U.S. patent application number 10/693030 was filed with the patent office on 2004-04-01 for methods for diagnosis of cancer using erbb-3.
Invention is credited to Aaronson, Stuart A., Kraus, Matthias H..
Application Number | 20040063140 10/693030 |
Document ID | / |
Family ID | 23764745 |
Filed Date | 2004-04-01 |
United States Patent
Application |
20040063140 |
Kind Code |
A1 |
Kraus, Matthias H. ; et
al. |
April 1, 2004 |
Methods for diagnosis of cancer using erbB-3
Abstract
A DNA fragment distinct from the epidermal growth factor
receptor (EGFR) and erbB-2 genes was detected by reduced stringency
hybridization of v-erbB to normal genomic human DNk
Characterization of the cloned DNA fragment mapped the region of
v-erbB homology to three exons with closest homology of 64% and 67%
to a contiguous region within the tyrosine kinase domains of the
EGFR and erbB-2 proteins, respectively. cDNA cloning revealed a
predicted 148 kd transmembrane polypeptide with structural features
identifying it as a member of the erbB family, prompting
designation of the new gene as erbB-3. It was mapped to human
chromosome 12q11-13 and was shown to be expressed as a 6.2 kb
transcript in a variety of normal tissues of epithelial origin.
Markedly elevated erbB-3 mRNA levels were demonstrated in certain
human mammary tumor cell lines. These findings indicate that
increased erbB-3 expression, as in the case of EGFR and erbB-2,
plays a role in some human malignancies. Using erbB-3 specific
antibodies (polyclonal or monoclonal), the erbB-3 protein was
identified as a 180 kDa glycoprotein, gp180.sup.erbB-3. The
intrinsic catalytic function of gp180.sup.erbB-3 was uncovered by
its ability to autophosphorylate in vitro. Ligand-dependent
signaling of its cytoplasmic domain was established employing
transfectants which express a chimeric EGFR/erbB-3 protein,
gp180.sup.EGFR/erbB-3. EGF induced tyrosine phosphorylation of the
chimera and promoted soft agar colony formation of such
transfectants. These findings, combined with the detection of
constitutive tyrosine phosphorylation of gp180.sup.erbB-3 in 4 out
of 12 human mammary tumor cell lines, implicate the activated
erbB-3 product in the pathogenesis of some human malignancies.
Thus, this invention also relates to a method for detecting a
receptor ligand capable of either activating or down-regulating the
receptor protein, as well as procedures for purifying the resultant
ligand; a method of screening potential ligand analogs for their
ability to activate the receptor protein; and procedures for
targeting a therapeutic drug to cells having a high level of the
receptor protein.
Inventors: |
Kraus, Matthias H.;
(Bethesda, MD) ; Aaronson, Stuart A.; (Vienna,
VA) |
Correspondence
Address: |
NATIONAL INSTITUTE OF HEALTH
C/O NEEDLE & ROSENBERG, P.C.
SUITE 1000
999 PEACHTREE STREET
ATLANTA
GA
30303
US
|
Family ID: |
23764745 |
Appl. No.: |
10/693030 |
Filed: |
October 24, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10693030 |
Oct 24, 2003 |
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09170699 |
Oct 13, 1998 |
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6639060 |
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09170699 |
Oct 13, 1998 |
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08473119 |
Jun 7, 1995 |
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5820859 |
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08473119 |
Jun 7, 1995 |
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07978895 |
Nov 10, 1992 |
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5480968 |
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07978895 |
Nov 10, 1992 |
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07444406 |
Dec 1, 1989 |
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5183884 |
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Current U.S.
Class: |
435/6.14 ;
435/320.1; 435/325; 435/69.1; 530/350; 536/23.5 |
Current CPC
Class: |
C07K 2317/34 20130101;
G01N 2500/02 20130101; A61K 47/6843 20170801; C07K 16/32 20130101;
A61K 38/00 20130101; G01N 33/5748 20130101; A61P 35/00 20180101;
G01N 33/57484 20130101; C07K 14/82 20130101; G01N 2500/04
20130101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/320.1; 435/325; 530/350; 536/023.5 |
International
Class: |
C12Q 001/68; C07H
021/04; C07K 014/71; C12P 021/02; C12N 005/06 |
Claims
What is claimed is:
1. An isolated polypeptide having the amino acid sequence as given
by SEQ ID NO: 4, or a unique portion thereof.
2. The isolated polypeptide of claim 1, wherein the unique portion
is selected from the group consisting of an amino acid sequence
given by SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and
SEQ ID NO: 9.
3. An isolated polypeptide having an amino acid sequence encoded by
the DNA isolate consisting essentially of a nucleotide sequence
that encodes an amino acid sequence differing in at least one amino
acid from the amino acid sequence of human erbB-3, or a unique
portion thereof, and having greater overall similarity to the amino
acid sequence of human erbB-3 than to that of any other
polypeptide.
