Note: Descriptions are shown in the official language in which they were submitted.
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SYNTHETIC PEPTIDE IMMUNOGENS AND ANTIBODIES THERETO
FIELD OF THE INVENTION
The present invention generally relates to methods for
selecting peptide immunogens to produce antibodies to a target
protein which exhibits amino acid variation between species.
More specifically, the present invention relates to methods of
selecting peptide immunogens and producing species-specific
antibodies to a mammalian tissue factor, and antibodies produced
by those methods.
DESCRIPTION OF RELATED ART
Antibodies to protein antigens have been used for many years
for numerous research and diagnostic purposes. Antibodies can be
made to specific regions of a protein by injecting a small
peptide portion of the protein (5-50 amino acids) which has been
conjugated to an immunogenic carrier (such as keyhole limpet
hemocyanin [KLH]). The immunized animal will recognize the
peptide as an epitope of the immunogenic carrier. This approach
has advantages over the more common methods involving the
injection of the whole protein. For example, this approach
allows antibody reagents to be made which bind to specific,
predictable domains of the protein of interest. These anti-
peptide antibodies are useful for epitope mapping in
structure/function studies, immunohistochemical localization, and
immunoaffinity purification of the protein of interest. One
particular protein of interest for this invention is mammalian
tissue factor.
Tissue factor (TF) is a transmembrane glycoprotein which
plays a critical role in coagulation (blood clotting). Normal
coagulation begins when vascular endothelium is damaged. Damaged
vascular endothelium exposes TF to plasma, allowing TF to bind to
factor VII, a serine protease zymogen. The binding of TF to
factor VII, and subsequent activation to factor VIIa, is the
primary step for coagulation initiation. Subsequent
conformational changes and Ca2+ bridge formation allow the
TF/FVIIa complex to bind to and to activate factor X. Activated
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factor X converts prothrombin into its active serine protease,
thrombin. Thrombin converts fibrinogen into fibrin. Finally,
fibrin fibers associate with one another to form a clot, or a
platelet-fibrin network that prevents further bleeding.
The amino acid sequence of TF protein between mice, rabbits,
and humans is highly conserved. Pawashe et al., 1991, Thromb.
Haemost. 66:315. The crystal structure of TF consists of two
immunoglobulin-like domains with an extensive interdomain
interface region which contains the binding site for factor VII.
Harlos et al., 1994, Nature 370:662.
TF is expressed in various tissues such as cardiac myocytes,
renal glomeruli, the granular layer of the epidermis, the
epithelium of oropharynx and vagina, and intestinal, urinary
bladder, respiratory mucosa, and at tissue barriers between the
body and the environment. Ruf et al., 1994, FASEB J. 8:385.
Tissue factor has also been implicated in the pathogenesis of
various infectious, cardiovascular and neoplastic diseases.
Excessive coagulation, for example, leads to stroke and ischemia,
resulting in infarction of tissues. Acute myeloblastic leukemias
are known to express TF, resulting in disseminated intravascular
coagulation (DIC), a disorder characterized by excessive bleeding
due to rapid consumption of platelets and disruption of fibrin
polymerization. Id. A better understanding of TF, therefore,
allows researchers to design more effective therapies for
controlling coagulation. Such control can potentially alleviate
complications in patients suffering from these and other
diseases.
Several antibodies have been made to TF. Most of these
anti-TF antibodies were made to the whole TF protein. For
example, monoclonal antibodies to native, factor VII-affinity
purified human TF were used to study the essential regions of the
extracellular domain of TF. Magdolen et al., 1998, Biological
Chemistry 379:157. Other antibodies have been made to purified
or recombinant TF from humans and rabbits. See, e.g., Taylor et
al., 1991, Circulatory Shock 33:127; Morissey et el., 1988,
Thrombosis Res. 52:247; Pawashe et al., 1994, Circulation Res.
