal to the predefined value, e.g.,
20%. Those
amino acid satisfying 3(a) and 3(b) are selected.
(4) Repeating step 3 for all amino acids in the epitope pattern
Further, a limit of 25 A was set as the maximum distance between any two
epitope residues.
This procedure was carried out for all 357 epitope patterns for each of the
following settings for
surface accessibility cutoff: 30, 40, 50, 60, 70 and 80%. Epitope patterns
finding a match on
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the 3 dimensional structure of SEQ ID NO: 1 according this procedure are
predicted as epi-
topes.
Finally, for each of the seven settings for solvent accessibility, a table of
all Der p 1 amino ac-
s ids was created, in which each amino acid residue was given a score by
adding up the number
of times it appeared in one of the epitopes (at that solvent setting). This
score will be an indi-
cation of the likelyhood that modification (substitution, insertion, deletion,
glycosylation or
chemical conjugation) of that amino acid will, result in a variant of lower
antigenicity. All amino
acids of the protein can then be ranked according to this score and those with
highest scores
1 o can be selected for mutagenesis.
The 10% highest scoring amino acids (i.e. the 22 highest scoring ones), at
each solvent
accessibility setting, are shown in the table below (however for solvent
accessibility settings of
70 and 80%, less than 22 residues scored at all, so in those two cases all
residues that scored
are listed). Cysteines are omitted, as they are often involved in maintaining
three-dimensional
15 structure.
Minimum SolventTop scoring 10% of amino acids
Accessibility
20% G32, A46, Y47, L55, D64, A66, S67, G73, T75, 180,
Q84, N86, G87, A98,
8105, Q 109, 8110, F 111, 6112, 1113
30% G30, G32, D64, A66, S67, Q84, N86, G87, Y93, A98,
R99, Q101, 8105,
P 106, F 111, G 112, I 113, I 158, A205
40% G29, G30, G 32, L 55, A 66, S 67, Q84, S 92, Y 93,
Y 96, A 98, R 99, E 100,
Q101, 8104, Q109, F111, 1113, 1144, K145, D146, N163
50% G29, G30, G32, S67, R99, E100, 1144, K145, D146, D148,
8151, 1159,
Q 160, 8161, D 162, N 163, 6164, Y165, Q 166, A180,
V 183, D 184
60% A10, A12, E 13, A132, 1144, K145, D 146, D 148, 8151,
1159, Q 160, 8161,
D 162, N 163, 6164, Y165, Q 166, N 179, A180, 6182,
D 184, 1208
70% A10, A12, 1144, K145, D146, D148, 1159, 8161, 6164,
Y165, Q166, D184,
1208
80% D146, D148, 6164, Y165, Q166
From this procedure it was found that residues A10, A12, E13, G29, G30, G32,
A46, Y47,
L55, D64, A66, S67, G73, T75, 180, Q84, N86, G87, S92, Y93, Y96, A98, R99,
E100,
20 Q101, 8104, 8105, P106, Q109, 8110, F111, 6112, 1113, A132, 1144, K145,
D146, D148,
8151, I 158, I 159, Q 160, 8161, D 162, N 163, G 164, Y165, Q 166, N 179,
A180, G 182, V 183,
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D184, A205, 1208 of SEQ ID NO:1, each belonged to the top 10% highest ranking
residues
at at least one solvent accesibility setting.
Residues A10, A12, G30, G32, A46, Y47, L55, D64, A66, S67, G87, S92, A98, R99,
E100,
Q 101, 8105, 8110, F 111, 6112, 1113, 1144, K145, D 146, D 148, 8151, 1159, Q
160, 8161,
D162, N163, 6164, Y165, Q166 of SEQ ID N0:1, each belonged to the top 5%
highest
ranking residues at at least one solvent accesibility setting.
By studying the positions of the top 10 % scoring amino acids on the three-
dimensional
1o model of Der p 1, it is possible to define 5 epitope containing regions on
the molecule. The
50% highest scoring amino acids within each region are then selected as best
candidates
for mutation: Residues A10, A12, G32, L55, A66, S67, G87, A98, R99, F111,
6112, 1113,
1144, D146, D148, 1159, 8161, 6164, Q166, A180, D184, A205 and 1208 of SEQ ID
N0:1
Example 3: Aligning group 1 mite polypeptides
The sequences of five different native group 1 mite prepro-polypeptides were
aligned:
-90 -80 -70 -60 -50 -40
__+____+__-_+____+___-+____+____+____+____+____+____+____+__
Eur m1 MKIILAIASLLVLSAVYARPASIKTFEEFKKAFNKTYATPEKEEVARKNFLESLKYVESN
Der f1 MKFVLAIASLLVLSTVYARPASIKTFEEFKKAFNKNYATVEEEEVARKNFLESLKYVEAN
Der p1 MKIVLAIASLLALSAVYARPSSIKTFEEYKKAFNKSYATFEDEEAARKNFLESVKYVQSN
Blo t1 ________-________-_________-_______--_______________________
Der ml ___-________-___________________-_________________-_________
-30 -20 -10 0 10 20
__+____+____+____+____+____+____+____+__-_+_____+____+___-+_
Eur m1 KGAINHLSDLSLDEFKNQFLMNANAFEQLKTQFDLNAETYACSINSVSLPSELDLRSLRT
Der_fl KGAINHLSDLSLDEFKNRYLMSAEAFEQLKTQFDLNAETSACRINSVNVPSELDLRSLRT
Der~l GGAINHLSDLSLDEFKNRFLMSAEAFEHLKTQFDLNAETNACSIN-GNAPAEIDLRQMRT
Blo_t1 ____________-_____________________________-_____IPANFDWRQKTH
Der m1 ----------------_-------_-------------TQACRINSGNVPSELDLRSLRT
30 40 50 60 70
___+____+____+____+____+____+____+____+____+____________+___
Eur ml VTPIRMQGGCGSCWAFSGVASTESAYLAYRNMSLDLAEQELVDCASQN--------GCHG
Der_fl VTPIRMQGGCGSCWAFSGVAATESAYLAYRNTSLDLSEQELVDCASQH--------GCHG
Der p1 VTPIRMQGGCGSCWAFSGVAATESAYLAYRNQSLDLAEQELVDCASQH--------GCHG
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Blo-tl VNPIRNQGGCGSCWAFAASSVAETLYAIHRHQNIILSEQELLDCTYHLYDPTYKCHGCQS
Der m1 VTPIRMQG-______________________________________-____________
80 90 100 110 120 130
_+____+____+____+____+____+____+_____+____+____+____+____+-_
Eur m1 DTIPRGIEYIQQNGVVQEHYYPYVAREQSCHR-PNAQRYGLKNYCQISPPDSNKIRQALT
Der f1 DTIPRGIEYIQQNGWEERSYPYVAREQRCRR-PNSQHYGISNYCQIYPPDVKQIREALT
Der p1 DTIPRGIEYIQHNGVVQESYYRYVAREQSCRR-PNAQRFGISNYCQIYPPNVNKIREALA
Blo-tl GMSPEAFKYNatQKGLLEESHYPYKMKLNQCQANARGTRYHVSSYNSLRYRAGDQEIQAAI
Der ml ____________________________________________________________
140 150 160 170 180 190
__+____+____+__-_+____+____+____+____+____+_-__+____+____+__
Eur ml QTHTAVAVIIGIKDLNAFRHYDGRTIMQHDNGYQPNYHAVNIVGYGNTQGVDYWIVRNSW
Der f1 QTHTAIAVIIGIKDLRAFQHYDGRTIIQHDNGYQPNYHAVNIVGYGSTQGDDYWIVRNSW
Der p1 QTHSAIAVIIGIKDLDAFRHYDGRTIIQRDNGYQPNYHAVNIVGYSNAQGVDYWIVRNSW
Blo-tl MNHGPWIYIHGTEA-HFRNLRKGILRGAGYNDAQIDHAVVLVGWGTQNGIDYWIVRTSW
Der ml _____________________-__________________-_____-_____________
200 210 220
__+____+____+____+____+____+__
Eur ml DTTWGDNGYGYFAANINLMMIEQYPYVVML
Der -fl DTTWGDSGYGYFQAGNNL1~IIEQYPYVVIM
Der p1 DTNWGDNGYGYFAANIDLNa2IEEYPYVVIL
Blo t1 GTQWGDAGYGFVERHHNSLGINNYPIYASL
Der ml ______________________________
Der p 1 holds an 18 amino acids signal peptide and an 80 amino acids
propeptide while the
mature Der p 1 is a 222 amino acid molecoule. In the alignement a gap has been
made in po-
sition 8 of Der p 1 because it lacks an amino acid here compared to other
group 1 mite poly-
peptides. Similar descriptions may be made for Eur m1, while for Der f 1 only
the mature poly-
peptide is shown and for Der m 1 only a fraction of the sequence has been
identified.