4. A purified antibody specific for a unique portion of SEQ ID NO:
4 or gp180.sup.erbB-3.
5. The antibody of claim 4, wherein the unique portion is selected
from the group consisting of an amino acid sequence given by SEQ ID
NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO:
9.
6. The antibody of claim 4, wherein the antibody is conjugated to a
therapeutic drug.
7. The antibody of claim 4, wherein the antibody is labeled with a
detectable moiety.
8. The antibody of claim 4, wherein the antibody is bound to a
solid support.
9. A bioassay for determining the amount of erbB-3 mRNA in a
biological sample comprising the steps of: a) contacting the
biological sample with a nucleic acid isolate consisting
essentially of a nucleotide sequence that encodes erbB-3 or a
unique portion thereof under conditions such that a hybrid molecule
can be formed; and b) determining the amount of hybrid molecule
present, the amount of hybrid molecule indicating the amount of
erbB-3 mRNA in the sample.
10. A bioassay for screening potential analogs of ligands of erbB-3
receptors for the ability to affect an activity mediated by the
erbB-3 receptors, comprising the steps of: a) contacting a molecule
suspected of being a ligand with erbB-3 receptors produced by a
cell that yields functional erbB-3 receptors; b) determining-the
amount of a biological activity mediated by the erbB-3 receptors;
and c) selecting those analogs which affect the biological activity
mediated by the erbB-3 receptor.
11. A bioassay for determining the amount of an erbB-3 antigen in a
biological sample comprising the steps of: a) contacting the sample
with an antibody according to claim 4 under conditions such that a
specific complex of the antibody and the antigen can be formed; and
b) determining the amount of the antibody present as the complex,
the amount of the complex indicating the amount of erbB-3 antigen
in the biological sample.
12. A method for targeting a therapeutic drug to cells having high
levels of erbB-3 receptors, comprising the steps of: a) conjugating
an antibody according to according to claim 4 or an active fragment
thereof to the drug; and b) administering the resulting conjugate
to an individual with cells having high levels of erbB-3 receptors
in an effective amount and by an effective route such that the
antibody is able to bind to the receptor on the cells.
13. A method for detecting the presence of an erbB-3 activating
ligand in a source containing a potential erbB-3 ligand, comprising
the steps of: a) contacting a first sample of cells from a cell
line that expresses erbB-3 protein with the source containing a
potential erbB-3 ligand for a time and under conditions sufficient
to allow erbB-3 ligand contained in the source to bind to erbB-3
protein to form a triggered sample, wherein the cell line expresses
erbB-3 protein having low level intrinsic tyrosine phosphorylation;
b) contacting a second sample of cells from the cell line with a
control medium for the time and under the conditions as given in
step a) to form a control sample; c) determining the level of
erbB-3 activation in the triggered sample and in the control
sample; and d) comparing the level of erbB-3 activation in the
triggered sample with the level of erbB-3 activation in the control
sample, wherein an increase in activation in the triggered sample
over the control sample indicates the presence of an erbB-3
activating ligand in the source containing a potential erbB-3
ligand.
14. The method of claim 13, wherein the determining step comprises
measuring the level of erbB-3 intrinsic tyrosine phosphorylation in
the triggered sample and in the control sample, wherein an increase
in the level of erbB-3 intrinsic tyrosine phosphorylation
correlates with an increase in the level of erbB-3 activation.
15. The method of claim 13, wherein the determining step comprises
measuring the level of cell growth in the triggered sample and in
the control sample, wherein an increase in the level of cell growth
correlates with an increase in the level of erbB-3 activation.
16. The method of claim 13, wherein the determining step comprises
measuring the level of DNA synthesis for the cells in the triggered
sample and in the control sample, wherein an increase in the level
of DNA synthesis for the cells correlates with an increase in the
level of erbB-3 activation.
17. The method of claim 13, wherein erbB-3 is overexpressed by the
cell line.
18. The method of claim 13, further comprising the step of
purifying the erbB-3 activating ligand.
19. A method for detecting the presence of an erbB-3 blocking
ligand in a source containing a potential erbB-3 ligand, comprising
the steps of: a) contacting a first sample of a cell line that
expresses erbB-3 protein with the source containing a potential
erbB-3 ligand for a time and under conditions sufficient to allow
erbB-3 ligand contained in the source to bind to erbB-3 protein to
form a blocked sample, wherein the cell line expresses erbB-3
protein having high level intrinsic tyrosine phosphorylation; b)
contacting a second sample of the cell line with a control medium
for the time and under the conditions as given in step a) to form a
control sample; c) determining the level of erbB-3 activation in
the blocked sample and in the control sample; and d) comparing the
level of erbB-3 activation in the blocked sample with the level of
erbB-3 activation in the control sample, wherein a decrease in
activation in the blocked sample over the control sample indicates
the presence of an erbB-3 blocking ligand in the source containing
a potential erbB-3 ligand.
