Note: Descriptions are shown in the official language in which they were submitted.
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SPEDIFIC BINDING PROTEINS FOR TREATING CANINE ALLERGY
FIELD OF THE INVENTION
This invention concerns peptides. More particularly, the
invention concerns compositions for administration to dogs,
which actively provide immunity to the dog's immunoglobulin E
molecules.
BACKGROUND ART
It is estimated that up to 30% of all dogs suffer from
allergies or allergy-related skin disorders. Specifically,
allergic dermatitis has been estimated to affect between 3 and
15% of the entire canine population. Given the prevalence of
allergies in dogs, there is a need to develop methods and
compositions to properly diagnose and treat canine allergies.
The substances most likely to cause an allergic reaction
vary from species to species. Common canine allergens include
fleas, pollens, molds and dust. Allergy to fleas is believed
to be the most common dog allergy. Typically, a flea's saliva
is the allergen, and a single fleabite can cause substantial
itching. An additional form of allergy in dogs is termed
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atopy. Atopy is a condition where a dog is allergic to
inhalants such as pollens, molds or microscopic mites such as
are found in house dust.
Antibody molecules play a role in allergic
manifestations. In mammals, antibody molecules are classified
into various isotypes referred to as IgA, IgD, IgE, IgG, and
IgM. Antibody molecules consist of both heavy and light chain
components. The heavy chains'of molecules of a'given isotype
have extensive regions of amino acid sequence homology, and
conversely have regions of difference from antibodies
belonging to other isotypes. The shared regions of the heavy
chains provide members of each isotype with common abilities
to bind to certain cell surface receptors or to other
macromolecules, such as complement. These heavy chain regions,
therefore, serve to activate particular immune effector
functions. Accordingly, separation of antibody molecules into
isotypes serves to separate the antibodies according to a set
of effector functions that they commonly activate.
In humans and dogs, immunoglobulin E (hereinafter IgE) is
involved in allergy. Thus, IgE is the antibody type that is
understood to be an important mediator of allergic responses,
including Type I immediate hypersensitivity.
IgE molecules bind to mast cells and basophils. This
binding occurs when the Fc region of the IgE molecule is bound
to Fc receptors on the mast cells. When such bound IgE
antibodies then bind to an allergen, the allergen cross-links
multiple IgE antibodies on the cell surface. This cross-
linking mediates Type I immediate hypersensitivity reactions
and causes release of histamines and other molecules that
produce symptoms associated with allergy.
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Monoclonal antibodies having different degrees of
sensitivity to canine IgE and IgG have been identified.
(DeBoer, et al. Immunology and Immunopathology 37, 183-199
(1993).) DeBoer, et al. identified several monoclonal
antibodies which had cross reactivity between IgG and IgE.
(See, e.g., DeBoer, et al., Table 4 and accompanying text.)
Three monoclonal antibodies (A5, D9, and B3) were identified
by DeBoer et al., as having some affinity for canine IgE. Of
the monoclonal antibodies identified in DeBoer et al.,
antibody D9 appeared to have the greatest degree of
neutralization of.Prausnitz-Kustner reactivity for atopic dog
serum. In the context of canine allergy, DeBoer et al.
proposed use of their monoclonal antibodies (MAbs) in the use
of antigen-specific IgE ELISA, and for quantifying canine IgE.
Additionally, they proposed use of their MAbs for
immunostaining of Western Blot assays, to evaluate the
molecular specificity of IgE antibodies, as well as for in
vitro studies on degranulation of mast cells.
In humans the serum level of total IgE is diagnostic of
allergic disease. To explore the possibility that the serum
level of IgE might also be diagnostic of allergy in dogs,
several studies were performed. (Hill and DeBoer Am. J. Yet.
Res., (July 1994) 55(7), 944-48). Publications following the
DeBoer article used a monoclonal antibody designated D9 in an
ELISA assay having the following configuration: D9 was bound
to a substrate, antibodies were captured by D9 and then D9
having a marker was used to flag the captured antibody. The
Hill and DeBoer ELISA was used to establish the total amount
of IgE in canine serum in an effort to diagnose canine
allergy. In contrast to humans, the quantity of IgE determined
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to exist in canine circulation was of no use whatsoever in the
diagnosis of allergy in dogs. (See, e.g., Abstract and
Discussion Sections of Hill and DeBoer) This finding was in
direct contrast to the situation in human immunology.
This divergent diagnostic result based on levels of IgE
in humans compared to such levels in dogs, points out the
difficulty of any attempt to correlate data between animals of
two different genera. This difficulty is further exacerbated
by the fact that dogs can be allergic to a different set of
antigens than humans are. Fleas, for instance, are a severe
problem for dogs, but not humans. Furthermore, in instances
where dogs and humans appear to be allergic to the same
allergen extract, studies by doctors Esch and Greer of Greer
Laboratories, have indicated that the specific allergens in an
allergen extract which produce canine disease are not
necessarily the same allergens that produce disease in humans.
For example, it is known that the immunodominant components of
dust mite extracts are different in dogs than in humans.
The genomic sequences encoding human and murine IgE heavy
chain constant region are known (For example, see Ishida et
al., "The Nucleotide Sequence of the Mouse Immunoglobulin E
Gene: Comparison with the Human Epsilon Gene Sequence", EMBO
Journal 1,1117-1123 (1982). A comparison of the human and
murine genes shows that they possess 60% homology within
exons, and 45-50% homology within introns, with various
insertions and deletions.
Patel et al. published the nucleotide and predicted amino
acid sequence for exons 1-4 of the heavy chain constant region
of canine IgE in the article entitled "Sequence of the Dog
Immunoglobulin Alpha and Epsilon Constant Region Genes,"
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Immunogenetics 41, 282-286 (March 22, 1995). The complete
sequence of the canine IgE heavy chain constant region, with
membrance bound portions encoded by exons 5 and 6 are
disclosed in the prior art.
Because IgE is believed to mediate allergic symptoms, it
may be desirable to decrease'IgE levels as a mechanism for
alleviating allergic symptoms. However, a patient's own IgE
molecules are self-proteins, and immune responses to such
proteins are usually suppressed. The suppression of immune
responses to self-proteins, i.e., tolerance to self-antigens,
is hypothesized to occur in a number of ways.
The current hypothesis for suppression of T cells
directed to self-antigens, involves an induction of "clonal
deletion" of such T cells in the thymus, whereby T cell
receptors which might recognize self-peptides in association
with MHC molecules are eliminated, and only those which
recognize foreign peptide and MHC molecules are allowed to
expand. In addition, suppressor T cells may also exist which
prevent the induction of immune responses to self-proteins.
In contrast to the situation with T cells, it is believed
that there are many B cells which express receptors (i.e.,
surface immunoglobulin) for self-proteins, and that the reason
these cells do not produce antibodies to self-proteins is
because the T cells required for the antigen presentation to
the B cell are normally missing.
A B cell which recognizes epitopes (antigen-binding
sites) on a patient's own IgE antibodies is capable of
generating antibodies, generally IgG, directed to this self-
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antigen, i.e., IgE. The existence of such B cells, therefore,
presents a unique opportunity to induce the production of
auto-antibody responses. There is an unmet need for such
antibodies in order to treat allergic disease.
The hypothesis regarding "antigen presentation" involves:
the recognition of antigen by surface immunoglobulin on the B
cell, the internalization and processing of this antigen, the
association of peptides derived from the antigen with MHC
molecules expressed on the surface of the B cell, and then,
the recognition of the associated antigen peptide and MHC
molecules by a particular T cell. The T-cell:B-cell
interaction then leads to signal transduction in both, cells
and the synthesis and elaboration of soluble cytokines which
eventually result in antibody production by the B cell.
Thus, in most circumstances, only when an antigen is
foreign does an immune response occur; otherwise the
internalization and processing of self-proteins would
regularly lead to the presentation of self peptide-MHC
complexes to T cells and thereby lead to autoimmune
antibodies.
Therefore, in order to induce an antibody response to a
self-peptide, such as IgE, the immune system must be
manipulated so as to allow an auto-reactive B cell to become
an antibody-secreting B cell. There is an unmet need to
manipulate the immune system in this way, particularly in the
context of allergic disease.
In general, there are several known approaches for
generating antibodies to peptide antigens. For example,
multiple antigenic peptides (MAPs), introduced by Dr. James
Tam (Tam, J.P., (1988) Proc. Natl. Acad. Sci. U.S.A. 85,
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5409-5413), have demonstrated several advantages for inducing
anti-peptide antibodies. The MAP approach is an improved
alternative to the conventional technique of conjugating a
peptide antigen to a protein carrier. One of the primary
limitations associated with the use of protein carriers is the
large mass of the carrier relative to the attached peptide
antigen. This relative size disparity may result in a low
ratio of anti-peptide antibodies compared to anti-carrier
antibodies. MAPS typically have 4 or 8 peptide arms branching
out from a lysine core matrix as depicted in Fig.1 A-B. The
peptide antigen is conjugated to each arm. Thus there is a
much higher ratio of antigen to carrier molecule in a MAP
system compared to traditional protein conjugation. This
design maximizes the concentration of the antigen for a
specific immunogenic response. Moreover, the central lysine
core of the MAP-peptide has been shown to be non-immunogenic.
(Tam, J.P., Proc. Natl. Acad. Sci. U.S.A. 85, 5409-5413
(1988); Posnett, D.N., McGrath, H., and Tam, J.P., J. Biol.
Chem. 263, 1719-1725 (1988)) Therefore, antibodies induced to
MAP-peptides are a direct response to the antigen. Accurate
knowledge of the chemical composition, structure, and quantity
of the peptide prior to immunization is possible by directly
synthesizing the antigen onto the branching lysine core. Also,
because the-MAP approach removes the need to conjugate
peptides to carrier proteins, which may alter the antigenic
determinants, chemical ambiguity is eliminated. Thus MAPs are
believed to induce antibody responses of high purity,
increased avidity, accurate chemical definition, and improved
safety.
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Fmoc MAP resins (available from Applied Biosystems,
Foster City, Calif.) are Fmoc-compatible resins connected to a
small core matrix of branching lysine residues. The core
matrix comprises several levels of lysine residues attached to
the previous lysine at both the N-a and N-E amino groups, as
depicted in Figure 1A-B.
