U.S. patent application number 17/122593 was filed with the patent office on 2021-05-06 for modular antigen transportation molecules and uses thereof in animals.
The applicant listed for this patent is Boehringer Ingelheim Vetmedica GmbH. Invention is credited to Dania Birte REICHE, Horst ROSE, Harald TAMMEN.
Application Number | 20210128719 17/122593 |
Document ID | / |
Family ID | 1000005341475 |
Filed Date | 2021-05-06 |
United States Patent
Application |
20210128719 |
Kind Code |
A1 |
ROSE; Horst ; et
al. |
May 6, 2021 |
MODULAR ANTIGEN TRANSPORTATION MOLECULES AND USES THEREOF IN
ANIMALS
Abstract
The present invention relates to (isolated) recombinant
proteins, also referred to as improved MAT (iMAT) molecules,
comprising at least one translocation module, at least one
targeting module and at least one antigen module, wherein at least
one cysteine residue is substituted with a different amino acid
residue. Such iMAT molecules are useful specifically as vaccines,
e.g. for therapy and/or prevention of allergies and/or infectious
diseases and/or prevention of transmission of infectious diseases
in animals, more preferably ruminants, pigs, dogs and/or cats, but
excluding equines. The present invention further relates to nucleic
acids encoding such iMAT molecules, corresponding vectors and
primary cells or cell lines.
Inventors: |
ROSE; Horst; (Burgdorf,
DE) ; REICHE; Dania Birte; (Bingen am Rhein, DE)
; TAMMEN; Harald; (Hannover, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Boehringer Ingelheim Vetmedica GmbH |
Ingelheim am Rhein |
|
DE |
|
|
Family ID: |
1000005341475 |
Appl. No.: |
17/122593 |
Filed: |
December 15, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
15276922 |
Sep 27, 2016 |
10898568 |
|
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17122593 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 39/35 20130101;
A61K 2039/575 20130101; C07K 14/47 20130101; A61K 2039/55505
20130101; A61K 2039/622 20130101; C12N 2740/16071 20130101; A61K
39/385 20130101; A61K 2039/577 20130101; C12N 7/00 20130101; C07K
7/06 20130101; C07K 2319/21 20130101; C07K 14/4359 20130101; A61K
38/00 20130101; C12N 2740/16033 20130101; C07K 2319/33 20130101;
C07K 2319/10 20130101; C07K 14/43531 20130101; A61K 2039/552
20130101 |
International
Class: |
A61K 39/35 20060101
A61K039/35; A61K 39/385 20060101 A61K039/385; C07K 7/06 20060101
C07K007/06; C07K 14/435 20060101 C07K014/435; C07K 14/47 20060101
C07K014/47; C12N 7/00 20060101 C12N007/00 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 30, 2015 |
EP |
15187810.5 |
Claims
1. An improved MAT (iMAT) molecule, comprising: (i) at least one
first module being an amino acid sequence allowing the
translocation of the iMAT molecule from the extracellular space
into the interior of cells, (ii) at least one second module being
an amino acid sequence allowing species-specific intracellular
targeting of the iMAT molecule to the cell organelles which are
involved in the processing of antigens and/or the loading of MHC
molecules with antigens, and (iii) at least one third module as
antigen module being an amino acid sequence derived from at least
one epitope of at least one antigen, determining the specificity of
an immune response modulated by such iMAT molecule, wherein in the
entire iMAT molecule all cysteine residues are substituted with a
different amino acid residue, wherein the at least one second
module is selected from the group consisting of: the invariant
chain selected from the canine and/or feline species' or a partial
sequence thereof, and wherein the at least one antigen module
comprises at least one epitope derived from at least one allergen
eliciting an immune response in animals is selected from members of
the genus Canis or Felis, in response to flea bites, allergens
derived from fleas and/or mites food allergies, atopic dermatitis,
atopic dermatitis caused by flea bites, allergic asthma, allergic
airway inflammation and/or obstruction.
2. (canceled)
3. The iMAT molecule according to claim 1, wherein the modules are
covalently linked to each other, and wherein no additional spacer
module(s) between two or more adjacent modules of said first,
second, and/or third modules are present.
4. The iMAT molecule according to claim 1, wherein the at least one
second module comprises, one or more of the amino acid sequences of
SEQ ID NO: 4 (canine) or SEQ ID NO: 5 (feline) or fragments
thereof.
5-7. (canceled)
8. The iMAT molecule according to claim 1, wherein said at least
one allergen comprises Der f 15 allergen according to SEQ ID NO: 11
and/or 18, and/or Cte f1 allergen according to SEQ ID NO: 13 and/or
20.
9. (canceled)
10. The iMAT molecule according to claim 1, further comprising at
least one tag module, wherein said at least one tag module is
present N-terminally and/or C-terminally.
11. (canceled)
12. The iMAT molecule according to claim 10, wherein said at least
one tag module is a His-tag module which is present N-terminally
after one methionine residue.
13. The iMAT molecule according to claim 1, wherein the at least
one first module comprises the amino acid sequence of HIV-tat, VP22
and/or Antennapedia or a partial sequence thereof.
14. The iMAT molecule according to claim 13, wherein, such at least
one first module comprises SEQ ID NO: 1.
15. The iMAT molecule according to claim 1, wherein the third
module comprises any one of SEQ ID NOs: 14 to 23.
16. The iMAT molecule according to claim 1, comprising any one of
SEQ ID NOS: 24 to 83.
17-18. (canceled)
19. An amino acid sequence of an improved MAT (iMAT) molecule,
comprising: (i) at least one first module being an amino acid
sequence allowing the translocation of the iMAT molecule from the
extracellular space into the interior of cells, (ii) at least one
second module being an amino acid sequence allowing
species-specific intracellular targeting of the iMAT molecule to
the cell organelles which are involved in the processing of
antigens and/or the loading of MHC molecules with antigens, and
(iii) at least one third module as antigen module being an amino
acid sequence derived from at least one epitope of at least one
antigen, determining the specificity of an immune response
modulated by such iMAT molecule, wherein said first module
comprises SEQ ID NO. 1, wherein the second module consists of SEQ
ID NOs: 4 or 5, and wherein the third module consists of any one of
SEQ ID NOs: 14 to 23.
20-22. (canceled)
23. The amino acid sequence according to claim 19, wherein the
third module comprises SEQ ID NO: 18.
24. The amino acid sequence according to claim 19, wherein the
modules are covalently linked to each other, and wherein no
additional spacer module(s) between two or more adjacent modules of
such first, second and/or third modules are present at all.
25. The amino acid sequence according to claim 19, further
comprising at least one His-tag module, wherein said Hid-tag module
is present N-terminally and/or C-terminally.
26. (canceled)
27. The amino acid sequence according to claim 25, wherein said at
least one His-tag module is present N-terminally after one
methionine residue.
28. The amino acid sequence according to claim 19, wherein said
amino acid sequence comprises any one of SEQ ID NOs: 24-83.
29-31. (canceled)
32. The amino acid sequence according to claim 28, wherein said
amino acid sequence comprises SEQ ID NO: 36 (Der f15 iMAT molecule
cat), SEQ ID NO: 37 (Der f15 iMAT molecule cat), SEQ ID NO: 38 (Der
f15 iMAT molecule cat), SEQ ID NO: 57 (Der f15 iMAT molecule dog),
SEQ ID NO: 58 (Der f15 iMAT molecule dog), or SEQ ID NO: 59 (Der
f15 iMAT molecule dog).
33. The amino acid sequence according to claim 28, wherein said
amino acid sequence/iMAT molecule comprises SEQ ID NO: 68 or SEQ ID
NO: 77.
34. An amino acid sequence, comprising: (i) at least one first
module being an amino acid sequence allowing the translocation of
the iMAT molecule from the extracellular space into the interior of
cells, comprising SEQ ID NO: 1, (ii) at least one second module
being an amino acid sequence allowing species-specific
intracellular targeting of the iMAT molecule to the cell organelles
which are involved in the processing of antigens and/or the loading
of MHC molecules with antigens, comprising SEQ ID NO: 4 or 5, and
(iii) at least one third module as antigen module being an amino
acid sequence derived from at least one full or partial amino acid
sequence of any combination of two or more antigens selected from
the group consisting of: SEQ ID NO: 7, 8, 9, 10, 11, 12, 84, 85,
86, 87, and 88, determining the specificity of an immune response
modulated by such iMAT molecule, wherein at least in the antigen
modules all cysteine residue are substituted with a different amino
acid residue.
35. The amino acid sequence/improved MAT (iMAT) molecule according
to claim 34, wherein the antigen module is an amino acid sequence
derived from at least one full or partial amino acid sequence of
any combination of two or more antigens selected from the group
consisting of: SEQ ID NO: 10, 11, 84, 85, 86, 87, and 88 (Hybrid
1), or wherein the antigen module is an amino acid sequence derived
from at least one full or partial amino acid sequence of any
combination of two or more antigens selected from the group
consisting of: SEQ ID NO: 7, 8, 9,10, 11, and 12 (Hybrid 2) or
wherein the antigen module is an amino acid sequence derived from
at least one full or partial amino acid sequence of any combination
of two or more antigens selected from the group consisting of: SEQ
ID NO: 7, 8, 10, and 11 (Hybrid 3).
36. The amino acid sequence according to claim 35, wherein the
antigen module is an amino acid sequence based on a backbone
derived from SEQ ID NO: 84 comprising any combination of one or
more of the peptides according to SEQ ID NOs: 91-96 embedded into
said backbone sequence, or wherein the antigen module is an amino
acid sequence derived from any combination of two or more of the
peptides according to SEQ ID NOs: 97-102, or wherein the antigen
module is an amino acid sequence derived from any combination of
two or more of the peptides according to SEQ ID NO: 97, SEQ ID NO:
98, SEQ ID NO: 101, and SEQ ID NO: 102.
37. An amino acid sequence comprising any of SEQ ID NO: 36 (Der f15
iMAT molecule cat), SEQ ID NO: 37 (Der f15 iMAT molecule cat), SEQ
ID NO: 38 (Der f15 iMAT molecule cat), SEQ ID NO: 57 (Der f15 iMAT
molecule dog), SEQ ID NO: 58 (Der f15 iMAT molecule dog), SEQ ID
NO: 59 (Der f15 iMAT molecule dog), SEQ ID NO: 66 (hybrid 1 iMAT
cat), SEQ ID NO: 67 (hybrid 1 iMAT cat), SEQ ID NO: 68 (hybrid 1
iMAT cat), SEQ ID NO: 75 (hybrid 1 iMAT dog), SEQ ID NO: 76 (hybrid
1 iMAT dog) and/or SEQ ID NO: 77 (hybrid 1 iMAT dog).
38-42. (canceled)
Description
SEQUENCE LISTING
[0001] This application contains a sequence listing in accordance
with 37 C.F.R. 1.821-1.825. The sequence listing accompanying this
application is hereby incorporated by reference in its
entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to (isolated) recombinant
proteins, also referred to as improved MAT (iMAT) molecules,
comprising at least one translocation module, at least one
targeting module and at least one antigen module, wherein at least
one cysteine residue is substituted with a different amino acid
residue. The iMAT molecules can be produced with substantially
reduced manufacturing efforts and are species-specific, safer and
immunologically very effective. Such (isolated) recombinant
proteins are useful specifically as vaccines, e.g. for therapy
and/or prevention of allergies and/or infections and/or prevention
of transmission of infections in animals, preferably ruminants,
pigs, humans, dogs and/or cats, but excluding equines.
BACKGROUND OF THE INVENTION
[0003] Prior art publication from Crameri et al. describes the
background in more detail (Crameri R. et al., Allergy 2007, 62:
197-206). Briefly, the processing of antigens by antigen-presenting
cells (APCs) takes place by two different routes. Antigens
occurring inside the cell are presented by MHC I (major
histocompatibility complex class I, MHC class I) molecules on the
cell surface, whereas extracellular antigens are presented by MHC
II (major histocompatibility complex class II, MHC class II)
molecules on the cell surface.
[0004] Both mechanisms initiate an immune response by the host to
the antigen. Major histocompatibility class-II molecules are cell
surface glycoproteins that present peptides to CD4+ T cells. In the
endoplasmic reticulum (ER), MHC-II molecules become associated with
a type II transmembrane protein termed the invariant chain (Ii)
preventing peptide binding to MHC-II in the ER. Ii homotrimers
associate in the ER with 3 MHC class II .alpha. dimers and this
prevents the binding of endogenous peptides to class II molecules.
The N-terminal cytoplasmic domain of Ii contains targeting motifs
which leads to its retention in the ER, or to targeting of class II
.alpha. dimers into the endosomal-lysosomal pathway via the Golgi.
Subsequent proteolytic degradation of Ii leaves a small fragment
CLIP (Class II associated Ii peptide) bound to class II .alpha.
dimers in the peptide-binding groove. The interaction of class II
.alpha. /CLIP complexes with HLA-DM, a class II-related .alpha.
dimer, in a specialized compartment releases the CLIP and allows
the class II molecules to bind peptides derived from exogenous
proteins. It has been shown that endogenously synthesized proteins,
generally excluded from the MHC-II presentation pathway, can be
efficiently presented as peptide-MHC-II complexes when they are
expressed as Ii fusion proteins. This property has been exploited
to clone genes encoding MHC-II-restricted antigens from cell lines
transfected with Ii-cDNA fusion libraries. Efficient allergy
vaccines targeting the MHC-II processing and presentation pathway
were achieved using translocable Ii-allergen fusions. The concept,
termed modular antigen translocation (MAT) technology bases on a
fusion protein consisting of a TAT-derived translocation peptide
converting extracellular into cytoplasmic proteins, the first 110
amino-acids of Ii for targeting the fusion proteins to
endosomal/lysosomal compartments, and an antigen for induction of
specific immune responses.
[0005] The concept of providing modular antigen transportation
(MAT) molecules for modulating immune responses, associated
constructs, method and uses thereof is disclosed in WO 2004/035793
(US equivalent US 2005/0281816). This document describes the
usefulness of a three-part-molecule, the MAT molecule, for
introducing epitopes of antigens into cells, thus, determining the
immune response to be modulated by such MAT molecule. Therein,
various translocation modules, targeting modules as well as antigen
modules are described. This technology and its underlying method
make it possible, firstly, to convey antigens efficiently from the
extracellular space into the intracellular space of a target cell,
and, secondly, make it possible for the antigens, after arrival in
the interior of the cell, to reach efficiently cell organelles in
order to be subsequently processed for antigen presentation.
Generally, the two-stage process can be utilized for the targeted,
efficient modulation of the immune response in a subject. The use
of MAT molecules is disclosed for example in Martinez-Gomez J M et
al. [Allergy 2009, 64(1): 172-178]; Rose H (Arb Paul Ehrlich Inst
Bundesinstitut Impfstoffe Biomed Arzneim Langen Hess, 2009, 96,
319-327) as well as recently in Senti G et al. [J Allergy Clin
Immunol., 2012, 129(5): 1290-1296]. Based on the MAT technology,
the major cat allergen Fel d1 was fused to a TAT-derived protein
translocation domain and to a truncated invariant human chain for
targeting the MHC class II pathway. Immunogenicity was evaluated in
mice, while potential safety issues were assessed by suitable tests
based on basophil reactivities from cat-dander-allergic patients.
The possible use of this model compound has been demonstrated. It
is described therein, that it is expected that MAT molecules are
safer and more efficient in inducing the desired immune response,
namely hyposensitization, than recombinant allergens or allergen
extracts in conventional allergen-specific immunotherapy (SIT). In
the recent publication by Senti G. et al. intralymphatic
immunotherapy for cat dander allergy in humans inducing tolerance
after three injections was described. Therein, a first-in-human
clinical study with the MAT-Fel d1 was described, demonstrating
safety and induction of allergen tolerance after intralymphatic
injection of three injections, only.
[0006] Further prior art is as follows:
[0007] Gadermaier G et al. (Molecular Immunology 2010, 47:
1292-1298) described the targeting of the cysteine-stabilized fold
of Art v1 for immunotherapy of Artemisia pollen allergy. The
authors used genetic engineering approaches for targeting Art v1
posttranslational modifications aiming at the creation of
hypoallergenic molecules: (i) disulfide bridges of the defensin
domain were disrupted by site-directed mutagenesis and (ii) the
mutant constructs expressed in E. coli for the production of
non-glycosylated proteins. However, the objective was clearly only
manipulating the three-dimensional fold of the Art v1 defensin
domain to abrogate IgE-binding (i.e. creating a hypoallergenic
molecule) by exchanging single cysteine residues for serine--while
keeping intact (i.e. unmodified) the recognized T-cell epitopes
(even if such contain cysteine residues).
[0008] The report of the 3.sup.rd Havemeyer workshop on allergenic
diseases of the Horse (Holar, Iceland, June 2007, Veterinary
Immunology and Immunotherapy 2008, 126: 351-361) focused on
immunological and genetic aspects of insect's bite hypersensitivity
(IBH) and recurrent airway obstruction (RAO). At this workshop
novel approaches for SIT against IBH were discussed, among others
the use of viral vectors or protein vaccination with allergens
coupled to modular antigen translocating (MAT) molecules.
[0009] In SIAF Annual Reports 2010 and 2011 Crameri R reports the
use of MAT technology for vaccination of IBH-affected horses.
[0010] Zhao et al. (Int J Clin Exp Med 2015; 8(4):6436-6443)
reported results of experiments with mosaic fusion proteins with
the MAT structure disclosed in WO 2004/035793 and 3 segments of T
cell epitope coding for Der p1 as antigen module. They reassembled
these sequences in linear manner to form a fusion gene for protein
expression. They describe their construct to exhibit a stronger
allergenicity (hyperallergenicity) as compared to the Der p1
protein.
[0011] However, major problems arose when producing and
manufacturing the MAT molecules described in the prior art. In
particular standard methods used in developing a downstream process
(DSP) for manufacturing of the MAT molecules under good
manufacturing practice (GMP) could not be applied. It was not
possible to purify a homogeneous molecular species of the MAT
molecules, evidently due to their anomalous physicochemical
properties.
[0012] Several methods of purification could not be applied (see
Example 4 herein) with the MAT molecules described in the prior art
although different separation principles (e.g., size exclusion
chromatography, RP-HPLC) were tested. Methods applied for
determination of purity for recombinant proteins in general include
chromatographic separation, e.g. RP-HPLC and electrophoretic
separation (e.g. capillary zone electrophoresis, isoelectric
focusing, SDS-PAGE under reducing or non-reducing conditions).
Also, these analytical methods could not be applied on MAT
molecules without molecule-specific adaptations. For the assessment
of purity, an adapted specific SDS-PAGE test procedure had to be
developed. This test procedure includes sample preparation with
reducing agent and lithium dodecyl sulfate (LDS) and heating up to
75.degree. C., resulting in multiple, reproducible sharp bands
after electrophoretic separation. Staining with Coomassie blue dye
leads to linear quantitative behavior (densitometry) in gels. Using
a monoclonal antibody that allows for detection of the allergen
module in a MAT molecule exhibited a main band and several minor
bands. All bands migrate reproducibly to the same position as in
the original gel also after re-loading a second gel with excised
bands of the first gel. Surprisingly, in all of these bands with
apparent lower and higher molecular weight, the full length MAT
molecules were identified by excision of bands out of the gel,
their tryptic digestion and subsequent analysis by mass
spectrometry (nanoLC/ESI-MS-MS). From these experiments an
untypical, anomalous behavior of different folding variants of MAT
molecules in the SDS-PAGE can be concluded ("gel shifting").
Furthermore, in all batches of MAT molecules multimeric forms of
the protein could be detected which were difficult to separate from
monomeric forms.
[0013] For e.g. economic aspects, but also for regulatory
requirements, it is necessary to improve (i) the manufacturing
process of the MAT molecules and (ii) their suitability for
standard analytical methods of purity determination. Additionally,
for adapting the MAT molecules to specific target species, such as
ruminants, pigs, dogs and/or cats, adaption of the immunological
targeting within the MAT technology is required. This species
specificity is necessary to be represented in said iMAT molecules
since differences in homologues Ii amino acid sequences between
mammalian species exist (FIG. 10). However, a proper binding of the
Ii fusion protein (iMAT) to the .alpha. subunits of MHC class II,
specifically in the CLIP region is required for an optimal
immunological function with respect to the antigen in said iMAT
molecules. A proper binding to said MHC class II molecules is
achieved if the Ii sequence in iMAT does resemble the original as
far as possible.
[0014] Additionally, the MAT molecules are readily employed in
allergies elicited by a known major allergen (e.g. cat dander
allergy in humans by Fel d1). However, it seems difficult to employ
the MAT molecules of the prior art in clinical settings, such as
allergies, where for instance a variety of non-cross-reactive
allergens are known to be involved, but the importance of such
allergens in eliciting the allergy is unknown (i.e. the major
allergens are unknown).
[0015] Furthermore, the prior art does not describe how more than
one (e.g. 2, 3, 4 or more) allergen can be embedded into (i)MAT
molecules without exceeding a certain size of the fusion protein
that hinders protein manufacturing.
[0016] The objective underlying the invention is to provide
improved MAT molecules useful as active agents in pharmaceutical
composition, such as vaccines, and their corresponding therapeutic
and/or preventive uses in animals, excluding equines, which
overcome the problems of the prior art.
SUMMARY OF THE INVENTION
[0017] In one aspect, the objective underlying the invention has
surprisingly been solved by providing a(n) (isolated) recombinant
protein, preferably an improved MAT (iMAT) molecule,
comprising:
[0018] (i) at least one first module being an amino acid sequence
allowing the translocation of the iMAT molecule from the
extracellular space into the interior of cells,
[0019] (ii) at least one second module being an amino acid sequence
allowing species-specific intracellular targeting of the iMAT
molecule to the cell organelles which are involved in the
processing of antigens and/or the loading of MHC molecules with
antigens, preferably processed antigens, and
[0020] (iii) at least one third module as antigen module being an
amino acid sequence derived from at least one full or partial
epitope of at least one antigen, preferably at least one allergen,
determining the specificity of an immune response modulated by such
iMAT molecule,
characterized in that at least in the antigen module(s) at least
one cysteine residue is substituted with a different amino acid
residue, preferably serine, leucine, isoleucine, arginine,
methionine, and/or aspartic acid, for use in a method of prevention
and/or therapy of one or more allergies in animals excluding
equines and/or for use in a method of prevention and/or therapy of
one or more infectious diseases in animals excluding equines and/or
for use in a method of prevention of transmission of one or more
infectious diseases in animals excluding equines and/or for use in
a method of prevention of transmission of one or more infectious
diseases in animals excluding equines by vectors.
[0021] Corresponding methods of prevention and/or treatment of
animals excluding equines, in need thereof and uses for the
preparation of a pharmaceutical composition/medicament for the
prevention and/or treatment of animals excluding equines, are also
intended to be within the scope of the present invention.
[0022] Preferably, in the at least one antigen module all cysteine
residues are substituted with a different amino acid residue,
preferably serine, leucine, isoleucine, arginine, methionine,
and/or aspartic acid. More preferably in the entire iMAT molecule
all cysteine residues are substituted with a different amino acid
residue, preferably serine, leucine, isoleucine, arginine,
methionine, and/or aspartic acid.
[0023] Preferably, if not all cysteine residues are substituted
with a different amino acid residue, preferably serine, leucine,
isoleucine, arginine, methionine, and/or aspartic acid, an even
number of cysteine residues remains in the entire iMAT
molecule.
[0024] Preferably, all of such modules are covalently linked to
each other, optionally by additional spacer module(s) between two
or more adjacent, optionally all, of such first, second and/or
third modules.
[0025] More preferably, all of such modules are covalently linked
to each other and no additional spacer module(s) are present
between two or more adjacent modules of such first, second and/or
third modules at all.
[0026] In another aspect, the objective underlying the invention
has surprisingly been solved by providing an iMAT molecule relating
to one or more of the amino acid sequences according to SEQ ID
NOs:2 or 3, preferably comprising one or more of the amino acid
sequences according to SEQ ID NOs:4 or 5. In a further aspect, the
objective underlying the invention has surprisingly been solved by
providing an iMAT molecule comprising one or more of the amino acid
sequences according to SEQ ID NOs:14-23. In a preferred aspect, the
objective underlying the invention has surprisingly been solved by
providing an iMAT molecule comprising, preferably consisting of,
one or more of the amino acid sequences according to SEQ ID NOs:
24-83.
[0027] In another aspect, the objective underlying the invention
has surprisingly been solved by providing a vaccine or immunogenic
composition or pharmaceutical composition comprising the (isolated)
recombinant protein as herein disclosed and claimed.
[0028] In another aspect, the objective underlying the invention
has surprisingly been solved by providing the (isolated)
recombinant protein as herein disclosed and claimed or the vaccine
or immunogenic composition or pharmaceutical composition as herein
disclosed and claimed for use in a method of prevention and/or
therapy of one or more allergies in animals, preferably dogs and/or
cats, but excluding equines; preferably allergies to flea bites
preferably in dogs and/or cats; allergies to certain food
components preferably in dogs and/or cats; atopic dermatitis
preferably in dogs and/or cats; allergic airway inflammation and/or
obstruction preferably in cats. Corresponding methods of prevention
and/or treatment of animals, preferably dogs and/or cats, but
excluding equines, in need thereof and uses for the preparation of
a pharmaceutical composition/medicament for the prevention and/or
treatment of animals, preferably dogs and/or cats, but excluding
equines, are also intended to be within the scope of the present
invention.
[0029] In another aspect, the objective underlying the invention
has surprisingly been solved by providing the (isolated)
recombinant protein as herein disclosed and claimed or the vaccine
or immunogenic composition or pharmaceutical composition as herein
disclosed and claimed for use in a method of prevention and/or
therapy of one or more infectious diseases in animals, preferably
ruminants, pigs, dogs and/or cats, but excluding equines, and/or
prevention of transmission of one or more infectious diseases in
animals, preferably ruminants, pigs, dogs and/or cats, but
excluding equines, by vectors, preferably by blood feeding bugs,
flies, midges, ticks and/or mosquitos. The infectious pathogen
and/or infectious disease may be one or more selected from
Campylobacter, heartworm, ehrlichiosis, leishmaniosis,
trypanomiasis, borreliosis, Schmallenberg-, blue tongue- and/or
west nile virus infection, dermatophytosis and/or infections of the
digestive tract and/or other organs by viruses (e.g. rota-,
coronavirus) and/or parasites (e.g., helminths) and/or protozoa
(e.g., coccidiosis, cryptosporidiosis) and/or their pre-patent
stages. Corresponding methods of prevention and/or treatment of
animals, preferably ruminants, pigs, humans, dogs and/or cats, but
excluding equines, in need thereof and uses for the preparation of
a pharmaceutical composition/medicament for the prevention and/or
treatment of animals, preferably ruminants, pigs, humans, dogs
and/or cats, but excluding equines, are also intended to be within
the scope of the present invention.
[0030] In a further aspect, the objective underlying the invention
has surprisingly been solved by providing a nucleic acid encoding
the (isolated) recombinant protein as herein disclosed and
claimed.
[0031] In a further aspect, the objective underlying the invention
has surprisingly been solved by providing a vector comprising at
least one nucleic acid as herein disclosed and claimed.
[0032] In yet a further aspect, the objective underlying the
invention has surprisingly been solved by providing a primary cell
or cell line comprising at least one nucleic acid as herein
disclosed and claimed and/or at least one vector as herein
disclosed and claimed.
[0033] Surprisingly, the iMAT molecules according to the present
invention, as herein disclosed and claimed, do possess
physicochemical and/or immunological characteristics that render
them superior to the MAT molecules of the pertinent prior art:
[0034] (i) The substitution of at least one cysteine residue,
preferably all cysteine residues, with a different amino acid
residue in the antigen module(s), preferably entire iMAT molecule,
which was selected in silico not to compromise the stability of the
final iMAT molecule, renders the iMAT molecule according to the
present invention surprisingly suitable for the application of
standard purification procedures for biopharmaceuticals as well as
for standard analytical methods.
[0035] (ii) The preferred direct covalent linkage of the modules of
the iMAT molecule, i.e. first translocation module, second
targeting module and third antigen module, without any additional
spacer modules between such modules, i.e. no additional spacer
modules between two or more adjacent modules of such first, second
and/or third modules at all, contributes in addition to the
superior characteristics of the iMAT molecules according to the
present invention: such iMAT molecules which are thought to be more
rigid in three-dimensional structure and hence unable to form
conformational IgE epitopes, are even more
hypoallergenic--virtually to the extent of no allergenicity at
all.
[0036] (iii) The preferred presence of a(n) (quasi) N-terminal or
C-terminal His-tag results in iMAT molecules according to the
present invention that can be used as a surrogate marker for
monitoring immunity and/or duration of immunity since such tag
module, optionally together with one or more adjacent amino acid
residues from the translocation module, can be used to induce a
specific immunologically detectable signal (e.g. an antibody) in
the target subject, that is specific to the structure of the iMAT
molecules (see Example 1 herein). Additionally, the presence of
such tag module can be used for separating proteins in a sample
containing the iMAT molecules according to the present invention,
e.g. using zinc- or cobalt-charged solid supports, and hence
further improves the possibility to produce iMAT molecules
according to the present invention without aggregation during the
purification process. A(n) (quasi) N-terminal His-tag is
preferred.
[0037] (iv) The at least one targeting module in the iMAT molecules
according to the present invention is preferably species-specific,
i.e. in case of an intended application of such iMAT molecules to
canine, feline, bovine, ovine, caprine or porcine a targeting
module, e.g. the canine invariant chain, is chosen accordingly. By
this species-specific targeting optimized binding characteristics
of the iMAT molecules according to the present invention to the MHC
Class II molecules can successfully be achieved.
[0038] (v) The at least one antigen module in the iMAT molecules
according to the present invention is preferably an allergen. This
could be derived from food and/or mold (fungi and/or their spores),
pollen, house dust or forage mites (and/or their feces) and/or
fleas, preferably pollen from tree, grass, herbaceous, Ambrosia
and/or brassicaceae pollen and/or fungi and/or their spores of the
genera Aspergillus, Alternaria, Botrytis, Cercospora, Cladosporium,
Curvularia, Drechslera, Eurotium, Helminthosporium, Epicoccum,
Erysipheloidium, Fusarium, Lichtheimia, Nigrospora, Penicillium,
Periconia, Peronospora, Polythrincium, Saccharopolyspora (formerly
also Faenia or Micropolyspora), Thermoactinomyces, Stemphylium,
torula and/or mites (or their feces) of the genera Acarus,
Glycophagus, Tyrophagus, Dermatophagoides, Euroglyphus,
Lepidoglyphus, Blomia and/or fleas of the genera Ceratophyllus,
Ctenocephalides, Pulex, Archaeopsylla. The at least one antigen
module in the iMAT molecules according to the present invention is
more preferably a Dermatophagoides allergen. The allergen can be
selected according to the following criteria: when major allergens
eliciting an allergy in subjects are unknown, the at least one
antigen module in the iMAT molecules according to the present
invention can be selected by a bioinformatics approach as described
exemplarily and in detail in Examples 5 and 6 herein. By this means
improved MAT molecules can be achieved which are useful
specifically as vaccines, e.g. for therapy and/or prevention of
allergies in animals, preferably dogs and/or cats, but excluding
equines. The at least one antigen module in the iMAT molecules
according to the present invention may also be an antigen of a
pathogen involved in one or more infectious disease(s). This could
be derived from the genera Campylobacter, Dirofilaria, Ehrlichia,
Leishmania, Trypanosoma, Borrelia, Orthobunyavirus, Orbivirus,
Flavivirus, Rotavirus, Coronavirus, Trichophyton, Microsporum;
other helminths like Cooperia, Haemonchus, Ostertagia,
Trichostrongylus, Dictyocaulus, Metastrongylus; and/or protozoa
with gastrointestinal infestation like Eimeria, Isospora,
Cryptosporidium, Giardia--in case of parasites also antigens
derived of the pre-patent stages might be employed. The at least
one antigen module in the iMAT molecules according to the present
invention may also be an antigen (e.g. saliva component) of a
vector involved in the transmission of one or more infectious
disease(s), e.g., belonging to the families Culicidae,
Ceratopogonidae, Phlebotominae Ixodidae and/or Cimicidae. By this
means improved MAT molecules can be achieved which are useful
specifically as vaccines, e.g. for therapy and/or prevention and/or
prevention of transmission of infectious diseases in animals,
preferably ruminants, pigs, dogs and/or cats, but excluding
equines.
[0039] (vi) Production of novel iMAT molecules that comprise
sequence motifs of more than one allergen in only one of said iMAT
molecule, i.e. mosaic fusion proteins, can be achieved. The
selection of the peptide sequences embedded in the allergen module
is based on pan allergenic motifs detected in the relevant major
allergens by bioinformatics tools. By this approach the size of
such iMAT molecules can be kept low enough to allow efficient
production in suitable expression systems.
[0040] In particular, the present invention provides (isolated)
recombinant proteins exhibiting an improved solubility, that are
readily applicable to chromatographic separation techniques and
that show an improved stability.
[0041] In addition, the (isolated) recombinant proteins according
to the present invention preferably display high activities and
efficacies in inducing the desired immunological effects, namely,
advantageously species specific modulating the immune response
against allergens in a subject becomes feasible, e.g. in animals,
more preferably dogs and/or cats, but excluding equines, allergen
specific IgE mediated hypersensitivity reactions may be modulated
in different target organs as skin, respiratory and/or the
gastrointestinal system. And/or prevention and/or therapy of
infectious diseases and/or prevention of transmission of infectious
diseases by vectors in animals, preferably ruminants, pigs, humans,
dogs and/or cats, but excluding equines, becomes feasible.
[0042] The allergenicity of a therapeutic allergen is of utmost
importance, it is a measure of the potential to induce adverse
events, e.g. provoke anaphylaxis. With regard to prior art MAT
molecules conflicting results about their allergenicity in
comparison to the corresponding native allergens have been reported
in the prior art. Senti G et al. (J Allergy Clin Immunol. 2012,
129(5): 1290-1296) demonstrated hypoallergenicity of a MAT-Fel d1
in the Cellular Antigen Stimulation Test (CAST) assay as well as in
the intradermal and in the intracutaneous test. The quantitative
difference in sensitivity between the allergen and the MAT molecule
comprising the Fel d1 was 100-, 23- and 16-fold, respectively.
Though MAT-Fel d1 was clearly hypoallergenic, some allergenicity
remained. In contrast Zhao et al. (Int J Clin Exp Med 2015; 8(4):
6436-6443) describe their MAT-Der p1 construct to exhibit an even
stronger allergenicity (hyperallergenicity) as compared to the
native Der p1 protein.
[0043] Surprisingly, the safety of the improved MAT molecules, as
disclosed and claimed herein, is superior in this respect. In
contrast to native allergens, which usually elicit a strong
histamine release, surprisingly the iMAT molecules according to the
present invention show virtually no histamine release response at
all. Thus, iMAT molecules show superiority in respect to safety as
compared with the MAT molecule as described in the prior art (see
above).
[0044] The consequence of this surprising safety property of iMAT
molecules in contrast to prior art MAT molecules is, that iMAT
molecules used as desensitizing proteins can be used similar to
vaccines against pathogens. No up-dosing as with classical
therapeutic allergens is needed, since vaccines comprising iMAT
molecules do not show allergen properties with respect to allergic
adverse events. Already the dose of the first injection of the iMAT
molecule in a treatment course is selected based on efficacy
considerations only and one does not have to consider potential
allergic adverse reactions. This could not be performed using MAT
molecules described in the prior art since the allergenicity of
MAT, compared to the native allergen, is only reduced to a certain
level. However, MAT molecules described in the prior art are still
allergens; iMAT molecules in contrast are not. The advantage of
this improved property renders a more efficacious treatment regime
possible with e.g. three subcutaneous or intralymphatic injections
with a high biopharmaceutical content (e.g. 3 times 1 .mu.g to 100
.mu.g, preferably 3 times 10 .mu.g to 50 .mu.g iMAT protein).
[0045] The lack of allergenicity of the iMAT molecules can be
explained by the fact that in contrast to the MAT molecules
described in the prior art no linker amino acid residues [i.e.
spacer module(s) between the first, second and/or third module(s)]
are used to separate the different modules in such iMAT
molecules.
[0046] It is known in the prior art that engineered fusion proteins
containing two or more functional polypeptides joined by a peptide
or protein linker, such linker is important for the function (e.g.
epitope recognition by the immune system) of the proteins [Klein J
S et al., Protein Eng Des Sel. 2014, 27(10): 325-330]. The
separation distance between functional units can impact epitope
access and the ability to bind with avidity.
[0047] It is assumed that in iMAT molecules of the present
invention, which are missing the amino acid residue linkers between
the modules, in particular between the targeting domain and the
antigen module, lead to a more rigid structure, conformational
epitopes of the allergen module might not be formed due to
incorrect folding. A cross linking of antibodies bound on the
surface of basophils (e.g. IgE) by its high affinity receptors is
necessary to induce activation and histamine release. However,
misfolded allergens might not be able to induce such cross linking.
Thus, an iMAT molecule without additional spacer modules/linkers
between the first, second and third module may not form
conformational IgE epitopes, which renders the iMAT molecules
according to the present invention non-allergenic. Thus, in a
specific embodiment the iMAT molecules of the present invention
lack any additional spacer modules or linkers between the first,
second and third module.
DETAILED DESCRIPTION OF THE INVENTION
[0048] Before the embodiments of the present invention are
described in further details it shall be noted that as used herein
and in the appended claims, the singular forms "a", "an", and "the"
include plural reference unless the context clearly dictates
otherwise.
[0049] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this invention belongs. All
given ranges and values may vary by 1 to 5% unless indicated
otherwise or known otherwise by the person skilled in the art,
therefore, the term "about" was usually omitted from the
description and claims. Although any methods and materials similar
or equivalent to those described herein can be used in the practice
or testing of the present invention, the preferred methods,
devices, and materials are now described. All publications
mentioned herein are incorporated herein by reference for the
purpose of describing and disclosing the substances, excipients,
carriers, and methodologies as reported in the publications which
might be used in connection with the invention. Nothing herein is
to be construed as an admission that the invention is not entitled
to antedate such disclosure by virtue of prior invention.
[0050] The terms "isolated recombinant protein", "recombinant
protein" and/or "improved MAT (iMAT) molecule" are interchangeably
used in the course of the present invention. They all have the
identical meaning.
[0051] The term "module" in the course of the present invention
refers to a specific amino acid sequence, e.g. a part, a unit or a
moiety of a polypeptide, usually short amino acid/peptide
sequences, having a defined function.
[0052] The term "first module being an amino acid sequence allowing
the translocation of the (isolated) recombinant protein, preferably
improved MAT (iMAT) molecule, from the extracellular space into the
interior of cells", herein also interchangeably referred to as
"translocation module" or "translocation sequence", in the course
of the present invention refers to a specific amino acid sequence
that promotes the transport of the cargo molecule, e.g. amino acid
sequence, peptide, polypeptide, protein and other classes of
substances, such as nucleic acids or pharmaceutically active
ingredients (API), to the interior of cells, in particular
eukaryotic cells, more particular, cells expressing the MHC class
II molecules on the surface and/or the MHC class I molecules on the
surface, as known in the literature.
[0053] By the presence of the translocation module it is possible
to promote the entry of said cargo molecule into the cells.
[0054] Amino acid sequences useful as translocation modules are
described in the prior art. For example, U.S. Pat. No. 7,653,866
discloses several useful translocation sequences including the
HIV-tat molecule or the protein VP22, which is derived from herpes
simplex virus. This principal of promoting the entry of a given
target molecule into the interior of cells is described numerously
in various studies in the pertinent patent and non-patent
literature. In addition, suitable translocation sequences include
homeoprotein sequences, leucine zipper sequences, arginine-rich
and/or lysine-rich sequences, and various other sequences of
proteins or polypeptides which are secreted despite the absence of
a secretion signal sequence. Particularly useful are viral peptide
sequences, e.g. the protein HIV transcriptional activator protein
(HIV tat). The Tat sequence or Tat peptide has been described in
the prior art including various modifications. All the variations
described in the prior art for peptide sequences of Tat are
generally suitable as translocation modules. Other examples include
the VP22 peptide as well as antennapedia peptides derived from the
Drosophila homeotic protein antennapedia. In addition, other
homeoproteins may be used. Various examples of suitable
homeoproteins are described in the prior art. In addition, leucine
zipper proteins, like human cFos-(139-164), or human cJun-(252-279)
can be used. Moreover, arginine-rich and/or lysine-rich peptides
are suitable as translocation modules including sequences like
HIV-1 rev (34-50) or other peptides derived from virus or yeast. Of
course, the polyarginine-rich and/or polylysine-rich peptides can
be produced synthetically. Said polyarginine-rich and/or
polylysine-rich peptides may comprise further amino acids. Suitable
examples are described in the pertinent prior art.
[0055] In a preferred embodiment, the at least one translocation
module comprises, preferably consists of, an amino acid sequence
which does not consist of a complete protein sequence, as
illustrated above, but instead of a minimal sequence still being
functional, i.e. capable of effectively promoting cell entry. A
suitable minimal sequence is for instance the amino acid sequence
YGRKKRRQRRR (SEQ ID NO: 1).
[0056] In another preferred embodiment, the at least one
translocation module comprises, preferably consists of, HIV-tat,
VP22 and/or Antennapedia or a partial sequence thereof, provided
that such at least one translocation module is functional as a
module for translocation from the extracellular space into the
interior of cells.
[0057] The term "second module being an amino acid sequence
allowing species-specific intracellular targeting of the (isolated)
recombinant protein, preferably improved MAT (iMAT) molecule, to
the cell organelles which are involved in the processing of
antigens and/or the loading of MHC molecules with antigens,
preferably processed antigens", herein also interchangeably
referred to as "targeting module" or "targeting sequence", in the
course of the present invention refers to a specific amino acid
sequence that allows/promotes the intracellular transport of the
(isolated) recombinant proteins, as disclosed and claimed herein,
to such cell organelles that are involved in the processing of
antigens and/or the loading of MHC molecules with antigens.
[0058] In particular, such cell organelles include the endoplasmic
reticulum, the Golgi apparatus, the trans-Golgi network, lysosomes,
endosomes and MHC II compartments. These intracellular organelles
are involved in processes such as, for example, the transport
and/or processing of antigens, the preparation and/or loading of
MHC II molecules with antigens or processed antigens, and/or the
transport of the MHC II molecules loaded with such antigens to the
cell surface.
[0059] A number of sequences are known in the prior art. A
prominent example of useful targeting sequences includes the
invariant chain of MHC class II molecules also known as Ii
invariant chain or MHC II gamma chain. Various variants of the
invariant chain are described in the patent and non-patent
literature.
[0060] In a preferred embodiment of the present invention, the
invariant chain is chosen from the species in which the immune
response should be modulated and/or from the species in which the
iMAT molecule should be intracellularly targeted. This species
specificity is necessary to be represented in said iMAT molecules
since differences in homologues Ii amino acid sequences between
mammalian species exist (FIG. 10). However, a proper binding of the
Ii fusion protein (iMAT) to the .alpha. subunits of MHC class II,
specifically in the CLIP region is required for an optimal
immunological function with respect to the antigen in said iMAT
molecules. A proper binding to said MHC class II molecules is
achieved if the Ii sequence in iMAT does resemble the original as
much as possible.
[0061] For example, for dogs, cats, cattle, sheep, goats or pigs
the preferred invariant chain chosen is the canine, feline, bovine,
ovine, caprine or porcine invariant chain. For dogs and cats, the
preferred invariant chain is the amino acid sequence according to
SEQ ID NO: 2 (canine) and SEQ ID NO: 3 (feline) or fragments
thereof, provided such fragments maintain their intracellular
transport function (e.g. the first 110 amino acids as shown in FIG.
10).
[0062] Other suitable examples for targeting sequences include
lysosomal membrane proteins, comprising sequences suitable as
targeting modules. That are, a number of membrane proteins
occurring in lysosomes which have sequence motifs that allow
targeting the lysosome. These groups of proteins include inter alia
lamp 1, lamp 2, lamp 3, limp II and lap. In addition, tetraspan
proteins are known in the prior art as targeting modules.
Additional proteins can be found in the endosomal/lysomal
compartments that show targeting properties. A skilled person is
aware how to determine suitable targeting sequences
accordingly.
[0063] In another embodiment, the at least one targeting module
comprises, preferably consists of, the canine, feline, bovine,
ovine, caprine or porcine invariant chain or fragments thereof
provided such fragments maintain their intracellular transport
function.
[0064] In a preferred embodiment, the at least one targeting module
is the canine invariant chain, comprising, preferably consisting of
SEQ ID NO: 4 (canine).
[0065] In a further preferred embodiment, the at least one
targeting module is the feline invariant chain, comprising,
preferably consisting of SEQ ID NO: 5 (feline).
[0066] The term "third module as antigen module being an amino acid
sequence derived from at least one full or partial epitope of at
least one antigen, preferably at least one allergen, determining
the specificity of an immune response modulated by such (isolated)
recombinant protein, preferably improved MAT (iMAT) molecule (in a
subject, preferably an animal, more preferably a ruminant, pig, dog
and/or cat, but excluding an equine)", herein also interchangeably
referred to as "antigen module" or "antigen sequence", in the
course of the present invention refers to a specific amino acid
sequence that allows modulating the immune response against the
epitope/antigen and determining the specificity of the immune
response in a subject, preferably an animal, more preferably a
ruminant, pig, dog and/or cat, but excluding an equine.
[0067] In this context, such antigen module(s) comprise(s) at least
one cysteine residue that is substituted with a different amino
acid residue, preferably serine, leucine, isoleucine, arginine,
methionine, and/or aspartic acid. Thus, the immune response is
different compared to the immune response of a subject, preferably
an animal, more preferably a ruminant, pig, dog and/or cat, but
excluding an equine, exposed to the unchanged amino acid sequence
of the antigen.
[0068] There are no restrictions relating to the antigens on the
basis of the method. The method can be used for example for
activating the immune system of a subject against pathogens such
as, for example, against viruses, bacteria, fungi, parasites,
protozoa etc., i.e. very generally as vaccine. Additionally, the
method can be used not only directly against such pathogens, but
also to activate the host immune system to prevent the transmission
in vector-borne diseases involving viruses, bacteria, fungi,
parasites, protozoa, etc. In addition, the method can be used to
activate the immune system against degenerated cells such as, for
example, tumor cells, etc. However, it can also be used on the
other hand for desensitization of the immune system of a subject
against allergens such as, for example derived from food and/or
aeroallergens, e.g. mold (fungi and/or their spores), pollen,
animal hair, house dust or forage mites (and/or their feces),
insect toxins, etc. or for targeted suppression of the immune
system, e.g. if autoimmune reactions are present, such as, for
example, arthritis, rheumatism, diabetes, SLE (systemic lupus
erythematosus), etc., and for suppressing transplant rejection
reactions. Further disorders which are not expressly mentioned and
which are associated with an immune reaction which is too strong or
too weak can likewise be treated with the iMAT molecules, as
disclosed and claimed herein.
[0069] It is possible to employ as antigen modules for the purposes
of the invention in principle all types of antigens able to
modulate an immune response. Both, antigens currently already known
and antigens to be discovered in future are suitable. In some
circumstances, the antigens may also be those which do not lead to
an immune response with conventional immunization methods known in
the art at present, but which lead on application of the novel
method described in the present invention to an immune response by
the subject. Further, the term antigen encompasses antigenic
fragments comprising the antigenic determinant/the antigenic
determinants which are also known as epitope(s). Thus, the antigen
module may be the whole molecule, e.g. the protein, or is a part of
the molecule, i.e. a fragment thereof, like a peptide, encompassing
at least one antigenic determinant or epitope. The at least one
antigenic determinant or epitope is able to elicit an immune
response against the antigen. The epitope can comprise one or more
than one amino acid or peptide or other structure capable of
eliciting an immune response such as sugar structures,
phosphorylated amino acids, etc. or combinations thereof. The
antigen can be a continuous epitope (i.e. not dependent on
conformation, e.g. present in for example native and denatured
proteins) or a discontinuous epitope (i.e. dependent on
conformation, e.g. only present in native, folded, but not present
in denatured proteins). It is possible to use not only proteins and
peptides, but also sugar structures, lipids, e.g.
lipopolysaccharides, lipoteichoic acids and other constituents of
bacterial membranes (CD1b binds, for example, sugar structures and
lipids), nucleic acids such as, for example, DNA comprising CpG
motifs, organic substances such as, for example, latex or
pharmaceutically active substances as antigen for the purposes of
the present invention. The antigen may be derived from all possible
life forms, such as e.g. animals, plants, fungi, parasites,
unicellular or multicellular microorganisms, viruses and other life
forms. The antigens may have been isolated from biological
material, have been prepared as recombinant antigens or have been
prepared by synthesis, e.g. by peptide synthesis. Synthetically
prepared antigens may be substances which occur in nature or which
do not occur in nature but are obtainable by chemical synthesis.
Examples of non-naturally occurring substances which are, however,
suitable as antigen in some circumstances are, for example,
synthetically prepared substances which are present in medicaments,
or synthetic peptides having amino acid sequences which do not
occur in nature, or peptidomimetics, etc. Naturally occurring or
synthetic or recombinant antigens can be modified by molecular
biology, enzymatic, chemical and/or other methods in order to
confer on them properties which are more advantageous for the
particular application. These advantageous properties may be, inter
alia, a higher or lower activity as antigen, a broader or a more
specific action as antigen, a better solubility in hydrophilic or
hydrophobic solvents, a greater permeability of the antigen modules
for cell membranes, for membranes of organelles, for the
blood-brain barrier, for the blood-CSF barrier etc., a higher or
lower half-life in vivo or in vitro, a lower or higher toxicity, a
better detectability of the antigen in vivo or in vitro after
application of the antigen in the form of an iMAT molecule etc. It
is additionally possible for the purposes of the present invention
to combine a plurality of antigens in one antigen module. For this
it is possible for identical antigens to be present in more than
one copy in the antigen module, or it is possible for example for
different variants of the same antigen to be combined in an antigen
module. Combination of antigens, e.g. of antigen 1, and other
antigens, e.g. of antigen 2, in an antigen module is also possible,
etc. Further combinations, such as, for example, antigen 1 in more
than one copy and antigen 2 in a single copy may also be combined
in an antigen module, etc. It is additionally possible also for one
or more different and/or one or more identical antigen modules to
be present in an iMAT molecule. In principle, it is conceivable
that for all possible combinations of single or multiple, identical
or altered, copies of antigens derived from one or more different
antigens can be combined for the purposes of the invention.
[0070] In a preferred embodiment, the antigen module comprises at
least one full or partial epitope derived from at least one
antigen, wherein such antigen is an allergen. At least one epitope
is able to elicit an immune response against the allergen whereby
the epitope can comprise one or more than one structure, e.g. a
peptide, capable of eliciting an immune response. The epitope may
be a continuous epitope or a discontinuous epitope of the allergen.
Epitopes are preferably at least eight amino acids in length
preferably are at least ten amino acids in length, more preferably
are at least 13 amino acids in length. The antigen module comprises
at least one full or partial epitope, but may also comprise two or
more full or partial epitopes which may be identical or different
from each other. Furthermore, the antigen module may comprise
additional amino acid sequences adjacent to the at least one full
or partial epitope. The epitope may be the natural occurring
epitope or may be a modified epitope, either modified in its amino
acid sequence and/or by one or more post-translational
modifications.
[0071] In an embodiment, the at least one (third) antigen module
comprises at least one full or partial epitope derived from at
least one antigen of a pathogen involved in one or more infectious
disease(s) of animals, more preferably ruminants, pigs, dogs and/or
cats, but excluding equines. This could be derived from the genera
Campylobacter, Dirofilaria, Ehrlichia, Leishmania, Trypanosoma,
Borrelia, Orthobunyavirus, Orbivirus, Flavivirus, Rotavirus,
Coronavirus, Trichophyton, Microsporum; other helminths like
Cooperia, Haemonchus, Ostertagia, Trichostrongylus, Dictyocaulus,
Metastrongylus; and/or protozoa with gastrointestinal infestation
like Eimeria, Isospora, Cryptosporidium, Giardia--in case of
parasites also antigens derived of the pre-patent stages might be
employed. The at least one antigen module in the iMAT molecules
according to the present invention may also be an antigen (e.g.
saliva component) of a vector involved in the transmission of one
or more infectious disease(s), e.g. belonging to the families
Culicidae, Ceratopogonidae, Phlebotominae, Ixodidae and/or
Cimicidae.
[0072] In a further preferred embodiment, the at least one (third)
antigen module comprises at least one full or partial epitope
derived from at least one allergen eliciting one or more allergies
in animals, more preferably dogs and/or cats, but excluding
equines. This could be at least one full or partial epitope of at
least one allergen derived from food and/or mold (fungi and/or
their spores), pollen, house dust or forage mites (and/or their
feces), preferably pollen from tree, grass, herbaceous, Ambrosia
and/or brassicaceae pollen and/or fungi and/or there spores of the
genera Aspergillus, Alternaria, Botrytis, Cercospora, Cladosporium,
Curvularia, Drechslera, Eurotium, Helminthosporium, Epicoccum,
Erysipheloidium, Fusarium, Lichtheimia, Nigrospora, Penicillium,
Periconia, Peronospora, Polythrincium, Saccharopolyspora (formerly
also Faenia or Micropolyspora), Thermoactinomyces, Stemphylium,
torula and/or mites (or their feces) of the genera Acarus,
Glycophagus, Tyrophagus, Dermatophagoides, Euroglyphus,
Lepidoglyphus, Blomia and/or fleas of the genera Ceratophyllus,
Ctenocephalides, Pulex, Archaeopsylla.
[0073] In a preferred embodiment this is, preferably at least one
full or partial epitope of at least one allergen derived from mites
more preferably from the genus Dermatophagoides.
[0074] Examples of allergens of the genus Dermatophagoides are
shown in FIG. 9 (9A and 9B) identifying the species, the allergen
and the UNIPROT accession number.
[0075] In a further preferred embodiment, such at least one
allergen is Der f 15 according to SEQ ID NO: 11 (full) and SEQ ID
NO: 18 (iMAT form). Preferred specific sequences of the antigen
modules are the amino acid sequences according to SEQ ID NOS: 7-23,
preferably SEQ ID NOs: 14-23 (iMAT forms).
[0076] The term "immune response modulated by" or interchangeably
"immunomodulatory immune response", in connection with "(isolated)
recombinant protein" and/or "iMAT molecule" in the course of the
present invention refers to immunogenic and/or tolerogenic immune
responses.
[0077] The term "hybrid iMAT" or "iMAT hybrid" or "mosaic-like
iMAT" are used interchangeably. These terms refer to an iMAT
molecule, which comprises in its third module more than one full or
partial epitope sequence from two or more antigens. Preferably said
antigens are two or more allergens, more preferably two or more
short peptide sequences from different allergens, determining the
specificity of an immune response modulated by such iMAT molecule
(in a subject, preferably an animal).
[0078] The term "allergen" in the course of the present invention
refers to a type of antigen that in the native form produces an
abnormally vigorous immune response in which the immune system
fights off all perceived threats that would otherwise be harmless
to the subject. Typically, these kinds of reactions result in the
phenotype known as allergy. Various types of allergens are
described in the prior art, including foods, drugs, animal products
or natural or synthetic materials. Preferably, a protein is
considered to be an allergen, when it elicits in its native form a
specific IgE response in at least five subjects, preferably
animals, more preferably dogs and/or cats, but excluding equines.
For the avoidance of doubt, an "allergen" in connection with the
"at least one (third) antigen module comprising at least one full
or partial epitope derived from at least one allergen" does not
need to be in the native form any longer, which is also
preferred--in other words, the term "allergen" in the course of the
present invention also explicitly refers to non-native amino acid
sequences as part of the iMAT molecules, as described and claimed
herein, that do not elicit a specific IgE response in at least five
subjects, preferably animals, more preferably dogs and/or cats, but
excluding equines, any longer.
[0079] The term "allergenicity" of a therapeutic allergen is a
measure of the potential to induce adverse events, e.g. provoke
anaphylaxis. Exemplarily for an allergy in mammals, an assay to
measure allergenicity in dogs is described in Griffin et al.
[Griffin C E. Diagnosis of canine atopic dermatitis DOI:
10.1002/9781118738818.ch10]. This document describes measurement of
allergen specific IgE mediated hypersensitivity in procedures as
the allergen provocation tests, in particular such tests targeting
the skin. Intradermal skin tests are used for the biological
evaluation of recombinant allergens and for validation of
genetically engineered hypoallergenic derivatives. Intradermal
testing in a dog is performed by administering injections of small
amounts of allergen solutions directly into the dog's dermis. This
is done with small-gauge (27 gauge) needles and injections of 0.05
to 0.1 mL at each site. The positive reactions are arbitrarily
interpreted by the presence of erythema, turgidity, height, and
size of the wheal. The advantage of the intradermal test is a high
sensitivity. This is of particular importance if the test shall
deliver a quantitative measure for allergenicity. Performing said
test by Griffin et al. with the iMAT molecules according to the
present invention, those iMAT molecules show a 10-, 100- to
1000-times or even higher molar concentration of the allergenic
component as compared to the corresponding natural, native allergen
applied in the same test to reach a positive reaction in sensitized
individuals, as for example cats and dogs.
[0080] The term "epitope", herein also interchangeably referred to
as "antigenic determinant", in the course of the present invention
refers to the part of an antigen that is recognized by the immune
system, either by B-cells or T-cells. Epitopes are presented on the
surface of antigen presenting cells by means of MEW molecules to
which they are bound.
[0081] The term "subject", herein also interchangeably referred to
as "individual" and/or "organism" and/or "host", in the course of
the present invention preferably refers to animals and/or humans,
e.g. ruminants, pigs, more preferably dogs and/or cats, but
excluding equines. The term "ruminant", in the course of the
present invention encompasses ruminating mammals including cattle,
goats, and sheep. Thus, members of the genus Bos, Capra and/or
Ovis, are interchangeably referred to as "bovine", "caprine" and/or
"ovine" species. The term "animal" as used herein includes mammals.
The animal may be selected from the group consisting of ruminants
or members of the genus Canis or interchangeably referred to as
"canine" species (e.g. dogs, wolves, foxes, coyotes, jackals), or
members of the genus Felis or interchangeably referred to as
"feline" species (e.g. lions, tigers, domestic cats, wild cats,
other big cats, and other felines including cheetahs and lynx) or
pigs, i.e. members of the genus Sus interchangeably referred to as
"porcine" species.
[0082] The terms "peptide" and "protein" are used side by side as
equivalent in the course of the present invention. A peptide or a
protein means for the purposes of the present invention a covalent
bond of at least two amino acids via a peptide linkage. The term
"amino acid" and the term "amino acid residue" are used as
equivalents in the present application, i.e. the meaning of the two
terms is identical. The terms amino acid/amino acid residue and
peptide/protein are used in the present application in the form of
the widest possible definition.
[0083] In this connection, the term "recombinant protein" refers to
a polypeptide which may be obtained by genetic engineering and
expression in eukaryotic or prokaryotic systems. In addition, said
term encompasses polypeptides obtained by artificial (e.g.
solid-phase) synthesis.
[0084] As used herein, the term "different amino acid residue"
refers to a known amino acid residue other than cysteine unless
otherwise indicated. For example, said amino acid residue may be a
naturally occurring amino acid residue, such as serine or
isoleucine.
[0085] As used herein, the term "linear form" refers to proteins
according to the present invention that lack secondary structure.
Such proteins are often assumed to exhibit a random-coil
conformation in which the only fixed relationship is the joining of
adjacent amino acid residues by a peptide bond.
[0086] In a preferred embodiment, the (isolated) recombinant
protein as herein disclosed and claimed is present in monomeric
form and/or linear form.
[0087] As used herein, the term "treatment" refers to the
administration of the (isolated) recombinant protein, as disclosed
and claimed herein, and/or the corresponding vaccines and/or
immunogenic compositions and/or pharmaceutical compositions in
order to obtain the desired clinical results including prophylactic
and/or therapeutic treatment.
[0088] As used herein, the term "immunotherapy" refers to a
therapeutic and/or prophylactic treatment of a subject, e.g. by
prophylactic and/or therapeutic vaccination.
[0089] As used herein, the term "vector" in connection with
"transmission of one or more infectious disease(s)" refers to an
alive organism and is herein interchangeably used with terms
"biological vector", "biological carrier" and/or "disease carrier",
such as blood feeding bugs, flies, midges, ticks and/or
mosquitos.
[0090] Furthermore, it is possible, and preferred, that the
(isolated) recombinant protein, as disclosed and claimed herein,
contains in addition at least one tag module. That is, it is
possible, and preferred, that one or more different and/or
identical tag modules are part of the (isolated) recombinant
protein, as disclosed and claimed herein. Tag modules may be short
peptides, frequently consisting of up to 20 amino acids or
functional groups which are not composed of amino acids, such as
for example biotin or digoxigenin. Suitable tag modules include the
well-known and preferred His-tag containing a histidine sequence of
4 to 12 or more, preferably directly consecutive histidine
residues, preferably 5, 6 or 7 consecutive histidine residues.
Other suitable tag modules include HA-tag, FLAG-tag, GST-tag or
Strep-tag. Although the tag can be present anywhere in the
(isolated) recombinant protein, as disclosed and claimed herein, in
a preferred embodiment, the tag module is present at the (quasi)
N-terminus and/or at the C-terminus of the (isolated) recombinant
protein.
[0091] The tag modules are useful for isolating the (isolated)
recombinant proteins, as disclosed and claimed herein, and in
addition allow detecting the presence of such (isolated)
recombinant proteins in vitro or in vivo. Furthermore, the tag
module optionally together with one or more adjacent amino acid
residues from an adjacent module or a linker spacing apart the
different modules can be used in order to induce a specific
immunologically detectable signal, (e.g. an antibody) in the target
subject that can be used as a surrogate marker for immunity and/or
duration of immunity. Immunotherapies with the (isolated)
recombinant proteins, as disclosed and claimed herein, elicit an
antigen-specific, preferably allergen-specific immune response in
the target subjects that is qualitatively indistinguishable from
the natural immune response after exposure to naturally existing
antigens, preferably allergens. Thus, antibodies binding to the
antigen module are not suitable for the purpose of being a
surrogate marker for the efficiency of the iMAT induced immune
modulatory effect. This obstacle can be eliminated by determining
the unique, antigen-specific immunological signal obtained by the
C-terminal and/or (quasi) N-terminal tag module--optionally
together with the adjacent amino acid residues. Hence, it is
possible to provide suitable surrogate markers accordingly.
[0092] In a preferred embodiment, the (isolated) recombinant
protein, as disclosed and claimed herein, further comprises at
least one tag module, preferably at least one His-tag, wherein such
at least one tag module preferably is present N-terminally and/or
C-terminally of the (isolated) recombinant protein, more preferably
N-terminally after one methionine residue.
[0093] Moreover, the modules of the (isolated) recombinant protein,
as disclosed and claimed herein, namely, the at least one
translocation module, the at least one targeting module and the at
least one antigen module may optionally be spaced apart by one or
more spacer modules located between at least two of such
modules.
[0094] The spacer modules may be, in particular, peptide sequences
or organic molecules. Numerous spacer molecules which can be used
for the purposes of the invention are known in the art. In
addition, it is also possible to use spacer molecules which will be
developed or discovered in future for the purposes of the
invention. Suitable spacer modules are, inter alia, peptide
spacers, crosslinkers, natural or synthetic polymers such as, for
example, nucleic acids, substituted or unsubstituted hydrocarbons,
etc.
[0095] The coupling can take place both by covalent (preferred) and
by non-covalent linkages. The spacer modules have the task inter
alia of separating the various modules of the (isolated)
recombinant protein, as disclosed and claimed herein, from each
other in space so that they do not have adverse effects on each
other with regard to their functionality. Modules of the (isolated)
recombinant protein for the purposes of the invention can be
coupled by one or more spacer modules which can be cleaved by
chemical and/or enzymatic reactions, e.g. by proteases. It is thus
possible to separate the modules of the (isolated) recombinant
protein, as disclosed and claimed herein, which are connected by
the spacer modules, from each other as required.
[0096] In a preferred embodiment, however, in particular if the
antigen module is an amino acid sequence derived from at least one
full or partial epitope of at least one antigen being at least one
allergen, no any such additional spacer modules, i.e. no additional
spacer modules between two or more adjacent modules of such first,
second and/or third modules at all are present.