20. The method of claim 19, wherein the determining step comprises
measuring the level of erbB-3 intrinsic tyrosine phosphorylation in
the blocked sample and in the control sample, wherein an decrease
in the level of erbB-3 intrinsic tyrosine phosphorylation
correlates with an decrease in the level of erbB-3 activation.
21. The method of claim 19, wherein the determining step comprises
measuring the level of cell growth in the blocked sample and in the
control sample, wherein an decrease in the level of cell growth
correlates with an decrease in the level of erbB-3 activation.
22. The method of claim 19, wherein the determining step comprises
measuring the level of DNA synthesis for the cells in the blocked
sample and in the control sample, wherein an decrease in the level
of DNA synthesis for the cells correlates with an decrease in the
level of erbB-3 activation.
23. The method of claim 19, wherein erbB-3 is overexpressed by the
cell line.
24. The method of claim 19, further comprising the step of
purifying the erbB-3 blocking ligand.
25. A purified erbB-3 activating ligand.
26. A purified erbB-3 blocking ligand.
27. A method of decreasing a biochemical or biological activity
mediated by the erbB-3 receptor, comprising blocking the binding of
an erbB-3 activating ligand with the erbB-3 receptor.
28. The method of claim 27, wherein the blocking is accomplished by
an antibody reactive with the ligand binding domain of the erbB-3
receptor.
29. The method of claim 27, wherein the blocking is accomplished by
an erbB-3 blocking ligand.
30. A method of promoting a biochemical or biological activity
mediated by the erbB-3 receptor, comprising contacting an erbB-3
activating ligand with the erbB-3 receptor.
31. A method of detecting the overexpression of erbB-3 in a sample
from a subject, comprising detecting the amount of erbB-3 in the
sample and comparing the amount in the sample to the amount in an
equivalent sample having normal expression, the presence of erbB-3
in a greater amount indicating overexpression of erbB-3.
32. The method of claim 31, further comprising correlating the
overexpression of erbB-3 to a tumor.
33. A method of detecting the activation of erbB-3 in a test sample
from a subject, comprising detecting the presence of
phosphorylation of erbB-3, the presence of phosphorylation of
erbB-3 indicating the presence of erbB-3 activation in the
sample.
34. The method of claim 33, further comprising comparing the amount
of erbB-3 phosphorylation in the test sample to the amount of
erbB-3 phosphorylation in a sample from a normal subject and
correlating an increase in phosphorylation in the test sample, with
the presence of a neoplastic condition in the subject.
Description
[0001] This application is a continuation-in-part of applicant's
pending U.S. Ser. No. 07/444,406, which was filed on Dec. 4,
1989.
FIELD OF THE INVENTION
[0002] The present invention relates to genes which encode novel
proteins related to a family of receptor proteins typified by two
related membrane spanning tyrosine kinases: the Epidermal Growth
Factor receptor. (EGFR), which is encoded by the erbB gene, the
normal human counterpart of an oncogene (v-erbB) that was first
recognized in the proviral DNA of avian erythroblastosis virus; and
the receptor encoded by the related gene erbB-2. In particular, the
present invention relates to a DNA segment encoding the coding
sequence, or a unique portion thereof, for a third member of this
receptor gene family, herein designated erbB-3.
BACKGROUND OF THE INVENTION
[0003] Proto-oncogenes encoding growth factor receptors constitute
several distinct families with close overall structural homology.
The highest degree of homology is observed in their catalytic
domains, essential for the intrinsic tyrosine kinase activity of
these proteins. Examples of such receptor families include: the
EGFR and the related product of the erbB-2 oncogene; the Colony
Stimulating Factor 1 receptor (CSF-1-R) and the related
Platelet-Derived Growth Factor receptor (PDGF-R); the insulin
receptor (IR) and the related Insulin-like Growth factor 1 receptor
(IGF-1R); and the receptors encoded by the related oncogenes eph
and elk.
[0004] It is well established that growth factor receptors in
several of these families play critical roles in regulation of
normal growth and development. Recent studies in Drosophila have
emphasized how critical and multifunctional are developmental
processes mediated by ligand-receptor interactions. An increasing
number of Drosophila mutants with often varying phenotypes have now
been identified as being due to lesions in genes encoding such
proteins. The genetic locus of the Drosophila EGFR homologue,
designated DER, has recently been identified as being allelic to
the zygotic embryonic lethal faint little ball exhibiting a complex
phenotype with deterioration of multiple tissue components of
ectodermal origin. Furthermore, other mutants appear to lack DER
function either in the egg or the surrounding maternal tissue.