74:56; U.S. Pat. App. 5,223,427. However, membrane-bound
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proteins such as TF are difficult to purify, especially in its
native folding conformation. In another approach, monoclonal
antibodies were made from mice injected with murine cultured
cells expressing recombinant rabbit TF. Speidel et al., 1995,
Circulation 92:3323.
Anti-peptide antibodies to TF has been made. In one case,
polyclonal antibodies were made to human TF regions which allow
them to inhibit TF binding to factor VII. Ruf et al., 1991,
Biochem. J. 278:729. In another case, anti-peptide monoclonal
antibodies were made to the C-terminal cytoplasmic domain of
human TF. Carson et al., 1992, Blood Coagulation and
Fibrinolysis 3:779.
Antibodies made to proteins such as TF which have extensive
homology between species would not be expected to exhibit
species-specificity. This has been shown to be the case with TF,
where monoclonal antibodies to human tissue factor cross-react
and inhibit baboon TF. Taylor et al., 1991, Circulation Shock
33:127. This could be a problem, e.g., where the antibodies are
used to diagnose diseases and are required to distinguish between
two pathogenic species. Proteins such as TF with extensive
sequence homology among vertebrates are also limited in the range
of antibodies they would be expected to induce. Since there is
little sequence difference between the protein from the species
of interest and the same protein in the immunizing animal, the
immune system of the immunizing animal would recognize the
homologous regions of the injected protein as a "self" epitope
and would thus only produce antibodies to immunodominant regions
of the injected protein which are not homologous with the
injected animal's analogous protein. For example, murine
monoclonal antibodies to whole human TF could only be produced to
regions of the human TF which are dissimilar to mouse TF. If
antibodies are desired to many regions of the TF protein, for
example for structure-function studies, the results would likely
be disappointing. Therefore, there is a need for improved
methods for making antibodies to proteins which show extensive
amino acid homology between species, where the antibodies need to
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be species-specific or where antibodies are desired to several
regions of vertebrate proteins.
SUNIr2ARY OF THE INVENTION
Among the objects of the invention may be noted the
provision of methods for determining peptides of a target protein
for use as immunogens in order to reliably produce antibodies
which are species-specific. A more specific object of the
invention is the provision of methods for determining regions of
tissue factor for use as immunogens to produce antibodies which
are species-specific. Another object of the invention is the
provision of antibodies to tissue factor made using the above
methods.
Briefly, therefore, the present invention is directed to a
method for selecting a peptide immunogen of a target protein from
a first species, wherein the target protein comprises an amino
acid sequence which varies between the first species and a second
species. The method comprises (a)identifying peptide regions of
the amino acid sequence of the target protein from the first
species which has a hydrophilicity value greater than 0, wherein
the regions are 5-50 amino acids in length, and (b) selecting a
peptide immunogen from regions identified in step (a) which have
at least 1 nonhomologous amino acid between the first species and
the second species.
The present invention is also directed to a method of making
an antibody which is specific for a target protein of a first
species, wherein the target protein comprises an amino acid
sequence which varies between the first species and the second
species. The method comprises selecting a peptide immunogen of
the target protein by the above-described method, synthesizing
the peptide immunogen, conjugating the peptide immunogen to an
immunogenic carrier molecule to make a peptide-carrier antigen,
and producing antibodies to the antigen.
Additionally, the present invention is directed to an
antibody made by the above-described method.
Furthermore, the present invention is directed to a peptide
which consists of a sequence from a tissue factor extracellular
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region from a first species which is 5-50 amino acids in length,
has a hydrophilicity value greater than 0, and has at least 1
nonhomologous amino acid from a tissue factor from a second
species.
5 The present invention is also directed to an antigen, and
antibodies made to an antigen, where the antigen comprises an
immunogenic carrier molecule conjugated to a peptide which
consists of a sequence from a tissue factor extracellular region
from a first species which is 5-50 amino acids in length, has a
l0 hydrophilicity value greater than 0, and has at least 1
nonhomologous amino acid from a tissue factor from a second
species.