[0097] Any desired arrangement of the individual modules of the
(isolated) recombinant protein, as disclosed and claimed herein, is
in general possible. Each module may be present one or more times
in the (isolated) recombinant protein. The minimum requirement is
the presence of at least one translocation module, at least one
targeting module and at least one antigen module. Additional
modules, such as tag modules, spacer modules, etc. may optionally
be present but do not need to be present. All modules may be
present one or more times in the (isolated) recombinant protein, as
disclosed and claimed herein. If modules are present more than
once, they may be present in the form of identical copies, or
different versions of a module may be present in each case in a
single copy or in more than one copy. It is also possible for
entirely different modules of the same class of modules, e.g. a
His-tag module and a biotin-tag module, to be present in the
(isolated) recombinant protein, as disclosed and claimed herein.
Both modules undertake functionally the same task (tag module) in
the (isolated) recombinant protein, but do not need to have
anything in common in terms of their molecular structure.
[0098] In a preferred embodiment, it is possible that two or more
copies of one of the modules are present in the (isolated)
recombinant protein, as disclosed and claimed herein. That is, two
or more copies of identical or different antigen modules may be
present. Alternatively, the (isolated) recombinant protein may
contain two different antigen modules, for modulating the immune
response in a subject.
[0099] Two or more identical copies of an antigen module in a
recombinant protein may for example cause an enhanced immune
response to such relevant antigen. Two or more different antigen
modules may for example be combined in one (isolated) recombinant
protein in order to modulate simultaneously the immune response
towards two or more different antigens. Two or more different
translocation modules can be used in the (isolated) recombinant
protein, as disclosed and claimed herein. For example, a Tat
sequence and a VP22 sequence can serve to make translocation more
efficient since the translocation of the (isolated) recombinant
protein then takes place efficiently in a broader spectrum of
different cell types or tissue types. It is also possible for
example to use two or more tag modules in one (isolated)
recombinant protein, e.g. a His-tag and a FLAG-tag, in which case
for example the His-tag is used to isolate the recombinant protein
and for example the FLAG-tag serves to detect the (isolated)
recombinant protein. It is possible to use two or more different
targeting modules in one (isolated) recombinant protein, e.g. a
sequence from the invariant chain of the MHC II molecule and as a
further targeting module a mannose 6-phosphate group. For example,
the invariant chain acts as targeting module into the MIICs, and
the mannose 6-phosphate group mediates targeting into the lysosome,
thus it being possible to increase the efficiency of antigen
presentation or the number of different epitopes of the antigen
presented by the antigen-presenting cells overall. In addition, the
iMAT molecule of the present invention may encompass two or more
different invariant chains stemming from identical or different
species, thus, allowing using the proteins according to the present
invention in different species.
[0100] The position of the individual modules within the (isolated)
recombinant proteins, as disclosed and claimed herein, can also be
varied as desired, as long as at least one translocation module, at
least one targeting module and at least one antigen module are
present. It is also possible for all or some of the modules of the
(isolated) recombinant protein for example to be present not in the
form of a linear sequential arrangement of modules, but as circular
or as branched module structure or else in the form of dendrimers,
or as a combination of linear and/or branched and/or circular
and/or dendrimeric molecule portions. There are commercial
suppliers of expression vectors which supply specific vectors which
make it possible to prepare circular fusion proteins by these
mechanisms, such as, for example, the IMPACT''-TWIN system from New
England Biolabs, Beverly, Mass., USA. Branched modules might be
prepared for example by synthesizing peptides in which, starting
from poly L-lysine, a new lysine residue is attached to both free
amino groups of each of the subsequent lysine residues. In this way
it is possible to create a peptide structure with virtually any
extent of branching. It is then possible to synthesize, for
example, translocation modules and/or targeting modules
subsequently onto the branched peptide basic structure. Further
modules can also be coupled onto a linear, circular or branched
peptide basic structure by protein ligation. It is also possible to
introduce for example biotin groups into the peptide basic
structure during the peptide synthesis, and modules can then be
attached to these biotin groups via, for example, streptavidin, the
Strep tag system or via the PINPOINT.TM. system (respectively IBA
GmbH, Gottingen, Germany and Promega Biosciences Inc., San Louis
Obispo, Calif., USA). Modules attached in this way are then coupled
via non-covalent linkages to the peptide's basic structure.
[0101] The antigen module of iMAT molecules according to the
present invention can be selected by a bioinformatics approach as
described exemplarily and in detail in Examples 5 and 6 herein. By
this means iMAT molecules can be rendered useful specifically as
vaccines, e.g. for therapy and/or prevention of allergic diseases
on the bases of the participation of allergen specific IgE mediated
hypersensitivity reactions in different target organs as skin,
respiratory--as well as gastrointestinal system, like atopic
dermatitis (AD), food allergies and/or allergic asthma in subjects,
preferably animals, more preferably dogs and/or cats, but excluding
equines.
[0102] In a preferred embodiment, the (isolated) recombinant
protein relates to, comprises, preferably consists of one or more
of the amino acid sequences according to SEQ ID NOs:24 to 83.
[0103] The (isolated) recombinant proteins, as disclosed and
claimed herein, are in particular useful in a method for
specifically addressing the therapy and/or prophylaxis in subjects,
such animals, more preferably dogs and/or cats, but excluding
equines, suffering from allergic diseases on the bases of the
participation of allergen specific IgE mediated hypersensitivity
reactions in different target organs, e.g. skin, respiratory--as
well as gastrointestinal system.
[0104] In veterinary medicine allergies are of major importance in
particular in the field of companion animals.
[0105] Dogs and cats suffer from such diseases, as e.g. canine
atopic dermatitis or feline atopic dermatitis or feline asthma.
[0106] Canine or feline atopic dermatitis (AD) has been defined as
a genetically predisposed inflammatory and pruritic allergic skin
disease with characteristic clinical features. It is most commonly
associated with IgE antibodies to environmental allergens. The
atopic phenotype can be seen in animals with IgE-mediated skin
disease, food allergy, or a condition called "atopic-like
dermatitis" (ALD). ALD is defined as a pruritic skin disease in
dogs with characteristic features of AD but negative tests for IgE
antibodies. Feline atopic dermatitis has many similarities to
canine atopic dermatitis. Common clinical signs in canine atopic
dermatitis include a history of seasonal or non-seasonal pruritus,
otitis externa, recurrent and chronic inflammatory dermatitis
especially in the axillary, inguinal, and flexor skin surfaces,
recurrent bacterial infections, face rubbing and/or foot licking
and chewing.
[0107] The etiology and pathogenesis of AD is complex and involves
a genetic predisposition, impairment of the normal barrier function
of the skin, and immunologic aberrations. Animals with AD are
thought to be genetically predisposed to become sensitized to
allergens in the environment. Allergens are proteins that, when
inhaled or absorbed through the skin, respiratory tract, or GI
tract, evoke allergen-specific IgE production. These
allergen-specific IgE molecules affix themselves to tissue mast
cells or basophils via the FCC receptors on such cells. When these
primed cells come in contact with the specific allergen again, mast
cell degranulation results in the release of proteolytic enzymes,
histamine, bradykinins, and other vasoactive amines, leading to
inflammation (erythema, edema, and pruritus). The skin is the
primary target organ in dogs and cats, but rhinitis and asthma can
also occur in .about.15% of affected animals.
[0108] Mites are known as major causes of allergic diseases such as
atopic dermatitis and asthma in e.g. dogs and cats. Conventionally,
desensitization therapy that uses causative substances of allergies
as therapeutic agents for allergic diseases is regarded as the most
important basic remedy. In particular, the desensitization therapy
is broadly conducted for diseases such as pollinosis, house dust
allergies, and fungal allergies, which are induced by antigens such
as inhalant allergens that are difficult to avoid. However, the
desensitization therapy involves the risk of adverse events in
particular anaphylaxis due to the action of sensitizing antigens,
so that administration of safe therapeutic antigens like iMAT
molecules is required.
[0109] Regarding mite allergic diseases, several species have been
described to be of relevance Dermatophagoides pteronyssinus,
Dermatophagoides farinae, Euroglyphus maynei, Dermatophagoides
siboney, Dermatophagoides microceras, Lepidoglyphus destructor,
Blomia tropicalis, Tyrophagus putrescentiae, Glycophagus
domesticus, Acarus siro. However, two types of mites,
Dermatophagoides pteronyssinus and Dermatophagoides farinae, have
been reported as main allergen sources in house dust (Thomas, W R.
et al., Chang Gung Med J 2004; 27:563-569). Major mite allergens
have been fractionated from these mites.
[0110] The group 1 and 2 allergens of Dermatophagoides sp. induce
high titers of IgE and Th2 cytokines in 80% of allergic patients.
The allergens Der p 3, 5, 6, 7 and 8 induce IgE in about 50% of
subjects usually at lower titers. The 92/98 kDa paramyosin (group
11) allergens binds IgE in 80% of allergic subjects and the 98 and
60 kDa chitinase enzymes (Der f 15 and 18) bind IgE from about 70%
and 54% of allergic subjects and are important allergens for
allergic dogs (McCall C et al., Vet Immunol Immunopath 2001; 78:
231-247).
[0111] In general, a variety of environmental allergens as pollens
of grasses, trees, weeds, house dust, dust and storage mites and
mold and/or mold spores, but also epidermal and insect antigens
have been described to contribute to the sensitization of dogs in
canine atopic dermatitis (Hill et al. Vet Immunol Immunopathol,
2001; 81(3-4):169-186).
[0112] As described above--the atopic phenotype may also be induced
by food allergies, a hypersensitivity with a number of clinical
manifestations. Besides gastrointestinal alterations (e.g.
gastroenteritis, diarrhea or vomiting), food hypersensitivity often
manifests in animals as a pruritic dermatitis and/or dermatosis of
the face and neck, miliary dermatitis, generalized scaling or
symmetrical alopecia. Especially in cats all the entities of the
eosinophilic granuloma may be a consequence of hypersensitivity to
certain food allergens.
[0113] The most common food allergens derive from meat, milk, fish,
but also soybeans and/or more in general tinned food and dried
food. Especially in the latter also involvement of allergies to
additives and/or storage mites are reported (Guaguere E et al.
EJCAP, 2009, 19 (3), 234-241; Jackson H A, EJCAP, 2009, 19 (3),
230-233).
[0114] Currently veterinary therapeutic options are restricted to
symptomatic (e.g. corticoid) treatments and/or eliminating the
foodstuff(s) responsible. However, care must be taken to not
adversely affect the nutritional balance of the diet. More recently
diets containing hydrolyzed proteins are available. Proteins are
broken down--thus being less allergenic, which is effective and
well-tolerated. However, these diets tend to be costly and little
palatable, which can be a mayor limitation for compliance of the
animals.
[0115] Allergen specific immunotherapy to treat food allergic
animals, preferably dogs and/or cats has not yet been applied.
[0116] In a further embodiment the (isolated) recombinant proteins,
as disclosed and claimed herein, are in particular useful as a
method for specifically addressing the therapy and/or prophylaxis
in cats suffering from allergic airway inflammation and/or
obstruction (allergic asthma).
[0117] Cats spontaneously develop eosinophilic airway inflammation
and airway hyper-reactivity that is very similar to human allergic
asthma, i.e. feline allergic asthma is a chronic inflammatory
disorder of the lower airways that may manifest with acute,
life-threatening clinical signs.
[0118] Typical treatment involves palliative treatment only (e.g.
bronchodilators and/or corticosteroids), but currently no causative
treatment is available. Some pilot studies of the university group
around Carol Reinero have addressed allergen specific immunotherapy
in an animal model of induced Bermuda grass allergic asthma in
cats.
[0119] Feline allergic asthma is a complex disease, but clearly
exposure to airborne allergens plays a pivotal role in the
etiology. Clinical remission can be achieved by eliminating the
exposure to the aeroallergens. Though, the major antigens involved
in triggering feline allergic asthma have not been clearly
identified so far. Numerous potential agents are present in the
cat's habitual environment e.g. pollen, molds, dust from cat
litter, perfumes, room fresheners, carpet deodorizers, hairspray,
aerosol cleaners or cigarette smoke. When screened using serum or
intradermal skin tests, naturally allergic cats kept in-house had
IgE reactivity to many of the same allergens implicated in human
allergic asthma, i.e. mainly to house dust and storage mites and/or
pollen (Prost C, Rev Fr Allergol Immunol Clin, 2008, 48 (5),
409-413).
[0120] In a further embodiment the (isolated) recombinant proteins,
as disclosed and claimed herein, are in particular useful in a
method for specifically addressing the therapy and/or prophylaxis
in cats and/or dogs suffering from AD caused by flea bites
(FAD).
[0121] FAD is one of the most severe skin allergies caused by flea
infestations in dogs and cats. FAD can have manifestations of both
immediate and delayed-type hypersensitivity. Typically, an
immediate hypersensitive response in an animal susceptible to FAD
includes wheal formation at the site of a fleabite. Such wheals can
develop into a papule with a crust, representative of delayed-type
hypersensitivity. Hypersensitive reactions to fleabites can occur
in genetically pre-disposed animals as well as in animals
sensitized by previous exposure to fleabites. Furthermore, flea
bites can cause scratch-related secondary infections as a
consequence of the inflammatory irritation of host's erythema,
papules, crusts, and alopecia. Previous work found that pets rarely
became desensitized to bites of flea once they have been made
allergic to them. So besides the elimination of the flea, the
alleviation of the animal's distress becomes the challenging
problem. Current therapies for this disease include desensitization
therapy or using some types of pharmacological intervention.
However, each of these therapeutic approaches has some
disadvantages. For example, anti-histamine medications could
increase drowsiness, dry mouth, difficulty in urination, and
constipation; whereas, classical desensitization therapy may cause
life-threatening anaphylactic shock. Other disadvantages of these
therapies are the high possibility of recrudescence and requirement
for long-term treatments. Therefore, novel, effective therapeutic
approaches are needed and should be developed in order to overcome
unwanted adverse reactions. Effective treatment of FAD has been
difficult if not impossible to achieve. FAD afflicts about 15% of
cats and dogs in flea endemic areas and the frequency is increasing
each year. In a geographical area, effective flea control requires
treatment of all animals. One treatment investigators have proposed
includes desensitization of animals using flea allergens. However,
reliable, defined preparations of flea allergens are needed for
such treatments. Whole flea antigen preparations have been used to
diagnose and desensitize animals with FAD. Available commercial
whole flea extracts, however, comprise only as a minor part saliva
proteins and thus are unpredictable in their specific allergen
content and, therefore, have limited usefulness. Prior art U.S.
Pat. No. 7,629,446 as well as McDermott M J et al. (Molecular
Immunology 2000, 37: 361-375) describe the discovery of the
allergen Cte f 1 as major allergen in FAD. In this publication,
they describe the cloning of the cDNA and characterization of a
flea saliva protein, Cte f 1, a major allergen for flea allergic
dogs and cats. Native Cte f 1 has a calculated molecular weight of
18 kDa and p1 of 9.3. Mass spectrometry analysis indicates that the
native molecule has no post-translational modifications and that
all of the 16 cysteines are involved in intramolecular disulfide
bonds. However, in the same publication the authors show several
isoforms of the recombinant protein. The 16 cysteines in the
secreted protein lead to the occurrence of these isoforms which
renders purification and thus manufacturing of such a product
difficult or even impossible under GMP conditions.
[0122] In research dogs experimentally sensitized to flea bite, Cte
f 1 is a major allergen. Using E. coli produced rCte f 1 as the
antigen in intradermal skin test and in solid phase ELISA, IgE can
be detected in 100% of these experimentally sensitized FAD dogs. In
addition, competition ELISA performed using sera from 14 sensitized
dogs demonstrated that rCte f 1 produced in three different
expression systems (E. coli, P. pastoris and baculovirus infected
insect cells) could inhibit approximately 95% of the binding of
antigen specific IgE to native Cte f1.
[0123] The therapeutic potential of immunotherapeutic approaches
comprising the Cte f 1 have been demonstrated by J. Jin et al. (Jin
J. et al. Vaccine 28 (2010) 1997-2004). They reported that the
simultaneous co-immunization with a DNA vaccine and its cognate
coded protein antigen (Cte f 1) exhibit the potential to protect
animals from FAD in a murine model. Furthermore, in their study
they clinically tested this protocol to treat established FAD in
cats following flea infestations. They presented data showing
therapeutic improvement of dermatitis in these FAD cats following
two co-immunizations.
[0124] iMAT molecules comprising the modified Cte f 1 sequence
substituting the cysteines with other amino acid residues can be
included into the iMAT molecule as allergen module together with
either the cat or the dog invariant chain in order to achieve an
optimized species specific immune-modulatory effect. Surprisingly
these molecules (i) allow efficient recombinant protein production
in suitable expression systems, (ii) have a substantially reduced
safety risk since effector cells as mast cells are activated at
much higher concentrations as compared to the unmodified Cte f 1
and (iii) induce a sustained immunological effect and a long
lasting clinical improvement after only three injections.
[0125] In a further embodiment the (isolated) recombinant proteins,
as disclosed and claimed herein, are in particular useful in a
method for specifically addressing the therapy and/or prophylaxis
in animals, more preferably ruminants, pigs, dogs and/or cats, but
excluding equines, suffering from infectious diseases caused by
bacteria.
[0126] The Gram-negative bacterium Campylobacter is the most common
bacterial cause of gastroenteritis in domesticated animals, e.g.
dogs, cats, pigs, ruminants. Clinical signs are mostly more severe
in young mammals. Besides enteritis also abortions and infertility
in various species has been reported. The infection is primarily
through ingestion--on entry the bacterium needs to overcome the
host defense with the expression of a variety of colonization and
virulence determinants. A number of those are antigenic surface or
outer membrane proteins, their interaction with e.g. epithelial
cells of the gastrointestinal tract is essential for colonization,
i.e. infection of the host.
[0127] In order to solve this medical problem, the at least one
antigen module in a campylobacteriosis dedicated (isolated)
recombinant protein, as disclosed and claimed herein, may be
selected from antigens derived from Campylobacter spp. e.g.
flagellin, surface-exposed proteins (CadF and PEB1), or other
surface proteins. Improved MAT molecules may be useful specifically
as vaccines, e.g. for therapy and/or prevention of
campylobacteriosis. The treatment according to the present
invention can involve the administration of the iMAT molecule to
the offspring and/or can also comprise a treatment of the pregnant
mother.
[0128] In a further embodiment the (isolated) recombinant proteins,
as disclosed and claimed herein, are in particular useful in a
method for specifically addressing the therapy and/or prophylaxis
in animals more preferably ruminants, pigs, dogs and/or cats, but
excluding equines, suffering from infectious diseases caused by
viruses.
[0129] For example, West Nile virus (WNV) is a mosquito-transmitted
positive-stranded RNA virus grouped within the Japanese
encephalitis virus serocomplex of the genus Flavivirus in the
family Flaviviridae. The WNV is the causative agent of the disease
syndrome also named West Nile Fever. Birds are the natural
reservoir hosts, and WNV is maintained in nature in a
mosquito-bird-mosquito transmission cycle. However, man, horses,
dogs, cats, but also ruminants have been described to be
susceptible. Though most WNV infections remain clinically
latent--WNV being a potentially neuroinvasive virus, it may cause
meningitis or encephalitis. In animals WNV remains frequently
unrecognized, but animals might be euthanatized due to severe
neurologic signs caused by WNV--including paresis, ataxia,
recumbency, and muscle fasciculation, whereas others exhibit mild
to severe polioencephalomyelitis.
[0130] In a further embodiment the (isolated) recombinant proteins,
as disclosed and claimed herein, are in particular useful in a
method for specifically addressing the prevention of transmission
of infectious diseases in animals, more preferably ruminants, pigs,
dogs and/or cats, but excluding equines, by vectors, e.g. blood
feeding bugs, flies, midges, ticks and/or mosquitos.
[0131] The pathogens are delivered into the skin of the mammalian
host along with arthropod saliva, which contains a wide variety of
bioactive molecules. These saliva components are capable of
altering hemostasis and immune responses and may contribute to the
ability of the pathogen to induce an infection. The presence of
infectious microorganisms in the salivary glands of blood-feeding
arthropods itself alters saliva composition, such as changes in the
concentration of e.g. apyrase or anti-thrombinase in infected
mosquitoes. Vector-associated or saliva components can e.g. alter
vaso-activity and/or modulate the immune response of a host and be
of crucial importance for transmission of infectious diseases.
Vaccination of the host against arthropod saliva components can
interfere with viral transmission, as shown for sandfly salivary
proteins and transmission of leishmania spp. These vaccines have
however not passed preclinical research up to date (WHO PD-VAC
2014--Status of Vaccine Research and Development of Vaccines for
Leishmaniasis).
[0132] Arthropod saliva antigens are well suited to be employed in
the antigen module of iMAT molecules according to the present
invention and may be useful specifically as vaccines to activate
the host immune system to prevent the transmission of infectious
pathogens by vectors e.g. viruses, fungi and/or parasites and/or
their pre-patent stages in animals, more preferably ruminants,
pigs, dogs and/or cats, but excluding equines.
[0133] In a further embodiment the (isolated) recombinant proteins,
as disclosed and claimed herein, are in particular useful in a
method for specifically addressing the therapy and/or prophylaxis
in animals, more preferably ruminants, pigs, dogs and/or cats, but
excluding equines, suffering from infectious diseases caused by
fungi.
[0134] Dermatophytosis or ringworm is a fungal infection of the
hair and of the superficial keratinized cell layers of the skin
occurring in animals and man. Several species of genus Microsporum
or genus Trichophyton belonging to the groups of zoophilic or
geophilic dermatophytes can cause clinical infections in mammals. A
variety of surface antigens of the fungi and/or their spores are
well suited to be employed in the antigen module of iMAT molecules
according to the present invention and may be useful specifically
as vaccines, e.g. for therapy and/or prevention of dermatophytosis
in animals, more preferably ruminants, pigs, dogs and/or cats, but
excluding equines.
[0135] In a further embodiment the (isolated) recombinant proteins,
as disclosed and claimed herein, are in particular useful in a
method for specifically addressing the therapy and/or prophylaxis
in animals more preferably ruminants, pigs, dogs and/or cats, but
excluding equines, suffering from infectious diseases caused by
parasites.
[0136] Parasites infecting mammals are ubiquitous and clinically
important across the world. The major parasitic threats to
ruminants, pigs, dogs and/or cats are e.g. Cooperia, Haemonchus,
Ostertagia, Trichostrongylus, Dictyocaulus, Metastrongylus.
Increasing levels of anthelmintic resistance is reported worldwide
in parasites. For protozoa, e.g. Cryptosporidium few drugs
consistently inhibit parasite infestation and/or reproduction in
the host. Mainly neonatal or young mammals are affected and outcome
relies on innate and adaptive immune responses.
[0137] Antigens deriving from adult parasites as well as prepatent
stages are another example to be employed in the antigen module of
iMAT molecules according to the present invention and may be useful
specifically as vaccines, e.g. for therapy and/or prevention of
parasite infection in animals, more preferably ruminants, pigs,
dogs and/or cats, but excluding equines.
[0138] The treatment according to the present invention can involve
the administration of the iMAT molecule to the offspring and/or can
also comprise a treatment of the pregnant mother.
[0139] In particular, in case of modulating the immune response in
canine, feline, bovine, ovine, caprine or porcine species the at
least one targeting module preferably is the respective invariant
chain.
[0140] In a preferred embodiment the at least one targeting module
is the canine invariant chain according to SEQ ID NO: 2 or 4. In
another preferred embodiment the at least one targeting module is
the feline invariant chain according to SEQ ID NO: 3 or 5.
[0141] In an advantageous embodiment, the (isolated) recombinant
protein, as disclosed and claimed herein, is present in a monomeric
form since, for instance, recombinant allergens tend to aggregate
formation, particularly, if produced via inclusion bodies. By
substituting at least one, preferably all cysteine residues in the
entire sequence of the (isolated) recombinant protein, preferably
by substituting for serine, leucine, isoleucine, arginine,
methionine and/or aspartic acid, it is possible to prevent
intermolecular disulfide bond formation, thus, avoiding any
aggregation, in particular, any non-native formation of inter-
and/or intramolecular bonds. That is, the (isolated) recombinant
protein being entire devoid of cysteine residues does not
aggregate. Consequently, the protein is easily to express and
demonstrates improved targeting and MHC presentation.
[0142] Furthermore, such cysteine-free variants of, for instance,
allergens, in which the cysteine residues in the amino acid
sequence of wild-type allergens have been mutated solely or in
combinations, show a reduced IgE reactivity compared to the
corresponding wild-type allergens and at the same time have
substantially retained reactivity towards T-lymphocytes and are
thus hypoallergenic.
[0143] The invention accordingly relates to such hypoallergenic
variants of allergens eliciting for example allergies to flea
bites; to certain food components and/or atopic dermatitis and/or
allergic airway inflammation and/or obstruction in animals, more
preferably ruminants, pigs, dogs and/or cats, but excluding
equines, wherein in the variants the cysteine residues of wild-type
allergens have been mutated solely or in combination.
[0144] Furthermore, the presence of a tag module for separating
proteins in a sample, comprising the iMAT molecules according to
the present invention, e.g. using zinc- or cobalt-charged solid
support further improve the possibility to produce fusion proteins
without aggregates during the purification process. Typically, the
tag module includes a polyhistidine tag of five to six consecutive
histidine residues.
[0145] The (isolated) recombinant proteins, as disclosed and
claimed herein, are useful in a pharmaceutical composition. For
example, the (isolated) recombinant proteins are for use in a
vaccine. Hence, the present invention provides vaccine compositions
containing one or more (isolated) recombinant proteins, as
disclosed and claimed herein. Such vaccine composition can be used
therapeutically and/or prophylactically in animals, more preferably
dogs and/or cats, but excluding equines, suffering from allergic
diseases on the basis of participation of allergen specific IgE
mediated hypersensitivity reactions in different target organs such
as skins, respiratory and gastrointestinal systems; or such vaccine
composition can be used therapeutically and/or prophylactically in
animals, more preferably ruminants, pigs, dogs and/or cats, but
excluding equines, suffering from infectious diseases induced by
pathogens, e.g. viruses, fungi and/or parasites and/or their
prepatent stages. Additionally, the method is not only directed
against such pathogens, but also capable to activate the host
immune system to prevent the transmission of a disease by vectors,
e.g. blood feeding bugs, flies, midges, ticks and/or mosquitos.
[0146] Thus, in a preferred embodiment of the present invention a
vaccine for subjects, such as animals, more preferably dogs and/or
cats, but excluding equines, is provided in order to treat and/or
prevent atopic dermatitis and/or allergic asthma caused by a
response of e.g. exposure to Dermatophagoides mites.
[0147] In a further embodiment of the present invention a vaccine
for subjects, such as animals, more preferably cats, but excluding
equines, is provided in order to treat and/or prevent allergic
asthma caused by a response to e.g. mold (fungi and/or their
spores), pollen, house dust or forage mites (and/or their
feces).
[0148] In a further embodiment of the present invention a vaccine
for subjects, such as animals, more preferably ruminants, pigs,
dogs and/or cats, but excluding equines, is provided in order to
treat and/or prevent infectious diseases involving e.g. the genera
Campylobacter, Dirofilaria, Ehrlichia, Leishmania, Trypanosoma,
Borrelia, Orthobunyavirus, Orbivirus, Flavivirus, Rotavirus,
Coronavirus, Trichophyton, Microsporum; other helminths like
Cooperia, Haemonchus, Ostertagia, Trichostrongylus, Dictyocaulus,
Metastrongylus; and/or protozoa with gastrointestinal infestation
like Eimeria, Isospora, Cryptosporidium, Giardia--in case of
parasites also antigens derived from the prepatent stages might be
employed. Additionally, the vaccine can be provided to activate the
host immune system to prevent transmission of diseases by vectors,
e.g. belonging to the families Culicidae, Ceratopogonidae,
Phlebotominae Ixodidae and/or Cimicidae and/or other blood feeding
insects.
[0149] The pharmaceutical composition, e.g. in form of a vaccine,
of the (isolated) recombinant proteins is preferably designed for
sublingual administration, subcutaneous and/or intradermal
injection, injection into a lymph node and/or for administration
via the mucous membranes, in particular, via the mucous membranes
of the gastrointestinal tract or of the respiratory system.
[0150] In a preferred embodiment of the present invention, the
pharmaceutical compositions are parenterally administered.
[0151] The iMAT molecules according to the present invention can be
used as a pharmaceutical or as a vaccine to modify, for instance,
allergic disorders. For example, atopic dermatitis and/or allergic
asthma can be treated by such iMAT molecules.
[0152] Low amounts (1 to 1000 .mu.g referring to the weight of
solely the one or more antigen modules) of recombinant iMAT
molecules comprising allergens of atopic dermatitis and/or allergic
asthma eliciting from mites of the genus Dermatophagoides e.g.
injected 1 to 5 times subcutaneously, intradermally or directly
into the lymph node, induce a strong and long lasting immune
response in cat and/or dog leading to prevention of the disease
and/or an amelioration of clinical symptoms.
[0153] In one preferred embodiment, the iMAT molecules of the
present invention are administered in combination with at least one
adjuvant. The adjuvant includes, but is not limited to, one or more
of the following: alum, BCG, aluminium hydroxide, aluminium
phosphate, calcium phosphate, lipid emulsions, lipid or polymeric
nano- or microspheres, micelles, liposomes, saponin, lipid A, or
muramyl dipeptide, bacterial products, chemokines, cytokines and
hormones, chitosan, starch, alginate, cellulose derivatives (e.g.,
ethyl cellulose, hydroxypropylmethyl cellulose), nucleic acids, or
a nucleic acid construct. One or more of these components may be
added to enhance or modify the immune response. Alternatively, the
iMAT molecule may be administered without an adjuvant or in an
aqueous form.
[0154] The iMAT molecules may be administered in a dose of about 1
.mu.g to 1000 .mu.g (this and the subsequent doses referring to the
weight of solely the one or more antigen modules) and more
preferably in a dose from about 10 .mu.g to about 100 .mu.g and
even more preferably in a dose from about 20 .mu.g to about 50
although the optimal dose may vary depending on the antigen,
preferably allergen, being injected, the weight of the subject, the
immune system of the subject, and alike. Effective treatment in
many cases may be accomplished with one administration. In some
embodiments, treatment includes 1 to 15 administrations. In
preferred embodiments, treatment includes 1 to 5 administrations
and more preferably 1 to 3 administrations. For initial treatment
administrations may be periodically, e.g. over a course of days,
once or twice per month or year, or several times a year. For
maintenance of immune response, administrations may be done in
intervals of several months to several years.
[0155] In a preferred embodiment of the present invention, the
(isolated) recombinant proteins, as disclosed and claimed herein,
are designed for lymphatic intranodal administration. In the course
of direct injection into a lymph node, the respective lymph node
may be visualized during the injection procedure e.g. by
ultrasound, in order to monitor the location of the needle and
changes in the lymph node, such as swelling. Injection into the
mandibular, axillary, inguinal and/or popliteus lymph nodes is
preferred due to ease of ultrasound guided location and
injection.
[0156] It is known in the art that several of the identified
proteins from mites in feces and from whole mite bodies induce IgE
reactivity and pathological dermal and respiratory reactions in
dogs and/or cats [Allergome (www.allergome.org)]. Thus, it is
expected that a treatment can only be successful, if most of the
relevant allergens are included in a medicine for specific
immunotherapy. However, surprisingly only 1, 2, 3 or 4 of such iMAT
molecules according to the present invention each comprising
different antigen modules e.g. of mites or a mosaic-like construct
of epitopes are sufficient to induce an immunomodulation and/or
clinical improvement of the diseased subjects if such iMAT
molecules are injected 1 to 5 times subcutaneously, intradermally
and/or directly into the lymph node.
[0157] In a preferred embodiment, a single iMAT molecule is
employed and is sufficient for the induction of a therapeutic
effect and/or prevention of development of allergen specific IgE
mediated hypersensitivity reactions in different target organs as
skin, respiratory and/or the gastrointestinal system e.g. in
animals, more preferably dogs and/or cats, but excluding equines.
Prevention and/or therapy of infectious diseases and/or prevention
of transmission of infectious diseases by vectors can be achieved
in animals, preferably ruminants, pigs, dogs and/or cats, but
excluding equines.
[0158] In another preferred embodiment, a combination of 2, 3 or 4
iMAT molecules is employed by means of simultaneous, sequential
and/or a chronologically staggered co-administrations.
[0159] In a preferred embodiment, a single iMAT molecule is
employed and is sufficient for the induction of a therapeutic
effect and/or prevention and/or prevention of transmission of
infectious diseases in animals, more preferably ruminants, pigs,
dogs and/or cats, but excluding equines, e.g. induced by viruses,
fungi and/or parasites and/or their prepatent stages and/or the
transmission of such infectious diseases by e.g. blood feeding
bugs, flies, midges, ticks and/or mosquitos.
[0160] In another preferred embodiment, a combination of 2, 3 or 4
iMAT molecules is employed by means of simultaneous, sequential
and/or a chronologically staggered co-administrations.
[0161] Depending on the thermodynamic evaluation of the (isolated)
recombinant protein, as herein disclosed and claimed, stability is
influenced by the cysteine mutation, namely the substituting
different amino acid residue(s). For example, the substitution may
be a Cys to Ser substitution. However, the stability may be higher
using a different amino acid residue than the Ser amino acid to
achieve the desired stability and solubility. That is, while a
first choice may be substitution of Cys with Ser, in case of
instability other amino acid residues than Ser should replace
Cys.
[0162] In order to select stabilizing amino acid residues as
replacements for cysteine residues in targeted sequences a three
step approach is chosen: [0163] 1. Modeling the tertiary structure
of the targeted protein including terminal Hexa-Histidine tags,
iMAT sequence and the primary amino acid sequence of the protein of
interest. Modeling can be conducted with the native sequence and
with cysteine substitutions. [0164] 2. Iterative determination of
protein stabilities based on single point substitutions, such as
substitution of a cysteine residue with a different amino acid
residue, e.g. Ser and/or Ile, and scoring to determine stabilizing
replacements by analyzing all available three dimensional
structures. [0165] 3. Re-Modeling of the stabilized structure and
validation of the stability by repeating step 1 and 2.
[0166] Three-dimensional protein structures are crucial for
understanding protein function on a molecular level and are of
great interest for the rational design of many different types of
biological experiments, such as site-directed mutagenesis. However,
the number of structurally characterized proteins is small compared
to the number of known protein sequences. It is possible to
identify a homologous protein with a known structure (template) for
a given protein sequence (target). In these cases, homology
modeling has proven to be the method of choice to generate a
reliable 3D model of a protein from its primary amino acid
sequence. Building a homology model comprises four main steps: (1)
identification of structural template(s), (2) alignment of target
sequence and template structure(s), (3) model building and (4)
model quality evaluation. These steps can be repeated until a
satisfying modeling result is achieved. In cases where no accurate
homologue model can be determined, computational approaches to
determine a protein structure from the primary structure (amino
acid sequences) are used. "De novo" or "ab initio" methods are
based on physical principles and try to imitate the folding
process. Such methods have to sample a large number of
conformations and require very accurate energy functions to
identify structures in the global minima of free energy. Many
methods use a combination of these described principles.
[0167] The availability of computational tools yielding reasonably
accurate estimations of the impact of amino acid substitutions on
the stability of proteins is of crucial importance in a wide range
of applications. In particular, such tools have the potential of
stimulating and supporting protein engineering and design projects
dedicated to the creation of modified proteins.
[0168] Protein stability can be regarded in terms of the
thermodynamic stability of a protein that unfolds and refolds
rapidly, reversibly. In these cases, the stability of the protein
is the difference in Gibbs free energy between the folded and the
unfolded states. The only factors affecting stability are the
relative free energies of the folded and the unfolded states. The
larger and more positive the folding free energy difference is, the
more stable the protein is to denaturation. The folding free energy
difference is typically small, of the order of 5-15 kcal/mol for a
globular protein. However, on the other hand, protein stability can
be regarded as a protein property to withstand harsh temperature or
solvent conditions. This is related to the thermodynamic stability
but also to reversibility or irreversibly of folding/unfolding
(kinetic stability).
[0169] To predict the thermodynamic stability changes caused by
single site substitutions in proteins, several different approaches
can be applied to study the impacts of such substitutions on
protein structure and function [Pires D E et al., Bioinformatics
2014, 30(3):335-342]. Such approaches can be broadly classified
into those that seek to understand the effects of substitutions
from the amino acid sequence of a protein alone, and those that
exploit the extensive structural information. Structure-based
approaches typically attempt to predict either the direction of
change in protein stability on substitutions or the actual free
energy value (.DELTA..DELTA.G).
[0170] The results for each specific protein and its corresponding
models are statistically analyzed in terms of the number of
appearances of specific substitutions using a scoring system, that
grades the replacement based on occurrences in used models and the
protein stability free energy change (.DELTA..DELTA.G) thereby
determining most destabilizing residues (if any) within the input
structure and possible replacements. The score is calculated by
determination of the lowest .DELTA..DELTA.G (.DELTA..DELTA.G<0)
at each position of interest in each model and assigning
corresponding linear values and cumulative .DELTA..DELTA.G's values
for each potential replacement position and results are than
evaluated to determine consistency among models. Since calculated
protein models possess different qualities (the probability of
predicting the correct three-dimensional structure) a weighting
factor can be implemented to prioritize results from more accurate
models. A significant (based on the .DELTA..DELTA.G) destabilizing
residue (if any) is substituted; new models are generated and
re-analyzed iteratively until a steady state is reached.
Additionally, generated models are evaluated by analyzing the xyz
coordinates via principal component analysis to determine potential
structural misfoldings due to replacement of amino acids at
specific positions.
[0171] In yet a further aspect, the objective underlying the
invention has surprisingly been solved by providing a method of
identifying amino acid substitutions in a predetermined amino acid
sequence allowing the stabilization of such predetermined amino
acid sequence comprising the steps of: [0172] i. modeling of the
three-dimensional/tertiary structure of the targeted predetermined
amino acid sequence, [0173] ii. iterative determination of protein
stabilities based on single point substitutions, such as
substitutions of cysteine residues with different amino acid
residues, preferably serine and/or isoleucine residues, and a
scoring system based on the protein stability free energy changes
(.DELTA..DELTA.G) to determine stabilizing substitutions by
analyzing all available three-dimensional/tertiary amino acid
sequence structures, [0174] iii. re-modeling of the substituted
three-dimensional/tertiary amino acid sequence structure and
calculation of its stability by repeating steps (ii) and (iii)
until a steady state is reached;
[0175] In yet a further aspect, the objective underlying the
invention has surprisingly been solved by providing an (isolated)
recombinant protein as herein disclosed and claimed, wherein its
full or partial amino acid sequence was stabilized by the method of
identifying amino acid substitutions in a predetermined amino acid
sequence as herein disclosed and claimed (see Example 5 as well as
Example 6 herein).
Sequences
[0176] The following protein sequences are detailed and disclosed
hereby in the present invention (aa or AA being short for amino
acids):
SEQ ID NO: 1 relates to a suitable minimal amino acid sequence for
one translocation module according to the present invention which
is still being functional, i.e. capable of effectively promoting
cell entry->TAT sequence;
SEQ ID NOs: 2-5 Invariant Chains (Full Sequences and 110aa):
[0177] SEQ ID NO: 2 relates to the full canine invariant chain
amino acid sequence;
>gi|545496086|Canis_lupus_familiaris|XP_536468.5| PREDICTED: HLA
class II histocompatibility antigen gamma chain isoform X1 [Canis
lupus familiaris] SEQ ID NO: 3 relates to the full feline invariant
chain amino acid sequence;
>gi|410949651|Felis_catus|XP_003981534.1| PREDICTED: HLA class
II histocompatibility antigen gamma chain isoform X1 [Felis catus]
SEQ ID NO: 4 relates to the first 110 aa of the canine invariant
chain amino acid sequence; SEQ ID NO: 5 relates to the first 110 aa
of the feline invariant chain amino acid sequence; SEQ ID NO: 6
relates to the N-Terminal Marker of 22 aa
SEQ ID NOs: 7-13 Allergens, Full Sequences:
[0178] SEQ ID NO: 7 relates to the full Der f 1 allergen amino acid
sequence; >Q58A71 Der f 1 allergen preproenzyme Dermatophagoides
farinae (American house dust mite) SEQ ID NO: 8 relates to the full
Der f 2 allergen amino acid sequence; >Q00855 Mite group 2
allergen Der f 2 (Allergen Der f II) (allergen Der f 2)
Dermatophagoides farinae (American house dust mite) SEQ ID NO: 9
relates to the full Der f 23 allergen amino acid sequence;
>A0A088SAW7 Der f 23 allergen Dermatophagoides farinae (American
house dust mite) SEQ ID NO: 10 relates to the full Der f 18p
allergen amino acid sequence; >Q86R84 60 kDa allergen Der f 18p
Dermatophagoides farinae (American house dust mite) SEQ ID NO: 11
relates to the full Der f 15 allergen amino acid sequence;
>Q9U6R7 98 kDa HDM allergen (Der f 15 allergen) (Group 15
allergen Der f 15) Dermatophagoides farinae (American house dust
mite) SEQ ID NO: 12 relates to the full Zen 1 protein allergen
amino acid sequence; >I7HDR2 Zen 1 protein Dermatophagoides
farinae (American house dust mite) SEQ ID NO: 13 relates to the
full Cte f 1 allergen amino acid sequence; >Q94424 Salivary
antigen 1 (FS-I) (allergen Cte f 1) Ctenocephalides felis (Cat
flea)
SEQ ID NOs: 14-20 Allergens, IMAT Forms (Short):
[0179] SEQ ID NO: 14 relates to the iMAT Form (short) of Der f 1
allergen amino acid sequence; SEQ ID NO: 15 relates to the iMAT
Form (short) Der f 2 allergen amino acid sequence; SEQ ID NO: 16
relates to the iMAT Form (short) Der f 23 allergen amino acid
sequence; SEQ ID NO: 17 relates to the iMAT Form (short) Der f 18p
allergen amino acid sequence; SEQ ID NO: 18 relates to the iMAT
Form (short) Der f 15 allergen amino acid sequence; SEQ ID NO: 19
relates to the iMAT Form (short) Zen 1 protein allergen amino acid
sequence; SEQ ID NO: 20 relates to the iMAT Form (short) Cte f 1
allergen amino acid sequence; SEQ ID NOs: 21-23 Allergens from
Hybrids 1, 2, and 3-IMAT Forms (Short): SEQ ID NO: 21 relates to
the iMAT Form (short) Hybrid 1 allergen SEQ ID NO: 22 relates to
the iMAT Form (short) Hybrid 2 allergen SEQ ID NO: 23 relates to
the iMAT Form (short) Hybrid 3 allergen SEQ ID NOs: 24-44 iMATs for
Cat (with N-Terminal or C-Terminal Hexa-Histidine or without
Hexa-Histidine/Methionine (IMAT_Pure)): SEQ ID NO: 24 relates to a
Der f 1 iMAT molecule (cat) with N-terminal Hexa-Histidine tag; SEQ
ID NO: 25 relates to a Der f 1 iMAT molecule (cat) with C-terminal
Hexa-Histidine tag; SEQ ID NO: 26 relates to a Der f 1 iMAT
molecule (cat) without tag; SEQ ID NO: 27 relates to a Der f 2 iMAT
molecule (cat) with N-terminal Hexa-Histidine tag; SEQ ID NO: 28
relates to a Der f 2 iMAT molecule (cat) with C-terminal
Hexa-Histidine tag; SEQ ID NO: 29 relates to a Der f 2 iMAT
molecule (cat) without tag; SEQ ID NO: 30 relates to a Der f 23
iMAT molecule (cat) with N-terminal Hexa-Histidine tag; SEQ ID NO:
31 relates to a Der f 23 iMAT molecule (cat) with C-terminal
Hexa-Histidine tag; SEQ ID NO: 32 relates to a Der f 23 iMAT
molecule (cat) without tag; SEQ ID NO: 33 relates to a Der f 18p
iMAT molecule (cat) with N-terminal Hexa-Histidine tag; SEQ ID NO:
34 relates to a Der f 18p iMAT molecule (cat) with C-terminal
Hexa-Histidine tag; SEQ ID NO: 35 relates to a Der f 18p iMAT
molecule (cat) without tag; SEQ ID NO: 36 relates to a Der f 15
iMAT molecule (cat) with N-terminal Hexa-Histidine tag; SEQ ID NO:
37 relates to a Der f 15 iMAT molecule (cat) with C-terminal
Hexa-Histidine tag; SEQ ID NO: 38 relates to a Der f 15 iMAT
molecule (cat) without tag; SEQ ID NO: 39 relates to a Zen 1
protein iMAT molecule (cat) with N-terminal Hexa-Histidine tag; SEQ
ID NO: 40 relates to a Zen 1 protein iMAT molecule (cat) with
C-terminal Hexa-Histidine tag; SEQ ID NO: 41 relates to a Zen 1
protein iMAT molecule (cat) without tag; SEQ ID NO: 42 relates to a
Cte f 1 protein iMAT molecule (cat) with N-terminal Hexa-Histidine
tag; SEQ ID NO: 43 relates to a Cte f 1 protein iMAT molecule (cat)
with C-terminal Hexa-Histidine tag; SEQ ID NO: 44 relates to a Cte
f 1 protein iMAT molecule (cat) without tag; SEQ ID NOs: 45-65
iMATs for Dog (with N-Terminal or C-Terminal Hexa-Histidine or
without Hexa-Histidine/Methionine (IMAT_Pure)): SEQ ID NO: 45
relates to a Der f 1 iMAT molecule (dog) with N-terminal
Hexa-Histidine tag; SEQ ID NO: 46 relates to a Der f 1 iMAT
molecule (dog) with C-terminal Hexa-Histidine tag; SEQ ID NO: 47
relates to a Der f 1 iMAT molecule (dog) without tag; SEQ ID NO: 48
relates to a Der f 2 iMAT molecule (dog) with N-terminal
Hexa-Histidine tag; SEQ ID NO: 49 relates to a Der f 2 iMAT
molecule (dog) with C-terminal Hexa-Histidine tag; SEQ ID NO: 50
relates to a Der f 2 iMAT molecule (dog) without tag; SEQ ID NO: 51
relates to a Der f 23 iMAT molecule (dog) with N-terminal
Hexa-Histidine tag; SEQ ID NO: 52 relates to a Der f 23 iMAT
molecule (dog) with C-terminal Hexa-Histidine tag; SEQ ID NO: 53
relates to a Der f 23 iMAT molecule (dog) without tag; SEQ ID NO:
54 relates to a Der f 18p iMAT molecule (dog) with N-terminal
Hexa-Histidine tag; SEQ ID NO: 55 relates to a Der f 18p iMAT
molecule (dog) with C-terminal Hexa-Histidine tag; SEQ ID NO: 56
relates to a Der f 18p iMAT molecule (dog) without tag; SEQ ID NO:
57 relates to a Der f 15 iMAT molecule (dog) with N-terminal
Hexa-Histidine tag; SEQ ID NO: 58 relates to a Der f 15 iMAT
molecule (dog) with C-terminal Hexa-Histidine tag; SEQ ID NO: 59
relates to a Der f 15 iMAT molecule (dog) without tag; SEQ ID NO:
60 relates to a Zen 1 protein iMAT molecule (dog) with N-terminal
Hexa-Histidine tag; SEQ ID NO: 61 relates to a Zen 1 protein iMAT
molecule (dog) with C-terminal Hexa-Histidine tag; SEQ ID NO: 62
relates to a Zen 1 protein iMAT molecule (dog) without tag; SEQ ID
NO: 63 relates to a Cte f 1 protein iMAT molecule (dog) with
N-terminal Hexa-Histidine tag; SEQ ID NO: 64 relates to a Cte f 1
protein iMAT molecule (dog) with C-terminal Hexa-Histidine tag; SEQ
ID NO: 65 relates to a Cte f 1 protein iMAT molecule (dog) without
tag; SEQ ID NOs: 66-74 Hybrid/Mosaic-Like iMATs for Cat (N-Terminal
or C-Terminal Hexa-Histidine or without Hexa-Histidine/Methionine
(IMAT_Pure)): SEQ ID NO: 66 relates to a Hybrid 1 iMAT molecule
(cat) with N-terminal Hexa-Histidine tag; SEQ ID NO: 67 relates to
a Hybrid 1 iMAT molecule (cat) with C-terminal Hexa-Histidine tag;
SEQ ID NO: 68 relates to a Hybrid 1 iMAT molecule (cat) without tag
(consisting of SEQ ID NO: 1, SEQ ID NO: 5 and SEQ ID NO: 21); SEQ
ID NO: 69 relates to a Hybrid 2 iMAT molecule (cat) with N-terminal
Hexa-Histidine tag; SEQ ID NO: 70 relates to a Hybrid 2 iMAT
molecule (cat) with C-terminal Hexa-Histidine tag; SEQ ID NO: 71
relates to a Hybrid 2 iMAT molecule (cat) without tag (consisting
of SEQ ID NO: 1, SEQ ID NO: 5 and SEQ ID NO: 22); SEQ ID NO: 72
relates to a Hybrid 3 iMAT molecule (cat) with N-terminal
Hexa-Histidine tag; SEQ ID NO: 73 relates to a Hybrid 3 iMAT
molecule (cat) with C-terminal Hexa-Histidine tag; SEQ ID NO: 74
relates to a Hybrid 3 iMAT molecule (cat) without tag (consisting
of SEQ ID NO: 1, SEQ ID NO: 5 and SEQ ID NO: 23); SEQ ID NOs: 75-83
Hybrid/Mosaic-Like iMATs for Dog (N-Terminal or C-Terminal
Hexa-Histidine or without Hexa-Histidine/Methionine (IMAT_Pure)):
SEQ ID NO: 75 relates to a Hybrid 1 iMAT molecule (dog) with
N-terminal Hexa-Histidine tag; SEQ ID NO: 76 relates to a Hybrid 1
iMAT molecule (dog) with C-terminal Hexa-Histidine tag; SEQ ID NO:
77 relates to a Hybrid 1 iMAT molecule (dog) without tag
(consisting of SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 21); SEQ
ID NO: 78 relates to a Hybrid 2 iMAT molecule (dog) with N-terminal
Hexa-Histidine tag; SEQ ID NO: 79 relates to a Hybrid 2 iMAT
molecule (dog) with C-terminal Hexa-Histidine tag; SEQ ID NO: 80
relates to a Hybrid 2 iMAT molecule (dog) without tag (consisting
of SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 22); SEQ ID NO: 81
relates to a Hybrid 3 iMAT molecule (dog) with N-terminal
Hexa-Histidine tag; SEQ ID NO: 82 relates to a Hybrid 3 iMAT
molecule (dog) with C-terminal Hexa-Histidine tag; SEQ ID NO: 83
relates to a Hybrid 3 iMAT molecule (dog) without tag (consisting
of SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 23);
SEQ ID NOs: 84-88 Non-redundant Hybrid Allergens:
[0180] SEQ ID NO: 84 relates to A1KXC1_DERFA_DFP1 (UNIPROT
database); SEQ ID NO: 85 relates to A0A088SAS1_DERFA Der f 28
allergen (UNIPROT database); SEQ ID NO: 86 relates to B7U5T1_DERFA
Der f 6 allergen (UNIPROT database); SEQ ID NO: 87 relates to
T2B4F3_DERPT LytFM (UNIPROT database); SEQ ID NO: 88 relates to
A7XXV2_DERFA Der f 2 allergen (UNIPROT database);
SEQ ID NOs: 89-90 Miscellaneous Sequences:
[0181] SEQ ID NO: 89 relates to a Cul o2 iMAT molecule with
N-terminal HEXA-HISTIDINE; SEQ ID NO: 90 relates to a Cul o3 iMAT
molecule with N-terminal HEXA-HISTIDINE.
SEQ ID NOs: 91-102 Peptide Components of Hybrids:
[0182] SEQ ID NO: 91 relates to a peptide part of a hybrid derived
from SEQ ID NO: 10, SEQ ID NO: 92 relates to a peptide part of a
hybrid derived from SEQ ID NO: 85, SEQ ID NO: 93 relates to a
peptide part of a hybrid derived from SEQ ID NO: 88, SEQ ID NO: 94
relates to a peptide part of a hybrid derived from SEQ ID NO: 86,
SEQ ID NO: 95 relates to a peptide part of a hybrid derived from
SEQ ID NO: 87, SEQ ID NO: 96 relates to a peptide part of a hybrid
derived from SEQ ID NO: 11, SEQ ID NO: 97 relates to a peptide part
of a hybrid derived from SEQ ID NO: 7, SEQ ID NO: 98 relates to a
peptide part of a hybrid derived from SEQ ID NO: 10, SEQ ID NO: 99
relates to a peptide part of a hybrid derived from SEQ ID NO: 12,
SEQ ID NO: 100 relates to a peptide part of a hybrid derived from
SEQ ID NO: 9, SEQ ID NO: 101 relates to a peptide part of a hybrid
derived from SEQ ID NO: 11, SEQ ID NO: 102 relates to a peptide
part of a hybrid derived from SEQ ID NO: 8.
[0183] Hybrid/Mosaic-Like iMAT Molecules:
[0184] In a specific aspect of the present invention the iMAT
molecules are further improved if components (amino acid
sequences/epitopes) of more than one allergen are included into the
antigen module. For this purpose it is possible to apply the basic
principle of the described bioinformatics selection approach
(Example 5) in a different way. Instead of selecting complete
allergens based on the hit count of allergen peptides found in the
allergen database, only the most abundant peptides of several of
such allergens are used to engineer an iMAT antigen module (see
e.g. Example 6). Thus, such an iMAT molecule consists of an antigen
module of peptides that stem from several allergens. This allows
broadening of the spectrum of a single iMAT molecule with respect
to its targeted immunological profile and is thus beneficial for
pharmacological drug development.
[0185] As a further step in engineering hybrid iMAT molecules the
TAT and the targeting domain, and optionally a His-Tag, are added.
Finally cysteine residues are replaced by most stabilizing residues
as described in examples 5 and 6.
Hybrid I
[0186] A protein precursor is chosen from the list of precursor
proteins corresponding to top ranking peptides and used as a
scaffold protein (SEQ ID NO: 84) for embedding other top ranking
peptides from other antigens (SEQ ID NOS: 10, 11, 85, 86, 87, 88).
The signal peptide sequence is removed from the scaffold protein.
Optionally additional adjacent N- or C-terminal amino acids are
inserted within the original sequence of the scaffold protein and
replace parts of the original sequence of the scaffold protein as
described below.
[0187] Components of the following proteins are employed to
construct hybrid 1:
SEQ ID NO: 84 (A1KXC1_DERFA DFP1 OS=Dermatophagoides farina) SEQ ID
NO: 10 (Q86R84 DERFA 60 kDa allergen Der f 18p OS=Dermatophagoides
farinae GN=Der f 18 PE=2 SV=1) SEQ ID NO: 85 (A0A088SAS1_DERFA Der
f 28 allergen OS=Dermatophagoides farinae PE=2 SV=1) SEQ ID NO: 88:
(A7XXV2_DERFA Der f 2 allergen OS=Dermatophagoides farinae PE=4
SV=1) SEQ ID NO: 86 (B7U5T1_DERFA Der f 6 allergen
OS=Dermatophagoides farinae PE=2 SV=1) SEQ ID NO: 87 (T2B4F3_DERPT
LytFM OS=Dermatophagoides pteronyssinus GN=lytFM PE=4 SV=1) SEQ ID
NO: 11 (Q9U6R7_DERFA 98 kDa HDM allergen OS=Dermatophagoides
farinae PE=2 SV=1)
[0188] Backbone of Hybrid 1:
SEQ ID NO: 84 (A1KXC1 18-400, without replacement parts below)
[0189] [Replacement 1 for: SEQ ID NO: 84 AA 39-52]:
TABLE-US-00001 SEQ ID NO: 10 (Q86R84_AA 97-110) i.e. (SEQ ID NO:
91) GNAKAMIAVGGSTM
[0190] [Replacement 2 for: SEQ ID NO: 84 AA 261-274]:
TABLE-US-00002 SEQ ID NO: 85 (A0A088SAS1_AA 611-624) i.e. (SEQ ID
NO: 92) MMKIYQQQQQQHHP
[0191] [Replacement 3 for: SEQ ID NO: 84 AA 234-246]:
TABLE-US-00003 SEQ ID NO: 88 (A7XXV2 AA_48-61) i.e. (SEQ ID NO: 93)
FLVYIHIANNEIKK
[0192] [Replacement 4 for: SEQ ID NO: 84 AA 53-65]:
TABLE-US-00004 SEQ ID NO: 86 (B7U5T1 AA_166-178) i.e. (SEQ ID NO:
94) IVDGDKVTIYGWG
[0193] [Replacement 5 for: SEQ ID NO: 84 AA 276-289]:
TABLE-US-00005 SEQ ID 87: (T2B4F3_AA 134-147) i.e. (SEQ ID NO: 95)
REENIWSDHIANVA
[0194] [Replacement 6 for: SEQ ID NO: 84 AA 203-216]:
TABLE-US-00006 SEQ ID NO: 11 (Q9U6R7_AA 469-482) i.e. (SEQ ID NO:
96) TPTTPTPAPTTSTP
[0195] After cysteine residues are replaced by most stabilizing
residues applying the bioengineering process described in Examples
5 and 6, this results in SEQ ID NO: 21.
[0196] The backbone of hybrid 1 is formed by the amino acid
sequence SEQ ID NO: 84. The further peptide components of hybrid 1
antigen module SEQ ID NOs: 91-96 are embedded into the backbone
sequence derived from SEQ ID NO: 84. These further peptide
components of hybrid 1 antigen module SEQ ID NOs: 91-96 can be
arranged in any order/in a different order (if compared to the
order of replacement described above). Any such re-arranged peptide
order based on the above described antigens/allergens is envisaged
by the present invention.
Hybrid 2
[0197] Complete allergens and/or top ranking peptides are chosen
and spliced together to obtain the allergen module for a given IMAT
molecule described below.
[0198] Components of the following proteins are employed to
construct hybrid 2 in the depicted particular order, whereby part 1
is the N-terminus. The further parts are added according to the
below order to the C-terminus of the previous part. In case of
hybrid 2 these are 5 further parts. There are 6 parts in total.
[0199] Part 1: SEQ ID NO 7 (Q58A71 Der f 1 allergen preproenzyme
Dermatophagoides farinae (American house dust mite) AA 99-321)
i.e.
TABLE-US-00007 (SEQ ID NO: 97) TSACRINSVNVPSELDLRSLRTVTPIRMQG
GCGSCWAFSGVAATESAYLAYRNTSLDLSE QELVDCASQHGCHGDTIPRGIEYIQQNGVV
EERSYPYVAREQQCRRPNSQHYGISNYCQI YPPDVKQIREALTQTHTAIAVIIGIKDLRA
FQHYDGRTIIQHDNGYQPNYHAVNIVGYGS TQGVDYWIVRNSWDTTWGDSGYGYFQAGNN
LMMIEQYPYVVIM
[0200] Part 2: SEQ ID NO 10 (Q86R84 60 kDa allergen Der f 18p
Dermatophagoides farinae (American house dust mite) AA 277-304)
i.e.
TABLE-US-00008 (SEQ ID NO: 98) FTQTDGFLSYNELCVQIQAETNAFTITR
[0201] Part 3: SEQ ID NO 12 (I7HDR2 Zen 1 protein Dermatophagoides
farinae (American house dust mite) AA 181-220) i.e.
TABLE-US-00009 (SEQ ID NO: 99)
EPTTPTPEPTTKTPEPTTKTPEPSTPTPEPTTKTPEPTTK
[0202] Part 4: SEQ ID NO 9 (A0A088SAW7 Der f 23 allergen
Dermatophagoides farinae (American house dust mite) AA 22-91)
i.e.
TABLE-US-00010 (SEQ ID NO: 100) DIDHDDDPTTMIDVQTTTVQPSDEFECPTR
FGYFADPKDPCKFYICSNWEAIHKSCPGNT RWNEKELTCT
[0203] Part 5: SEQ ID NO 11 (Q9U6R7 98 kDa HDM allergen (Der f 15
allergen) (Group 15 allergen Der f 15) Dermatophagoides farinae
(American house dust mite) AA 437-463) i.e.
TABLE-US-00011 (SEQ ID NO: 101) SPTTPTTTPSPTTPTTTPSPTTPTTTP
[0204] Part 6: SEQ ID NO 8 (Q00855 Mite group 2 allergen Der f 2
(Allergen Der f II) (allergen Der f 2) Dermatophagoides farinae
(American house dust mite) AA 18-146) i.e.
TABLE-US-00012 (SEQ ID NO: 102) DQVDVKDCANNEIKKVMVDGCHGSDPCIIH
RGKPFTLEALFDANQNTKTAKIEIKASLDG LEIDVPGIDTNACHFMKCPLVKGQQYDIKY
TWNVPKIAPKSENVVVTVKLIGDNGVLAC AIATHGKIRD
[0205] After cysteine residues are replaced by most stabilizing
residues applying the bioengineering process described in Examples
5 and 6, this results in SEQ ID NO: 22.
[0206] However, the above peptide components of hybrid 2 antigen
module can be arranged in any order/in a different order (if
compared to the order of replacement described above). Any such
re-arranged peptide order based on the above described
antigens/allergens is envisaged by the present invention.
Hybrid 3
[0207] Components of the following proteins are employed to
construct hybrid 3 in the depicted particular order, whereby part 1
is the N-terminus. The further parts are added according to the
below order to the C-terminus of the previous part. In case of
hybrid 3 these are 4 further parts. There are 5 parts in total.
[0208] Part 1: SEQ ID NO 7 (Q58A71 Der f 1 allergen preproenzyme
Dermatophagoides farinae (American house dust mite) AA 99-321)
i.e.
TABLE-US-00013 (SEQ ID NO: 97) TSACRINSVNVPSELDLRSLRTVTPIRMQG
GCGSCWAFSGVAATESAYLAYRNTSLDLSE QELVDCASQHGCHGDTIPRGIEYIQQNGVV
EERSYPYVAREQQCRRPNSQHYGISNYCQI YPPDVKQIREALTQTHTAIAVIIGIKDLRA
FQHYDGRTIIQHDNGYQPNYHAVNIVGYGS TQGVDYWIVRNSWDTTWGDSGYGYFQAGNN
LMMIEQYPYVVIM
[0209] Part 2: SEQ ID NO 10 (Q86R84 60 kDa allergen Der f 18p
Dermatophagoides farinae (American house dust mite) AA 277-304)
i.e. FTQTDGFLSYNELCVQIQAETNAFTITR (SEQ ID NO: 98)
[0210] Part 3: SEQ ID NO 11 (Q9U6R7 98 kDa HDM allergen (Der f 15
allergen) (Group 15 allergen Der f 15) Dermatophagoides farinae
(American house dust mite) AA 437-463) i.e.
TABLE-US-00014 (SEQ ID NO: 101) SPTTPTTTPSPTTPTTTPSPTTPTTTP
[0211] Part 4: SEQ ID NO 8 (Q00855 Mite group 2 allergen Der f 2
(Allergen Der f II) (allergen Der f 2) Dermatophagoides farinae
(American house dust mite) AA 18-146) i.e.
TABLE-US-00015 (SEQ ID NO: 102) DQVDVKDCANNEIKKVMVDGCHGSDPCIIH
RGKPFTLEALFDANQNTKTAKIEIKASLD GLEIDVPGIDTNACHFMKCPLVKGQQYDIK
YTWNVPKIAPKSENVVVTVKLIGDNGVLA CAIATHGKIRD
[0212] After cysteine residues are replaced by most stabilizing
residues applying the bioengineering process described in Examples
5 and 6, this results in SEQ ID NO: 23.
[0213] However, the above peptide components of hybrid 3 antigen
module can be arranged in any order/in a different order (if
compared to the order of the parts described above). Any such
re-arranged peptide order based on the above described
antigens/allergens is envisaged by the present invention.