MAP-peptides used in experimental vaccine design have
elicited high titers of anti-peptide antibodies that recognize
the native protein. (Tam, J.P., (1988) Proc. Natl. Acad. Sci.
U.S.A. 85, 5409-5413; Posnett, D.N., McGrath, H., and Tam,
J.P., (1988) J. Biol. Chem. 263, 1719-1725; Auriault, C.,
Wolowczuk, I., Gras-Masse, H., Maguerite, M., Boulanger, D.,
Capron, A., and Tartar, A., (1991) Peptide Res.4, 6-11).
Additionally, increased sensitivity and reliability of
antibody-antigen interactions in solid-phase immunoassays have
been observed with MAP-peptides due to enhanced coating
capacity and avidity. (Tam, J.P., and Zavala, F., (1989) J.
Immunol. Meth., 124, 53-61).
An additional approach that is known to be useful for
generating antibodies to peptide antigens involves placing
multiple copies of peptides on the surface of plant virus
particles. EPICOATTM technology (Axis Genetics plc, Cambridge,
England) is one such example. The EPICOATTM technology is based
on chimeric virus particle (CVP) technology that utilizes the
recombinant genetic modification of plant viruses.
The EPICOATTM technology involves insertion of a small
portion of a foreign protein (a peptide) into a plant virus in
such a way that multiple copies of the peptide are displayed
on the surface of the virus particle. The EPICOATTM technology
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is currently based on the cow pea mosaic virus (CPMV) a plant
virus that infects the cow pea plant, also known as the
"black-eyed" bean. The unmodified CPMV particle is
icosahedral, and about twenty-eight nanometers (nm.) in
diameter. CPMV particles are composed of two proteins,
referred to as the large and small coat proteins. Studies have
revealed a site within the small coat protein which allows
presentation of a foreign peptide in a prominent position on
the virus surface, whereby up to sixty copies of a particular
peptide can be presented on each virus particle.
With the EPICOATTM technology, DNA copies of the plant
virus's genetic material are used. A minute quantity of the
DNA encoding the virus protein, including the inserted foreign
peptide, is applied to the leaves of young cow pea plant,
along with an abrasive powder. Upon gentle rubbing, DNA enters
the leaves and utilizes the plant's own cellular mechanisms to
initiate generation of functional virus particles. The virus
replicates within the inoculated leaves and spreads throughout
the growing plant.
After two to three weeks, leaf material containing large
quantities of the virus is harvested. Chimeric virus particles
(CVPs) are isolated by centrifugation and selective
precipitation of homogenized plant material. Between 1 to 2
grams of CVPs can be obtained per kilogram fresh weight of
leaf material. Cow pea plants are readily grown in abundance
in controlled environments, allowing generation of large
quantities of CVPs.
It has been reported that peptides of up to thirty-six
amino acids in size have been successfully incorporated into
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CVPs. The resulting particles are extremely robust with a
thermal inactivation point of 65 C. The CV-Ps have been shown
to withstand acidic pH as well as protein degrading enzymes.
It has been reported that the expressed peptides are capable
of eliciting specific immune responses in animals. It has been
hypothesized that surface presentation of peptides may enhance
the recognition by the host immune system, and may provide a
route for development of recombinant sub-unit vaccines and
immuno-therapeutics.
DISCLOSURE OF THE INVENTION
Disclosed is a specific binding protein which
specifically binds to native canine free or B cell-bound IgE
exon 3, and which does not bind to IgE exon 3 when the IgE is
bound to receptor on a mast cell. The surface-bound IgE can
be. IgE expressed on the surface of a canine B cell
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In a related aspect, disclosed is a specific binding
protein selected from the group consisting of a monoclonal
antibody, a polyclonal antibody, an antigen-binding fragment of
a monoclonal antibody, an antigen-binding fragment of a
polyclonal antibody, a hybrid antibody, and a single chain
antibody, which specifically binds to native canine free or
B cell-bound IgE by binding to an amino acid sequence
comprising any one of SEQ ID NO:1-12 and 16-17.
In another related aspect, disclosed is a specific
binding protein selected from the group consisting of a
monoclonal antibody, a polyclonal antibody, an antigen-binding
fragment of a monoclonal antibody, antigen-binding fragment of
a polyclonal antibody, a hybrid antibody, and a single chain
antibody, which specifically binds to an isolated and purified
peptide consisting of SEQ ID NOs:1-25.
Disclosed is a specific binding protein which
specifically binds to an isolated and purified peptide
comprising a leucine positioned two peptide bonds away from a
tyrosine-arginine pair; e.g., SEQ ID NO:1 leucine-blank-blank-
tyrosine-arginine, SEQ ID NO:2 tyrosine-arginine-blank-blank-
leucine, or SEQ ID NO:3 leucine-blank-blank-tyrosine-arginine-
blank-blank-leucine. The peptide can consist of from 5 to 71
amino acids.
Disclosed is an antibody which binds to a defined epitope
and which is raised to an isolated and purified peptide
comprising an amino acid sequence, or a conservative variant
thereof, which comprises: SEQ ID NO:4 Thr-Leu-Leu-Glu-Tyr-Arg-
Met; also disclosed is a recombinant binding molecule which
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specifically binds to the defined epitope bound by the
antibody.
Disclosed is an antibody which binds to a defined epitope
and which is raised to an isolated and purified peptide
comprising an amino acid sequence, or a conservative variant
thereof, which comprises: SEQ ID NO:5 Gly-Met-Asn-Leu-Thr-Trp-
Tyr-Arg-Glu-Ser-Lys; also disclosed is a recombinant binding
molecule which specifically'binds to the defined epitope bound
by the antibody.
Also disclosed is a specific binding protein which is
raised to a multiply antigenic peptide comprising multiple
copies of an isolated and purified peptide which comprises a
leucine positioned two peptide bonds away from a tyrosine-
arginine pair; the specific binding protein specifically binds
to a defined epitope. The isolated and purified peptide can
be from 5-71 amino acids. Also disclosed is a recombinant
binding molecule which specifically binds to the defined
epitope bound by the binding protein.
Disclosed is a specific binding protein which
specifically binds to an isolated and purified peptide
comprising a cysteine positioned two peptide bonds away from a
proline-histidine pair positioned three peptide bonds from a
cysteine. The peptide can have the form: SEQ ID NO:6
cysteine-blandk-blank-proline-histidine-blank-blank-blank-
cysteine. The cysteines may form a covalent bond through a
reduction reaction, cyclizing the peptide and becoming
cystine. The peptide can comprise from 9 to 71 amino acids.
Disclosed is an antibody that binds to a defined epitope
and which is raised to an isolated and purified peptide that
has the amino acid sequence, or a conservative variant
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thereof, which comprises: SEQ ID NO:7 serine-valine-threonine-
leucine-cysteine-proline-asparagine-proline-histidine-
isoleucine-proline-methionine-cysteine-glycine-glycine-
glycine. The cysteines may form a covalent bond through a
reduction reaction, cyclizing the peptide and becoming
cystine. Also disclosed is a recombinant binding molecule
that specifically binds to the defined epitope bound by the
antibody.
Disclosed is an antibody that binds to a defined epitope
and which is raised to an isolated and purified peptide that
has the amino acid sequence, or a conservative variant
thereof, which comprises: SEQ ID NO:8 serine-alanine-cysteine-
proline-asparagine-proline-histidine-asparagine-proline-
tyrosine-cysteine-glycine-glycine-glycine. The cysteines may
form a covalent bond through a reduction reaction, cyclizing
the peptide changing the amino acid to cystine. Also
disclosed is a recombinant binding molecule that specifically
binds to the defined epitope bound by the antibody.
Disclosed is a specific binding protein that specifically
binds to an isolated and purified peptide comprising a
cysteine positioned one peptide bond from a proline-histidine
pair, positioned one peptide bond from a proline, positioned
two peptide bonds from a cysteine; the peptide can have the
form: SEQ ID. NO:9 cysteine-blank-proline-histidine-blank-
proline-blank-blank-cysteine. The cysteines may form a
convalent bond through a reduction reaction, cyclizing the
peptide and becoming cystine. The peptide can comprise from 9
to 71 amino acids.
Disclosed is an antibody that binds to a defined epitope
and which is raised to an isolated and purified peptide that
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Also disclosed is use of a specific binding
protein for treatment or prophylaxis of canine allergy,
wherein the specific binding protein specifically binds to
native canine free or B cell-bound IgE, and does not bind to
IgE when the IgE is bound to a receptor on a mast cell, and
wherein the specific binding protein specifically binds to
an amino sequence comprising SEQ ID NO:l-12, 16 or 17.
Also disclosed is use of a specific binding
protein for treatment or prophylaxis of canine allergy,
wherein the specific binding protein specifically binds to
an isolated and purified peptide comprising a leucine
positioned two peptide bonds away from a tyrosine-arginine
pair comprising the form leucine-blank-blank-tyrosine-
arginine (SEQ ID NO:1), tyrosine-arginine-blank-blank-
leucine (SEQ ID NO:2), or leucine-blank-blank-tyrosine-
arginine-blank-blank-leucine (SEQ ID NO:3), where at least
one blank is an amino acid with an aromatic ring.
Also disclosed is use of an antibody for treatment
or prophylaxis of canine allergy, wherein the antibody
specifically binds to an isolated and purified peptide
comprising an amino acid sequence which comprises Thr-Leu-
Leu-Glu-Tyr-Arg-Met (SEQ ID NO:4), or a conservative variant
thereof.
Also disclosed is use of an antibody for treatment
or prophylaxis of canine allergy, wherein the antibody
specifically binds to an isolated and purified peptide
comprising an amino acid sequence which comprises Gly-Met-
Asn-Leu-Thr-Trp-Tyr-Arg-Glu-Ser-Lys (SEQ ID NO:5), or a
conservative variant thereof.
Also disclosed is use of a specific binding
protein for the treatment or prophylaxis of canine allergy,
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wherein the specific binding protein specifically binds to a
multiply antigenic peptide comprising multiple copies of an
isolated and purified peptide which comprises a leucine
positioned two peptide bonds away from a tyrosine-arginine
pair.