The alignment confirms high homology among group 1 mite polypeptides and excep
tionally high homology or conservatism in the amino acids identified as
involved in epitopes.
Thus it seems safe to say that the epitopes and thus mutations suggested for
Der p 1 are also
suitable for the remaining members of group 1 mite polypeptides.
Example 4: Construction and Expression of Enzyme Variants
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Der p 1 variants of the invention comprising specific substitutions were made
by cloning
of DNA fragments (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd
Ed., Cold
Spring Harbor, 1989) produced by PCR with oligos containing the desired
mutations.
Recombinant Der p 1 and all variants were expressed with the Der p 1
propeptide on
and they all had the mutation S54N which disrupts the only N-glycosylation
motif within the
mature sequence.
The template plasmid DNA may be pSteD212, or an analogue of this containing
Der p
1 or a variant of Der p 1. Mutations were introduced by oligo directed
mutagenesis to the con-
struction of variants. The Der p 1 plasmid constructs were transformed into S.
cerevisiae,
strain JG169, as described by Becker and Guarente (1991, Methods Enzymology,
194: 182-
187).
The Cystein protease or variants hereof of the present invention were located
in vector
pSteD212, which is derived from yeast expression vector pYES 2.0 (Kofod et al.
1994 J. Biol.
Chem. 269: 29182-29189 and Christgau et al. 1994, Biochem. Mol. Biol. Int. 33:
917-925).
This plasmid replicated both in E. coli and in S. cerevisiae. In S. cerevisiae
Der p 1 or
variants hereof according to the invention were expressed from this plasmid.
Example 5: Fermentation
Fermentations for the production of Der p 1 enzyme/Der p 1 variants were
performed at
degree C on a rotary shaking table (250 r.p.m.) in 500 ml baffled Erlenmeyer
flasks contain-
ing 100 ml SC medium for 5 days.
Consequently, in order to make e.g. a 2 litre broth 20 Erlenmeyer flasks were
fer-
mented simultaneously.
SC Medium (per litre):
Yeast Nitrogen Base without amino acids 7.5 g
Succinic acid 11.3 g
Casamino acid without vitamine 5.6 g
Tryptophan 0.1 g
Add H20. Autoclave and cool before adding glucose and L-threonin to a final
concentration of
4 % and 0.02 %, respectively.
For agar plates, 20 g bactoagar was added to the medium before autoclave.
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Example 6: Screening for Der p 1 variants
For screening of yeast transformants expressing Der p 1 or Der p 1 variants,
the trans
formation solution was plated on SC-agar plates for colony formation at 30
degree C, 3 days.
Colonies were inoculated in 96 micro-well plates, each well containing 200
microL SC medium.
The plates were fermented at 30 degree C, 250 r.p.m. for 5 days.
50 microL culture broth was diluted 1:1 in 0.15 M Phosphate Buffered Saline
(PBS) be-
fore OD450 measurement in sandwich ELISA. Culture broth of yeast transformed
with a plas-
mid without the Der p 1 gene was used as background with an average OD450 of
0.55. Vari-
ants tested in sandwich ELISA with OD450 > 0.55 were sequenced and approved as
correctly
folded and yeast expressed Der p 1 variants.
A p art o f the variants w ere c oncentration d etermined d irectly i n c
ulture b roth b y t he
sandwich ELISA technique with natural Der p 1 as a standard.
Der p 1 variants identified and determined by both OD450>0.55 and in a
quantitative
sandwich ELISA (concentrations given as microg/mL) are shown in table below.
Variant
no. Mutations concentration
DP003 S54N 11
DP009 G32E, S54N, A66V 0,2
DP010 G32D, S54N, A66V 2,9
DP017 S54N, G87V 0,016
DP018 S54N, G87E, 1113E 0,032
DP019 S54N, 1113V 7,05
DP020 S54N, G87E, 1113V 0,032
DP021 S54N, 1113D 0,032
DP024 S54N, T75D 5,4
DP025 S54N, T75A 8
DP026 S54N, T75V 7,3
DP033 S54N, D184A 1,62
DP034 S54N, D184A, A205D 0,027
DP035 S54N, A205D 0,048
DP049 A10Y, A12Y, S54N 0,81
DP050 A10N, A12N, S54N 0,027
DP054 S54N, 1159V 0,97
DP065 S54N, R99G 3,9
DP067 S54N, 1144L 0,1
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DP068 S54N, R161S 0,1
DP070 S54N, F1111 0,6
DP071 S54N, G87V, G112D 1,5
Variant
no. Mutations OD450>0.55
DP073 G32V, S54N, A66V, S67C, A98G 1,20
DP074 G32D, S54N, A98V, 1144T 1,01
DP075 S54N, D64N, A66D 0,66
DP077 A66D, R99G 0,72
DP078 G32V, S54N, A66G, A98V, R99D 1,15
DP079 S54N, S67H, A98V 0,69
DP080 S54N, 1144L, D148Y 0,64
DP081 S54N, 1144L, D148N, R161S, G164D1,10
S54N, D148N, 1159L, R161G, G164D,
DP082 Q166L 0,62
DP083 S54N, D148Y, R161S, G164V 0,62
DP084 S54N, 1144V, D148Y, R161G 1,19
DP085 S54N, D148N, R161S, G164V 1,53
DP086 S54N, D148H, 1159F, R161S, Q166R1,16
DP087 S54N, D148H 0,66
DP088 S54N, A180V 1,12
DP089 S54N, D184H 1,27
DP090 S54N, A180G 1,30
DP091 S54N, A180V, D184H 0,65
DP092 S54N, A180G, D184N 0,67
DP094 S54N, L55V, F111W, G112D 1,15
DP095 S54N, G112V, 1113L 1,24
DP096 S54N, F1111, G112D 1,36
Example 7: Purification method for Pro form and mature form of Der P1 antigen
Assay for detection of Der P1 and Pro-Der P1
F
Qualitative ELISA (Enzyme linked immunosorbent assay) for detection of Der P1
and
Pro-Derp 1.