[0214] Further specific aspects of the present invention:
The invention concerns an amino acid sequence/improved MAT (iMAT)
molecule, comprising: (i) at least one first module being an amino
acid sequence allowing the translocation of the iMAT molecule from
the extracellular space into the interior of cells, (ii) at least
one second module being an amino acid sequence allowing
species-specific intracellular targeting of the iMAT molecule to
the cell organelles which are involved in the processing of
antigens and/or the loading of MI-IC molecules with antigens,
preferably processed antigens, and (iii) at least one third module
as antigen module being an amino acid sequence derived from at
least one full or partial amino acid sequence, preferably an
epitope, of at least one antigen, preferably at least one allergen,
determining the specificity of an immune response modulated by such
iMAT molecule, characterized in that at least in the antigen
modules at least one cysteine residue is substituted with a
different amino acid residue, preferably serine, leucine,
isoleucine, arginine, methionine, and/or aspartic acid, for use in
a method of prevention and/or therapy of one or more allergies in
animals excluding equines and/or for use in a method of prevention
and/or therapy of one or more infectious diseases in animals
excluding equines and/or for use in a method of prevention of
transmission of one or more infectious diseases in animals
excluding equines and/or for use in a method of prevention of
transmission of one or more infectious diseases in animals
excluding equines by vectors.
[0215] The invention further concerns a method of prevention and/or
therapy of one or more allergies in animals excluding equines
and/or a method of prevention and/or therapy of one or more
infectious diseases in animals excluding equines and/or a method of
prevention of transmission of one or more infectious diseases in
animals excluding equines, preferably by vectors, comprising
administering a therapeutic effective amount of an amino acid
sequence/improved MAT (iMAT) molecule, comprising:
(i) at least one first module being an amino acid sequence allowing
the translocation of the iMAT molecule from the extracellular space
into the interior of cells, (ii) at least one second module being
an amino acid sequence allowing species-specific intracellular
targeting of the iMAT molecule to the cell organelles which are
involved in the processing of antigens and/or the loading of MI-IC
molecules with antigens, preferably processed antigens, and (iii)
at least one third module as antigen module being an amino acid
sequence derived from at least one full or partial amino acid
sequence, preferably an epitope, of at least one antigen,
preferably at least one allergen, determining the specificity of an
immune response modulated by such iMAT molecule, characterized in
that at least in the antigen modules at least one cysteine residue
is substituted with a different amino acid residue, preferably
serine, leucine, isoleucine, arginine, methionine, and/or aspartic
acid.
[0216] In a specific aspect the at least one antigen module all
cysteine residues are substituted with a different amino acid
residue, preferably serine, leucine, isoleucine, arginine,
methionine, and/or aspartic acid. Preferably all cysteine residues
in the entire iMAT molecule are substituted with a different amino
acid residue, preferably serine, leucine, isoleucine, arginine,
methionine, and/or aspartic acid.
[0217] In a further aspect all of such modules are covalently
linked to each other, optionally by additional spacer module(s)
between two or more adjacent modules, optionally between all of
such first, second and/or third modules.
[0218] In a preferred aspect all of such modules are covalently
linked to each other, and no additional spacer module(s) between
two or more adjacent modules of such first, second and/or third
modules are present at all.
[0219] In another aspect the at least one second module comprises
the invariant chain selected from the canine, feline, bovine,
ovine, caprine and/or porcine species' or a partial sequence
thereof, provided that such at least one second module is
functional as a module allowing species-specific intracellular
targeting of the iMAT molecule to the cell organelles which are
involved in the processing of antigens and/or loading of MHC
molecules with antigens, preferably processed antigens. A preferred
canine invariant chain sequence is SEQ ID NO: 2. A preferred feline
invariant chain sequence is SEQ ID NO: 3.
[0220] In a preferred aspect the at least one second module
comprises, preferably consisting of, the amino acid sequence
according to SEQ ID NO: 4 (canine) or SEQ ID NO: 5 (feline). SEQ ID
NOs: 4 and 5 represent 110 amino acid residues of the invariant
chains of SEQ ID NOs: 2 and 3, respectively.
[0221] In another preferred aspect the at least one second module
comprises, preferably consisting of, the amino acid sequence
according to SEQ ID NO: 4 (canine) or SEQ ID NO: 5 (feline) or
fragments thereof, provided such fragments maintain their
intracellular transport function.
[0222] In a further aspect the at least one antigen module
comprises at least one full or partial amino acid sequence,
preferably an epitope, derived from at least one allergen eliciting
an allergy in animals excluding equines, preferably at least one
full or partial amino acid sequence, preferably an epitope, of at
least one allergen derived from preferably allergies to flea bites,
preferably in dogs and/or cats; allergies to certain food
components, preferably in dogs and/or cats; atopic dermatitis,
preferably in dogs and/or cats; allergic airway inflammation and/or
obstruction, preferably in cats.
[0223] In a preferred aspect such at least one antigen, preferably
at least one allergen, is Der f 15 allergen according to SEQ ID NO:
11 or 18. In a preferred aspect such at least one antigen module
comprises, preferably consists of, SEQ ID NO: 18.
[0224] In a specific aspect such at least one antigen, preferably
at least one allergen, is derived from food and/or mold (fungi
and/or their spores), pollen, house dust or forage mites (and/or
their feces) and/or fleas, preferably from pollen from tree, grass,
herbaceous, Ambrosia and/or brassicaceae pollen and/or fungi and/or
their spores of the genera Aspergillus, Alternaria, Botrytis,
Cercospora, Cladosporium, Curvularia, Drechslera, Eurotium,
Helminthosporium, Epicoccum, Erysipheloidium, Fusarium,
Lichtheimia, Nigrospora, Penicillium, Periconia, Peronospora,
Polythrincium, Saccharopolyspora (formerly also Faenia or
Micropolyspora), Thermoactinomyces, Stemphylium, torula and/or
mites (or their feces) of the genera Acarus, Glycophagus,
Tyrophagus, Dermatophagoides, Euroglyphus, Lepidoglyphus, Blomia
and/or fleas of the genera Ceratophyllus, Ctenocephalides, Pulex,
Archaeopsylla, and more preferably is a Dermatophagoides
allergen.
[0225] In yet another specific aspect the at least one antigen
module comprises at least one full or partial amino acid sequence,
preferably an epitope, derived from at least one antigen of a
pathogen eliciting one or more infectious diseases in animals
excluding equines, preferably at least one full or partial amino
acid sequence, preferably an epitope, of at least one antigen of a
pathogen eliciting one or more infectious diseases in animals
excluding equines selected from the genera Campylobacter,
Dirofilaria, Ehrlichia, Leishmania, Trypanosoma, Borrelia,
Orthobunyavirus, Orbivirus, Flavivirus, Rotavirus, Coronavirus,
Trichophyton, Microsporum; Cooperia, Haemonchus, Ostertagia,
Trichostrongylus, Dictyocaulus, Metastrongylus; Eimeria, Isospora,
Cryptosporidium, Giardia, wherein preferably the at least one
antigen module may also be an antigen of a vector involved in the
transmission of one or more infectious diseases in animals
excluding equines, preferably an antigen selected from saliva
components of vectors selected from blood feeding bugs, flies,
midges, ticks and/or mosquitos.
[0226] In a preferred aspect the iMAT molecule further comprises at
least one tag module, preferably at least one His-tag. Preferably
such at least one tag module is present N-terminally. In another
preferred specific aspect such at least one tag module is present
C-terminally. In another preferred specific aspect such at least
one tag module is present N-terminally and C-terminally.
[0227] In a specific preferred aspect the iMAT molecule comprises
one tag module, preferably one His-tag, N-terminally after one
methionine residue.
[0228] In another specific preferred aspect there is no tag module
present in the iMAT molecules.
[0229] In a further aspect the at least one first module comprises,
preferably consists of, the amino acid sequence of HIV-tat, VP22
and/or Antennapedia or a partial sequence thereof, provided that
such at least one first module is functional as a module for
translocation of the iMAT molecule from the extracellular space
into the interior of cells, most preferably is amino acid sequence
YGRKKRRQRRR (SEQ ID NO: 1).
[0230] In another aspect the iMAT molecule is present in monomeric
form and/or in linear form.
[0231] In a further aspect at least one third module comprises,
preferably consists of, any one of SEQ ID NOs: 14 to 23.
[0232] In a preferred aspect the iMAT molecule comprises,
preferably consists of, one or more of the amino acid sequences
according to SEQ ID NOS: 24 to 83.
[0233] A specifically preferred iMAT molecule comprises, preferably
consists of, SEQ ID NO: 36 (Der f15 iMAT molecule cat) or SEQ ID
NO: 57 (Der f15 iMAT molecule dog). Further preferred iMAT
molecules (comprising an N-terminal His-tag) are selected from the
group consisting of: SEQ ID NOs: 24, 27, 30, 33, 36, 39, 42, 45,
48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81. Further preferred
iMAT molecules (comprising a C-terminal His-tag) are selected from
the group consisting of: SEQ ID NOs: 25, 28, 31, 34, 37, 40, 43,
46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82. Further
preferred iMAT molecules (without His-tag) are selected from the
group consisting of: SEQ ID NOs: 26, 29, 32, 35, 38, 41, 44, 47,
50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83.
[0234] Further preferred iMAT molecules (hybrid/mosaic-like iMATs)
are selected from the group consisting of: SEQ ID NOs: 66-83.
Further preferred hybrid iMAT molecules are 66, 69, 72, 75, 78, 81
(with N-terminal His-tag). Especially preferred is SEQ ID NO: 66
and/or 75. Further preferred specific hybrid iMAT molecules are 67,
70, 73, 76, 79, 82 (with C-terminal His-tag). Especially preferred
is SEQ ID NO: 67 and/or 76. Further preferred specific hybrid iMAT
molecules are 68, 71, 74, 77, 80, 83 (without His-tag). Especially
preferred is SEQ ID NO: 68 and/or 77.
[0235] In a further aspect the animal is selected from ruminants,
including cattle, goats, sheep, such as members of the genus Bos,
Capra and/or Ovis, members of the genus Canis, such as dogs,
wolves, foxes, coyotes, jackals, members of the genus Felis, such
as lions, tigers, domestic cats, wild cats, other big cats, and
other felines including cheetahs and lynx, and/or members of the
genus Sus, such as pigs, wherein preferably the animal is selected
from cats and/or dogs.
[0236] The invention concerns an amino acid sequence,
comprising:
(i) at least one first module being an amino acid sequence allowing
the translocation of the iMAT molecule from the extracellular space
into the interior of cells, preferably said first module comprises,
more preferably consists of SEQ ID NO: 1, (ii) at least one second
module being an amino acid sequence allowing species-specific
intracellular targeting of the iMAT molecule to the cell organelles
which are involved in the processing of antigens and/or the loading
of WIC molecules with antigens, preferably processed antigens,
preferably said second module comprises, more preferably consists
of, SEQ ID NO: 4 or 5 (iii) at least one third module as antigen
module being an amino acid sequence derived from at least one full
or partial amino acid sequence, preferably an epitope, of at least
one antigen, preferably at least one allergen, determining the
specificity of an immune response modulated by such iMAT molecule,
characterized in that at least in the antigen modules at least one
cysteine residue is substituted with a different amino acid
residue, preferably serine, leucine, isoleucine, arginine,
methionine, and/or aspartic acid. The third module preferably
comprises, more preferably consists of any one of SEQ ID NOs: 14 to
23.
[0237] The invention concerns an improved MAT (iMAT) molecule,
comprising:
(i) at least one first module being an amino acid sequence allowing
the translocation of the iMAT molecule from the extracellular space
into the interior of cells, preferably said first module comprises,
more preferably consists of SEQ ID NO: 1, (ii) at least one second
module being an amino acid sequence allowing species-specific
intracellular targeting of the iMAT molecule to the cell organelles
which are involved in the processing of antigens and/or the loading
of WIC molecules with antigens, preferably processed antigens,
preferably said second module comprises, more preferably consists
of, SEQ ID NO: 4 or 5 (iii) at least one third module as antigen
module being an amino acid sequence derived from at least one full
or partial amino acid sequence, preferably an epitope, of at least
one antigen, preferably at least one allergen, determining the
specificity of an immune response modulated by such iMAT molecule,
characterized in that at least in the antigen modules at least one
cysteine residue is substituted with a different amino acid
residue, preferably serine, leucine, isoleucine, arginine,
methionine, and/or aspartic acid. The third module preferably
comprises, more preferably consists of any one of SEQ ID NOs: 14 to
23.
[0238] In a specific aspect the at least one antigen module all
cysteine residues are substituted with a different amino acid
residue, preferably serine, leucine, isoleucine, arginine,
methionine, and/or aspartic acid, Preferably all cysteine residues
in the entire iMAT molecule are substituted with a different amino
acid residue, preferably serine, leucine, isoleucine, arginine,
methionine, and/or aspartic acid.
[0239] In a further aspect all of such modules are covalently
linked to each other, optionally by additional spacer module(s)
between two or more adjacent modules, optionally between all of
such first, second and/or third modules.
[0240] In a preferred aspect all of such modules are covalently
linked to each other, and no additional spacer module(s) between
two or more adjacent modules of such first, second and/or third
modules are present at all.
[0241] In another preferred aspect the at least one second module
comprises, preferably consisting of, the amino acid sequence
according to SEQ ID NO: 4 (canine) or SEQ ID NO: 5 (feline) or
fragments thereof, provided such fragments maintain their
intracellular transport function. SEQ ID NOs: 4 and 5 represent 110
amino acid residues of the invariant chains of SEQ ID NOs: 2 and 3,
respectively. In a more preferred aspect the at least one second
module comprises, preferably consists of, the amino acid sequence
according to SEQ ID NO: 4 (canine) or SEQ ID NO: 5 (feline).
[0242] In a further aspect the at least one antigen module
comprises at least one full or partial amino acid sequence,
preferably an epitope, derived from at least one allergen eliciting
an allergy in animals excluding equines, preferably at least one
full or partial amino acid sequence, preferably an epitope, of at
least one allergen derived from preferably allergies to flea bites,
preferably in dogs and/or cats; allergies to certain food
components, preferably in dogs and/or cats; atopic dermatitis,
preferably in dogs and/or cats; allergic airway inflammation and/or
obstruction, preferably in cats.
[0243] In a specific aspect such at least one antigen comprises the
amino acid sequence according to SEQ ID NO: 11 or 18. In a
preferred aspect such at least one antigen, preferably at least one
allergen, is Der f 15 allergen according to SEQ ID NO: 11 or 18. In
another preferred aspect such at least one antigen module
comprises, preferably consists of, SEQ ID NO: 18.
[0244] In a further preferred aspect the iMAT molecule comprises,
preferably consists of, an (one) amino acid sequence according to
SEQ ID NOs: 24 to 83 (one of those alternatives). In a further
preferred aspect the iMAT molecule comprises, preferably consists
of, any one of SEQ ID NOs: 36-38 (Der f15 iMAT molecule cat), most
preferably SEQ ID NO: 36, or the iMAT molecule comprises,
preferably consists of, any one of SEQ ID NOs: 57-59 (Der f15 iMAT
molecule dog), most preferably SEQ ID NO: 57.
[0245] In a preferred aspect the iMAT molecule further comprises at
least one tag module, preferably at least one His-tag. Preferably
such at least one tag module is present N-terminally. In a specific
preferred aspect the iMAT molecule comprises one tag module,
preferably one His-tag, N-terminally after one methionine
residue.
[0246] Preferred iMAT molecules with N-terminal His-tag comprise,
preferably consist of, any one of SEQ ID NOs: 24, 27, 30, 33, 36,
39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81.
[0247] In another specific preferred aspect such at least one tag
module is present C-terminally. In another specific preferred
aspect such at least one tag module is present N-terminally and
C-terminally. Preferred iMAT molecules with C-terminal His-tag
comprise, preferably consist of, any one of SEQ ID NOs: 25, 28, 31,
34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79,
82.
[0248] In another specific preferred aspect there is no tag module
present. Preferred iMAT molecules without any tag at all comprise,
preferably consist of, any one of SEQ ID NOs: 26, 29, 32, 35, 38,
41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83.
[0249] In a further aspect the at least one first module comprises,
preferably consists of, the amino acid sequence of HIV-tat, VP22
and/or Antennapedia or a partial sequence thereof, provided that
such at least one first module is functional as a module for
translocation of the iMAT molecule from the extracellular space
into the interior of cells, most preferably is amino acid sequence
YGRKKRRQRRR (SEQ ID NO: 1).
[0250] In another aspect the iMAT molecule is present in monomeric
form and/or in linear form.
[0251] In a further specific aspect the iMAT molecule is a hybrid
iMAT molecule (mosaic-like iMAT molecule). Preferably such hybrid
iMAT molecule comprises an amino acid sequence according to any one
of SEQ ID NOs: 21-23. More specifically such hybrid iMAT molecule
comprises, preferably consists of, an amino acid sequence according
to any one of SEQ ID NOs: 66-83.
[0252] The invention furthermore concerns an amino acid sequence
comprising, preferably consisting of, one or more of the amino acid
sequences according to SEQ ID NOs: 24 to 83.
[0253] The invention further concerns an iMAT molecule comprising,
preferably consisting of, one or more of the amino acid sequences
according to SEQ ID NOs: 24 to 83.
[0254] The invention further concerns an amino acid sequence/iMAT
molecule comprising, preferably consisting of, one or more amino
acid sequences selected from the group consisting of: SEQ ID NOs:
24 to 83.
[0255] A specifically preferred amino acid sequence/iMAT molecule
comprises, preferably consists of, SEQ ID NO: 36 (Der f15 iMAT
molecule cat) or SEQ ID NO: 57 (Der f15 iMAT molecule dog). Further
preferred amino acid sequences/iMAT molecules (comprising an
N-terminal His-tag) are selected from the group consisting of: SEQ
ID NOs: 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66,
69, 72, 75, 78, 81. Further preferred amino acid sequences/iMAT
molecules (comprising a C-terminal His-tag) are selected from the
group consisting of: SEQ ID NOs: 25, 28, 31, 34, 37, 40, 43, 46,
49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82. Further preferred
amino acid sequences/iMAT molecules (without His-tag) are selected
from the group consisting of: SEQ ID NOs: 26, 29, 32, 35, 38, 41,
44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83.
[0256] Further preferred amino acid sequences/iMAT molecules
(hybrid/mosaic-like iMATs) are selected from the group consisting
of: SEQ ID NOs: 66-83. Further preferred hybrid iMAT molecules are
66, 69, 72, 75, 78, 81 (with N-terminal His-tag). Further preferred
hybrid iMAT molecules are 67, 70, 73, 76, 79, 82 (with C-terminal
His-tag). Further preferred hybrid iMAT molecules are 68, 71, 74,
77, 80, 83 (without His-tag).
[0257] The invention concerns an amino acid sequence/improved MAT
(iMAT) molecule, comprising:
(i) at least one first module being an amino acid sequence allowing
the translocation of the iMAT molecule from the extracellular space
into the interior of cells, preferably said first module comprises,
more preferably consists of SEQ ID NO: 1, (ii) at least one second
module being an amino acid sequence allowing species-specific
intracellular targeting of the iMAT molecule to the cell organelles
which are involved in the processing of antigens and/or the loading
of MHC molecules with antigens, preferably processed antigens,
preferably said second module comprises, more preferably consists
of, SEQ ID NO: 4 or 5 (iii) at least one third module as antigen
module being an amino acid sequence derived from at least one full
or partial amino acid sequence, preferably an epitope sequence, of
any combination of two or more antigens selected from the group
consisting of: SEQ ID NO: 7, 8, 9, 10, 11, 12, 84, 85, 86, 87, and
88 determining the specificity of an immune response modulated by
such iMAT molecule, characterized in that at least in the antigen
modules at least one cysteine residue is substituted with a
different amino acid residue, preferably serine, leucine,
isoleucine, arginine, methionine, and/or aspartic acid.
[0258] In a preferred aspect the antigen module is an amino acid
sequence derived from at least one full or partial amino acid
sequence, preferably an epitope sequence, of any combination of two
or more antigens selected from the group consisting of: SEQ ID NO:
10, 11, 84, 85, 86, 87, and 88 (Hybrid 1).
[0259] In a specific aspect the antigen module is an amino acid
sequence derived from at least one full or partial amino acid
sequence, preferably an epitope sequence, of any combination of two
or more antigens selected from the group consisting of: SEQ ID NO:
7, 8, 9,10, 11, and 12 (Hybrid 2).
[0260] In a specific aspect the antigen module is an amino acid
sequence derived from at least one full or partial amino acid
sequence, preferably an epitope sequence, of any combination of two
or more antigens selected from the group consisting of: SEQ ID NO:
7, 8, 10, and 11 (Hybrid 3).
[0261] Thus, the invention specifically concerns an amino acid
sequence/improved MAT (iMAT) molecule, comprising:
(i) at least one first module being an amino acid sequence allowing
the translocation of the iMAT molecule from the extracellular space
into the interior of cells, preferably said first module comprises,
more preferably consists of SEQ ID NO: 1, (ii) at least one second
module being an amino acid sequence allowing species-specific
intracellular targeting of the iMAT molecule to the cell organelles
which are involved in the processing of antigens and/or the loading
of MHC molecules with antigens, preferably processed antigens,
preferably said second module comprises, more preferably consists
of, SEQ ID NO: 4 or 5 (iii) at least one third module as antigen
module being an amino acid sequence based on a backbone derived
from SEQ ID NO: 84 comprising any combination of one or more of the
peptides according to SEQ ID NOs: 91-96 (in any order) embedded
into said backbone sequence, determining the specificity of an
immune response modulated by such iMAT molecule, characterized in
that at least in the antigen modules at least one cysteine residue
is substituted with a different amino acid residue, preferably
serine, leucine, isoleucine, arginine, methionine, and/or aspartic
acid.
[0262] In a preferred aspect the order of peptide sequences within
the backbone derived from SEQ ID NO: 84 in the third module is SEQ
ID NO: 91, followed by SEQ ID NO: 92, followed by SEQ ID NO: 93,
followed by SEQ ID NO: 94, followed by SEQ ID NO: 95, followed by
SEQ ID NO: 96 starting from the N-terminus. In a most preferred
aspect the third module comprises, preferably consist of, SEQ ID
NO: 21.
[0263] In a further specific aspect the order of peptide sequences
within the backbone derived from SEQ ID NO: 84 in the third module
is SEQ ID NO: 93, followed by SEQ ID NO: 91, followed by SEQ ID NO:
92, followed by SEQ ID NO: 94, followed by SEQ ID NO: 96, followed
by SEQ ID NO: 95 starting from the N-terminus. Further aspects of
the present invention relate to any further combination and order
of the peptides according to SEQ ID NOs: 91-96 embedded into the
backbone sequence derived from SEQ ID NO: 84.
[0264] Thus, the invention further concerns an amino acid
sequence/improved MAT (iMAT) molecule, comprising:
(i) at least one first module being an amino acid sequence allowing
the translocation of the iMAT molecule from the extracellular space
into the interior of cells, preferably said first module comprises,
more preferably consists of SEQ ID NO: 1, (ii) at least one second
module being an amino acid sequence allowing species-specific
intracellular targeting of the iMAT molecule to the cell organelles
which are involved in the processing of antigens and/or the loading
of WIC molecules with antigens, preferably processed antigens,
preferably said second module comprises, more preferably consists
of, SEQ ID NO: 4 or 5 (iii) at least one third module as antigen
module being an amino acid sequence derived from any combination of
two or more of the peptides according to SEQ ID NOs: 97-102 (in any
order), determining the specificity of an immune response modulated
by such iMAT molecule, characterized in that at least in the
antigen modules at least one cysteine residue is substituted with a
different amino acid residue, preferably serine, leucine,
isoleucine, arginine, methionine, and/or aspartic acid. In a
preferred aspect the order of peptide sequences within the third
module is SEQ ID NO: 97, followed by SEQ ID NO: 98, followed by SEQ
ID NO: 99, followed by SEQ ID NO: 100, followed by SEQ ID NO: 101,
followed by SEQ ID NO: 102 starting from the N-terminus. In a most
preferred aspect the third module comprises, preferably consist of,
SEQ ID NO: 22.
[0265] In a further specific aspect the order of peptide sequences
within the third module is SEQ ID NO: 99, followed by SEQ ID NO:
98, followed by SEQ ID NO: 102, followed by SEQ ID NO: 100,
followed by SEQ ID NO: 101, followed by SEQ ID NO: 97 starting from
the N-terminus. Further aspects of the present invention relate to
any further combination and order of the peptides according to SEQ
ID NOs: 97-102.
[0266] Thus, the invention additionally concerns an amino acid
sequence/improved MAT (iMAT) molecule, comprising:
(i) at least one first module being an amino acid sequence allowing
the translocation of the iMAT molecule from the extracellular space
into the interior of cells, preferably said first module comprises,
more preferably consists of SEQ ID NO: 1, (ii) at least one second
module being an amino acid sequence allowing species-specific
intracellular targeting of the iMAT molecule to the cell organelles
which are involved in the processing of antigens and/or the loading
of WIC molecules with antigens, preferably processed antigens,
preferably said second module comprises, more preferably consists
of, SEQ ID NO: 4 or 5 (iii) at least one third module as antigen
module being an amino acid sequence derived from any combination of
two or more of the peptides according to SEQ ID NO: 97, SEQ ID NO:
98, SEQ ID NO: 101, and SEQ ID NO: 102 (in any order), determining
the specificity of an immune response modulated by such iMAT
molecule, characterized in that at least in the antigen modules at
least one cysteine residue is substituted with a different amino
acid residue, preferably serine, leucine, isoleucine, arginine,
methionine, and/or aspartic acid. In a preferred aspect the order
of peptide sequences within the third module is SEQ ID NO: 97,
followed by SEQ ID NO: 98, followed by SEQ ID NO: 101, followed by
SEQ ID NO: 102 starting from the N-terminus. In a most preferred
aspect the third module comprises, preferably consist of, SEQ ID
NO: 23.
[0267] In a further specific aspect the order of peptide sequences
within the third module is SEQ ID NO: 102, followed by SEQ ID NO:
98, followed by SEQ ID NO: 101, followed by SEQ ID NO: 97 starting
from the N-terminus. Further aspects of the present invention
relate to any further combination and order of the peptides
according to SEQ ID NOs: 97, 98, 101, and 102.
[0268] The invention further concerns a vaccine or immunogenic
composition or a pharmaceutical composition comprising the amino
acid sequence/iMAT molecule according to the present invention.
[0269] The invention furthermore concerns a nucleic acid encoding
the amino acid/iMAT molecule according to the present
invention.
[0270] Additionally, the invention concerns a vector comprising at
least one nucleic acid according to the present invention.
[0271] Furthermore, the invention concerns a primary cell or cell
line comprising at least one nucleic acid according to the present
invention and/or at least one vector according to the present
invention.
[0272] The invention additionally concerns a method of identifying
improved MAT molecules comprising the steps of:
a) selecting a protein as allergen module in iMAT molecules, and b)
constructing an iMAT molecule with said allergens that is
thermodynamically stable and can be produced efficiently by protein
engineering.
[0273] The invention specifically concerns a method of identifying
improved MAT molecules comprising the steps of:
a) selecting a protein as allergen module in iMAT molecules that is
an allergen and thus has a high potential to cause hypersensitivity
in affected subjects and thus can also be the target for tolerance
induction, and b) constructing an iMAT molecule with said allergens
that is thermodynamically stable and can be produced efficiently by
protein engineering and can additionally be analyzed with standard
methods to ensure sufficient enough quality (i.e. identity, purity
and potency).
[0274] In a specific aspect of the method step a) comprises:
[0275] selecting (an) allergen(s) based on local homology searches
of peptides derived from given proteins to known allergenic
proteins,
[0276] exporting amino acid sequences of proteins suspected to have
allergenic properties from publicly available databases (e.g.
UNIPROT),
[0277] determining redundancies by analysis of sequences homologies
within the exported dataset,
[0278] eliminating highly homologue sequence counterparts
(remaining sequences serve as the canonical sequence database of
probable valid antigens for subsequent analyses),
[0279] in silico cleaving proteins into peptides with lengths of 6
to 15 amino acids (with a one amino acid shifting)
[0280] performing local-pairwise alignments of proteins and the
corresponding peptides,
[0281] scaling of obtained alignment hits by setting the
self-alignment score of a given protein to one and aligning hits of
the corresponding peptides accordingly,
[0282] counting the number of alignment hits exceeding a given
threshold for each peptide,
[0283] compared the local-pairwise alignment to a randomly
generated database of protein sequences with no known allergenic
properties,
[0284] scaling and counting of hits,
[0285] subtracting the "non-allergic protein" counts from those of
the allergen results,
[0286] calculating cumulative hit scores for each protein based on
the number of hits for all corresponding peptides,
[0287] selecting proteins with highest counts as iMAT antigen
module candidates.
[0288] In a specific aspect of the method step b) comprises
identifying amino acid substitutions in a predetermined amino acid
sequence allowing the stabilization of such predetermined amino
acid sequence, preferably comprising the steps of:
(i) modeling of the three-dimensional/tertiary structure of the
targeted predetermined amino acid sequence, (ii) iterative
determination of protein stabilities based on single point
substitutions, such as substitutions of cysteine residues with
different amino acid residues, preferably serine and/or isoleucine
residues, and a scoring system based on the protein stability free
energy changes (.DELTA..DELTA.G) to determine stabilizing
substitutions by analyzing all available three-dimensional/tertiary
amino acid sequence structures, (iii) re-modeling of the
substituted three-dimensional/tertiary amino acid sequence
structure and calculation of its stability by repeating steps (ii)
and (iii) until a steady state is reached.
BRIEF DESCRIPTION OF THE FIGURES
[0289] FIG. 1A: shows an SDS-PAGE of MAT-Fel d1 (IVN201) under
reducing conditions; FIG. 1A shows the Original SDS-PAGE (10 .mu.g
protein per lane, Coomassie staining).
[0290] FIG. 1B: shows an SDS-PAGE of MAT-Fel d1 (IVN201) under
reducing conditions with bands that have been cut out of the
SDS-PAGE gel shown in FIG. 1A and reloaded on SDS-PAGE (silver
staining).
[0291] FIG. 2: shows an SDS-PAGE of Fel d1 under reducing
conditions; NUPAGE.RTM. 4-12% Bis-Tris Gel. Lanes: 1) Marker:
SeeBlue Plus2 Pre-Stained Standard 2) 5 .mu.g Fel d1; 3) 5 .mu.g
Fel d1.
[0292] FIG. 3A: shows an RP-HPLC chromatogram 0.1% TFA/Acetonitrile
gradient of native protein (MAT-Fel d1) without additives.
[0293] FIG. 3B: shows an RP-HPLC chromatogram 0.1% TFA/Acetonitrile
gradient of MAT-Fel d1 denaturated by addition of guanidinium
chloride plus DTT.
[0294] FIG. 4: depicts MAT-Fel d1 molecule and its Kyte Doolittle
hydrophobicity plot.
[0295] FIG. 5: shows a NUPAGE.RTM. SDS-PAGE-System (4-12% Bis-Tris
Gels, 1.times.MES-Running buffer, 35 min, 200 V). Lanes: 1)
Lysozyme, 1 .mu.g Protein ox. 2) PageRuler Prestained Protein
Ladder; 3) MAT-Fel d1 (5 .mu.g) oxidized with Iodacetamide (ox.);
4) iMAT-Cul o4 (ox.); 5) PageRuler Prestained Protein Ladder; 6)
MAT-Fel d1 (5 .mu.g) reduced; 7) iMAT-Cul o4 reduced; 8) Lysozyme
reduced.
[0296] FIG. 6: shows an RP-HPLC chromatogram 0.1% TFA/Acetonitrile
gradient. The peak reflects native (oxidized) protein (iMAT-Cul o4)
without additives.
[0297] FIG. 7: shows the results of the following experiment:
Proteins and ADJU-PHOS.RTM. that were incubated at RT for 30 min
while mixing gently. After incubation, the samples were centrifuged
for 3 min. and subsequently analyzed by SDS-Page Lane 1) pageRuler
Prestained Protein Ladder; 2) iMAT-Cul o3 Supernatant; 3) iMAT-Cul
o3 Pellet in Urea 4) iMAT-Cul o3 Supernatant; 5) iMAT-Cul o3 Pellet
in Urea; 6) Empty; 7) iMAT-Cul o2 Supernatant 8) iMAT-Cul o2 Pellet
in Urea 9) iMAT-Cul o2 Supernatant 10) iMAT-Cul o2 Pellet in Urea;
11) Empty; 12) iMAT-Cul o4 Supernatant; 13) iMAT-Cul o4 Pellet in
Urea 14) iMAT-Cul o4 Supernatant; 15) iMAT-Cul o4 Pellet in Urea;
(2, 3, 7, 8, 12, 13 w/o freeze thaw); (4, 5, 9, 10, 14, 15 after
two times freeze thaw process).
[0298] FIG. 8: shows the Kyte-Doolittle hydrophobicity plot of MAT
molecules versus iMAT molecules in the generic part of the
respective fusion proteins [i.e. without the respective antigen
module(s)]. The hydrophobicity index is shown on the Y-axis versus
the amino acid position on the X-axis. Positive numbers in the
index indicate hydrophobicity. The shift of the graph of the iMAT
molecule at the beginning of the hydrophobicity plot as compared to
the MAT molecule is due to the additional N-terminal presence of a
His-tag and one methionine residue in the iMAT molecule as well as
the absence of any spacer module(s).
[0299] FIG. 9A: shows examples of D. pteronyssinus allergens of the
genus Dermatophagoides identifying the species, the allergen and
the uniprot accession number.
[0300] FIG. 9B: shows examples of D. farinae allergens of the genus
Dermatophagoides identifying the species, the allergen and the
uniprot accession number.
[0301] FIG. 10: shows the N-terminus sequence alignment of
invariant chains from dog and cat. The CLIP sequence is shaded in
grey.
[0302] FIG. 11A: shows Histamine Release Test (Details of assay in
Example 2) of 5 concentrations of iMAT-Cul o2 and the respective
allergens in a polysensitized horse (Horse 1).
[0303] FIG. 11B: shows Histamine Release Test (Details of assay in
Example 2) of 5 concentrations of iMAT-Cul o3 and the respective
allergens in a polysensitized horse (Horse 1).
EXAMPLES
[0304] The following examples serve to further illustrate the
present invention; but the same should not be construed as a
limitation of the scope of the invention disclosed herein.
Example 1--Surrogate Marker for Immunity/Duration of Immunity
[0305] Administration of an (isolated) recombinant protein, as
disclosed and claimed herein, to an animal, e.g. ruminants, pigs,
more preferably a dog and/or a cat, but excluding an equine,
produces an immunological response to the allergen and/or epitope
present in the antigen module. Additionally, the C- or N-terminal
tag, e.g. a HIS-tag, the TAT module together with the adjacent
amino acid residues from the adjacent module is used in order to
detect a unique product-specific immunological signal (e.g. an
antibody--or a T-cell response) in a target subject that is used as
a surrogate marker for immunity or duration of immunity. This
surrogate marker, as a treatment-specific immunological parameter,
enables the assessment of immunity or immune modulation or the
duration of immunity or the duration of immune modulation after the
administration. Thus, a specific indicator for an immune response
triggered by the (isolated) recombinant protein according to the
invention is the induction of terminal-tag (optionally with the
adjacent amino acid residues) specific antibodies, such as IgG
antibodies. Alternatively or additionally, the indicator is the
induction of antibodies specific to the junction of a spacer and a
module as described and claimed herein or the junction between two
modules.
[0306] A single iMAT molecule such as SEQ ID NO: 57 (Der f 15 iMAT
molecule (dog) with N-terminal Hexa-Histidine tag) or a combination
of one or several iMAT molecules selected from SEQ ID NOs: 45, 51,
54, 57, 60, 63, 66, 69, 72 containing different antigen modules
according to the present invention are employed for treating
prophylactically or therapeutically a dog suffering from or being
at risk of allergic diseases, especially atopic dermatitis. In such
dogs the above iMAT molecules are administered as described in
Example 3. In serum samples derived from a blood draw from these
iMAT treated dogs, the immunological reaction upon iMAT treatment
against the N-terminus of the iMAT proteins with respect to
antibody production more specifically the specific IgG can be
measured. The measurement employs standard ELISA (Enzyme Linked
Immunosorbent Assay) techniques whereby sufficient amounts of a
synthetized peptide comprising SEQ ID NO: 6 is coated on the
surface of ELISA plates. Serum samples of treated animals are then
incubated on such plates and the specific binding of IgG to the
said peptide is detected by a secondary biotinylated antibody,
specific for IgG in cats or dogs respectively, followed by
application of the corresponding detection system e.g.
streptavidin-peroxidase and 3,3',5,5'-tetramethylbenzidine (TMB) as
substrate.
[0307] With such ELISA test the onset of immunity elicited by iMAT
immunotherapy as well as the duration of immunity is determined and
observed over time. The therapeutic vaccination regime, i.e. number
and schedule of booster injections, is determined by this surrogate
parameter during clinical development.
Example 2--Hypoallergenicity
[0308] The allergenicity of a therapeutic allergen is of utmost
importance, it is a measure of the potential to induce adverse
events, e.g. provoke anaphylaxis. Exemplarily for an allergy in
mammals, allergen specific IgE mediated hypersensitivity is studied
in procedures as the allergen provocation tests, in particular such
tests targeting the skin [Griffin C E. Diagnosis of canine atopic
dermatitis DOI: 10.1002/9781118738818.ch10].
[0309] Intradermal skin tests have been used for the biological
evaluation of recombinant allergens and for validation of
genetically engineered hypoallergenic derivatives.
[0310] Intradermal testing in a dog is performed by administering
injections of small amounts of allergen solutions directly into the
dog's dermis. This is usually done with small-gauge (27 gauge)
needles and injections of 0.05 to 0.1 mL at each site. The positive
reactions are arbitrarily interpreted by the presence of erythema,
turgidity, height, and size of the wheal.
[0311] The advantages of the intradermal tests are high
sensitivities. This is of particular importance if the test shall
deliver a quantitative measure for hypoallergenicity. In said tests
the MAT molecules show a 10-, 100- to 1000-times or even higher
molar concentration of the allergenic component as compared to the
natural, native allergen applied in the same test to reach a
positive reaction in sensitized individuals, as cats and dogs.
[0312] With regard to MAT molecules conflicting results about their
allergenicity in comparison to the corresponding native allergens
have been reported in the prior art. Senti G et al. (J Allergy Clin
Immunol. 2012, 129(5): 1290-1296) demonstrated hypoallergenicity of
a MAT-Fel d1 in the Cellular Antigen Stimulation Test (CAST) assay
as well as in the intradermal and in the intracutaneous test. The
quantitative difference in sensitivity between the allergen and the
MAT molecule comprising the Fel d1 was 100-, 23- and 16-fold,
respectively. Though MAT-Fel d1 was clearly hypoallergenic, some
allergenicity remained. In contrast Zhao et al. Int J Clin Exp Med
2015; 8(4):6436-6443 describe their MAT-Der p1 construct to exhibit
an even stronger allergenicity (hyperallergenicity) as compared to
the native Der p1 protein.
[0313] Surprisingly the improved MAT molecules, as disclosed and
claimed herein show clear superiority in this respect. The safety
of 2 iMAT molecules manufactured according to the present invention
comprising Cul o2 and Cul o3 in the antigen module, respectively,
is tested. Freshly withdrawn blood of a horse sensitized to these
allergens and suffering from insect bite hypersensitivity (IBH) is
employed in the histamine release test (HRT) as described
below.
[0314] As shown in FIG. 11B and FIG. 11A, respectively, the native
allergens elicit a strong histamine release whereas surprisingly
the two different iMAT molecules (iMAT-Cul o3 and iMAT-Cul o2) show
virtually no response at all.
[0315] Thus, iMAT molecules show clear superiority in respect to
safety as compared with the MAT molecule as described in the prior
art (see above).
[0316] Histamine release test (HRT): Freshly withdrawn blood of
subjects is prepared to test the basophil reactivity to (isolated)
recombinant proteins/iMAT molecules, as disclosed and claimed
herein. Briefly, a 10-fold dilution series (e.g. ranging from 10 nM
to 0.001 nM final allergen concentration) of iMAT molecules and/or
recombinant allergens is prepared in PIPES buffer (AppliChem,
Darmstadt, Germany), pH 7.4. Washed red and white blood cells
obtained from Na-EDTA coagulation-inhibited blood are incubated
with individual dilutions for 1 h at 37.degree. C. The reaction is
stopped by incubation on ice for 20 min and the supernatant
containing the released histamine is collected from each sample
after centrifugation. Maximal histamine content is obtained by
boiling the blood cells for 10 min in a water bath (maximal
release). The incubation of releasing buffer with washed blood
cells serves as a negative control (spontaneous release). Histamine
concentrations are determined using a competitive RIA (LDN
Nordhorn, Germany) as per the manufacturer's instructions.
[0317] Alternatively, another basophil activation test is the
Cellular Antigen Stimulation Test CAST.RTM. ELISA which can also be
considered as an in vitro allergy provocation test. This assay is
done according to the manufacturer's instructions (Bilhlmann
Laboratories AG, Allschwil, Switzerland). In the CAST.RTM.,
sedimented leukocytes from allergic subjects' blood are
simultaneously primed with the cytokine IL-3 and stimulated with
iMAT molecules and/or recombinant allergens. Basophilic cells among
others generate the allergic mediator, sulfidoleukotriene LTC4, and
its metabolites LTD4 and LTE4. These freshly synthesized
sulfidoleukotrienes (sLT) are subsequently measured in an ELISA
test (Enzyme Linked Immunosorbent Assay).
[0318] The potential of iMAT molecules to induce adverse events,
e.g. provoke anaphylaxis as a side effect of administration can be
evaluated in vitro with these assays by comparing the effects of
the iMAT molecules (containing an allergen) to the respective
recombinant allergen alone.
[0319] A reduced basophil degranulation, e.g. histamine and/or
sulfidoleukotriene release by iMAT molecules as compared to the
recombinant allergen indicates a lower potential for adverse
effects, i.e. a better safety profile of the iMAT molecules.
[0320] Said HRT (or CAST.RTM.) can be used as an in-vitro
provocation test for type 1 allergic reactions in a subject.
Allergen specific histamine release indicates the relevance of the
respective allergen for the basophilic cell activation and thus can
be used as a quantitative parameter for the allergen specific
sensitization of a subject.
[0321] It can be expected that no allergen related adverse
reactions occur if subjects suffering from allergen specific IgE
mediated hypersensitivity are treated with the corresponding iMAT
molecules comprising relevant allergens in the iMAT-antigen module.
This makes a desensitization therapy applying iMAT proteins
specifically appropriate for treatment of life threatening
diseases.
[0322] The consequence of this surprising safety property of iMAT
molecules in contrast to MAT molecules is, that iMAT molecules used
as desensitizing proteins can be used similar to vaccines against
pathogens. No up-dosing as with classical therapeutic allergens is
needed, since vaccines comprising iMAT molecules do not show
allergen properties with respect to allergic adverse events.
Already the dose of the first injection of the iMAT molecule in a
treatment course is selected based on efficacy considerations only
and one does not have to consider potential allergic adverse
reactions. This could not be performed using MAT molecules
described in the prior art since the allergenicity of MAT, compared
to the native allergen, was only reduced to a certain level.
However, MAT molecules still are allergens; iMAT molecules in
contrast are not. The advantage of this improved property renders a
more efficacious treatment regime possible with e.g. three
subcutaneous or intralymphatic injections with a high
biopharmaceutical content (e.g. 3 times 1 .mu.g to 100 .mu.g,
preferably 3 times 10 .mu.g to 50 .mu.g iMAT protein).
[0323] The lack of allergenicity of the iMAT molecules can be
explained by the fact that in contrast to the MAT molecules
described in the prior art no linker amino acid residues [i.e.
spacer module(s) between the first, second and/or third module(s)]
are used to separate the different modules in such iMAT molecules.
It is known in the prior art that engineered fusion proteins
containing two or more functional polypeptides joined by a peptide
or protein linker are important for the function (e.g. epitope
recognition by the immune system) of the proteins [Klein J S et
al., Protein Eng Des Sel. 2014, 27(10): 325-330]. The separation
distance between functional units can impact epitope access and the
ability to bind with avidity. If the missing amino acid residue
linkers between the modules, in particular between the targeting
domain and the antigen module, lead to a more rigid structure,
conformational epitopes of the allergen module might not be formed
due to incorrect folding. A cross linking of antibodies bound on
the surface of basophils (e.g. IgE) by its high affinity receptors
is necessary to induce activation and histamine release. However,
misfolded allergens might not be able to induce such cross linking.
Thus, an iMAT molecule without linker may not form conformational
IgE epitopes which renders the iMAT molecules non-allergenic.
Example 3--Therapeutic Vaccine/Prophylaxis of Atopic Dermatitis in
Dogs and/or Cats
[0324] A single iMAT molecule or a combination of iMAT molecules
containing different antigen modules according to the present
invention is employed for treating prophylactically or
therapeutically a dog and/or a cat suffering from or to be at risk
of atopic dermatitis (AD).
[0325] In first example the iMAT molecule according to SEQ ID NO:
36 (Der f 15) is administered into the popliteal lymph node of cats
suffering from atopic dermatitis.
[0326] In a second example the iMAT molecule according to SEQ ID
NO: 66 (Hybrid 1) is administered into the popliteal lymph node of
cats suffering from atopic dermatitis.
[0327] In a third example the iMAT molecule according to SEQ ID NO:
57 (Der f 15) is administered into the popliteal lymph node of dogs
suffering from atopic dermatitis.
[0328] In a fourth example the iMAT molecule according to SEQ ID
NO: 81 (Hybrid 3) is administered into the popliteal lymph node of
dogs suffering from atopic dermatitis.
[0329] In each case, the hair over the lymph node of the affected
animal is clipped and surgically prepared. Using palpation and/or
ultrasound for guidance a 25 G needle is inserted into the lymph
node. The injected iMAT molecule is adsorbed to an adjuvant. The
adjuvant consists e.g. of aluminum phosphate (ADJU-PHOS.RTM.,
Brenntag Biosector, Denmark). The iMAT molecule stock is a frozen
solution of e.g. 375 .mu.g/mL protein concentration in vials, each
containing 500 .mu.L to be thawed before use.
[0330] After thawing the iMAT molecule solution, 400 .mu.L of the
solution are mixed with e.g. 200 .mu.L of the adjuvant. This final
formulation is left at room temperature e.g. for 60 minutes prior
to the intralymphatic injection to allow for absorption of the iMAT
molecule to e.g. ADJU-PHOS.RTM., e.g. 50 .mu.L of the mixture
containing 12.5 .mu.g iMAT molecule is removed into a 500 .mu.L
syringe for lymph node injection. This preparation is first
administered typically on day 0, day 28 and day 56 in a dose
between 10 .mu.g and 50 .mu.g (referring to the weight of solely
the one or more antigen modules) per injection and iMAT
molecule.
[0331] Throughout the treatment period and/or thereafter the
efficacy of a therapy or the prevention of AD is investigated
clinically by quantitative, semi-quantitative or qualitative
assessment of pruritus, skin lesions and a medication score (Hobi
S, Mueller R S; Tierarztliche Praxis. Ausgabe K,
Kleintiere/Heimtiere 2014, 42(3):167-173).
[0332] These clinical parameters are compared to clinical signs of
the individual dog and/or cat prior to the start of a therapeutic
intervention. Alternatively, a comparison to AD affected dogs
and/or cats that are not treated or treated with placebo can
demonstrate the efficacy of the iMAT molecule-mediated treatment
and/or prevention of clinical signs of AD.
[0333] Alternatively or in addition an intradermal provocation test
with certain Dermatophagoides allergens can be employed in said
dogs and/or cats. A reduced response (immediate and/or late phase
reactivity) indicates a therapy and/or prevention effect of the
iMAT molecule administration.
[0334] Furthermore, the modulation of the different components of
the immune system are monitored, e.g. changes in allergen specific
IgE and IgG antibody titers indicate therapy and/or prevention
effects.
[0335] Apart from changes in IgE levels, an increase in
allergen-specific IgG is surprisingly found when treating a dog
and/or cat for AD with such iMAT molecules. These antibodies block
IgE-mediated anaphylaxis in vivo and seem to inhibit not only the
allergen-induced release of inflammatory mediators from basophils
and mast cells, but also IgE-mediated allergen presentation to T
cells. Among the iMAT-induced IgG antibodies specifically binding
to the allergen, some allergen-specific subtypes have been
suggested to play an important "protective" role, as they compete
with allergen-specific IgE antibodies and can prevent the
activation of CD4+ T cells, by inhibiting the IgE-mediated antigen
presentation. Furthermore, the IgG subset which is secreted
promotes a significant reduction in mast cells and eosinophils,
accompanied by a diminished release of inflammatory mediators.
[0336] Allergen-specific immunotherapy can modulate different
components of the immune system. Cellular modifications consist of
a reduction in allergen-induced T-cell proliferation, indicating
the induction of peripheral tolerance in allergen-specific T cells
and a decrease in antigen-specific Th2-dominated immune response in
favor of a Th1 reaction with increased IFN-.gamma. production. The
key cell type responsible for coordinating this immunological
switch is a heterogeneous T-cell population, called regulatory T
cells (T.sub.reg). At the cellular level, the crucial factor for
successful allergen immunotherapy is the peripheral induction of
type 1 T.sub.reg cells. Functional studies on type 1 T.sub.reg
cells, specific in recognizing antigens, revealed that the
modulation of Th1 and Th2 responses by type 1 T.sub.reg cells
mostly depends on the secretion of the cytokine IL-10, which has
immunosuppressive properties. In fact, IL-10 inhibits the
proliferative response of peripheral T cells against specific
allergens and plays a central role in the induction of T-cell
anergy. In vitro, IL-10 enhances the expression of the regulatory
factor FoxP3, modulates eosinophilic function and reduces
pro-inflammatory mediators released by mast cells.
[0337] Another possible marker of the outstanding clinical efficacy
of said iMAT molecules-mediated immunotherapy is the detection of
changes in the number or the nature of allergen-specific T cells.
On the basis of, for example, Bet v1 tetramer staining studies, the
levels and characteristics of circulating birch pollen-specific
CD4.sup.+ T cells can potentially be compared before and after SIT.
Recently, transforming growth factor (TGF)-.beta. has also been
identified as a key cytokine in successful SIT. Many actions may
account for its relevance, such as the suppression of specific Th1,
Th2 and Th17 cells, the induction of FoxP3 and the suppressive
function of Tregs. In addition, TGF-.beta. downregulates
Fc.epsilon.RI expression on Langerhans cells and suppresses IgE
synthesis. These immunological relevant T cell mediated
immunological responses can be measured in ex vivo experiments
applying peripheral blood mononuclear cell (PBMC) cultures and
cytokine detection thereof as described by Nuttall et al. (T. J.
Nuttall et al., Veterinary immunology and Immunopathology 84 (2002)
143-150).
Example 4--Comparison of iMAT Molecules According to the Present
Invention with Prior Art MAT Molecules According to WO 2004/035793
(US Equivalent US 2005/0281816)
[0338] For assessment of purity of a MAT protein, a sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) test
procedure has been established (Thompson J et al., J Biol Chem
2002, 277: 34310-34331). The method, including sample preparation
with a reducing agent, Lithium Dodecylsulphate (LDS) and heating at
75.degree. C., resulted in reproducible multiple sharp bands after
electrophoretic separation. Staining with Coomassie blue gives
linear quantitative (densitometry) features in gels loaded with 200
to 1000 ng protein. Using a monoclonal antibody, detecting the
allergen module in a MAT molecule with Fel d 1 as allergen module
(MAT-Fel d1) it has been shown, that the main band and 13 minor
bands all contain the MAT-Fel d1 protein. The small bands migrate
at the same position as on the original gel also after re-loading
on the gel (FIG. 1A and FIG. 1B). Several different methods, such
as different temperature and buffer composition protocols, for
sample preparation prior to PAGE generated the same band
pattern.
[0339] In all of these bands the presence of the full length
(complete) MAT-Fel d1 protein and only traces of host cell proteins
could be demonstrated after each band was cut out of the gel,
digested by trypsin and subsequently analyzed by mass spectrometry
(nanoLC/ESI-MS). From these experiments an anomalous feature (gel
shifting), e.g. of different folding variants, of MAT-Fel d1 in the
SDS-PAGE can be concluded. This means that MAT-Fel d1 in the
analyzed preparation is not suitable as biopharmaceutical molecule,
in particular for clinical and/or commercial biopharmaceutical
manufacturing, since its purity could not be determined with
standard methods (e.g. SDS-PAGE), but only with the modified
procedure explained above.
[0340] In contrast to this gel shifting phenomenon of the MAT-Fel
d1 molecule, the Fel d1 as such does not show such anomalous
feature in SDS PAGE (FIG. 2, lanes 2 and 3). The Fel d1 displays a
single sharp band at the expected molecular weight (19.6 kD).
[0341] A further anomalous feature could be observed in RP-HPLC
analysis. No single peak of the MAT-Fel d1 was seen in this
analytical method (FIG. 3A and FIG. 3B), neither in the native
conformation of the protein (FIG. 3A) nor in the denatured form
(FIG. 3B) induced by chaotropic and reducing conditions. However,
for GMP certified biopharmaceutical manufacturing a single isoform
of the biomolecule in marketed pharmaceutical preparations is
mandatory.
[0342] These observations in SDS-PAGE and RP-HPLC analysis may be
explained by the physicochemical properties based on the amino acid
sequence. Analysis in the Kyte & Doolittle hydrophobicity plot
[Kyte J, Doolittle R F, Journal of Molecular Biology 1982, 157(1),
105-132] revealed adjacent extreme hydrophobic and hydrophilic
domains (FIG. 4) which may be responsible for this anomalous
behavior.
[0343] In particular, the hydrophobic region of the targeting
domain of the fusion protein is similar to the transmembrane
segments of membrane proteins which are known in the art to cause
such anomalous feature [Rath A et al., Proc Natl Acad Sci USA.
2009, 106(6): 1760-1765].
[0344] Migration on SDS-PAGE, which does not correlate with
formula's molecular weights, termed "gel shifting" appears to be
common for membrane proteins. This means, that the prerequisite of
the SDS-PAGE method, which is a separation of molecules solely
according to their molecular weight, independent on their native
2D- or 3D-structure does not apply in these cases. In the above
cited work (PNAS article), the authors investigate the anomalous
gel mobility of helical membrane proteins using a library of
wild-type and mutant helix-loop-helix ("hairpin") sequences derived
from transmembrane segments 3 and 4 of the human cystic fibrosis
transmembrane conductance regulator (CFTR), including
disease-phenotypic residue substitutions. They found that these
hairpins migrate at rates of minus 10% to plus 30% vs. their actual
formula's molecular weights on SDS-PAGE and load detergent at
ratios ranging from 3.4-10 g SDS/g protein. They additionally
demonstrated that mutant gel shifts strongly correlate with changes
in hairpin SDS loading capacity, and with hairpin helicity,
indicating that gel shift behavior originates in altered detergent
binding. In some cases, this differential solvation by SDS may
result from replacing protein-detergent contacts with
protein-protein contacts, implying that detergent binding and
folding are intimately linked.
[0345] The SDS PAGE (FIG. 5) as well as the RP-HPLC analysis (FIGS.
3A, 3B, and 6) of MAT and iMAT proteins reveal substantial
differences in the migration pattern or elution, respectively. The
oxidized form of MAT-Fel d1 does not show a single sharp band on
the SDS-PAGE gel (Lane 3) but several diffuse bands with larger and
smaller apparent molecular weights than the actual 32.2 kD of
MAT-Fel d1. In contrast, as an example an iMAT molecule with an
antigen module of a Culicoides obsoletus allergen (iMAT-Cul o4)
exhibits a single sharp band (M=41.6 kD) under oxidized conditions.
Also the RP-HPLC chromatogram shows a single peak (FIG. 6).
[0346] Under reducing conditions, the MAT-Fel d1 in the SDS-PAGE
reveals a main band migrating approximately at the known molecular
weight but in addition some of the minor bands described in FIG. 1A
and FIG. 1B known to contain the complete sequence of MAT-Fel d1
emerge again (FIG. 5, lane 6). This is an attribute, which is
characteristic for the anomalous feature of MAT-Fel d1.
Additionally, the RP-HPLC chromatogram of MAT-Fel d1 under reducing
conditions (FIG. 3B) exhibits at least 3 different isoforms of the
MAT-Fel d1. In contrast the iMAT molecules reveal characteristics
evidently indicative for a single isoform in the SDS-PAGE (FIG. 5,
lane 7) as well as in the RP-HPLC (FIG. 6).
[0347] The reducing conditions lead to a cleavage of the disulfide
bridges in the MAT molecule, thus the MAT and the iMAT molecules
should behave alike under reducing conditions if the disulphide
bridges are solely responsible for the anomalous feature of MAT.
However, this is not the case, since the anomalous gel shifting and
the occurrence of isoforms in RP-HPLC of MAT molecules is still
present under reducing conditions.
[0348] However, the iMAT molecule does not show such gel shifting
and exhibits a peak in RP-HPLC chromatogram in the native
(oxidized) form of the protein. Furthermore, the Kyte-Doolittle
plots of MAT and iMAT molecules are nearly identical at the
N-terminus covering the sequence of His-tag, TAT and targeting
domain (FIG. 8). Consequently, a person skilled in the art would
not be motivated to construct a MAT molecule according to the prior
art with cysteine residues substituted with other amino acid
residues in order to overcome the disadvantages of the prior
art.
[0349] iMAT molecules can be constructed by the "bioinformatical
engineering" procedures according to Example 5 below and produced
by recombinant expression technology in E. coli. As an example
three iMAT molecules, as shown in FIG. 7 are stable in buffer (20
mM citrate, 1 M arginine, pH 6.0) after freezing and thawing twice
and could be adsorbed to ADJU-PHOS.RTM. (Brenntag, Denmark) as
adjuvant, so that the iMAT molecules can be used as a vaccine. The
proteins are desorbed from ADJU-PHOS.RTM. without degradation in
the same buffer system (FIG. 7).
Example 5--"Bioinformatical Engineering" of iMAT Molecules:
Selection of Proteins for the Antigen Module and Optimization of
the Full iMAT Molecule
[0350] In order to, for example, treat dogs and/or cats with
allergic disorders using the iMAT technology effectively, it is
further advisable:
a) to select a protein as allergen module in iMAT molecules that is
an allergen and thus has a high potential to cause hypersensitivity
in affected subjects and thus can also be the target for tolerance
induction, and b) to construct an iMAT molecule with said allergens
that is thermodynamically stable and can be produced efficiently by
protein engineering and can additionally be analyzed with standard
methods to ensure sufficient enough quality (i.e. identity, purity
and potency).
[0351] In order to fulfil these requirements a bioinformatics
approach is chosen for the selection of the allergen to be included
into the iMAT molecules according to the invention. The objective
of the selection is (i) to choose one or more allergens to be
expected to be of relevance in a given allergic disorder, i.e. that
the majority of individuals suffering from allergic disorder are
sensitized to the respective allergen, and (ii) to choose the
allergen with the highest probability of comprising linear epitopes
of allergen characteristics, i.e. comprising high numbers of short
peptide sequences (7 to 13 amino acid residues) homologue to those
in published allergens.
[0352] To select appropriate antigens for pharmaceutical
preparations, a homology comparison based on local sequence
alignments to known allergens is chosen. Often epitope detection
for antibody recognition (mostly conformational epitopes) is
achieved by functional analysis (e.g. peptide microarrays) or for
T-cells epitopes (linear epitopes) by calculation of peptide
binding probabilities to MHC molecules. The therapeutic principle
of the iMAT technology inter alia is based on endocytosis and
degradation by acid-dependent proteases in endosomes followed by
MHC Class II binding and antigen presentation.
[0353] Thus a different--non experimental but
bioinformatics--approach for allergen selection is chosen that is
based on local homology searches of peptides derived from given
proteins to known allergenic proteins, most of which are known to
raise allergies in humans. Amino acid sequences of proteins
suspected to have allergenic properties are exported from publicly
available databases (e.g. UNIPROT) and redundancies are determined
by analysis of sequences homologies within the exported dataset.
Highly homologue sequence counterparts are eliminated and the
resulting remainder of sequences served as the canonical sequence
database of probable valid antigens for subsequent analyses. To
determine proteins with putative high allergenic potential,
proteins are in silico cleaved into peptides with lengths of 6 to
15 amino acids with a one amino acid shifting. Next, local-pairwise
alignments of e.g. Dermatophagoides proteins and the corresponding
peptides to the canonical sequence database are performed.
Following this, a scaling of obtained alignment hits is conducted
by setting the self-alignment score of a given protein to one and
alignment hits of the corresponding peptides accordingly.
Thereafter the number of alignment hits exceeding a given threshold
are counted for each peptide and compared by local-pairwise
alignment to a randomly generated database of protein sequences
with no known allergenic properties, and subsequently scaled and
counted. The resulting "non allergic protein" counts are subtracted
from those of the allergen results and cumulative hit scores for
each protein based on the number of hits for all corresponding
peptides are calculated. Proteins with highest counts are selected
as iMAT antigen module candidates.
[0354] Each of the selected allergens are integrated into separate
iMAT molecules as the antigen module and subsequently the full iMAT
molecule is optimized for thermodynamic stability by iterative
modeling of three-dimensional protein structures and calculation of
changes of free energies after substitution of single amino acids.
Physicochemical properties and stability is influenced by
substituting different amino acid residue(s) within the primary
amino acid sequence.
[0355] The results of the herein described analyses (antigen search
and modeling) are transformed into an iMAT amino acid sequence
suitable for pharmacological production and application.
[0356] In a specific example, this bioinformatics engineering
approach identifies Zen 1 and Der f 15 to be of relevance in the
allergic disorder atopic dermatitis elicited by proteins derived
from the mite species Dermatophagoides farinae. Furthermore, the
bioinformatics analysis reveals stable iMAT molecules with
cysteines substituted by other amino acid residues. Examples of
such stable iMAT molecules are SEQ ID NO: 39 (Dog Zen1) and SEQ ID
NO: 57 (Dog Der f 15).
Example 6--Construction of Mosaic-Like iMAT Molecules According to
the Invention
[0357] It is expected that iMAT molecules according to the
invention is further improved if components of more than one
allergen are included into the antigen module. For this purpose, it
is possible to apply the basic principle of the above described
bioinformatics selection approach (Example 5) in a different way.
Instead of selecting complete allergens based on the hit count of
allergen peptides found in the allergen data base, only the most
abundant peptides of several of such allergens are used to engineer
an iMAT antigen module. Thus, such an iMAT molecule consists of an
antigen module of peptides that stem from several allergens. This
allows broadening of the spectrum of a single iMAT molecule with
respect to its targeted allergic profile and is thus beneficial for
pharmacological drug development.
[0358] In order to find short peptide sequences that qualify for
such mosaic-like iMAT molecule proteins from e.g. Dermatophagoides
are analyzed by homology comparison as described above. Briefly in
silico cleaved proteins with peptide lengths of 6-15 amino acid
residues are locally aligned to a canonical sequence database of
allergen-related proteins and a random database of
non-allergy-related proteins. The number of differences of
significant homologies for each peptide found within the canonical
database is determined. Subsequently, each peptide is locally
aligned to a random database triple the size of the canonical
database to reduce false positive hits. The top (e.g. tenth
percentile) of remaining homologies for each peptide length is
especially suitable to serve as a base for construction of a
mosaic-like or hybrid allergen carrying iMAT molecule. To construct
a mosaic-like iMAT molecule a protein precursor is chosen (for
example from the list of precursor proteins corresponding to top
ranking peptides) as a scaffold protein for embedding top ranking
peptides. The signal peptide sequence is removed from the scaffold
protein and top ranking peptides, optionally with additional
adjacent N- or C-terminal amino acids, can be inserted within the
original sequence of the scaffold protein or can replace parts of
the original sequence of the scaffold protein. The position for
insertion or replacement is determined using similarity alignments
or the reference position of the peptide in the corresponding
precursor protein. As a next step, His-Tag, the TAT and targeting
domain are added. Finally, cysteine residues are replaced by most
stabilizing residues as described above.