Thus, the DER receptor may play an important role in the
ligand-receptor interaction between egg and follicle cells
necessary for determination of correct shape of eggshell and
embryo. It is not yet known whether DER represents the sole
Drosophila counterpart of known mammalian erbB-related genes.
[0005] Some of these receptor molecules have been implicated in the
neoplastic process as well. In particular, both the erbB and erbB-2
genes have been shown to be activated as oncogenes by mechanisms
involving overexpression or mutations that constitutively activate
the catalytic activity of their encoded receptor proteins
(Bargmann, C. I., Hung, M. C. & Weinberg, R. A., 1986, Cell
45:649-657; Di Fiore, P. P., Pierce, J. H., Kraus, M. H., Segatto,
O., King, C. R. & Aaronson, S. A., 1987, Science 237:178-182;
Di Fiore, P. P., Pierce, J. H., Fleming, T. P., Hazan, R., Ullrich,
A, King, C. R., Schlessinger, J. & Aaronson, S. A., 1987, Cell
51:1063-1070; Velu, T. J., Beguinot, L., Vass, W. C., Willingham,
M. C., Merlino, G. T., Pastan, I. & Lowy, D. R., 1987, Science
238:1408-1410). Both erbB and erbB-2 have been causally implicated
in human malignancy. erbB gene amplification or overexpression, or
a combination of both, has been demonstrated in squamous cell
carcinomas and glioblastomas (Libermann, T. A, Nusbaum, H. R.,
Razon, N., Kris, R., Lax, I., Sorcq, H., Whittle, N., Waterfield,
M. D., Ullrich, A & Schlessinger, J., 1985, Nature
313:144-147). erbB-2 amplification and overexpression have been
observed in human breast and ovarian carcinomas (King, C. R.,
Kraus, M. H. & Aaronson, S. A, 1985, Science 229:974-976;
Slamon, D. I., Godolphin, W., Jones, L. A., Holt, J. A., Wong, S.
G., Keith, D. E., Ievin, W. J., Stuart, S. G., Udove, J., UlIrich,
A & Press, M. F., 1989, Science 244:707-712), and erbB-2
overexpression has been reported to be an important prognostic
indicator of particularly aggressive tumors (Slamon, D. J., et al.,
1989, supra). Yet, not all such tumors have been found to
overexpress erbB-2, and many human tumors have not yet been
associated with any known oncogene. Thus, there has been a
continuing need to search for additional oncogenes which would
provide knowledge and methods for diagnosis and, ultimately, for
rational molecular therapy of human cancers.
[0006] Throughout this application, various publications are
referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference into this
application in order to more fully describe the state of the art to
which this invention pertains.
SUMMARY OF THE INVENTION
[0007] It is an object of present invention to provide a DNA
segment encoding a receptor protein related to the erbB
proto-oncogene family which previously has not been known or even
suspected to exist. Further, it is an object of the present
invention to develop assays for expression of the RNA and protein
products of such genes to enable determining whether abnormal
expression of such genes is involved in human cancers. Thus,
further objects of this invention include providing antibodies,
either polyclonal or monoclonal, specific to a unique portion of
the receptor protein; a method for detecting the presence of an
erbB-3 ligand that is capable of either activating or
down-regulating the receptor protein as well as procedures for
purifying the resultant ligand; a method of screening potential
ligand analogs for their ability to activate the receptor protein;
and procedures for targeting a therapeutic drug to cells having a
high level of the receptor protein.
[0008] In pursuit of the above objects, the present inventors have
discovered a human genomic DNA fragment that is produced by
cleavage with the SacI restriction enzyme, has a size of about 9
kbp, and is detectable by nucleic acid hybridization with a probe
derived from the v-erbB gene only under reduced stringency
hybridization conditions. Thus, this DNA fragment is distinct from
those known to encode the epidermal growth factor receptor (EGFR)
(i.e.,t the erbB gene) and from the related erbB-2 gene.
Characterization of this DNA fragment after partial purification
and molecular cloning showed that the region of v-erbB homology
mapped to three exons that encode amino acid sequences having
homologies of 64% and 67% to contiguous regions within the tyrosine
kinase domains of the EGFR and erbB-2 proteins, respectively. A
probe derived from the genomic DNA clone identified cDNA clones of
the related mRNA which encode a predicted 148 kd transmembrane
polypeptide with structural features identifying it as a member of
the erbB family, prompting designation of the new gene as erbB-3.
This gene was mapped to human chromosome 12q11-13 and was shown to
be expressed as a 6.2 kb transcript in a variety of normal tissues
of epithelial origin. Markedly elevated erbB-3 mRNA levels were
demonstrated in certain human mammary tumor cell lines.