Ployclonal antibodies were raised in Rabbits against Native Der P1 bought from
Indoor
technologies. The polyconal antibodies were purified by ammonium sulphate
precipitation and
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on Protein a column as described in literature and finally dialyzed against 50
mM Borate pH 8
buffer.
The purified antibodies against Der P1 were labelled with Biotin using NHS-
Biotin as
described in Product sheet described by Pierce Chemicals 3747 N.Meridian Rd.PO
Box117.
Rockford, 1161105 USA, and the labelled antibodies were used as detecting
antibodies.
Method for fast qualitative detection of Der P1 or Pro Der P1 was as follows.
Immunosrop microtiter plates were bought from NUNC and microtiter wells were
coated
with 100 microlitres of 10 microgram per ml unlabelled polyclonal antibodies
against Der P1 for
overnight at 4 degree centigrade. The microtiter wells were then washed with
PBS Tween
buffer as described in literature. Microtiter wells were then saturated with
200 microlitres of
PBS buffer containing 10 milligrams per millilitres BSA and 0.05 % Tween 20
and incubated for
30 minutes at room temperature.
Microtiter wells were then washed thrice with PBS buffer containing 0.05 %
Tween 20.
Microtiter wells were then coated with 100 microlitres fractions containing
Der P1 or
Pro-Der P1 and incubated for 20 minutes with gentle shaking. Microtiter wells
were then
2o washed thrice with PBS buffer containing 0.05 % Tween 20. Microtiter wells
were then coated
with 100 microlitres of biotin labelled polyclonal antibodies around 1
microgram per millilitres
diluted in PBS buffer with 0.05 % Tween 20 and incubated for 20 minutes at
room temperature
with gentle shaking.
Microtiter wells were then washed thrice with PBS buffer and coated with 100
micro-
litres of properly diluted Immunopure Avidin Horse radish peroxidase conjugate
which was
purchased from Pierce chemicals. After 20 minutes incubation wait room
temperature the
wells were then washed with PBS buffer containing 0.05 % Tween 20.
One hundred microlitres of Horse Radish peroxidase substrate TMB One purchased
form Kem EN Tec was then added to the microtiter wells and incubated for few
minutes and
reaction was stopped by adding Phosphoric acid as described by KEM EN TEC. For
blank ex-
act same procedure was carried out but no antigen was included in the wells.
This method can be used as qualitative assay for detection of Der P1 or Pro
Der P1.
Method for Purification of Der P1 and Pro Der P1.
One litre fermentation supernatant of Pro form of Der P1 antigen
(Dermatophagoides
pteronyssinus) expressed in Yeast or A.oryzae was centrifuged and precipitate
containing cell
debris was discarded. The cell supernatants were then sterile filtered under
pressure through
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0.22p sterile filter Seitz-EKS obtained from Pall Corporation (Pall Seitz
Schenk Filter system
GmbH Pianiger Str.137D Cad Kreuznach Germany).
Sterile filtered cell supernatant containing the desired protein was then
concentrated
using Ultra filtration technique using 10 kDa cut off membrane purchased from
from Millipore
Corporation, Bedford. MA 01730 USA: The small molecules under 10 kDa were then
removed
by dig filtration using 50 mM Borate pH 8 as buffer.
To the concentrated and dig filtrated supernatant containing the desired
protein solid
ammonium sulphate was gradually added under gentle stirring to a final
concentration of one
M ammonium sulphate and pH was adjusted to 8.
Hydrophobic interaction chromatography was carried out on 50 ml XK26 column
pur-
chased from Amersham - Pharmacia which was packed with Toyopearl Phenyl -650
matrix
purchased from TOSOH Bioscience GmbH Zettacchring 6, 70567 Stuttgart, Germany.
The column was washed then equilibrated with 1 M ammonium sulphate dissolved
in 50
mM Borate pH 8.
The concentrated fermentation supernatant was then applied on the column with
a flow
of 20 mL per minute. Unbound material was then washed out using 1 M ammonium
sulphate
dissolved in the borate pH 8 buffer (Buffer A). When all the unbound material
was washed out
from the column which was monitored using UV detector attached to fraction
collector from
Amesham Pharmacia.
Bound proteins were then eluted with buffer B which contained 50 mM Borate pH
8
without any other salt and 10 ml fractions were collected. Fractions contain
desired protein
were checked by SDS-PAGE. Fractions containing Protein with molecular weights
between 33
kDa and 22 kDa and found immunoreactive in the qualitative as described above
were then
pooled and further purified on anion exchange chromatography.
Anion exchange chromatography of Der P1 and Pro Der P1
Anion exchanger fast flow Q sepharose 50 ml column XK26 pre-packed by Amersham
Pharmacia was washed and equilibrated with 50 mM Borate pH 8 buffer.
Pool containing Der P1 and or Pro Der P1 from Hydrophobic chromatography was
then
diluted to adjust ionic strength below 4 mSi and pH was adjusted to 8. The
diluted pool was
then applied on the Fast flow Q sepharose column with flow rate 20 ml per
minute and un-
bound material was washed with the 50 mM Borate buffer pH 8 as buffer A.
Bound proteins were then eluted with linear gradient using buffer B containing
50 mM
Borate pH 8 with 1 M salt as Sodium chloride. Total buffer used was 20 column
volumes
All the fractions were then analysed by SDS-PAGE and qualitative ELISA assay.
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Proteins with molecular weight around 30 kDa were then pooled as Pro-Der P1
and
mature Der P1 due to slight processing was observed as 20 kDa Protein. The
purified proteins
were then analysed for N-terminal after SDS-PAGE and blotting on PVDF membrane
by Using
applied Bio system equipment.
Example 8: In vivo assessment of allergenicity of an enzyme variant (MINT
assay)
Mouse intranasal (MINT) model (Robinson et al., Fund. Appl. Toxicol. Vol. 34,
pp. 15-
24, 1996) can be used to verify allergenicity of group 1 mite polypeptide
variants.
Mice are dosed intranasally with the group 1 mite polypeptide variant on the
first and
third day of the experiment and from thereon on a weekly basis for a period of
6 weeks. Blood
samples are taken 15, 31 and 45 days after the start of the study, and the
serum can subse-
quently be analysed for IgE levels.
Measurement of the concentration of specific IaE in mouse serum by ELISA:
The relative concentrations of specific IgE antibodies in serum samples from
mice are
measured by a three layer sandwich ELISA according to the following procedure:
1 ) The ELISA-plate (Nunc Maxisorp) is coated with 100 microlitre/well rat
anti-mouse IgE
Heavy chain (HD-212-85-IgE3 diluted 1:100 in 0.05 M Carbonate buffer pH 9.6).
Incu-
bated over night at 4°C.
2) The wells are emptied and blocked with 200 microlitre/well 2% skim milk in
0.15 M PBS
buffer pH 7.5 for 1 hour at 4°C. The plates are washed as before.
3) The plates are incubated with dilutions of mouse sera (100 microUwell),
starting from an
8-fold dilution and 2-fold dilutions hereof in 0.15 M PBS buffer with 0.5%
skim milk and
0.05% Tween20. Appropriate dilutions of positive and negative control serum
samples
plus buffer controls are included. Incubated for 1 hour at room temperature.