[0359] In a specific example (hybrid 1), this bioinformatics
engineering approach identifies a combination of SEQ ID NO: 84
(A1KXC1_DERFA DFP1 OS=Dermatophagoides farinae), SEQ ID NO: 10
(Q86R84_DERFA 60 kDa allergen Der f 18p OS=Dermatophagoides farinae
GN=Der f 18 PE=2 SV=1), SEQ ID NO: 85 (A0A088SAS1_DERFA Der f 28
allergen OS=Dermatophagoides farinae PE=2 SV=1), SEQ ID NO: 88:
(A7XXV2_DERFA Der f 2 allergen OS=Dermatophagoides farinae PE=4
SV=1), SEQ ID NO: 86 (B7U5T1_DERFA Der f 6 allergen
OS=Dermatophagoides farinae PE=2 SV=1), SEQ ID NO: 87 (T2B4F3_DERPT
LytFM OS=Dermatophagoides pteronyssinus GN=lytFM PE=4 SV=1), SEQ ID
NO: 11 (Q9U6R7_DERFA 98 kDa HDM allergen OS=Dermatophagoides
farinae PE=2 SV=1)
[0360] to be of relevance in the allergic disorder atopic
dermatitis elicited by proteins derived from the mite species
Dermatophagoides farinae. Furthermore, the bioinformatics analysis
reveals stable iMAT molecules with cysteines substituted by other
amino acid residues. Examples of such stable mosaic-like iMAT
molecules are
[0361] SEQ ID NOS: 75 (Dog Hybrid 1) and SEQ ID NO: 66 (Cat Hybrid
1).
Example 7--Therapeutic Vaccine/Prophylaxis of Allergic Asthma in
Cats
[0362] A single iMAT molecule or a combination of iMAT molecules
containing different antigen modules according to the present
invention can be employed for treating prophylactically or
therapeutically a cat suffering from or being at risk of allergic
asthma. In cats iMAT molecules according to the present invention
are administered as described in Example 3.
[0363] Adult cats with a known history of allergic asthma will be
included in the study, i.e. cats reported to exhibit clinical signs
as spastic coughing episodes, wheezing and expiratory dyspnea.
[0364] Bronchioalveolar lavage fluids (BALF) are collected prior to
treatment start and e.g. 2, 3, and 6 months during/after treatment.
BALF is used for cytologic examination and nucleated cell
counts.
[0365] Cats are sedated with e.g. Ketamine HCl intravenously.
Bronchoalveolar lavage fluid is collected by gently inserting e.g.
a 7 Fr polypropylene catheter through the endotracheal tube. When
resistance is felt, an up to 20 ml aliquot of warmed sterile saline
is lavaged through the catheter and retrieved by manual suction.
After centrifugation and resuspension, a smear cytology of the
collected BALF cells is prepared, the presence of significant
numbers of eosinophils support a diagnosis of feline asthma.
Differential cell counts can quantitatively evaluate the ratio (%)
of eosinophils in BAL fluids.
[0366] Alternatively or in addition, employing certain recombinant
allergens an intradermal provocation test, skin prick test or also
allergen specific IgE and/or IgG determination in BAL fluid or
serum can be monitored in said cats (Norris et al., Vet Immunol
Immunopathol. 2003, 96(3-4): 119-127). A reduced response
(immediate and/or late phase reactivity) and/or changes of the
antibody titers indicate a therapy and/or prevention effects of the
iMAT molecule treatment.
[0367] Clinical signs as the respiratory rate and scores to account
for respiratory effort/difficulty are employed. Said "respiratory
scoring system" can be employed also e.g. in response to an aerosol
challenge. Briefly, awake, spontaneously breathing cats in a sealed
chamber are exposed for different time length and/or different
concentrations of aerosolized recombinant allergens. Alternatively,
quantitative measures of the airway hyper-responsiveness can be
performed in anesthetized cats. Pneumotachograph measurements can
be done baseline and in a broncho-provocation protocol e.g. a dose
response of the pulmonary resistance to methacholine and/or
selected recombinant allergens.
[0368] Thus, throughout the treatment period and/or thereafter the
efficacy of a therapy or the prevention of allergic asthma is
investigated clinically by quantitative, semi-quantitative or
qualitative assessments.
[0369] The parameters can be compared in the individual cat to the
severity prior to the start of a therapeutic intervention.
Alternatively, a comparison to cats with allergic asthma that are
not treated or treated with placebo demonstrates the efficacy of
the iMAT molecule-mediated treatment and/or prevention of clinical
signs of feline allergic asthma.
Example 8--Therapeutic Vaccine/Prophylaxis of Flea Allergy in Cats
and/or Dogs
[0370] A single iMAT molecule or a combination of iMAT molecules
containing different antigen modules according to the present
invention can be employed for treating prophylactically or
therapeutically a cat and/or a dog suffering from or being at risk
of flea atopic dermatitis.
[0371] In a first example the iMAT molecule according to SEQ ID NO:
42 (Cat Cte f 1) is administered into the popliteal lymph node of
cats suffering from or being at risk of flea atopic dermatitis.
[0372] In a second example the iMAT molecule according to SEQ ID
NO: 63 (Dog Cte f 1) is administered into the popliteal lymph node
of dogs suffering from or being at risk of flea atopic
dermatitis.
[0373] The further treatment details are as described in Example 3
above.
[0374] The efficacy of said iMAT treatment is evaluated by an
intradermal test (IDT), T-cell analyses and measurement of flea
allergen specific IgE and IgG (Gerber, J. D. Vaccine
1990-12-8(6):536-542) in treated cats and/or dogs before and after
iMAT treatment as described by Jin (Jin J et al., Vaccine 28 (2010)
1997-2004). Intradermal tests (IDTs) are done following the
protocol from Hillier and DeBoer, (DeBoer, D. J., Hillier, A.
Veterinary Immunology and Immunopathology 2001, 81 (3-4), 271-276).
4 weeks after the last immunization, the cats and/or dogs are
injected with 100 .mu.l PBS containing 100 .mu.g of flea extract on
the lateral thorax skin of the cats and/or dogs intradermally;
histamine is used as positive control, BSA used as an irrelevant
stimulator, and saline used as the negative control. The size of
reactive bleb on the skin is marked with a marker pen and measured
perpendicularly and horizontally within 20 min after the challenge
by a micrometer. The results are calculated as an average of the
three measurements. A reduced response (immediate and/or late phase
reactivity) and/or changes of the antibody titers or Th1 or Treg
skewed T-cell responses indicate a therapy and/or prevention
effects of the iMAT molecule treatment.
REFERENCES
[0375] (1) 3.sup.rd Havemeyer workshop, Holar, Iceland, June 2007,
Veterinary Immunology and Immunotherapy 2008, 126: 351-361 [0376]
(2) Allergome (www.allergome.org) [0377] (3) Crameri R. et al.,
Allergy 2007, 62: 197-206 [0378] (4) DeBoer, D. J., Hillier, A.
Veterinary Immunology and Immunopathology 2001, 81(3-4): 271-276
[0379] (5) Gadermaier G et al., Molecular Immunology 2010, 47:
1292-1298 [0380] (6) Gerber, J. D. Vaccine 1990-12-8(6):536-542
[0381] (7) Griffin C E. Diagnosis of canine atopic dermatitis in
Veterinary Allergy DOI: 10.1002/9781118738818.ch10 [0382] (8)
Guaguere E et al. EJCAP, 2009, 19 (3), 234-241 [0383] (9) Hill et
al. Vet Immunol Immunopathol, 2001; 81(3-4): 169-186 [0384] (10)
Hobi S, Mueller R S; Tierarztliche Praxis. Ausgabe K, Kleintiere
Heimtiere 2014, 42(3):167-173 [0385] (11) Jackson H A, EJCAP, 2009,
19 (3), 230-233 [0386] (12) Jin J. et al. Vaccine 2010, 28:
1997-2004 [0387] (13) Klein J S et al., Protein Eng Des Sel 2014,
27(10): 325-330 [0388] (14) Kyte J, Doolittle R F, Journal of
Molecular Biology 1982, 157(1): 105-132 [0389] (15) Martinez-Gomez
J M et al., Allergy 2009, 64(1): 172-178 [0390] (16) McCall C et
al., Vet Immunol Immunopath 2001; 78:231-247 [0391] (17) McDermott
M J et al. Molecular Immunology 2000, 37: 361-375 [0392] (18)
Norris et al., Vet Immunol Immunopathol. 2003, 96(3-4): 119-127
[0393] (19) Nuttall T. J. et al., Veterinary immunology and
Immunopathology 2002; 84: 143-150 [0394] (20) Pires D E et al.
Bioinformatics 2014, 30(3): 335-342 [0395] (21) Prost C, Rev Fr
Allergol Immunol Clin, 2008, 48(5), 409-413 [0396] (22) Rath A et
al., PNAS 2009, 106(6): 1760-1765 [0397] (23) Rose H, Arb Paul
Ehrlich Inst Bundesinstitut Impfstoffe Biomed Arzneim Langen Hess
2009, 96: 319-327 [0398] (24) Senna G et al., Curr Opin Allergy
Clin Immunol. 2011, 11(4): 375-380 [0399] (25) Senti G et al., J
Allergy Clin Immunol. 2012, 129(5): 1290-1296 [0400] (26) SIAF
Annual Report 2010 [0401] (27) SIAF Annual Report 2011 [0402] (28)
Thomas W R et al., Chang Gung Med J 2004; 27: 563-569 [0403] (29)
Thompson J et al., J Biol Chem 2002, 277: 34310-34331 [0404] (30)
US 2005/0281816 [0405] (31) U.S. Pat. No. 7,629,446 [0406] (32)
U.S. Pat. No. 7,653,866 [0407] (33) WHO PD-VAC 2014--Status of
Vaccine Research and Development of Vaccines for Leishmaniasis
[0408] (34) WO 2004/035793 [0409] (35) Zhao et al. Int J Clin Exp
Med 2015; 8(4):6436-6443
Sequence CWU 1
1
102111PRTArtificial SequenceTAT sequence 1Tyr Gly Arg Lys Lys Arg
Arg Gln Arg Arg Arg1 5 102272PRTCanis lupus familiaris 2Met Glu Asp
Gln Arg Asp Leu Ile Ser Asn His Glu Gln Leu Pro Ile1 5 10 15Leu Gly
Gln Arg Pro Gly Ala Pro Glu Ser Lys Cys Ser Arg Gly Ala 20 25 30Leu
Tyr Thr Gly Phe Ser Val Leu Val Ala Leu Leu Leu Ala Gly Gln 35 40
45Ala Thr Thr Ala Tyr Phe Leu Tyr Gln Gln Gln Gly Arg Leu Asp Lys
50 55 60Leu Thr Val Thr Ser Gln Asn Leu Gln Leu Glu Ser Leu Arg Met
Lys65 70 75 80Leu Pro Lys Pro Pro Lys Pro Leu Ser Lys Met Arg Val
Ala Thr Pro 85 90 95Met Met Met Gln Ala Leu Pro Ile Gln Ser Leu Pro
Gln Gly Pro Thr 100 105 110Gln Asn Ala Thr Glu Tyr Gly Asn Met Thr
Gln Asp His Val Met His 115 120 125Leu Leu Leu Glu Ala Asp Pro Leu
Lys Val Tyr Pro Lys Leu Lys Gly 130 135 140Ser Phe Pro Glu Asn Leu
Lys His Leu Lys Asn Thr Met Glu Thr Leu145 150 155 160Asp Trp Lys
Val Phe Glu Asn Trp Met Tyr Gln Trp Leu Leu Phe Glu 165 170 175Met
Ser Lys Asn Ser Leu Glu Lys Lys Ser Thr Glu Val Pro Leu Lys 180 185
190Ala Leu Thr Lys Cys Gln Glu Glu Val Ser Arg Ile Pro Asp Val His
195 200 205Pro Gly Met Phe Arg Pro Gln Cys Asp Glu Asn Gly Asn Tyr
Lys Pro 210 215 220Leu Gln Cys Tyr Gly Ser Thr Gly Tyr Cys Trp Cys
Val Phe Pro Asn225 230 235 240Gly Thr Glu Val Pro His Thr Arg Ser
His Gly His His Asn Cys Ser 245 250 255Glu Pro Leu Asp Met Glu Asp
Leu Ser Ser Gly Leu Gly Met Ala Lys 260 265 2703280PRTFelis catus
3Met Glu Asp Gln Arg Asp Leu Ile Ser Asn His Glu Gln Leu Pro Ile1 5
10 15Leu Gly Gln Arg Pro Ala Ala Pro Glu Ser Lys Cys Ser Arg Gly
Ala 20 25 30Leu Tyr Thr Gly Phe Ser Val Leu Val Ala Leu Leu Leu Ala
Gly Gln 35 40 45Ala Thr Thr Ala Tyr Phe Leu Tyr Gln Gln Gln Gly Arg
Leu Asp Lys 50 55 60Leu Thr Val Thr Ala Gln Asn Leu Gln Leu Glu Asn
Leu Arg Met Lys65 70 75 80Leu Pro Lys Pro Ala Lys Pro Leu Asn Lys
Leu Arg Val Ala Thr Pro 85 90 95Met Leu Met Gln Thr Met Pro Val Arg
Gly Leu Leu Gln Ala Pro Met 100 105 110Gln Asn Ala Thr Lys Tyr Gly
Asn Met Thr Gln Asp His Val Met His 115 120 125Met Leu Leu Glu Gly
Asp Pro Leu Lys Val Tyr Pro Gln Leu Lys Gly 130 135 140Asn Phe Pro
Glu Asn Leu Lys His Leu Lys Asn Thr Met Gly Thr Leu145 150 155
160Asp Trp Lys Val Phe Glu Asn Trp Met Tyr Gln Trp Leu Leu Phe Glu
165 170 175Met Ser Lys Asn Ser Leu Glu Lys His Pro Ala Asp Ile Pro
Leu Lys 180 185 190Val Leu Thr Lys Cys Gln Glu Glu Val Ser Arg Ile
Pro Ala Val His 195 200 205Pro Gly Thr Phe Arg Pro Gln Cys Asp Glu
Asn Gly Asn Tyr Lys Pro 210 215 220Leu Gln Cys Tyr Gly Ser Thr Gly
Tyr Cys Trp Cys Val Phe Pro Asn225 230 235 240Gly Thr Glu Val Pro
His Ser Arg Ser His Gly His Arg Asn Cys Ser 245 250 255Glu Ser Val
Asp Val Glu Asp Leu Ser Ser Gly Leu Gly Met Thr Lys 260 265 270Pro
Asp Leu Gly Gln Val Val Met 275 2804110PRTArtificial
sequenceInvariant Chain Dog [110 AA] 4Met Glu Asp Gln Arg Asp Leu
Ile Ser Asn His Glu Gln Leu Pro Ile1 5 10 15Leu Gly Gln Arg Pro Gly
Ala Pro Glu Ser Lys Cys Ser Arg Gly Ala 20 25 30Leu Tyr Thr Gly Phe
Ser Val Leu Val Ala Leu Leu Leu Ala Gly Gln 35 40 45Ala Thr Thr Ala
Tyr Phe Leu Tyr Gln Gln Gln Gly Arg Leu Asp Lys 50 55 60Leu Thr Val
Thr Ser Gln Asn Leu Gln Leu Glu Ser Leu Arg Met Lys65 70 75 80Leu
Pro Lys Pro Pro Lys Pro Leu Ser Lys Met Arg Val Ala Thr Pro 85 90
95Met Met Met Gln Ala Leu Pro Ile Gln Ser Leu Pro Gln Gly 100 105
1105110PRTArtificial SequenceInvariant Chain Cat [110 AA] 5Met Glu
Asp Gln Arg Asp Leu Ile Ser Asn His Glu Gln Leu Pro Ile1 5 10 15Leu
Gly Gln Arg Pro Ala Ala Pro Glu Ser Lys Cys Ser Arg Gly Ala 20 25
30Leu Tyr Thr Gly Phe Ser Val Leu Val Ala Leu Leu Leu Ala Gly Gln
35 40 45Ala Thr Thr Ala Tyr Phe Leu Tyr Gln Gln Gln Gly Arg Leu Asp
Lys 50 55 60Leu Thr Val Thr Ala Gln Asn Leu Gln Leu Glu Asn Leu Arg
Met Lys65 70 75 80Leu Pro Lys Pro Ala Lys Pro Leu Asn Lys Leu Arg
Val Ala Thr Pro 85 90 95Met Leu Met Gln Thr Met Pro Val Arg Gly Leu
Leu Gln Ala 100 105 110622PRTArtificial sequenceN-Term Marker [22
AA] 6Met His His His His His His Tyr Gly Arg Lys Lys Arg Arg Gln
Arg1 5 10 15Arg Arg Met Glu Asp Gln 207321PRTDermatophagoides
farinae 7Met Lys Phe Val Leu Ala Ile Ala Ser Leu Leu Val Leu Ser
Thr Val1 5 10 15Tyr Ala Arg Pro Ala Ser Ile Lys Thr Phe Glu Glu Phe
Lys Lys Ala 20 25 30Phe Asn Lys Asn Tyr Ala Thr Val Glu Glu Glu Glu
Val Ala Arg Lys 35 40 45Asn Phe Leu Glu Ser Leu Lys Tyr Val Glu Ala
Asn Lys Gly Ala Ile 50 55 60Asn His Leu Ser Asp Leu Ser Leu Asp Glu
Phe Lys Asn Arg Tyr Leu65 70 75 80Met Ser Ala Glu Ala Phe Glu Gln
Leu Lys Thr Gln Phe Asp Leu Asn 85 90 95Ala Glu Thr Ser Ala Cys Arg
Ile Asn Ser Val Asn Val Pro Ser Glu 100 105 110Leu Asp Leu Arg Ser
Leu Arg Thr Val Thr Pro Ile Arg Met Gln Gly 115 120 125Gly Cys Gly
Ser Cys Trp Ala Phe Ser Gly Val Ala Ala Thr Glu Ser 130 135 140Ala
Tyr Leu Ala Tyr Arg Asn Thr Ser Leu Asp Leu Ser Glu Gln Glu145 150
155 160Leu Val Asp Cys Ala Ser Gln His Gly Cys His Gly Asp Thr Ile
Pro 165 170 175Arg Gly Ile Glu Tyr Ile Gln Gln Asn Gly Val Val Glu
Glu Arg Ser 180 185 190Tyr Pro Tyr Val Ala Arg Glu Gln Gln Cys Arg
Arg Pro Asn Ser Gln 195 200 205His Tyr Gly Ile Ser Asn Tyr Cys Gln
Ile Tyr Pro Pro Asp Val Lys 210 215 220Gln Ile Arg Glu Ala Leu Thr
Gln Thr His Thr Ala Ile Ala Val Ile225 230 235 240Ile Gly Ile Lys
Asp Leu Arg Ala Phe Gln His Tyr Asp Gly Arg Thr 245 250 255Ile Ile
Gln His Asp Asn Gly Tyr Gln Pro Asn Tyr His Ala Val Asn 260 265
270Ile Val Gly Tyr Gly Ser Thr Gln Gly Val Asp Tyr Trp Ile Val Arg
275 280 285Asn Ser Trp Asp Thr Thr Trp Gly Asp Ser Gly Tyr Gly Tyr
Phe Gln 290 295 300Ala Gly Asn Asn Leu Met Met Ile Glu Gln Tyr Pro
Tyr Val Val Ile305 310 315 320Met8146PRTDermatophagoides farinae
8Met Ile Ser Lys Ile Leu Cys Leu Ser Leu Leu Val Ala Ala Val Val1 5
10 15Ala Asp Gln Val Asp Val Lys Asp Cys Ala Asn Asn Glu Ile Lys
Lys 20 25 30Val Met Val Asp Gly Cys His Gly Ser Asp Pro Cys Ile Ile
His Arg 35 40 45Gly Lys Pro Phe Thr Leu Glu Ala Leu Phe Asp Ala Asn
Gln Asn Thr 50 55 60Lys Thr Ala Lys Ile Glu Ile Lys Ala Ser Leu Asp
Gly Leu Glu Ile65 70 75 80Asp Val Pro Gly Ile Asp Thr Asn Ala Cys
His Phe Met Lys Cys Pro 85 90 95Leu Val Lys Gly Gln Gln Tyr Asp Ile
Lys Tyr Thr Trp Asn Val Pro 100 105 110Lys Ile Ala Pro Lys Ser Glu
Asn Val Val Val Thr Val Lys Leu Ile 115 120 125Gly Asp Asn Gly Val
Leu Ala Cys Ala Ile Ala Thr His Gly Lys Ile 130 135 140Arg
Asp145991PRTDermatophagoides farinae 9Met Lys Phe Asn Ile Thr Ile
Ala Phe Val Ser Leu Ala Ile Leu Ile1 5 10 15His Ser Ser Tyr Ala Asp
Ile Asp His Asp Asp Asp Pro Thr Thr Met 20 25 30Ile Asp Val Gln Thr
Thr Thr Val Gln Pro Ser Asp Glu Phe Glu Cys 35 40 45Pro Thr Arg Phe
Gly Tyr Phe Ala Asp Pro Lys Asp Pro Cys Lys Phe 50 55 60Tyr Ile Cys
Ser Asn Trp Glu Ala Ile His Lys Ser Cys Pro Gly Asn65 70 75 80Thr
Arg Trp Asn Glu Lys Glu Leu Thr Cys Thr 85
9010462PRTDermatophagoides farinae 10Met Thr Arg Phe Ser Leu Thr
Val Leu Ala Val Leu Ala Ala Cys Phe1 5 10 15Gly Ser Asn Ile Arg Pro
Asn Val Ala Thr Leu Glu Pro Lys Thr Val 20 25 30Cys Tyr Tyr Glu Ser
Trp Val His Trp Arg Gln Gly Glu Gly Lys Met 35 40 45Asp Pro Glu Asp
Ile Asp Thr Ser Leu Cys Thr His Ile Val Tyr Ser 50 55 60Tyr Phe Gly
Ile Asp Ala Ala Thr His Glu Ile Lys Leu Leu Asp Glu65 70 75 80Tyr
Leu Met Lys Asp Leu His Asp Met Glu His Phe Thr Gln His Lys 85 90
95Gly Asn Ala Lys Ala Met Ile Ala Val Gly Gly Ser Thr Met Ser Asp
100 105 110Gln Phe Ser Lys Thr Ala Ala Val Glu His Tyr Arg Glu Thr
Phe Val 115 120 125Val Ser Thr Val Asp Leu Met Thr Arg Tyr Gly Phe
Asp Gly Val Met 130 135 140Ile Asp Trp Ser Gly Met Gln Ala Lys Asp
Ser Asp Asn Phe Ile Lys145 150 155 160Leu Leu Asp Lys Phe Asp Glu
Lys Phe Ala His Thr Ser Phe Val Met 165 170 175Gly Val Thr Leu Pro
Ala Thr Ile Ala Ser Tyr Asp Asn Tyr Asn Ile 180 185 190Pro Ala Ile
Ser Asn Tyr Val Asp Phe Met Asn Val Leu Ser Leu Asp 195 200 205Tyr
Thr Gly Ser Trp Ala His Thr Val Gly His Ala Ser Pro Phe Pro 210 215
220Glu Gln Leu Lys Thr Leu Glu Ala Tyr His Lys Arg Gly Ala Pro
Arg225 230 235 240His Lys Met Val Met Ala Val Pro Phe Tyr Ala Arg
Thr Trp Ile Leu 245 250 255Glu Lys Met Asn Lys Gln Asp Ile Gly Asp
Lys Ala Ser Gly Pro Gly 260 265 270Pro Arg Gly Gln Phe Thr Gln Thr
Asp Gly Phe Leu Ser Tyr Asn Glu 275 280 285Leu Cys Val Gln Ile Gln
Ala Glu Thr Asn Ala Phe Thr Ile Thr Arg 290 295 300Asp His Asp Asn
Thr Ala Ile Tyr Ala Val Tyr Val His Ser Asn His305 310 315 320Ala
Glu Trp Ile Ser Phe Glu Asp Arg His Thr Leu Gly Glu Lys Ala 325 330
335Lys Asn Ile Thr Gln Gln Gly Tyr Ala Gly Met Ser Val Tyr Thr Leu
340 345 350Ser Asn Glu Asp Val His Gly Val Cys Gly Asp Lys Asn Pro
Leu Leu 355 360 365His Ala Ile Gln Ser Asn Tyr Tyr His Gly Val Val
Thr Glu Pro Thr 370 375 380Val Val Thr Leu Pro Pro Val Thr His Thr
Thr Glu His Val Thr Asp385 390 395 400Ile Pro Gly Val Phe His Cys
His Glu Glu Gly Phe Phe Arg Asp Lys 405 410 415Thr Tyr Cys Ala Thr
Tyr Tyr Glu Cys Lys Lys Gly Asp Phe Gly Leu 420 425 430Glu Lys Thr
Val His His Cys Ala Asn His Leu Gln Ala Phe Asp Glu 435 440 445Val
Ser Arg Thr Cys Ile Asp His Thr Lys Ile Pro Gly Cys 450 455
46011555PRTDermatophagoides farinae 11Met Lys Thr Ile Tyr Ala Ile
Leu Ser Ile Met Ala Cys Ile Gly Leu1 5 10 15Met Asn Ala Ser Ile Lys
Arg Asp His Asn Asp Tyr Ser Lys Asn Pro 20 25 30Met Arg Ile Val Cys
Tyr Val Gly Thr Trp Ser Val Tyr His Lys Val 35 40 45Asp Pro Tyr Thr
Ile Glu Asp Ile Asp Pro Phe Lys Cys Thr His Leu 50 55 60Met Tyr Gly
Phe Ala Lys Ile Asp Glu Tyr Lys Tyr Thr Ile Gln Val65 70 75 80Phe
Asp Pro Tyr Gln Asp Asp Asn His Asn Ser Trp Glu Lys Arg Gly 85 90
95Tyr Glu Arg Phe Asn Asn Leu Arg Leu Lys Asn Pro Glu Leu Thr Thr
100 105 110Met Ile Ser Leu Gly Gly Trp Tyr Glu Gly Ser Glu Lys Tyr
Ser Asp 115 120 125Met Ala Ala Asn Pro Thr Tyr Arg Gln Gln Phe Ile
Gln Ser Val Leu 130 135 140Asp Phe Leu Gln Glu Tyr Lys Phe Asp Gly
Leu Asp Leu Asp Trp Glu145 150 155 160Tyr Pro Gly Ser Arg Leu Gly
Asn Pro Lys Ile Asp Lys Gln Asn Tyr 165 170 175Leu Ala Leu Val Arg
Glu Leu Lys Asp Ala Phe Glu Pro His Gly Tyr 180 185 190Leu Leu Thr
Ala Ala Val Ser Pro Gly Lys Asp Lys Ile Asp Arg Ala 195 200 205Tyr
Asp Ile Lys Glu Leu Asn Lys Leu Phe Asp Trp Met Asn Val Met 210 215
220Thr Tyr Asp Tyr His Gly Gly Trp Glu Asn Phe Tyr Gly His Asn
Ala225 230 235 240Pro Leu Tyr Lys Arg Pro Asp Glu Thr Asp Glu Leu
His Thr Tyr Phe 245 250 255Asn Val Asn Tyr Thr Met His Tyr Tyr Leu
Asn Asn Gly Ala Thr Arg 260 265 270Asp Lys Leu Val Met Gly Val Pro
Phe Tyr Gly Arg Ala Trp Ser Ile 275 280 285Glu Asp Arg Ser Lys Leu
Lys Leu Gly Asp Pro Ala Lys Gly Met Ser 290 295 300Pro Pro Gly Phe
Ile Ser Gly Glu Glu Gly Val Leu Ser Tyr Ile Glu305 310 315 320Leu
Cys Gln Leu Phe Gln Lys Glu Glu Trp His Ile Gln Tyr Asp Glu 325 330
335Tyr Tyr Asn Ala Pro Tyr Gly Tyr Asn Asp Lys Ile Trp Val Gly Tyr
340 345 350Asp Asp Leu Ala Ser Ile Ser Cys Lys Leu Ala Phe Leu Lys
Glu Leu 355 360 365Gly Val Ser Gly Val Met Val Trp Ser Leu Glu Asn
Asp Asp Phe Lys 370 375 380Gly His Cys Gly Pro Lys Asn Pro Leu Leu
Asn Lys Val His Asn Met385 390 395 400Ile Asn Gly Asp Glu Lys Asn
Ser Phe Glu Cys Ile Leu Gly Pro Ser 405 410 415Thr Thr Thr Pro Thr
Pro Thr Thr Thr Pro Thr Thr Pro Thr Thr Thr 420 425 430Pro Thr Thr
Pro Ser Pro Thr Thr Pro Thr Thr Thr Pro Ser Pro Thr 435 440 445Thr
Pro Thr Thr Thr Pro Ser Pro Thr Thr Pro Thr Thr Thr Pro Ser 450 455
460Pro Thr Thr Pro Thr Pro Thr Thr Pro Thr Pro Ala Pro Thr Thr
Ser465 470 475 480Thr Pro Ser Pro Thr Thr Thr Glu His Thr Ser Glu
Thr Pro Lys Tyr 485 490 495Thr Thr Tyr Val Asp Gly His Leu Ile Lys
Cys Tyr Lys Glu Gly Asp 500 505 510Ile Pro His Pro Thr Asn Ile His
Lys Tyr Leu Val Cys Glu Phe Val 515 520 525Asn Gly Gly Trp Trp Val
His Ile Met Pro Cys Pro Pro Gly Thr Ile 530 535 540Trp Cys Gln Glu
Lys Leu Thr Cys Ile Gly Glu545 550 55512500PRTDermatophagoides
farinae 12Met Lys Leu Thr Ala Thr Leu Leu Leu Ile Leu Thr Leu Ser
Trp Ala1 5 10 15Gly Ile Phe Val Asp Ala Asn Pro Arg Phe Lys Arg Asp
Asn Arg Asp 20 25 30Asp Val Leu Lys Gln Thr Glu Glu Leu Ile
Lys Ser Ala Gln Asp Val 35 40 45Leu Glu Lys Leu Pro Asp Ser Asp Leu
Lys Asp Glu Ile Ala Glu Lys 50 55 60Leu Ala Thr Met Lys His Tyr Lys
His Glu Leu Glu Asn Ala Lys Asn65 70 75 80Pro Ile Lys Ile Ala His
Phe Glu Leu Glu Leu Leu Thr Met Phe Lys 85 90 95Lys Phe Gln Ser Leu
Leu Asn Glu Ala Asn Glu Ile Ile Lys Ser Leu 100 105 110Thr Thr Thr
Thr Thr Glu Pro Thr Thr Pro Thr Pro Glu Pro Thr Thr 115 120 125Thr
Thr Pro Glu Pro Thr Thr Lys Thr Pro Glu Pro Thr Thr Lys Thr 130 135
140Pro Glu Pro Thr Thr Pro Thr Pro Glu Pro Thr Thr Lys Thr Pro
Glu145 150 155 160Pro Thr Thr Lys Thr Pro Glu Pro Thr Thr Pro Thr
Pro Glu Pro Thr 165 170 175Thr Lys Thr Pro Glu Pro Thr Thr Pro Thr
Pro Glu Pro Thr Thr Lys 180 185 190Thr Pro Glu Pro Thr Thr Lys Thr
Pro Glu Pro Ser Thr Pro Thr Pro 195 200 205Glu Pro Thr Thr Lys Thr
Pro Glu Pro Thr Thr Lys Thr Pro Glu Pro 210 215 220Ser Thr Pro Thr
Pro Glu Pro Thr Thr Lys Thr Pro Glu Pro Ser Thr225 230 235 240Pro
Thr Pro Glu Pro Thr Thr Lys Thr Pro Glu Pro Thr Thr Lys Thr 245 250
255Pro Glu Pro Ser Thr Pro Thr Pro Glu Pro Thr Thr Lys Thr Pro Glu
260 265 270Pro Ser Thr Pro Thr Pro Glu Pro Thr Thr Lys Thr Pro Glu
Pro Ser 275 280 285Thr Thr Lys Lys Pro Asn Arg Asp Asp Val Leu Lys
Gln Ala Glu Glu 290 295 300Leu Ile Lys Arg Ala Glu Asp Val Phe Glu
Lys Leu Pro Asp Ser Asp305 310 315 320Leu Lys Asn Glu Ile Ala Glu
Lys Leu Ala Thr Met Lys Asn Tyr Lys 325 330 335His Glu Leu Glu Asn
Ala Lys Asn Pro Ile Lys Ile Ala His Leu Glu 340 345 350Ser Glu Leu
Leu Thr Met Phe Lys Met Phe Gln Ser Leu Leu Asn Glu 355 360 365Ala
Asp Glu Ile Ile Arg Ser Leu Thr Thr Thr Thr Glu Pro Thr Thr 370 375
380Leu Asn Ser Thr Thr Pro Glu Pro Thr Thr Leu Asn Ser Thr Thr
Pro385 390 395 400Glu Pro Thr Thr Leu Asn Ser Thr Thr Pro Glu Pro
Thr Thr Leu Asn 405 410 415Ser Thr Thr Pro Glu Pro Thr Thr Leu Asn
Ser Thr Thr Pro Glu Pro 420 425 430Thr Thr Leu Asn Ser Thr Thr Pro
Glu Pro Thr Thr Leu Asn Ser Thr 435 440 445Thr Pro Glu Pro Thr Thr
Leu Asn Ser Thr Thr Pro Glu Pro Thr Thr 450 455 460Ser Asn Ser Thr
Thr Ser Glu Pro Thr Asn Ser Ile Asn Arg Lys Thr465 470 475 480Ser
Glu Ile Ser Phe Leu Ser Asp Trp Phe His Lys Ile Arg Thr Arg 485 490
495Phe Asn Ile Phe 50013176PRTCtenocephalides felis 13Met Asn Tyr
Cys Phe Leu Val Phe Leu Val Tyr Leu Val Phe Ala Val1 5 10 15Asn Gly
Glu Asp Ile Trp Lys Val Asn Lys Lys Cys Thr Ser Gly Gly 20 25 30Lys
Asn Gln Asp Arg Lys Leu Asp Gln Ile Ile Gln Lys Gly Gln Gln 35 40
45Val Lys Ile Gln Asn Ile Cys Lys Leu Ile Arg Asp Lys Pro His Thr
50 55 60Asn Gln Glu Lys Glu Lys Cys Met Lys Phe Cys Lys Lys Val Cys
Lys65 70 75 80Gly Tyr Arg Gly Ala Cys Asp Gly Asn Ile Cys Tyr Cys
Ser Arg Pro 85 90 95Ser Asn Leu Gly Pro Asp Trp Lys Val Ser Lys Glu
Cys Lys Asp Pro 100 105 110Asn Asn Lys Asp Ser Arg Pro Thr Glu Ile
Val Pro Tyr Arg Gln Gln 115 120 125Leu Ala Ile Pro Asn Ile Cys Lys
Leu Lys Asn Ser Glu Thr Asn Glu 130 135 140Asp Ser Lys Cys Lys Lys
His Cys Lys Glu Lys Cys Arg Gly Gly Asn145 150 155 160Asp Ala Gly
Cys Asp Gly Asn Phe Cys Tyr Cys Arg Pro Lys Asn Lys 165 170
17514223PRTArtificial sequenceIMAT_Allergen from Q58A71 Der f 1
allergen preproenzyme Dermatophagoides farinae (American house dust
mite) 14Thr Ser Ala Ile Arg Ile Asn Ser Val Asn Val Pro Ser Glu Leu
Asp1 5 10 15Leu Arg Ser Leu Arg Thr Val Thr Pro Ile Arg Met Gln Gly
Gly Ile 20 25 30Gly Ser Leu Trp Ala Phe Ser Gly Val Ala Ala Thr Glu
Ser Ala Tyr 35 40 45Leu Ala Tyr Arg Asn Thr Ser Leu Asp Leu Ser Glu
Gln Glu Leu Val 50 55 60Asp Arg Ala Ser Gln His Gly Arg His Gly Asp
Thr Ile Pro Arg Gly65 70 75 80Ile Glu Tyr Ile Gln Gln Asn Gly Val
Val Glu Glu Arg Ser Tyr Pro 85 90 95Tyr Val Ala Arg Glu Gln Gln Ser
Arg Arg Pro Asn Ser Gln His Tyr 100 105 110Gly Ile Ser Asn Tyr Ile
Gln Ile Tyr Pro Pro Asp Val Lys Gln Ile 115 120 125Arg Glu Ala Leu
Thr Gln Thr His Thr Ala Ile Ala Val Ile Ile Gly 130 135 140Ile Lys
Asp Leu Arg Ala Phe Gln His Tyr Asp Gly Arg Thr Ile Ile145 150 155
160Gln His Asp Asn Gly Tyr Gln Pro Asn Tyr His Ala Val Asn Ile Val
165 170 175Gly Tyr Gly Ser Thr Gln Gly Val Asp Tyr Trp Ile Val Arg
Asn Ser 180 185 190Trp Asp Thr Thr Trp Gly Asp Ser Gly Tyr Gly Tyr
Phe Gln Ala Gly 195 200 205Asn Asn Leu Met Met Ile Glu Gln Tyr Pro
Tyr Val Val Ile Met 210 215 22015129PRTArtificial
sequenceIMAT_Allergen from Q00855 Mite group 2 allergen Der f 2
(Allergen Der f II) (allergen Der f 2) Dermatophagoides farinae
(American house dust mite) 15Asp Gln Val Asp Val Lys Asp Ser Ala
Asn Asn Glu Ile Lys Lys Val1 5 10 15Met Val Asp Gly Ile His Gly Ser
Asp Pro Leu Ile Ile His Arg Gly 20 25 30Lys Pro Phe Thr Leu Glu Ala
Leu Phe Asp Ala Asn Gln Asn Thr Lys 35 40 45Thr Ala Lys Ile Glu Ile
Lys Ala Ser Leu Asp Gly Leu Glu Ile Asp 50 55 60Val Pro Gly Ile Asp
Thr Asn Ala Ile His Phe Met Lys Leu Pro Leu65 70 75 80Val Lys Gly
Gln Gln Tyr Asp Ile Lys Tyr Thr Trp Asn Val Pro Lys 85 90 95Ile Ala
Pro Lys Ser Glu Asn Val Val Val Thr Val Lys Leu Ile Gly 100 105
110Asp Asn Gly Val Leu Ala Leu Ala Ile Ala Thr His Gly Lys Ile Arg
115 120 125Asp1670PRTArtificial sequenceIMAT_Allergen from
A0A088SAW7 Der f 23 allergen Dermatophagoides farinae (American
house dust mite) 16Asp Ile Asp His Asp Asp Asp Pro Thr Thr Met Ile
Asp Val Gln Thr1 5 10 15Thr Thr Val Gln Pro Ser Asp Glu Phe Glu Leu
Pro Thr Arg Phe Gly 20 25 30Tyr Phe Ala Asp Pro Lys Asp Pro Leu Lys
Phe Tyr Ile Ile Ser Asn 35 40 45Trp Glu Ala Ile His Lys Ser Leu Pro
Gly Asn Thr Arg Trp Asn Glu 50 55 60Lys Glu Leu Thr Ile Thr65
7017445PRTArtificial sequenceIMAT_Allergen from Q86R84 60 kDa
allergen Der f 18p Dermatophagoides farinae (American house dust
mite) 17Ser Asn Ile Arg Pro Asn Val Ala Thr Leu Glu Pro Lys Thr Val
Leu1 5 10 15Tyr Tyr Glu Ser Trp Val His Trp Arg Gln Gly Glu Gly Lys
Met Asp 20 25 30Pro Glu Asp Ile Asp Thr Ser Leu Met Thr His Ile Val
Tyr Ser Tyr 35 40 45Phe Gly Ile Asp Ala Ala Thr His Glu Ile Lys Leu
Leu Asp Glu Tyr 50 55 60Leu Met Lys Asp Leu His Asp Met Glu His Phe
Thr Gln His Lys Gly65 70 75 80Asn Ala Lys Ala Met Ile Ala Val Gly
Gly Ser Thr Met Ser Asp Gln 85 90 95Phe Ser Lys Thr Ala Ala Val Glu
His Tyr Arg Glu Thr Phe Val Val 100 105 110Ser Thr Val Asp Leu Met
Thr Arg Tyr Gly Phe Asp Gly Val Met Ile 115 120 125Asp Trp Ser Gly
Met Gln Ala Lys Asp Ser Asp Asn Phe Ile Lys Leu 130 135 140Leu Asp
Lys Phe Asp Glu Lys Phe Ala His Thr Ser Phe Val Met Gly145 150 155
160Val Thr Leu Pro Ala Thr Ile Ala Ser Tyr Asp Asn Tyr Asn Ile Pro
165 170 175Ala Ile Ser Asn Tyr Val Asp Phe Met Asn Val Leu Ser Leu
Asp Tyr 180 185 190Thr Gly Ser Trp Ala His Thr Val Gly His Ala Ser
Pro Phe Pro Glu 195 200 205Gln Leu Lys Thr Leu Glu Ala Tyr His Lys
Arg Gly Ala Pro Arg His 210 215 220Lys Met Val Met Ala Val Pro Phe
Tyr Ala Arg Thr Trp Ile Leu Glu225 230 235 240Lys Met Asn Lys Gln
Asp Ile Gly Asp Lys Ala Ser Gly Pro Gly Pro 245 250 255Arg Gly Gln
Phe Thr Gln Thr Asp Gly Phe Leu Ser Tyr Asn Glu Leu 260 265 270Ser
Val Gln Ile Gln Ala Glu Thr Asn Ala Phe Thr Ile Thr Arg Asp 275 280
285His Asp Asn Thr Ala Ile Tyr Ala Val Tyr Val His Ser Asn His Ala
290 295 300Glu Trp Ile Ser Phe Glu Asp Arg His Thr Leu Gly Glu Lys
Ala Lys305 310 315 320Asn Ile Thr Gln Gln Gly Tyr Ala Gly Met Ser
Val Tyr Thr Leu Ser 325 330 335Asn Glu Asp Val His Gly Val Arg Gly
Asp Lys Asn Pro Leu Leu His 340 345 350Ala Ile Gln Ser Asn Tyr Tyr
His Gly Val Val Thr Glu Pro Thr Val 355 360 365Val Thr Leu Pro Pro
Val Thr His Thr Thr Glu His Val Thr Asp Ile 370 375 380Pro Gly Val
Phe His Leu His Glu Glu Gly Phe Phe Arg Asp Lys Thr385 390 395
400Tyr Ser Ala Thr Tyr Tyr Glu Ile Lys Lys Gly Asp Phe Gly Leu Glu
405 410 415Lys Thr Val His His Leu Ala Asn His Leu Gln Ala Phe Asp
Glu Val 420 425 430Ser Arg Thr Arg Ile Asp His Thr Lys Ile Pro Gly
Ser 435 440 44518536PRTArtificial sequenceIMAT_Allergen from Q9U6R7
98kDa HDM allergen (Der f 15 allergen) (Group 15 allergen Der f 15)
Dermatophagoides farinae (American house dust mite) 18Ser Ile Lys
Arg Asp His Asn Asp Tyr Ser Lys Asn Pro Met Arg Ile1 5 10 15Val Leu
Tyr Val Gly Thr Trp Ser Val Tyr His Lys Val Asp Pro Tyr 20 25 30Thr
Ile Glu Asp Ile Asp Pro Phe Lys Ser Thr His Leu Met Tyr Gly 35 40
45Phe Ala Lys Ile Asp Glu Tyr Lys Tyr Thr Ile Gln Val Phe Asp Pro
50 55 60Tyr Gln Asp Asp Asn His Asn Ser Trp Glu Lys Arg Gly Tyr Glu
Arg65 70 75 80Phe Asn Asn Leu Arg Leu Lys Asn Pro Glu Leu Thr Thr
Met Ile Ser 85 90 95Leu Gly Gly Trp Tyr Glu Gly Ser Glu Lys Tyr Ser
Asp Met Ala Ala 100 105 110Asn Pro Thr Tyr Arg Gln Gln Phe Ile Gln
Ser Val Leu Asp Phe Leu 115 120 125Gln Glu Tyr Lys Phe Asp Gly Leu
Asp Leu Asp Trp Glu Tyr Pro Gly 130 135 140Ser Arg Leu Gly Asn Pro
Lys Ile Asp Lys Gln Asn Tyr Leu Ala Leu145 150 155 160Val Arg Glu
Leu Lys Asp Ala Phe Glu Pro His Gly Tyr Leu Leu Thr 165 170 175Ala
Ala Val Ser Pro Gly Lys Asp Lys Ile Asp Arg Ala Tyr Asp Ile 180 185
190Lys Glu Leu Asn Lys Leu Phe Asp Trp Met Asn Val Met Thr Tyr Asp
195 200 205Tyr His Gly Gly Trp Glu Asn Phe Tyr Gly His Asn Ala Pro
Leu Tyr 210 215 220Lys Arg Pro Asp Glu Thr Asp Glu Leu His Thr Tyr
Phe Asn Val Asn225 230 235 240Tyr Thr Met His Tyr Tyr Leu Asn Asn
Gly Ala Thr Arg Asp Lys Leu 245 250 255Val Met Gly Val Pro Phe Tyr
Gly Arg Ala Trp Ser Ile Glu Asp Arg 260 265 270Ser Lys Leu Lys Leu
Gly Asp Pro Ala Lys Gly Met Ser Pro Pro Gly 275 280 285Phe Ile Ser
Gly Glu Glu Gly Val Leu Ser Tyr Ile Glu Leu Ser Gln 290 295 300Leu
Phe Gln Lys Glu Glu Trp His Ile Gln Tyr Asp Glu Tyr Tyr Asn305 310
315 320Ala Pro Tyr Gly Tyr Asn Asp Lys Ile Trp Val Gly Tyr Asp Asp
Leu 325 330 335Ala Ser Ile Ser Ser Lys Leu Ala Phe Leu Lys Glu Leu
Gly Val Ser 340 345 350Gly Val Met Val Trp Ser Leu Glu Asn Asp Asp
Phe Lys Gly His Leu 355 360 365Gly Pro Lys Asn Pro Leu Leu Asn Lys
Val His Asn Met Ile Asn Gly 370 375 380Asp Glu Lys Asn Ser Phe Glu
Ile Ile Leu Gly Pro Ser Thr Thr Thr385 390 395 400Pro Thr Pro Thr
Thr Thr Pro Thr Thr Pro Thr Thr Thr Pro Thr Thr 405 410 415Pro Ser
Pro Thr Thr Pro Thr Thr Thr Pro Ser Pro Thr Thr Pro Thr 420 425
430Thr Thr Pro Ser Pro Thr Thr Pro Thr Thr Thr Pro Ser Pro Thr Thr
435 440 445Pro Thr Pro Thr Thr Pro Thr Pro Ala Pro Thr Thr Ser Thr
Pro Ser 450 455 460Pro Thr Thr Thr Glu His Thr Ser Glu Thr Pro Lys
Tyr Thr Thr Tyr465 470 475 480Val Asp Gly His Leu Ile Lys Ser Tyr
Lys Glu Gly Asp Ile Pro His 485 490 495Pro Thr Asn Ile His Lys Tyr
Leu Val Ile Glu Phe Val Asn Gly Gly 500 505 510Trp Trp Val His Ile
Met Pro Ser Pro Pro Gly Thr Ile Trp Ser Gln 515 520 525Glu Lys Leu
Thr Ser Ile Gly Glu 530 53519478PRTArtificial sequenceIMAT_Allergen
from I7HDR2 Zen 1 protein Dermatophagoides farinae (American house
dust mite) 19Asn Pro Arg Phe Lys Arg Asp Asn Arg Asp Asp Val Leu
Lys Gln Thr1 5 10 15Glu Glu Leu Ile Lys Ser Ala Gln Asp Val Leu Glu
Lys Leu Pro Asp 20 25 30Ser Asp Leu Lys Asp Glu Ile Ala Glu Lys Leu
Ala Thr Met Lys His 35 40 45Tyr Lys His Glu Leu Glu Asn Ala Lys Asn
Pro Ile Lys Ile Ala His 50 55 60Phe Glu Leu Glu Leu Leu Thr Met Phe
Lys Lys Phe Gln Ser Leu Leu65 70 75 80Asn Glu Ala Asn Glu Ile Ile
Lys Ser Leu Thr Thr Thr Thr Thr Glu 85 90 95Pro Thr Thr Pro Thr Pro
Glu Pro Thr Thr Thr Thr Pro Glu Pro Thr 100 105 110Thr Lys Thr Pro
Glu Pro Thr Thr Lys Thr Pro Glu Pro Thr Thr Pro 115 120 125Thr Pro
Glu Pro Thr Thr Lys Thr Pro Glu Pro Thr Thr Lys Thr Pro 130 135
140Glu Pro Thr Thr Pro Thr Pro Glu Pro Thr Thr Lys Thr Pro Glu
Pro145 150 155 160Thr Thr Pro Thr Pro Glu Pro Thr Thr Lys Thr Pro
Glu Pro Thr Thr 165 170 175Lys Thr Pro Glu Pro Ser Thr Pro Thr Pro
Glu Pro Thr Thr Lys Thr 180 185 190Pro Glu Pro Thr Thr Lys Thr Pro
Glu Pro Ser Thr Pro Thr Pro Glu 195 200 205Pro Thr Thr Lys Thr Pro
Glu Pro Ser Thr Pro Thr Pro Glu Pro Thr 210 215 220Thr Lys Thr Pro
Glu Pro Thr Thr Lys Thr Pro Glu Pro Ser Thr Pro225 230 235 240Thr
Pro Glu Pro Thr Thr Lys Thr Pro Glu Pro Ser Thr Pro Thr Pro 245 250
255Glu Pro Thr Thr Lys Thr Pro Glu Pro Ser Thr Thr Lys Lys Pro Asn
260 265 270Arg Asp Asp Val Leu Lys Gln Ala Glu Glu Leu Ile Lys Arg
Ala Glu 275 280 285Asp Val Phe Glu Lys Leu Pro Asp Ser Asp Leu Lys
Asn Glu Ile Ala 290
295 300Glu Lys Leu Ala Thr Met Lys Asn Tyr Lys His Glu Leu Glu Asn
Ala305 310 315 320Lys Asn Pro Ile Lys Ile Ala His Leu Glu Ser Glu
Leu Leu Thr Met 325 330 335Phe Lys Met Phe Gln Ser Leu Leu Asn Glu
Ala Asp Glu Ile Ile Arg 340 345 350Ser Leu Thr Thr Thr Thr Glu Pro
Thr Thr Leu Asn Ser Thr Thr Pro 355 360 365Glu Pro Thr Thr Leu Asn
Ser Thr Thr Pro Glu Pro Thr Thr Leu Asn 370 375 380Ser Thr Thr Pro
Glu Pro Thr Thr Leu Asn Ser Thr Thr Pro Glu Pro385 390 395 400Thr
Thr Leu Asn Ser Thr Thr Pro Glu Pro Thr Thr Leu Asn Ser Thr 405 410
415Thr Pro Glu Pro Thr Thr Leu Asn Ser Thr Thr Pro Glu Pro Thr Thr
420 425 430Leu Asn Ser Thr Thr Pro Glu Pro Thr Thr Ser Asn Ser Thr
Thr Ser 435 440 445Glu Pro Thr Asn Ser Ile Asn Arg Lys Thr Ser Glu
Ile Ser Phe Leu 450 455 460Ser Asp Trp Phe His Lys Ile Arg Thr Arg
Phe Asn Ile Phe465 470 47520158PRTArtificial sequenceIMAT_Allergen
from Q94424 Salivary antigen 1 (FS-I) (allergen Cte f 1)
Ctenocephalides felis (Cat flea) 20Glu Asp Ile Trp Lys Val Asn Lys
Lys Arg Thr Ser Gly Gly Lys Asn1 5 10 15Gln Asp Arg Lys Leu Asp Gln
Ile Ile Gln Lys Gly Gln Gln Val Lys 20 25 30Ile Gln Asn Ile Leu Lys
Leu Ile Arg Asp Lys Pro His Thr Asn Gln 35 40 45Glu Lys Glu Lys Leu
Met Lys Phe Leu Lys Lys Val Leu Lys Gly Tyr 50 55 60Arg Gly Ala Arg
Asp Gly Asn Ile Leu Tyr Leu Ser Arg Pro Ser Asn65 70 75 80Leu Gly
Pro Asp Trp Lys Val Ser Lys Glu Arg Lys Asp Pro Asn Asn 85 90 95Lys
Asp Ser Arg Pro Thr Glu Ile Val Pro Tyr Arg Gln Gln Leu Ala 100 105
110Ile Pro Asn Ile Leu Lys Leu Lys Asn Ser Glu Thr Asn Glu Asp Ser
115 120 125Lys Leu Lys Lys His Met Lys Glu Lys Ser Arg Gly Gly Asn
Asp Ala 130 135 140Gly Ile Asp Gly Asn Phe Leu Tyr Leu Arg Pro Lys
Asn Lys145 150 15521383PRTArtificial sequenceIMAT_Allergen from
Hybrid1 21Gln Ser His Gln Tyr Tyr His Thr Ser Gly Leu Arg Asn Leu
Gly Gly1 5 10 15Ser Tyr Tyr Arg Ser Gly Asn Ala Lys Ala Met Ile Ala
Val Gly Gly 20 25 30Ser Thr Met Ile Val Asp Gly Asp Lys Val Thr Ile
Tyr Gly Trp Gly 35 40 45Ser Gly Leu Gly Tyr Gly Leu Gly Tyr Gly Leu
Gly Tyr Gly Gln Ala 50 55 60Val Ala Leu Ala Pro Ala Gln Ala Val Gly
Tyr Val Ala Ala Ala Pro65 70 75 80Ala Val Ala Val Gln Ala Pro Ala
Val Ser Tyr Ala Ala Ala Ala Pro 85 90 95Ala Val Gln Thr Val Ala Val
Gln Ala Pro Ala Val Ser Tyr Ala Ala 100 105 110Ala Pro Ala Val Ala
Val Gln Ala His Thr Ala Gln Val Ser Gly Pro 115 120 125Ile His Ala
Ala Ile Glu Ser Arg Arg Thr Val Glu Val Ile Asp Gly 130 135 140Pro
Ser Thr Gly Asp Ala Pro Val Ala Ser Thr Val Val Ile Gly Pro145 150
155 160Asn Val Gln Pro Ile Asn Leu Glu Phe Gln Thr Gln Ala Ser Pro
Leu 165 170 175Ala Ala Thr Gln Asn His Val Pro Thr Thr Pro Thr Thr
Pro Thr Pro 180 185 190Ala Pro Thr Thr Ser Thr Pro Asp Leu Leu Arg
Gln Asp Ile Val Lys 195 200 205Pro Val Val Gln Asp Val His Glu Phe
Leu Val Tyr Ile His Ile Ala 210 215 220Asn Asn Glu Ile Lys Lys Val
Gln Glu Ser Val His Gln Ile Leu Pro225 230 235 240Arg Gly Gln Met
Met Lys Ile Tyr Gln Gln Gln Gln Gln Gln His His 245 250 255Pro Gln
Arg Glu Glu Asn Ile Trp Ser Asp His Ile Ala Asn Val Ala 260 265
270Gln Ala Ala Pro Ala Ile Ser Ala Val Arg Val Ala Ala Ala Pro Ala
275 280 285Val Ala Tyr Ala Ala Pro Ala Val Ser Thr Val Ser Ala Ala
Pro Ala 290 295 300Ala Ile Gly Val Ile Gly Val Gln Pro Val Ala Gly
Tyr Ile Gly Tyr305 310 315 320Gly Ala Gly Tyr Gly Thr Gly Tyr Gly
Thr Gly Tyr Gly Val Ala Lys 325 330 335Tyr Gly Thr Gly Tyr Gly Leu
Thr Ser Gly Leu Ile Gly Gly Gly Ser 340 345 350Tyr Gly Ser Ser Tyr
Ser Val Gln Pro Ala Ser Tyr Gly Thr Gly Tyr 355 360 365Gly Tyr Thr
Thr Tyr Ser Ser Asp Ala Tyr Pro Ile Arg Lys Lys 370 375
38022517PRTArtificial sequenceIMAT_Allergen from Hybrid2 22Thr Ser
Ala Ser Arg Ile Asn Ser Val Asn Val Pro Ser Glu Leu Asp1 5 10 15Leu
Arg Ser Leu Arg Thr Val Thr Pro Ile Arg Met Gln Gly Gly Ser 20 25
30Gly Ser Ser Trp Ala Phe Ser Gly Val Ala Ala Thr Glu Ser Ala Tyr
35 40 45Leu Ala Tyr Arg Asn Thr Ser Leu Asp Leu Ser Glu Gln Glu Leu
Val 50 55 60Asp Ser Ala Ser Gln His Gly Ser His Gly Asp Thr Ile Pro
Arg Gly65 70 75 80Ile Glu Tyr Ile Gln Gln Asn Gly Val Val Glu Glu
Arg Ser Tyr Pro 85 90 95Tyr Val Ala Arg Glu Gln Gln Ser Arg Arg Pro
Asn Ser Gln His Tyr 100 105 110Gly Ile Ser Asn Tyr Ser Gln Ile Tyr
Pro Pro Asp Val Lys Gln Ile 115 120 125Arg Glu Ala Leu Thr Gln Thr
His Thr Ala Ile Ala Val Ile Ile Gly 130 135 140Ile Lys Asp Leu Arg
Ala Phe Gln His Tyr Asp Gly Arg Thr Ile Ile145 150 155 160Gln His
Asp Asn Gly Tyr Gln Pro Asn Tyr His Ala Val Asn Ile Val 165 170
175Gly Tyr Gly Ser Thr Gln Gly Val Asp Tyr Trp Ile Val Arg Asn Ser
180 185 190Trp Asp Thr Thr Trp Gly Asp Ser Gly Tyr Gly Tyr Phe Gln
Ala Gly 195 200 205Asn Asn Leu Met Met Ile Glu Gln Tyr Pro Tyr Val
Val Ile Met Phe 210 215 220Thr Gln Thr Asp Gly Phe Leu Ser Tyr Asn
Glu Leu Ser Val Gln Ile225 230 235 240Gln Ala Glu Thr Asn Ala Phe
Thr Ile Thr Arg Glu Pro Thr Thr Pro 245 250 255Thr Pro Glu Pro Thr
Thr Lys Thr Pro Glu Pro Thr Thr Lys Thr Pro 260 265 270Glu Pro Ser
Thr Pro Thr Pro Glu Pro Thr Thr Lys Thr Pro Glu Pro 275 280 285Thr
Thr Lys Asp Ile Asp His Asp Asp Asp Pro Thr Thr Met Ile Asp 290 295
300Val Gln Thr Thr Thr Val Gln Pro Ser Asp Glu Phe Glu Ser Pro
Thr305 310 315 320Arg Phe Gly Tyr Phe Ala Asp Pro Lys Asp Pro Ile
Lys Phe Tyr Ile 325 330 335Ser Ser Asn Trp Glu Ala Ile His Lys Ser
Ser Pro Gly Asn Thr Arg 340 345 350Trp Asn Glu Lys Glu Leu Thr Ser
Thr Ser Pro Thr Thr Pro Thr Thr 355 360 365Thr Pro Ser Pro Thr Thr
Pro Thr Thr Thr Pro Ser Pro Thr Thr Pro 370 375 380Thr Thr Thr Pro
Asp Gln Val Asp Val Lys Asp Leu Ala Asn Asn Glu385 390 395 400Ile
Lys Lys Val Met Val Asp Gly Ile His Gly Ser Asp Pro Ser Ile 405 410
415Ile His Arg Gly Lys Pro Phe Thr Leu Glu Ala Leu Phe Asp Ala Asn
420 425 430Gln Asn Thr Lys Thr Ala Lys Ile Glu Ile Lys Ala Ser Leu
Asp Gly 435 440 445Leu Glu Ile Asp Val Pro Gly Ile Asp Thr Asn Ala
Leu His Phe Met 450 455 460Lys Ile Pro Leu Val Lys Gly Gln Gln Tyr
Asp Ile Lys Tyr Thr Trp465 470 475 480Asn Val Pro Lys Ile Ala Pro
Lys Ser Glu Asn Val Val Val Thr Val 485 490 495Lys Leu Ile Gly Asp
Asn Gly Val Leu Ala Ser Ala Ile Ala Thr His 500 505 510Gly Lys Ile
Arg Asp 51523407PRTArtificial sequenceIMAT_Allergen from Hybrid3
23Thr Ser Ala Ser Arg Ile Asn Ser Val Asn Val Pro Ser Glu Leu Asp1
5 10 15Leu Arg Ser Leu Arg Thr Val Thr Pro Ile Arg Met Gln Gly Gly
Ser 20 25 30Gly Ser Leu Trp Ala Phe Ser Gly Val Ala Ala Thr Glu Ser
Ala Tyr 35 40 45Leu Ala Tyr Arg Asn Thr Ser Leu Asp Leu Ser Glu Gln
Glu Leu Val 50 55 60Asp Ile Ala Ser Gln His Gly Leu His Gly Asp Thr
Ile Pro Arg Gly65 70 75 80Ile Glu Tyr Ile Gln Gln Asn Gly Val Val