Also disclosed is use of a specific binding
protein for the treatment or prophylaxis of canine allergy,
wherein the specific binding protein specifically binds to a
recombinant plant virus particle comprising at least one
copy of an isolated and purified peptide comprising a
leucine positioned two peptide bonds away from a tyrosine-
arginine pair.
Also disclosed is use of a monoclonal antibody
which is 8H.8, which binds to SEQ ID NO:39, inhibits binding
of IgE to high affinity IgE receptor on mast cells and
basophils, and does not bind to canine IgE bound by high
affinity IgE receptor, for the treatment or prophylaxis of
canine allergy.
Also disclosed is use of a specific binding
protein for the treatment or prophylaxis of canine allergy,
wherein the specific binding protein specifically binds to
an isolated and purified peptide comprising cysteine-blank-
proline-histidine-blank-proline-blank-blank-cysteine, (SEQ
ID NO:9) where blank is any amino acid.
Also disclosed is use of an antibody for the
treatment or prophylaxis of canine allergy, wherein the
antibody specifically binds to an isolated and purified
peptide comprising an amino acid sequence which comprises
Cys-His-Pro-His-Leu-Pro-Lys-Ser-Cys (SEQ ID NO:18), or a
conservative variant thereof.
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Also disclosed is use of a specific binding
protein for the treatment or prophylaxis of canine allergy,
wherein the specific binding protein specifically binds to
an isolated and purified peptide comprising cysteine-blank-
blank-proline-histidine-blank-blank-blank-cysteine (SEQ ID
NO:6), where blank is any amino acid.
Also disclosed is use of an antibody for the
treatment or prophylaxis of canine allergy, wherein the
antibody specifically binds to an isolated and purified
peptide comprising an amino acid sequence which comprises
Cys-Pro-Asn-Pro-His-Ile-Pro-Met-Cys (SEQ ID NO:16) or a
conservative variant thereof.
Also disclosed is use of an antibody for the
treatment or prophylaxis of canine allergy, wherein the
antibody specifically binds to an isolated and purified
peptide comprising an amino acid sequence which comprises
Cys-Pro-Asn-Pro-His-Asn-Pro-Tyr-Cys (SEQ ID NO:17) or a
conservative variant thereof.
Also disclosed is use of a monoclonal antibody
which is 15A.2, which binds to SEQ ID NO:26, binds canine
IgE exon 3, inhibits binding of IgE to high affinity IgE
receptor on mast cells and basophils, and does not bind to
canine IgE bound by high affinity IgE receptor, for the
treatment or prophylaxis of canine allergy.
Also disclosed is use of a specific binding
protein for the treatment or prophylaxis of canine allergy,
wherein the specific binding protein specifically binds to a
multiply antigenic peptide comprising multiple copies of an
isolated and purified peptide which comprises Cys-blank-
blank-Pro-His-blank-blank-blank-Cys (SEQ ID NO:6).
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Also disclosed is use of a specific binding
protein for the treatment or prophylaxis of canine allergy,
wherein the specific binding protein specifically binds to a
recombinant plant virus particle comprising at least one
copy of an isolated and purified peptide comprising
Cys-blank-blank-Pro-His-blank-blank-blank-Cys (SEQ ID NO:6).
Also disclosed is use of a specific binding
protein for the treatment or prophylaxis of canine allergy,
wherein the specific binding protein specifically binds to a
multiply antigenic peptide comprising multiple copies of an
isolated and purified peptide which comprises Cys-blank-Pro-
His-blank-Pro-blank-blank-Cys (SEQ ID NO:9).
Also disclosed is use of a specific binding
protein for the treatment or prophylaxis of canine allergy,
wherein the specific binding protein specifically binds to a
recombinant plant virus particle comprising at least one
copy of an isolated and purified peptide comprising
Cys-blank-Pro-His-blank-Pro-blank-blank-Cys (SEQ ID NO:9).
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DEFINITIONS
Amino Acids:
TABLE 1: AMINO ACID ABBREVIATIONS
Amino Acid One-Letter Symbol Three-Letter Symol
alanine A ala
arginine R arg
asparagine N asn
aspartic acid D asp
cysteine C cys
glutamic acid E glu
glutamine Q gln
glycine G gly
histidine H his
isoleucine I ile
leucine L leu
lysine K lys
methionine M met
phenylalanine F phe
proline P pro
serine S ser
threonine T thr
tryptophan w trp
tyrosine Y tyr
valine V val
cDNA clone: A duplex DNA sequence representing an RNA,
carried in a cloning vector.
Cloning: The selection and propagation of a single DNA
species.
Cloning Vector: A plasmid, phage DNA or other DNA
sequence, able. to replicate in a host cell and capable of
carrying exogenously added DNA sequence for purposes of
amplification or expression of the added DNA sequence.
Codon: A triplet of nucleotides that represents an amino
acid or termination signal.
Conservative variants: Conservative variants of
nucleotide sequences include nucleotide substitutions that do
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not result in changes in the amino acid sequence encoded by
such nucleotides, as well as nucleotide substitutions that
result in conservative amino acid substitutions, e.g., amino
acid substitutions which do not substantially affect the
character of the polypeptide translated from said nucleotides.
For example, the character of a peptide derived from IgE is
not substantially affected if the substitutions do not
preclude specific binding of the peptide to canine IgE
receptor or other canine IgE binding ligands.
Conservative variants of amino acid sequences include
amino acid substitutions or deletions that do not
substantially affect the character of the variant polypeptide
relative to the starting peptide. For example, polypeptide
character is not substantially affected if the substitutions
or deletions do not preclude specific binding of the variant
peptide to a specific binding partner of the starting peptide.
The term mimotope refers to a conservative variant of an amino
acid sequence, to which antibody specificity has been raised.
The mimotope. comprises a variant of the epitope of the
starting peptide such that it is able to bind antibodies that
cross-react with the original epitope.
DNA Sequence: A linear series of nucleotides connected
one to the other by phosphodiester bonds between the 3' and 5'
carbons of adjacent pentoses.
Expression: The process undergone by a structural gene to
produce a polypeptide. It is a combination of transcription
and translation.
Expression Control Sequence: A DNA sequence of
nucleotides that controls and regulates expression of
structural genes when operatively linked to those genes.
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Exon: A contiguous region of DNA encoding a portion of a
polypeptide. Reference to any exon, e.g. "DNA sequence of exon
6", refers to the complete exon or any portion thereof.
Genome: The entire DNA of a substance. It includes
inter alia the structural genes encoding for the polypeptides
of the substance, as well as operator, promoter and ribosome
binding and interaction sequences such as the Shine-Dalgarno
sequences.
Nucleotide: A monomeric unit of DNA or RNA consisting of
a sugar moiety (pentose), a phosphate, and a nitrogenous
heterocyclic base. The four DNA bases are adenine ("A"),
guanine ("G"), cytosine ("C") and thymine ("T"). The four RNA
bases are A, G, C and uracil ("U"). A and G are purines, and
C, T, and U are pyrimidines.
Phage or Bacteriophage: Bacterial virus, many of which
include DNA sequences encapsidated in a protein envelope or
coat ("capsid").
Plasmid: An autonomous self-replicating extrachromosomal
circular DNA.
Polymerase Chain Reaction (PCR): A method of amplifying a
target DNA sequence contained in a mixture of DNA sequences,
by using oligonucleotide primers that flank the target DNA
sequence for repeated cycles of DNA synthesis of the target
DNA sequence.
Polypeptide: A linear series of amino acids connected
one to the other by peptide bonds between the a-amino and
carboxyl groups of adjacent amino acids.
Reading Frame: The grouping of codons during translation
of mRNA into amino acid sequences. For example, the sequence
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GCTGGTGTAAG may be translated in three reading frames or
phases, each of which affords a different amino acid sequence:
GCT GGT TGT AAG-Ala-Gly-Cys-Lys
G CTG GTT GTA AG-Leu-Val-Val
GC TGG TTG TAA A-Trp-Leu-(STOP).
Recombinant DNA Molecule: A hybrid DNA sequence
comprising at least two nucleotide sequences, the first
sequence not normally being found together in nature with the
second.
Specific binding: Binding of one substance to another at
greater binding affinity than background binding. Two
substances that exhibit specific binding are referred to as
specific binding partners, or as a specific binding pair. An
antibody and its antigen are one example of a specific binding
pair.
Specific Binding Molecule: A molecule that exhibits
specific binding to its corresponding binding partner to form
a specific binding pair. As used herein, this definition of
specific binding molecule covers monoclonal and polyclonal
antibodies, antigen-binding fragments of these antibodies,
hybrid antibodies, single-chain antibodies, and recombinant
molecules capable of specific binding to a ligand.
Structural Gene: A DNA sequence that encodes through its
template or.messenger RNA ("mRNAII) a sequence of amino acids
characteristic of a specific polypeptide.,
Transcription: Synthesis of RNA on a DNA template.
Translation: Synthesis of peptides on the mRNA template.
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DESCRIPTION OF FIGURES
Fig. 1A depicts the core structure of a MAP protein with
four arms, i.e., a 4-MAP protein; Fig. 1B depicts the core
structure of a MAP protein with eight arms, i.e., an 8-MAP
protein.
Fig. 2 depicts data comparing the binding characteristics
of several monoclonal antibodies to surface bound IgE (e.g.,
analogous to IgE expressed on the surface of B cells); IgE
receptor (e.g., analogous to the Fc receptor on mast cells);
and to a combination of TgE when bound by the IgE receptor.
Thus each component assayed was immobilized on a solid
surface.
Fig. 3 depicts the ability of recombinant exon 3 to
inhibit conjugated antibody 8H.8 or conjugated antibody 15A.2
from binding to an IgE solid phase. Data for 8H.8 is depicted
by diagonal hatched bar graphs, data for 15A.2 is depicted by
speckled bar graphs.
Fig. 4 depicts the ability of soluble, purified canine
IgE to inhibit monoclonal antibody 8H.8 (2.5 g/ml) or
monoclonal antibody 15A.2 (2.5 g/ml) from binding to a canine
IgE solid phase. Data for 8H.8 is depicted by diagonal hatched
bar graphs; data for 15A.2 is depicted by speckled bar graphs.