Gently
shaken. The plates are washed 3 times in 0.15 M PBS buffer with 0.05% Tween20.
4) 100 microlitrelwell of group 1 mite polypeptide variant diluted to 1
microgram protein/ml in
0.15 M PBS buffer with 0.5% skim milk and 0.05% Tween20 is added to the
plates. The
plates are incubated for 1 hour at 4°C. The plates are washed as
before.
5) Specific polyclonal anti-group 1 mite polypeptide variant antiserum serum
(plg) for detect-
ing bound antigen is diluted in 0.15 M PBS buffer with 0.15% skim milk and
0.05%
Tween20. 100 microUwell and incubated for 1 hour at 4°C. The plates are
washed as be-
fore.
6) 100 microlitre/well pig anti-rabbit Ig conjugated with peroxidase diluted
1:1000 in 0.15
M PBS buffer with 0.5% skim milk and 0.05% Tween20 is added to the plates.
Incu-
bated for 1 hour at 4°C. The plates are washed as before.
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7) 100 p1/ well TMB (Plus (Ready-to-go substrate; Kem-En-Tec, Cat. No.: 4390A)
is
added, and the reaction is allowed to run for 10 min.
8) The reaction is stopped by adding 100 microlitre/well 1 M H2S04,
9) The plates are read at 450 nm with 620 nm as reference.
The dose response curves are graphed, and fitted to a sigmoid curve using non-
linear re-
gression, and the EC50 is calculated for the group 1 mite polypeptide variant.
Measurement of the concentration of specific IaG1 in mouse serum by ELISA
The relative concentrations of specific IgG1 antibodies in serum samples from
mice are
measured by a three layer sandwich ELISA according to the following procedure:
1 ) The ELISA-plate (Nunc Maxisorp) is coated with 100 microlitrelwell of
group 1 mite poly-
peptide variant diluted in PBS to 10 microg/ml. Incubated over night at
4°C.
2) The wells are emptied and blocked with 200 microlitrelwell 2% skim milk in
0.15 M PBS
buffer pH 7.5 for 1 hour at 4°C. The plates are washed 3 times with
0.15 M PBS buffer
with 0.05% Tween20.
3) The plates are incubated with dilutions of mouse sera (100 microUwell),
starting from an
20-fold dilution and 3-fold dilutions hereof in 0.15 M PBS buffer with 0.5%
skim milk and
0.05% Tween20. Appropriate dilutions of positive and negative control serum
samples
plus buffer controls are included. Incubated for 1 hour at room temperature.
Gently
2o shaken. The plates were washed as before.
4) 100 microlitre/well biotinylated Rat-anti-mouse IgG~ (Serotec, Cat. No.:
MCA 336B), di-
luted 2000x in 0.15 M PBS buffer with 0.5% skim milk and 0.05% Tween20 is
added to
the plates. The plates are incubated for 1 hour at 4°C. The plates are
washed as before.
5) 100 microlitre/well Horseradish Peroxidase-conjugated Streptavidin
(Kierkegaard &
Perry, Cat. No.: 14-30-00), diluted 1000x in 0.15 M PBS buffer with 0.15% skim
milk and
0.05% Tween20. Incubated for 1 hour at 4°C. The plates are washed as
before.
6) 100 p1/ well TMB (Plus (Ready-to-go substrate; Kem-En-Tec, Cat. No.: 4390A)
is
added, and the reaction is allowed to run for 10 min.
7) The reaction is stopped by adding 100 microlitre/well 1 M HzSO4,
8) The plates are read at 450 nm with 620 nm as reference.
The dose response curves are plotted and fitted to a sigmoid curve using non-
linear re-
gression, and the EC50 is calculated for the group 1 mite polypeptide variant.
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Example 9: In vitro assessment of IgE-antigenicity of an enzyme variant
Reduced IgE binding was verified in vitro by direct or competitive ELISA and
Basophil
histamine release. Group 1 mite polypeptide variants with reduced IgE-
antigenicity can then be
tested further in vivo, by skin prick testing.
Direct ELISA:
Immunoplates (Nunc Maxisorp; Nunc-Nalgene) were coated overnight at 4°C
with a
suitable dose, or dose-range, of group 1 mite polypeptide allergen or with
recombinant group 1
mite polypeptide variant allergen. T he plates were then washed thoroughly
with Phosphate
Buffered Saline (PBS) containing 0,05% Tween 20 (PBST), and remaining binding
sites were
blocked with PBS containing 2% Skim Milk Powder (SMP). Sera from patients
allergic to dust
mites, (dust mite allergy was diagnosed on the basis of case history, skin
prick testing and de-
termination of specific IgE to dust mite extracts (CAP-RAST measurements)),
were then di-
luted 1/4 in PBST and added to the plates and incubated at room temperature
(RT) for 1 hour
or overnight at 4degree C. Following a thorough wash with PBST, the allergen-
IgE complexes
were detected, by incubation with a rabbit anti-human I gE antibody
(DakoCytomation), and
swine anti-rabbit Ig coupled to horseradish peroxidase. The enzymatic activity
was measured
by adding TMB from Kem-En-Tec, and the reaction was stopped by adding an equal
volume of
0.2 M H2S04, and quantitaing colour development by measuring optical density
at 450 nm
(0D450) in an ELISA reader. 0D450 reflect IgE binding to the allergen.
First, suitable donors for competitive ELISA were identified as follows: nDer
p 1-specific
IgE-binding in serum isolated from 23 patients allergic to dust mite allergens
were analysed in
a dose response curve to nDer p 1 in a direct ELISA. OD450 values obtaining a
coating con-
centration of 5 00 n g/well a Ilergen were d etermined, a nd s era that d id
not reach a n OD450
equal or higher than 0.5 were excluded from further analysis in the
competitive ELISA. Sera
from donor 1, 3, 6, 7, 9, 13, 14, 22 and 23 were chosen for analysis in
competitive ELISA
Table 1. 0D450 titer obtaining a coating concentration of 500 ng/well group 1
mite wild type
polypeptide was determined in sera isolated from 23 patients allergic to dust
mite and are
shown in the table below.
nDer p 1 specific IgE
titer
Donor 1 0,9741
Donor 2 0,1815
Donor 3 1,7443
Donor 4 0,2672
Donor 5 0,2207
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Donor 6 0,8607
Donor 7 1,0819
Donor 8 0,2219
Donor 9 1,0168
Donor 10 0,3519
Donor 11 0,4384
Donor 12 0,2385
Donor 13 1,2593
Donor 14 2,3644
Donor 15 0,2345
Donor 16 0,3490
Donor 17 0,1829
Donor 18 0,1237
Donor 19 0,4820
Donor 20 0,1087
Donor 21 0,4195
Donor 22 1,3897
Donor 23 1,9637
nDer p 1-specific IgE-binding in serum isolated from 23 patients allergic to
dust mite al-
lergens were analysed in a dose response curve to nDer p 1 in a direct ELISA.
0D450 values
correlating to a coating concentration of 500 ng/well allergen was determined
and sera that did
not reach an OD450 equal or higher than 0.5 were excluded from further
analysis in the com-
petitive ELISA. Based on the data shown in Table x1, sera from donor 1, 3, 6,
7, 9, 13, 14, 22
and 23 were chosen for analysis of the individual variants by competitive
ELISA. Data from the
full dose-response curve for different variant concentrations confirmed this
selection (data not
1o shown).