Glu Glu Arg Ser Tyr Pro 85 90 95Tyr Val Ala Arg Glu Gln Gln Ser Arg
Arg Pro Asn Ser Gln His Tyr 100 105 110Gly Ile Ser Asn Tyr Leu Gln
Ile Tyr Pro Pro Asp Val Lys Gln Ile 115 120 125Arg Glu Ala Leu Thr
Gln Thr His Thr Ala Ile Ala Val Ile Ile Gly 130 135 140Ile Lys Asp
Leu Arg Ala Phe Gln His Tyr Asp Gly Arg Thr Ile Ile145 150 155
160Gln His Asp Asn Gly Tyr Gln Pro Asn Tyr His Ala Val Asn Ile Val
165 170 175Gly Tyr Gly Ser Thr Gln Gly Val Asp Tyr Trp Ile Val Arg
Asn Ser 180 185 190Trp Asp Thr Thr Trp Gly Asp Ser Gly Tyr Gly Tyr
Phe Gln Ala Gly 195 200 205Asn Asn Leu Met Met Ile Glu Gln Tyr Pro
Tyr Val Val Ile Met Phe 210 215 220Thr Gln Thr Asp Gly Phe Leu Ser
Tyr Asn Glu Leu Ser Val Gln Ile225 230 235 240Gln Ala Glu Thr Asn
Ala Phe Thr Ile Thr Arg Ser Pro Thr Thr Pro 245 250 255Thr Thr Thr
Pro Ser Pro Thr Thr Pro Thr Thr Thr Pro Ser Pro Thr 260 265 270Thr
Pro Thr Thr Thr Pro Asp Gln Val Asp Val Lys Asp Ser Ala Asn 275 280
285Asn Glu Ile Lys Lys Val Met Val Asp Gly Ile His Gly Ser Asp Pro
290 295 300Ser Ile Ile His Arg Gly Lys Pro Phe Thr Leu Glu Ala Leu
Phe Asp305 310 315 320Ala Asn Gln Asn Thr Lys Thr Ala Lys Ile Glu
Ile Lys Ala Ser Leu 325 330 335Asp Gly Leu Glu Ile Asp Val Pro Gly
Ile Asp Thr Asn Ala Ser His 340 345 350Phe Met Lys Ser Pro Leu Val
Lys Gly Gln Gln Tyr Asp Ile Lys Tyr 355 360 365Thr Trp Asn Val Pro
Lys Ile Ala Pro Lys Ser Glu Asn Val Val Val 370 375 380Thr Val Lys
Leu Ile Gly Asp Asn Gly Val Leu Ala Ser Ala Ile Ala385 390 395
400Thr His Gly Lys Ile Arg Asp 40524351PRTArtificial
sequenceIMAT_NTERM Cat from Q58A71 Der f 1 allergen preproenzyme
Dermatophagoides farinae (American house dust mite) 24Met His His
His His His His Tyr Gly Arg Lys Lys Arg Arg Gln Arg1 5 10 15Arg Arg
Met Glu Asp Gln Arg Asp Leu Ile Ser Asn His Glu Gln Leu 20 25 30Pro
Ile Leu Gly Gln Arg Pro Ala Ala Pro Glu Ser Lys Ser Ser Arg 35 40
45Gly Ala Leu Tyr Thr Gly Phe Ser Val Leu Val Ala Leu Leu Leu Ala
50 55 60Gly Gln Ala Thr Thr Ala Tyr Phe Leu Tyr Gln Gln Gln Gly Arg
Leu65 70 75 80Asp Lys Leu Thr Val Thr Ala Gln Asn Leu Gln Leu Glu
Asn Leu Arg 85 90 95Met Lys Leu Pro Lys Pro Ala Lys Pro Leu Asn Lys
Leu Arg Val Ala 100 105 110Thr Pro Met Leu Met Gln Thr Met Pro Val
Arg Gly Leu Leu Gln Ala 115 120 125Thr Ser Ala Ile Arg Ile Asn Ser
Val Asn Val Pro Ser Glu Leu Asp 130 135 140Leu Arg Ser Leu Arg Thr
Val Thr Pro Ile Arg Met Gln Gly Gly Ile145 150 155 160Gly Ser Leu
Trp Ala Phe Ser Gly Val Ala Ala Thr Glu Ser Ala Tyr 165 170 175Leu
Ala Tyr Arg Asn Thr Ser Leu Asp Leu Ser Glu Gln Glu Leu Val 180 185
190Asp Arg Ala Ser Gln His Gly Arg His Gly Asp Thr Ile Pro Arg Gly
195 200 205Ile Glu Tyr Ile Gln Gln Asn Gly Val Val Glu Glu Arg Ser
Tyr Pro 210 215 220Tyr Val Ala Arg Glu Gln Gln Ser Arg Arg Pro Asn
Ser Gln His Tyr225 230 235 240Gly Ile Ser Asn Tyr Ile Gln Ile Tyr
Pro Pro Asp Val Lys Gln Ile 245 250 255Arg Glu Ala Leu Thr Gln Thr
His Thr Ala Ile Ala Val Ile Ile Gly 260 265 270Ile Lys Asp Leu Arg
Ala Phe Gln His Tyr Asp Gly Arg Thr Ile Ile 275 280 285Gln His Asp
Asn Gly Tyr Gln Pro Asn Tyr His Ala Val Asn Ile Val 290 295 300Gly
Tyr Gly Ser Thr Gln Gly Val Asp Tyr Trp Ile Val Arg Asn Ser305 310
315 320Trp Asp Thr Thr Trp Gly Asp Ser Gly Tyr Gly Tyr Phe Gln Ala
Gly 325 330 335Asn Asn Leu Met Met Ile Glu Gln Tyr Pro Tyr Val Val
Ile Met 340 345 35025351PRTArtificial sequenceIMAT_CTERM Cat from
Q58A71 Der f 1 allergen preproenzyme Dermatophagoides farinae
(American house dust mite) 25Met Tyr Gly Arg Lys Lys Arg Arg Gln
Arg Arg Arg Met Glu Asp Gln1 5 10 15Arg Asp Leu Ile Ser Asn His Glu
Gln Leu Pro Ile Leu Gly Gln Arg 20 25 30Pro Ala Ala Pro Glu Ser Lys
Ser Ser Arg Gly Ala Leu Tyr Thr Gly 35 40 45Phe Ser Val Leu Val Ala
Leu Leu Leu Ala Gly Gln Ala Thr Thr Ala 50 55 60Tyr Phe Leu Tyr Gln
Gln Gln Gly Arg Leu Asp Lys Leu Thr Val Thr65 70 75 80Ala Gln Asn
Leu Gln Leu Glu Asn Leu Arg Met Lys Leu Pro Lys Pro 85 90 95Ala Lys
Pro Leu Asn Lys Leu Arg Val Ala Thr Pro Met Leu Met Gln 100 105
110Thr Met Pro Val Arg Gly Leu Leu Gln Ala Thr Ser Ala Ile Arg Ile
115 120 125Asn Ser Val Asn Val Pro Ser Glu Leu Asp Leu Arg Ser Leu
Arg Thr 130 135 140Val Thr Pro Ile Arg Met Gln Gly Gly Ile Gly Ser
Leu Trp Ala Phe145 150 155 160Ser Gly Val Ala Ala Thr Glu Ser Ala
Tyr Leu Ala Tyr Arg Asn Thr 165 170 175Ser Leu Asp Leu Ser Glu Gln
Glu Leu Val Asp Arg Ala Ser Gln His 180 185 190Gly Arg His Gly Asp
Thr Ile Pro Arg Gly Ile Glu Tyr Ile Gln Gln 195 200 205Asn Gly Val
Val Glu Glu Arg Ser Tyr Pro Tyr Val Ala Arg Glu Gln 210 215 220Gln
Ser Arg Arg Pro Asn Ser Gln His Tyr Gly Ile Ser Asn Tyr Ile225 230
235 240Gln Ile Tyr Pro Pro Asp Val Lys Gln Ile Arg Glu Ala Leu Thr
Gln 245 250 255Thr His Thr Ala Ile Ala Val Ile Ile Gly Ile Lys Asp
Leu Arg Ala 260 265 270Phe Gln His Tyr Asp Gly Arg Thr Ile Ile Gln
His Asp Asn Gly Tyr 275 280 285Gln Pro Asn Tyr His Ala Val Asn Ile
Val Gly Tyr Gly Ser Thr Gln 290 295 300Gly Val Asp Tyr Trp Ile Val
Arg Asn Ser Trp Asp Thr Thr Trp Gly305 310 315 320Asp Ser Gly Tyr
Gly Tyr Phe Gln Ala Gly Asn Asn Leu Met Met Ile 325 330 335Glu Gln
Tyr Pro Tyr Val Val Ile Met His His His His His His 340 345
35026344PRTArtificial sequenceIMAT_PURE Cat from Q58A71 Der f 1
allergen preproenzyme Dermatophagoides farinae (American house dust
mite) 26Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Met Glu Asp Gln
Arg1 5 10 15Asp Leu Ile Ser Asn His Glu Gln Leu Pro Ile Leu Gly Gln
Arg Pro 20 25
30Ala Ala Pro Glu Ser Lys Ser Ser Arg Gly Ala Leu Tyr Thr Gly Phe
35 40 45Ser Val Leu Val Ala Leu Leu Leu Ala Gly Gln Ala Thr Thr Ala
Tyr 50 55 60Phe Leu Tyr Gln Gln Gln Gly Arg Leu Asp Lys Leu Thr Val
Thr Ala65 70 75 80Gln Asn Leu Gln Leu Glu Asn Leu Arg Met Lys Leu
Pro Lys Pro Ala 85 90 95Lys Pro Leu Asn Lys Leu Arg Val Ala Thr Pro
Met Leu Met Gln Thr 100 105 110Met Pro Val Arg Gly Leu Leu Gln Ala
Thr Ser Ala Ile Arg Ile Asn 115 120 125Ser Val Asn Val Pro Ser Glu
Leu Asp Leu Arg Ser Leu Arg Thr Val 130 135 140Thr Pro Ile Arg Met
Gln Gly Gly Ile Gly Ser Leu Trp Ala Phe Ser145 150 155 160Gly Val
Ala Ala Thr Glu Ser Ala Tyr Leu Ala Tyr Arg Asn Thr Ser 165 170
175Leu Asp Leu Ser Glu Gln Glu Leu Val Asp Arg Ala Ser Gln His Gly
180 185 190Arg His Gly Asp Thr Ile Pro Arg Gly Ile Glu Tyr Ile Gln
Gln Asn 195 200 205Gly Val Val Glu Glu Arg Ser Tyr Pro Tyr Val Ala
Arg Glu Gln Gln 210 215 220Ser Arg Arg Pro Asn Ser Gln His Tyr Gly
Ile Ser Asn Tyr Ile Gln225 230 235 240Ile Tyr Pro Pro Asp Val Lys
Gln Ile Arg Glu Ala Leu Thr Gln Thr 245 250 255His Thr Ala Ile Ala
Val Ile Ile Gly Ile Lys Asp Leu Arg Ala Phe 260 265 270Gln His Tyr
Asp Gly Arg Thr Ile Ile Gln His Asp Asn Gly Tyr Gln 275 280 285Pro
Asn Tyr His Ala Val Asn Ile Val Gly Tyr Gly Ser Thr Gln Gly 290 295
300Val Asp Tyr Trp Ile Val Arg Asn Ser Trp Asp Thr Thr Trp Gly
Asp305 310 315 320Ser Gly Tyr Gly Tyr Phe Gln Ala Gly Asn Asn Leu
Met Met Ile Glu 325 330 335Gln Tyr Pro Tyr Val Val Ile Met
34027257PRTArtificial sequenceIMAT_NTERM Cat from Q00855 Mite group
2 allergen Der f 2 (Allergen Der f II) (allergen Der f 2)
Dermatophagoides farinae (American house dust mite) 27Met His His
His His His His Tyr Gly Arg Lys Lys Arg Arg Gln Arg1 5 10 15Arg Arg
Met Glu Asp Gln Arg Asp Leu Ile Ser Asn His Glu Gln Leu 20 25 30Pro
Ile Leu Gly Gln Arg Pro Ala Ala Pro Glu Ser Lys Ser Ser Arg 35 40
45Gly Ala Leu Tyr Thr Gly Phe Ser Val Leu Val Ala Leu Leu Leu Ala
50 55 60Gly Gln Ala Thr Thr Ala Tyr Phe Leu Tyr Gln Gln Gln Gly Arg
Leu65 70 75 80Asp Lys Leu Thr Val Thr Ala Gln Asn Leu Gln Leu Glu
Asn Leu Arg 85 90 95Met Lys Leu Pro Lys Pro Ala Lys Pro Leu Asn Lys
Leu Arg Val Ala 100 105 110Thr Pro Met Leu Met Gln Thr Met Pro Val
Arg Gly Leu Leu Gln Ala 115 120 125Asp Gln Val Asp Val Lys Asp Ser
Ala Asn Asn Glu Ile Lys Lys Val 130 135 140Met Val Asp Gly Ile His
Gly Ser Asp Pro Leu Ile Ile His Arg Gly145 150 155 160Lys Pro Phe
Thr Leu Glu Ala Leu Phe Asp Ala Asn Gln Asn Thr Lys 165 170 175Thr
Ala Lys Ile Glu Ile Lys Ala Ser Leu Asp Gly Leu Glu Ile Asp 180 185
190Val Pro Gly Ile Asp Thr Asn Ala Ile His Phe Met Lys Leu Pro Leu
195 200 205Val Lys Gly Gln Gln Tyr Asp Ile Lys Tyr Thr Trp Asn Val
Pro Lys 210 215 220Ile Ala Pro Lys Ser Glu Asn Val Val Val Thr Val
Lys Leu Ile Gly225 230 235 240Asp Asn Gly Val Leu Ala Leu Ala Ile
Ala Thr His Gly Lys Ile Arg 245 250 255Asp28257PRTArtificial
sequenceIMAT_CTERM Cat from Q00855 Mite group 2 allergen Der f 2
(Allergen Der f II) (allergen Der f 2) Dermatophagoides farinae
(American house dust mite) 28Met Tyr Gly Arg Lys Lys Arg Arg Gln
Arg Arg Arg Met Glu Asp Gln1 5 10 15Arg Asp Leu Ile Ser Asn His Glu
Gln Leu Pro Ile Leu Gly Gln Arg 20 25 30Pro Ala Ala Pro Glu Ser Lys
Ser Ser Arg Gly Ala Leu Tyr Thr Gly 35 40 45Phe Ser Val Leu Val Ala
Leu Leu Leu Ala Gly Gln Ala Thr Thr Ala 50 55 60Tyr Phe Leu Tyr Gln
Gln Gln Gly Arg Leu Asp Lys Leu Thr Val Thr65 70 75 80Ala Gln Asn
Leu Gln Leu Glu Asn Leu Arg Met Lys Leu Pro Lys Pro 85 90 95Ala Lys
Pro Leu Asn Lys Leu Arg Val Ala Thr Pro Met Leu Met Gln 100 105
110Thr Met Pro Val Arg Gly Leu Leu Gln Ala Asp Gln Val Asp Val Lys
115 120 125Asp Ser Ala Asn Asn Glu Ile Lys Lys Val Met Val Asp Gly
Ile His 130 135 140Gly Ser Asp Pro Leu Ile Ile His Arg Gly Lys Pro
Phe Thr Leu Glu145 150 155 160Ala Leu Phe Asp Ala Asn Gln Asn Thr
Lys Thr Ala Lys Ile Glu Ile 165 170 175Lys Ala Ser Leu Asp Gly Leu
Glu Ile Asp Val Pro Gly Ile Asp Thr 180 185 190Asn Ala Ile His Phe
Met Lys Leu Pro Leu Val Lys Gly Gln Gln Tyr 195 200 205Asp Ile Lys
Tyr Thr Trp Asn Val Pro Lys Ile Ala Pro Lys Ser Glu 210 215 220Asn
Val Val Val Thr Val Lys Leu Ile Gly Asp Asn Gly Val Leu Ala225 230
235 240Leu Ala Ile Ala Thr His Gly Lys Ile Arg Asp His His His His
His 245 250 255His29250PRTArtificial sequenceIMAT_PURE Cat from
Q00855 Mite group 2 allergen Der f 2 (Allergen Der f II) (allergen
Der f 2) Dermatophagoides farinae (American house dust mite) 29Tyr
Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Met Glu Asp Gln Arg1 5 10
15Asp Leu Ile Ser Asn His Glu Gln Leu Pro Ile Leu Gly Gln Arg Pro
20 25 30Ala Ala Pro Glu Ser Lys Ser Ser Arg Gly Ala Leu Tyr Thr Gly
Phe 35 40 45Ser Val Leu Val Ala Leu Leu Leu Ala Gly Gln Ala Thr Thr
Ala Tyr 50 55 60Phe Leu Tyr Gln Gln Gln Gly Arg Leu Asp Lys Leu Thr
Val Thr Ala65 70 75 80Gln Asn Leu Gln Leu Glu Asn Leu Arg Met Lys
Leu Pro Lys Pro Ala 85 90 95Lys Pro Leu Asn Lys Leu Arg Val Ala Thr
Pro Met Leu Met Gln Thr 100 105 110Met Pro Val Arg Gly Leu Leu Gln
Ala Asp Gln Val Asp Val Lys Asp 115 120 125Ser Ala Asn Asn Glu Ile
Lys Lys Val Met Val Asp Gly Ile His Gly 130 135 140Ser Asp Pro Leu
Ile Ile His Arg Gly Lys Pro Phe Thr Leu Glu Ala145 150 155 160Leu
Phe Asp Ala Asn Gln Asn Thr Lys Thr Ala Lys Ile Glu Ile Lys 165 170
175Ala Ser Leu Asp Gly Leu Glu Ile Asp Val Pro Gly Ile Asp Thr Asn
180 185 190Ala Ile His Phe Met Lys Leu Pro Leu Val Lys Gly Gln Gln
Tyr Asp 195 200 205Ile Lys Tyr Thr Trp Asn Val Pro Lys Ile Ala Pro
Lys Ser Glu Asn 210 215 220Val Val Val Thr Val Lys Leu Ile Gly Asp
Asn Gly Val Leu Ala Leu225 230 235 240Ala Ile Ala Thr His Gly Lys
Ile Arg Asp 245 25030198PRTArtificial sequenceIMAT_NTERM Cat from
A0A088SAW7 Der f 23 allergen Dermatophagoides farinae (American
house dust mite) 30Met His His His His His His Tyr Gly Arg Lys Lys
Arg Arg Gln Arg1 5 10 15Arg Arg Met Glu Asp Gln Arg Asp Leu Ile Ser
Asn His Glu Gln Leu 20 25 30Pro Ile Leu Gly Gln Arg Pro Ala Ala Pro
Glu Ser Lys Glu Ser Arg 35 40 45Gly Ala Leu Tyr Thr Gly Phe Ser Val
Leu Val Ala Leu Leu Leu Ala 50 55 60Gly Gln Ala Thr Thr Ala Tyr Phe
Leu Tyr Gln Gln Gln Gly Arg Leu65 70 75 80Asp Lys Leu Thr Val Thr
Ala Gln Asn Leu Gln Leu Glu Asn Leu Arg 85 90 95Met Lys Leu Pro Lys
Pro Ala Lys Pro Leu Asn Lys Leu Arg Val Ala 100 105 110Thr Pro Met
Leu Met Gln Thr Met Pro Val Arg Gly Leu Leu Gln Ala 115 120 125Asp
Ile Asp His Asp Asp Asp Pro Thr Thr Met Ile Asp Val Gln Thr 130 135
140Thr Thr Val Gln Pro Ser Asp Glu Phe Glu Leu Pro Thr Arg Phe
Gly145 150 155 160Tyr Phe Ala Asp Pro Lys Asp Pro Leu Lys Phe Tyr
Ile Ile Ser Asn 165 170 175Trp Glu Ala Ile His Lys Ser Leu Pro Gly
Asn Thr Arg Trp Asn Glu 180 185 190Lys Glu Leu Thr Ile Thr
19531198PRTArtificial sequenceIMAT_CTERM Cat from A0A088SAW7 Der f
23 allergen Dermatophagoides farinae (American house dust mite)
31Met Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Met Glu Asp Gln1
5 10 15Arg Asp Leu Ile Ser Asn His Glu Gln Leu Pro Ile Leu Gly Gln
Arg 20 25 30Pro Ala Ala Pro Glu Ser Lys Glu Ser Arg Gly Ala Leu Tyr
Thr Gly 35 40 45Phe Ser Val Leu Val Ala Leu Leu Leu Ala Gly Gln Ala
Thr Thr Ala 50 55 60Tyr Phe Leu Tyr Gln Gln Gln Gly Arg Leu Asp Lys
Leu Thr Val Thr65 70 75 80Ala Gln Asn Leu Gln Leu Glu Asn Leu Arg
Met Lys Leu Pro Lys Pro 85 90 95Ala Lys Pro Leu Asn Lys Leu Arg Val
Ala Thr Pro Met Leu Met Gln 100 105 110Thr Met Pro Val Arg Gly Leu
Leu Gln Ala Asp Ile Asp His Asp Asp 115 120 125Asp Pro Thr Thr Met
Ile Asp Val Gln Thr Thr Thr Val Gln Pro Ser 130 135 140Asp Glu Phe
Glu Leu Pro Thr Arg Phe Gly Tyr Phe Ala Asp Pro Lys145 150 155
160Asp Pro Leu Lys Phe Tyr Ile Ile Ser Asn Trp Glu Ala Ile His Lys
165 170 175Ser Leu Pro Gly Asn Thr Arg Trp Asn Glu Lys Glu Leu Thr
Ile Thr 180 185 190His His His His His His 19532191PRTArtificial
sequenceIMAT_PURE Cat from A0A088SAW7 Der f 23 allergen
Dermatophagoides farinae (American house dust mite) 32Tyr Gly Arg
Lys Lys Arg Arg Gln Arg Arg Arg Met Glu Asp Gln Arg1 5 10 15Asp Leu
Ile Ser Asn His Glu Gln Leu Pro Ile Leu Gly Gln Arg Pro 20 25 30Ala
Ala Pro Glu Ser Lys Glu Ser Arg Gly Ala Leu Tyr Thr Gly Phe 35 40
45Ser Val Leu Val Ala Leu Leu Leu Ala Gly Gln Ala Thr Thr Ala Tyr
50 55 60Phe Leu Tyr Gln Gln Gln Gly Arg Leu Asp Lys Leu Thr Val Thr
Ala65 70 75 80Gln Asn Leu Gln Leu Glu Asn Leu Arg Met Lys Leu Pro
Lys Pro Ala 85 90 95Lys Pro Leu Asn Lys Leu Arg Val Ala Thr Pro Met
Leu Met Gln Thr 100 105 110Met Pro Val Arg Gly Leu Leu Gln Ala Asp
Ile Asp His Asp Asp Asp 115 120 125Pro Thr Thr Met Ile Asp Val Gln
Thr Thr Thr Val Gln Pro Ser Asp 130 135 140Glu Phe Glu Leu Pro Thr
Arg Phe Gly Tyr Phe Ala Asp Pro Lys Asp145 150 155 160Pro Leu Lys
Phe Tyr Ile Ile Ser Asn Trp Glu Ala Ile His Lys Ser 165 170 175Leu
Pro Gly Asn Thr Arg Trp Asn Glu Lys Glu Leu Thr Ile Thr 180 185
19033573PRTArtificial sequenceIMAT_NTERM Cat from Q86R84 60 kDa
allergen Der f 18p Dermatophagoides farinae (American house dust
mite) 33Met His His His His His His Tyr Gly Arg Lys Lys Arg Arg Gln
Arg1 5 10 15Arg Arg Met Glu Asp Gln Arg Asp Leu Ile Ser Asn His Glu
Gln Leu 20 25 30Pro Ile Leu Gly Gln Arg Pro Ala Ala Pro Glu Ser Lys
Ser Ser Arg 35 40 45Gly Ala Leu Tyr Thr Gly Phe Ser Val Leu Val Ala
Leu Leu Leu Ala 50 55 60Gly Gln Ala Thr Thr Ala Tyr Phe Leu Tyr Gln
Gln Gln Gly Arg Leu65 70 75 80Asp Lys Leu Thr Val Thr Ala Gln Asn
Leu Gln Leu Glu Asn Leu Arg 85 90 95Met Lys Leu Pro Lys Pro Ala Lys
Pro Leu Asn Lys Leu Arg Val Ala 100 105 110Thr Pro Met Leu Met Gln
Thr Met Pro Val Arg Gly Leu Leu Gln Ala 115 120 125Ser Asn Ile Arg
Pro Asn Val Ala Thr Leu Glu Pro Lys Thr Val Leu 130 135 140Tyr Tyr
Glu Ser Trp Val His Trp Arg Gln Gly Glu Gly Lys Met Asp145 150 155
160Pro Glu Asp Ile Asp Thr Ser Leu Met Thr His Ile Val Tyr Ser Tyr
165 170 175Phe Gly Ile Asp Ala Ala Thr His Glu Ile Lys Leu Leu Asp
Glu Tyr 180 185 190Leu Met Lys Asp Leu His Asp Met Glu His Phe Thr
Gln His Lys Gly 195 200 205Asn Ala Lys Ala Met Ile Ala Val Gly Gly
Ser Thr Met Ser Asp Gln 210 215 220Phe Ser Lys Thr Ala Ala Val Glu
His Tyr Arg Glu Thr Phe Val Val225 230 235 240Ser Thr Val Asp Leu
Met Thr Arg Tyr Gly Phe Asp Gly Val Met Ile 245 250 255Asp Trp Ser
Gly Met Gln Ala Lys Asp Ser Asp Asn Phe Ile Lys Leu 260 265 270Leu
Asp Lys Phe Asp Glu Lys Phe Ala His Thr Ser Phe Val Met Gly 275 280
285Val Thr Leu Pro Ala Thr Ile Ala Ser Tyr Asp Asn Tyr Asn Ile Pro
290 295 300Ala Ile Ser Asn Tyr Val Asp Phe Met Asn Val Leu Ser Leu
Asp Tyr305 310 315 320Thr Gly Ser Trp Ala His Thr Val Gly His Ala
Ser Pro Phe Pro Glu 325 330 335Gln Leu Lys Thr Leu Glu Ala Tyr His
Lys Arg Gly Ala Pro Arg His 340 345 350Lys Met Val Met Ala Val Pro
Phe Tyr Ala Arg Thr Trp Ile Leu Glu 355 360 365Lys Met Asn Lys Gln
Asp Ile Gly Asp Lys Ala Ser Gly Pro Gly Pro 370 375 380Arg Gly Gln
Phe Thr Gln Thr Asp Gly Phe Leu Ser Tyr Asn Glu Leu385 390 395
400Ser Val Gln Ile Gln Ala Glu Thr Asn Ala Phe Thr Ile Thr Arg Asp
405 410 415His Asp Asn Thr Ala Ile Tyr Ala Val Tyr Val His Ser Asn
His Ala 420 425 430Glu Trp Ile Ser Phe Glu Asp Arg His Thr Leu Gly
Glu Lys Ala Lys 435 440 445Asn Ile Thr Gln Gln Gly Tyr Ala Gly Met
Ser Val Tyr Thr Leu Ser 450 455 460Asn Glu Asp Val His Gly Val Arg
Gly Asp Lys Asn Pro Leu Leu His465 470 475 480Ala Ile Gln Ser Asn
Tyr Tyr His Gly Val Val Thr Glu Pro Thr Val 485 490 495Val Thr Leu
Pro Pro Val Thr His Thr Thr Glu His Val Thr Asp Ile 500 505 510Pro
Gly Val Phe His Leu His Glu Glu Gly Phe Phe Arg Asp Lys Thr 515 520
525Tyr Ser Ala Thr Tyr Tyr Glu Ile Lys Lys Gly Asp Phe Gly Leu Glu
530 535 540Lys Thr Val His His Leu Ala Asn His Leu Gln Ala Phe Asp
Glu Val545 550 555 560Ser Arg Thr Arg Ile Asp His Thr Lys Ile Pro
Gly Ser 565 57034573PRTArtificial sequenceIMAT_CTERM Cat from
Q86R84 60 kDa allergen Der f 18p Dermatophagoides farinae (American
house dust mite) 34Met Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg
Met Glu Asp Gln1 5 10 15Arg Asp Leu Ile Ser Asn His Glu Gln Leu Pro
Ile Leu Gly Gln Arg 20 25 30Pro Ala Ala Pro Glu Ser Lys Ser Ser Arg
Gly Ala Leu Tyr Thr Gly 35 40 45Phe Ser Val Leu Val Ala Leu Leu Leu
Ala Gly Gln Ala Thr Thr Ala 50 55 60Tyr Phe Leu Tyr Gln Gln Gln
Gly
Arg Leu Asp Lys Leu Thr Val Thr65 70 75 80Ala Gln Asn Leu Gln Leu
Glu Asn Leu Arg Met Lys Leu Pro Lys Pro 85 90 95Ala Lys Pro Leu Asn
Lys Leu Arg Val Ala Thr Pro Met Leu Met Gln 100 105 110Thr Met Pro
Val Arg Gly Leu Leu Gln Ala Ser Asn Ile Arg Pro Asn 115 120 125Val
Ala Thr Leu Glu Pro Lys Thr Val Leu Tyr Tyr Glu Ser Trp Val 130 135
140His Trp Arg Gln Gly Glu Gly Lys Met Asp Pro Glu Asp Ile Asp
Thr145 150 155 160Ser Leu Met Thr His Ile Val Tyr Ser Tyr Phe Gly
Ile Asp Ala Ala 165 170 175Thr His Glu Ile Lys Leu Leu Asp Glu Tyr
Leu Met Lys Asp Leu His 180 185 190Asp Met Glu His Phe Thr Gln His
Lys Gly Asn Ala Lys Ala Met Ile 195 200 205Ala Val Gly Gly Ser Thr
Met Ser Asp Gln Phe Ser Lys Thr Ala Ala 210 215 220Val Glu His Tyr
Arg Glu Thr Phe Val Val Ser Thr Val Asp Leu Met225 230 235 240Thr
Arg Tyr Gly Phe Asp Gly Val Met Ile Asp Trp Ser Gly Met Gln 245 250
255Ala Lys Asp Ser Asp Asn Phe Ile Lys Leu Leu Asp Lys Phe Asp Glu
260 265 270Lys Phe Ala His Thr Ser Phe Val Met Gly Val Thr Leu Pro
Ala Thr 275 280 285Ile Ala Ser Tyr Asp Asn Tyr Asn Ile Pro Ala Ile
Ser Asn Tyr Val 290 295 300Asp Phe Met Asn Val Leu Ser Leu Asp Tyr
Thr Gly Ser Trp Ala His305 310 315 320Thr Val Gly His Ala Ser Pro
Phe Pro Glu Gln Leu Lys Thr Leu Glu 325 330 335Ala Tyr His Lys Arg
Gly Ala Pro Arg His Lys Met Val Met Ala Val 340 345 350Pro Phe Tyr
Ala Arg Thr Trp Ile Leu Glu Lys Met Asn Lys Gln Asp 355 360 365Ile
Gly Asp Lys Ala Ser Gly Pro Gly Pro Arg Gly Gln Phe Thr Gln 370 375
380Thr Asp Gly Phe Leu Ser Tyr Asn Glu Leu Ser Val Gln Ile Gln
Ala385 390 395 400Glu Thr Asn Ala Phe Thr Ile Thr Arg Asp His Asp
Asn Thr Ala Ile 405 410 415Tyr Ala Val Tyr Val His Ser Asn His Ala
Glu Trp Ile Ser Phe Glu 420 425 430Asp Arg His Thr Leu Gly Glu Lys
Ala Lys Asn Ile Thr Gln Gln Gly 435 440 445Tyr Ala Gly Met Ser Val
Tyr Thr Leu Ser Asn Glu Asp Val His Gly 450 455 460Val Arg Gly Asp
Lys Asn Pro Leu Leu His Ala Ile Gln Ser Asn Tyr465 470 475 480Tyr
His Gly Val Val Thr Glu Pro Thr Val Val Thr Leu Pro Pro Val 485 490
495Thr His Thr Thr Glu His Val Thr Asp Ile Pro Gly Val Phe His Leu
500 505 510His Glu Glu Gly Phe Phe Arg Asp Lys Thr Tyr Ser Ala Thr
Tyr Tyr 515 520 525Glu Ile Lys Lys Gly Asp Phe Gly Leu Glu Lys Thr
Val His His Leu 530 535 540Ala Asn His Leu Gln Ala Phe Asp Glu Val
Ser Arg Thr Arg Ile Asp545 550 555 560His Thr Lys Ile Pro Gly Ser
His His His His His His 565 57035566PRTArtificial sequenceIMAT_PURE
Cat from Q86R84 60 kDa allergen Der f 18p Dermatophagoides farinae
(American house dust mite) 35Tyr Gly Arg Lys Lys Arg Arg Gln Arg
Arg Arg Met Glu Asp Gln Arg1 5 10 15Asp Leu Ile Ser Asn His Glu Gln
Leu Pro Ile Leu Gly Gln Arg Pro 20 25 30Ala Ala Pro Glu Ser Lys Ser
Ser Arg Gly Ala Leu Tyr Thr Gly Phe 35 40 45Ser Val Leu Val Ala Leu
Leu Leu Ala Gly Gln Ala Thr Thr Ala Tyr 50 55 60Phe Leu Tyr Gln Gln
Gln Gly Arg Leu Asp Lys Leu Thr Val Thr Ala65 70 75 80Gln Asn Leu
Gln Leu Glu Asn Leu Arg Met Lys Leu Pro Lys Pro Ala 85 90 95Lys Pro
Leu Asn Lys Leu Arg Val Ala Thr Pro Met Leu Met Gln Thr 100 105
110Met Pro Val Arg Gly Leu Leu Gln Ala Ser Asn Ile Arg Pro Asn Val
115 120 125Ala Thr Leu Glu Pro Lys Thr Val Leu Tyr Tyr Glu Ser Trp
Val His 130 135 140Trp Arg Gln Gly Glu Gly Lys Met Asp Pro Glu Asp
Ile Asp Thr Ser145 150 155 160Leu Met Thr His Ile Val Tyr Ser Tyr
Phe Gly Ile Asp Ala Ala Thr 165 170 175His Glu Ile Lys Leu Leu Asp
Glu Tyr Leu Met Lys Asp Leu His Asp 180 185 190Met Glu His Phe Thr
Gln His Lys Gly Asn Ala Lys Ala Met Ile Ala 195 200 205Val Gly Gly
Ser Thr Met Ser Asp Gln Phe Ser Lys Thr Ala Ala Val 210 215 220Glu
His Tyr Arg Glu Thr Phe Val Val Ser Thr Val Asp Leu Met Thr225 230
235 240Arg Tyr Gly Phe Asp Gly Val Met Ile Asp Trp Ser Gly Met Gln
Ala 245 250 255Lys Asp Ser Asp Asn Phe Ile Lys Leu Leu Asp Lys Phe
Asp Glu Lys 260 265 270Phe Ala His Thr Ser Phe Val Met Gly Val Thr
Leu Pro Ala Thr Ile 275 280 285Ala Ser Tyr Asp Asn Tyr Asn Ile Pro
Ala Ile Ser Asn Tyr Val Asp 290 295 300Phe Met Asn Val Leu Ser Leu
Asp Tyr Thr Gly Ser Trp Ala His Thr305 310 315 320Val Gly His Ala
Ser Pro Phe Pro Glu Gln Leu Lys Thr Leu Glu Ala 325 330 335Tyr His
Lys Arg Gly Ala Pro Arg His Lys Met Val Met Ala Val Pro 340 345
350Phe Tyr Ala Arg Thr Trp Ile Leu Glu Lys Met Asn Lys Gln Asp Ile
355 360 365Gly Asp Lys Ala Ser Gly Pro Gly Pro Arg Gly Gln Phe Thr
Gln Thr 370 375 380Asp Gly Phe Leu Ser Tyr Asn Glu Leu Ser Val Gln
Ile Gln Ala Glu385 390 395 400Thr Asn Ala Phe Thr Ile Thr Arg Asp
His Asp Asn Thr Ala Ile Tyr 405 410 415Ala Val Tyr Val His Ser Asn
His Ala Glu Trp Ile Ser Phe Glu Asp 420 425 430Arg His Thr Leu Gly
Glu Lys Ala Lys Asn Ile Thr Gln Gln Gly Tyr 435 440 445Ala Gly Met
Ser Val Tyr Thr Leu Ser Asn Glu Asp Val His Gly Val 450 455 460Arg
Gly Asp Lys Asn Pro Leu Leu His Ala Ile Gln Ser Asn Tyr Tyr465 470
475 480His Gly Val Val Thr Glu Pro Thr Val Val Thr Leu Pro Pro Val
Thr 485 490 495His Thr Thr Glu His Val Thr Asp Ile Pro Gly Val Phe
His Leu His 500 505 510Glu Glu Gly Phe Phe Arg Asp Lys Thr Tyr Ser
Ala Thr Tyr Tyr Glu 515 520 525Ile Lys Lys Gly Asp Phe Gly Leu Glu
Lys Thr Val His His Leu Ala 530 535 540Asn His Leu Gln Ala Phe Asp
Glu Val Ser Arg Thr Arg Ile Asp His545 550 555 560Thr Lys Ile Pro
Gly Ser 56536664PRTArtificial sequenceIMAT_NTERM Cat from Q9U6R7
98kDa HDM allergen (Der f 15 allergen) (Group 15 allergen Der f 15)
Dermatophagoides farinae (American house dust mite) 36Met His His
His His His His Tyr Gly Arg Lys Lys Arg Arg Gln Arg1 5 10 15Arg Arg
Met Glu Asp Gln Arg Asp Leu Ile Ser Asn His Glu Gln Leu 20 25 30Pro
Ile Leu Gly Gln Arg Pro Ala Ala Pro Glu Ser Lys Ser Ser Arg 35 40
45Gly Ala Leu Tyr Thr Gly Phe Ser Val Leu Val Ala Leu Leu Leu Ala
50 55 60Gly Gln Ala Thr Thr Ala Tyr Phe Leu Tyr Gln Gln Gln Gly Arg
Leu65 70 75 80Asp Lys Leu Thr Val Thr Ala Gln Asn Leu Gln Leu Glu
Asn Leu Arg 85 90 95Met Lys Leu Pro Lys Pro Ala Lys Pro Leu Asn Lys
Leu Arg Val Ala 100 105 110Thr Pro Met Leu Met Gln Thr Met Pro Val
Arg Gly Leu Leu Gln Ala 115 120 125Ser Ile Lys Arg Asp His Asn Asp
Tyr Ser Lys Asn Pro Met Arg Ile 130 135 140Val Leu Tyr Val Gly Thr
Trp Ser Val Tyr His Lys Val Asp Pro Tyr145 150 155 160Thr Ile Glu
Asp Ile Asp Pro Phe Lys Ser Thr His Leu Met Tyr Gly 165 170 175Phe
Ala Lys Ile Asp Glu Tyr Lys Tyr Thr Ile Gln Val Phe Asp Pro 180 185
190Tyr Gln Asp Asp Asn His Asn Ser Trp Glu Lys Arg Gly Tyr Glu Arg
195 200 205Phe Asn Asn Leu Arg Leu Lys Asn Pro Glu Leu Thr Thr Met
Ile Ser 210 215 220Leu Gly Gly Trp Tyr Glu Gly Ser Glu Lys Tyr Ser
Asp Met Ala Ala225 230 235 240Asn Pro Thr Tyr Arg Gln Gln Phe Ile
Gln Ser Val Leu Asp Phe Leu 245 250 255Gln Glu Tyr Lys Phe Asp Gly
Leu Asp Leu Asp Trp Glu Tyr Pro Gly 260 265 270Ser Arg Leu Gly Asn
Pro Lys Ile Asp Lys Gln Asn Tyr Leu Ala Leu 275 280 285Val Arg Glu
Leu Lys Asp Ala Phe Glu Pro His Gly Tyr Leu Leu Thr 290 295 300Ala
Ala Val Ser Pro Gly Lys Asp Lys Ile Asp Arg Ala Tyr Asp Ile305 310
315 320Lys Glu Leu Asn Lys Leu Phe Asp Trp Met Asn Val Met Thr Tyr
Asp 325 330 335Tyr His Gly Gly Trp Glu Asn Phe Tyr Gly His Asn Ala
Pro Leu Tyr 340 345 350Lys Arg Pro Asp Glu Thr Asp Glu Leu His Thr
Tyr Phe Asn Val Asn 355 360 365Tyr Thr Met His Tyr Tyr Leu Asn Asn
Gly Ala Thr Arg Asp Lys Leu 370 375 380Val Met Gly Val Pro Phe Tyr
Gly Arg Ala Trp Ser Ile Glu Asp Arg385 390 395 400Ser Lys Leu Lys
Leu Gly Asp Pro Ala Lys Gly Met Ser Pro Pro Gly 405 410 415Phe Ile
Ser Gly Glu Glu Gly Val Leu Ser Tyr Ile Glu Leu Ser Gln 420 425
430Leu Phe Gln Lys Glu Glu Trp His Ile Gln Tyr Asp Glu Tyr Tyr Asn
435 440 445Ala Pro Tyr Gly Tyr Asn Asp Lys Ile Trp Val Gly Tyr Asp
Asp Leu 450 455 460Ala Ser Ile Ser Ser Lys Leu Ala Phe Leu Lys Glu
Leu Gly Val Ser465 470 475 480Gly Val Met Val Trp Ser Leu Glu Asn
Asp Asp Phe Lys Gly His Leu 485 490 495Gly Pro Lys Asn Pro Leu Leu
Asn Lys Val His Asn Met Ile Asn Gly 500 505 510Asp Glu Lys Asn Ser
Phe Glu Ile Ile Leu Gly Pro Ser Thr Thr Thr 515 520 525Pro Thr Pro
Thr Thr Thr Pro Thr Thr Pro Thr Thr Thr Pro Thr Thr 530 535 540Pro
Ser Pro Thr Thr Pro Thr Thr Thr Pro Ser Pro Thr Thr Pro Thr545 550
555 560Thr Thr Pro Ser Pro Thr Thr Pro Thr Thr Thr Pro Ser Pro Thr
Thr 565 570 575Pro Thr Pro Thr Thr Pro Thr Pro Ala Pro Thr Thr Ser
Thr Pro Ser 580 585 590Pro Thr Thr Thr Glu His Thr Ser Glu Thr Pro
Lys Tyr Thr Thr Tyr 595 600 605Val Asp Gly His Leu Ile Lys Ser Tyr
Lys Glu Gly Asp Ile Pro His 610 615 620Pro Thr Asn Ile His Lys Tyr
Leu Val Ile Glu Phe Val Asn Gly Gly625 630 635 640Trp Trp Val His
Ile Met Pro Ser Pro Pro Gly Thr Ile Trp Ser Gln 645 650 655Glu Lys
Leu Thr Ser Ile Gly Glu 66037664PRTArtificial sequenceIMAT_CTERM
Cat from Q9U6R7 98kDa HDM allergen (Der f 15 allergen) (Group 15
allergen Der f 15) Dermatophagoides farinae (American house dust
mite) 37Met Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Met Glu Asp
Gln1 5 10 15Arg Asp Leu Ile Ser Asn His Glu Gln Leu Pro Ile Leu Gly
Gln Arg 20 25 30Pro Ala Ala Pro Glu Ser Lys Ser Ser Arg Gly Ala Leu
Tyr Thr Gly 35 40 45Phe Ser Val Leu Val Ala Leu Leu Leu Ala Gly Gln
Ala Thr Thr Ala 50 55 60Tyr Phe Leu Tyr Gln Gln Gln Gly Arg Leu Asp
Lys Leu Thr Val Thr65 70 75 80Ala Gln Asn Leu Gln Leu Glu Asn Leu
Arg Met Lys Leu Pro Lys Pro 85 90 95Ala Lys Pro Leu Asn Lys Leu Arg
Val Ala Thr Pro Met Leu Met Gln 100 105 110Thr Met Pro Val Arg Gly
Leu Leu Gln Ala Ser Ile Lys Arg Asp His 115 120 125Asn Asp Tyr Ser
Lys Asn Pro Met Arg Ile Val Leu Tyr Val Gly Thr 130 135 140Trp Ser
Val Tyr His Lys Val Asp Pro Tyr Thr Ile Glu Asp Ile Asp145 150 155
160Pro Phe Lys Ser Thr His Leu Met Tyr Gly Phe Ala Lys Ile Asp Glu
165 170 175Tyr Lys Tyr Thr Ile Gln Val Phe Asp Pro Tyr Gln Asp Asp
Asn His 180 185 190Asn Ser Trp Glu Lys Arg Gly Tyr Glu Arg Phe Asn
Asn Leu Arg Leu 195 200 205Lys Asn Pro Glu Leu Thr Thr Met Ile Ser
Leu Gly Gly Trp Tyr Glu 210 215 220Gly Ser Glu Lys Tyr Ser Asp Met
Ala Ala Asn Pro Thr Tyr Arg Gln225 230 235 240Gln Phe Ile Gln Ser
Val Leu Asp Phe Leu Gln Glu Tyr Lys Phe Asp 245 250 255Gly Leu Asp
Leu Asp Trp Glu Tyr Pro Gly Ser Arg Leu Gly Asn Pro 260 265 270Lys
Ile Asp Lys Gln Asn Tyr Leu Ala Leu Val Arg Glu Leu Lys Asp 275 280
285Ala Phe Glu Pro His Gly Tyr Leu Leu Thr Ala Ala Val Ser Pro Gly
290 295 300Lys Asp Lys Ile Asp Arg Ala Tyr Asp Ile Lys Glu Leu Asn
Lys Leu305 310 315 320Phe Asp Trp Met Asn Val Met Thr Tyr Asp Tyr
His Gly Gly Trp Glu 325 330 335Asn Phe Tyr Gly His Asn Ala Pro Leu
Tyr Lys Arg Pro Asp Glu Thr 340 345 350Asp Glu Leu His Thr Tyr Phe
Asn Val Asn Tyr Thr Met His Tyr Tyr 355 360 365Leu Asn Asn Gly Ala
Thr Arg Asp Lys Leu Val Met Gly Val Pro Phe 370 375 380Tyr Gly Arg
Ala Trp Ser Ile Glu Asp Arg Ser Lys Leu Lys Leu Gly385 390 395
400Asp Pro Ala Lys Gly Met Ser Pro Pro Gly Phe Ile Ser Gly Glu Glu
405 410 415Gly Val Leu Ser Tyr Ile Glu Leu Ser Gln Leu Phe Gln Lys
Glu Glu 420 425 430Trp His Ile Gln Tyr Asp Glu Tyr Tyr Asn Ala Pro
Tyr Gly Tyr Asn 435 440 445Asp Lys Ile Trp Val Gly Tyr Asp Asp Leu
Ala Ser Ile Ser Ser Lys 450 455 460Leu Ala Phe Leu Lys Glu Leu Gly
Val Ser Gly Val Met Val Trp Ser465 470 475 480Leu Glu Asn Asp Asp
Phe Lys Gly His Leu Gly Pro Lys Asn Pro Leu 485 490 495Leu Asn Lys
Val His Asn Met Ile Asn Gly Asp Glu Lys Asn Ser Phe 500 505 510Glu
Ile Ile Leu Gly Pro Ser Thr Thr Thr Pro Thr Pro Thr Thr Thr 515 520
525Pro Thr Thr Pro Thr Thr Thr Pro Thr Thr Pro Ser Pro Thr Thr Pro
530 535 540Thr Thr Thr Pro Ser Pro Thr Thr Pro Thr Thr Thr Pro Ser
Pro Thr545 550 555 560Thr Pro Thr Thr Thr Pro Ser Pro Thr Thr Pro
Thr Pro Thr Thr Pro 565 570 575Thr Pro Ala Pro Thr Thr Ser Thr Pro
Ser Pro Thr Thr Thr Glu His 580 585 590Thr Ser Glu Thr Pro Lys Tyr
Thr Thr Tyr Val Asp Gly His Leu Ile 595 600 605Lys Ser Tyr Lys Glu
Gly Asp Ile Pro His Pro Thr Asn Ile His Lys 610 615 620Tyr Leu Val
Ile Glu Phe Val Asn Gly Gly Trp Trp Val His Ile Met625 630 635
640Pro Ser Pro Pro Gly Thr Ile Trp Ser Gln Glu Lys Leu Thr Ser Ile
645 650 655Gly Glu His His His His His His 66038657PRTArtificial
sequenceIMAT_PURE Cat
from Q9U6R7 98kDa HDM allergen (Der f 15 allergen) (Group 15
allergen Der f 15) Dermatophagoides farinae (American house dust
mite) 38Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Met Glu Asp Gln
Arg1 5 10 15Asp Leu Ile Ser Asn His Glu Gln Leu Pro Ile Leu Gly Gln
Arg Pro 20 25 30Ala Ala Pro Glu Ser Lys Ser Ser Arg Gly Ala Leu Tyr
Thr Gly Phe 35 40 45Ser Val Leu Val Ala Leu Leu Leu Ala Gly Gln Ala
Thr Thr Ala Tyr 50 55 60Phe Leu Tyr Gln Gln Gln Gly Arg Leu Asp Lys
Leu Thr Val Thr Ala65 70 75 80Gln Asn Leu Gln Leu Glu Asn Leu Arg
Met Lys Leu Pro Lys Pro Ala 85 90 95Lys Pro Leu Asn Lys Leu Arg Val
Ala Thr Pro Met Leu Met Gln Thr 100 105 110Met Pro Val Arg Gly Leu
Leu Gln Ala Ser Ile Lys Arg Asp His Asn 115 120 125Asp Tyr Ser Lys
Asn Pro Met Arg Ile Val Leu Tyr Val Gly Thr Trp 130 135 140Ser Val
Tyr His Lys Val Asp Pro Tyr Thr Ile Glu Asp Ile Asp Pro145 150 155
160Phe Lys Ser Thr His Leu Met Tyr Gly Phe Ala Lys Ile Asp Glu Tyr
165 170 175Lys Tyr Thr Ile Gln Val Phe Asp Pro Tyr Gln Asp Asp Asn
His Asn 180 185 190Ser Trp Glu Lys Arg Gly Tyr Glu Arg Phe Asn Asn
Leu Arg Leu Lys 195 200 205Asn Pro Glu Leu Thr Thr Met Ile Ser Leu
Gly Gly Trp Tyr Glu Gly 210 215 220Ser Glu Lys Tyr Ser Asp Met Ala
Ala Asn Pro Thr Tyr Arg Gln Gln225 230 235 240Phe Ile Gln Ser Val
Leu Asp Phe Leu Gln Glu Tyr Lys Phe Asp Gly 245 250 255Leu Asp Leu
Asp Trp Glu Tyr Pro Gly Ser Arg Leu Gly Asn Pro Lys 260 265 270Ile
Asp Lys Gln Asn Tyr Leu Ala Leu Val Arg Glu Leu Lys Asp Ala 275 280
285Phe Glu Pro His Gly Tyr Leu Leu Thr Ala Ala Val Ser Pro Gly Lys
290 295 300Asp Lys Ile Asp Arg Ala Tyr Asp Ile Lys Glu Leu Asn Lys
Leu Phe305 310 315 320Asp Trp Met Asn Val Met Thr Tyr Asp Tyr His
Gly Gly Trp Glu Asn 325 330 335Phe Tyr Gly His Asn Ala Pro Leu Tyr
Lys Arg Pro Asp Glu Thr Asp 340 345 350Glu Leu His Thr Tyr Phe Asn
Val Asn Tyr Thr Met His Tyr Tyr Leu 355 360 365Asn Asn Gly Ala Thr
Arg Asp Lys Leu Val Met Gly Val Pro Phe Tyr 370 375 380Gly Arg Ala
Trp Ser Ile Glu Asp Arg Ser Lys Leu Lys Leu Gly Asp385 390 395
400Pro Ala Lys Gly Met Ser Pro Pro Gly Phe Ile Ser Gly Glu Glu Gly
405 410 415Val Leu Ser Tyr Ile Glu Leu Ser Gln Leu Phe Gln Lys Glu
Glu Trp 420 425 430His Ile Gln Tyr Asp Glu Tyr Tyr Asn Ala Pro Tyr
Gly Tyr Asn Asp 435 440 445Lys Ile Trp Val Gly Tyr Asp Asp Leu Ala
Ser Ile Ser Ser Lys Leu 450 455 460Ala Phe Leu Lys Glu Leu Gly Val
Ser Gly Val Met Val Trp Ser Leu465 470 475 480Glu Asn Asp Asp Phe
Lys Gly His Leu Gly Pro Lys Asn Pro Leu Leu 485 490 495Asn Lys Val
His Asn Met Ile Asn Gly Asp Glu Lys Asn Ser Phe Glu 500 505 510Ile
Ile Leu Gly Pro Ser Thr Thr Thr Pro Thr Pro Thr Thr Thr Pro 515 520
525Thr Thr Pro Thr Thr Thr Pro Thr Thr Pro Ser Pro Thr Thr Pro Thr
530 535 540Thr Thr Pro Ser Pro Thr Thr Pro Thr Thr Thr Pro Ser Pro
Thr Thr545 550 555 560Pro Thr Thr Thr Pro Ser Pro Thr Thr Pro Thr
Pro Thr Thr Pro Thr 565 570 575Pro Ala Pro Thr Thr Ser Thr Pro Ser
Pro Thr Thr Thr Glu His Thr 580 585 590Ser Glu Thr Pro Lys Tyr Thr
Thr Tyr Val Asp Gly His Leu Ile Lys 595 600 605Ser Tyr Lys Glu Gly
Asp Ile Pro His Pro Thr Asn Ile His Lys Tyr 610 615 620Leu Val Ile
Glu Phe Val Asn Gly Gly Trp Trp Val His Ile Met Pro625 630 635
640Ser Pro Pro Gly Thr Ile Trp Ser Gln Glu Lys Leu Thr Ser Ile Gly
645 650 655Glu39606PRTArtificial sequenceIMAT_NTERM Cat from I7HDR2
Zen 1 protein Dermatophagoides farinae (American house dust mite)
39Met His His His His His His Tyr Gly Arg Lys Lys Arg Arg Gln Arg1
5 10 15Arg Arg Met Glu Asp Gln Arg Asp Leu Ile Ser Asn His Glu Gln
Leu 20 25 30Pro Ile Leu Gly Gln Arg Pro Ala Ala Pro Glu Ser Lys Ser
Ser Arg 35 40 45Gly Ala Leu Tyr Thr Gly Phe Ser Val Leu Val Ala Leu
Leu Leu Ala 50 55 60Gly Gln Ala Thr Thr Ala Tyr Phe Leu Tyr Gln Gln
Gln Gly Arg Leu65 70 75 80Asp Lys Leu Thr Val Thr Ala Gln Asn Leu
Gln Leu Glu Asn Leu Arg 85 90 95Met Lys Leu Pro Lys Pro Ala Lys Pro
Leu Asn Lys Leu Arg Val Ala 100 105 110Thr Pro Met Leu Met Gln Thr
Met Pro Val Arg Gly Leu Leu Gln Ala 115 120 125Asn Pro Arg Phe Lys
Arg Asp Asn Arg Asp Asp Val Leu Lys Gln Thr 130 135 140Glu Glu Leu
Ile Lys Ser Ala Gln Asp Val Leu Glu Lys Leu Pro Asp145 150 155
160Ser Asp Leu Lys Asp Glu Ile Ala Glu Lys Leu Ala Thr Met Lys His
165 170 175Tyr Lys His Glu Leu Glu Asn Ala Lys Asn Pro Ile Lys Ile
Ala His 180 185 190Phe Glu Leu Glu Leu Leu Thr Met Phe Lys Lys Phe
Gln Ser Leu Leu 195 200 205Asn Glu Ala Asn Glu Ile Ile Lys Ser Leu
Thr Thr Thr Thr Thr Glu 210 215 220Pro Thr Thr Pro Thr Pro Glu Pro
Thr Thr Thr Thr Pro Glu Pro Thr225 230 235 240Thr Lys Thr Pro Glu
Pro Thr Thr Lys Thr Pro Glu Pro Thr Thr Pro 245 250 255Thr Pro Glu
Pro Thr Thr Lys Thr Pro Glu Pro Thr Thr Lys Thr Pro 260 265 270Glu
Pro Thr Thr Pro Thr Pro Glu Pro Thr Thr Lys Thr Pro Glu Pro 275 280
285Thr Thr Pro Thr Pro Glu Pro Thr Thr Lys Thr Pro Glu Pro Thr Thr
290 295 300Lys Thr Pro Glu Pro Ser Thr Pro Thr Pro Glu Pro Thr Thr
Lys Thr305 310 315 320Pro Glu Pro Thr Thr Lys Thr Pro Glu Pro Ser
Thr Pro Thr Pro Glu 325 330 335Pro Thr Thr Lys Thr Pro Glu Pro Ser
Thr Pro Thr Pro Glu Pro Thr 340 345 350Thr Lys Thr Pro Glu Pro Thr
Thr Lys Thr Pro Glu Pro Ser Thr Pro 355 360 365Thr Pro Glu Pro Thr
Thr Lys Thr Pro Glu Pro Ser Thr Pro Thr Pro 370 375 380Glu Pro Thr
Thr Lys Thr Pro Glu Pro Ser Thr Thr Lys Lys Pro Asn385 390 395
400Arg Asp Asp Val Leu Lys Gln Ala Glu Glu Leu Ile Lys Arg Ala Glu
405 410 415Asp Val Phe Glu Lys Leu Pro Asp Ser Asp Leu Lys Asn Glu
Ile Ala 420 425 430Glu Lys Leu Ala Thr Met Lys Asn Tyr Lys His Glu
Leu Glu Asn Ala 435 440 445Lys Asn Pro Ile Lys Ile Ala His Leu Glu
Ser Glu Leu Leu Thr Met 450 455 460Phe Lys Met Phe Gln Ser Leu Leu
Asn Glu Ala Asp Glu Ile Ile Arg465 470 475 480Ser Leu Thr Thr Thr
Thr Glu Pro Thr Thr Leu Asn Ser Thr Thr Pro 485 490 495Glu Pro Thr
Thr Leu Asn Ser Thr Thr Pro Glu Pro Thr Thr Leu Asn 500 505 510Ser
Thr Thr Pro Glu Pro Thr Thr Leu Asn Ser Thr Thr Pro Glu Pro 515 520
525Thr Thr Leu Asn Ser Thr Thr Pro Glu Pro Thr Thr Leu Asn Ser Thr
530 535 540Thr Pro Glu Pro Thr Thr Leu Asn Ser Thr Thr Pro Glu Pro
Thr Thr545 550 555 560Leu Asn Ser Thr Thr Pro Glu Pro Thr Thr Ser
Asn Ser Thr Thr Ser 565 570 575Glu Pro Thr Asn Ser Ile Asn Arg Lys
Thr Ser Glu Ile Ser Phe Leu 580 585 590Ser Asp Trp Phe His Lys Ile
Arg Thr Arg Phe Asn Ile Phe 595 600 60540606PRTArtificial
sequenceIMAT_CTERM Cat from I7HDR2 Zen 1 protein Dermatophagoides
farinae (American house dust mite) 40Met Tyr Gly Arg Lys Lys Arg
Arg Gln Arg Arg Arg Met Glu Asp Gln1 5 10 15Arg Asp Leu Ile Ser Asn
His Glu Gln Leu Pro Ile Leu Gly Gln Arg 20 25 30Pro Ala Ala Pro Glu
Ser Lys Ser Ser Arg Gly Ala Leu Tyr Thr Gly 35 40 45Phe Ser Val Leu
Val Ala Leu Leu Leu Ala Gly Gln Ala Thr Thr Ala 50 55 60Tyr Phe Leu
Tyr Gln Gln Gln Gly Arg Leu Asp Lys Leu Thr Val Thr65 70 75 80Ala
Gln Asn Leu Gln Leu Glu Asn Leu Arg Met Lys Leu Pro Lys Pro 85 90
95Ala Lys Pro Leu Asn Lys Leu Arg Val Ala Thr Pro Met Leu Met Gln
100 105 110Thr Met Pro Val Arg Gly Leu Leu Gln Ala Asn Pro Arg Phe
Lys Arg 115 120 125Asp Asn Arg Asp Asp Val Leu Lys Gln Thr Glu Glu
Leu Ile Lys Ser 130 135 140Ala Gln Asp Val Leu Glu Lys Leu Pro Asp
Ser Asp Leu Lys Asp Glu145 150 155 160Ile Ala Glu Lys Leu Ala Thr
Met Lys His Tyr Lys His Glu Leu Glu 165 170 175Asn Ala Lys Asn Pro
Ile Lys Ile Ala His Phe Glu Leu Glu Leu Leu 180 185 190Thr Met Phe
Lys Lys Phe Gln Ser Leu Leu Asn Glu Ala Asn Glu Ile 195 200 205Ile
Lys Ser Leu Thr Thr Thr Thr Thr Glu Pro Thr Thr Pro Thr Pro 210 215
220Glu Pro Thr Thr Thr Thr Pro Glu Pro Thr Thr Lys Thr Pro Glu
Pro225 230 235 240Thr Thr Lys Thr Pro Glu Pro Thr Thr Pro Thr Pro
Glu Pro Thr Thr 245 250 255Lys Thr Pro Glu Pro Thr Thr Lys Thr Pro
Glu Pro Thr Thr Pro Thr 260 265 270Pro Glu Pro Thr Thr Lys Thr Pro
Glu Pro Thr Thr Pro Thr Pro Glu 275 280 285Pro Thr Thr Lys Thr Pro
Glu Pro Thr Thr Lys Thr Pro Glu Pro Ser 290 295 300Thr Pro Thr Pro
Glu Pro Thr Thr Lys Thr Pro Glu Pro Thr Thr Lys305 310 315 320Thr
Pro Glu Pro Ser Thr Pro Thr Pro Glu Pro Thr Thr Lys Thr Pro 325 330
335Glu Pro Ser Thr Pro Thr Pro Glu Pro Thr Thr Lys Thr Pro Glu Pro
340 345 350Thr Thr Lys Thr Pro Glu Pro Ser Thr Pro Thr Pro Glu Pro
Thr Thr 355 360 365Lys Thr Pro Glu Pro Ser Thr Pro Thr Pro Glu Pro
Thr Thr Lys Thr 370 375 380Pro Glu Pro Ser Thr Thr Lys Lys Pro Asn
Arg Asp Asp Val Leu Lys385 390 395 400Gln Ala Glu Glu Leu Ile Lys
Arg Ala Glu Asp Val Phe Glu Lys Leu 405 410 415Pro Asp Ser Asp Leu
Lys Asn Glu Ile Ala Glu Lys Leu Ala Thr Met 420 425 430Lys Asn Tyr
Lys His Glu Leu Glu Asn Ala Lys Asn Pro Ile Lys Ile 435 440 445Ala
His Leu Glu Ser Glu Leu Leu Thr Met Phe Lys Met Phe Gln Ser 450 455
460Leu Leu Asn Glu Ala Asp Glu Ile Ile Arg Ser Leu Thr Thr Thr
Thr465 470 475 480Glu Pro Thr Thr Leu Asn Ser Thr Thr Pro Glu Pro
Thr Thr Leu Asn 485 490 495Ser Thr Thr Pro Glu Pro Thr Thr Leu Asn
Ser Thr Thr Pro Glu Pro 500 505 510Thr Thr Leu Asn Ser Thr Thr Pro
Glu Pro Thr Thr Leu Asn Ser Thr 515 520 525Thr Pro Glu Pro Thr Thr
Leu Asn Ser Thr Thr Pro Glu Pro Thr Thr 530 535 540Leu Asn Ser Thr
Thr Pro Glu Pro Thr Thr Leu Asn Ser Thr Thr Pro545 550 555 560Glu
Pro Thr Thr Ser Asn Ser Thr Thr Ser Glu Pro Thr Asn Ser Ile 565 570
575Asn Arg Lys Thr Ser Glu Ile Ser Phe Leu Ser Asp Trp Phe His Lys
580 585 590Ile Arg Thr Arg Phe Asn Ile Phe His His His His His His
595 600 60541599PRTArtificial sequenceIMAT_PURE Cat from I7HDR2 Zen
1 protein Dermatophagoides farinae (American house dust mite) 41Tyr
Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Met Glu Asp Gln Arg1 5 10
15Asp Leu Ile Ser Asn His Glu Gln Leu Pro Ile Leu Gly Gln Arg Pro
20 25 30Ala Ala Pro Glu Ser Lys Ser Ser Arg Gly Ala Leu Tyr Thr Gly
Phe 35 40 45Ser Val Leu Val Ala Leu Leu Leu Ala Gly Gln Ala Thr Thr
Ala Tyr 50 55 60Phe Leu Tyr Gln Gln Gln Gly Arg Leu Asp Lys Leu Thr
Val Thr Ala65 70 75 80Gln Asn Leu Gln Leu Glu Asn Leu Arg Met Lys