Fig. 5 depicts the ability of monoclonal antibody 15A.2
or monoclonal antibody 8H.8 to inhibit IgE from binding to
recombinant IgE receptor immobilized on a solid phase as
detected by monoclonal antibody D9.
Fig. 6 depicts the amino acid sequences of the New
England Biolabs PhDc7c phage display library that were bound
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by the monoclonal antibody 15A.2. These sequences are shown
in alignment with the protein sequence of canine IgE.
Fig. 7 depicts the alignment of the 15A.2 binding region
from seven different mammals: dog, human, green monkey, cat,
swine, mouse and horse.
Fig. 8 depicts the ability of phage displaying 15A.2
mimotope peptides to inhibit canine IgE from binding the 15A.2
monoclonal antibody on the solid phase.
Fig. 9 depicts the ability of a specific 15A.2 mimotope
peptide sequence attached to the solid phase to be able to
bind the 15A.2 mimotope monoclonal antibody.
Fig. 1OA and 10B depict the ability of a 15A.2 mimotope
peptide to be able to prevent the 15A.2 monoclonal antibody
from binding canine IgE on the solid phase.
Fig. 11 depicts the ability of monclonal antibody 8H.8
and monoclonal antibody 15A.2 to bind to purified canine IgE
immobilized on a solid phase.
Fig. 12 depicts data from a study evaluating the ability
of antibody 15A.2 conjugated to a signal moiety to bind to
solid phases having various peptides immobilized thereon.
Fig. 13 depicts data from a study evaluating the ability
of monoclonal antibody 14K2, known to bind with canine IgE
exon 4, to bind with various peptides immobilized on a solid
phase.
Fig. 14 depicts data from a study evaluating the ability
of monoclonal antibody 15A.2, known to bind to canine IgE exon
3, to bind to various peptides immobilized on a solid phase.
Fig. 15 depicts data for studies that compared the
binding of monclonal antibody 8H.8, or a polyclonal antibody
raised to recombinant IgE (Re-hu IgE), to bind to a solid
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phase having peptide E3a.5 or substituted peptide E3a.5
(designated peptide S3a.5) immobilized thereon.
Fig. 16 depicts data from a study evaluating the ability
of antibody 8H.8 conjugated to a signal moiety to bind to
solid phases having various peptide immobilized thereon.
Fig. 17 depicts the ability of polyclonal anti-human IgE
antibodies to bind to various peptides immobilized on a solid
phase.
MODES FOR CARRYING OUT INVENTION
Proposed Mechanism of Action of Anti-IgE Monoclonal
Antibodies in Causing Persistent Serum IgE Depletion
A hypothesis in accordance with the present invention is
that anti-IgE monoclonal antibodies affect IgE levels by
directly binding with the resident circulating IgE, after
which the bound complex is removed from the circulation.
Moreover, it is hypothesized that anti-IgE monoclonal
antibodies affect IgE levels by interfering with the
production of new IgE by B-cells. Memory B-cells, cells that
upon interacting with antigen and T-cell become antibody
producing cells (i.e., "memory IgE B-cells"), are involved in
replenishing serum IgE, and these cells contain IgE as their
cell surface receptors. Thus, it is possible that anti-IgE
monoclonal antibodies bind to the IgE on these antibody
producing cells and interfere with their function, such as by
down-regulation or by one or more mechanisms which lead to
cell destruction. Two general ways have been proposed for how
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the binding of a monoclonal antibody to a memory B cell might
interfere with production of IgE.
First, activation of a memory B cell, may also require
the involvement of a surface IgE-binding factor designated
CD23. Binding of certain monoclonal antibodies to the cell
surface IgE may preclude CD23 binding, and may thus lead to an
inability to mount an IgE response.
Second, IgE antibody production by a memory IgE B cell
may be reduced or eliminated by binding of a monoclonal
antibody that is directed against, or blocks, the receptor for
the IgE molecule which is located on the surface of the memory
B cell.
In the absence of a monoclonal antibody that leads to
memory IgE B cell elimination or inactivation, however, it
would be expected that the memory IgE B-cells would be
replenished, leading to the eventual replenishment of
circulating IgE.
The serum levels of IgE are elevated in patients
experiencing allergic disease. As disclosed herein, when
antibodies were generated that specifically bind to a species
IgE, and these antibodies were administered to a patient who
is a member of the species ("passive immunization"), that
patient's serum levels of IgE declined. As appreciated by one
of ordinary-skill in the art, the specific binding proteins in
accordance with the invention are administered in
pharmacologically accepted dosages, and can be administered
with suitable biocompatible diluents. Also disclosed herein
are a method and related compositions that serve to induce a
response to a self-peptide, such as an IgE molecule, whereby
the immune system is manipulated so as to allow an
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auto-reactive B cell to become an antibody-secreting B cell
("active immunization").
Receptors are present on mast cells that bind to IgE from
the circulation. When IgE has become bound to the receptors on
mast cells, the IgE molecules can become crosslinked. Upon
cross-linking these IgE molecules, the mast cell is induced to
release histamine. Histamine is an agent that induces the
manifestation of allergic symptoms. Generally, cross-linking
occurs by binding of the IgE molecules to an antigen, e.g., an
allergen. However, a monoclonal antibody that binds to IgE
could serve as a means for cross-linking IgE which has become
bound to the surface of mast cells.
For allergic individuals, if anti-IgE antibodies were
present in circulation, it becomes easy to envisage that the
binding of such auto-anti-IgE to IgE molecules on mast cells
could serve to cross-link the IgE molecules on those cells and
exacerbate an ongoing allergic response even in the absence of
allergen. This occurrence is clearly undesirable in the
context of allergy treatment. An antibody that achieved this
function would therefore not be preferred for use as a
therapeutic in accordance with the invention. Therefore, an
antibody that achieves such crosslinking is disadvantageous,
and is not preferred in accordance with the invention.
Two approaches exist for producing monoclonal antibodies
which target IgE, but which do not cross-link IgE molecules
which have become bound to a mast cell. The first is to
produce monoclonal antibodies that are directed to an epitope
of an IgE molecule that is only accessible when the IgE is in
circulation. Since the monoclonal antibody can only bind to
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IgE when it is in circulation, it will not be able to bind to
IgE that has become bound to the surface of a mast cell.
An alternative approach for producing anti-IgE antibodies
has been hypothesized by Stanworth et al, e.g., U.S. Patent
No. 5,601,821 issued 11 February 1997. The antibodies of
Stanworth are disclosed to cross-link IgE on mast cells, but
in a manner that does not induce histamine release. Stanworth
has disclosed an epitope of the human IgE molecule which must
be available or accessible after IgE molecules have become
cross-linked on the surface of a mast cell, in order for the
mast cell to release histamine. Thus, Stanworth disclosed an
antibody that binds to that particular epitope. Accordingly,
monoclonal antibodies that are directed to this IgE epitope
will serve to cross-link the IgE, but will not permit the mast
cell to release histamine.
The approach followed in accordance with the present
invention is the development of antibodies, either ex vivo
monoclonal or in vivo anti-self antibodies, directed to
epitopes which are accessible on circulating IgE but which are
not accessible when such IgE becomes bound to a mast cell.
Passive Immunity
Monoclonal Antibodies 15A.2 and 8H.8
Monoclonal antibody 15A.2 binds to canine IgE. The 15A.2
monoclonal antibody was derived by immunizing mice with
affinity-purified canine IgE. The epitope bound by 15A.2 is
conformational, not linear. As indicated by data depicted in
Figure 2, 15A.2 did not bind to the IgE receptor. Nor did it
bind to IgE when bound to receptor. However, 15A.2 exhibited
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affinity for free IgE. It binds to recombinant canine exon 3
fusion proteins in an enzyme linked immunosorbent assay
(ELISA) and by Western blot, but not to other recombinant
canine IgE fusion proteins.
Characterization experiments of the 15A.2 monoclonal
antibody showed that 15A.2 did not bind to IgE that was
already bound by the IgE receptor on mast cells. Thus, it
appeared that access to the epitope bound by 15A.2 was
hindered by IgE binding to the Fc receptor on mast cells.
Accordingly, 15A.2 will not crosslink IgE bound to mast cells.
This finding was demonstrated by binding studies with a
recombinant receptor discussed herein.
Monoclonal antibody 8H.8 also binds to canine IgE. The
8H8 monoclonal antibody was derived by immunizing mice with a
shortened version of exon 3 of the canine IgE molecule,
designated exon 3a. Exon 3a contains the C-terminal 71 amino
acids of the full length exon 3. Previous studies (data not
presented herein) had shown that immunizing mice with the full
length exon 3 did not generate antibodies having specificity
such as that ultimately found with the 8H8 antibody.
Characterization experiments of the 8H8 monoclonal
antibody showed that, unlike monoclonal antibodies derived by
immunization with all other recombinant IgE sequences, 8H8
would also bind to native canine IgE. Additionally, like
15A.2 and as depicted in Figure 2, it was found that 8H.8 did
not bind to IgE that was already bound by the IgE receptor on
mast cells. Thus, it appeared that access to the epitope
bound by 8H.8 was also hindered by IgE binding to the Fc
receptor on mast cells. As a result, BH.B will not crosslink
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IgE bound to mast cells. This finding was demonstrated by
binding studies with a recombinant receptor discussed herein.
Competitive assays were performed between an antibody,
8H.8 or 15A.2, and soluble exon 3 to identify the level of
inhibition of binding of these antibodies to affinity purified
native IgE immobilized on the solid phase of an ELISA. The
results from this study are depicted in Figure 3. In Figure 3
it is seen that increasing the concentration of recombinant
exon 3 inhibited the binding of monoclonal antibody 8H.8, but
did not inhibit antibody 15A.2.
In addition, the ability of these monoclonal antibodies
to inhibit IgE from binding to recombinant IgE receptor on an
ELISA solid phase was examined.
Additionally, competitive assays were performed with
antibody 8H.8, and analogous assays were performed with 15A.2,
where the antibody was in competition with affinity-purified
canine IgE in solution and IgE on an ELISA solid phase. Data
from these assays is depicted in Figure 4.