Comaetetive ELISA:
Was carried out like direct ELISA, with two exeptions: the immunoplates were
coated
with a fixed concentration (500 ng/well) of wild type popypeptide, and the
diluted serum from
allergic patients was pre-incubated with a dose range of group 1 mite wild
type polypeptide,
recombinant group 1 mite polypeptide wild type or group 1 mite polypeptide
variant allergen.
When IgE binds to the polypeptide in solution, binding to the platebound wild
type polypeptide
is reduced, thus reducing the OD450. The reduced IgE binding was interpreted
using Graph
Pad Prism software: OD450 was plotted against the logarithm of dose of variant
allergen,
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thereby creating a sigmodal dose-response curve-fit. By using a model of four-
parameter logis-
tic (bottom, top, log EC50 and Hill slope) sigmoidal curve fit in Prism,
comparison of EC50
found by incubation with group 1 mite wild type polypeptide and variant
allergen was per-
formed. Differences in binding affinity are expressed as an X-fold increase or
decrease of the
amount of variant required obtaining a 50% inhibition of IgE binding to the
group 1 mite wild
type polypeptide.
Table 2: Dose-response curves in nDer p 1-specific IgE serum isolated from 9
dust mite aller-
gic patients were plotted and fitted to a sigmoid curve, and the EC50 was
calculated for the
group 1 mite polypeptide variants.
nDer rec- rec- DP DP DP DP DP DP DP
donorp1 proDerp1Derp1019 024 025 026 033 065 071
1 1 5 2 2 8 2 10 2 3 3
3 1 2 4 2 11 3 10 3 4 4
6 1 3 2 1 10 1 5 2 3 3
7 1 2 2 2 9 1 10 2 4 3
9 1 2 3 2 10 2 8 2 3 3
13 1 1 3 2 22 2 39 3 4 4
14 1 2 1 1 8 1 4 2 2 2
22 1 1 1 1 9 1 3 2 2 2
23 1 2 1 1 9 1 9 2 2
Table 3: Dose response curves in nDer p 1-specific IgE serum isolated from 6
patients with
dust-mite allergy were plotted and fitted to a sigmoid curve, and the EC50 was
calculated for
group 1 mite polypeptide variants.
nDer p 1 rec-proDerp1rec-Derp1DP070
Donor 1 1 1 2 46
Donor 3 1 2 2 43
Donor 7 1 1 2 33
Donor 9 1 1 2 26
Donor 14 1 2 2 23
Donor 23 1 1 2 37
The data disclosed in Table 2 and Table 3 show that the three variants DP024,
DP026, and
DP070 have a highly reduced IgE binding as compared to the group 1 mite
polypeptide. The
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mutations introduced in the DP024 variant lowers the affinity for specific
serum IgE by a factor
of about 8 to 22-fold, the mutations introduced in DP026 lowers the affinity
for specific serum
IgE by a factor of 3 to 39-fold, and the mutations introduced in the DP070
lowers the affinity for
specific serum IgE by a factor of about 23-46, compared to the response of
nDer p 1. The data
further indicates that the variants DP065 and DP071 have a reduced IgE binding
compared to
the response of nDer p 1.
Bas~ahil histamine release:
Histamine release from basophil leukocytes was performed as follows.
Heparinized
1 o blood (40 mL) was drawn from each dust-mite allergic patient, stored at
room temperature, and
used within 24 hours. Twenty-five microliters of heparinized blood was applied
to glass fibre
coated microtitre wells (Reference Laboratory, Copenhagen, Denmark) and
incubated with 25
microliters of a dose-range of wild type polypeptide, recombinant wild type,
variant allergen,
House Dust mite (HDM) extract or anti-IgE for 1 hour at 37degree C. All serial-
dilutions of al-
lergen were made in PIPES-buffer (Reference L aboratory, Denmark). T hereafter
the plates
were rinsed with water and interfering substances were removed. Finally,
histamine bound to
the microfibres was measured spectrophotofluometrically. The results are
interpreted using the
following formula:
of Allergen-induced histamine release = (histamine in allergen-stimulated
super-
natants - basal value) / (total histamine release - basal value) x 100,
where
basal value = spontaneous histamine release in supernatants without allergen
stimuli,
and
total histamine release = total histamine contents in blood sample measured
after treat-
ment with perchloric acid 2 %.
Specific histamine release greater than 10% was considered as positive.
The above procedure was applied on a number of group 1 mite polypeptide
variant al-
lergen. % Histamine release as a function of allergen concentration was
plotted (see figure 1 ),
and t he E C50 w as d etermined. V ariant a Ilergens w ith r educed b asophile
h istamine r elease
were selected based upon a shift of the EC50 to higher concentrations as
reflecting differences
in the induction of histamine release.
Basophile cells from 23 patients allergic to dust-mite and 3 negative donors
(negative
to histamine release on stimulation with house d ust-mite ( HDM) extract (ALK-
Abello)) were
analysed in a histamine release assay on stimulation with group 1 mite
polypeptide and group
1 mite polypeptide variants. Of the 23 dust mite allergic patients, only 14
patients were found
to induce histamine release in response to stimulation with nDer p 1,
demonstrating that ap-
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proximately 61 % o f t he p atients a Ilergic t o d ust-mite h ave n Der p 1-
specific I gE a ntibodies
(data not shown).
The data disclosed in Figure 1 shows the potency of rec-proper p 1, rec-Der p
1 and DP070
variants to induce histamine release in a human basophile cell assay from one
dust-mite aller-
gic patient. It is seen that the release curve of DP070 variant is clearly
shifted to the right com-
pared to the release curve of nDer p 1, rec-proper p 1 and rec-Der p 1. The
shift indicates that
the potency of DP070 variant to induce histamine release is reduced about a 22-
fold fold rela-
tive to nDer p 1. The shift of the DP070 release curve to the right was found
in all the 14 pa-
tients with nDer p 1-specific IgE-mediated responses with the corresponding
potency reduc-
tions ranging from 2 to 22-fold.
Basophile cells from the 9 remaining dust mite allergic patients did not
respond to stimulation
with concentrations of the group 1 mite polypeptide up to 1.67 pg/ml (data not
shown). How-
ever, at the highest concentration of group 1 mite polypeptide (20 Nglml),
histamine release
was observed from basophile cells from these patients. This induction of
histamine release in
high concentration of nDer p 1 may be due to low levels of impurities of
commercial nDer p 1
and thus, the presence of other dust mite allergens. No histamine release was
observed in ba
sophile cells from these 9 patients in response to stimulation with group 1
mite polypeptide
variants (data not shown).
Basophile cells from the 3 negative donors did not respond to stimulation with
group 1 mite
polypeptide or to group 1 mite polypeptide variants, demonstrating no
unspecific stimulation of
the crude extract.
The data disclosed in Figure 2 demonstrate the overall reduction in IgE
antigenicity as meas-
ured by histamine release assay. For each variant, in each of the nine
patients, the ratio of
EC50 value for the variant to the EC50 value of nDer p 1 was calculated. Thus,
for the nDer p
1 sample used as control, all donors show a normalized value of 1 (left
column). A control se-
ries of nDer p 1 samples were included and treated as a normal sample. The
result for this se-
ries is shown in the rightmost column, and demonstrates a relatively low
variability, considering
this is a rather sensitive biological response assay. The variants DP024 and
DP070 show av-
erage improvement factors of around 5 and around 6,7, respectively. Further,
the variants
DP071, DP065, DP033, and DP026 show improvents in IgE-based antigenicity, as
measured
by the increase in EC50 value, in most of the donors.