Leu Pro Lys Pro Ala 85 90 95Lys Pro Leu Asn Lys Leu Arg Val Ala Thr
Pro Met Leu Met Gln Thr 100 105 110Met Pro Val Arg Gly Leu Leu Gln
Ala Asn Pro Arg Phe Lys Arg Asp 115 120 125Asn Arg Asp Asp Val Leu
Lys Gln Thr Glu Glu Leu Ile Lys Ser Ala 130 135 140Gln Asp Val Leu
Glu Lys Leu Pro Asp Ser Asp Leu Lys Asp Glu Ile145 150 155 160Ala
Glu Lys Leu Ala Thr Met Lys His Tyr Lys His Glu Leu Glu Asn 165 170
175Ala Lys Asn Pro Ile Lys Ile Ala His Phe Glu Leu Glu Leu Leu Thr
180 185 190Met Phe Lys Lys Phe Gln Ser Leu Leu Asn Glu Ala Asn Glu
Ile Ile 195 200 205Lys Ser Leu Thr Thr Thr Thr Thr Glu Pro Thr Thr
Pro Thr Pro Glu 210 215 220Pro Thr Thr Thr Thr Pro Glu Pro Thr Thr
Lys Thr Pro Glu Pro Thr225 230 235 240Thr Lys Thr Pro Glu Pro Thr
Thr Pro Thr Pro Glu Pro Thr Thr Lys 245 250 255Thr Pro Glu Pro Thr
Thr Lys Thr Pro Glu Pro Thr Thr Pro Thr Pro 260 265 270Glu Pro Thr
Thr Lys Thr Pro Glu Pro Thr Thr Pro Thr Pro Glu Pro 275 280 285Thr
Thr Lys Thr Pro Glu Pro Thr Thr Lys Thr Pro Glu Pro Ser Thr 290 295
300Pro Thr Pro Glu Pro Thr Thr Lys Thr Pro Glu Pro Thr Thr Lys
Thr305 310 315 320Pro Glu Pro Ser Thr Pro Thr Pro Glu Pro Thr Thr
Lys Thr Pro Glu 325 330 335Pro Ser Thr Pro Thr Pro Glu Pro Thr Thr
Lys Thr Pro Glu Pro Thr 340 345 350Thr Lys Thr Pro Glu Pro Ser Thr
Pro Thr Pro Glu Pro Thr Thr Lys 355 360 365Thr Pro Glu Pro Ser Thr
Pro Thr Pro Glu Pro Thr Thr Lys Thr Pro 370 375 380Glu Pro Ser Thr
Thr Lys Lys Pro Asn Arg Asp Asp Val Leu Lys Gln385 390 395 400Ala
Glu Glu Leu Ile Lys Arg Ala Glu Asp Val Phe Glu Lys Leu Pro 405 410
415Asp Ser Asp Leu Lys Asn Glu Ile Ala Glu Lys Leu Ala Thr Met Lys
420 425 430Asn Tyr Lys His Glu Leu Glu Asn Ala Lys Asn Pro Ile Lys
Ile Ala 435 440 445His Leu Glu Ser Glu Leu Leu Thr Met Phe Lys Met
Phe Gln Ser Leu 450 455 460Leu Asn Glu Ala Asp Glu Ile Ile Arg Ser
Leu Thr Thr Thr Thr Glu465 470 475 480Pro Thr Thr Leu Asn Ser Thr
Thr Pro Glu Pro Thr Thr Leu Asn Ser 485 490 495Thr Thr Pro Glu Pro
Thr Thr Leu Asn Ser Thr Thr Pro Glu Pro Thr 500 505 510Thr Leu Asn
Ser Thr Thr Pro Glu Pro Thr Thr Leu Asn Ser Thr Thr 515 520 525Pro
Glu Pro Thr
Thr Leu Asn Ser Thr Thr Pro Glu Pro Thr Thr Leu 530 535 540Asn Ser
Thr Thr Pro Glu Pro Thr Thr Leu Asn Ser Thr Thr Pro Glu545 550 555
560Pro Thr Thr Ser Asn Ser Thr Thr Ser Glu Pro Thr Asn Ser Ile Asn
565 570 575Arg Lys Thr Ser Glu Ile Ser Phe Leu Ser Asp Trp Phe His
Lys Ile 580 585 590Arg Thr Arg Phe Asn Ile Phe
59542286PRTArtificial sequenceIMAT_NTERM Cat from Q94424 Salivary
antigen 1 (FS-I) (allergen Cte f 1) Ctenocephalides felis (Cat
flea) 42Met His His His His His His Tyr Gly Arg Lys Lys Arg Arg Gln
Arg1 5 10 15Arg Arg Met Glu Asp Gln Arg Asp Leu Ile Ser Asn His Glu
Gln Leu 20 25 30Pro Ile Leu Gly Gln Arg Pro Ala Ala Pro Glu Ser Lys
Arg Ser Arg 35 40 45Gly Ala Leu Tyr Thr Gly Phe Ser Val Leu Val Ala
Leu Leu Leu Ala 50 55 60Gly Gln Ala Thr Thr Ala Tyr Phe Leu Tyr Gln
Gln Gln Gly Arg Leu65 70 75 80Asp Lys Leu Thr Val Thr Ala Gln Asn
Leu Gln Leu Glu Asn Leu Arg 85 90 95Met Lys Leu Pro Lys Pro Ala Lys
Pro Leu Asn Lys Leu Arg Val Ala 100 105 110Thr Pro Met Leu Met Gln
Thr Met Pro Val Arg Gly Leu Leu Gln Ala 115 120 125Glu Asp Ile Trp
Lys Val Asn Lys Lys Arg Thr Ser Gly Gly Lys Asn 130 135 140Gln Asp
Arg Lys Leu Asp Gln Ile Ile Gln Lys Gly Gln Gln Val Lys145 150 155
160Ile Gln Asn Ile Leu Lys Leu Ile Arg Asp Lys Pro His Thr Asn Gln
165 170 175Glu Lys Glu Lys Leu Met Lys Phe Leu Lys Lys Val Leu Lys
Gly Tyr 180 185 190Arg Gly Ala Arg Asp Gly Asn Ile Leu Tyr Leu Ser
Arg Pro Ser Asn 195 200 205Leu Gly Pro Asp Trp Lys Val Ser Lys Glu
Arg Lys Asp Pro Asn Asn 210 215 220Lys Asp Ser Arg Pro Thr Glu Ile
Val Pro Tyr Arg Gln Gln Leu Ala225 230 235 240Ile Pro Asn Ile Leu
Lys Leu Lys Asn Ser Glu Thr Asn Glu Asp Ser 245 250 255Lys Leu Lys
Lys His Met Lys Glu Lys Ser Arg Gly Gly Asn Asp Ala 260 265 270Gly
Ile Asp Gly Asn Phe Leu Tyr Leu Arg Pro Lys Asn Lys 275 280
28543286PRTArtificial sequenceIMAT_CTERM Cat from Q94424 Salivary
antigen 1 (FS-I) (allergen Cte f 1) Ctenocephalides felis (Cat
flea) 43Met Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Met Glu Asp
Gln1 5 10 15Arg Asp Leu Ile Ser Asn His Glu Gln Leu Pro Ile Leu Gly
Gln Arg 20 25 30Pro Ala Ala Pro Glu Ser Lys Arg Ser Arg Gly Ala Leu
Tyr Thr Gly 35 40 45Phe Ser Val Leu Val Ala Leu Leu Leu Ala Gly Gln
Ala Thr Thr Ala 50 55 60Tyr Phe Leu Tyr Gln Gln Gln Gly Arg Leu Asp
Lys Leu Thr Val Thr65 70 75 80Ala Gln Asn Leu Gln Leu Glu Asn Leu
Arg Met Lys Leu Pro Lys Pro 85 90 95Ala Lys Pro Leu Asn Lys Leu Arg
Val Ala Thr Pro Met Leu Met Gln 100 105 110Thr Met Pro Val Arg Gly
Leu Leu Gln Ala Glu Asp Ile Trp Lys Val 115 120 125Asn Lys Lys Arg
Thr Ser Gly Gly Lys Asn Gln Asp Arg Lys Leu Asp 130 135 140Gln Ile
Ile Gln Lys Gly Gln Gln Val Lys Ile Gln Asn Ile Leu Lys145 150 155
160Leu Ile Arg Asp Lys Pro His Thr Asn Gln Glu Lys Glu Lys Leu Met
165 170 175Lys Phe Leu Lys Lys Val Leu Lys Gly Tyr Arg Gly Ala Arg
Asp Gly 180 185 190Asn Ile Leu Tyr Leu Ser Arg Pro Ser Asn Leu Gly
Pro Asp Trp Lys 195 200 205Val Ser Lys Glu Arg Lys Asp Pro Asn Asn
Lys Asp Ser Arg Pro Thr 210 215 220Glu Ile Val Pro Tyr Arg Gln Gln
Leu Ala Ile Pro Asn Ile Leu Lys225 230 235 240Leu Lys Asn Ser Glu
Thr Asn Glu Asp Ser Lys Leu Lys Lys His Met 245 250 255Lys Glu Lys
Ser Arg Gly Gly Asn Asp Ala Gly Ile Asp Gly Asn Phe 260 265 270Leu
Tyr Leu Arg Pro Lys Asn Lys His His His His His His 275 280
28544279PRTArtificial sequenceIMAT_PURE Cat from Q94424 Salivary
antigen 1 (FS-I) (allergen Cte f 1) Ctenocephalides felis (Cat
flea) 44Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Met Glu Asp Gln
Arg1 5 10 15Asp Leu Ile Ser Asn His Glu Gln Leu Pro Ile Leu Gly Gln
Arg Pro 20 25 30Ala Ala Pro Glu Ser Lys Arg Ser Arg Gly Ala Leu Tyr
Thr Gly Phe 35 40 45Ser Val Leu Val Ala Leu Leu Leu Ala Gly Gln Ala
Thr Thr Ala Tyr 50 55 60Phe Leu Tyr Gln Gln Gln Gly Arg Leu Asp Lys
Leu Thr Val Thr Ala65 70 75 80Gln Asn Leu Gln Leu Glu Asn Leu Arg
Met Lys Leu Pro Lys Pro Ala 85 90 95Lys Pro Leu Asn Lys Leu Arg Val
Ala Thr Pro Met Leu Met Gln Thr 100 105 110Met Pro Val Arg Gly Leu
Leu Gln Ala Glu Asp Ile Trp Lys Val Asn 115 120 125Lys Lys Arg Thr
Ser Gly Gly Lys Asn Gln Asp Arg Lys Leu Asp Gln 130 135 140Ile Ile
Gln Lys Gly Gln Gln Val Lys Ile Gln Asn Ile Leu Lys Leu145 150 155
160Ile Arg Asp Lys Pro His Thr Asn Gln Glu Lys Glu Lys Leu Met Lys
165 170 175Phe Leu Lys Lys Val Leu Lys Gly Tyr Arg Gly Ala Arg Asp
Gly Asn 180 185 190Ile Leu Tyr Leu Ser Arg Pro Ser Asn Leu Gly Pro
Asp Trp Lys Val 195 200 205Ser Lys Glu Arg Lys Asp Pro Asn Asn Lys
Asp Ser Arg Pro Thr Glu 210 215 220Ile Val Pro Tyr Arg Gln Gln Leu
Ala Ile Pro Asn Ile Leu Lys Leu225 230 235 240Lys Asn Ser Glu Thr
Asn Glu Asp Ser Lys Leu Lys Lys His Met Lys 245 250 255Glu Lys Ser
Arg Gly Gly Asn Asp Ala Gly Ile Asp Gly Asn Phe Leu 260 265 270Tyr
Leu Arg Pro Lys Asn Lys 27545351PRTArtificial sequenceIMAT_NTERM
Dog from Q58A71 Der f 1 allergen preproenzyme Dermatophagoides
farinae (American house dust mite) 45Met His His His His His His
Tyr Gly Arg Lys Lys Arg Arg Gln Arg1 5 10 15Arg Arg Met Glu Asp Gln
Arg Asp Leu Ile Ser Asn His Glu Gln Leu 20 25 30Pro Ile Leu Gly Gln
Arg Pro Gly Ala Pro Glu Ser Lys Ser Ser Arg 35 40 45Gly Ala Leu Tyr
Thr Gly Phe Ser Val Leu Val Ala Leu Leu Leu Ala 50 55 60Gly Gln Ala
Thr Thr Ala Tyr Phe Leu Tyr Gln Gln Gln Gly Arg Leu65 70 75 80Asp
Lys Leu Thr Val Thr Ser Gln Asn Leu Gln Leu Glu Ser Leu Arg 85 90
95Met Lys Leu Pro Lys Pro Pro Lys Pro Leu Ser Lys Met Arg Val Ala
100 105 110Thr Pro Met Met Met Gln Ala Leu Pro Ile Gln Ser Leu Pro
Gln Gly 115 120 125Thr Ser Ala Ile Arg Ile Asn Ser Val Asn Val Pro
Ser Glu Leu Asp 130 135 140Leu Arg Ser Leu Arg Thr Val Thr Pro Ile
Arg Met Gln Gly Gly Ile145 150 155 160Gly Ser Leu Trp Ala Phe Ser
Gly Val Ala Ala Thr Glu Ser Ala Tyr 165 170 175Leu Ala Tyr Arg Asn
Thr Ser Leu Asp Leu Ser Glu Gln Glu Leu Val 180 185 190Asp Arg Ala
Ser Gln His Gly Arg His Gly Asp Thr Ile Pro Arg Gly 195 200 205Ile
Glu Tyr Ile Gln Gln Asn Gly Val Val Glu Glu Arg Ser Tyr Pro 210 215
220Tyr Val Ala Arg Glu Gln Gln Ser Arg Arg Pro Asn Ser Gln His
Tyr225 230 235 240Gly Ile Ser Asn Tyr Ile Gln Ile Tyr Pro Pro Asp
Val Lys Gln Ile 245 250 255Arg Glu Ala Leu Thr Gln Thr His Thr Ala
Ile Ala Val Ile Ile Gly 260 265 270Ile Lys Asp Leu Arg Ala Phe Gln
His Tyr Asp Gly Arg Thr Ile Ile 275 280 285Gln His Asp Asn Gly Tyr
Gln Pro Asn Tyr His Ala Val Asn Ile Val 290 295 300Gly Tyr Gly Ser
Thr Gln Gly Val Asp Tyr Trp Ile Val Arg Asn Ser305 310 315 320Trp
Asp Thr Thr Trp Gly Asp Ser Gly Tyr Gly Tyr Phe Gln Ala Gly 325 330
335Asn Asn Leu Met Met Ile Glu Gln Tyr Pro Tyr Val Val Ile Met 340
345 35046351PRTArtificial sequenceIMAT_CTERM Dog from Q58A71 Der f
1 allergen preproenzyme Dermatophagoides farinae (American house
dust mite) 46Met Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Met
Glu Asp Gln1 5 10 15Arg Asp Leu Ile Ser Asn His Glu Gln Leu Pro Ile
Leu Gly Gln Arg 20 25 30Pro Gly Ala Pro Glu Ser Lys Ser Ser Arg Gly
Ala Leu Tyr Thr Gly 35 40 45Phe Ser Val Leu Val Ala Leu Leu Leu Ala
Gly Gln Ala Thr Thr Ala 50 55 60Tyr Phe Leu Tyr Gln Gln Gln Gly Arg
Leu Asp Lys Leu Thr Val Thr65 70 75 80Ser Gln Asn Leu Gln Leu Glu
Ser Leu Arg Met Lys Leu Pro Lys Pro 85 90 95Pro Lys Pro Leu Ser Lys
Met Arg Val Ala Thr Pro Met Met Met Gln 100 105 110Ala Leu Pro Ile
Gln Ser Leu Pro Gln Gly Thr Ser Ala Ile Arg Ile 115 120 125Asn Ser
Val Asn Val Pro Ser Glu Leu Asp Leu Arg Ser Leu Arg Thr 130 135
140Val Thr Pro Ile Arg Met Gln Gly Gly Ile Gly Ser Leu Trp Ala
Phe145 150 155 160Ser Gly Val Ala Ala Thr Glu Ser Ala Tyr Leu Ala
Tyr Arg Asn Thr 165 170 175Ser Leu Asp Leu Ser Glu Gln Glu Leu Val
Asp Arg Ala Ser Gln His 180 185 190Gly Arg His Gly Asp Thr Ile Pro
Arg Gly Ile Glu Tyr Ile Gln Gln 195 200 205Asn Gly Val Val Glu Glu
Arg Ser Tyr Pro Tyr Val Ala Arg Glu Gln 210 215 220Gln Ser Arg Arg
Pro Asn Ser Gln His Tyr Gly Ile Ser Asn Tyr Ile225 230 235 240Gln
Ile Tyr Pro Pro Asp Val Lys Gln Ile Arg Glu Ala Leu Thr Gln 245 250
255Thr His Thr Ala Ile Ala Val Ile Ile Gly Ile Lys Asp Leu Arg Ala
260 265 270Phe Gln His Tyr Asp Gly Arg Thr Ile Ile Gln His Asp Asn
Gly Tyr 275 280 285Gln Pro Asn Tyr His Ala Val Asn Ile Val Gly Tyr
Gly Ser Thr Gln 290 295 300Gly Val Asp Tyr Trp Ile Val Arg Asn Ser
Trp Asp Thr Thr Trp Gly305 310 315 320Asp Ser Gly Tyr Gly Tyr Phe
Gln Ala Gly Asn Asn Leu Met Met Ile 325 330 335Glu Gln Tyr Pro Tyr
Val Val Ile Met His His His His His His 340 345
35047344PRTArtificial sequenceIMAT_PURE Dog from Q58A71 Der f 1
allergen preproenzyme Dermatophagoides farinae (American house dust
mite) 47Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Met Glu Asp Gln
Arg1 5 10 15Asp Leu Ile Ser Asn His Glu Gln Leu Pro Ile Leu Gly Gln
Arg Pro 20 25 30Gly Ala Pro Glu Ser Lys Ser Ser Arg Gly Ala Leu Tyr
Thr Gly Phe 35 40 45Ser Val Leu Val Ala Leu Leu Leu Ala Gly Gln Ala
Thr Thr Ala Tyr 50 55 60Phe Leu Tyr Gln Gln Gln Gly Arg Leu Asp Lys
Leu Thr Val Thr Ser65 70 75 80Gln Asn Leu Gln Leu Glu Ser Leu Arg
Met Lys Leu Pro Lys Pro Pro 85 90 95Lys Pro Leu Ser Lys Met Arg Val
Ala Thr Pro Met Met Met Gln Ala 100 105 110Leu Pro Ile Gln Ser Leu
Pro Gln Gly Thr Ser Ala Ile Arg Ile Asn 115 120 125Ser Val Asn Val
Pro Ser Glu Leu Asp Leu Arg Ser Leu Arg Thr Val 130 135 140Thr Pro
Ile Arg Met Gln Gly Gly Ile Gly Ser Leu Trp Ala Phe Ser145 150 155
160Gly Val Ala Ala Thr Glu Ser Ala Tyr Leu Ala Tyr Arg Asn Thr Ser
165 170 175Leu Asp Leu Ser Glu Gln Glu Leu Val Asp Arg Ala Ser Gln
His Gly 180 185 190Arg His Gly Asp Thr Ile Pro Arg Gly Ile Glu Tyr
Ile Gln Gln Asn 195 200 205Gly Val Val Glu Glu Arg Ser Tyr Pro Tyr
Val Ala Arg Glu Gln Gln 210 215 220Ser Arg Arg Pro Asn Ser Gln His
Tyr Gly Ile Ser Asn Tyr Ile Gln225 230 235 240Ile Tyr Pro Pro Asp
Val Lys Gln Ile Arg Glu Ala Leu Thr Gln Thr 245 250 255His Thr Ala
Ile Ala Val Ile Ile Gly Ile Lys Asp Leu Arg Ala Phe 260 265 270Gln
His Tyr Asp Gly Arg Thr Ile Ile Gln His Asp Asn Gly Tyr Gln 275 280
285Pro Asn Tyr His Ala Val Asn Ile Val Gly Tyr Gly Ser Thr Gln Gly
290 295 300Val Asp Tyr Trp Ile Val Arg Asn Ser Trp Asp Thr Thr Trp
Gly Asp305 310 315 320Ser Gly Tyr Gly Tyr Phe Gln Ala Gly Asn Asn
Leu Met Met Ile Glu 325 330 335Gln Tyr Pro Tyr Val Val Ile Met
34048257PRTArtificial sequenceIMAT_NTERM Dog from Q00855 Mite group
2 allergen Der f 2 (Allergen Der f II) (allergen Der f 2)
Dermatophagoides farinae (American house dust mite) 48Met His His
His His His His Tyr Gly Arg Lys Lys Arg Arg Gln Arg1 5 10 15Arg Arg
Met Glu Asp Gln Arg Asp Leu Ile Ser Asn His Glu Gln Leu 20 25 30Pro
Ile Leu Gly Gln Arg Pro Gly Ala Pro Glu Ser Lys Ser Ser Arg 35 40
45Gly Ala Leu Tyr Thr Gly Phe Ser Val Leu Val Ala Leu Leu Leu Ala
50 55 60Gly Gln Ala Thr Thr Ala Tyr Phe Leu Tyr Gln Gln Gln Gly Arg
Leu65 70 75 80Asp Lys Leu Thr Val Thr Ser Gln Asn Leu Gln Leu Glu
Ser Leu Arg 85 90 95Met Lys Leu Pro Lys Pro Pro Lys Pro Leu Ser Lys
Met Arg Val Ala 100 105 110Thr Pro Met Met Met Gln Ala Leu Pro Ile
Gln Ser Leu Pro Gln Gly 115 120 125Asp Gln Val Asp Val Lys Asp Ser
Ala Asn Asn Glu Ile Lys Lys Val 130 135 140Met Val Asp Gly Ile His
Gly Ser Asp Pro Leu Ile Ile His Arg Gly145 150 155 160Lys Pro Phe
Thr Leu Glu Ala Leu Phe Asp Ala Asn Gln Asn Thr Lys 165 170 175Thr
Ala Lys Ile Glu Ile Lys Ala Ser Leu Asp Gly Leu Glu Ile Asp 180 185
190Val Pro Gly Ile Asp Thr Asn Ala Ile His Phe Met Lys Leu Pro Leu
195 200 205Val Lys Gly Gln Gln Tyr Asp Ile Lys Tyr Thr Trp Asn Val
Pro Lys 210 215 220Ile Ala Pro Lys Ser Glu Asn Val Val Val Thr Val
Lys Leu Ile Gly225 230 235 240Asp Asn Gly Val Leu Ala Leu Ala Ile
Ala Thr His Gly Lys Ile Arg 245 250 255Asp49257PRTArtificial
sequenceIMAT_CTERM Dog from Q00855 Mite group 2 allergen Der f 2
(Allergen Der f II) (allergen Der f 2) Dermatophagoides farinae
(American house dust mite) 49Met Tyr Gly Arg Lys Lys Arg Arg Gln
Arg Arg Arg Met Glu Asp Gln1 5 10 15Arg Asp Leu Ile Ser Asn His Glu
Gln Leu Pro Ile Leu Gly Gln Arg 20 25 30Pro Gly Ala Pro Glu Ser Lys
Ser Ser Arg Gly Ala Leu Tyr Thr Gly 35 40 45Phe Ser Val Leu Val Ala
Leu Leu Leu Ala Gly Gln Ala Thr Thr Ala 50 55 60Tyr Phe Leu Tyr Gln
Gln Gln Gly Arg Leu Asp Lys Leu Thr Val Thr65 70 75 80Ser Gln Asn
Leu Gln Leu Glu Ser Leu Arg Met
Lys Leu Pro Lys Pro 85 90 95Pro Lys Pro Leu Ser Lys Met Arg Val Ala
Thr Pro Met Met Met Gln 100 105 110Ala Leu Pro Ile Gln Ser Leu Pro
Gln Gly Asp Gln Val Asp Val Lys 115 120 125Asp Ser Ala Asn Asn Glu
Ile Lys Lys Val Met Val Asp Gly Ile His 130 135 140Gly Ser Asp Pro
Leu Ile Ile His Arg Gly Lys Pro Phe Thr Leu Glu145 150 155 160Ala
Leu Phe Asp Ala Asn Gln Asn Thr Lys Thr Ala Lys Ile Glu Ile 165 170
175Lys Ala Ser Leu Asp Gly Leu Glu Ile Asp Val Pro Gly Ile Asp Thr
180 185 190Asn Ala Ile His Phe Met Lys Leu Pro Leu Val Lys Gly Gln
Gln Tyr 195 200 205Asp Ile Lys Tyr Thr Trp Asn Val Pro Lys Ile Ala
Pro Lys Ser Glu 210 215 220Asn Val Val Val Thr Val Lys Leu Ile Gly
Asp Asn Gly Val Leu Ala225 230 235 240Leu Ala Ile Ala Thr His Gly
Lys Ile Arg Asp His His His His His 245 250
255His50250PRTArtificial sequenceIMAT_PURE Dog from Q00855 Mite
group 2 allergen Der f 2 (Allergen Der f II) (allergen Der f 2)
Dermatophagoides farinae (American house dust mite) 50Tyr Gly Arg
Lys Lys Arg Arg Gln Arg Arg Arg Met Glu Asp Gln Arg1 5 10 15Asp Leu
Ile Ser Asn His Glu Gln Leu Pro Ile Leu Gly Gln Arg Pro 20 25 30Gly
Ala Pro Glu Ser Lys Ser Ser Arg Gly Ala Leu Tyr Thr Gly Phe 35 40
45Ser Val Leu Val Ala Leu Leu Leu Ala Gly Gln Ala Thr Thr Ala Tyr
50 55 60Phe Leu Tyr Gln Gln Gln Gly Arg Leu Asp Lys Leu Thr Val Thr
Ser65 70 75 80Gln Asn Leu Gln Leu Glu Ser Leu Arg Met Lys Leu Pro
Lys Pro Pro 85 90 95Lys Pro Leu Ser Lys Met Arg Val Ala Thr Pro Met
Met Met Gln Ala 100 105 110Leu Pro Ile Gln Ser Leu Pro Gln Gly Asp
Gln Val Asp Val Lys Asp 115 120 125Ser Ala Asn Asn Glu Ile Lys Lys
Val Met Val Asp Gly Ile His Gly 130 135 140Ser Asp Pro Leu Ile Ile
His Arg Gly Lys Pro Phe Thr Leu Glu Ala145 150 155 160Leu Phe Asp
Ala Asn Gln Asn Thr Lys Thr Ala Lys Ile Glu Ile Lys 165 170 175Ala
Ser Leu Asp Gly Leu Glu Ile Asp Val Pro Gly Ile Asp Thr Asn 180 185
190Ala Ile His Phe Met Lys Leu Pro Leu Val Lys Gly Gln Gln Tyr Asp
195 200 205Ile Lys Tyr Thr Trp Asn Val Pro Lys Ile Ala Pro Lys Ser
Glu Asn 210 215 220Val Val Val Thr Val Lys Leu Ile Gly Asp Asn Gly
Val Leu Ala Leu225 230 235 240Ala Ile Ala Thr His Gly Lys Ile Arg
Asp 245 25051198PRTArtificial sequenceIMAT_NTERM Dog from
A0A088SAW7 Der f 23 allergen Dermatophagoides farinae (American
house dust mite) 51Met His His His His His His Tyr Gly Arg Lys Lys
Arg Arg Gln Arg1 5 10 15Arg Arg Met Glu Asp Gln Arg Asp Leu Ile Ser
Asn His Glu Gln Leu 20 25 30Pro Ile Leu Gly Gln Arg Pro Gly Ala Pro
Glu Ser Lys Glu Ser Arg 35 40 45Gly Ala Leu Tyr Thr Gly Phe Ser Val
Leu Val Ala Leu Leu Leu Ala 50 55 60Gly Gln Ala Thr Thr Ala Tyr Phe
Leu Tyr Gln Gln Gln Gly Arg Leu65 70 75 80Asp Lys Leu Thr Val Thr
Ser Gln Asn Leu Gln Leu Glu Ser Leu Arg 85 90 95Met Lys Leu Pro Lys
Pro Pro Lys Pro Leu Ser Lys Met Arg Val Ala 100 105 110Thr Pro Met
Met Met Gln Ala Leu Pro Ile Gln Ser Leu Pro Gln Gly 115 120 125Asp
Ile Asp His Asp Asp Asp Pro Thr Thr Met Ile Asp Val Gln Thr 130 135
140Thr Thr Val Gln Pro Ser Asp Glu Phe Glu Leu Pro Thr Arg Phe
Gly145 150 155 160Tyr Phe Ala Asp Pro Lys Asp Pro Leu Lys Phe Tyr
Ile Ile Ser Asn 165 170 175Trp Glu Ala Ile His Lys Ser Leu Pro Gly
Asn Thr Arg Trp Asn Glu 180 185 190Lys Glu Leu Thr Ile Thr
19552198PRTArtificial sequenceIMAT_CTERM Dog from A0A088SAW7 Der f
23 allergen Dermatophagoides farinae (American house dust mite)
52Met Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Met Glu Asp Gln1
5 10 15Arg Asp Leu Ile Ser Asn His Glu Gln Leu Pro Ile Leu Gly Gln
Arg 20 25 30Pro Gly Ala Pro Glu Ser Lys Glu Ser Arg Gly Ala Leu Tyr
Thr Gly 35 40 45Phe Ser Val Leu Val Ala Leu Leu Leu Ala Gly Gln Ala
Thr Thr Ala 50 55 60Tyr Phe Leu Tyr Gln Gln Gln Gly Arg Leu Asp Lys
Leu Thr Val Thr65 70 75 80Ser Gln Asn Leu Gln Leu Glu Ser Leu Arg
Met Lys Leu Pro Lys Pro 85 90 95Pro Lys Pro Leu Ser Lys Met Arg Val
Ala Thr Pro Met Met Met Gln 100 105 110Ala Leu Pro Ile Gln Ser Leu
Pro Gln Gly Asp Ile Asp His Asp Asp 115 120 125Asp Pro Thr Thr Met
Ile Asp Val Gln Thr Thr Thr Val Gln Pro Ser 130 135 140Asp Glu Phe
Glu Leu Pro Thr Arg Phe Gly Tyr Phe Ala Asp Pro Lys145 150 155
160Asp Pro Leu Lys Phe Tyr Ile Ile Ser Asn Trp Glu Ala Ile His Lys
165 170 175Ser Leu Pro Gly Asn Thr Arg Trp Asn Glu Lys Glu Leu Thr
Ile Thr 180 185 190His His His His His His 19553191PRTArtificial
sequenceIMAT_PURE Dog from A0A088SAW7 Der f 23 allergen
Dermatophagoides farinae (American house dust mite) 53Tyr Gly Arg
Lys Lys Arg Arg Gln Arg Arg Arg Met Glu Asp Gln Arg1 5 10 15Asp Leu
Ile Ser Asn His Glu Gln Leu Pro Ile Leu Gly Gln Arg Pro 20 25 30Gly
Ala Pro Glu Ser Lys Glu Ser Arg Gly Ala Leu Tyr Thr Gly Phe 35 40
45Ser Val Leu Val Ala Leu Leu Leu Ala Gly Gln Ala Thr Thr Ala Tyr
50 55 60Phe Leu Tyr Gln Gln Gln Gly Arg Leu Asp Lys Leu Thr Val Thr
Ser65 70 75 80Gln Asn Leu Gln Leu Glu Ser Leu Arg Met Lys Leu Pro
Lys Pro Pro 85 90 95Lys Pro Leu Ser Lys Met Arg Val Ala Thr Pro Met
Met Met Gln Ala 100 105 110Leu Pro Ile Gln Ser Leu Pro Gln Gly Asp
Ile Asp His Asp Asp Asp 115 120 125Pro Thr Thr Met Ile Asp Val Gln
Thr Thr Thr Val Gln Pro Ser Asp 130 135 140Glu Phe Glu Leu Pro Thr
Arg Phe Gly Tyr Phe Ala Asp Pro Lys Asp145 150 155 160Pro Leu Lys
Phe Tyr Ile Ile Ser Asn Trp Glu Ala Ile His Lys Ser 165 170 175Leu
Pro Gly Asn Thr Arg Trp Asn Glu Lys Glu Leu Thr Ile Thr 180 185
19054573PRTArtificial sequenceIMAT_NTERM Dog from Q86R84 60 kDa
allergen Der f 18p Dermatophagoides farinae (American house dust
mite) 54Met His His His His His His Tyr Gly Arg Lys Lys Arg Arg Gln
Arg1 5 10 15Arg Arg Met Glu Asp Gln Arg Asp Leu Ile Ser Asn His Glu
Gln Leu 20 25 30Pro Ile Leu Gly Gln Arg Pro Gly Ala Pro Glu Ser Lys
Ser Ser Arg 35 40 45Gly Ala Leu Tyr Thr Gly Phe Ser Val Leu Val Ala
Leu Leu Leu Ala 50 55 60Gly Gln Ala Thr Thr Ala Tyr Phe Leu Tyr Gln
Gln Gln Gly Arg Leu65 70 75 80Asp Lys Leu Thr Val Thr Ser Gln Asn
Leu Gln Leu Glu Ser Leu Arg 85 90 95Met Lys Leu Pro Lys Pro Pro Lys
Pro Leu Ser Lys Met Arg Val Ala 100 105 110Thr Pro Met Met Met Gln
Ala Leu Pro Ile Gln Ser Leu Pro Gln Gly 115 120 125Ser Asn Ile Arg
Pro Asn Val Ala Thr Leu Glu Pro Lys Thr Val Leu 130 135 140Tyr Tyr
Glu Ser Trp Val His Trp Arg Gln Gly Glu Gly Lys Met Asp145 150 155
160Pro Glu Asp Ile Asp Thr Ser Leu Met Thr His Ile Val Tyr Ser Tyr
165 170 175Phe Gly Ile Asp Ala Ala Thr His Glu Ile Lys Leu Leu Asp
Glu Tyr 180 185 190Leu Met Lys Asp Leu His Asp Met Glu His Phe Thr
Gln His Lys Gly 195 200 205Asn Ala Lys Ala Met Ile Ala Val Gly Gly
Ser Thr Met Ser Asp Gln 210 215 220Phe Ser Lys Thr Ala Ala Val Glu
His Tyr Arg Glu Thr Phe Val Val225 230 235 240Ser Thr Val Asp Leu
Met Thr Arg Tyr Gly Phe Asp Gly Val Met Ile 245 250 255Asp Trp Ser
Gly Met Gln Ala Lys Asp Ser Asp Asn Phe Ile Lys Leu 260 265 270Leu
Asp Lys Phe Asp Glu Lys Phe Ala His Thr Ser Phe Val Met Gly 275 280
285Val Thr Leu Pro Ala Thr Ile Ala Ser Tyr Asp Asn Tyr Asn Ile Pro
290 295 300Ala Ile Ser Asn Tyr Val Asp Phe Met Asn Val Leu Ser Leu
Asp Tyr305 310 315 320Thr Gly Ser Trp Ala His Thr Val Gly His Ala
Ser Pro Phe Pro Glu 325 330 335Gln Leu Lys Thr Leu Glu Ala Tyr His
Lys Arg Gly Ala Pro Arg His 340 345 350Lys Met Val Met Ala Val Pro
Phe Tyr Ala Arg Thr Trp Ile Leu Glu 355 360 365Lys Met Asn Lys Gln
Asp Ile Gly Asp Lys Ala Ser Gly Pro Gly Pro 370 375 380Arg Gly Gln
Phe Thr Gln Thr Asp Gly Phe Leu Ser Tyr Asn Glu Leu385 390 395
400Ser Val Gln Ile Gln Ala Glu Thr Asn Ala Phe Thr Ile Thr Arg Asp
405 410 415His Asp Asn Thr Ala Ile Tyr Ala Val Tyr Val His Ser Asn
His Ala 420 425 430Glu Trp Ile Ser Phe Glu Asp Arg His Thr Leu Gly
Glu Lys Ala Lys 435 440 445Asn Ile Thr Gln Gln Gly Tyr Ala Gly Met
Ser Val Tyr Thr Leu Ser 450 455 460Asn Glu Asp Val His Gly Val Arg
Gly Asp Lys Asn Pro Leu Leu His465 470 475 480Ala Ile Gln Ser Asn
Tyr Tyr His Gly Val Val Thr Glu Pro Thr Val 485 490 495Val Thr Leu
Pro Pro Val Thr His Thr Thr Glu His Val Thr Asp Ile 500 505 510Pro
Gly Val Phe His Leu His Glu Glu Gly Phe Phe Arg Asp Lys Thr 515 520
525Tyr Ser Ala Thr Tyr Tyr Glu Ile Lys Lys Gly Asp Phe Gly Leu Glu
530 535 540Lys Thr Val His His Leu Ala Asn His Leu Gln Ala Phe Asp
Glu Val545 550 555 560Ser Arg Thr Arg Ile Asp His Thr Lys Ile Pro
Gly Ser 565 57055573PRTArtificial sequenceIMAT_CTERM Dog from
Q86R84 60 kDa allergen Der f 18p Dermatophagoides farinae (American
house dust mite) 55Met Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg
Met Glu Asp Gln1 5 10 15Arg Asp Leu Ile Ser Asn His Glu Gln Leu Pro
Ile Leu Gly Gln Arg 20 25 30Pro Gly Ala Pro Glu Ser Lys Ser Ser Arg
Gly Ala Leu Tyr Thr Gly 35 40 45Phe Ser Val Leu Val Ala Leu Leu Leu
Ala Gly Gln Ala Thr Thr Ala 50 55 60Tyr Phe Leu Tyr Gln Gln Gln Gly
Arg Leu Asp Lys Leu Thr Val Thr65 70 75 80Ser Gln Asn Leu Gln Leu
Glu Ser Leu Arg Met Lys Leu Pro Lys Pro 85 90 95Pro Lys Pro Leu Ser
Lys Met Arg Val Ala Thr Pro Met Met Met Gln 100 105 110Ala Leu Pro
Ile Gln Ser Leu Pro Gln Gly Ser Asn Ile Arg Pro Asn 115 120 125Val
Ala Thr Leu Glu Pro Lys Thr Val Leu Tyr Tyr Glu Ser Trp Val 130 135
140His Trp Arg Gln Gly Glu Gly Lys Met Asp Pro Glu Asp Ile Asp
Thr145 150 155 160Ser Leu Met Thr His Ile Val Tyr Ser Tyr Phe Gly
Ile Asp Ala Ala 165 170 175Thr His Glu Ile Lys Leu Leu Asp Glu Tyr
Leu Met Lys Asp Leu His 180 185 190Asp Met Glu His Phe Thr Gln His
Lys Gly Asn Ala Lys Ala Met Ile 195 200 205Ala Val Gly Gly Ser Thr
Met Ser Asp Gln Phe Ser Lys Thr Ala Ala 210 215 220Val Glu His Tyr
Arg Glu Thr Phe Val Val Ser Thr Val Asp Leu Met225 230 235 240Thr
Arg Tyr Gly Phe Asp Gly Val Met Ile Asp Trp Ser Gly Met Gln 245 250
255Ala Lys Asp Ser Asp Asn Phe Ile Lys Leu Leu Asp Lys Phe Asp Glu
260 265 270Lys Phe Ala His Thr Ser Phe Val Met Gly Val Thr Leu Pro
Ala Thr 275 280 285Ile Ala Ser Tyr Asp Asn Tyr Asn Ile Pro Ala Ile
Ser Asn Tyr Val 290 295 300Asp Phe Met Asn Val Leu Ser Leu Asp Tyr
Thr Gly Ser Trp Ala His305 310 315 320Thr Val Gly His Ala Ser Pro
Phe Pro Glu Gln Leu Lys Thr Leu Glu 325 330 335Ala Tyr His Lys Arg
Gly Ala Pro Arg His Lys Met Val Met Ala Val 340 345 350Pro Phe Tyr
Ala Arg Thr Trp Ile Leu Glu Lys Met Asn Lys Gln Asp 355 360 365Ile
Gly Asp Lys Ala Ser Gly Pro Gly Pro Arg Gly Gln Phe Thr Gln 370 375
380Thr Asp Gly Phe Leu Ser Tyr Asn Glu Leu Ser Val Gln Ile Gln
Ala385 390 395 400Glu Thr Asn Ala Phe Thr Ile Thr Arg Asp His Asp
Asn Thr Ala Ile 405 410 415Tyr Ala Val Tyr Val His Ser Asn His Ala
Glu Trp Ile Ser Phe Glu 420 425 430Asp Arg His Thr Leu Gly Glu Lys
Ala Lys Asn Ile Thr Gln Gln Gly 435 440 445Tyr Ala Gly Met Ser Val
Tyr Thr Leu Ser Asn Glu Asp Val His Gly 450 455 460Val Arg Gly Asp
Lys Asn Pro Leu Leu His Ala Ile Gln Ser Asn Tyr465 470 475 480Tyr
His Gly Val Val Thr Glu Pro Thr Val Val Thr Leu Pro Pro Val 485 490
495Thr His Thr Thr Glu His Val Thr Asp Ile Pro Gly Val Phe His Leu
500 505 510His Glu Glu Gly Phe Phe Arg Asp Lys Thr Tyr Ser Ala Thr
Tyr Tyr 515 520 525Glu Ile Lys Lys Gly Asp Phe Gly Leu Glu Lys Thr
Val His His Leu 530 535 540Ala Asn His Leu Gln Ala Phe Asp Glu Val
Ser Arg Thr Arg Ile Asp545 550 555 560His Thr Lys Ile Pro Gly Ser
His His His His His His 565 57056566PRTArtificial sequenceIMAT_PURE
Dog from Q86R84 60 kDa allergen Der f 18p Dermatophagoides farinae
(American house dust mite) 56Tyr Gly Arg Lys Lys Arg Arg Gln Arg
Arg Arg Met Glu Asp Gln Arg1 5 10 15Asp Leu Ile Ser Asn His Glu Gln
Leu Pro Ile Leu Gly Gln Arg Pro 20 25 30Gly Ala Pro Glu Ser Lys Ser
Ser Arg Gly Ala Leu Tyr Thr Gly Phe 35 40 45Ser Val Leu Val Ala Leu
Leu Leu Ala Gly Gln Ala Thr Thr Ala Tyr 50 55 60Phe Leu Tyr Gln Gln
Gln Gly Arg Leu Asp Lys Leu Thr Val Thr Ser65 70 75 80Gln Asn Leu
Gln Leu Glu Ser Leu Arg Met Lys Leu Pro Lys Pro Pro 85 90 95Lys Pro
Leu Ser Lys Met Arg Val Ala Thr Pro Met Met Met Gln Ala 100 105
110Leu Pro Ile Gln Ser Leu Pro Gln Gly Ser Asn Ile Arg Pro Asn Val
115 120 125Ala Thr Leu Glu Pro Lys Thr Val Leu Tyr Tyr Glu Ser Trp
Val His 130 135 140Trp Arg Gln Gly Glu Gly Lys Met Asp Pro Glu Asp
Ile Asp Thr Ser145 150 155 160Leu Met Thr His Ile Val Tyr Ser Tyr
Phe Gly Ile Asp Ala Ala Thr 165 170 175His Glu Ile Lys Leu Leu Asp
Glu Tyr Leu Met Lys Asp Leu His Asp
180 185 190Met Glu His Phe Thr Gln His Lys Gly Asn Ala Lys Ala Met
Ile Ala 195 200 205Val Gly Gly Ser Thr Met Ser Asp Gln Phe Ser Lys
Thr Ala Ala Val 210 215 220Glu His Tyr Arg Glu Thr Phe Val Val Ser
Thr Val Asp Leu Met Thr225 230 235 240Arg Tyr Gly Phe Asp Gly Val
Met Ile Asp Trp Ser Gly Met Gln Ala 245 250 255Lys Asp Ser Asp Asn
Phe Ile Lys Leu Leu Asp Lys Phe Asp Glu Lys 260 265 270Phe Ala His
Thr Ser Phe Val Met Gly Val Thr Leu Pro Ala Thr Ile 275 280 285Ala
Ser Tyr Asp Asn Tyr Asn Ile Pro Ala Ile Ser Asn Tyr Val Asp 290 295
300Phe Met Asn Val Leu Ser Leu Asp Tyr Thr Gly Ser Trp Ala His
Thr305 310 315 320Val Gly His Ala Ser Pro Phe Pro Glu Gln Leu Lys
Thr Leu Glu Ala 325 330 335Tyr His Lys Arg Gly Ala Pro Arg His Lys
Met Val Met Ala Val Pro 340 345 350Phe Tyr Ala Arg Thr Trp Ile Leu
Glu Lys Met Asn Lys Gln Asp Ile 355 360 365Gly Asp Lys Ala Ser Gly
Pro Gly Pro Arg Gly Gln Phe Thr Gln Thr 370 375 380Asp Gly Phe Leu
Ser Tyr Asn Glu Leu Ser Val Gln Ile Gln Ala Glu385 390 395 400Thr
Asn Ala Phe Thr Ile Thr Arg Asp His Asp Asn Thr Ala Ile Tyr 405 410
415Ala Val Tyr Val His Ser Asn His Ala Glu Trp Ile Ser Phe Glu Asp
420 425 430Arg His Thr Leu Gly Glu Lys Ala Lys Asn Ile Thr Gln Gln
Gly Tyr 435 440 445Ala Gly Met Ser Val Tyr Thr Leu Ser Asn Glu Asp
Val His Gly Val 450 455 460Arg Gly Asp Lys Asn Pro Leu Leu His Ala
Ile Gln Ser Asn Tyr Tyr465 470 475 480His Gly Val Val Thr Glu Pro
Thr Val Val Thr Leu Pro Pro Val Thr 485 490 495His Thr Thr Glu His
Val Thr Asp Ile Pro Gly Val Phe His Leu His 500 505 510Glu Glu Gly
Phe Phe Arg Asp Lys Thr Tyr Ser Ala Thr Tyr Tyr Glu 515 520 525Ile
Lys Lys Gly Asp Phe Gly Leu Glu Lys Thr Val His His Leu Ala 530 535
540Asn His Leu Gln Ala Phe Asp Glu Val Ser Arg Thr Arg Ile Asp
His545 550 555 560Thr Lys Ile Pro Gly Ser 56557664PRTArtificial
sequenceIMAT_NTERM Dog from Q9U6R7 98kDa HDM allergen (Der f 15
allergen) (Group 15 allergen Der f 15) Dermatophagoides farinae
(American house dust mite) 57Met His His His His His His Tyr Gly
Arg Lys Lys Arg Arg Gln Arg1 5 10 15Arg Arg Met Glu Asp Gln Arg Asp
Leu Ile Ser Asn His Glu Gln Leu 20 25 30Pro Ile Leu Gly Gln Arg Pro
Gly Ala Pro Glu Ser Lys Ser Ser Arg 35 40 45Gly Ala Leu Tyr Thr Gly
Phe Ser Val Leu Val Ala Leu Leu Leu Ala 50 55 60Gly Gln Ala Thr Thr
Ala Tyr Phe Leu Tyr Gln Gln Gln Gly Arg Leu65 70 75 80Asp Lys Leu
Thr Val Thr Ser Gln Asn Leu Gln Leu Glu Ser Leu Arg 85 90 95Met Lys
Leu Pro Lys Pro Pro Lys Pro Leu Ser Lys Met Arg Val Ala 100 105
110Thr Pro Met Met Met Gln Ala Leu Pro Ile Gln Ser Leu Pro Gln Gly
115 120 125Ser Ile Lys Arg Asp His Asn Asp Tyr Ser Lys Asn Pro Met
Arg Ile 130 135 140Val Leu Tyr Val Gly Thr Trp Ser Val Tyr His Lys
Val Asp Pro Tyr145 150 155 160Thr Ile Glu Asp Ile Asp Pro Phe Lys
Ser Thr His Leu Met Tyr Gly 165 170 175Phe Ala Lys Ile Asp Glu Tyr
Lys Tyr Thr Ile Gln Val Phe Asp Pro 180 185 190Tyr Gln Asp Asp Asn
His Asn Ser Trp Glu Lys Arg Gly Tyr Glu Arg 195 200 205Phe Asn Asn
Leu Arg Leu Lys Asn Pro Glu Leu Thr Thr Met Ile Ser 210 215 220Leu
Gly Gly Trp Tyr Glu Gly Ser Glu Lys Tyr Ser Asp Met Ala Ala225 230
235 240Asn Pro Thr Tyr Arg Gln Gln Phe Ile Gln Ser Val Leu Asp Phe
Leu 245 250 255Gln Glu Tyr Lys Phe Asp Gly Leu Asp Leu Asp Trp Glu
Tyr Pro Gly 260 265 270Ser Arg Leu Gly Asn Pro Lys Ile Asp Lys Gln
Asn Tyr Leu Ala Leu 275 280 285Val Arg Glu Leu Lys Asp Ala Phe Glu
Pro His Gly Tyr Leu Leu Thr 290 295 300Ala Ala Val Ser Pro Gly Lys
Asp Lys Ile Asp Arg Ala Tyr Asp Ile305 310 315 320Lys Glu Leu Asn
Lys Leu Phe Asp Trp Met Asn Val Met Thr Tyr Asp 325 330 335Tyr His
Gly Gly Trp Glu Asn Phe Tyr Gly His Asn Ala Pro Leu Tyr 340 345
350Lys Arg Pro Asp Glu Thr Asp Glu Leu His Thr Tyr Phe Asn Val Asn
355 360 365Tyr Thr Met His Tyr Tyr Leu Asn Asn Gly Ala Thr Arg Asp
Lys Leu 370 375 380Val Met Gly Val Pro Phe Tyr Gly Arg Ala Trp Ser
Ile Glu Asp Arg385 390 395 400Ser Lys Leu Lys Leu Gly Asp Pro Ala
Lys Gly Met Ser Pro Pro Gly 405 410 415Phe Ile Ser Gly Glu Glu Gly
Val Leu Ser Tyr Ile Glu Leu Ser Gln 420 425 430Leu Phe Gln Lys Glu
Glu Trp His Ile Gln Tyr Asp Glu Tyr Tyr Asn 435 440 445Ala Pro Tyr
Gly Tyr Asn Asp Lys Ile Trp Val Gly Tyr Asp Asp Leu 450 455 460Ala
Ser Ile Ser Ser Lys Leu Ala Phe Leu Lys Glu Leu Gly Val Ser465 470
475 480Gly Val Met Val Trp Ser Leu Glu Asn Asp Asp Phe Lys Gly His
Leu 485 490 495Gly Pro Lys Asn Pro Leu Leu Asn Lys Val His Asn Met
Ile Asn Gly 500 505 510Asp Glu Lys Asn Ser Phe Glu Ile Ile Leu Gly
Pro Ser Thr Thr Thr 515 520 525Pro Thr Pro Thr Thr Thr Pro Thr Thr
Pro Thr Thr Thr Pro Thr Thr 530 535 540Pro Ser Pro Thr Thr Pro Thr
Thr Thr Pro Ser Pro Thr Thr Pro Thr545 550 555 560Thr Thr Pro Ser
Pro Thr Thr Pro Thr Thr Thr Pro Ser Pro Thr Thr 565 570 575Pro Thr
Pro Thr Thr Pro Thr Pro Ala Pro Thr Thr Ser Thr Pro Ser 580 585
590Pro Thr Thr Thr Glu His Thr Ser Glu Thr Pro Lys Tyr Thr Thr Tyr
595 600 605Val Asp Gly His Leu Ile Lys Ser Tyr Lys Glu Gly Asp Ile
Pro His 610 615 620Pro Thr Asn Ile His Lys Tyr Leu Val Ile Glu Phe
Val Asn Gly Gly625 630 635 640Trp Trp Val His Ile Met Pro Ser Pro
Pro Gly Thr Ile Trp Ser Gln 645 650 655Glu Lys Leu Thr Ser Ile Gly
Glu 66058664PRTArtificial sequenceIMAT_CTERM Dog from Q9U6R7 98kDa
HDM allergen (Der f 15 allergen) (Group 15 allergen Der f 15)
Dermatophagoides farinae (American house dust mite) 58Met Tyr Gly
Arg Lys Lys Arg Arg Gln Arg Arg Arg Met Glu Asp Gln1 5 10 15Arg Asp
Leu Ile Ser Asn His Glu Gln Leu Pro Ile Leu Gly Gln Arg 20 25 30Pro
Gly Ala Pro Glu Ser Lys Ser Ser Arg Gly Ala Leu Tyr Thr Gly 35 40
45Phe Ser Val Leu Val Ala Leu Leu Leu Ala Gly Gln Ala Thr Thr Ala
50 55 60Tyr Phe Leu Tyr Gln Gln Gln Gly Arg Leu Asp Lys Leu Thr Val
Thr65 70 75 80Ser Gln Asn Leu Gln Leu Glu Ser Leu Arg Met Lys Leu
Pro Lys Pro 85 90 95Pro Lys Pro Leu Ser Lys Met Arg Val Ala Thr Pro
Met Met Met Gln 100 105 110Ala Leu Pro Ile Gln Ser Leu Pro Gln Gly
Ser Ile Lys Arg Asp His 115 120 125Asn Asp Tyr Ser Lys Asn Pro Met
Arg Ile Val Leu Tyr Val Gly Thr 130 135 140Trp Ser Val Tyr His Lys
Val Asp Pro Tyr Thr Ile Glu Asp Ile Asp145 150 155 160Pro Phe Lys
Ser Thr His Leu Met Tyr Gly Phe Ala Lys Ile Asp Glu 165 170 175Tyr
Lys Tyr Thr Ile Gln Val Phe Asp Pro Tyr Gln Asp Asp Asn His 180 185
190Asn Ser Trp Glu Lys Arg Gly Tyr Glu Arg Phe Asn Asn Leu Arg Leu
195 200 205Lys Asn Pro Glu Leu Thr Thr Met Ile Ser Leu Gly Gly Trp
Tyr Glu 210 215 220Gly Ser Glu Lys Tyr Ser Asp Met Ala Ala Asn Pro
Thr Tyr Arg Gln225 230 235 240Gln Phe Ile Gln Ser Val Leu Asp Phe
Leu Gln Glu Tyr Lys Phe Asp 245 250 255Gly Leu Asp Leu Asp Trp Glu
Tyr Pro Gly Ser Arg Leu Gly Asn Pro 260 265 270Lys Ile Asp Lys Gln
Asn Tyr Leu Ala Leu Val Arg Glu Leu Lys Asp 275 280 285Ala Phe Glu
Pro His Gly Tyr Leu Leu Thr Ala Ala Val Ser Pro Gly 290 295 300Lys
Asp Lys Ile Asp Arg Ala Tyr Asp Ile Lys Glu Leu Asn Lys Leu305 310
315 320Phe Asp Trp Met Asn Val Met Thr Tyr Asp Tyr His Gly Gly Trp
Glu 325 330 335Asn Phe Tyr Gly His Asn Ala Pro Leu Tyr Lys Arg Pro
Asp Glu Thr 340 345 350Asp Glu Leu His Thr Tyr Phe Asn Val Asn Tyr
Thr Met His Tyr Tyr 355 360 365Leu Asn Asn Gly Ala Thr Arg Asp Lys
Leu Val Met Gly Val Pro Phe 370 375 380Tyr Gly Arg Ala Trp Ser Ile
Glu Asp Arg Ser Lys Leu Lys Leu Gly385 390 395 400Asp Pro Ala Lys
Gly Met Ser Pro Pro Gly Phe Ile Ser Gly Glu Glu 405 410 415Gly Val
Leu Ser Tyr Ile Glu Leu Ser Gln Leu Phe Gln Lys Glu Glu 420 425
430Trp His Ile Gln Tyr Asp Glu Tyr Tyr Asn Ala Pro Tyr Gly Tyr Asn
435 440 445Asp Lys Ile Trp Val Gly Tyr Asp Asp Leu Ala Ser Ile Ser
Ser Lys 450 455 460Leu Ala Phe Leu Lys Glu Leu Gly Val Ser Gly Val
Met Val Trp Ser465 470 475 480Leu Glu Asn Asp Asp Phe Lys Gly His
Leu Gly Pro Lys Asn Pro Leu 485 490 495Leu Asn Lys Val His Asn Met
Ile Asn Gly Asp Glu Lys Asn Ser Phe 500 505 510Glu Ile Ile Leu Gly
Pro Ser Thr Thr Thr Pro Thr Pro Thr Thr Thr 515 520 525Pro Thr Thr
Pro Thr Thr Thr Pro Thr Thr Pro Ser Pro Thr Thr Pro 530 535 540Thr
Thr Thr Pro Ser Pro Thr Thr Pro Thr Thr Thr Pro Ser Pro Thr545 550
555 560Thr Pro Thr Thr Thr Pro Ser Pro Thr Thr Pro Thr Pro Thr Thr
Pro 565 570 575Thr Pro Ala Pro Thr Thr Ser Thr Pro Ser Pro Thr Thr
Thr Glu His 580 585 590Thr Ser Glu Thr Pro Lys Tyr Thr Thr Tyr Val
Asp Gly His Leu Ile 595 600 605Lys Ser Tyr Lys Glu Gly Asp Ile Pro
His Pro Thr Asn Ile His Lys 610 615 620Tyr Leu Val Ile Glu Phe Val
Asn Gly Gly Trp Trp Val His Ile Met625 630 635 640Pro Ser Pro Pro
Gly Thr Ile Trp Ser Gln Glu Lys Leu Thr Ser Ile 645 650 655Gly Glu
His His His His His His 66059657PRTArtificial sequenceIMAT_PURE Dog
from Q9U6R7 98kDa HDM allergen (Der f 15 allergen) (Group 15
allergen Der f 15) Dermatophagoides farinae (American house dust
mite) 59Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Met Glu Asp Gln
Arg1 5 10 15Asp Leu Ile Ser Asn His Glu Gln Leu Pro Ile Leu Gly Gln
Arg Pro 20 25 30Gly Ala Pro Glu Ser Lys Ser Ser Arg Gly Ala Leu Tyr
Thr Gly Phe 35 40 45Ser Val Leu Val Ala Leu Leu Leu Ala Gly Gln Ala
Thr Thr Ala Tyr 50 55 60Phe Leu Tyr Gln Gln Gln Gly Arg Leu Asp Lys
Leu Thr Val Thr Ser65 70 75 80Gln Asn Leu Gln Leu Glu Ser Leu Arg
Met Lys Leu Pro Lys Pro Pro 85 90 95Lys Pro Leu Ser Lys Met Arg Val
Ala Thr Pro Met Met Met Gln Ala 100 105 110Leu Pro Ile Gln Ser Leu
Pro Gln Gly Ser Ile Lys Arg Asp His Asn 115 120 125Asp Tyr Ser Lys
Asn Pro Met Arg Ile Val Leu Tyr Val Gly Thr Trp 130 135 140Ser Val
Tyr His Lys Val Asp Pro Tyr Thr Ile Glu Asp Ile Asp Pro145 150 155
160Phe Lys Ser Thr His Leu Met Tyr Gly Phe Ala Lys Ile Asp Glu Tyr
165 170 175Lys Tyr Thr Ile Gln Val Phe Asp Pro Tyr Gln Asp Asp Asn
His Asn 180 185 190Ser Trp Glu Lys Arg Gly Tyr Glu Arg Phe Asn Asn
Leu Arg Leu Lys 195 200 205Asn Pro Glu Leu Thr Thr Met Ile Ser Leu
Gly Gly Trp Tyr Glu Gly 210 215 220Ser Glu Lys Tyr Ser Asp Met Ala
Ala Asn Pro Thr Tyr Arg Gln Gln225 230 235 240Phe Ile Gln Ser Val
Leu Asp Phe Leu Gln Glu Tyr Lys Phe Asp Gly 245 250 255Leu Asp Leu
Asp Trp Glu Tyr Pro Gly Ser Arg Leu Gly Asn Pro Lys 260 265 270Ile
Asp Lys Gln Asn Tyr Leu Ala Leu Val Arg Glu Leu Lys Asp Ala 275 280
285Phe Glu Pro His Gly Tyr Leu Leu Thr Ala Ala Val Ser Pro Gly Lys
290 295 300Asp Lys Ile Asp Arg Ala Tyr Asp Ile Lys Glu Leu Asn Lys
Leu Phe305 310 315 320Asp Trp Met Asn Val Met Thr Tyr Asp Tyr His
Gly Gly Trp Glu Asn 325 330 335Phe Tyr Gly His Asn Ala Pro Leu Tyr
Lys Arg Pro Asp Glu Thr Asp 340 345 350Glu Leu His Thr Tyr Phe Asn
Val Asn Tyr Thr Met His Tyr Tyr Leu 355 360 365Asn Asn Gly Ala Thr
Arg Asp Lys Leu Val Met Gly Val Pro Phe Tyr 370 375 380Gly Arg Ala
Trp Ser Ile Glu Asp Arg Ser Lys Leu Lys Leu Gly Asp385 390 395
400Pro Ala Lys Gly Met Ser Pro Pro Gly Phe Ile Ser Gly Glu Glu Gly
405 410 415Val Leu Ser Tyr Ile Glu Leu Ser Gln Leu Phe Gln Lys Glu
Glu Trp 420 425 430His Ile Gln Tyr Asp Glu Tyr Tyr Asn Ala Pro Tyr
Gly Tyr Asn Asp 435 440 445Lys Ile Trp Val Gly Tyr Asp Asp Leu Ala
Ser Ile Ser Ser Lys Leu 450 455 460Ala Phe Leu Lys Glu Leu Gly Val
Ser Gly Val Met Val Trp Ser Leu465 470 475 480Glu Asn Asp Asp Phe
Lys Gly His Leu Gly Pro Lys Asn Pro Leu Leu 485 490 495Asn Lys Val
His Asn Met Ile Asn Gly Asp Glu Lys Asn Ser Phe Glu 500 505 510Ile
Ile Leu Gly Pro Ser Thr Thr Thr Pro Thr Pro Thr Thr Thr Pro 515 520
525Thr Thr Pro Thr Thr Thr Pro Thr Thr Pro Ser Pro Thr Thr Pro Thr
530 535 540Thr Thr Pro Ser Pro Thr Thr Pro Thr Thr Thr Pro Ser Pro
Thr Thr545 550 555 560Pro Thr Thr Thr Pro Ser Pro Thr Thr Pro Thr
Pro Thr Thr Pro Thr 565 570 575Pro Ala Pro Thr Thr Ser Thr Pro Ser
Pro Thr Thr Thr Glu His Thr 580 585 590Ser Glu Thr Pro Lys Tyr Thr
Thr Tyr Val Asp Gly His Leu Ile Lys 595 600 605Ser Tyr Lys Glu Gly
Asp Ile Pro His Pro Thr Asn Ile His Lys Tyr 610 615 620Leu Val Ile
Glu Phe Val Asn Gly Gly Trp Trp Val His Ile Met Pro625 630 635
640Ser Pro Pro Gly Thr Ile Trp Ser Gln Glu Lys Leu Thr Ser Ile Gly
645 650 655Glu60606PRTArtificial sequenceIMAT_NTERM Dog from I7HDR2
Zen 1 protein Dermatophagoides farinae (American house dust mite)
60Met His His His His His His Tyr Gly Arg Lys Lys Arg Arg Gln Arg1
5
10 15Arg Arg Met Glu Asp Gln Arg Asp Leu Ile Ser Asn His Glu Gln
Leu 20 25 30Pro Ile Leu Gly Gln Arg Pro Gly Ala Pro Glu Ser Lys Ser
Ser Arg 35 40 45Gly Ala Leu Tyr Thr Gly Phe Ser Val Leu Val Ala Leu
Leu Leu Ala 50 55 60Gly Gln Ala Thr Thr Ala Tyr Phe Leu Tyr Gln Gln
Gln Gly Arg Leu65 70 75 80Asp Lys Leu Thr Val Thr Ser Gln Asn Leu
Gln Leu Glu Ser Leu Arg 85 90 95Met Lys Leu Pro Lys Pro Pro Lys Pro
Leu Ser Lys Met Arg Val Ala 100 105 110Thr Pro Met Met Met Gln Ala
Leu Pro Ile Gln Ser Leu Pro Gln Gly 115 120 125Asn Pro Arg Phe Lys
Arg Asp Asn Arg Asp Asp Val Leu Lys Gln Thr 130 135 140Glu Glu Leu
Ile Lys Ser Ala Gln Asp Val Leu Glu Lys Leu Pro Asp145 150 155
160Ser Asp Leu Lys Asp Glu Ile Ala Glu Lys Leu Ala Thr Met Lys His
165 170 175Tyr Lys His Glu Leu Glu Asn Ala Lys Asn Pro Ile Lys Ile
Ala His 180 185 190Phe Glu Leu Glu Leu Leu Thr Met Phe Lys Lys Phe
Gln Ser Leu Leu 195 200 205Asn Glu Ala Asn Glu Ile Ile Lys Ser Leu
Thr Thr Thr Thr Thr Glu 210 215 220Pro Thr Thr Pro Thr Pro Glu Pro