ABBREVIATIONS AND DEFINITIONS
To facilitate understanding of the invention, a number of
terms as used herein are defined below:
As used herein, "TF" shall mean tissue factor.
As used herein, the term "species-specific", when used to
describe an antibody, denotes that the antibody will bind to a
protein of one species but not the same protein of another
species. An antibody which binds to (or "reacts" or
"recognizes") a protein from a first species but not the same
protein from a second species is said to be species-specific to
the protein from the first species, because it does not "cross-
react" to the protein from the second species.
As used herein, the term "synthetic peptide" or "peptide" is
a chain of 5 to 50 amino acids covalently linked to one another
by peptide bonds.
As used herein, an "antigen" is a macromolecule to which
antibodies are made. The antigen is usually injected into a
vertebrate, which then elicits production of antibodies to
regions ("epitopes") of the antigen.
As used herein, the term "immunogen" is a portion of an
antigen to which antibodies are desired. An immunogen may be an
epitope of the antigen or it may be comprised of several
epitopes. More specifically, the term "immunogen" is applied
herein to mean a peptide from a protein of interest which is
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conjugated to an immunogenic carrier molecule for the purpose of
eliciting antibodies to the immunogen portion of the antigen.
As used herein, the term "hydrophilic" when used in
reference to amino acids refers to those amino acids which have
polar and/or charged side chains. Hydrophilic amino acids
include lysine, arginine, histidine, aspartate (i.e., aspartic
acid), glutamate (i.e., glutamic acid), serine, threonine,
cysteine, tyrosine, asparagine and glutamine. The terms
"hydrophilicity value" or "Hydrophilicity index value" as used
herein means a number value assigned to each amino acid from 3.0
to -3.4, where the larger numbers are more hydrophilic. The
hydrophilicity values are calculated using the method of Hopp et
al., 1981, Proc. Natl. Acad. Sci. U.S.A. 76:3824. Also, a moving
hydrophilicity average of these values for six amino acids have
been calculated using the Hopp et al. method as described above.
As used herein, the term "hydrophobic" when used in
reference to amino acids refers to those amino acids which have
nonpolar side chains. Hydrophobic amino acids include valine,
leucine, isoleucine, cysteine and methionine. Three hydrophobic
amino acids have aromatic side chains. Accordingly, the term
"aromatic" when used in reference to amino acids refers to the
three aromatic hydrophobic amino acids phenylalanine, tyrosine
and tryptophan.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE 1 is a sequence homology comparison of the rabbit,
human, and mouse tissue factor proteins. The figure was taken
from Pawashe et al., 1991, Thromb. Haemost. 66:315.
FIGURE 2 is a list of candidate immunogenic peptides from
rabbit tissue factor selected using the methods of the present
invention.
FIGURE 3 is a list of candidate immunogenic peptides from
human tissue factor selected using the methods of the present
invention.
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DETAILED DESCRIPTION OF THE INVENTION
The contents of each of the references cited herein are
herein incorporated by reference. The procedures disclosed
herein which involve the molecular manipulation of nucleic acids
are known to those skilled in the art. See generally Fredrick M.
Ausubel et al. (1995), "Short Protocols in Molecular Biology",
John Wiley and Sons, and Joseph Sambrook et al. (1989),
"Molecular Cloning, A Laboratory Manual", second ed., Cold Spring
Harbor Laboratory Press, which are both incorporated by
reference.
The present invention is directed to methods of selecting a
peptide immunogen consisting of a region of a target protein from
a first species, where the target protein has a different amino
acid sequence in a second species. The immunogen is selected for
the purpose of eliciting antibodies to the region of the target
protein containing the peptide. The method comprises the
following steps: (a) the amino acid sequence of the target
protein is analyzed for hydrophilicity, and regions from 5-50
amino acids long are identified which are hydrophilic, for
example having a value greater than 0 using the method of Hopp et
al., 1981, Proc. Natl. Acad. Sci. U.S.A. 76:3824; and (b) the
hydrophilic amino acid regions of the target protein are compared
to the same regions from the same protein of the second species,
and a region is selected as the peptide immunogen which has at
least 1 nonhomologous amino acid between the first species and
the second species. Antibodies are then made to the selected
immunogen after conjugating the immunogen to an immunogenic
carrier molecule. Antibodies which are generated to the
immunogen generally do not bind to the same protein of the second
species.