[0009] The predicted human erbB-3 gene product is closely related
to EGFR and erbB-2, which have been implicated as oncogenes in
model systems and human neoplasia The erbB-3 coding sequence was
expressed in NIH/3T3 fibroblasts and its product was identified as
a 180 kDa glycoprotein, gp180.sup.erbB-3. Tunicamycin and
pulse-chase experiments revealed that the mature protein was
processed by N-linked glycosylation of a 145 kDa erbB-3 core
polypeptide. The intrinsic catalytic function of gp180.sup.erbB-3
was uncovered by its ability to autophosphorylate in vitro.
Ligand-dependent signaling of its cytoplasmic domain was
established employing transfectants which express a chimeric
EGFR/erbB-3 protein, gp180.sup.EGFR/erbB-3. EGF induced tyrosine
phosphorylation of the chimera and promoted soft agar colony
formation of such transfectants. These findings, combined with the
detection of constitutive tyrosine phosphorylation of
gp180.sup.erbB-3 in 4 out of 12 human mammary tumor cell lines,
implicates the activated erbB-3 product in the pathogenesis of some
human malignancies.
[0010] Accordingly, in a principal embodiment, the present
invention relates to a DNA segment having a nucleotide sequence
that encodes an erbB-3 gene or a unique portion thereof. This
portion of an erbB-3 gene includes at least about 12 to 14
nucleotides which are sufficient to allow formation of a stable
duplex with a DNA or RNA segment having sequences complementary to
those in this portion of an erbB-3 gene. Further, this unique
portion of an erbB-3 gene, of course, has a sequence not present in
an erbB or an erbB-2 gene. In other words, the sequence of this
portion of an erbB-3 gene differs in at least one nucleotide from
the sequence of any other DNA segment. In one embodiment, this DNA
segment is exemplified by a human genomic DNA fragment that is
produced by cleavage with the SacI restriction enzyme, has a size
of about 9 kbp, and is detectable by nucleic acid hybridization
with a probe derived from the v-erbB gene only under reduced
stringency hybridization conditions, as described in Example 1. By
application of the nucleic acid hybridization and cloning methods
described in the present disclosure, without undue experimentation,
one of ordinary skill in the art of recombinant DNA is enabled to
identify and isolate DNA fragments related to the present human DNA
fragment comprising a nucleotide sequence that encodes at least a
portion of a mammalian erbB-3 gene other than the human erbB-3
gene. Application of the genomic DNA fragment of the erbB-3 gene as
a probe in hybridization methods also enables one of ordinary skill
in the art to obtain an entire erbB-3 gene, by sequential isolation
of overlapping fragments adjoining the present fragment, i.e., by
an approach known in the art as chromosome walking.
[0011] The present disclosure describes the partial nucleotide
sequence of the human genomic 9 kbp SacI DNA fragment, within the
region of homology to the v-erbB gene; however, the methods in the
present disclosure further enable the isolation and determination
of the sequence of the entire 9 kbp human genomic DNA fragment
according to the present invention. Accordingly, the present
invention further relates to a DNA segment having the nucleotide
sequence, or a unique portion thereof, of a human genomic DNA
fragment that is produced by cleavage with the SacI restriction
enzyme, has a size of about 9 kbp, and is detectable by nucleic
acid hybridization with a probe derived from the v-erbB gene only
under reduced stringency hybridization conditions, as described in
Example 1. By extension of the chromosome walking approach noted
above, the present invention further enables one of ordinary skill
in the art to determine the sequences of related DNA fragments
comprising the complete human erbB-3 gene as well as erbB-3 genes
of, for example, mammals other than human.
[0012] In the application of the present SacI DNA fragment or any
portion thereof as a probe for nucleic acid hybridization, the
fragment is amplified, for example, by the in vitro polymerase
chain reaction method (PCR; see U.S. Pat. No. 4,683,202; U.S. Pat.
No. 4,683,195; and Saiki et al., 1985, Science 230:1350-54) or by
standard methods of molecular cloning. For example, a clone of the
human erbB-3 gene DNA segment according to the present invention is
exemplified by a recombinant clone of a normal human thymus DNA
fragment, herein designated as the E3-1 genomic clone, having the
partial restriction enzyme map defined in FIG. 2 and the partial
DNA sequence defined in FIG. 3 and SEQ ID NO: 1 of the present
application. Isolation and characterization of genomic clone E3-1
is described in Example 2, below.
[0013] Analysis of the nucleotide sequences of the human genomic
DNA segment according to the present invention reveals that the
nucleotide sequence encodes three open reading frames bordered by
splice junction consensus sequences which define the boundaries
between nontranslated intron sequences and the translated exons
(shown in FIG. 2 and SEQ ID NO: 1). The predicted amino acid
sequences of the three exons (SEQ ID NOS: 1 and 2) are highly
similar to three regions which are contiguous in the tyrosine
kinase domains of v-erbB, as well as human EGFR and erbB-2
proteins. Moreover, the predicted amino acid sequences of this
human genomic clone are included in a larger open reading frame in
complementary DNA (cDNA) clones of an mRNA species that is detected
by hybridization of a probe derived from the human genomic DNA
clone.