These studies suggested that 15A.2 had a high affinity
for soluble native canine IgE because as the concentration of
the soluble IgE was increased, the binding of 15A.2 to the
immobilized immunoglobulin dropped appreciably, indicating
that 15A.2 was now binding to the soluble IgE. In contrast,
it is seen that antibody 8H.8 has a much lower affinity for
soluble IgE, because increasingly high concentrations of the
soluble IgE did not inhibit the ability of 8H.8 to bind to the
immobilized IgE. Thus, the data suggest that soluble IgE was
not effective at inhibiting 8H.8 from binding to IgE
immobilized on a solid phase. Thus, the affinity of 8H.8 for
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soluble canine IgE was lower than the affinity of this
monoclonal antibody for IgE on a solid phase.
These studies further suggested that 8H.8 had a low
affinity for soluble native canine IgE, because increasingly
high concentrations of the soluble IgE did not inhibit the
ability of 8H.8 to bind to the immobilized IgE. In contrast,
it is seen that antibody 15A.2 has a much higher affinity for
soluble IgE, since as to the concentration of the soluble IgE
was increased, the binding of 15A.2 to the immobilized
immunoglobulin dropped appreciably, indicating the 15A.2 was
now binding to the soluble IgE. Thus, the data suggested that
soluble IgE was not effective at inhibiting 8H.8 from binding
to IgE immobilized on a solid phase. Thus, the affinity of
8H.8 for soluble canine IgE was lower than the affinity of
this monoclonal for IgE on a solid phase.
To further substantiate the finding that 15A.2 has a
higher affinity for soluble native IgE, and that 8H.8 had a
low affinity for soluble native IgE, studies were performed
where 8H.8 was used, and analogous studies were conducted with
15A.2, to determine the extent to which each antibody inhibits
binding of the soluble IgE to a recombinant IgE receptor solid
phase. A labeled antibody D9 was used as a means for
detection in the studies depicted in Figure 5. The monoclonal
antibody D9-is known to bind to IgE when bound to receptor.
For the studies depicted in Figure 5, the native IgE was
present at a concentration of 2.5 micrograms per milliliter.
As shown in Figure 5, 15A.2 was effective at inhibiting
binding of the native IgE to recombinant IgE receptor at
antibody concentrations as low as 190 nanograms per
milliliter. In contrast, 3,125 nanograms per milliliter of
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8H.8 were necessary to inhibit the binding of native IgE to
the recombinant IgE receptor. Thus, since more 8H.8 than
15A.2 was required to inhibit the binding of native IgE to the
recombinant receptor, it was seen that 8H.8 had a lower
affinity for the soluble IgE. It appears, therefore, that
when a sufficient concentration of either 8H.8 or 15A.2
antibodies is provided, that the antibodies impair the binding
of soluble IgE to the IgE receptor.
Furthermore, as indicated by the data depicted in Figure
2, the avidity of 8H8 binding for native canine IgE was
greatly increased when the IgE was immobilized. These studies
suggested that 8H8 had a low affinity for soluble native
canine IgE, but that when the IgE is immobilized on a surface,
or when it is expressed on the surface of a memory B cell., the
avidity of 8H.8 binding for native canine IgE was greatly
increased.
Accordingly, in view of these findings, it was
hypothesized that the region within native IgE recognized by
8H.8 might be partially hidden, particularly when the IgE is
in serum. A partially sequestered amino acid sequence of IgE
may not be readily exposed to the native canine immune system
and its normal protective mechanisms. Furthermore, since the
full length exon 3 did not lead to the generation of
antibodies having 8H.8-like specificity, the full length
sequence might contain suppressor peptides capable of down-
regulating or eliminating an auto-anti-IgE response which is
specific against the epitope recognized by the 8H.8 antibody.
Either mechanism could serve to avoid production of an
autoimmune response to self-antigens.
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The implications of these findings are that 8H.8
antibody, when used as an allergy therapeutic in dogs, would
preferentially bind to IgE on memory B cells rather than to
IgE in solution. Moreover, 8H.8 has a low binding affinity for
IgE when bound by Fc receptor on mast cells.
Phage display technology was used to map the epitope,
bound by the 8H.8 antibody. A preferred method for performing
phage display technology is accomplished by use of a Ph.D.
Phage Display Peptide Library Kit (New England BioLabs,
Beverly Mass.). It was found that a 7 amino acid peptide
(Thr-Leu-Leu-Glu-Tyr-Arg-Met) (SEQ ID NO:4) inhibited
monoclonal antibody 8H.8 from competitive binding to either
native or recombinant canine IgE. This sequence contained 6
amino acids common in form and/or spacing to the C-terminal
region of exon 3. An 11 amino acid peptide synthesized from
this region (Gly-Met-Asn-Leu-Thr-Trp-Tyr-Arg-Glu-Ser-Lys)(SEQ
ID NO:5) designated E3a.5 also inhibited monoclonal antibody
8H8 from competitive binding to native or recombinant IgE.
It is believed that antibody responses with specificity
such as that of 8H.8 do not occur naturally, even upon
occurrence of events which would induce auto-anti-IgE
responses to other epitopes, because the 8H8 epitope is only
partially available for recognition. Nevertheless, it is
hypothesized that antibodies to an 8H.8-type epitope,
generated from peptide immunization in accordance with the
invention do bind to the partially available epitope, as does
the 8H.8 monoclonal antibody.
It is further hypothesized that the region within native
IgE recognized by 15A.2 might serve as an immunogen to induce
auto-anti-IgE responses and might generate antibodies capable
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of binding to IgE+ B cells and of down regulating IgE
synthesis. The implications of these findings are that 15A.2
antibody, when used as an allergy therapeutic in dogs, would
prevent IgE from binding to mast cells and potentially affect
the synthesis of IgE by B cells.
Phage display technology was used to map the epitope
bound by the 15A.2 antibody. As noted above, a preferred
method for performing phage display technology is accomplished
by use of a Ph.D. Phage Display Peptide Library Kit (New
England BioLabs, Beverly Mass.). Figure 6 depicts the amino
acid sequences of the PhDc7c library that were bound by the
monoclonal antibody 15A.2. These sequences are shown in
alignment with the protein sequence of canine IgE. Figure 7
depicts the alignment of the 15A.2 epitope from seven
different mammals: dogs, human, green monkey, cat, swine,
mouse and horse.
Figure 8 portrays the ability of different phage
displaying 15A.2 mimotope peptides to inhibit canine IgE from
binding the 15A.2 monoclonal antibody on the solid phase. 466
l of biotinylated 15A.2 antibody (10 g/ml) were mixed with
466 l of phage from a fresh overnight culture. Into each
well of a microtiter plate 100 l of the mixture was added.
The antibodies and phage were allowed to bind for 2 % hours.
The wells were then coated with strepavidin. The plates were
washed three times with standard wash buffer to remove loosely
bound material. A serial dilution of canine IgE was prepared,
starting with the concentration of 1 g/100 l of PBS, o.l%
Tween. 100 l of dilution IgE was added per well and allowed
to bind for 10 minutes. The competition reaction was stopped
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by washing the plates five times with wash solution. The
plate was then developed with HRPO linked D9 monoclonal
antibody, which binds to domain 4 of IgE, to visualize the IgE
in the solid phase. Loss-of signal indicates the phage
successfully repelled canine IgE competition. Table 1 shows
the concentrations of the reactants at the various points on
the x-axis of figure B.
TABLE 1
IgE Phage
1 1.0 0
2 1.0 50 1
3 0.5 g 50 1
4 0.25 g 50 1
5 0.125 g 50 l
Using the New England Biolabs PhD12 phage display
library, it was found that a 12 amino acid peptide (SEQ ID
NO:11 Val-Thr-Leu-Cys-Pro-Asn-Pro-His-Ile-Pro-Met-Cys)
inhibited monoclonal antibody 15A.2 from competitive binding
to either native or recombinant canine IgE. This sequence
contained 4 amino acids common in form and/or spacing to the
N-terminal region of exon 3.
Figure 9 displays the ability of 15A.2 monoclonal
antibody to bind a synthetic peptide. The peptide SEQ ID
NO:12 Ser-Val-Thr-Leu-Cys-Pro-Asn-Pro-His-Ile-Pro-Met-Cys-Gly-
Gly-Gly-Lys was synthesized and biotinylated on the epsilon
carbon of the Lys residue. This sequence corresponds to the
isolate M13-48. The peptide was treated in a manner to
promote reduction and cyclization of the cysteines to form a
cyclic peptide. A serial dilution of the peptide was prepared
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and bound to strepavidin-coated microtiter plates (5 g
strepavidin per well). Bound peptide was detected with HRPO
conjugated 15A.2 monoclonal antibody. Figure 9 shows the
results of this experiment. This solid phase specific 15A.2
mimotope peptide bound the 15A.2 monoclonal antibody in a
concentration dependent manner.
Figures l0A and 10B depict the ability of a 15A.2
mimotope peptide to prevent the 15A.2 monoclonal antibody from
binding canine IgE on the solid phase. Plates were coated
with canine IgE at a concentration of 1 g/ml. A serial
dilution of the synthetic peptide was added to HRPO conjugated
15A.2 monoclonal antibody (final concentration 1 g/ml) and
allowed to bind for two hours. 100 l of the 15A.2/peptide
mixture was added to the wells and allowed to bind 1 hour.
The reaction was stopped by washing the plates six times with
wash buffer. The plates. were developed with HRPO substrate,
the reactions stopped with stop mix, and the plates were
measured for O.D. using a plate reader.
Apart from active immunization with antibody 15A.2 or
8H.8 to induce auto-anti-IgE production in vivo, this
invention also comprises a specific binding molecule that
specifically binds a ligand of the type bound by 15A.2 or
8H.8, as well as the therapeutic or prophylactic use thereof.
Thus, the invention comprises a monoclonal antibody specific
for a conservative variant of a sequence bound by 15A.2, for a
conservative variant of a sequence bound by 8H.8, or for a
native sequence of canine IgE up to 100 amino acids from the
C' terminal portion of exon 3. The specific binding molecules
of the invention can be used in a method in accordance with
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the invention as a treatment for or as prophylaxis for allergy
symptoms, i.e., passive immunization.