The method of the current invention may be applied for the
production of antibodies to any target protein in a species of
interest to which there exists an analogous protein in another
species which has an amino acid sequence which is different from
the amino acid sequence in the target protein in the species of
interest. Included are proteins from any prokaryotic,
eukaryotic, or viral source, including but not limited to,
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vertebrates, invertebrates, protozoans, bacteria, fungi, and
plants.
The amino acid sequences of the target protein and the
analogous protein from the second species c.an be obtained from
various sources, such as published material, as well as protein
sequencing (e. g. Edman degradation). The amino acid sequences
may also be deduced from the genes encoding the target proteins
which can be obtained from published sources or by cloning the
genes encoding the target proteins from both species and
determining the start codon by known methods.
The regions of the target protein which are hydrophilic are
utilized because those regions tend to be more antigenic than
hydrophobic regions. Hydrophilic peptide regions also tend to
localize on the outer surface of the target protein, and are
therefore more accessible for eliciting an antibody response and
for being exposed to an antibody in in situ studies such as
immunocytochemical investigations. Using hydrophilic peptides to
produce immunogens also have the advantage of being more soluble
in aqueous solution than hydrophobic peptides. This makes the
procedure for conjugating the peptides to carrier proteins
simpler with preferred conjugation procedures. Additionally,
antigens comprising hydrophilic peptides are more soluble than
antigens comprising hydrophobic peptides, making immunization
procedures with the former antigen simpler.
A hydrophilicity analysis provides a determination of the
moving average of the polar portion, preferably a hexapeptide, of
the protein. The hydrophilic epitopes of tissue factor may be
determined by the method of Hopp et al., 1981, Proc. Natl. Acad.
Sci. U.S.A. 76:3824. Computer software, such as MacVector from
International Biotechnologies, Inc., are also available to
analyze hydrophilicity of a target protein. Using the Hopp
method where hydrophilicity values for individual amino acids
range from -3.4 (for tryptophan) to 3.0 (for lysine, glutamic
acid, aspartic acid, and arginine), the hydrophilicity is
preferably determined by averaging the mean hexapeptide
hydrophilicity value over the length of the peptide. Peptides
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may be selected which have a hydrophilicity greater than 0.
Preferred peptides have a hydrophilicity greater than 0.2.
After selecting hydrophilic peptides from the regions of
interest in the target protein, the sequence of each peptide is
compared with the analogous sequence in the same protein from the
second species. To make this comparison, the amino acid
sequences of the target protein and the same protein from the
second species are preferably aligned by 3-dimensional structural
similarity. The structural similarity between the two sequences
can be determined by comparing predicted 3-dimensional structure
which can be determined by a number of computer programs, or by
determining the crystal structure of both of the proteins by
known methods (see, e.g., Harlos et al., 1994, Nature 370:662 for
the crystal structure of TF). However, 3-dimensional structural
similarity can generally be ascertained by aligning the sequences
of the two proteins by introducing gaps in one or the other
sequence to minimize mismatches. This task can also be performed
by a number of well-known computer programs (see, e.g., Altschul
et al., 1997, Nucl. Acids Res. 25:3389). See Figure 1 for such
an alignment of mouse, rabbit, and human TF.