[0014] Accordingly, the present invention also relates to a DNA
segment having a nucleotide sequence of an erbB-3 gene in which
that nucleotide sequence encodes the amino acid sequence of an
erbB-3 protein or a unique portion thereof In other words, the
sequence of this portion of an erbB-3 amino acid sequence differs
in at least one amino acid residue from the amino acid sequence
encoded by any other DNA segment. This portion of an erbB-3 amino
acid sequence includes at least about 4 to 6 amino acids which are
sufficient to provide a binding site for an antibody specific for
this portion of the erbB-3 polypeptide. Further, this unique
portion of an erbB-3 amino acid sequence, of course, includes
sequences not present in an erbB or an erbB-2 gene. In particular,
the present invention relates to such a DNA segment for which this
amino acid sequence or unique portion thereof is that of the
polypeptide product of the human erbB-3 gene. This DNA segment is
exemplified by the human genomic DNA clone E3-1, above, as well as
by human cDNA cones designated E3-6, E3-8, E3-9, E3-11 and E3-16,
which are described in Example 3 below. A preferred embodiment of
this DNA segment that encodes the amino acid sequence of the entire
polypeptide product of the human erbB-3 gene is human cDNA clone E3
-16 having the nucleotide sequence defined in SEQ ID NO: 3 and
having the predicted amino acid sequence defined in SEQ ID NOS: 3
and 4.
[0015] The DNA segments according to this invention are useful for
detection of expression of erbB-3 genes in normal and tumor
tissues, as described in Example 5 below. Therefore, in yet another
aspect, the present invention relates to a bioassay for determining
the amount of erbB-3 mRNA in a biological sample comprising the
steps of: i) contacting that biological sample with a nucleic acid
isolate consisting essentially of a nucleotide sequence that
encodes erbB-3 or a unique portion thereof under conditions such
that a nucleic acid:RNA hybrid molecule, such as a DNA:RNA hybrid
molecule, can be formed; and ii) determining the amount of hybrid
molecule present, the amount of hybrid molecule indicating the
amount of erbB-3 mRNA in the sample. Findings described in Example
5, below, indicate that increased erbB-3 expression, as detected by
this method of this invention, plays a role in some human
malignancies, as is the case for the EGFR- (erbB) and erbB-2
genes.
[0016] Of course, it will be understood by one skilled in the art
of genetic engineering that in relation to production of erbB-3
polypeptide products, the present invention also includes DNA
segments having DNA sequences other., than those in the present
examples that also encode the amino acid sequence of the
polypeptide product of an erbB-3 gene. For example, it is known
that by reference to the universal genetic code, standard genetic
engineering methods can be used to produce synthetic DNA segments
having various sequences that encode any given amino acid sequence.
Such synthetic DNA segments encoding at least a portion of the
amino acid sequence of the polypeptide product of the human erbB-3
gene also fall within the scope of the present invention. Further,
it is known that different individuals may have slightly different
DNA sequences for any given human gene and, in some cases, such
mutant or variant genes encode polypeptide products having amino
acid sequences which differ among individuals without affecting the
essential function of the polypeptide product.. Still further, it
is also known that many amino acid substitutions can be made in a
polypeptide product by genetic engineering methods without
affecting the essential function of that polypeptide. Accordingly,
the present invention further relates to a DNA segment having a
nucleotide sequence that encodes an amino acid sequence differing
in at least one amino acid from the amino acid sequence of human
erbB-3, or a unique portion thereof, and having greater overall
similarity to the amino acid sequence of human erbB-3 than to that
of any other polypeptide. The amino acid sequence of this DNA
segment includes at least about 4 to 6 amino acids which are
sufficient to provide a binding site for an antibody specific for
the portion of a polypeptide containing this sequence. In a
preferred embodiment, this DNA segment encodes an amino acid
sequence having substantially the function of the human erbB-3
polypeptide. As noted above, the predicted erbB-3 polypeptide is a
148 kd transmembrane polypeptide with structural features
identifying it as a member of the erbB receptor family.
[0017] The similarity of the amino acid sequence of the present
invention with that of an erbB-3 amino acid sequence is determined
by the method of analysis defined by the sequence alignment and
comparison algorithms described by Pearson and Lipman (Pearson, W.
R. & Lipman, D. J., 1988, Proc Nat. Acad. Sci. U.S.A.
85:2444-48). This comparison contemplates not only precise homology
of amino acid sequences, but also substitutions of one residue for
another which are known to occur frequently in families of
evolutionarily related proteins sharing a conserved function.
[0018] The present invention further relates to a recombinant DNA
molecule comprising a DNA segment of this invention and a vector.