The use of basophil histamine release is described in Nolte H, Schiotz O, Skov
PS. A new
glass microfibre-based histamine analysis for allergy testing in children.
Results compared with
67
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conventional leukocyte histamine release assay, skin prick test, bronchial
provocation test and
RAST. Allergy. 1987 Ju1;42(5):366-73; and in: Winther L, Moseholm L, Reimert
CM, Stahl Skov
P, Kaergaard Poulsen L. Basophil histamine release, IgE, eosinophil counts,
ECP, and EPX
are related to the severity of symptoms in seasonal allergic rhinitis.
Allergy. 1999
May;54(5):436-45.
Skin prick testinc,~
Is carried out on patients allergic to group 1 mite polypeptides, and the
technique is
well known in the art, e.g., Kronquist et al., Clin. Exp. Allergy, 2000, vol.
30, pp. 670-676).
Briefly, 15 microL of solutions containing the recombinant wild type or
variant allergens
is placed on the patient's forearm. Thereafter, the skin is pricked with a
Prick-Lancette (ALK).
The test sites are placed 3 cm apart to avoid false-positive results. From an
initial solution of
recombinant allergens (e.g., 1 mg protein/mL), suitable serial dilutions
(e.g., from 100 pg to 0.1
microg/mLl) are made in sterile physiologic saline solution. These dilutions
are selected ac-
cording to the concentration allergens, which elicited significant histamine
release by sensi-
tized basophils. It has been shown that the thresholds of positivity for
histamine release tests
and intradermal reactions are in the same range; and it is assumed that the
sensitivity of prick
tests is 102 to 103 times lower than that of intradermal tests. A negative
control test is per-
formed with saline solution, and a positive control test is done with
histamine at e.g., 1 mg/mL.
The diameter of the weal is used as a measure of allergenic reactivity towards
that variant, and
this allows for comparison of the variant allergens to the parent or native
type allergen.
Example 10: Assessing retained ability to stimulate T cells
The lymphocyte fraction from heparinized blood from patients allergic to group
1 mite
polypeptides was purified by density gradient centrifugation on Lymphoprep
(Axix-Shield PoC,
Norway) and resuspended in AIM-V (Invitrogene) and plated at a cellular
density of 2.5 x 105
cells/well in a 96 sterile tissue-culture plate (Nunclon Delta). Serial
dilution of wild type group 1
mite polypeptide and group 1 mite polypeptide variant allergens were made up
in growth me-
dia and added to the cells, together with a media-only control. The plates
were then incubated
for 7 days at 37degree C, 5% C02, 100% humidity. At the end of the incubation,
T cell prolif-
eration was measured by the incorporation of 3[H]-thymidine. 20 hour prior to
harvest, 3[H]
thymidine (0.5 NCi) per well was added. The cells were harvested onto glass
fiber filters, and
3[H]-thymidine incorporation was measured in scintillate counter.
Proliferation was expressed
as mean counts per miute (cpm) of 3[H]-thymidine incorporation of triplicate
or duplicate wells.
The stimulation index (S1) was calculated as the quotient of the cpm obtained
by allergen
stimulation and the unstimulated control (media-only control).
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SI is shown for each donor and a selection of group 1 mite polypeptide
variants in table 4. Do-
nor 1 - 23 represents the 23 dust mite allergic patients, whereas donor 2.4-26
represents the 3
negative donors.
Table 4: Stimulation indexes for T cell proliferation in response to
stimulation with group 1 mite
polypeptide and group 1 mite polypeptide variants. Unless otherwise stated in
the table, analy-
sis of T cell proliferatian was carried out on stimulation with 1.0 Ng/ml
group 1 mite polypeptide
or group 1 mite polypeptide variants.
nDer Rec-properRec-Derp1DP015 DP019 DP024 DP025 DP026 ppp33
p 1
1 (0.64 (0.44
pg/ml) ug/ml)
Donor 8.9 4.0 5.0 3.9 6.6 1.9 2.8 2.8 2.7
1
Donor 3.0 1.5 1.6 0.7 n.da 0.7 0,2 1.8 1.4
2
Donor 7.1 5.3 5.6 3.7 4.8 0.7 1.0 7.8 4.8
3
Donor 10.4 5.3 6.7 1.1 n.d 2.1 0.5 3.9 1.5
4
Donor 27.8 1.8 43.8 1.0 18.2 9.7 1.7 26.5 4.0
5
Donor 1.3 3.4 1.5 2.9 0.6 0.6 0.2 2.7 5.5
6
Donor 41.3 19.5 19.8 2.7 n.d 5.4 1.0 7.0 7.6
7
Donor 1.8 0.9 0.6 0.5 0.7 0.5 0.2 0.5 0.5
8
Donor 13.7 5.2 9.0 1.1 8.7 1.9 0,1 9.4 2.7
9
Donor 35.9 25.8 25.2 3.4 30.7 15.3 1.7 13.2 48.3
Donor 4.3 1.3 8.8 0.9 11.1 1.9 0.7 2.3 1.2
11
Donor 12.6 8.1 8.5 1.3 n.d 9.8 2.3 14.1 5.2
12
Donor 30.9 41.1 34.0 1.6 n.d 8.1 1.0 33.5 14.3
13
Donor 42.7 9.0 13.3 5.3 14.9 5.9 0.7 9.4 7.8
14
Donor 6.6 5.9 6.1 1.3 9.9 5.1 1.5 4.0 2.8
Donor 7.0 3.1 6.1 1.7 n.d 1.6 0.3 6.0 3.4
16
Donor 3.2 2.4 3.8 6.6 5.1 2.9 0.6 3.1 6.1
17
Donor 15.8 3.1 4.5 3.0 n.d 1.6 0.5 3.5 3.2
18
Donor 12.2 8.6 33.7 9.8 29.2 9.1 2.1 23.7 6.3
19
Donor 2.6 1.3 1.6 1.2 n.d 0.4 n.d n.d 1.2
Donor 2.3 1.9 1.9 1.5 n.d 0.3 0.04 n.d 2.6
21
Donor 8.8 5.0 5.5 3.1 7.3 2.7 0.5 8.0 4.0
22
Donor 3.6 3.3 3.7 2.9 2.6 1.1 0.05 3.8 2.1
23
Donor 1.1 2.7 2.0 0.7 n.d 1.0 n.d n.d 1.7
24
Donor 1.0 1.9 1.5 1.6 1.4 0.7 0.2 1.6 1.2
Donor 3.1 6.7 22.5 0.8 5.4 3.3 0.3 6.7 3.1
26
n.d. not determined
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The data disclosed in the table above shows that group 1 mite polypeptide
variants were able
to induce proliferation in primary T cells from dust mite allergic patients.
This suggests that the
variants can initiate the cellular immune response necessary for antibody
production.