The aligned sequences of the candidate hydrophilic peptides
are compared for sequence homology. Peptides which have at least
1 nonhomologous amino acid between the target protein and the
analogous protein from the second species may be used as
immunogens according to the present invention. Preferably, the
candidate hydrophilic peptides have at least 3 nonhomologous
amino acids between the target protein and the analogous protein
from the second species. The nonhomologous amino acids of the
candidate hydrophilic peptides may or may not be consecutive in
the peptide chain. Preferably, the homology between the two
peptide sequences is less than 75%. Even more preferably the
homology between the two peptide sequences is less than 60%. As
the sequence homology between the two peptide sequences
decreases, the species-specific properties of the antibodies
generated from the immunogen of the protein of the first species
increases, thereby decreasing the possibility that the antibody
will cross-react with the same protein of the second species.
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After the peptide immunogen is selected, it is synthesized
in preparation for conjugation to the carrier molecule.
Synthesis of the peptide can be by any convenient method, for
example by translating a nucleic acid sequence encoding the
5 peptide in a suitable recombinant host cell, e.g., E. coli, then
purifying the peptide by routine methods (e. g., reversed phase
HPLC). However, the peptide is preferably synthesized using
chemical methods, preferably solid phase methods. The preferred
method for this employs Fmoc chemistry (Fields et al., 1990, Int.
10 J. Pept. Protein Res. 35:161; Wellings et al., 1997, Meth.
Enzymol. 289:44). Preferably, the peptide is selected with a
terminal amino acid residue suitable for coupling to the carrier
with a crosslinking reagent, or synthesized with an additional
amino acid residue at the terminus for this purpose. Examples of
such amino acids are tyrosine, for a bis-diazobenzidine (BDB)-
activated carrier; aspartic acid, glutamic acid, or a free
carboxy terminus, for a 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide hydrochloride (EDC)-activated carrier; or cysteine,
lysine, or a free amino terminus, for a m-maleimidobenzoyl-N-
hydrosuccinamide (MBS)-activated carrier. Preferred is cysteine
and MBS carrier activation.
After the peptide immunogen is synthesized, it is conjugated
to a carrier molecule. The carrier is preferably an antigenic,
soluble protein. Preferably, the carrier is selected which would
not elicit antibodies which would bind to antigens in the first
or second species. A preferred carrier is keyhole limpet
hemocyanin (KLH).
Alternatively, the peptide immunogen can be conjugated to a
resin-polylysine carrier, to follow the multiple antigenic
peptide strategy. See, e.g., McClean et a1.,1991, J. Immunol.
Meth. 137:149.
Production of the antigen is achieved by conjugating the
peptide immunogen to the carrier molecule. This conjugation
preferably proceeds by activation of the carrier, e.g., by BDB,
EDC, or, preferably, MBS, then addition of the peptide.
The antigen is then used to produce an antibody which binds
to the immunogen. The antibody may be a mono-specific antibody.
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The monospecific antibody may be a monoclonal antibody produced,
for example, by the method of Galfre et al., 1977, Nature
266:550. Alternatively, the monospecific antibody may be a
recombinant antibody produced, for example, by the method of
Lowman et al., 1991, Biochemistry 30:10832.
The antibody can also be a polyclonal antibody. The
polyclonal antibody can be prepared, for example, by immunizing a
mammal such as a mouse, goat, sheep, or rabbit with the peptide-
carrier antigen and subsequently isolating the serum therefrom to
obtain an antiserum that contains the polyclonal antibodies.
The antibody is preferably characterized by determining its
ability to bind to the immunogen and the target protein by, e.g.,
ELISA, hemagglutination, fluorescent antibody binding to tissue,
western blot, or any other suitable procedure. Tissue factor
antibodies made by the invention method can be tested, e.g., for
reactivity to the immunogen, and to recombinant and/or native TF
from the first and second species, or other species.
The peptide antigens, immunogens, and antibodies of the
present invention can be used in various applications. Among
other uses, the peptide antigens can be employed to purify the
antibodies as noted above. The synthetic peptides of the present
invention can be used to test antibody specificity as noted
above. The antigens can also be used in inhibition assays. For
example, a peptide from TF can be added to, e.g., thromboplatin
assays to determine if it can inhibit TF function. Such work
could help characterize TF structure and function.