Thr Thr Thr Thr Pro Glu Pro Thr225 230 235 240Thr Lys Thr Pro Glu
Pro Thr Thr Lys Thr Pro Glu Pro Thr Thr Pro 245 250 255Thr Pro Glu
Pro Thr Thr Lys Thr Pro Glu Pro Thr Thr Lys Thr Pro 260 265 270Glu
Pro Thr Thr Pro Thr Pro Glu Pro Thr Thr Lys Thr Pro Glu Pro 275 280
285Thr Thr Pro Thr Pro Glu Pro Thr Thr Lys Thr Pro Glu Pro Thr Thr
290 295 300Lys Thr Pro Glu Pro Ser Thr Pro Thr Pro Glu Pro Thr Thr
Lys Thr305 310 315 320Pro Glu Pro Thr Thr Lys Thr Pro Glu Pro Ser
Thr Pro Thr Pro Glu 325 330 335Pro Thr Thr Lys Thr Pro Glu Pro Ser
Thr Pro Thr Pro Glu Pro Thr 340 345 350Thr Lys Thr Pro Glu Pro Thr
Thr Lys Thr Pro Glu Pro Ser Thr Pro 355 360 365Thr Pro Glu Pro Thr
Thr Lys Thr Pro Glu Pro Ser Thr Pro Thr Pro 370 375 380Glu Pro Thr
Thr Lys Thr Pro Glu Pro Ser Thr Thr Lys Lys Pro Asn385 390 395
400Arg Asp Asp Val Leu Lys Gln Ala Glu Glu Leu Ile Lys Arg Ala Glu
405 410 415Asp Val Phe Glu Lys Leu Pro Asp Ser Asp Leu Lys Asn Glu
Ile Ala 420 425 430Glu Lys Leu Ala Thr Met Lys Asn Tyr Lys His Glu
Leu Glu Asn Ala 435 440 445Lys Asn Pro Ile Lys Ile Ala His Leu Glu
Ser Glu Leu Leu Thr Met 450 455 460Phe Lys Met Phe Gln Ser Leu Leu
Asn Glu Ala Asp Glu Ile Ile Arg465 470 475 480Ser Leu Thr Thr Thr
Thr Glu Pro Thr Thr Leu Asn Ser Thr Thr Pro 485 490 495Glu Pro Thr
Thr Leu Asn Ser Thr Thr Pro Glu Pro Thr Thr Leu Asn 500 505 510Ser
Thr Thr Pro Glu Pro Thr Thr Leu Asn Ser Thr Thr Pro Glu Pro 515 520
525Thr Thr Leu Asn Ser Thr Thr Pro Glu Pro Thr Thr Leu Asn Ser Thr
530 535 540Thr Pro Glu Pro Thr Thr Leu Asn Ser Thr Thr Pro Glu Pro
Thr Thr545 550 555 560Leu Asn Ser Thr Thr Pro Glu Pro Thr Thr Ser
Asn Ser Thr Thr Ser 565 570 575Glu Pro Thr Asn Ser Ile Asn Arg Lys
Thr Ser Glu Ile Ser Phe Leu 580 585 590Ser Asp Trp Phe His Lys Ile
Arg Thr Arg Phe Asn Ile Phe 595 600 60561606PRTArtificial
sequenceIMAT_CTERM Dog from I7HDR2 Zen 1 protein Dermatophagoides
farinae (American house dust mite) 61Met Tyr Gly Arg Lys Lys Arg
Arg Gln Arg Arg Arg Met Glu Asp Gln1 5 10 15Arg Asp Leu Ile Ser Asn
His Glu Gln Leu Pro Ile Leu Gly Gln Arg 20 25 30Pro Gly Ala Pro Glu
Ser Lys Ser Ser Arg Gly Ala Leu Tyr Thr Gly 35 40 45Phe Ser Val Leu
Val Ala Leu Leu Leu Ala Gly Gln Ala Thr Thr Ala 50 55 60Tyr Phe Leu
Tyr Gln Gln Gln Gly Arg Leu Asp Lys Leu Thr Val Thr65 70 75 80Ser
Gln Asn Leu Gln Leu Glu Ser Leu Arg Met Lys Leu Pro Lys Pro 85 90
95Pro Lys Pro Leu Ser Lys Met Arg Val Ala Thr Pro Met Met Met Gln
100 105 110Ala Leu Pro Ile Gln Ser Leu Pro Gln Gly Asn Pro Arg Phe
Lys Arg 115 120 125Asp Asn Arg Asp Asp Val Leu Lys Gln Thr Glu Glu
Leu Ile Lys Ser 130 135 140Ala Gln Asp Val Leu Glu Lys Leu Pro Asp
Ser Asp Leu Lys Asp Glu145 150 155 160Ile Ala Glu Lys Leu Ala Thr
Met Lys His Tyr Lys His Glu Leu Glu 165 170 175Asn Ala Lys Asn Pro
Ile Lys Ile Ala His Phe Glu Leu Glu Leu Leu 180 185 190Thr Met Phe
Lys Lys Phe Gln Ser Leu Leu Asn Glu Ala Asn Glu Ile 195 200 205Ile
Lys Ser Leu Thr Thr Thr Thr Thr Glu Pro Thr Thr Pro Thr Pro 210 215
220Glu Pro Thr Thr Thr Thr Pro Glu Pro Thr Thr Lys Thr Pro Glu
Pro225 230 235 240Thr Thr Lys Thr Pro Glu Pro Thr Thr Pro Thr Pro
Glu Pro Thr Thr 245 250 255Lys Thr Pro Glu Pro Thr Thr Lys Thr Pro
Glu Pro Thr Thr Pro Thr 260 265 270Pro Glu Pro Thr Thr Lys Thr Pro
Glu Pro Thr Thr Pro Thr Pro Glu 275 280 285Pro Thr Thr Lys Thr Pro
Glu Pro Thr Thr Lys Thr Pro Glu Pro Ser 290 295 300Thr Pro Thr Pro
Glu Pro Thr Thr Lys Thr Pro Glu Pro Thr Thr Lys305 310 315 320Thr
Pro Glu Pro Ser Thr Pro Thr Pro Glu Pro Thr Thr Lys Thr Pro 325 330
335Glu Pro Ser Thr Pro Thr Pro Glu Pro Thr Thr Lys Thr Pro Glu Pro
340 345 350Thr Thr Lys Thr Pro Glu Pro Ser Thr Pro Thr Pro Glu Pro
Thr Thr 355 360 365Lys Thr Pro Glu Pro Ser Thr Pro Thr Pro Glu Pro
Thr Thr Lys Thr 370 375 380Pro Glu Pro Ser Thr Thr Lys Lys Pro Asn
Arg Asp Asp Val Leu Lys385 390 395 400Gln Ala Glu Glu Leu Ile Lys
Arg Ala Glu Asp Val Phe Glu Lys Leu 405 410 415Pro Asp Ser Asp Leu
Lys Asn Glu Ile Ala Glu Lys Leu Ala Thr Met 420 425 430Lys Asn Tyr
Lys His Glu Leu Glu Asn Ala Lys Asn Pro Ile Lys Ile 435 440 445Ala
His Leu Glu Ser Glu Leu Leu Thr Met Phe Lys Met Phe Gln Ser 450 455
460Leu Leu Asn Glu Ala Asp Glu Ile Ile Arg Ser Leu Thr Thr Thr
Thr465 470 475 480Glu Pro Thr Thr Leu Asn Ser Thr Thr Pro Glu Pro
Thr Thr Leu Asn 485 490 495Ser Thr Thr Pro Glu Pro Thr Thr Leu Asn
Ser Thr Thr Pro Glu Pro 500 505 510Thr Thr Leu Asn Ser Thr Thr Pro
Glu Pro Thr Thr Leu Asn Ser Thr 515 520 525Thr Pro Glu Pro Thr Thr
Leu Asn Ser Thr Thr Pro Glu Pro Thr Thr 530 535 540Leu Asn Ser Thr
Thr Pro Glu Pro Thr Thr Leu Asn Ser Thr Thr Pro545 550 555 560Glu
Pro Thr Thr Ser Asn Ser Thr Thr Ser Glu Pro Thr Asn Ser Ile 565 570
575Asn Arg Lys Thr Ser Glu Ile Ser Phe Leu Ser Asp Trp Phe His Lys
580 585 590Ile Arg Thr Arg Phe Asn Ile Phe His His His His His His
595 600 60562599PRTArtificial sequenceIMAT_PURE Dog from I7HDR2 Zen
1 protein Dermatophagoides farinae (American house dust mite) 62Tyr
Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Met Glu Asp Gln Arg1 5 10
15Asp Leu Ile Ser Asn His Glu Gln Leu Pro Ile Leu Gly Gln Arg Pro
20 25 30Gly Ala Pro Glu Ser Lys Ser Ser Arg Gly Ala Leu Tyr Thr Gly
Phe 35 40 45Ser Val Leu Val Ala Leu Leu Leu Ala Gly Gln Ala Thr Thr
Ala Tyr 50 55 60Phe Leu Tyr Gln Gln Gln Gly Arg Leu Asp Lys Leu Thr
Val Thr Ser65 70 75 80Gln Asn Leu Gln Leu Glu Ser Leu Arg Met Lys
Leu Pro Lys Pro Pro 85 90 95Lys Pro Leu Ser Lys Met Arg Val Ala Thr
Pro Met Met Met Gln Ala 100 105 110Leu Pro Ile Gln Ser Leu Pro Gln
Gly Asn Pro Arg Phe Lys Arg Asp 115 120 125Asn Arg Asp Asp Val Leu
Lys Gln Thr Glu Glu Leu Ile Lys Ser Ala 130 135 140Gln Asp Val Leu
Glu Lys Leu Pro Asp Ser Asp Leu Lys Asp Glu Ile145 150 155 160Ala
Glu Lys Leu Ala Thr Met Lys His Tyr Lys His Glu Leu Glu Asn 165 170
175Ala Lys Asn Pro Ile Lys Ile Ala His Phe Glu Leu Glu Leu Leu Thr
180 185 190Met Phe Lys Lys Phe Gln Ser Leu Leu Asn Glu Ala Asn Glu
Ile Ile 195 200 205Lys Ser Leu Thr Thr Thr Thr Thr Glu Pro Thr Thr
Pro Thr Pro Glu 210 215 220Pro Thr Thr Thr Thr Pro Glu Pro Thr Thr
Lys Thr Pro Glu Pro Thr225 230 235 240Thr Lys Thr Pro Glu Pro Thr
Thr Pro Thr Pro Glu Pro Thr Thr Lys 245 250 255Thr Pro Glu Pro Thr
Thr Lys Thr Pro Glu Pro Thr Thr Pro Thr Pro 260 265 270Glu Pro Thr
Thr Lys Thr Pro Glu Pro Thr Thr Pro Thr Pro Glu Pro 275 280 285Thr
Thr Lys Thr Pro Glu Pro Thr Thr Lys Thr Pro Glu Pro Ser Thr 290 295
300Pro Thr Pro Glu Pro Thr Thr Lys Thr Pro Glu Pro Thr Thr Lys
Thr305 310 315 320Pro Glu Pro Ser Thr Pro Thr Pro Glu Pro Thr Thr
Lys Thr Pro Glu 325 330 335Pro Ser Thr Pro Thr Pro Glu Pro Thr Thr
Lys Thr Pro Glu Pro Thr 340 345 350Thr Lys Thr Pro Glu Pro Ser Thr
Pro Thr Pro Glu Pro Thr Thr Lys 355 360 365Thr Pro Glu Pro Ser Thr
Pro Thr Pro Glu Pro Thr Thr Lys Thr Pro 370 375 380Glu Pro Ser Thr
Thr Lys Lys Pro Asn Arg Asp Asp Val Leu Lys Gln385 390 395 400Ala
Glu Glu Leu Ile Lys Arg Ala Glu Asp Val Phe Glu Lys Leu Pro 405 410
415Asp Ser Asp Leu Lys Asn Glu Ile Ala Glu Lys Leu Ala Thr Met Lys
420 425 430Asn Tyr Lys His Glu Leu Glu Asn Ala Lys Asn Pro Ile Lys
Ile Ala 435 440 445His Leu Glu Ser Glu Leu Leu Thr Met Phe Lys Met
Phe Gln Ser Leu 450 455 460Leu Asn Glu Ala Asp Glu Ile Ile Arg Ser
Leu Thr Thr Thr Thr Glu465 470 475 480Pro Thr Thr Leu Asn Ser Thr
Thr Pro Glu Pro Thr Thr Leu Asn Ser 485 490 495Thr Thr Pro Glu Pro
Thr Thr Leu Asn Ser Thr Thr Pro Glu Pro Thr 500 505 510Thr Leu Asn
Ser Thr Thr Pro Glu Pro Thr Thr Leu Asn Ser Thr Thr 515 520 525Pro
Glu Pro Thr Thr Leu Asn Ser Thr Thr Pro Glu Pro Thr Thr Leu 530 535
540Asn Ser Thr Thr Pro Glu Pro Thr Thr Leu Asn Ser Thr Thr Pro
Glu545 550 555 560Pro Thr Thr Ser Asn Ser Thr Thr Ser Glu Pro Thr
Asn Ser Ile Asn 565 570 575Arg Lys Thr Ser Glu Ile Ser Phe Leu Ser
Asp Trp Phe His Lys Ile 580 585 590Arg Thr Arg Phe Asn Ile Phe
59563286PRTArtificial sequenceIMAT_NTERM Dog from Q94424 Salivary
antigen 1 (FS-I) (allergen Cte f 1) Ctenocephalides felis (Cat
flea) 63Met His His His His His His Tyr Gly Arg Lys Lys Arg Arg Gln
Arg1 5 10 15Arg Arg Met Glu Asp Gln Arg Asp Leu Ile Ser Asn His Glu
Gln Leu 20 25 30Pro Ile Leu Gly Gln Arg Pro Gly Ala Pro Glu Ser Lys
Arg Ser Arg 35 40 45Gly Ala Leu Tyr Thr Gly Phe Ser Val Leu Val Ala
Leu Leu Leu Ala 50 55 60Gly Gln Ala Thr Thr Ala Tyr Phe Leu Tyr Gln
Gln Gln Gly Arg Leu65 70 75 80Asp Lys Leu Thr Val Thr Ser Gln Asn
Leu Gln Leu Glu Ser Leu Arg 85 90 95Met Lys Leu Pro Lys Pro Pro Lys
Pro Leu Ser Lys Met Arg Val Ala 100 105 110Thr Pro Met Met Met Gln
Ala Leu Pro Ile Gln Ser Leu Pro Gln Gly 115 120 125Glu Asp Ile Trp
Lys Val Asn Lys Lys Arg Thr Ser Gly Gly Lys Asn 130 135 140Gln Asp
Arg Lys Leu Asp Gln Ile Ile Gln Lys Gly Gln Gln Val Lys145 150 155
160Ile Gln Asn Ile Leu Lys Leu Ile Arg Asp Lys Pro His Thr Asn Gln
165 170 175Glu Lys Glu Lys Leu Met Lys Phe Leu Lys Lys Val Leu Lys
Gly Tyr 180 185 190Arg Gly Ala Arg Asp Gly Asn Ile Leu Tyr Leu Ser
Arg Pro Ser Asn 195 200 205Leu Gly Pro Asp Trp Lys Val Ser Lys Glu
Arg Lys Asp Pro Asn Asn 210 215 220Lys Asp Ser Arg Pro Thr Glu Ile
Val Pro Tyr Arg Gln Gln Leu Ala225 230 235 240Ile Pro Asn Ile Leu
Lys Leu Lys Asn Ser Glu Thr Asn Glu Asp Ser 245 250 255Lys Leu Lys
Lys His Met Lys Glu Lys Ser Arg Gly Gly Asn Asp Ala 260 265 270Gly
Ile Asp Gly Asn Phe Leu Tyr Leu Arg Pro Lys Asn Lys 275 280
28564286PRTArtificial sequenceIMAT_CTERM Dog from Q94424 Salivary
antigen 1 (FS-I) (allergen Cte f 1) Ctenocephalides felis (Cat
flea) 64Met Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Met Glu Asp
Gln1 5 10 15Arg Asp Leu Ile Ser Asn His Glu Gln Leu Pro Ile Leu Gly
Gln Arg 20 25 30Pro Gly Ala Pro Glu Ser Lys Arg Ser Arg Gly Ala Leu
Tyr Thr Gly 35 40 45Phe Ser Val Leu Val Ala Leu Leu Leu Ala Gly Gln
Ala Thr Thr Ala 50 55 60Tyr Phe Leu Tyr Gln Gln Gln Gly Arg Leu Asp
Lys Leu Thr Val Thr65 70 75 80Ser Gln Asn Leu Gln Leu Glu Ser Leu
Arg Met Lys Leu Pro Lys Pro 85 90 95Pro Lys Pro Leu Ser Lys Met Arg
Val Ala Thr Pro Met Met Met Gln 100 105 110Ala Leu Pro Ile Gln Ser
Leu Pro Gln Gly Glu Asp Ile Trp Lys Val 115 120 125Asn Lys Lys Arg
Thr Ser Gly Gly Lys Asn Gln Asp Arg Lys Leu Asp 130 135 140Gln Ile
Ile Gln Lys Gly Gln Gln Val Lys Ile Gln Asn Ile Leu Lys145 150 155
160Leu Ile Arg Asp Lys Pro His Thr Asn Gln Glu Lys Glu Lys Leu Met
165 170 175Lys Phe Leu Lys Lys Val Leu Lys Gly Tyr Arg Gly Ala Arg
Asp Gly 180 185 190Asn Ile Leu Tyr Leu Ser Arg Pro Ser Asn Leu Gly
Pro Asp Trp Lys 195 200 205Val Ser Lys Glu Arg Lys Asp Pro Asn Asn
Lys Asp Ser Arg Pro Thr 210 215 220Glu Ile Val Pro Tyr Arg Gln Gln
Leu Ala Ile Pro Asn Ile Leu Lys225 230 235 240Leu Lys Asn Ser Glu
Thr Asn Glu Asp Ser Lys Leu Lys Lys His Met 245 250 255Lys Glu Lys
Ser Arg Gly Gly Asn Asp Ala Gly Ile Asp Gly Asn Phe 260 265 270Leu
Tyr Leu Arg Pro Lys Asn Lys His His His His His His 275 280
28565279PRTArtificial sequenceIMAT_PURE Dog from Q94424 Salivary
antigen 1 (FS-I) (allergen Cte f 1) Ctenocephalides felis (Cat
flea) 65Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Met Glu Asp Gln
Arg1
5 10 15Asp Leu Ile Ser Asn His Glu Gln Leu Pro Ile Leu Gly Gln Arg
Pro 20 25 30Gly Ala Pro Glu Ser Lys Arg Ser Arg Gly Ala Leu Tyr Thr
Gly Phe 35 40 45Ser Val Leu Val Ala Leu Leu Leu Ala Gly Gln Ala Thr
Thr Ala Tyr 50 55 60Phe Leu Tyr Gln Gln Gln Gly Arg Leu Asp Lys Leu
Thr Val Thr Ser65 70 75 80Gln Asn Leu Gln Leu Glu Ser Leu Arg Met
Lys Leu Pro Lys Pro Pro 85 90 95Lys Pro Leu Ser Lys Met Arg Val Ala
Thr Pro Met Met Met Gln Ala 100 105 110Leu Pro Ile Gln Ser Leu Pro
Gln Gly Glu Asp Ile Trp Lys Val Asn 115 120 125Lys Lys Arg Thr Ser
Gly Gly Lys Asn Gln Asp Arg Lys Leu Asp Gln 130 135 140Ile Ile Gln
Lys Gly Gln Gln Val Lys Ile Gln Asn Ile Leu Lys Leu145 150 155
160Ile Arg Asp Lys Pro His Thr Asn Gln Glu Lys Glu Lys Leu Met Lys
165 170 175Phe Leu Lys Lys Val Leu Lys Gly Tyr Arg Gly Ala Arg Asp
Gly Asn 180 185 190Ile Leu Tyr Leu Ser Arg Pro Ser Asn Leu Gly Pro
Asp Trp Lys Val 195 200 205Ser Lys Glu Arg Lys Asp Pro Asn Asn Lys
Asp Ser Arg Pro Thr Glu 210 215 220Ile Val Pro Tyr Arg Gln Gln Leu
Ala Ile Pro Asn Ile Leu Lys Leu225 230 235 240Lys Asn Ser Glu Thr
Asn Glu Asp Ser Lys Leu Lys Lys His Met Lys 245 250 255Glu Lys Ser
Arg Gly Gly Asn Asp Ala Gly Ile Asp Gly Asn Phe Leu 260 265 270Tyr
Leu Arg Pro Lys Asn Lys 27566511PRTArtificial sequenceIMAT_NTERM
Cat Hybrid1 66Met His His His His His His Tyr Gly Arg Lys Lys Arg
Arg Gln Arg1 5 10 15Arg Arg Met Glu Asp Gln Arg Asp Leu Ile Ser Asn
His Glu Gln Leu 20 25 30Pro Ile Leu Gly Gln Arg Pro Ala Ala Pro Glu
Ser Lys Ser Ser Arg 35 40 45Gly Ala Leu Tyr Thr Gly Phe Ser Val Leu
Val Ala Leu Leu Leu Ala 50 55 60Gly Gln Ala Thr Thr Ala Tyr Phe Leu
Tyr Gln Gln Gln Gly Arg Leu65 70 75 80Asp Lys Leu Thr Val Thr Ala
Gln Asn Leu Gln Leu Glu Asn Leu Arg 85 90 95Met Lys Leu Pro Lys Pro
Ala Lys Pro Leu Asn Lys Leu Arg Val Ala 100 105 110Thr Pro Met Leu
Met Gln Thr Met Pro Val Arg Gly Leu Leu Gln Ala 115 120 125Gln Ser
His Gln Tyr Tyr His Thr Ser Gly Leu Arg Asn Leu Gly Gly 130 135
140Ser Tyr Tyr Arg Ser Gly Asn Ala Lys Ala Met Ile Ala Val Gly
Gly145 150 155 160Ser Thr Met Ile Val Asp Gly Asp Lys Val Thr Ile
Tyr Gly Trp Gly 165 170 175Ser Gly Leu Gly Tyr Gly Leu Gly Tyr Gly
Leu Gly Tyr Gly Gln Ala 180 185 190Val Ala Leu Ala Pro Ala Gln Ala
Val Gly Tyr Val Ala Ala Ala Pro 195 200 205Ala Val Ala Val Gln Ala
Pro Ala Val Ser Tyr Ala Ala Ala Ala Pro 210 215 220Ala Val Gln Thr
Val Ala Val Gln Ala Pro Ala Val Ser Tyr Ala Ala225 230 235 240Ala
Pro Ala Val Ala Val Gln Ala His Thr Ala Gln Val Ser Gly Pro 245 250
255Ile His Ala Ala Ile Glu Ser Arg Arg Thr Val Glu Val Ile Asp Gly
260 265 270Pro Ser Thr Gly Asp Ala Pro Val Ala Ser Thr Val Val Ile
Gly Pro 275 280 285Asn Val Gln Pro Ile Asn Leu Glu Phe Gln Thr Gln
Ala Ser Pro Leu 290 295 300Ala Ala Thr Gln Asn His Val Pro Thr Thr
Pro Thr Thr Pro Thr Pro305 310 315 320Ala Pro Thr Thr Ser Thr Pro
Asp Leu Leu Arg Gln Asp Ile Val Lys 325 330 335Pro Val Val Gln Asp
Val His Glu Phe Leu Val Tyr Ile His Ile Ala 340 345 350Asn Asn Glu
Ile Lys Lys Val Gln Glu Ser Val His Gln Ile Leu Pro 355 360 365Arg
Gly Gln Met Met Lys Ile Tyr Gln Gln Gln Gln Gln Gln His His 370 375
380Pro Gln Arg Glu Glu Asn Ile Trp Ser Asp His Ile Ala Asn Val
Ala385 390 395 400Gln Ala Ala Pro Ala Ile Ser Ala Val Arg Val Ala
Ala Ala Pro Ala 405 410 415Val Ala Tyr Ala Ala Pro Ala Val Ser Thr
Val Ser Ala Ala Pro Ala 420 425 430Ala Ile Gly Val Ile Gly Val Gln
Pro Val Ala Gly Tyr Ile Gly Tyr 435 440 445Gly Ala Gly Tyr Gly Thr
Gly Tyr Gly Thr Gly Tyr Gly Val Ala Lys 450 455 460Tyr Gly Thr Gly
Tyr Gly Leu Thr Ser Gly Leu Ile Gly Gly Gly Ser465 470 475 480Tyr
Gly Ser Ser Tyr Ser Val Gln Pro Ala Ser Tyr Gly Thr Gly Tyr 485 490
495Gly Tyr Thr Thr Tyr Ser Ser Asp Ala Tyr Pro Ile Arg Lys Lys 500
505 51067511PRTArtificial sequenceIMAT_CTERM Cat Hybrid1 67Met Tyr
Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Met Glu Asp Gln1 5 10 15Arg
Asp Leu Ile Ser Asn His Glu Gln Leu Pro Ile Leu Gly Gln Arg 20 25
30Pro Ala Ala Pro Glu Ser Lys Ser Ser Arg Gly Ala Leu Tyr Thr Gly
35 40 45Phe Ser Val Leu Val Ala Leu Leu Leu Ala Gly Gln Ala Thr Thr
Ala 50 55 60Tyr Phe Leu Tyr Gln Gln Gln Gly Arg Leu Asp Lys Leu Thr
Val Thr65 70 75 80Ala Gln Asn Leu Gln Leu Glu Asn Leu Arg Met Lys
Leu Pro Lys Pro 85 90 95Ala Lys Pro Leu Asn Lys Leu Arg Val Ala Thr
Pro Met Leu Met Gln 100 105 110Thr Met Pro Val Arg Gly Leu Leu Gln
Ala Gln Ser His Gln Tyr Tyr 115 120 125His Thr Ser Gly Leu Arg Asn
Leu Gly Gly Ser Tyr Tyr Arg Ser Gly 130 135 140Asn Ala Lys Ala Met
Ile Ala Val Gly Gly Ser Thr Met Ile Val Asp145 150 155 160Gly Asp
Lys Val Thr Ile Tyr Gly Trp Gly Ser Gly Leu Gly Tyr Gly 165 170
175Leu Gly Tyr Gly Leu Gly Tyr Gly Gln Ala Val Ala Leu Ala Pro Ala
180 185 190Gln Ala Val Gly Tyr Val Ala Ala Ala Pro Ala Val Ala Val
Gln Ala 195 200 205Pro Ala Val Ser Tyr Ala Ala Ala Ala Pro Ala Val
Gln Thr Val Ala 210 215 220Val Gln Ala Pro Ala Val Ser Tyr Ala Ala
Ala Pro Ala Val Ala Val225 230 235 240Gln Ala His Thr Ala Gln Val
Ser Gly Pro Ile His Ala Ala Ile Glu 245 250 255Ser Arg Arg Thr Val
Glu Val Ile Asp Gly Pro Ser Thr Gly Asp Ala 260 265 270Pro Val Ala
Ser Thr Val Val Ile Gly Pro Asn Val Gln Pro Ile Asn 275 280 285Leu
Glu Phe Gln Thr Gln Ala Ser Pro Leu Ala Ala Thr Gln Asn His 290 295
300Val Pro Thr Thr Pro Thr Thr Pro Thr Pro Ala Pro Thr Thr Ser
Thr305 310 315 320Pro Asp Leu Leu Arg Gln Asp Ile Val Lys Pro Val
Val Gln Asp Val 325 330 335His Glu Phe Leu Val Tyr Ile His Ile Ala
Asn Asn Glu Ile Lys Lys 340 345 350Val Gln Glu Ser Val His Gln Ile
Leu Pro Arg Gly Gln Met Met Lys 355 360 365Ile Tyr Gln Gln Gln Gln
Gln Gln His His Pro Gln Arg Glu Glu Asn 370 375 380Ile Trp Ser Asp
His Ile Ala Asn Val Ala Gln Ala Ala Pro Ala Ile385 390 395 400Ser
Ala Val Arg Val Ala Ala Ala Pro Ala Val Ala Tyr Ala Ala Pro 405 410
415Ala Val Ser Thr Val Ser Ala Ala Pro Ala Ala Ile Gly Val Ile Gly
420 425 430Val Gln Pro Val Ala Gly Tyr Ile Gly Tyr Gly Ala Gly Tyr
Gly Thr 435 440 445Gly Tyr Gly Thr Gly Tyr Gly Val Ala Lys Tyr Gly
Thr Gly Tyr Gly 450 455 460Leu Thr Ser Gly Leu Ile Gly Gly Gly Ser
Tyr Gly Ser Ser Tyr Ser465 470 475 480Val Gln Pro Ala Ser Tyr Gly
Thr Gly Tyr Gly Tyr Thr Thr Tyr Ser 485 490 495Ser Asp Ala Tyr Pro
Ile Arg Lys Lys His His His His His His 500 505
51068504PRTArtificial sequenceHybrid 1 iMAT molecule (cat) without
tag 68Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Met Glu Asp Gln
Arg1 5 10 15Asp Leu Ile Ser Asn His Glu Gln Leu Pro Ile Leu Gly Gln
Arg Pro 20 25 30Ala Ala Pro Glu Ser Lys Cys Ser Arg Gly Ala Leu Tyr
Thr Gly Phe 35 40 45Ser Val Leu Val Ala Leu Leu Leu Ala Gly Gln Ala
Thr Thr Ala Tyr 50 55 60Phe Leu Tyr Gln Gln Gln Gly Arg Leu Asp Lys
Leu Thr Val Thr Ala65 70 75 80Gln Asn Leu Gln Leu Glu Asn Leu Arg
Met Lys Leu Pro Lys Pro Ala 85 90 95Lys Pro Leu Asn Lys Leu Arg Val
Ala Thr Pro Met Leu Met Gln Thr 100 105 110Met Pro Val Arg Gly Leu
Leu Gln Ala Gln Ser His Gln Tyr Tyr His 115 120 125Thr Ser Gly Leu
Arg Asn Leu Gly Gly Ser Tyr Tyr Arg Ser Gly Asn 130 135 140Ala Lys
Ala Met Ile Ala Val Gly Gly Ser Thr Met Ile Val Asp Gly145 150 155
160Asp Lys Val Thr Ile Tyr Gly Trp Gly Ser Gly Leu Gly Tyr Gly Leu
165 170 175Gly Tyr Gly Leu Gly Tyr Gly Gln Ala Val Ala Leu Ala Pro
Ala Gln 180 185 190Ala Val Gly Tyr Val Ala Ala Ala Pro Ala Val Ala
Val Gln Ala Pro 195 200 205Ala Val Ser Tyr Ala Ala Ala Ala Pro Ala
Val Gln Thr Val Ala Val 210 215 220Gln Ala Pro Ala Val Ser Tyr Ala
Ala Ala Pro Ala Val Ala Val Gln225 230 235 240Ala His Thr Ala Gln
Val Ser Gly Pro Ile His Ala Ala Ile Glu Ser 245 250 255Arg Arg Thr
Val Glu Val Ile Asp Gly Pro Ser Thr Gly Asp Ala Pro 260 265 270Val
Ala Ser Thr Val Val Ile Gly Pro Asn Val Gln Pro Ile Asn Leu 275 280
285Glu Phe Gln Thr Gln Ala Ser Pro Leu Ala Ala Thr Gln Asn His Val
290 295 300Pro Thr Thr Pro Thr Thr Pro Thr Pro Ala Pro Thr Thr Ser
Thr Pro305 310 315 320Asp Leu Leu Arg Gln Asp Ile Val Lys Pro Val
Val Gln Asp Val His 325 330 335Glu Phe Leu Val Tyr Ile His Ile Ala
Asn Asn Glu Ile Lys Lys Val 340 345 350Gln Glu Ser Val His Gln Ile
Leu Pro Arg Gly Gln Met Met Lys Ile 355 360 365Tyr Gln Gln Gln Gln
Gln Gln His His Pro Gln Arg Glu Glu Asn Ile 370 375 380Trp Ser Asp
His Ile Ala Asn Val Ala Gln Ala Ala Pro Ala Ile Ser385 390 395
400Ala Val Arg Val Ala Ala Ala Pro Ala Val Ala Tyr Ala Ala Pro Ala
405 410 415Val Ser Thr Val Ser Ala Ala Pro Ala Ala Ile Gly Val Ile
Gly Val 420 425 430Gln Pro Val Ala Gly Tyr Ile Gly Tyr Gly Ala Gly
Tyr Gly Thr Gly 435 440 445Tyr Gly Thr Gly Tyr Gly Val Ala Lys Tyr
Gly Thr Gly Tyr Gly Leu 450 455 460Thr Ser Gly Leu Ile Gly Gly Gly
Ser Tyr Gly Ser Ser Tyr Ser Val465 470 475 480Gln Pro Ala Ser Tyr
Gly Thr Gly Tyr Gly Tyr Thr Thr Tyr Ser Ser 485 490 495Asp Ala Tyr
Pro Ile Arg Lys Lys 50069645PRTArtificial sequenceIMAT_NTERM Cat
Hybrid2 69Met His His His His His His Tyr Gly Arg Lys Lys Arg Arg
Gln Arg1 5 10 15Arg Arg Met Glu Asp Gln Arg Asp Leu Ile Ser Asn His
Glu Gln Leu 20 25 30Pro Ile Leu Gly Gln Arg Pro Ala Ala Pro Glu Ser
Lys Ser Ser Arg 35 40 45Gly Ala Leu Tyr Thr Gly Phe Ser Val Leu Val
Ala Leu Leu Leu Ala 50 55 60Gly Gln Ala Thr Thr Ala Tyr Phe Leu Tyr
Gln Gln Gln Gly Arg Leu65 70 75 80Asp Lys Leu Thr Val Thr Ala Gln
Asn Leu Gln Leu Glu Asn Leu Arg 85 90 95Met Lys Leu Pro Lys Pro Ala
Lys Pro Leu Asn Lys Leu Arg Val Ala 100 105 110Thr Pro Met Leu Met
Gln Thr Met Pro Val Arg Gly Leu Leu Gln Ala 115 120 125Thr Ser Ala
Ser Arg Ile Asn Ser Val Asn Val Pro Ser Glu Leu Asp 130 135 140Leu
Arg Ser Leu Arg Thr Val Thr Pro Ile Arg Met Gln Gly Gly Ser145 150
155 160Gly Ser Ser Trp Ala Phe Ser Gly Val Ala Ala Thr Glu Ser Ala
Tyr 165 170 175Leu Ala Tyr Arg Asn Thr Ser Leu Asp Leu Ser Glu Gln
Glu Leu Val 180 185 190Asp Ser Ala Ser Gln His Gly Ser His Gly Asp
Thr Ile Pro Arg Gly 195 200 205Ile Glu Tyr Ile Gln Gln Asn Gly Val
Val Glu Glu Arg Ser Tyr Pro 210 215 220Tyr Val Ala Arg Glu Gln Gln
Ser Arg Arg Pro Asn Ser Gln His Tyr225 230 235 240Gly Ile Ser Asn
Tyr Ser Gln Ile Tyr Pro Pro Asp Val Lys Gln Ile 245 250 255Arg Glu
Ala Leu Thr Gln Thr His Thr Ala Ile Ala Val Ile Ile Gly 260 265
270Ile Lys Asp Leu Arg Ala Phe Gln His Tyr Asp Gly Arg Thr Ile Ile
275 280 285Gln His Asp Asn Gly Tyr Gln Pro Asn Tyr His Ala Val Asn
Ile Val 290 295 300Gly Tyr Gly Ser Thr Gln Gly Val Asp Tyr Trp Ile
Val Arg Asn Ser305 310 315 320Trp Asp Thr Thr Trp Gly Asp Ser Gly
Tyr Gly Tyr Phe Gln Ala Gly 325 330 335Asn Asn Leu Met Met Ile Glu
Gln Tyr Pro Tyr Val Val Ile Met Phe 340 345 350Thr Gln Thr Asp Gly
Phe Leu Ser Tyr Asn Glu Leu Ser Val Gln Ile 355 360 365Gln Ala Glu
Thr Asn Ala Phe Thr Ile Thr Arg Glu Pro Thr Thr Pro 370 375 380Thr
Pro Glu Pro Thr Thr Lys Thr Pro Glu Pro Thr Thr Lys Thr Pro385 390
395 400Glu Pro Ser Thr Pro Thr Pro Glu Pro Thr Thr Lys Thr Pro Glu
Pro 405 410 415Thr Thr Lys Asp Ile Asp His Asp Asp Asp Pro Thr Thr
Met Ile Asp 420 425 430Val Gln Thr Thr Thr Val Gln Pro Ser Asp Glu
Phe Glu Ser Pro Thr 435 440 445Arg Phe Gly Tyr Phe Ala Asp Pro Lys
Asp Pro Ile Lys Phe Tyr Ile 450 455 460Ser Ser Asn Trp Glu Ala Ile
His Lys Ser Ser Pro Gly Asn Thr Arg465 470 475 480Trp Asn Glu Lys
Glu Leu Thr Ser Thr Ser Pro Thr Thr Pro Thr Thr 485 490 495Thr Pro
Ser Pro Thr Thr Pro Thr Thr Thr Pro Ser Pro Thr Thr Pro 500 505
510Thr Thr Thr Pro Asp Gln Val Asp Val Lys Asp Leu Ala Asn Asn Glu
515 520 525Ile Lys Lys Val Met Val Asp Gly Ile His Gly Ser Asp Pro
Ser Ile 530 535 540Ile His Arg Gly Lys Pro Phe Thr Leu Glu Ala Leu
Phe Asp Ala Asn545 550 555 560Gln Asn Thr Lys Thr Ala Lys Ile Glu
Ile Lys Ala Ser Leu Asp Gly 565 570 575Leu Glu Ile Asp Val Pro Gly
Ile Asp Thr Asn Ala Leu His Phe Met 580 585 590Lys Ile Pro Leu Val
Lys Gly Gln Gln Tyr Asp Ile Lys Tyr Thr Trp 595 600 605Asn Val Pro
Lys Ile Ala Pro Lys Ser Glu Asn Val Val Val Thr Val 610 615 620Lys
Leu Ile Gly Asp Asn Gly Val Leu Ala Ser Ala Ile Ala Thr His625 630
635 640Gly Lys Ile Arg Asp 64570645PRTArtificial sequenceIMAT_CTERM
Cat
Hybrid2 70Met Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Met Glu
Asp Gln1 5 10 15Arg Asp Leu Ile Ser Asn His Glu Gln Leu Pro Ile Leu
Gly Gln Arg 20 25 30Pro Ala Ala Pro Glu Ser Lys Ser Ser Arg Gly Ala
Leu Tyr Thr Gly 35 40 45Phe Ser Val Leu Val Ala Leu Leu Leu Ala Gly
Gln Ala Thr Thr Ala 50 55 60Tyr Phe Leu Tyr Gln Gln Gln Gly Arg Leu
Asp Lys Leu Thr Val Thr65 70 75 80Ala Gln Asn Leu Gln Leu Glu Asn
Leu Arg Met Lys Leu Pro Lys Pro 85 90 95Ala Lys Pro Leu Asn Lys Leu
Arg Val Ala Thr Pro Met Leu Met Gln 100 105 110Thr Met Pro Val Arg
Gly Leu Leu Gln Ala Thr Ser Ala Ser Arg Ile 115 120 125Asn Ser Val
Asn Val Pro Ser Glu Leu Asp Leu Arg Ser Leu Arg Thr 130 135 140Val
Thr Pro Ile Arg Met Gln Gly Gly Ser Gly Ser Ser Trp Ala Phe145 150
155 160Ser Gly Val Ala Ala Thr Glu Ser Ala Tyr Leu Ala Tyr Arg Asn
Thr 165 170 175Ser Leu Asp Leu Ser Glu Gln Glu Leu Val Asp Ser Ala
Ser Gln His 180 185 190Gly Ser His Gly Asp Thr Ile Pro Arg Gly Ile
Glu Tyr Ile Gln Gln 195 200 205Asn Gly Val Val Glu Glu Arg Ser Tyr
Pro Tyr Val Ala Arg Glu Gln 210 215 220Gln Ser Arg Arg Pro Asn Ser
Gln His Tyr Gly Ile Ser Asn Tyr Ser225 230 235 240Gln Ile Tyr Pro
Pro Asp Val Lys Gln Ile Arg Glu Ala Leu Thr Gln 245 250 255Thr His
Thr Ala Ile Ala Val Ile Ile Gly Ile Lys Asp Leu Arg Ala 260 265
270Phe Gln His Tyr Asp Gly Arg Thr Ile Ile Gln His Asp Asn Gly Tyr
275 280 285Gln Pro Asn Tyr His Ala Val Asn Ile Val Gly Tyr Gly Ser
Thr Gln 290 295 300Gly Val Asp Tyr Trp Ile Val Arg Asn Ser Trp Asp
Thr Thr Trp Gly305 310 315 320Asp Ser Gly Tyr Gly Tyr Phe Gln Ala
Gly Asn Asn Leu Met Met Ile 325 330 335Glu Gln Tyr Pro Tyr Val Val
Ile Met Phe Thr Gln Thr Asp Gly Phe 340 345 350Leu Ser Tyr Asn Glu
Leu Ser Val Gln Ile Gln Ala Glu Thr Asn Ala 355 360 365Phe Thr Ile
Thr Arg Glu Pro Thr Thr Pro Thr Pro Glu Pro Thr Thr 370 375 380Lys
Thr Pro Glu Pro Thr Thr Lys Thr Pro Glu Pro Ser Thr Pro Thr385 390
395 400Pro Glu Pro Thr Thr Lys Thr Pro Glu Pro Thr Thr Lys Asp Ile
Asp 405 410 415His Asp Asp Asp Pro Thr Thr Met Ile Asp Val Gln Thr
Thr Thr Val 420 425 430Gln Pro Ser Asp Glu Phe Glu Ser Pro Thr Arg
Phe Gly Tyr Phe Ala 435 440 445Asp Pro Lys Asp Pro Ile Lys Phe Tyr
Ile Ser Ser Asn Trp Glu Ala 450 455 460Ile His Lys Ser Ser Pro Gly
Asn Thr Arg Trp Asn Glu Lys Glu Leu465 470 475 480Thr Ser Thr Ser
Pro Thr Thr Pro Thr Thr Thr Pro Ser Pro Thr Thr 485 490 495Pro Thr
Thr Thr Pro Ser Pro Thr Thr Pro Thr Thr Thr Pro Asp Gln 500 505
510Val Asp Val Lys Asp Leu Ala Asn Asn Glu Ile Lys Lys Val Met Val
515 520 525Asp Gly Ile His Gly Ser Asp Pro Ser Ile Ile His Arg Gly
Lys Pro 530 535 540Phe Thr Leu Glu Ala Leu Phe Asp Ala Asn Gln Asn
Thr Lys Thr Ala545 550 555 560Lys Ile Glu Ile Lys Ala Ser Leu Asp
Gly Leu Glu Ile Asp Val Pro 565 570 575Gly Ile Asp Thr Asn Ala Leu
His Phe Met Lys Ile Pro Leu Val Lys 580 585 590Gly Gln Gln Tyr Asp
Ile Lys Tyr Thr Trp Asn Val Pro Lys Ile Ala 595 600 605Pro Lys Ser
Glu Asn Val Val Val Thr Val Lys Leu Ile Gly Asp Asn 610 615 620Gly
Val Leu Ala Ser Ala Ile Ala Thr His Gly Lys Ile Arg Asp His625 630
635 640His His His His His 64571638PRTArtificial sequenceHybrid 2
iMAT molecule (cat) without tag 71Tyr Gly Arg Lys Lys Arg Arg Gln
Arg Arg Arg Met Glu Asp Gln Arg1 5 10 15Asp Leu Ile Ser Asn His Glu
Gln Leu Pro Ile Leu Gly Gln Arg Pro 20 25 30Ala Ala Pro Glu Ser Lys
Cys Ser Arg Gly Ala Leu Tyr Thr Gly Phe 35 40 45Ser Val Leu Val Ala
Leu Leu Leu Ala Gly Gln Ala Thr Thr Ala Tyr 50 55 60Phe Leu Tyr Gln
Gln Gln Gly Arg Leu Asp Lys Leu Thr Val Thr Ala65 70 75 80Gln Asn
Leu Gln Leu Glu Asn Leu Arg Met Lys Leu Pro Lys Pro Ala 85 90 95Lys
Pro Leu Asn Lys Leu Arg Val Ala Thr Pro Met Leu Met Gln Thr 100 105
110Met Pro Val Arg Gly Leu Leu Gln Ala Thr Ser Ala Ser Arg Ile Asn
115 120 125Ser Val Asn Val Pro Ser Glu Leu Asp Leu Arg Ser Leu Arg
Thr Val 130 135 140Thr Pro Ile Arg Met Gln Gly Gly Ser Gly Ser Ser
Trp Ala Phe Ser145 150 155 160Gly Val Ala Ala Thr Glu Ser Ala Tyr
Leu Ala Tyr Arg Asn Thr Ser 165 170 175Leu Asp Leu Ser Glu Gln Glu
Leu Val Asp Ser Ala Ser Gln His Gly 180 185 190Ser His Gly Asp Thr
Ile Pro Arg Gly Ile Glu Tyr Ile Gln Gln Asn 195 200 205Gly Val Val
Glu Glu Arg Ser Tyr Pro Tyr Val Ala Arg Glu Gln Gln 210 215 220Ser
Arg Arg Pro Asn Ser Gln His Tyr Gly Ile Ser Asn Tyr Ser Gln225 230
235 240Ile Tyr Pro Pro Asp Val Lys Gln Ile Arg Glu Ala Leu Thr Gln
Thr 245 250 255His Thr Ala Ile Ala Val Ile Ile Gly Ile Lys Asp Leu
Arg Ala Phe 260 265 270Gln His Tyr Asp Gly Arg Thr Ile Ile Gln His
Asp Asn Gly Tyr Gln 275 280 285Pro Asn Tyr His Ala Val Asn Ile Val
Gly Tyr Gly Ser Thr Gln Gly 290 295 300Val Asp Tyr Trp Ile Val Arg
Asn Ser Trp Asp Thr Thr Trp Gly Asp305 310 315 320Ser Gly Tyr Gly
Tyr Phe Gln Ala Gly Asn Asn Leu Met Met Ile Glu 325 330 335Gln Tyr
Pro Tyr Val Val Ile Met Phe Thr Gln Thr Asp Gly Phe Leu 340 345
350Ser Tyr Asn Glu Leu Ser Val Gln Ile Gln Ala Glu Thr Asn Ala Phe
355 360 365Thr Ile Thr Arg Glu Pro Thr Thr Pro Thr Pro Glu Pro Thr
Thr Lys 370 375 380Thr Pro Glu Pro Thr Thr Lys Thr Pro Glu Pro Ser
Thr Pro Thr Pro385 390 395 400Glu Pro Thr Thr Lys Thr Pro Glu Pro
Thr Thr Lys Asp Ile Asp His 405 410 415Asp Asp Asp Pro Thr Thr Met
Ile Asp Val Gln Thr Thr Thr Val Gln 420 425 430Pro Ser Asp Glu Phe
Glu Ser Pro Thr Arg Phe Gly Tyr Phe Ala Asp 435 440 445Pro Lys Asp
Pro Ile Lys Phe Tyr Ile Ser Ser Asn Trp Glu Ala Ile 450 455 460His
Lys Ser Ser Pro Gly Asn Thr Arg Trp Asn Glu Lys Glu Leu Thr465 470
475 480Ser Thr Ser Pro Thr Thr Pro Thr Thr Thr Pro Ser Pro Thr Thr
Pro 485 490 495Thr Thr Thr Pro Ser Pro Thr Thr Pro Thr Thr Thr Pro
Asp Gln Val 500 505 510Asp Val Lys Asp Leu Ala Asn Asn Glu Ile Lys
Lys Val Met Val Asp 515 520 525Gly Ile His Gly Ser Asp Pro Ser Ile
Ile His Arg Gly Lys Pro Phe 530 535 540Thr Leu Glu Ala Leu Phe Asp
Ala Asn Gln Asn Thr Lys Thr Ala Lys545 550 555 560Ile Glu Ile Lys
Ala Ser Leu Asp Gly Leu Glu Ile Asp Val Pro Gly 565 570 575Ile Asp
Thr Asn Ala Leu His Phe Met Lys Ile Pro Leu Val Lys Gly 580 585
590Gln Gln Tyr Asp Ile Lys Tyr Thr Trp Asn Val Pro Lys Ile Ala Pro
595 600 605Lys Ser Glu Asn Val Val Val Thr Val Lys Leu Ile Gly Asp
Asn Gly 610 615 620Val Leu Ala Ser Ala Ile Ala Thr His Gly Lys Ile
Arg Asp625 630 63572535PRTArtificial sequenceIMAT_NTERM Cat Hybrid3
72Met His His His His His His Tyr Gly Arg Lys Lys Arg Arg Gln Arg1
5 10 15Arg Arg Met Glu Asp Gln Arg Asp Leu Ile Ser Asn His Glu Gln
Leu 20 25 30Pro Ile Leu Gly Gln Arg Pro Ala Ala Pro Glu Ser Lys Ser
Ser Arg 35 40 45Gly Ala Leu Tyr Thr Gly Phe Ser Val Leu Val Ala Leu
Leu Leu Ala 50 55 60Gly Gln Ala Thr Thr Ala Tyr Phe Leu Tyr Gln Gln
Gln Gly Arg Leu65 70 75 80Asp Lys Leu Thr Val Thr Ala Gln Asn Leu
Gln Leu Glu Asn Leu Arg 85 90 95Met Lys Leu Pro Lys Pro Ala Lys Pro
Leu Asn Lys Leu Arg Val Ala 100 105 110Thr Pro Met Leu Met Gln Thr
Met Pro Val Arg Gly Leu Leu Gln Ala 115 120 125Thr Ser Ala Ser Arg
Ile Asn Ser Val Asn Val Pro Ser Glu Leu Asp 130 135 140Leu Arg Ser
Leu Arg Thr Val Thr Pro Ile Arg Met Gln Gly Gly Ser145 150 155
160Gly Ser Leu Trp Ala Phe Ser Gly Val Ala Ala Thr Glu Ser Ala Tyr
165 170 175Leu Ala Tyr Arg Asn Thr Ser Leu Asp Leu Ser Glu Gln Glu
Leu Val 180 185 190Asp Ile Ala Ser Gln His Gly Leu His Gly Asp Thr
Ile Pro Arg Gly 195 200 205Ile Glu Tyr Ile Gln Gln Asn Gly Val Val
Glu Glu Arg Ser Tyr Pro 210 215 220Tyr Val Ala Arg Glu Gln Gln Ser
Arg Arg Pro Asn Ser Gln His Tyr225 230 235 240Gly Ile Ser Asn Tyr
Leu Gln Ile Tyr Pro Pro Asp Val Lys Gln Ile 245 250 255Arg Glu Ala
Leu Thr Gln Thr His Thr Ala Ile Ala Val Ile Ile Gly 260 265 270Ile
Lys Asp Leu Arg Ala Phe Gln His Tyr Asp Gly Arg Thr Ile Ile 275 280
285Gln His Asp Asn Gly Tyr Gln Pro Asn Tyr His Ala Val Asn Ile Val
290 295 300Gly Tyr Gly Ser Thr Gln Gly Val Asp Tyr Trp Ile Val Arg
Asn Ser305 310 315 320Trp Asp Thr Thr Trp Gly Asp Ser Gly Tyr Gly
Tyr Phe Gln Ala Gly 325 330 335Asn Asn Leu Met Met Ile Glu Gln Tyr
Pro Tyr Val Val Ile Met Phe 340 345 350Thr Gln Thr Asp Gly Phe Leu
Ser Tyr Asn Glu Leu Ser Val Gln Ile 355 360 365Gln Ala Glu Thr Asn
Ala Phe Thr Ile Thr Arg Ser Pro Thr Thr Pro 370 375 380Thr Thr Thr
Pro Ser Pro Thr Thr Pro Thr Thr Thr Pro Ser Pro Thr385 390 395
400Thr Pro Thr Thr Thr Pro Asp Gln Val Asp Val Lys Asp Ser Ala Asn
405 410 415Asn Glu Ile Lys Lys Val Met Val Asp Gly Ile His Gly Ser
Asp Pro 420 425 430Ser Ile Ile His Arg Gly Lys Pro Phe Thr Leu Glu
Ala Leu Phe Asp 435 440 445Ala Asn Gln Asn Thr Lys Thr Ala Lys Ile
Glu Ile Lys Ala Ser Leu 450 455 460Asp Gly Leu Glu Ile Asp Val Pro
Gly Ile Asp Thr Asn Ala Ser His465 470 475 480Phe Met Lys Ser Pro
Leu Val Lys Gly Gln Gln Tyr Asp Ile Lys Tyr 485 490 495Thr Trp Asn
Val Pro Lys Ile Ala Pro Lys Ser Glu Asn Val Val Val 500 505 510Thr
Val Lys Leu Ile Gly Asp Asn Gly Val Leu Ala Ser Ala Ile Ala 515 520
525Thr His Gly Lys Ile Arg Asp 530 53573535PRTArtificial
sequenceIMAT_CTERM Cat Hybrid3 73Met Tyr Gly Arg Lys Lys Arg Arg
Gln Arg Arg Arg Met Glu Asp Gln1 5 10 15Arg Asp Leu Ile Ser Asn His
Glu Gln Leu Pro Ile Leu Gly Gln Arg 20 25 30Pro Ala Ala Pro Glu Ser
Lys Ser Ser Arg Gly Ala Leu Tyr Thr Gly 35 40 45Phe Ser Val Leu Val
Ala Leu Leu Leu Ala Gly Gln Ala Thr Thr Ala 50 55 60Tyr Phe Leu Tyr
Gln Gln Gln Gly Arg Leu Asp Lys Leu Thr Val Thr65 70 75 80Ala Gln
Asn Leu Gln Leu Glu Asn Leu Arg Met Lys Leu Pro Lys Pro 85 90 95Ala
Lys Pro Leu Asn Lys Leu Arg Val Ala Thr Pro Met Leu Met Gln 100 105
110Thr Met Pro Val Arg Gly Leu Leu Gln Ala Thr Ser Ala Ser Arg Ile
115 120 125Asn Ser Val Asn Val Pro Ser Glu Leu Asp Leu Arg Ser Leu
Arg Thr 130 135 140Val Thr Pro Ile Arg Met Gln Gly Gly Ser Gly Ser
Leu Trp Ala Phe145 150 155 160Ser Gly Val Ala Ala Thr Glu Ser Ala
Tyr Leu Ala Tyr Arg Asn Thr 165 170 175Ser Leu Asp Leu Ser Glu Gln
Glu Leu Val Asp Ile Ala Ser Gln His 180 185 190Gly Leu His Gly Asp
Thr Ile Pro Arg Gly Ile Glu Tyr Ile Gln Gln 195 200 205Asn Gly Val
Val Glu Glu Arg Ser Tyr Pro Tyr Val Ala Arg Glu Gln 210 215 220Gln
Ser Arg Arg Pro Asn Ser Gln His Tyr Gly Ile Ser Asn Tyr Leu225 230
235 240Gln Ile Tyr Pro Pro Asp Val Lys Gln Ile Arg Glu Ala Leu Thr
Gln 245 250 255Thr His Thr Ala Ile Ala Val Ile Ile Gly Ile Lys Asp
Leu Arg Ala 260 265 270Phe Gln His Tyr Asp Gly Arg Thr Ile Ile Gln
His Asp Asn Gly Tyr 275 280 285Gln Pro Asn Tyr His Ala Val Asn Ile
Val Gly Tyr Gly Ser Thr Gln 290 295 300Gly Val Asp Tyr Trp Ile Val
Arg Asn Ser Trp Asp Thr Thr Trp Gly305 310 315 320Asp Ser Gly Tyr
Gly Tyr Phe Gln Ala Gly Asn Asn Leu Met Met Ile 325 330 335Glu Gln
Tyr Pro Tyr Val Val Ile Met Phe Thr Gln Thr Asp Gly Phe 340 345
350Leu Ser Tyr Asn Glu Leu Ser Val Gln Ile Gln Ala Glu Thr Asn Ala
355 360 365Phe Thr Ile Thr Arg Ser Pro Thr Thr Pro Thr Thr Thr Pro
Ser Pro 370 375 380Thr Thr Pro Thr Thr Thr Pro Ser Pro Thr Thr Pro
Thr Thr Thr Pro385 390 395 400Asp Gln Val Asp Val Lys Asp Ser Ala
Asn Asn Glu Ile Lys Lys Val 405 410 415Met Val Asp Gly Ile His Gly
Ser Asp Pro Ser Ile Ile His Arg Gly 420 425 430Lys Pro Phe Thr Leu
Glu Ala Leu Phe Asp Ala Asn Gln Asn Thr Lys 435 440 445Thr Ala Lys
Ile Glu Ile Lys Ala Ser Leu Asp Gly Leu Glu Ile Asp 450 455 460Val
Pro Gly Ile Asp Thr Asn Ala Ser His Phe Met Lys Ser Pro Leu465 470
475 480Val Lys Gly Gln Gln Tyr Asp Ile Lys Tyr Thr Trp Asn Val Pro
Lys 485 490 495Ile Ala Pro Lys Ser Glu Asn Val Val Val Thr Val Lys
Leu Ile Gly 500 505 510Asp Asn Gly Val Leu Ala Ser Ala Ile Ala Thr
His Gly Lys Ile Arg 515 520 525Asp His His His His His His 530
53574528PRTArtificial sequenceHybrid 3 iMAT molecule (cat) without
tag 74Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Met Glu Asp Gln
Arg1 5 10 15Asp Leu Ile Ser Asn His Glu Gln Leu Pro Ile Leu Gly Gln
Arg Pro 20 25 30Ala Ala Pro Glu Ser Lys Cys Ser Arg Gly Ala Leu Tyr
Thr Gly Phe 35 40 45Ser Val Leu Val Ala Leu Leu Leu Ala Gly Gln Ala
Thr Thr Ala Tyr 50 55 60Phe Leu Tyr Gln Gln Gln Gly Arg Leu Asp Lys
Leu Thr Val Thr Ala65 70 75 80Gln Asn Leu Gln Leu Glu Asn Leu Arg
Met Lys Leu Pro Lys Pro Ala
85 90 95Lys Pro Leu Asn Lys Leu Arg Val Ala Thr Pro Met Leu Met Gln
Thr 100 105 110Met Pro Val Arg Gly Leu Leu Gln Ala Thr Ser Ala Ser
Arg Ile Asn 115 120 125Ser Val Asn Val Pro Ser Glu Leu Asp Leu Arg
Ser Leu Arg Thr Val 130 135 140Thr Pro Ile Arg Met Gln Gly Gly Ser
Gly Ser Leu Trp Ala Phe Ser145 150 155 160Gly Val Ala Ala Thr Glu
Ser Ala Tyr Leu Ala Tyr Arg Asn Thr Ser 165 170 175Leu Asp Leu Ser
Glu Gln Glu Leu Val Asp Ile Ala Ser Gln His Gly 180 185 190Leu His
Gly Asp Thr Ile Pro Arg Gly Ile Glu Tyr Ile Gln Gln Asn 195 200
205Gly Val Val Glu Glu Arg Ser Tyr Pro Tyr Val Ala Arg Glu Gln Gln
210 215 220Ser Arg Arg Pro Asn Ser Gln His Tyr Gly Ile Ser Asn Tyr
Leu Gln225 230 235 240Ile Tyr Pro Pro Asp Val Lys Gln Ile Arg Glu
Ala Leu Thr Gln Thr 245 250 255His Thr Ala Ile Ala Val Ile Ile Gly
Ile Lys Asp Leu Arg Ala Phe 260 265 270Gln His Tyr Asp Gly Arg Thr
Ile Ile Gln His Asp Asn Gly Tyr Gln 275 280 285Pro Asn Tyr His Ala
Val Asn Ile Val Gly Tyr Gly Ser Thr Gln Gly 290 295 300Val Asp Tyr
Trp Ile Val Arg Asn Ser Trp Asp Thr Thr Trp Gly Asp305 310 315
320Ser Gly Tyr Gly Tyr Phe Gln Ala Gly Asn Asn Leu Met Met Ile Glu
325 330 335Gln Tyr Pro Tyr Val Val Ile Met Phe Thr Gln Thr Asp Gly
Phe Leu 340 345 350Ser Tyr Asn Glu Leu Ser Val Gln Ile Gln Ala Glu
Thr Asn Ala Phe 355 360 365Thr Ile Thr Arg Ser Pro Thr Thr Pro Thr
Thr Thr Pro Ser Pro Thr 370 375 380Thr Pro Thr Thr Thr Pro Ser Pro
Thr Thr Pro Thr Thr Thr Pro Asp385 390 395 400Gln Val Asp Val Lys
Asp Ser Ala Asn Asn Glu Ile Lys Lys Val Met 405 410 415Val Asp Gly
Ile His Gly Ser Asp Pro Ser Ile Ile His Arg Gly Lys 420 425 430Pro
Phe Thr Leu Glu Ala Leu Phe Asp Ala Asn Gln Asn Thr Lys Thr 435 440
445Ala Lys Ile Glu Ile Lys Ala Ser Leu Asp Gly Leu Glu Ile Asp Val
450 455 460Pro Gly Ile Asp Thr Asn Ala Ser His Phe Met Lys Ser Pro
Leu Val465 470 475 480Lys Gly Gln Gln Tyr Asp Ile Lys Tyr Thr Trp
Asn Val Pro Lys Ile 485 490 495Ala Pro Lys Ser Glu Asn Val Val Val
Thr Val Lys Leu Ile Gly Asp 500 505 510Asn Gly Val Leu Ala Ser Ala
Ile Ala Thr His Gly Lys Ile Arg Asp 515 520 52575511PRTArtificial
sequenceIMAT_NTERM Dog Hybrid1 75Met His His His His His His Tyr
Gly Arg Lys Lys Arg Arg Gln Arg1 5 10 15Arg Arg Met Glu Asp Gln Arg
Asp Leu Ile Ser Asn His Glu Gln Leu 20 25 30Pro Ile Leu Gly Gln Arg
Pro Gly Ala Pro Glu Ser Lys Ser Ser Arg 35 40 45Gly Ala Leu Tyr Thr
Gly Phe Ser Val Leu Val Ala Leu Leu Leu Ala 50 55 60Gly Gln Ala Thr
Thr Ala Tyr Phe Leu Tyr Gln Gln Gln Gly Arg Leu65 70 75 80Asp Lys
Leu Thr Val Thr Ser Gln Asn Leu Gln Leu Glu Ser Leu Arg 85 90 95Met
Lys Leu Pro Lys Pro Pro Lys Pro Leu Ser Lys Met Arg Val Ala 100 105
110Thr Pro Met Met Met Gln Ala Leu Pro Ile Gln Ser Leu Pro Gln Gly
115 120 125Gln Ser His Gln Tyr Tyr His Thr Ser Gly Leu Arg Asn Leu
Gly Gly 130 135 140Ser Tyr Tyr Arg Ser Gly Asn Ala Lys Ala Met Ile
Ala Val Gly Gly145 150 155 160Ser Thr Met Ile Val Asp Gly Asp Lys
Val Thr Ile Tyr Gly Trp Gly 165 170 175Ser Gly Leu Gly Tyr Gly Leu
Gly Tyr Gly Leu Gly Tyr Gly Gln Ala 180 185 190Val Ala Leu Ala Pro
Ala Gln Ala Val Gly Tyr Val Ala Ala Ala Pro 195 200 205Ala Val Ala
Val Gln Ala Pro Ala Val Ser Tyr Ala Ala Ala Ala Pro 210 215 220Ala
Val Gln Thr Val Ala Val Gln Ala Pro Ala Val Ser Tyr Ala Ala225 230
235 240Ala Pro Ala Val Ala Val Gln Ala His Thr Ala Gln Val Ser Gly
Pro 245 250 255Ile His Ala Ala Ile Glu Ser Arg Arg Thr Val Glu Val
Ile Asp Gly 260 265 270Pro Ser Thr Gly Asp Ala Pro Val Ala Ser Thr
Val Val Ile Gly Pro 275 280 285Asn Val Gln Pro Ile Asn Leu Glu Phe
Gln Thr Gln Ala Ser Pro Leu 290 295 300Ala Ala Thr Gln Asn His Val
Pro Thr Thr Pro Thr Thr Pro Thr Pro305 310 315 320Ala Pro Thr Thr
Ser Thr Pro Asp Leu Leu Arg Gln Asp Ile Val Lys 325 330 335Pro Val
Val Gln Asp Val His Glu Phe Leu Val Tyr Ile His Ile Ala 340 345
350Asn Asn Glu Ile Lys Lys Val Gln Glu Ser Val His Gln Ile Leu Pro
355 360 365Arg Gly Gln Met Met Lys Ile Tyr Gln Gln Gln Gln Gln Gln
His His 370 375 380Pro Gln Arg Glu Glu Asn Ile Trp Ser Asp His Ile
Ala Asn Val Ala385 390 395 400Gln Ala Ala Pro Ala Ile Ser Ala Val
Arg Val Ala Ala Ala Pro Ala 405 410 415Val Ala Tyr Ala Ala Pro Ala
Val Ser Thr Val Ser Ala Ala Pro Ala 420 425 430Ala Ile Gly Val Ile
Gly Val Gln Pro Val Ala Gly Tyr Ile Gly Tyr 435 440 445Gly Ala Gly
Tyr Gly Thr Gly Tyr Gly Thr Gly Tyr Gly Val Ala Lys 450 455 460Tyr
Gly Thr Gly Tyr Gly Leu Thr Ser Gly Leu Ile Gly Gly Gly Ser465 470
475 480Tyr Gly Ser Ser Tyr Ser Val Gln Pro Ala Ser Tyr Gly Thr Gly
Tyr 485 490 495Gly Tyr Thr Thr Tyr Ser Ser Asp Ala Tyr Pro Ile Arg
Lys Lys 500 505 51076511PRTArtificial sequenceIMAT_CTERM Dog