Example 11: Epitope Mapping based on human anti-Der p 1 antiserum
Preparation of human IgE beads with specificity for Der p 1 for targeting in
selection
experiments (anti-Der p 1 beads):
Rabbit anti-human IgE (anti-hlgE) antibodies were covalently linked to
commercially
available tosyl-activated paramagnetic beads. After inactivation of the
remaining linkage sites
and washing of the beads according to manufacturer's specification, the anti-
hlgE-beads were
incubated overnight at 4°C with pooled sera from patients sensitized to
Der p 1 (the sera were
3x diluted in dilution buffer (PBS at pH 6.6)). 200 microlitre beads were
washed 3x with 40m1
of washing buffer (PBS at pH6.6 plus 0.05% Tween20), then incubated with PBS
supple
mented with 2 % skim milk for an hour at room temperature and washed 3x as
before.
Selection of epitope mimics from commercially available phage-displayed
peptide li-
braries: Epitope mimicking peptides were isolated from commercially available
phage display
libraries of either 7mer, constrained 7mer or 12mer peptide libraries (New
England Biolabs).
The anti-Der p 1-beads were incubated with 2*10" library phages for 4h at room
temperature,
after which unbound phages were removed by extensive washing. To avoid the
enrichment of
peptides that were bound to either plain beads or to the anti-hlgE antibody, a
specific elution
procedure was implemented: After washing, beads were first incubated with PBS
supple-
mented with 0.5% skim milk for an hour at room temperature. After this
additional washing
step, phages were eluted from the beads by incubation with 25 microM purified
Der p 1 in PBS
supplemented with 0.5% skim milk, again lasting an hour at room temperature.
Only phages in
the supernatant of this elution were propagated further. Selected phages were
amplified ac-
cording to the guidelines of the library manufacturer (NEB user manual) after
a first round of
selection using ER2738 cells. After a second round, cells were infected and
spread out for iso-
lation of phages, which were subsequently tested for binding and sequenced.
Phage ELISA to test binding to serum IgE:
100 ng of anti-hlgE antibody in 100 microlitre coating buffer (50mM
sodiumcarbonate at
pH 9.0) were coated overnight at 4degree C on 96-well plates. Unoccupied
binding sites were
blocked for 2 hrs at room temperature with skimmed milk (2 (wt/vol) % in
washing buffer (PBS
at pH6.6 plus 0.05% Tween20). Human IgE are then selectively immobilized from
serum sam-
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pies that were diluted 50x in dilution buffer (PBS at pH 6.6) to 100
microlitre and incubated for
2 hr at room temperature. Phage preparations from isolated cells infected with
2"d round elu-
tion, which contained phages displaying putative epitope mimics, were sampled
in varying dilu-
tions for their binding. Bound phages were detected either with mouse anti M13-
phage anti-
s body-horseradish peroxidase (HRP) conjugates or with a mouse anti-pill
Antibody, Sheep anti-
mouse IgG antibody-HRP sandwich when cross-reactivity to the serum in absence
of any
phage preparation was detected.
71
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10317.ST25
SEQUENCE LISTING
<110> Soni, Nanna Kristensen
Friis, Esben Peter
Ludo, Erwin Roggen
<120> Group 1 mite polypeptide variants
<130> Nz 10317.000/dk
<160> 5
<170> Patentln version 3.2
<210> 1
<211> 320
<212> PRT
<213> Dermatophagoides pteronyssinus
<220>
<221> SIGNAL
<222> (1)..(18)
<220>
<221> PROPEP
<222> (19)..(98)
<220>
<221> mat_peptide
<222> (99)..(320)
<400> 1
Met Lys Ile Val Leu Ala Ile Ala Ser Leu Leu Ala Leu Ser Ala Val
-95 -90 -85
Tyr Ala Arg Pro Ser Ser Ile Lys Thr Phe Glu Glu Tyr Lys Lys Ala
-80 -75 -70
Phe Asn Lys Ser Tyr Ala Thr Phe Glu Asp Glu Glu Ala Ala Arg Lys
-65 -60 -55
Asn Phe Leu Glu Ser Val Lys Tyr Val Gln Ser Asn Gly Gly Ala Ile
-50 -45 -40 -35
Asn His Leu Ser Asp Leu Ser Leu Asp Glu Phe Lys Asn Arg Phe Leu
-30 -25 -20
Met Ser Ala Glu Ala Phe Glu His Leu Lys Thr Gln Phe Asp Leu Asn
-15 -10 -5
Ala Glu Thr Asn Ala Cys Ser Ile Asn Gly Asn Ala Pro Ala Glu Ile
-1 1 5 10
Asp Leu Arg Gln Met Arg Thr Val Thr Pro Ile Arg Met Gln Gly Gly
15 20 25 30
Cys Gly Ser Cys Trp Ala Phe Ser Gly Val Ala Ala Thr Glu Ser Ala
35 40 45
Page 1
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10317.ST25
Tyr Leu Ala Tyr Arg Asn Gln Ser Leu Asp Leu Ala Glu Gln Glu Leu
50 55 60
Val Asp Cys Ala Ser Gln His Gly Cys His Gly Asp Thr Ile Pro Arg
65 70 75
Gly Ile Glu Tyr Ile Gln His Asn Gly Val Val Gln Glu Ser Tyr Tyr
80 85 90
Arg Tyr Val Ala Arg Glu Gln Ser Cys Arg Arg Pro Asn Ala Gln Arg
95 100 105 110
Phe Gly Ile Ser Asn Tyr Cys Gln Ile Tyr Pro Pro Asn Val Asn Lys
115 120 125
Ile Arg Glu Ala Leu Ala Gln Thr His Ser Ala Ile Ala Val Ile Ile
130 135 140
Gly Ile Lys Asp Leu Asp Ala Phe Arg His Tyr Asp Gly Arg Thr Ile
145 150 155
Ile Gln Arg Asp Asn Gly Tyr Gln Pro Asn Tyr His Ala Val Asn Ile
160 165 170
Val Gly Tyr Ser Asn Ala Gln Gly Val Asp Tyr Trp Ile Val Arg Asn
175 180 185 190
Ser Trp Asp Thr Asn Trp Gly Asp Asn Gly Tyr Gly Tyr Phe Ala Ala
195 200 205
Asn Ile Asp Leu Met Met Ile Glu Glu Tyr Pro Tyr Val Val Ile Leu