Applications of antibodies of the present invention include
epitope mapping in structure/function studies,
immunohistochemical localization, and immunoaffinity purification
of the target protein. For example, anti-TF antibodies can be
used in a thromboplastin assays to determine if the antibody can
inhibit TF function. These antibodies can also be used to study
other aspects of TF, such as characteristics of the TF/substrate
complex.
The antibodies of the present invention can also be used in
diagnostic assays for antigens, particularly where it is required
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that the antibodies do not cross-react with antigens from another
species (e. g., assays for a particular bacteria in food, etc.)
EXAMPLES
Example 1
Identification of Peptide Immunogens to Rabbit and Human
Tissue Factor
The sequences of rabbit, human, and mouse tissue factor were
have been described (Pawashe et al., 1991, Thromb Haemost.
66:315). A hydrophilicity analysis was performed using the
method of Hopp et al., 1981, Proc. Natl. Acad. Sci. U.S.A.
76:3824. The analysis was performed using the moving averages
function on the Excel computer spreadsheet program. Briefly,
each amino acid of the rabbit and human sequence was assigned a
value from 3.0 to -3.4, where the larger numbers are more
hydrophilic. A moving hydrophilicity average of each hexapeptide
was established. A mean hydrophilicity average of the
hexapeptide averages was then established for regions of the
proteins with predominantly positive hexapeptide averages.
Regions with mean hexapeptide averages above 0.2 were selected as
candidate immunogens for further evaluation. Figures 2 and 3
shows these candidate immunogens.
The candidate immunogens were next evaluated for homology to
the mouse sequence. The human and rabbit sequence was aligned
with the mouse sequence as in Figure 1. The candidate human and
rabbit immunogens were then compared to the aligned mouse
sequence and immunogens were selected which have more than 3
nonhomologous amino acids from the analogous mouse sequence.
The selected immunogens are shown as the candidate
immunogens of Figures 2 and 3 which have 3 or more mismatches.
Eight of the ten candidate immunogens from rabbit TF and eight of
the eleven candidate immunogens from human T'F. All of those
immunogens are useful for producing anti-TF antibodies. However,
the most effective immunogens would be those which have a higher
mean hydrophilicity value and a larger number of mismatches.
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Example 2
Production and Characterization of Polyclonal Antibodies to
Rabbit Tissue Factor
Polyclonal antibodies were made to two regions of rabbit TF.
The 12 residue peptide TTGFPEEPPFRN from position 84 through 95
(84T-95S), located in the loop connecting domains 1F and 1G and
oriented toward the putative top of the molecule (Harlos et al.,
1994, Nature 370:662), demonstrated the most amino acids
mismatches of the candidate immunoqens (Figure 2). The 13
residue peptide VIPSRKRKQRSPE from position 190 through 202
(190V-202E), located within the 2F-2G loop constrained by a
disulfide bond, had the highest hydrophilicity index (Figure 2).
Both peptides are unique to the rabbit TF sequence when analyzed
by the BLASTP search engine with an E value of 100. These two
peptides were synthesized using standard FMOC chemistry. An
additional cysteine was added to the carboxy terminal. The
peptides were then conjugated to MBS-activated KLH by standard
methods. For each peptide immunogen, three goats were immunized
with the peptide-KLH antigens. Antisera collected from the goats
were then tested for immunoreactivity in a microtiter plate
format in which unconjugated peptides, rabbit recombinant TF,
human recombinant TF, and a Triton extract of rabbit brain
acetone powder (RBAP) were the coated antigens. The plates were
probed with rabbit anti-goat IgG alkaline phosphatase conjugate.
Each antisera demonstrated specific immunoreactivity to its
respective peptide immunogen. All antisera cross-reacted with
rabbit recombinant TF. Two antisera, one to each of the
peptides, also demonstrated reactivity to natural rabbit TF in
the RBAP extracts.