In yet another aspect, the present invention relates to a culture
of cells transformed with a DNA segment according to this
invention. These host cells transformed with DNAs of the invention
include both higher eukaryotes, including animal, plant and insect
cells, and lower eukaryotes, such as yeast cells, as well as
prokaryotic hosts including bacterial cells such as those of E.
coli and Bacilus subtilis. These aspects of the invention are
exemplified by recombinant DNAs and cells described in Examples 2,
3 and 6, below.
[0019] One particular embodiment of this aspect of this invention
comprises a cell, preferably a mammalian cell, transformed with a
DNA of the invention, wherein the transforming DNA is capable of
being expressed to produce the functional polypeptide of an erbB-3
gene. For example, mammalian cells (COS-1) transformed with the
pSV2 gpt vector carrying the E3-16 cDNA are prepared according to
well-known methods, such as those described in U.S. patent
application Ser. No. 07/308,302 of Matsui et al., filed Feb. 9,
1989; see also Pierce, J. H. et al., 1988, Science 239:628-631; and
Matsui, T., Heidaran, M., Miki, T., Popescu, N., La Rochelle, W.,
Kraus, M., Pierce, J. & Aaronson, S., 1989 Science
243:800-804). Briefly, cDNA expression plasmids are constructed by
introducing the erbB-3-related cDNA encompassing all the
nucleotides in the open reading frame into the pSV2 gpt vector into
which the simian sarcoma virus long-terminal-repeat (LTR) had been
engineered as the promotor, as previously described in detail.
Transient expression of the erbB-3 gene in such recombinant vectors
is achieved by transfection into COS-1 cells.
[0020] Stable expression of an erbB-3 gene can also be obtained
with mammalian expression vectors such as the pZIPNEOSVX vector
(Cepko, C. L., Roberts B. E. and Mulligan, R. C., 1984, Cell
37:1053-62). For example, a eukaryotic expression vector was
engineered by cloning the full-length erbB-3 coding sequence
derived from cDNA clone E3-16 into the BamHI site of the pZIPNEOSVX
vector DNA adapting the DNA fragments with synthetic
oligonucleotides. NIH/3T3 cells were transfected with 1 .mu.g of
recombinant expression vector DNA (LTRerbB-3) and selected with the
resistance marker antibiotic G418. To detect expression of erbB-3,
polyclonal rabbit antiserum was raised against a synthetic peptide
(such as amino acid (aa) positions 1191-1205 (SEQ ID NO: 5); aa
1254-1268 (SEQ ID NO: 6); aa 478492 (SEQ ID NO: 7); aa 1116-1130
(SEQ ID NO: 8) and aa 1199-1213 (SEQ ID NO: 9)). These peptide
epitopes are located intracellularly within the predicted carboxyl
terminus of the erbB-3 coding sequence with the exception of aa
478-492, which resides in the extracellular domain of the erbB-3
protein. For example, as shown in FIG. 7, immunoblotting analysis
using antiserum raised against aa 1191-1205 led to detection of the
erbB-3 protein (panel A). The specificity of erbB-3 protein
detection was demonstrated by preincubating the antiserum with the
homologous peptide (panel B). Moreover, the normal 180 kD erbB-3
protein was specifically detected with the polyclonal antiserum
only in cell transfected with the recombinant erbB-3 expression
vector, while control NIH3T3 cells that were not transfected with
the vector were negative. There was no cross-reactivity of the
above-listed antisera with the related EGFR or erbB-2 proteins
overexpressed in NIH/3T3 cells. The stably transfected NIH3T3 cells
are useful as erbB-3 receptor protein sources for testing potential
candidates for an erbB-3-specific ligand, analysis of the
biological activity, as well as generation of monoclonal antibodies
raised against the native erbB-3 protein. An erbB-3-specific ligand
is identified by detection of autophosphorylation of the erbB-3
receptor protein, stimulation of DNA synthesis or induction of the
transformed phenotype of the LTRerbB-3 transfected NIH3T3
cells.