Accordingly, administration to a dog of either an
antibody in accordance with the invention, such as 15A.2 or
8H.8, or a peptide in accordance with the invention, such as
the 7, 11 or 12 amino acid peptide, leads to a diminution of
further IgE production. This may occur, for example, by the
15A.2 or 8H.8 antibody binding to the memory B cell and
preventing further IgE production. For a peptide, its
administration would lead to production of antibodies of
comparable specificity and effect as 15A.2 or 8H.8.
EXAMPLES
Example 1
Figure 11 depicts results of the binding study where
antibody 8H.8 and antibody 15A.2 were separately assayed to
determine their ability to bind to a solid phase having
affinity purified canine IgE immobilized thereon. These
results showed that those 8H.8 and 15A.2 bind to native IgE
when immobilized on a solid phase. The results from these
studies suggest that the binding of each of these antibodies
to the surface bound IgE was of comparable affinity. To
determine whether the affinity of binding of each antibody
was, in fact, comparable, further studies were performed.
Figure 12 depicts results of the study where antibody
8H.8 conjugated to a signal moiety was reacted at various
concentration with solid phases having various peptides
immobilized thereon. The data depicted by (+) reflects a
solid phase having canine IgE immobilized thereon; data
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depicted by (0) depicts solid phase having E3a.5 immobilized
thereon; data depicted by (A) depicts solid phase having
E3a.5 extended peptide immobilized thereon; data depicted by
(X) depicts data for a solid phase having an E3a.5 scrambled
peptide immobilized thereon; and data depicted by (s) depicts
data for solid phase having the seven amino acid peptide
identified by phage display technology to which 8H.8 binds
immobilized thereon, Thus, it is seen that 8H.8 binds to each
solid phase with the exception solid phase having the E3a.5
scrambled peptide.
Monoclonal antibody 14K.2 is known to bind exclusive to
canine IgE exon 4. This monoclonal antibody was reacted with
various solid phases having different peptides immobilized
thereon. The data depicted by (=) reflects a solid phase
having canine IgE immobilized thereon; data depicted by (a)
depicts solid phase having E3a.5 immobilized thereon; data
depicted by (A) depicts solid phase having E3a.5 extended
peptide immobilized thereon; data depicted by (X) depicts data
for a solid phase having an E3a.5 scrambled peptide
immobilized thereon; and data depicted by (0) depicts data for
solid phase having the seven amino acid peptide identified by
phage display technology to which 8H.8 binds immobilized
thereon. Thus it is seen that 14K.2 only binds to a solid
phase having the entire canine IgE molecule immobilized
thereon. This data is depicted in Figure 13.
Monoclonal antibody 15A.2 is believed to bind only to
canine IgE exon 3. Figure 14 depicts data for studies which
evaluated the binding of 15A.2 to various peptides. The data
depicted by (+) reflects a solid phase having canine IgE
immobilized thereon; data depicted by (U) depicts solid phase
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having E3a.5 immobilized thereon; data depicted by (0) depicts
solid phase have E3a.5 extended peptide immobilized thereon;
data depicted by an (X) reflects data for a solid phase having
an E3a.5 scrambled peptide immobilized thereon; and data
depicted by (I)depicts data for solid phase having the seven
amino acid peptide identified by phage display technology to
which 8H.8 binds immobilized thereon. These binding studies
showed that 15A.2 bound only to a solid phase which had the
entire canine IgE molecule immobilized thereon. This data
indicated that 15A.2 does not bind the same epitope as that
bound by 8H.8.
Figure 15 depicts data for studies that compare the
binding of 8H.8 or polyclonal antibodies raised to recombinant
human IgE, to peptide E3a.5 or to peptide E3a.5 with an amino
acid substitution making the peptide more analogous to a
sequence in the human genome which encodes human IgE,
designated peptide S3a.5. In Figure 15 data depicted by the
(=) reflects substituted 3a.5 compared with 8H.8; data
depicted by (a) reflects substituted peptide 3a.5 and Re-Hu
IgE; data depeicted by (A) reflects peptide E3a.5 and 8H.8;
and data depicted by (X) reflects data regarding peptide E3a.5
and polyclonal Re-Hu IgE. These data indicated that only 8H.8
became bound to E3a.5 on a solid phase, no other combination
exhibited binding.
Figure 16 depicts the data on studies that compared the
binding of 8H.8 to various peptides immobilized on a solid
phase. Data depicted by (*) reflects data for a solid phase
having canine IgE immobilized thereon; data depicted by (0)
reflects data for a solid phase having a peptide E3a.5
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769.09-207
extended immobilized thereon; data depicted by (A) reflects
data for a solid phase having human IgE immobilized thereon;
and data depicted by an (X) reflects data for a solid phase
having a 8H.8 peptide immobilized thereon. Accordingly it is
seen that 8H.8 binds to canine IgE, peptide E3a.5 extended or
the seven amino acid peptide identified by phage display
technology as the binding epitope for 8H.8, when each of these
peptides were immobilized on a solid phase. Additionally, it
is seen that 8H.8 does not bind to human IgE.
Figure 17 depicts data for studies which compare the
ability of polyclonal anti-human IgE antibodies ability to
bind to solid phases having various peptides immobilized
thereon. Data depicted by ()reflects data for a solid phase
having canine IgE immobilized thereon; data depicted by (0)
reflects data for a solid phase having a peptide E3a.5
extended immobilized thereon; data depicted by (A)reflects
data for a solid phase having human IgE immobilized thereon;
and data depicted by an (X) reflects data for a solid phase
having a 8H.8 peptide immobilized thereon. Thus, it was seen
that the anti-human IgE polyclonal antibodies bound to human
IgE when immobilized on a solid phase. It was also seen that
the polyclonal antiserum exhibited limited binding to peptide
E3a.5 extended when the polyclonal antibodies were present at
very high concentrations; this was believed to be a binding
artifact due to the high antibody concentration. Thus, it was
found that antibodies to Human IgE do not specifically bind to
canine IgE or to peptides in accordance with the invention.
CA 02329148 2000-11-29
WO 00/58365 PCT/USOO/08347
Example 2
The amino acid sequences for the epitope bound by 8H.8,
the 11 amino acid sequence used in the immunization studies
presented herein, as well as analogous sequences from feline
and Human IgE sequences are set forth in Table 2.
Additionally, a substituted canine sequence was prepared
having an amino acid substitution which made the peptide more
closely analogous to the human peptide.
TABLE 2
Species/Source Sequence SEQ ID NO.
mimotope bound by 8H.8 T-L-L-E-Y-R-M 4
canine G-M-N-L-T-W-Y-R-E-S-K 5
human V-N-L-T-w-S-R 13
feline G-M-T-L-T-W-S-R-E-N-G 14
Substituted Canine Sequence G-M-N-L-T-W-S-R-E-S-K 15
Each of the peptides in Table 2 was placed on a solid
phase and studies were performed to identify if 8H.8 would
bind to the surface-bound peptide. It was found that 8H.8
bound to a surface having the mimotope peptide thereon, and to
a surface having the 11 amino acid peptide bound thereon. It
was found that 8H.8 did not bind to the human peptide, the
feline peptide or to the canine sequence which had an amino
acid substitution which made it more analogous to the human
sequence, when either of these peptides were surface bound.
Closing
As used herein and in the appended claims, the singular
forms "a," "and," and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example,
36
CA 02329148 2010-04-30
reference to "a formulation" includes mixtures of different
formulations and reference to "the method of treatment"
includes reference to equivalent steps and methods known to
those skilled in the art, and so forth.
Unless defined otherwise, all technical and scientific
terms used herein have the same meaning as commonly understood
by one of ordinary skill in the art to which this invention
belongs. Although any methods and materials similar to or
equivalent to those described herein can be used in the
practice or testing of the invention, the preferred methods
and materials are now described.
SEQUENCE LISTING IN ELECTRONIC FORM
In accordance with Section 111(1) of the Patent Rules, this description
contains a sequence listing in electronic form in ASCII text format
(file: 76909-207 Seq 23-APR-10 v2.txt).
A copy of the sequence listing in electronic form is available from the
Canadian Intellectual Property Office.
The sequences in the sequence listing in electronic form are reproduced
in the following table.