Other features, objects and advantages of the present
invention will be apparent to those skilled in the art. The
explanations and illustrations presented herein are intended to
acquaint others skilled in the art with the invention, its
principles, and its practical application. Those skilled in the
art may adapt and apply the invention in its numerous forms, as
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may be best suited to the requirements of a particular use.
Accordingly, the specific embodiments of the present invention as
set forth are not intended as being exhaustive or limiting of the
invention.
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Leu
65 70 75 80
Lys Thr Thr AsnGlu PheLeuIle AspVal AspLys GlyGlu His
Asn
85 90 95
Tyr Cys Ser ValGln AlaValIle ProSer ArgThr ValAsn Arg
Phe
100 105 110
Lys Ser Asp SerPro ValGluCys MetGly GlnGln LysGly Glu
Thr
115 120 125
Phe Arg Ile PheTyr IleIleGly AlaVal ValPhe ValVal Ile
Glu
130 135 140
CA 02339404 2001-02-07
WO 01/00237 PCT/US00/17609
4
Ile Leu Val Ile Ile Leu Ala Ile Ser Leu
145 150
<210> 8
<211> 22
<212> PRT
<213> Human
<400> 8
Ile Ile Lys Cys Arg Lys Ala Gly Val Gly Gln Ser Trp Lys Glu Asn
1 5 10 15
Ser Pro Leu Asn Val Ser
<210> 9
<211> 5
<212> PRT
15 <213> Mouse
<400> 9
Gly Ile Pro Glu Lys
1 5
<210> 10
20 <211> 240
<212> PRT
<213> Mouse
<400> 10
Ala Phe Asn Leu Thr Trp Ile Ser Thr Asp Phe Lys Thr Ile Leu Glu
1 5 10 15
Trp Gln Pro Lys Pro Thr Asn Tyr Thr Tyr Thr Val Gln Ile Ser Asp
20 25 30
Arg Ser Arg Asn Trp Lys Asn Lys Cys Phe Ser Thr Thr Asp Thr Glu
40 45
30 Cys Asp Leu Thr Asp Glu Ile Val Lys Asp Val Thr Trp Ala Tyr Glu
50 55 60
Ala Lys Val Leu Ser Val Pro Arg Arg Asn Ser Val His Gly Asp Gly
65 70 75 80
Asp Gln Leu Val Ile Asn Gly Glu Glu Pro Pro Phe Thr Asn Ala Pro
35 85 90 95
Lys Phe Leu Pro Tyr Arg Asp Thr Asn Leu Gly Gln Arg Val Ile Gln
100 105 110
Gln Phe Glu Gln Gln Gly Arg Lys Leu Asn Val Val Val Lys Asp Ser
115 120 125
CA 02339404 2001-02-07
WO 01100237 PCT/US00/17609
Leu Thr Leu Val Arg Lys Asn Gly Thr Phe Leu Thr Leu Arg Gln Val
130 135 140
Phe Gly Lys Asp Leu Gly Tyr Ile Ile Thr Tyr Arg Leu Gly Ser Ser
145 150 155 160
5 Thr Gly Lys Lys Thr Asn Ile Thr Asn Thr Asn Glu Phe Ser Ile Asp
165 170 175
Val Glu Glu Gly Val Ser Tyr Cys Phe Phe Val Gln Ala Met Ile Phe
180 185 190
Ser Arg Lys Thr Asn Gln Asn Ser Pro Gly Ser Ser Thr Val Cys Thr
195 200 205
Glu Asn Trp Ser Lys Phe Leu G1y Glu Thr Leu Ile Ile Val Gly Ala
210 215 220
Val Val Leu Leu Ala Thr Ile Phe Ile Ile Leu Leu Ser Ile Ser Leu
225 230 235 240
<210> 11
<211> 19
<212> PRT
<213> Mouse
<400> 11
Cys Lys Arg Arg Lys Asn Arg Ala Gly Gln Lys Gly Lys Asn Thr Pro
1 5 10 15
Ser Arg Leu