Hybrid1 76Met Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Met Glu
Asp Gln1 5 10 15Arg Asp Leu Ile Ser Asn His Glu Gln Leu Pro Ile Leu
Gly Gln Arg 20 25 30Pro Gly Ala Pro Glu Ser Lys Ser Ser Arg Gly Ala
Leu Tyr Thr Gly 35 40 45Phe Ser Val Leu Val Ala Leu Leu Leu Ala Gly
Gln Ala Thr Thr Ala 50 55 60Tyr Phe Leu Tyr Gln Gln Gln Gly Arg Leu
Asp Lys Leu Thr Val Thr65 70 75 80Ser Gln Asn Leu Gln Leu Glu Ser
Leu Arg Met Lys Leu Pro Lys Pro 85 90 95Pro Lys Pro Leu Ser Lys Met
Arg Val Ala Thr Pro Met Met Met Gln 100 105 110Ala Leu Pro Ile Gln
Ser Leu Pro Gln Gly Gln Ser His Gln Tyr Tyr 115 120 125His Thr Ser
Gly Leu Arg Asn Leu Gly Gly Ser Tyr Tyr Arg Ser Gly 130 135 140Asn
Ala Lys Ala Met Ile Ala Val Gly Gly Ser Thr Met Ile Val Asp145 150
155 160Gly Asp Lys Val Thr Ile Tyr Gly Trp Gly Ser Gly Leu Gly Tyr
Gly 165 170 175Leu Gly Tyr Gly Leu Gly Tyr Gly Gln Ala Val Ala Leu
Ala Pro Ala 180 185 190Gln Ala Val Gly Tyr Val Ala Ala Ala Pro Ala
Val Ala Val Gln Ala 195 200 205Pro Ala Val Ser Tyr Ala Ala Ala Ala
Pro Ala Val Gln Thr Val Ala 210 215 220Val Gln Ala Pro Ala Val Ser
Tyr Ala Ala Ala Pro Ala Val Ala Val225 230 235 240Gln Ala His Thr
Ala Gln Val Ser Gly Pro Ile His Ala Ala Ile Glu 245 250 255Ser Arg
Arg Thr Val Glu Val Ile Asp Gly Pro Ser Thr Gly Asp Ala 260 265
270Pro Val Ala Ser Thr Val Val Ile Gly Pro Asn Val Gln Pro Ile Asn
275 280 285Leu Glu Phe Gln Thr Gln Ala Ser Pro Leu Ala Ala Thr Gln
Asn His 290 295 300Val Pro Thr Thr Pro Thr Thr Pro Thr Pro Ala Pro
Thr Thr Ser Thr305 310 315 320Pro Asp Leu Leu Arg Gln Asp Ile Val
Lys Pro Val Val Gln Asp Val 325 330 335His Glu Phe Leu Val Tyr Ile
His Ile Ala Asn Asn Glu Ile Lys Lys 340 345 350Val Gln Glu Ser Val
His Gln Ile Leu Pro Arg Gly Gln Met Met Lys 355 360 365Ile Tyr Gln
Gln Gln Gln Gln Gln His His Pro Gln Arg Glu Glu Asn 370 375 380Ile
Trp Ser Asp His Ile Ala Asn Val Ala Gln Ala Ala Pro Ala Ile385 390
395 400Ser Ala Val Arg Val Ala Ala Ala Pro Ala Val Ala Tyr Ala Ala
Pro 405 410 415Ala Val Ser Thr Val Ser Ala Ala Pro Ala Ala Ile Gly
Val Ile Gly 420 425 430Val Gln Pro Val Ala Gly Tyr Ile Gly Tyr Gly
Ala Gly Tyr Gly Thr 435 440 445Gly Tyr Gly Thr Gly Tyr Gly Val Ala
Lys Tyr Gly Thr Gly Tyr Gly 450 455 460Leu Thr Ser Gly Leu Ile Gly
Gly Gly Ser Tyr Gly Ser Ser Tyr Ser465 470 475 480Val Gln Pro Ala
Ser Tyr Gly Thr Gly Tyr Gly Tyr Thr Thr Tyr Ser 485 490 495Ser Asp
Ala Tyr Pro Ile Arg Lys Lys His His His His His His 500 505
51077504PRTArtificial sequenceHybrid 1 iMAT molecule (dog) without
tag 77Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Met Glu Asp Gln
Arg1 5 10 15Asp Leu Ile Ser Asn His Glu Gln Leu Pro Ile Leu Gly Gln
Arg Pro 20 25 30Gly Ala Pro Glu Ser Lys Cys Ser Arg Gly Ala Leu Tyr
Thr Gly Phe 35 40 45Ser Val Leu Val Ala Leu Leu Leu Ala Gly Gln Ala
Thr Thr Ala Tyr 50 55 60Phe Leu Tyr Gln Gln Gln Gly Arg Leu Asp Lys
Leu Thr Val Thr Ser65 70 75 80Gln Asn Leu Gln Leu Glu Ser Leu Arg
Met Lys Leu Pro Lys Pro Pro 85 90 95Lys Pro Leu Ser Lys Met Arg Val
Ala Thr Pro Met Met Met Gln Ala 100 105 110Leu Pro Ile Gln Ser Leu
Pro Gln Gly Gln Ser His Gln Tyr Tyr His 115 120 125Thr Ser Gly Leu
Arg Asn Leu Gly Gly Ser Tyr Tyr Arg Ser Gly Asn 130 135 140Ala Lys
Ala Met Ile Ala Val Gly Gly Ser Thr Met Ile Val Asp Gly145 150 155
160Asp Lys Val Thr Ile Tyr Gly Trp Gly Ser Gly Leu Gly Tyr Gly Leu
165 170 175Gly Tyr Gly Leu Gly Tyr Gly Gln Ala Val Ala Leu Ala Pro
Ala Gln 180 185 190Ala Val Gly Tyr Val Ala Ala Ala Pro Ala Val Ala
Val Gln Ala Pro 195 200 205Ala Val Ser Tyr Ala Ala Ala Ala Pro Ala
Val Gln Thr Val Ala Val 210 215 220Gln Ala Pro Ala Val Ser Tyr Ala
Ala Ala Pro Ala Val Ala Val Gln225 230 235 240Ala His Thr Ala Gln
Val Ser Gly Pro Ile His Ala Ala Ile Glu Ser 245 250 255Arg Arg Thr
Val Glu Val Ile Asp Gly Pro Ser Thr Gly Asp Ala Pro 260 265 270Val
Ala Ser Thr Val Val Ile Gly Pro Asn Val Gln Pro Ile Asn Leu 275 280
285Glu Phe Gln Thr Gln Ala Ser Pro Leu Ala Ala Thr Gln Asn His Val
290 295 300Pro Thr Thr Pro Thr Thr Pro Thr Pro Ala Pro Thr Thr Ser
Thr Pro305 310 315 320Asp Leu Leu Arg Gln Asp Ile Val Lys Pro Val
Val Gln Asp Val His 325 330 335Glu Phe Leu Val Tyr Ile His Ile Ala
Asn Asn Glu Ile Lys Lys Val 340 345 350Gln Glu Ser Val His Gln Ile
Leu Pro Arg Gly Gln Met Met Lys Ile 355 360 365Tyr Gln Gln Gln Gln
Gln Gln His His Pro Gln Arg Glu Glu Asn Ile 370 375 380Trp Ser Asp
His Ile Ala Asn Val Ala Gln Ala Ala Pro Ala Ile Ser385 390 395
400Ala Val Arg Val Ala Ala Ala Pro Ala Val Ala Tyr Ala Ala Pro Ala
405 410 415Val Ser Thr Val Ser Ala Ala Pro Ala Ala Ile Gly Val Ile
Gly Val 420 425 430Gln Pro Val Ala Gly Tyr Ile Gly Tyr Gly Ala Gly
Tyr Gly Thr Gly 435 440 445Tyr Gly Thr Gly Tyr Gly Val Ala Lys Tyr
Gly Thr Gly Tyr Gly Leu 450 455 460Thr Ser Gly Leu Ile Gly Gly Gly
Ser Tyr Gly Ser Ser Tyr Ser Val465 470 475 480Gln Pro Ala Ser Tyr
Gly Thr Gly Tyr Gly Tyr Thr Thr Tyr Ser Ser 485 490 495Asp Ala Tyr
Pro Ile Arg Lys Lys 50078645PRTArtificial sequenceIMAT_NTERM Dog
Hybrid2 78Met His His His His His His Tyr Gly Arg Lys Lys Arg Arg
Gln Arg1 5 10 15Arg Arg Met Glu Asp Gln Arg Asp Leu Ile Ser Asn His
Glu Gln Leu 20 25 30Pro Ile Leu Gly Gln Arg Pro Gly Ala Pro Glu Ser
Lys Ser Ser Arg 35 40 45Gly Ala Leu Tyr Thr Gly Phe Ser Val Leu Val
Ala Leu Leu Leu Ala 50 55 60Gly Gln Ala Thr Thr Ala Tyr Phe Leu Tyr
Gln Gln Gln Gly Arg Leu65 70 75 80Asp Lys Leu Thr Val Thr Ser Gln
Asn Leu Gln Leu Glu Ser Leu Arg 85 90 95Met Lys Leu Pro Lys Pro Pro
Lys Pro Leu Ser Lys Met Arg Val Ala 100 105 110Thr Pro Met Met Met
Gln Ala Leu Pro Ile Gln Ser Leu Pro Gln Gly 115 120 125Thr Ser Ala
Ser Arg Ile Asn Ser Val Asn Val Pro Ser Glu Leu Asp 130 135 140Leu
Arg Ser Leu Arg Thr Val Thr Pro Ile Arg Met Gln Gly Gly Ser145 150
155 160Gly Ser Ser Trp Ala Phe Ser Gly Val Ala Ala Thr Glu Ser Ala
Tyr 165 170 175Leu Ala Tyr Arg Asn Thr Ser Leu Asp Leu Ser Glu Gln
Glu Leu Val 180 185 190Asp Ser Ala Ser Gln His Gly Ser His Gly Asp
Thr Ile Pro Arg Gly 195 200 205Ile Glu Tyr Ile Gln Gln Asn Gly Val
Val Glu Glu Arg Ser Tyr Pro 210 215 220Tyr Val Ala Arg Glu Gln Gln
Ser Arg Arg Pro Asn Ser Gln His Tyr225 230 235 240Gly Ile Ser Asn
Tyr Ser Gln Ile Tyr Pro Pro Asp Val Lys Gln Ile 245 250 255Arg Glu
Ala Leu Thr Gln Thr His Thr Ala Ile Ala Val Ile Ile Gly 260 265
270Ile Lys Asp Leu Arg Ala Phe Gln His Tyr Asp Gly Arg Thr Ile Ile
275 280 285Gln His Asp Asn Gly Tyr Gln Pro Asn Tyr His Ala Val Asn
Ile Val 290 295 300Gly Tyr Gly Ser Thr Gln Gly Val Asp Tyr Trp Ile
Val Arg Asn Ser305 310 315 320Trp Asp Thr Thr Trp Gly Asp Ser Gly
Tyr Gly Tyr Phe Gln Ala Gly 325 330 335Asn Asn Leu Met Met Ile Glu
Gln Tyr Pro Tyr Val Val Ile Met Phe 340 345 350Thr Gln Thr Asp Gly
Phe Leu Ser Tyr Asn Glu Leu Ser Val Gln Ile 355 360 365Gln Ala Glu
Thr Asn Ala Phe Thr Ile Thr Arg Glu Pro Thr Thr Pro 370 375 380Thr
Pro Glu Pro Thr Thr Lys Thr Pro Glu Pro Thr Thr Lys Thr Pro385 390
395 400Glu Pro Ser Thr Pro Thr Pro Glu Pro Thr Thr Lys Thr Pro Glu
Pro 405 410 415Thr Thr Lys Asp Ile Asp His Asp Asp Asp Pro Thr Thr
Met Ile Asp 420 425 430Val Gln Thr Thr Thr Val Gln Pro Ser Asp Glu
Phe Glu Ser Pro Thr 435 440 445Arg Phe Gly Tyr Phe Ala Asp Pro Lys
Asp Pro Ile Lys Phe Tyr Ile 450 455 460Ser Ser Asn Trp Glu Ala Ile
His Lys Ser Ser Pro Gly Asn Thr Arg465 470 475
480Trp Asn Glu Lys Glu Leu Thr Ser Thr Ser Pro Thr Thr Pro Thr Thr
485 490 495Thr Pro Ser Pro Thr Thr Pro Thr Thr Thr Pro Ser Pro Thr
Thr Pro 500 505 510Thr Thr Thr Pro Asp Gln Val Asp Val Lys Asp Leu
Ala Asn Asn Glu 515 520 525Ile Lys Lys Val Met Val Asp Gly Ile His
Gly Ser Asp Pro Ser Ile 530 535 540Ile His Arg Gly Lys Pro Phe Thr
Leu Glu Ala Leu Phe Asp Ala Asn545 550 555 560Gln Asn Thr Lys Thr
Ala Lys Ile Glu Ile Lys Ala Ser Leu Asp Gly 565 570 575Leu Glu Ile
Asp Val Pro Gly Ile Asp Thr Asn Ala Leu His Phe Met 580 585 590Lys
Ile Pro Leu Val Lys Gly Gln Gln Tyr Asp Ile Lys Tyr Thr Trp 595 600
605Asn Val Pro Lys Ile Ala Pro Lys Ser Glu Asn Val Val Val Thr Val
610 615 620Lys Leu Ile Gly Asp Asn Gly Val Leu Ala Ser Ala Ile Ala
Thr His625 630 635 640Gly Lys Ile Arg Asp 64579645PRTArtificial
sequenceIMAT_CTERM Dog Hybrid2 79Met Tyr Gly Arg Lys Lys Arg Arg
Gln Arg Arg Arg Met Glu Asp Gln1 5 10 15Arg Asp Leu Ile Ser Asn His
Glu Gln Leu Pro Ile Leu Gly Gln Arg 20 25 30Pro Gly Ala Pro Glu Ser
Lys Ser Ser Arg Gly Ala Leu Tyr Thr Gly 35 40 45Phe Ser Val Leu Val
Ala Leu Leu Leu Ala Gly Gln Ala Thr Thr Ala 50 55 60Tyr Phe Leu Tyr
Gln Gln Gln Gly Arg Leu Asp Lys Leu Thr Val Thr65 70 75 80Ser Gln
Asn Leu Gln Leu Glu Ser Leu Arg Met Lys Leu Pro Lys Pro 85 90 95Pro
Lys Pro Leu Ser Lys Met Arg Val Ala Thr Pro Met Met Met Gln 100 105
110Ala Leu Pro Ile Gln Ser Leu Pro Gln Gly Thr Ser Ala Ser Arg Ile
115 120 125Asn Ser Val Asn Val Pro Ser Glu Leu Asp Leu Arg Ser Leu
Arg Thr 130 135 140Val Thr Pro Ile Arg Met Gln Gly Gly Ser Gly Ser
Ser Trp Ala Phe145 150 155 160Ser Gly Val Ala Ala Thr Glu Ser Ala
Tyr Leu Ala Tyr Arg Asn Thr 165 170 175Ser Leu Asp Leu Ser Glu Gln
Glu Leu Val Asp Ser Ala Ser Gln His 180 185 190Gly Ser His Gly Asp
Thr Ile Pro Arg Gly Ile Glu Tyr Ile Gln Gln 195 200 205Asn Gly Val
Val Glu Glu Arg Ser Tyr Pro Tyr Val Ala Arg Glu Gln 210 215 220Gln
Ser Arg Arg Pro Asn Ser Gln His Tyr Gly Ile Ser Asn Tyr Ser225 230
235 240Gln Ile Tyr Pro Pro Asp Val Lys Gln Ile Arg Glu Ala Leu Thr
Gln 245 250 255Thr His Thr Ala Ile Ala Val Ile Ile Gly Ile Lys Asp
Leu Arg Ala 260 265 270Phe Gln His Tyr Asp Gly Arg Thr Ile Ile Gln
His Asp Asn Gly Tyr 275 280 285Gln Pro Asn Tyr His Ala Val Asn Ile
Val Gly Tyr Gly Ser Thr Gln 290 295 300Gly Val Asp Tyr Trp Ile Val
Arg Asn Ser Trp Asp Thr Thr Trp Gly305 310 315 320Asp Ser Gly Tyr
Gly Tyr Phe Gln Ala Gly Asn Asn Leu Met Met Ile 325 330 335Glu Gln
Tyr Pro Tyr Val Val Ile Met Phe Thr Gln Thr Asp Gly Phe 340 345
350Leu Ser Tyr Asn Glu Leu Ser Val Gln Ile Gln Ala Glu Thr Asn Ala
355 360 365Phe Thr Ile Thr Arg Glu Pro Thr Thr Pro Thr Pro Glu Pro
Thr Thr 370 375 380Lys Thr Pro Glu Pro Thr Thr Lys Thr Pro Glu Pro
Ser Thr Pro Thr385 390 395 400Pro Glu Pro Thr Thr Lys Thr Pro Glu
Pro Thr Thr Lys Asp Ile Asp 405 410 415His Asp Asp Asp Pro Thr Thr
Met Ile Asp Val Gln Thr Thr Thr Val 420 425 430Gln Pro Ser Asp Glu
Phe Glu Ser Pro Thr Arg Phe Gly Tyr Phe Ala 435 440 445Asp Pro Lys
Asp Pro Ile Lys Phe Tyr Ile Ser Ser Asn Trp Glu Ala 450 455 460Ile
His Lys Ser Ser Pro Gly Asn Thr Arg Trp Asn Glu Lys Glu Leu465 470
475 480Thr Ser Thr Ser Pro Thr Thr Pro Thr Thr Thr Pro Ser Pro Thr
Thr 485 490 495Pro Thr Thr Thr Pro Ser Pro Thr Thr Pro Thr Thr Thr
Pro Asp Gln 500 505 510Val Asp Val Lys Asp Leu Ala Asn Asn Glu Ile
Lys Lys Val Met Val 515 520 525Asp Gly Ile His Gly Ser Asp Pro Ser
Ile Ile His Arg Gly Lys Pro 530 535 540Phe Thr Leu Glu Ala Leu Phe
Asp Ala Asn Gln Asn Thr Lys Thr Ala545 550 555 560Lys Ile Glu Ile
Lys Ala Ser Leu Asp Gly Leu Glu Ile Asp Val Pro 565 570 575Gly Ile
Asp Thr Asn Ala Leu His Phe Met Lys Ile Pro Leu Val Lys 580 585
590Gly Gln Gln Tyr Asp Ile Lys Tyr Thr Trp Asn Val Pro Lys Ile Ala
595 600 605Pro Lys Ser Glu Asn Val Val Val Thr Val Lys Leu Ile Gly
Asp Asn 610 615 620Gly Val Leu Ala Ser Ala Ile Ala Thr His Gly Lys
Ile Arg Asp His625 630 635 640His His His His His
64580638PRTArtificial sequenceHybrid 2 iMAT molecule (dog) without
tag 80Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Met Glu Asp Gln
Arg1 5 10 15Asp Leu Ile Ser Asn His Glu Gln Leu Pro Ile Leu Gly Gln
Arg Pro 20 25 30Gly Ala Pro Glu Ser Lys Cys Ser Arg Gly Ala Leu Tyr
Thr Gly Phe 35 40 45Ser Val Leu Val Ala Leu Leu Leu Ala Gly Gln Ala
Thr Thr Ala Tyr 50 55 60Phe Leu Tyr Gln Gln Gln Gly Arg Leu Asp Lys
Leu Thr Val Thr Ser65 70 75 80Gln Asn Leu Gln Leu Glu Ser Leu Arg
Met Lys Leu Pro Lys Pro Pro 85 90 95Lys Pro Leu Ser Lys Met Arg Val
Ala Thr Pro Met Met Met Gln Ala 100 105 110Leu Pro Ile Gln Ser Leu
Pro Gln Gly Thr Ser Ala Ser Arg Ile Asn 115 120 125Ser Val Asn Val
Pro Ser Glu Leu Asp Leu Arg Ser Leu Arg Thr Val 130 135 140Thr Pro
Ile Arg Met Gln Gly Gly Ser Gly Ser Ser Trp Ala Phe Ser145 150 155
160Gly Val Ala Ala Thr Glu Ser Ala Tyr Leu Ala Tyr Arg Asn Thr Ser
165 170 175Leu Asp Leu Ser Glu Gln Glu Leu Val Asp Ser Ala Ser Gln
His Gly 180 185 190Ser His Gly Asp Thr Ile Pro Arg Gly Ile Glu Tyr
Ile Gln Gln Asn 195 200 205Gly Val Val Glu Glu Arg Ser Tyr Pro Tyr
Val Ala Arg Glu Gln Gln 210 215 220Ser Arg Arg Pro Asn Ser Gln His
Tyr Gly Ile Ser Asn Tyr Ser Gln225 230 235 240Ile Tyr Pro Pro Asp
Val Lys Gln Ile Arg Glu Ala Leu Thr Gln Thr 245 250 255His Thr Ala
Ile Ala Val Ile Ile Gly Ile Lys Asp Leu Arg Ala Phe 260 265 270Gln
His Tyr Asp Gly Arg Thr Ile Ile Gln His Asp Asn Gly Tyr Gln 275 280
285Pro Asn Tyr His Ala Val Asn Ile Val Gly Tyr Gly Ser Thr Gln Gly
290 295 300Val Asp Tyr Trp Ile Val Arg Asn Ser Trp Asp Thr Thr Trp
Gly Asp305 310 315 320Ser Gly Tyr Gly Tyr Phe Gln Ala Gly Asn Asn
Leu Met Met Ile Glu 325 330 335Gln Tyr Pro Tyr Val Val Ile Met Phe
Thr Gln Thr Asp Gly Phe Leu 340 345 350Ser Tyr Asn Glu Leu Ser Val
Gln Ile Gln Ala Glu Thr Asn Ala Phe 355 360 365Thr Ile Thr Arg Glu
Pro Thr Thr Pro Thr Pro Glu Pro Thr Thr Lys 370 375 380Thr Pro Glu
Pro Thr Thr Lys Thr Pro Glu Pro Ser Thr Pro Thr Pro385 390 395
400Glu Pro Thr Thr Lys Thr Pro Glu Pro Thr Thr Lys Asp Ile Asp His
405 410 415Asp Asp Asp Pro Thr Thr Met Ile Asp Val Gln Thr Thr Thr
Val Gln 420 425 430Pro Ser Asp Glu Phe Glu Ser Pro Thr Arg Phe Gly
Tyr Phe Ala Asp 435 440 445Pro Lys Asp Pro Ile Lys Phe Tyr Ile Ser
Ser Asn Trp Glu Ala Ile 450 455 460His Lys Ser Ser Pro Gly Asn Thr
Arg Trp Asn Glu Lys Glu Leu Thr465 470 475 480Ser Thr Ser Pro Thr
Thr Pro Thr Thr Thr Pro Ser Pro Thr Thr Pro 485 490 495Thr Thr Thr
Pro Ser Pro Thr Thr Pro Thr Thr Thr Pro Asp Gln Val 500 505 510Asp
Val Lys Asp Leu Ala Asn Asn Glu Ile Lys Lys Val Met Val Asp 515 520
525Gly Ile His Gly Ser Asp Pro Ser Ile Ile His Arg Gly Lys Pro Phe
530 535 540Thr Leu Glu Ala Leu Phe Asp Ala Asn Gln Asn Thr Lys Thr
Ala Lys545 550 555 560Ile Glu Ile Lys Ala Ser Leu Asp Gly Leu Glu
Ile Asp Val Pro Gly 565 570 575Ile Asp Thr Asn Ala Leu His Phe Met
Lys Ile Pro Leu Val Lys Gly 580 585 590Gln Gln Tyr Asp Ile Lys Tyr
Thr Trp Asn Val Pro Lys Ile Ala Pro 595 600 605Lys Ser Glu Asn Val
Val Val Thr Val Lys Leu Ile Gly Asp Asn Gly 610 615 620Val Leu Ala
Ser Ala Ile Ala Thr His Gly Lys Ile Arg Asp625 630
63581535PRTArtificial sequenceIMAT_NTERM Dog Hybrid3 81Met His His
His His His His Tyr Gly Arg Lys Lys Arg Arg Gln Arg1 5 10 15Arg Arg
Met Glu Asp Gln Arg Asp Leu Ile Ser Asn His Glu Gln Leu 20 25 30Pro
Ile Leu Gly Gln Arg Pro Gly Ala Pro Glu Ser Lys Ser Ser Arg 35 40
45Gly Ala Leu Tyr Thr Gly Phe Ser Val Leu Val Ala Leu Leu Leu Ala
50 55 60Gly Gln Ala Thr Thr Ala Tyr Phe Leu Tyr Gln Gln Gln Gly Arg
Leu65 70 75 80Asp Lys Leu Thr Val Thr Ser Gln Asn Leu Gln Leu Glu
Ser Leu Arg 85 90 95Met Lys Leu Pro Lys Pro Pro Lys Pro Leu Ser Lys
Met Arg Val Ala 100 105 110Thr Pro Met Met Met Gln Ala Leu Pro Ile
Gln Ser Leu Pro Gln Gly 115 120 125Thr Ser Ala Ser Arg Ile Asn Ser
Val Asn Val Pro Ser Glu Leu Asp 130 135 140Leu Arg Ser Leu Arg Thr
Val Thr Pro Ile Arg Met Gln Gly Gly Ser145 150 155 160Gly Ser Leu
Trp Ala Phe Ser Gly Val Ala Ala Thr Glu Ser Ala Tyr 165 170 175Leu
Ala Tyr Arg Asn Thr Ser Leu Asp Leu Ser Glu Gln Glu Leu Val 180 185
190Asp Ile Ala Ser Gln His Gly Leu His Gly Asp Thr Ile Pro Arg Gly
195 200 205Ile Glu Tyr Ile Gln Gln Asn Gly Val Val Glu Glu Arg Ser
Tyr Pro 210 215 220Tyr Val Ala Arg Glu Gln Gln Ser Arg Arg Pro Asn
Ser Gln His Tyr225 230 235 240Gly Ile Ser Asn Tyr Leu Gln Ile Tyr
Pro Pro Asp Val Lys Gln Ile 245 250 255Arg Glu Ala Leu Thr Gln Thr
His Thr Ala Ile Ala Val Ile Ile Gly 260 265 270Ile Lys Asp Leu Arg
Ala Phe Gln His Tyr Asp Gly Arg Thr Ile Ile 275 280 285Gln His Asp
Asn Gly Tyr Gln Pro Asn Tyr His Ala Val Asn Ile Val 290 295 300Gly
Tyr Gly Ser Thr Gln Gly Val Asp Tyr Trp Ile Val Arg Asn Ser305 310
315 320Trp Asp Thr Thr Trp Gly Asp Ser Gly Tyr Gly Tyr Phe Gln Ala
Gly 325 330 335Asn Asn Leu Met Met Ile Glu Gln Tyr Pro Tyr Val Val
Ile Met Phe 340 345 350Thr Gln Thr Asp Gly Phe Leu Ser Tyr Asn Glu
Leu Ser Val Gln Ile 355 360 365Gln Ala Glu Thr Asn Ala Phe Thr Ile
Thr Arg Ser Pro Thr Thr Pro 370 375 380Thr Thr Thr Pro Ser Pro Thr
Thr Pro Thr Thr Thr Pro Ser Pro Thr385 390 395 400Thr Pro Thr Thr
Thr Pro Asp Gln Val Asp Val Lys Asp Ser Ala Asn 405 410 415Asn Glu
Ile Lys Lys Val Met Val Asp Gly Ile His Gly Ser Asp Pro 420 425
430Ser Ile Ile His Arg Gly Lys Pro Phe Thr Leu Glu Ala Leu Phe Asp
435 440 445Ala Asn Gln Asn Thr Lys Thr Ala Lys Ile Glu Ile Lys Ala
Ser Leu 450 455 460Asp Gly Leu Glu Ile Asp Val Pro Gly Ile Asp Thr
Asn Ala Ser His465 470 475 480Phe Met Lys Ser Pro Leu Val Lys Gly
Gln Gln Tyr Asp Ile Lys Tyr 485 490 495Thr Trp Asn Val Pro Lys Ile
Ala Pro Lys Ser Glu Asn Val Val Val 500 505 510Thr Val Lys Leu Ile
Gly Asp Asn Gly Val Leu Ala Ser Ala Ile Ala 515 520 525Thr His Gly
Lys Ile Arg Asp 530 53582535PRTArtificial sequenceIMAT_CTERM Dog
Hybrid3 82Met Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Met Glu
Asp Gln1 5 10 15Arg Asp Leu Ile Ser Asn His Glu Gln Leu Pro Ile Leu
Gly Gln Arg 20 25 30Pro Gly Ala Pro Glu Ser Lys Ser Ser Arg Gly Ala
Leu Tyr Thr Gly 35 40 45Phe Ser Val Leu Val Ala Leu Leu Leu Ala Gly
Gln Ala Thr Thr Ala 50 55 60Tyr Phe Leu Tyr Gln Gln Gln Gly Arg Leu
Asp Lys Leu Thr Val Thr65 70 75 80Ser Gln Asn Leu Gln Leu Glu Ser
Leu Arg Met Lys Leu Pro Lys Pro 85 90 95Pro Lys Pro Leu Ser Lys Met
Arg Val Ala Thr Pro Met Met Met Gln 100 105 110Ala Leu Pro Ile Gln
Ser Leu Pro Gln Gly Thr Ser Ala Ser Arg Ile 115 120 125Asn Ser Val
Asn Val Pro Ser Glu Leu Asp Leu Arg Ser Leu Arg Thr 130 135 140Val
Thr Pro Ile Arg Met Gln Gly Gly Ser Gly Ser Leu Trp Ala Phe145 150
155 160Ser Gly Val Ala Ala Thr Glu Ser Ala Tyr Leu Ala Tyr Arg Asn
Thr 165 170 175Ser Leu Asp Leu Ser Glu Gln Glu Leu Val Asp Ile Ala
Ser Gln His 180 185 190Gly Leu His Gly Asp Thr Ile Pro Arg Gly Ile
Glu Tyr Ile Gln Gln 195 200 205Asn Gly Val Val Glu Glu Arg Ser Tyr
Pro Tyr Val Ala Arg Glu Gln 210 215 220Gln Ser Arg Arg Pro Asn Ser
Gln His Tyr Gly Ile Ser Asn Tyr Leu225 230 235 240Gln Ile Tyr Pro
Pro Asp Val Lys Gln Ile Arg Glu Ala Leu Thr Gln 245 250 255Thr His
Thr Ala Ile Ala Val Ile Ile Gly Ile Lys Asp Leu Arg Ala 260 265
270Phe Gln His Tyr Asp Gly Arg Thr Ile Ile Gln His Asp Asn Gly Tyr
275 280 285Gln Pro Asn Tyr His Ala Val Asn Ile Val Gly Tyr Gly Ser
Thr Gln 290 295 300Gly Val Asp Tyr Trp Ile Val Arg Asn Ser Trp Asp
Thr Thr Trp Gly305 310 315 320Asp Ser Gly Tyr Gly Tyr Phe Gln Ala
Gly Asn Asn Leu Met Met Ile 325 330 335Glu Gln Tyr Pro Tyr Val Val
Ile Met Phe Thr Gln Thr Asp Gly Phe 340 345 350Leu Ser Tyr Asn Glu
Leu Ser Val Gln Ile Gln Ala Glu Thr Asn Ala 355 360 365Phe Thr Ile
Thr Arg Ser Pro Thr Thr Pro Thr Thr Thr Pro Ser Pro 370 375 380Thr
Thr Pro Thr Thr Thr Pro Ser Pro Thr Thr Pro Thr Thr Thr Pro385 390
395 400Asp Gln Val Asp Val Lys Asp Ser Ala Asn Asn Glu Ile Lys Lys
Val 405 410 415Met Val Asp Gly Ile His Gly Ser Asp Pro Ser Ile Ile
His Arg Gly 420 425 430Lys Pro Phe Thr Leu Glu Ala Leu Phe Asp Ala
Asn Gln Asn Thr Lys 435 440 445Thr Ala Lys Ile Glu Ile Lys Ala Ser
Leu Asp Gly Leu Glu Ile Asp 450
455 460Val Pro Gly Ile Asp Thr Asn Ala Ser His Phe Met Lys Ser Pro
Leu465 470 475 480Val Lys Gly Gln Gln Tyr Asp Ile Lys Tyr Thr Trp
Asn Val Pro Lys 485 490 495Ile Ala Pro Lys Ser Glu Asn Val Val Val
Thr Val Lys Leu Ile Gly 500 505 510Asp Asn Gly Val Leu Ala Ser Ala
Ile Ala Thr His Gly Lys Ile Arg 515 520 525Asp His His His His His
His 530 53583528PRTArtificial sequenceHybrid 3 iMAT molecule (dog)
without tag 83Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Met Glu
Asp Gln Arg1 5 10 15Asp Leu Ile Ser Asn His Glu Gln Leu Pro Ile Leu
Gly Gln Arg Pro 20 25 30Gly Ala Pro Glu Ser Lys Cys Ser Arg Gly Ala
Leu Tyr Thr Gly Phe 35 40 45Ser Val Leu Val Ala Leu Leu Leu Ala Gly
Gln Ala Thr Thr Ala Tyr 50 55 60Phe Leu Tyr Gln Gln Gln Gly Arg Leu
Asp Lys Leu Thr Val Thr Ser65 70 75 80Gln Asn Leu Gln Leu Glu Ser
Leu Arg Met Lys Leu Pro Lys Pro Pro 85 90 95Lys Pro Leu Ser Lys Met
Arg Val Ala Thr Pro Met Met Met Gln Ala 100 105 110Leu Pro Ile Gln
Ser Leu Pro Gln Gly Thr Ser Ala Ser Arg Ile Asn 115 120 125Ser Val
Asn Val Pro Ser Glu Leu Asp Leu Arg Ser Leu Arg Thr Val 130 135
140Thr Pro Ile Arg Met Gln Gly Gly Ser Gly Ser Leu Trp Ala Phe
Ser145 150 155 160Gly Val Ala Ala Thr Glu Ser Ala Tyr Leu Ala Tyr
Arg Asn Thr Ser 165 170 175Leu Asp Leu Ser Glu Gln Glu Leu Val Asp
Ile Ala Ser Gln His Gly 180 185 190Leu His Gly Asp Thr Ile Pro Arg
Gly Ile Glu Tyr Ile Gln Gln Asn 195 200 205Gly Val Val Glu Glu Arg
Ser Tyr Pro Tyr Val Ala Arg Glu Gln Gln 210 215 220Ser Arg Arg Pro
Asn Ser Gln His Tyr Gly Ile Ser Asn Tyr Leu Gln225 230 235 240Ile
Tyr Pro Pro Asp Val Lys Gln Ile Arg Glu Ala Leu Thr Gln Thr 245 250
255His Thr Ala Ile Ala Val Ile Ile Gly Ile Lys Asp Leu Arg Ala Phe
260 265 270Gln His Tyr Asp Gly Arg Thr Ile Ile Gln His Asp Asn Gly
Tyr Gln 275 280 285Pro Asn Tyr His Ala Val Asn Ile Val Gly Tyr Gly
Ser Thr Gln Gly 290 295 300Val Asp Tyr Trp Ile Val Arg Asn Ser Trp
Asp Thr Thr Trp Gly Asp305 310 315 320Ser Gly Tyr Gly Tyr Phe Gln
Ala Gly Asn Asn Leu Met Met Ile Glu 325 330 335Gln Tyr Pro Tyr Val
Val Ile Met Phe Thr Gln Thr Asp Gly Phe Leu 340 345 350Ser Tyr Asn
Glu Leu Ser Val Gln Ile Gln Ala Glu Thr Asn Ala Phe 355 360 365Thr
Ile Thr Arg Ser Pro Thr Thr Pro Thr Thr Thr Pro Ser Pro Thr 370 375
380Thr Pro Thr Thr Thr Pro Ser Pro Thr Thr Pro Thr Thr Thr Pro
Asp385 390 395 400Gln Val Asp Val Lys Asp Ser Ala Asn Asn Glu Ile
Lys Lys Val Met 405 410 415Val Asp Gly Ile His Gly Ser Asp Pro Ser
Ile Ile His Arg Gly Lys 420 425 430Pro Phe Thr Leu Glu Ala Leu Phe
Asp Ala Asn Gln Asn Thr Lys Thr 435 440 445Ala Lys Ile Glu Ile Lys
Ala Ser Leu Asp Gly Leu Glu Ile Asp Val 450 455 460Pro Gly Ile Asp
Thr Asn Ala Ser His Phe Met Lys Ser Pro Leu Val465 470 475 480Lys
Gly Gln Gln Tyr Asp Ile Lys Tyr Thr Trp Asn Val Pro Lys Ile 485 490
495Ala Pro Lys Ser Glu Asn Val Val Val Thr Val Lys Leu Ile Gly Asp
500 505 510Asn Gly Val Leu Ala Ser Ala Ile Ala Thr His Gly Lys Ile
Arg Asp 515 520 52584400PRTArtificial
sequencetr|A1KXC1|A1KXC1_DERFA DFP1 OS=Dermatophagoides farinae
GN=DFP1 PE=2 SV=1 84Met Lys Phe Ala Leu Phe Val Val Ala Ser Leu Ile
Ala Thr Val Tyr1 5 10 15Gly Gln Ser His Gln Tyr Tyr His Thr Ser Gly
Leu Arg Asn Leu Gly 20 25 30Gly Ser Tyr Tyr Arg Ser Ala Gly Ile Ser
Gly Val Ala Gly Leu Gly 35 40 45Gly Leu Ala Tyr Gly Thr Gly Leu Gly
Tyr Gly Thr Arg Tyr Gly Tyr 50 55 60Gly Ser Gly Leu Gly Tyr Gly Leu
Gly Tyr Gly Leu Gly Tyr Gly Gln65 70 75 80Ala Val Ala Leu Ala Pro
Ala Gln Ala Val Gly Tyr Val Ala Ala Ala 85 90 95Pro Ala Val Ala Val
Gln Ala Pro Ala Val Ser Tyr Ala Ala Ala Ala 100 105 110Pro Ala Val
Gln Thr Val Ala Val Gln Ala Pro Ala Val Ser Tyr Ala 115 120 125Ala
Ala Pro Ala Val Ala Val Gln Ala His Thr Ala Gln Val Ser Gly 130 135
140Pro Ile His Ala Ala Ile Glu Ser Arg Arg Thr Val Glu Val Ile
Asp145 150 155 160Gly Pro Ser Thr Gly Asp Ala Pro Val Ala Ser Thr
Val Val Ile Gly 165 170 175Pro Asn Val Gln Pro Ile Asn Leu Glu Phe
Gln Thr Gln Ala Ser Pro 180 185 190Leu Ala Ala Thr Gln Asn His Val
Pro Thr Ala Pro Ala Glu Pro Gln 195 200 205Gln Ser Ser Tyr Glu Glu
Gln Pro Asp Leu Leu Arg Gln Asp Ile Val 210 215 220Lys Pro Val Val
Gln Asp Val His Glu Thr Ile Val Pro Phe Arg Arg225 230 235 240Ile
Thr Gln Glu Leu Lys Pro Val Gln Glu Ser Val His Gln Ile Leu 245 250
255Pro Arg Gly Gln Glu Arg Gly Phe Tyr Gln Gln Gln Gln Gln Val Arg
260 265 270Val Ala Gln His Val Ala Ala Pro Ala Ala Val Ala Val Gln
Pro Val 275 280 285Val Gln Ala Ala Pro Ala Ile Ser Ala Val Arg Val
Ala Ala Ala Pro 290 295 300Ala Val Ala Tyr Ala Ala Pro Ala Val Ser
Thr Val Ser Ala Ala Pro305 310 315 320Ala Ala Ile Gly Val Ile Gly
Val Gln Pro Val Ala Gly Tyr Ile Gly 325 330 335Tyr Gly Ala Gly Tyr
Gly Thr Gly Tyr Gly Thr Gly Tyr Gly Val Ala 340 345 350Lys Tyr Gly
Thr Gly Tyr Gly Leu Thr Ser Gly Leu Ile Gly Gly Gly 355 360 365Ser
Tyr Gly Ser Ser Tyr Ser Val Gln Pro Ala Ser Tyr Gly Thr Gly 370 375
380Tyr Gly Tyr Thr Thr Tyr Ser Ser Asp Ala Tyr Pro Ile Arg Lys
Lys385 390 395 40085654PRTArtificial
sequencetr|A0A088SAS1|A0A088SAS1_DERFA Der f 28 allergen
OS=Dermatophagoides farinae PE=2 SV=1 85Met Pro Ser Lys Thr Leu Lys
Ala Pro Ala Ile Gly Ile Asp Leu Gly1 5 10 15Thr Thr Tyr Ser Cys Val
Gly Val Phe Gln Asn Gly Ser Val Glu Ile 20 25 30Ile Ala Asn Asp Gln
Gly Asn Arg Thr Thr Pro Ser Tyr Val Ala Phe 35 40 45Asn Asp Thr Glu
Arg Leu Ile Gly Asp Ala Ala Lys Asn Gln Val Ser 50 55 60Met Asn Pro
Thr Asn Thr Ile Phe Asp Ala Lys Arg Leu Ile Gly Arg65 70 75 80Arg
Phe Asp Glu Ser Ser Val Lys Ser Asp Met Lys His Trp Pro Phe 85 90
95Lys Val Val Ser Glu Ser Gly Lys Pro Lys Leu Glu Val Glu Phe Lys
100 105 110Gly Glu Arg Lys Arg Phe Trp Pro Glu Glu Ile Ser Ala Met
Val Leu 115 120 125Thr Lys Met Lys Glu Thr Ala Glu Ala Tyr Leu Gly
Gln Lys Val Thr 130 135 140Asp Val Val Ile Thr Val Pro Ala Tyr Phe
Asn Asp Ser Gln Arg Gln145 150 155 160Ala Thr Lys Asp Ala Gly Val
Ile Ala Gly Leu Asn Val Leu Arg Ile 165 170 175Ile Asn Glu Pro Thr
Ala Ala Ala Ile Ala Tyr Gly Leu Asp Lys Lys 180 185 190Gly Gly Gly
Glu Lys Asn Val Leu Ile Phe Asp Leu Gly Gly Gly Thr 195 200 205Phe
Asp Val Ser Val Leu Thr Ile Asp Asn Gly Ile Phe Glu Val Lys 210 215
220Ser Thr Ala Gly Asp Thr His Leu Gly Gly Glu Asp Phe Asp Asn
Arg225 230 235 240Leu Val Asn His Phe Val Gln Glu Phe Lys Arg Lys
Phe Gly Lys Asp 245 250 255Ile Met Ser Asn Lys Arg Ala Leu Arg Arg
Leu Arg Thr Ser Cys Glu 260 265 270Arg Ala Lys Arg Thr Leu Ser Ser
Ser Thr Gln Thr Ser Ile Glu Ile 275 280 285Asp Ser Leu His Glu Gly
Ile Asp Phe Tyr Ser Thr Ile Thr Arg Ala 290 295 300Arg Phe Glu Glu
Leu Cys Ser Asp Leu Phe Arg Ser Thr Leu Glu Pro305 310 315 320Val
Glu Lys Ala Leu Arg Asp Ala Lys Leu Asp Lys Ser Lys Ile Asp 325 330
335Glu Ile Val Leu Val Gly Gly Ser Thr Arg Ile Pro Lys Ile Gln Lys
340 345 350Leu Leu Ser Asp Phe Phe Asn Gly Lys Glu Leu Asn Lys Ser
Ile Asn 355 360 365Pro Asp Glu Ala Val Ala Tyr Gly Ala Ala Val Gln
Ala Ala Ile Leu 370 375 380Thr Gly Asp Asn Ser Asn Asn Val Lys Asp
Leu Leu Leu Leu Asp Val385 390 395 400Ala Pro Leu Ser Leu Gly Ile
Glu Thr Ala Gly Gly Val Met Thr Thr 405 410 415Leu Ile Lys Arg Asn
Thr Thr Ile Pro Thr Lys Gln Thr Gln Thr Phe 420 425 430Thr Thr Tyr
Ala Asp Asn Gln Pro Ala Val Thr Ile Gln Val Tyr Glu 435 440 445Gly
Glu Arg Ala Met Thr Lys Asp Asn Asn Arg Leu Gly Thr Phe Asp 450 455
460Leu Thr Gly Ile Pro Pro Ala Pro Arg Gly Val Pro Gln Ile Glu
Val465 470 475 480Thr Phe Asp Val Asp Ala Asn Gly Ile Leu Asn Val
Ser Ala Val Asp 485 490 495Lys Ser Thr Gly Arg Gln Asn Lys Ile Thr
Ile Thr Asn Asp Lys Gly 500 505 510Arg Leu Ser Lys Ala Asp Ile Glu
Lys Met Val Gln Glu Ala Glu Gln 515 520 525Tyr Arg Glu Asp Asp Glu
Arg Gln Arg Glu Arg Ile Ala Ala Lys Asn 530 535 540Gln Leu Glu Ala
Tyr Ala Phe Gln Leu Lys Ser Thr Met Glu Glu Glu545 550 555 560Ala
Val Lys Ser Lys Leu Ser Glu Glu Asp Arg Lys Thr Val Leu Asn 565 570
575Lys Val Asp Glu Thr Leu Arg Trp Leu Asp Ser Asn Gln Leu Ala Asp
580 585 590Lys Glu Glu Phe Glu His Arg Gln Lys Glu Ile Glu Asn Ala
Cys Arg 595 600 605Pro Ile Met Met Lys Ile Tyr Gln Gln Gln Gln Gln
Gln His His Pro 610 615 620Gly Ala Asn Gly Ser Cys Gly Ser Asn Ala
Tyr Pro Gly Tyr Asn Gly625 630 635 640Phe Lys Ser Asn Asn Asp Gly
Pro Val Val Glu Glu Val Asn 645 65086279PRTArtificial
sequencetr|B7U5T1|B7U5T1_DERFA Der f 6 allergen OS=Dermatophagoides
farinae PE=2 SV=1 86Met Ile Lys Ile Phe Leu Val Thr Ile Leu Ile Val
Ile Thr Val Thr1 5 10 15Val Asp Ala Arg Phe Pro Arg Ser Leu Gln Pro
Lys Trp Ala Tyr Leu 20 25 30Asp Ser Asn Glu Phe Ser Arg Ser Lys Ile
Gly Asp Ser Pro Ile Ala 35 40 45Gly Val Val Gly Gly Gln Asp Ala Asp
Leu Ala Glu Ala Pro Phe Gln 50 55 60Ile Ser Leu Leu Lys Asp Tyr Leu
Ile Met Lys Ser His Met Cys Gly65 70 75 80Gly Ser Leu Ile Ser Glu
Ser Thr Val Val Thr Ala Ala His Cys Thr 85 90 95Tyr Gly Gln Lys Ala
Ser Ser Leu Ser Val Arg Tyr Gly Thr Asn Gln 100 105 110Arg Thr Ser
Ser Ser Tyr Gly Asp Leu Lys Val Lys Thr Ile Ile Gln 115 120 125His
Glu Ser Tyr Asp Pro Asp Thr Ile Gln Asn Asp Ile Ser Leu Leu 130 135
140Ile Leu Ser Lys Pro Val Val Pro Ser Thr Asn Val Gln Met Ile
Glu145 150 155 160Ile Glu Thr Asp Asp Ile Val Asp Gly Asp Lys Val
Thr Ile Tyr Gly 165 170 175Trp Gly Leu Thr Asp Gly Asn Gly Lys Asp
Leu Pro Asp Lys Leu Gln 180 185 190Lys Gly Ser Met Thr Ile Val Gly
Asn Asp Arg Cys Asn Glu Lys Trp 195 200 205Gly Ser Ile Asp Ala Ile
His Pro Gly Met Ile Cys Ala Leu Asp Lys 210 215 220Thr Gln Ser Gly
Cys Asn Gly Asp Ser Gly Gly Pro Leu Val Ser Ala225 230 235 240Asn
Arg Lys Leu Thr Gly Ile Val Ser Trp Gly Pro Ser Lys Cys Pro 245 250
255Pro Gly Glu Tyr Met Ser Val Phe Thr Arg Pro Lys Tyr Tyr Leu Asp
260 265 270Trp Ile Thr Lys Asn Ile Val 27587150PRTArtificial
sequencetr|T2B4F3|T2B4F3_DERPT LytFM OS=Dermatophagoides
pteronyssinus GN=lytFM PE=4 SV=1 87Met Lys Phe Phe Phe Thr Leu Ala
Leu Phe Cys Thr Leu Ala Ile Ser1 5 10 15Gln Val Tyr Cys Asn Gly Ala
Ala Ile Val Ser Ala Ala Arg Ser Gln 20 25 30Ile Gly Val Pro Tyr Ser
Trp Gly Gly Gly Gly Ile His Gly Lys Ser 35 40 45Arg Gly Ile Gly Glu
Gly Ala Asn Thr Val Gly Phe Asp Cys Ser Gly 50 55 60Leu Ala Gln Tyr
Ser Val Tyr Gln Gly Thr His Lys Val Leu Ala Arg65 70 75 80Val Ala
Ser Gly Gln Tyr Ser Asp Pro Lys Cys His His Val Ala Tyr 85 90 95Gly
Ser His Gln Pro Gly Asp Leu Val Phe Phe Gly Asn Pro Ile His 100 105
110His Val Gly Ile Val Ser Ala His Gly Arg Met Ile Asn Ala Pro His
115 120 125Thr Gly Thr Asn Val Arg Glu Glu Asn Ile Trp Ser Asp His
Ile Ala 130 135 140Asn Val Ala Arg Cys Trp145 15088175PRTArtificial
sequencetr|A7XXV2|A7XXV2_DERFA Der f 2 allergen OS=Dermatophagoides
farinae PE=4 SV=1 88Met Ile Ser Lys Ile Leu Cys Leu Ser Leu Leu Val
Ala Ala Val Val1 5 10 15Ala Asp Gln Val Asp Val Lys Asp Cys Gly Lys
Phe Val Cys Val Ile 20 25 30His Phe Phe Ser Phe His Leu Phe Asn Thr
Lys His Asn Phe Leu Phe 35 40 45Leu Val Tyr Ile His Ile Ala Asn Asn
Glu Ile Lys Lys Val Met Val 50 55 60Asp Gly Cys His Gly Ser Asp Pro
Cys Ile Ile His Arg Gly Lys Pro65 70 75 80Phe Thr Leu Glu Ala Leu
Phe Asp Ala Asn Gln Asn Thr Lys Thr Ala 85 90 95Lys Ile Glu Ile Lys
Ala Ser Leu Asp Gly Leu Glu Ile Asp Val Pro 100 105 110Gly Ile Asp
Thr Asn Ala Cys His Phe Met Lys Cys Pro Leu Val Lys 115 120 125Gly
Gln Gln Tyr Asp Ile Lys Tyr Thr Trp Asn Val Pro Lys Ile Ala 130 135
140Pro Lys Ser Glu Asn Val Val Val Thr Val Lys Leu Ile Gly Asp
Asn145 150 155 160Gly Val Leu Ala Cys Ala Ile Ala Thr His Gly Lys
Ile Arg Asp 165 170 17589493PRTArtificial sequenceiMAT-Cul o2
relates to a Cul o2 iMAT molecule [with n-terminal HEXA-HISTIDINE]
89Met His His His His His His Tyr Gly Arg Lys Lys Arg Arg Gln Arg1
5 10 15Arg Arg Met Glu Asp Gln Arg Asp Leu Ile Ser Asn His Glu Gln
Val 20 25 30Pro Ile Leu Gly Gln Arg Pro Ala Ala Pro Glu Arg Lys Ser
Ser Arg 35 40 45Gly Ala Leu Tyr Thr Gly Phe Ser Val Leu Val Ala Leu
Leu Leu Ala 50 55 60Gly Gln Ala Thr Thr Ala Tyr Phe Leu Phe Gln Gln
Gln Gly Arg Leu65 70 75 80Asp Lys Leu Thr Val Thr Ala Gln Asn Leu
Gln Leu Glu Lys Leu Arg 85 90 95Met Lys Leu Pro Lys Ser Ala Lys Pro
Val Ser Lys Ile Arg Val Ala 100 105
110Thr Pro Met Leu Met Gln Ala Leu Pro Met Glu Gly Leu Ser His Gly
115 120 125Gln His Leu Ile Gln Ala Glu Leu Pro Asp Ser Pro Leu Asn
Ile Val 130 135 140Lys Glu Phe Asp Asp Asp Gly Arg Asn Asn Asn Asn
Gln Asn Asp Phe145 150 155 160Asn Phe Tyr Trp Asn Ile Pro Ser Phe
Met Ser Ala Gln His Asn Ile 165 170 175Thr Phe Thr Asp Met Thr Ser
Ser Tyr Asn Ile Val Gln Asn Lys Asp 180 185 190Asp Lys Trp Arg Gly
Asp Lys Ile Val Ile Leu Tyr Asp Pro Gly Lys 195 200 205Phe Pro Ala
Leu Leu Glu His Gln Gly Gln Leu Tyr Arg Arg Asn Gly 210 215 220Gly
Val Pro Gln Glu Gly Asn Leu Gln Glu His Ile Asp Ile Leu Ala225 230
235 240Glu His Ile Asn Lys Leu Ile Pro Asp Thr Gln Phe Ser Gly Ile
Gly 245 250 255Val Ile Asp Phe Glu Ser Trp Arg Pro Ile Phe Arg Gln
Asn Ser Gly 260 265 270Val Leu Gln Pro Tyr Lys Asp Leu Ser Tyr Lys
Leu Val His Arg Asp 275 280 285His Lys Leu Trp Asn Arg Lys Arg Val
Glu Ile Glu Ala Ala Arg Leu 290 295 300Phe Glu Ala Ala Gly Arg Thr
Phe Val Glu Glu Thr Ile Asn Val Ala305 310 315 320Lys Ile Leu Arg
Pro Lys Ala Lys Trp Gly Tyr Tyr Gly Phe Pro Tyr 325 330 335Ser Phe
Asn Met Asn Gly Gly Ala Asn Met Asn Glu Asp Ser Pro Ala 340 345
350Asn Val Lys Glu Glu Asn Asp Gln Ile Lys Trp Leu Trp Asp Ile Val
355 360 365Asp Val Val Leu Pro Ser Val Tyr Leu Asn Asn Lys Ile Thr
Ser Ala 370 375 380Gln Arg Val Gln Phe Val Arg Gly Arg Met Arg Glu
Gly Tyr Arg Val385 390 395 400Ala Lys Met Ser Lys Lys Ser Pro Lys
Pro Pro Val Leu Ala Tyr Leu 405 410 415Arg Tyr Val Tyr Thr Asp Thr
Leu Lys Phe Leu Ser Asn Glu Asp Leu 420 425 430Lys Gln Ala Ile Lys
Val Ser Lys Glu Gln Lys Ser Lys Gly Met Ile 435 440 445Phe Trp Gly
Ser Ser Tyr Asp Val Lys Thr Lys Glu Gln Ser Ile Asp 450 455 460Phe
Arg Lys Tyr Val Asp Asn Asn Leu Gly Pro Ile Val Leu Leu Ala465 470
475 480Asn Asn Lys Ser Pro Lys Val Leu Thr Pro Asn Leu Ala 485
49090372PRTArtificial sequenceiMAT-Cul o3 relates to a Cul o3 iMAT
molecule [with n-terminal HEXA-HISTIDINE] 90Met His His His His His
His Tyr Gly Arg Lys Lys Arg Arg Gln Arg1 5 10 15Arg Arg Met Glu Asp
Gln Arg Asp Leu Ile Ser Asn His Glu Gln Val 20 25 30Pro Ile Leu Gly
Gln Arg Pro Ala Ala Pro Glu Arg Lys Ser Ser Arg 35 40 45Gly Ala Leu
Tyr Thr Gly Phe Ser Val Leu Val Ala Leu Leu Leu Ala 50 55 60Gly Gln
Ala Thr Thr Ala Tyr Phe Leu Phe Gln Gln Gln Gly Arg Leu65 70 75
80Asp Lys Leu Thr Val Thr Ala Gln Asn Leu Gln Leu Glu Lys Leu Arg
85 90 95Met Lys Leu Pro Lys Ser Ala Lys Pro Val Ser Lys Ile Arg Val
Ala 100 105 110Thr Pro Met Leu Met Gln Ala Leu Pro Met Glu Gly Leu
Ser His Gly 115 120 125Thr Asp Phe Ser Asp Arg Lys Leu Ser Arg Arg
Gln Ile Glu Pro Asn 130 135 140Val Tyr Gln Asn Ile Pro His Ile Gly
Ser Asn His Asp Gly Arg Asn145 150 155 160Ser Pro Ala Ser Pro Ser
Asp Ala Lys Ile Leu Pro Met Ser Thr Lys 165 170 175Arg Lys Asn Leu
Ile Leu Arg Val His Asn Arg Leu Arg Asn Lys Val 180 185 190Ala Leu
Gly Gln Leu Pro Gly Tyr Pro Lys Ala Val Arg Met Pro Ile 195 200
205Leu Arg Trp Asp Asp Glu Leu Ala Tyr Leu Ala Glu Leu Asn Val Lys
210 215 220Gln Ser Glu Met Lys His Asp Gln Ser Arg Asn Thr Asp Lys
Phe Arg225 230 235 240Tyr Ala Gly Gln Asn Leu Ala Tyr Ile Gly Gly
Gly Lys Glu Pro Asn 245 250 255Ala Val Arg Ile Lys Thr Leu Val Arg
Ala Trp Phe Asp Glu Tyr Lys 260 265 270Asp Ala Asn Ser Ser Phe Ile
Asp Lys Tyr Arg Ser His Pro Asn Gly 275 280 285Lys Ala Ile Gly His
Phe Thr Ala Met Val Gln Asp Arg Thr Asp Thr 290 295 300Val Gly Ile
Ala Ile Leu Arg His Thr Lys Asn Thr Tyr Phe Phe Leu305 310 315
320Ala Ile Asn Tyr Ser Phe Thr Asn Met Val Lys Asp Lys Val Tyr Thr
325 330 335Arg Gly Ala Lys Ser Ser Ser Lys Ser Arg Thr Gly Ser Ser
Pro Val 340 345 350Tyr Lys Gly Leu Ser Lys Pro His Glu Tyr Val Asn
Pro Asp Pro Asp 355 360 365Glu Asp Leu Asp 3709114PRTArtificial
sequenceartificial peptide derived from SEQ ID NO 10 (Q86R84_AA
97-110) i.e. 91Gly Asn Ala Lys Ala Met Ile Ala Val Gly Gly Ser Thr
Met1 5 109214PRTArtificial sequenceartificial peptide derived from
SEQ ID NO 85 (A0A088SAS1_AA 611-624) i.e. 92Met Met Lys Ile Tyr Gln
Gln Gln Gln Gln Gln His His Pro1 5 109314PRTArtificial
sequenceartificial peptide derived from SEQ ID NO 88 (A7XXV2) AA
48-61) i.e. 93Phe Leu Val Tyr Ile His Ile Ala Asn Asn Glu Ile Lys
Lys1 5 109413PRTArtificial sequenceartificial peptide derived from
SEQ ID NO 86 (B7U5T1_AA 166-178) i.e. 94Ile Val Asp Gly Asp Lys Val
Thr Ile Tyr Gly Trp Gly1 5 109514PRTArtificial sequenceartificial
peptide derived from SEQ ID 87 (T2B4F3_AA 134-147) i.e. 95Arg Glu
Glu Asn Ile Trp Ser Asp His Ile Ala Asn Val Ala1 5
109614PRTArtificial sequenceartificial peptide derived from SEQ ID
NO 11 (Q9U6R7_AA 469-482) i.e. 96Thr Pro Thr Thr Pro Thr Pro Ala
Pro Thr Thr Ser Thr Pro1 5 1097223PRTArtificial sequenceartificial
peptide derived from SEQ ID NO 7 (Q58A71 Der f 1 allergen
preproenzyme Dermatophagoides farinae (American house dust mite) AA
99-321) i.e. 97Thr Ser Ala Cys Arg Ile Asn Ser Val Asn Val Pro Ser
Glu Leu Asp1 5 10 15Leu Arg Ser Leu Arg Thr Val Thr Pro Ile Arg Met
Gln Gly Gly Cys 20 25 30Gly Ser Cys Trp Ala Phe Ser Gly Val Ala Ala
Thr Glu Ser Ala Tyr 35 40 45Leu Ala Tyr Arg Asn Thr Ser Leu Asp Leu
Ser Glu Gln Glu Leu Val 50 55 60Asp Cys Ala Ser Gln His Gly Cys His
Gly Asp Thr Ile Pro Arg Gly65 70 75 80Ile Glu Tyr Ile Gln Gln Asn
Gly Val Val Glu Glu Arg Ser Tyr Pro 85 90 95Tyr Val Ala Arg Glu Gln
Gln Cys Arg Arg Pro Asn Ser Gln His Tyr 100 105 110Gly Ile Ser Asn
Tyr Cys Gln Ile Tyr Pro Pro Asp Val Lys Gln Ile 115 120 125Arg Glu
Ala Leu Thr Gln Thr His Thr Ala Ile Ala Val Ile Ile Gly 130 135
140Ile Lys Asp Leu Arg Ala Phe Gln His Tyr Asp Gly Arg Thr Ile
Ile145 150 155 160Gln His Asp Asn Gly Tyr Gln Pro Asn Tyr His Ala
Val Asn Ile Val 165 170 175Gly Tyr Gly Ser Thr Gln Gly Val Asp Tyr
Trp Ile Val Arg Asn Ser 180 185 190Trp Asp Thr Thr Trp Gly Asp Ser
Gly Tyr Gly Tyr Phe Gln Ala Gly 195 200 205Asn Asn Leu Met Met Ile
Glu Gln Tyr Pro Tyr Val Val Ile Met 210 215 2209828PRTArtificial
sequenceartificial peptide derived from SEQ ID NO 10 (Q86R84 60 kDa
allergen Der f 18p Dermatophagoides farinae (American house dust
mite) AA 277-304) i.e. 98Phe Thr Gln Thr Asp Gly Phe Leu Ser Tyr
Asn Glu Leu Cys Val Gln1 5 10 15Ile Gln Ala Glu Thr Asn Ala Phe Thr
Ile Thr Arg 20 259940PRTArtificial sequenceartificial peptide
derived from SEQ ID NO 12 (I7HDR2 Zen 1 protein Dermatophagoides
farinae (American house dust mite) AA 181-220) i.e. 99Glu Pro Thr
Thr Pro Thr Pro Glu Pro Thr Thr Lys Thr Pro Glu Pro1 5 10 15Thr Thr
Lys Thr Pro Glu Pro Ser Thr Pro Thr Pro Glu Pro Thr Thr 20 25 30Lys
Thr Pro Glu Pro Thr Thr Lys 35 4010070PRTArtificial
sequenceartificial peptide derived from SEQ ID NO 9 (A0A088SAW7 Der
f 23 allergen Dermatophagoides farinae (American house dust mite)
AA 22-91) i.e. 100Asp Ile Asp His Asp Asp Asp Pro Thr Thr Met Ile
Asp Val Gln Thr1 5 10 15Thr Thr Val Gln Pro Ser Asp Glu Phe Glu Cys
Pro Thr Arg Phe Gly 20 25 30Tyr Phe Ala Asp Pro Lys Asp Pro Cys Lys
Phe Tyr Ile Cys Ser Asn 35 40 45Trp Glu Ala Ile His Lys Ser Cys Pro
Gly Asn Thr Arg Trp Asn Glu 50 55 60Lys Glu Leu Thr Cys Thr65
7010127PRTArtificial sequenceartificial peptide derived from SEQ ID
NO 11 (Q9U6R7 98kDa HDM allergen (Der f 15 allergen) (Group 15
allergen Der f 15) Dermatophagoides farinae (American house dust
mite) AA 437-463) i.e. 101Ser Pro Thr Thr Pro Thr Thr Thr Pro Ser
Pro Thr Thr Pro Thr Thr1 5 10 15Thr Pro Ser Pro Thr Thr Pro Thr Thr
Thr Pro 20 25102129PRTArtificial sequenceartificial peptide derived
from SEQ ID NO 8 (Q00855 Mite group 2 allergen Der f 2 (Allergen
Der f II) (allergen Der f 2) Dermatophagoides farinae (American
house dust mite) AA 18-146) i.e. 102Asp Gln Val Asp Val Lys Asp Cys
Ala Asn Asn Glu Ile Lys Lys Val1 5 10 15Met Val Asp Gly Cys His Gly
Ser Asp Pro Cys Ile Ile His Arg Gly 20 25 30Lys Pro Phe Thr Leu Glu
Ala Leu Phe Asp Ala Asn Gln Asn Thr Lys 35 40 45Thr Ala Lys Ile Glu
Ile Lys Ala Ser Leu Asp Gly Leu Glu Ile Asp 50 55 60Val Pro Gly Ile
Asp Thr Asn Ala Cys His Phe Met Lys Cys Pro Leu65 70 75 80Val Lys
Gly Gln Gln Tyr Asp Ile Lys Tyr Thr Trp Asn Val Pro Lys 85 90 95Ile
Ala Pro Lys Ser Glu Asn Val Val Val Thr Val Lys Leu Ile Gly 100 105
110Asp Asn Gly Val Leu Ala Cys Ala Ile Ala Thr His Gly Lys Ile Arg
115 120 125Asp
* * * * *