SEQUENCE TABLE
<110> Lawton, Robert
Mermer, Brion
Francoeur, Greg
<120> Specific Binding Protein for Treating
Canine Allergy
<130> 01-1275A
<140> CA 2,329,148
<141> 2000-03-30
<150> 09/281,760
<151> 1999-03-30
<150> 09/058,331
<151> 1998-04-09
37
CA 02329148 2010-04-30
<160> 39
<170> FastSEQ for Windows Version 3.0
<210> 1
<211> 5
<212> PRT
<213> Canis familiaris
<220>
<221> PEPTIDE
<222> (2) ... (3)
<223> Xaa = any amino acid
<400> 1
Leu Xaa Xaa Tyr Arg
1 5
<210> 2
<211> 5
<212> PRT
<213> Canis familiaris
<220>
<221> PEPTIDE
<222> (3) ... (4)
<223> Xaa = Any amino acid
<400> 2
Tyr Arg Xaa Xaa Leu
1 5
<210> 3
<211> 8
<212> PRT
<213> Canis familiaris
<220>
<221> PEPTIDE
<222> (2) ... (3)
<223> Xaa = Any amino acid
<220>
<221> PEPTIDE
<222> (6) ... (7)
<223> Xaa = Any amino acid
<400> 3
Leu Xaa Xaa Tyr Arg Xaa Xaa Leu
1 5
<210> 4
<211> 7
<212> PRT
<213> Canis familiaris
<400> 4
Thr Leu Leu Glu Tyr Arg Met
1 5
37a
CA 02329148 2010-04-30
<210> 5
<211> 11
<212> PRT
<213> Canis familiaris
<400> 5
Gly Met Asn Leu Thr Trp Tyr Arg Glu Ser Lys
1 5 10
<210> 6
<211> 9
<212> PRT
<213> Canis familiaris
<220>
<221> PEPTIDE
<222> (2)...(3)
<223> Xaa = Any amino acid
<220>
<221> PEPTIDE
<222> (6)...(8)
<223> Xaa = Any amino acid
<400> 6
Cys Xaa Xaa Pro His Xaa Xaa Xaa Cys
1 5
<210> 7
<211> 16
<212> PRT
<213> Canis familiaris
<400> 7
Ser Val Thr Leu Cys Pro Asn Pro His Ile Pro Met Cys Gly Gly Gly
1 5 10 15
<210> 8
<211> 14
<212> PRT
<213> Canis familiaris
<400> 8
Ser Ala Cys Pro Asn Pro His Asn Pro Tyr Cys Gly Gly Gly
1 5 10
<210> 9
<211> 9
<212> PRT
<213> Canis familiaris
<220>
<221> PEPTIDE
<222> (2)...(2)
<223> Xaa = Any amino acid
<220>
<221> PEPTIDE
<222> (5)...(5)
<223> Xaa = Any amino acid
37b
CA 02329148 2010-04-30
<220>
<221> PEPTIDE
<222> (7)...(8)
<223> Xaa = Any amino acid
<400> 9
Cys Xaa Pro His Xaa Pro Xaa Xaa Cys
1 5
<210> 10
<211> 14
<212> PRT
<213> Canis familiaris
<400> 10
Ser Ala Cys His Pro His Leu Pro Lys Ser Cys Gly Gly Gly
1 5 10
<210> 11
<211> 12
<212> PRT
<213> Canis familiaris
<400> 11
Val Thr Leu Cys Pro Asn Pro His Ile Pro Met Cys
1 5 10
<210> 12
<211> 17
<212> PRT
<213> Canis familiaris
<400> 12
Ser Val Thr Leu Cys Pro Asn Pro His Ile Pro Met Cys Gly Gly Gly
1 5 10 15
Lys
<210> 13
<211> 7
<212> PRT
<213> Homo sapiens
<400> 13
Val Asn Leu Thr Trp Ser Arg
1 5
<210> 14
<211> 11
<212> PRT
<213> Felis catus
<400> 14
Gly Met Thr Leu Thr Trp Ser Arg Glu Asn Gly
1 5 10
<210> 15
<211> 11
37c
CA 02329148 2010-04-30
<212> PRT
<213> Canis familiaris
<400> 15
Gly Met Asn Leu Thr Trp Ser Arg Glu Ser Lys
1 5 10
<210> 16
<211> 9
<212> PRT
<213> Canis familiaris
<400> 16
Cys Pro Asn Pro His Ile Pro Met Cys
1 5
<210> 17
<211> 9
<212> PRT
<213> Canis familiaris
<400> 17
Cys Pro Asn Pro His Asn Pro Tyr Cys
1 5
<210> 18
<211> 9
<212> PRT
<213> Canis familiaris
<400> 18
Cys His Pro His Leu Pro Lys Ser Cys
1 5
<210> 19
<211> 9
<212> PRT
<213> Canis familiaris
<400> 19
Cys Ser Asn Pro His Val Thr His Cys
1 5
<210> 20
<211> 9
<212> PRT
<213> Canis familiaris
<400> 20
Cys Ser His Pro His Leu Thr His Cys
1 5
<210> 21
<211> 9
<212> PRT
<213> Canis familiaris
37d
CA 02329148 2010-04-30
<400> 21
Cys Ser Asn Pro His Ile Thr Gln Cys
1 5
<210> 22
<211> 9
<212> PRT
<213> Canis familiaris
<400> 22
Cys Met Asn Pro His Ile Thr His Cys
1 5
<210> 23
<211> 9
<212> PRT
<213> Canis familiaris
<400> 23
Cys Thr Asn Pro His Asn Pro Tyr Cys
1 5
<210> 24
<211> 9
<212> PRT
<213> Canis familiaris
<400> 24
Cys Pro Asn Pro His Asn Pro Tyr Cys
1 5
<210> 25
<211> 9
<212> PRT
<213> Canis familiaris
<400> 25
Cys His Pro His Leu Pro Lys Arg Cys
1 5
<210> 26
<211> 17
<212> PRT
<213> Canis familiaris
<400> 26
Tyr Cys Arg Val Thr His Pro His Leu Pro Lys Asp Ile Val Arg Ser
1 5 10 15
Ile
<210> 27
<211> 17
<212> PRT
<213> Homo sapiens
37e
CA 02329148 2010-04-30
<400> 27
Gln Cys Arg Val Thr His Pro His Leu Pro Arg Ala Leu Met Arg Ser
1 5 10 15
Thr
<210> 28
<211> 17
<212> PRT
<213> Cercopithecus aethiops
<400> 28
Gln Cys Arg Val Thr His Pro His Leu Pro Arg Ala Leu Val Arg Ser
1 5 10 15
Thr
<210> 29
<211> 17
<212> PRT
<213> Felis catus
<400> 29
Gln Cys Lys Val Thr His Pro Asp Leu Pro Leu Val Ile Val Arg Ser
1 5 10 15
Ile
<210> 30
<211> 17
<212> PRT
<213> Sus scrofa
<400> 30
Tyr Cys Asn Val Thr His Pro Asp Leu Pro Lys Pro Ile Leu Arg Ser
1 5 10 15
Ile
<210> 31
<211> 15
<212> PRT
<213> Mus musculus
<400> 31
Gin Cys Ile Val Asp His Pro Asp Phe Pro Ile Val Arg Ser Ile
1 5 10 15
<210> 32
<211> 16
<212> PRT
<213> Equus caballus
<400> 32
Lys Cys Thr Val Ser His Pro Asp Leu Pro Arg Glu Trp Arg Ser Ile
1 5 10 15
<210> 33
<211> 1842
<212> DNA
<213> Canis familiaris
37f
CA 02329148 2010-04-30
<220>
<221> misc feature
<222> (136)..(136)
<223> "n" stands for any nucleic acid
<220>
<221> misc_feature
<222> (413)..(414)
<223> "n" stands for any nucleic acid
<220>
<221> misc_feature
<222> (451)..(451)
<223> "n" stands for any nucleic acid
<220>
<221> misc_feature
<222> (460)..(462)
<223> "n" stands for any nucleic acid
<220>
<221> misc_feature
<222> (500)..(500)
<223> "n" stands for any nucleic acid
<220>
<221> misc_feature
<222> (530)..(530)
<223> "n" stands for any nucleic acid
<220>
<221> misc_feature
<222> (568)..(568)
<223> "n" stands for any nucleic acid
<220>
<221> misc_feature
<222> (847)..(849)
<223> "n" stands for any nucleic acid
<220>
<221> misc_feature
<222> (853)..(853)
<223> "n" stands for any nucleic acid
<220>
<221> misc_feature
<222> (1382)..(1382)
<223> "n" stands for any nucleic acid
<220>
<221> misc_feature
<222> (1832)..(1832)
<223> "n" stands for any nucleic acid
<220>
<221> CDS
<222> (167)..(448)
<220>
<221> CDS
<222> (608)..(931)
37g
CA 02329148 2010-04-30
<220>
<221> CDS
<222> (1024)..(1344)
<220>
<221> CDS
<222> (1419)..(1742)
<400> 33
gtccagtgac ctccatctct gcccccatgc ttttccttct cagacgcccc ctggggccag 60
gagcaggata ccccaggtca acagcgggcc tggcatatga tggggtgaca gtcccaaggc 120
aggcactgac actggncctg tccccacagc caccagccag gacctg tct gtg ttc 175
Ser Val Phe
1
ccc ttg gcc tcc tgc tgt aaa gac aac atc gcc agt acc tct gtt aca 223
Pro Leu Ala Ser Cys Cys Lys Asp Asn Ile Ala Ser Thr Ser Val Thr
10 15
ctg ggc tgt ctg gtc acc ggc tat ctc ccc atg tcg aca act gtg acc 271
Leu Gly Cys Leu Val Thr Gly Tyr Leu Pro Met Ser Thr Thr Val Thr
20 25 30 35
tgg gac acg ggg tct cta aat aag aat gtc acg acc ttc ccc acc acc 319
Trp Asp Thr Gly Ser Leu Asn Lys Asn Val Thr Thr Phe Pro Thr Thr
40 45 50
ttc cac gag acc tac ggc ctc cac agc atc gtc agc cag gtg acc gcc 367
Phe His Glu Thr Tyr Gly Leu His Ser Ile Val Ser Gln Val Thr Ala
55 60 65
tcg ggc gag tgg gcc aaa cag agg ttc acc tgc agc gtg get cac not 415
Ser Gly Glu Trp Ala Lys Gln Arg Phe Thr Cys Ser Val Ala His Xaa
70 75 80
gag tcc acc gcc atc aac aag acc ttc agt get aanccagggt tnnntggcca 468
Glu Ser Thr Ala Ile Asn Lys Thr Phe Ser Ala
85 90
catgacactg gagggagaag ggacaggctg gngaatgcgc catggctggt aacgcccagc 528
anatgtgggg ctggggctga cacatgagtc ccgtgggctn agagacacca ctgccacatg 588
gctgcctcta cttctagca tgt gcc tta aac ttc att ccg cct acc gtg aag 640