210 215 220
<210> 2
<211> 321
<212> PRT
<213> Euroglyphus maynei
<220>
<221> PROPEP
<222> (1)..(98)
<223> Propeptide containing a signal peptide
<220>
<221> mat_peptide
<222> (99)..(321)
<400> 2
Met Lys Ile Ile Leu Ala Ile Ala Ser Leu Leu Val Leu Ser Ala Val
-95 -90 -85
Tyr Ala Arg Pro Ala Ser Ile Lys Thr Phe Glu Glu Phe Lys Lys Ala
Page 2
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10317.5T25
-80 -75 -70
Phe Asn Lys Thr Tyr Ala Thr Pro Glu Lys Glu Glu Val Ala Arg Lys
-65 -60 -55
Asn Phe Leu Glu Ser Leu Lys Tyr Val Glu Ser Asn Lys Gly Ala Ile
-50 -45 -40 -35
Asn His Leu Ser Asp Leu Ser Leu Asp Glu Phe Lys Asn Gln Phe Leu
-30 -25 -20
Met Asn Ala Asn Ala Phe Glu Gln Leu Lys Thr Gln Phe Asp Leu Asn
-15 -10 -5
Ala Glu Thr Tyr Ala Cys Ser Ile Asn Ser Val Ser Leu Pro Ser Glu
-1 1 5 10
Leu Asp Leu Arg Ser Leu Arg Thr Val Thr Pro Ile Arg Met Gln Gly
15 20 25 30
Gly Cys Gly Ser Cys Trp Ala Phe Ser Gly Val Ala Ser Thr Glu Ser
35 40 45
Ala Tyr Leu Ala Tyr Arg Asn Met Ser Leu Asp Leu Ala Glu Gln Glu
50 55 60
Leu Val Asp Cys Ala Ser Gln Asn Gly Cys His Gly Asp Thr Ile Pro
65 70 75
Arg Gly Ile Glu Tyr Ile Gln Gln Asn Gly Val Val Gln Glu His Tyr
80 85 90
Tyr Pro Tyr Val Ala Arg Glu Gln Ser Cys His Arg Pro Asn Ala Gln
95 100 105 110
Arg Tyr Gly Leu Lys Asn Tyr Cys Gln Ile Ser Pro Pro Asp Ser Asn
115 120 125
Lys Ile Arg Gln Ala Leu Thr Gln Thr His Thr Ala Val Ala Val Ile
130 135 140
Ile Gly Ile Lys Asp Leu Asn Ala Phe Arg His Tyr Asp Gly Arg Thr
145 150 155
Ile Met Gln His Asp Asn Gly Tyr Gln Pro Asn Tyr His Ala Val Asn
160 165 170
Ile Val Gly Tyr Gly Asn Thr Gln Gly Val Asp Tyr Trp Ile Val Arg
175 180 185 190
Asn Ser Trp Asp Thr Thr Trp Gly Asp Asn Gly Tyr Gly Tyr Phe Ala
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195 200 205
Ala Asn Ile Asn Leu Met Met Ile Glu Gln Tyr Pro Tyr Val Val Met
210 215 220
Leu
<210> 3
<211> 321
<212> PRT
<213> ~ermatophagoides farinae
<220>
<221> PROPEP
<222> (1)..(98)
<223> Propeptoide containing a signal peptide
<220>
<221> mat_peptide
<222> (99)..(321)
<400> 3
Met Lys Phe Val Leu Ala Ile Ala Ser Leu Leu Val Leu Ser Thr Val
-95 -90 -85
Tyr Ala Arg Pro Ala Ser Ile Lys Thr Phe Glu Glu Phe Lys Lys Ala
-80 -75 -70
Phe Asn Lys Asn Tyr Ala Thr Val Glu Glu Glu Glu Val Ala Arg Lys
-65 -60 -55
Asn Phe Leu Glu Ser Leu Lys Tyr Val Glu Ala Asn Lys Gly Ala Ile
-50 -45 -40 -35
Asn His Leu Ser Asp Leu Ser Leu Asp Glu Phe Lys Asn Arg Tyr Leu
-30 -25 -20
Met Ser Ala Glu Ala Phe Glu Gln Leu Lys Thr Gln Phe Asp Leu Asn
-15 -10 -5
Ala Glu Thr Ser Ala Cys Arg Ile Asn Ser Val Asn Val Pro Ser Glu
-1 1 5 10
Leu Asp Leu Arg Ser Leu Arg Thr Val Thr Pro Ile Arg Met Gln Gly
15 20 25 30
Gly Cys Gly Ser Cys Trp Ala Phe Ser Gly Val Ala Ala Thr Glu Ser
35 40 45
Ala Tyr Leu Ala Tyr Arg Asn Thr Ser Leu Asp Leu Ser Glu Gln Glu
50 55 60
Page 4
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Leu Val Asp Cys Ala Ser Gln His Gly Cys His Gly Asp Thr Ile Pro
65 70 75
Arg Gly Ile Glu Tyr Ile Gln Gln Asn Gly Val Val Glu Glu Arg Ser
80 85 90
Tyr Pro Tyr Val Ala Arg Glu Gln Arg Cys Arg Arg Pro Asn Ser Gln
95 100 105 110
His Tyr Gly Ile Ser Asn Tyr Cys Gln Ile Tyr Pro Pro Asp Val Lys
115 120 125
Gln Ile Arg Glu Ala Leu Thr Gln Thr His Thr Ala Ile Ala Val Ile
130 135 140
Ile Gly Ile Lys Asp Leu Arg Ala Phe Gln His Tyr Asp Gly Arg Thr
145 150 155
Ile Ile Gln His Asp Asn Gly Tyr Gln Pro Asn Tyr His Ala Val Asn
160 165 170
Ile Val Gly Tyr Gly Ser Thr Gln Gly Asp Asp Tyr Trp Ile Val Arg
175 180 185 190
Asn Ser Trp Asp Thr Thr Trp Gly Asp Ser Gly Tyr Gly Tyr Phe Gln
195 200 205
Ala Gly Asn Asn Leu Met Met Ile Glu Gln Tyr Pro Tyr Val Val Ile
210 215 220
Met
<210> 4
<211> 30
<212> PRT
<213> Dermatophagoides microceras
<220>
<221> MISC_FEATURE
<222> (1)..(30)
<223> N-terminal fragment
<400> 4
Thr Gln Ala Cys Arg Ile Asn Ser Gly Asn Val Pro Ser Glu Leu Asp
1 5 10 15
Leu Arg Ser Leu Arg Thr Val Thr Pro Ile Arg Met Gln Gly
20 25 30
<210> 5
<211> 221
<212> PRT
Page 5
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<213> Blomia tropicalis
<220>
<221> mat_peptide
<222> (1)..(221)
<400> 5
10317.ST25
Ile Pro Ala Asn Phe Asp Trp Arg Gln Lys Thr His Val Asn Pro Ile
1 5 10 15
Arg Asn Gln Gly Gly Cys Gly Ser Cys Trp Ala Phe Ala Ala Ser Ser
20 25 30
Val Ala Glu Thr Leu Tyr Ala Ile His Arg His Gln Asn Ile Ile Leu
35 40 45
Ser Glu Gln Glu Leu Leu Asp Cys Thr Tyr His Leu Tyr Asp Pro Thr
50 55 60
Tyr Lys Cys His Gly Cys Gln Ser Gly Met Ser Pro Glu Ala Phe Lys
65 70 75 g0
Tyr Met Lys Gln Lys Gly Leu Leu Glu Glu Ser His Tyr Pro Tyr Lys
85 90 95
Met Lys Leu Asn Gln Cys Gln Ala Asn Ala Arg Gly Thr Arg Tyr His
100 105 110
Val Ser Ser Tyr Asn Ser Leu Arg Tyr Arg Ala Gly Asp Gln Glu Ile
115 120 125
Gln Ala Ala Ile Met Asn His Gly Pro Val Val Ile Tyr Ile His Gly
130 135 140
Thr Glu Ala His Phe Arg Asn Leu Arg Lys Gly Ile Leu Arg Gly Ala
145 150 155 160
Gly Tyr Asn Asp Ala Gln Ile Asp His Ala Val Val Leu Val Gly Trp
165 170 175
Gly Thr Gln Asn Gly Ile Asp Tyr Trp Ile Val Arg Thr Ser Trp Gly
180 185 190
Thr Gln Trp Gly Asp Ala Gly Tyr Gly Phe Val Glu Arg His His Asn
195 200 205
Ser Leu Gly Ile Asn Asn Tyr Pro Ile Tyr Ala Ser Leu
210 215 220
Page 6