Cys Ala Leu Asn Phe Ile Pro Pro Thr Val Lys
95 100 105
ctc ttc cac tcc tcc tgc aac ccc gtc ggt gat acc cac acc acc atc 688
Leu Phe His Ser Ser Cys Asn Pro Val Gly Asp Thr His Thr Thr Ile
110 115 120
cag ctc ctg tgc etc atc tct ggc tac gtc cca ggt gac atg gag gtc 736
Gln Leu Leu Cys Leu Ile Ser Gly Tyr Val Pro Gly Asp Met Glu Val
125 130 135
atc tgg ctg gtg gat ggg caa aag get aca aac ata ttc cca tac act 784
Ile Trp Leu Val Asp Gly Gln Lys Ala Thr Asn Ile Phe Pro Tyr Thr
140 145 150
gca ccc ggc aca aag gag ggc aac gtg acc tct acc cac agc gag ctc 832
Ala Pro Gly Thr Lys Glu Gly Asn Val Thr Ser Thr His Ser Glu Leu
155 160 165
37h
CA 02329148 2010-04-30
aac atc acc cag ggn nng tgn gta tcc caa aaa acc tac acc tgc cag 880
Asn Ile Thr Gln Gly Xaa Xaa Val Ser Gln Lys Thr Tyr Thr Cys Gln
170 175 180 185
gtc acc tat caa ggc ttt acc ttt aaa gat gag get cgc aag tgc tca 928
Val Thr Tyr Gln Gly Phe Thr Phe Lys Asp Glu Ala Arg Lys Cys Ser
190 195 200
gag atggcccccc tgtcccccag aaacccagat gcgcgaggct cagagatgag 981
Glu
ggccaaggca cgccctcatg cagcctctca cacactgcag ag tcc gac ccc cga 1035
Ser Asp Pro Arg
205
ggc gtg agc agc tac ctg agc cca ccc agc ccc ctt gac ctg tat gtc 1083
Gly Val Ser Ser Tyr Leu Ser Pro Pro Ser Pro Leu Asp Leu Tyr Val
210 215 220
cac aag gcg ccc aag atc acc tgc ctg gta gtg gac ctg gcc acc atg 1131
His Lys Ala Pro Lys Ile Thr Cys Leu Val Val Asp Leu Ala Thr Met
225 230 235
gaa ggc atg aac ctg acc tgg tac cgg gag agc aaa gaa ccc gtg aac 1179
Glu Gly Met Asn Leu Thr Trp Tyr Arg Glu Ser Lys Glu Pro Val Asn
240 245 250
ccg gtc cct ttg aac aag aag gat cac ttc aat ggg acg atc aca gtc 1227
Pro Val Pro Leu Asn Lys Lys Asp His Phe Asn Gly Thr Ile Thr Val
255 260 265 270
acg tct acc ctg cca gtg aac acc aat gac tgg atc gag ggc gag acc 1275
Thr Ser Thr Leu Pro Val Asn Thr Asn Asp Trp Ile Glu Gly Glu Thr
275 280 285
tac tat tgc agg gtg acc cac ccg cac ctg ccc aag gac atc gtg cgc 1323
Tyr Tyr Cys Arg Val Thr His Pro His Leu Pro Lys Asp Ile Val Arg
290 295 300
tcc att gcc aag gcc cct ggt gagccacggg cccaggggag gtgggcgggc 1374
Ser Ile Ala Lys Ala Pro Gly
305
ctcctgancc ggagcctggg ctgaccccac acctatccac aggc aag cgt gcc ccc 1430
Lys Arg Ala Pro
310
ccg gat gtg tac ttg ttc ctg cca ccg gag gag gag cag ggg acc aag 1478
Pro Asp Val Tyr Leu Phe Leu Pro Pro Glu Glu Glu Gln Gly Thr Lys
315 320 325
gac aga gtc acc ctc acg tgc ctg atc cag aac ttc ttc ccc gag gac 1526
Asp Arg Val Thr Leu Thr Cys Leu Ile Gln Asn Phe Phe Pro Glu Asp
330 335 340 345
att tca gtg caa tgg ctg cga aac gac agc ccc atc cag aca gac cag 1574
Ile Ser Val Gln Trp Leu Arg Asn Asp Ser Pro Ile Gln Thr Asp Gin
350 355 360
tac acc acc acg ggg ccc cac aag gtc tcg ggc tcc agg cct gcc ttc 1622
Tyr Thr Thr Thr Gly Pro His Lys Val Ser Gly Ser Arg Pro Ala Phe
365 370 375
37i
CA 02329148 2010-04-30
ttc atc ttc agt cgc ctg gtg gac tgg gag cag aaa aac aaa ttc acc 1670
Phe Ile Phe Ser Arg Leu Val Asp Trp Glu Gln Lys Asn Lys Phe Thr
380 385 390
tgc caa gtg gtg cat gag gcg ctg tcc ggc tct agg atc ctc cag aaa 1718
Cys Gln Val Val His Glu Ala Leu Ser Gly Ser Arg Ile Leu Gln Lys
395 400 405
tgg gtg tcc aaa acc ccc ggt aaa tgatgcccac cctcctcccg ccgccacccc 1772
Trp Val Ser Lys Thr Pro Gly Lys
410 415
ccagggctcc acctgctggg gcaggggagg ggggctggca agaccctcca tctatccttn 1832
tcaataaaca 1842
<210> 34
<211> 94
<212> PRT
<213> Canis familiaris
<220>
<221> misc feature
<222> (83)_.(83)
<223> The 'Xaa' at location 83 stands for Asn, Ser, Thr, Ile, Asp, Gly,
Ala, Val, His, Arg, Pro, Leu, Tyr, Cys, or Phe.
<400> 34
Ser Val Phe Pro Leu Ala Ser Cys Cys Lys Asp Asn Ile Ala Ser Thr
1 5 10 15
Ser Val Thr Leu Gly Cys Leu Val Thr Gly Tyr Leu Pro Met Ser Thr
20 25 30
Thr Val Thr Trp Asp Thr Gly Ser Leu Asn Lys Asn Val Thr Thr Phe
35 40 45
Pro Thr Thr Phe His Glu Thr Tyr Gly Leu His Ser Ile Val Ser Gln
50 55 60
Val Thr Ala Ser Gly Glu Trp Ala Lys Gln Arg Phe Thr Cys Ser Val
65 70 75 80
Ala His Xaa Glu Ser Thr Ala Ile Asn Lys Thr Phe Ser Ala
85 90
<210> 35
<211> 108
<212> PRT
<213> Canis familiaris
<220>
<221> misc feature
<222> (81)_.(81)
<223> The 'Xaa' at location 81 stands for Lys, Arg, Thr, Met, Glu, Gly,
Ala, Val, Gln, Pro, Leu, a stop codon, Trp, or Ser.
<400> 35
Cys Ala Leu Asn Phe Ile Pro Pro Thr Val Lys Leu Phe His Ser Ser
1 5 10 15
Cys Asn Pro Val Gly Asp Thr His Thr Thr Ile Gln Leu Leu Cys Leu
20 25 30
Ile Ser Gly Tyr Val Pro Gly Asp Met Glu Val Ile Trp Leu Val Asp
35 40 45
Gly Gln Lys Ala Thr Asn Ile Phe Pro Tyr Thr Ala Pro Gly Thr Lys
50 55 60
Glu Gly Asn Val Thr Ser Thr His Ser Glu Leu Asn Ile Thr Gln Gly
65 70 75 80
37j
CA 02329148 2010-04-30
Xaa Xaa Val Ser Gln Lys Thr Tyr Thr Cys Gln Val Thr Tyr Gln Gly
85 90 95
Phe Thr Phe Lys Asp Glu Ala Arg Lys Cys Ser Glu
100 105
<210> 36
<211> 107
<212> PRT
<213> Canis familiaris
<400> 36
Ser Asp Pro Arg Gly Val Ser Ser Tyr Leu Ser Pro Pro Ser Pro Leu
1 5 10 15
Asp Leu Tyr Val His Lys Ala Pro Lys Ile Thr Cys Leu Val Val Asp
20 25 30
Leu Ala Thr Met Glu Gly Met Asn Leu Thr Trp Tyr Arg Glu Ser Lys
35 40 45
Glu Pro Val Asn Pro Val Pro Leu Asn Lys Lys Asp His Phe Asn Gly
50 55 60
Thr Ile Thr Val Thr Ser Thr Leu Pro Val Asn Thr Asn Asp Trp Ile
65 70 75 80
Glu Gly Glu Thr Tyr Tyr Cys Arg Val Thr His Pro His Leu Pro Lys
85 90 95
Asp Ile Val Arg Ser Ile Ala Lys Ala Pro Gly
100 105
<210> 37
<211> 108
<212> PRT
<213> Canis familiaris
<400> 37
Lys Arg Ala Pro Pro Asp Val Tyr Leu Phe Leu Pro Pro Glu Glu Glu
1 5 10 15
Gln Gly Thr Lys Asp Arg Val Thr Leu Thr Cys Leu Ile Gln Asn Phe
20 25 30
Phe Pro Glu Asp Ile Ser Val Gln Trp Leu Arg Asn Asp Ser Pro Ile
35 40 45
Gln Thr Asp Gln Tyr Thr Thr Thr Gly Pro His Lys Val Ser Gly Ser
50 55 60
Arg Pro Ala Phe Phe Ile Phe Ser Arg Leu Val Asp Trp Glu Gln Lys
65 70 75 80
Asn Lys Phe Thr Cys Gln Val Val His Glu Ala Leu Ser Gly Ser Arg
85 90 95
Ile Leu Gln Lys Trp Val Ser Lys Thr Pro Gly Lys
100 105
<210> 38
<211> 213
<212> DNA
<213> Canis familiaris
<220>
<221> CDS
<222> (1)..(213)
<400> 38
gaa ggc atg aac ctg acc tgg tac cgg gag agc aaa gaa ccc gtg aac 48
Glu Gly Met Asn Leu Thr Trp Tyr Arg Glu Ser Lys Glu Pro Val Asn
1 5 10 15
37k
CA 02329148 2010-04-30
ccg gtc cct ttg aac aag aag gat cac ttc aat ggg acg atc aca gtc 96
Pro Val Pro Leu Asn Lys Lys Asp His Phe Asn Gly Thr Ile Thr Val
20 25 30
acg tct acc ctg cca gtg aac acc aat gac tgg atc gag ggc gag acc 144
Thr Ser Thr Leu Pro Val Asn Thr Asn Asp Trp Ile Glu Gly Glu Thr
35 40 45
tac tat tgc agg gtg acc cac ccg cac ctg ccc aag gac atc gtg cgc 192
Tyr Tyr Cys Arg Val Thr His Pro His Leu Pro Lys Asp Ile Val Arg
50 55 60
tcc att gcc aag gcc cct ggt 213
Ser Ile Ala Lys Ala Pro Gly
65 70
<210> 39
<211> 71
<212> PRT
<213> Canis familiaris
<400> 39
Glu Gly Met Asn Leu Thr Trp Tyr Arg Glu Ser Lys Glu Pro Val Asn
1 5 10 15
Pro Val Pro Leu Asn Lys Lys Asp His Phe Asn Gly Thr Ile Thr Val
20 25 30
Thr Ser Thr Leu Pro Val Asn Thr Asn Asp Trp Ile Glu Gly Glu Thr
35 40 45
Tyr Tyr Cys Arg Val Thr His Pro His Leu Pro Lys Asp Ile Val Arg
50 55 60
Ser Ile Ala Lys Ala Pro Gly
65 70
371