[0021] Alternatively, other transformed cell systems are available
for functional expression of receptors of the erbB receptor family,
for example, a system based on the 32D cell line, a mouse
hematopoictic cell line normally dependent on interleukin-3 (I1-3)
for survival and proliferation. Recent studies have established
that introduction of an expression vector for the EGFR in these
cells leads to effective coupling with EGF mitogenic signal
transduction pathways, thereby allowing a ligand of the EGFR to
replace I1-3 in supporting survival and growth of the 32D cells. By
employing the known methods described for the EGFR, for example
(Pierce, J. H. et al., 1988, supra), the E3-16 cDNA of the present
invention is expressed to produce functional receptors in 32D cells
which are then useful for examining the biological function of
these erbB-3 receptors, for instance, the specificity of their
ligand binding capacity and coupling capacities to secondary
messenger systems. Thus, by so using gene expression methods
described herein with the DNAs of the present invention, especially
the preferred E3-16 cDNA clone, one of ordinary skill in the art,
without undue experimentation, can construct cell systems which
fall within the scope of this invention, for determining the
mechanisms of erbB-3 regulatory processes. Accordingly, the present
invention also relates to a bioassay for screening potential
analogs of ligands of erbB-3 receptors for the ability to affect an
activity mediated by erbB-3 receptors, comprising the steps of: i)
contacting a molecule suspected of being a ligand with erbB-3
receptors produced by a cell that yields functional erbB-3
receptors; ii) determining the amount of a biological activity
mediated by those erbB-3 receptors; and iii) selecting those
analogs which affect the biological activity mediated by the erbB-3
receptors. For example, a compound can be added to a cell having
normal or low level erbB-3 phosphorylation. The amount of erbB-3
phosphorylation is then measured and compared to the level prior to
adding the compound. The presence of increased activity can then be
selected. Alternatively, a cell with high or constitutive erbB-3
phosphorylation can be used to screen for compounds which decrease
activity. In addition, an erbB-3 ligand or analogs can be used in
this system to screen for the amount of ligand which is necessary
to promote or inhibit phosphorylation.
[0022] Various standard recombinant systems, such as those cited
above as well as others known in the art, are suitable as well for
production of large amounts of the novel erbB-3 receptor protein
using methods of isolation for receptor proteins that are well
known in the art. Therefore, the present invention also encompasses
an isolated polypeptide having at least a portion of the amino acid
sequence defined in FIG. 4 (SEQ ID NO: 4), such as those
polypeptides given by SEQ ID NOS: 5-9.
[0023] The invention further presents results undertaken in an
effort to identify and characterize the normal erbB-3 gene product
(Examples 6-8). By analysis of an EGFR/erbB-3 chimeric receptor,
this invention demonstrates that EGF-dependent activation of the
erbB-3 catalytic domain results in a proliferative response in
transfected NIH/3T3 cells. Further, the invention shows that some
human mammary tumor cell lines exhibit a dramatic elevation of
steady state erbB-3 tyrosine phosphorylation, implying functional
erbB-3 activation in these tumor cells.
[0024] The identification of erbB-3 ligands is of great importance
because, for instance, the availability of these ligands will
facilitate the complete characterization of erbB-3 biological
function as well as development of therapeutic strategies involving
the ligands. In particular, the instant observation of functional
erbB-3 activation in mammary tumor cells at steady state raises the
possibility that a role of erbB-3 in human tumors involves
autocrine activation. That is, the simultaneous expression of the
ligand by the tumor cell may constitutively activate erbB-3,
leading to an uncontrolled proliferative growth response.
Accordingly, this invention provides for the detection,
purification and characterization of erbB-3 ligands, particularly
erbB-3 ligands that are capable of either activating or
down-regulating (blocking the activation of) the erbB-3
protein.
[0025] The ligand detection and purification method of this
invention capitalizes on the erbB-3 expression and activation
characteristics in certain cell lines as well as the common
property of growth factor receptor tyrosine kinases to rapidly
autophosphorylate on tyrosine residues in response to ligand
triggering to detect activating or blocking ligand from source
containing potential erbB-3 ligands, as described in Example 9.
Therefore, in yet another aspect, the present invention relates to
a method for detecting the presence of an erbB-3 ligand in a source
containing a potential erbB-3 ligand, comprising the steps of a)
contacting a first sample of cells from a cell line that expresses
erbB-3 protein with the source containing a potential erbB-3 ligand
for a time and under conditions sufficient to allow erbB-3 ligand
contained in the source to bind to erbB-3 protein to form a
triggered sample, wherein the cell line expresses erbB-3 protein
having low level intrinsic tyrosine phosphorylation; b) contacting
a second sample of cells from the cell line with a control medium
(unconditioned serum free medium) for the time and under the
conditions as given in step a) above to form a control sample; c)
determining the level of erbB-3 activation in the triggered sample
and in the control sample; and d) comparing the level of erbB-3
activation in the triggered sample with the level of erbB-3
activation in the control sample, wherein an increase in activation
in the triggered sample over the control sample indicates the
presence of an erbB-3 activating ligand in the source containing a
potential erbB-3 ligand. Alternatively, chimeric receptors, as
shown in FIG. 11, can be utilized to screen for erbB-3 ligands. The
erbB-3 activation can be ascertained by measuring the level of
erbB-3 tyrosine phosphorylation in the triggered sample and in the
control sample (an increase in the level of erbB-3 tyrosine
phosphorylation correlates with an increase in the level of erbB-3
protein activation); measuring the level of cell growth in the
triggered sample and in the control sample (wherein an increase in
the level of cell growth correlates with an increase in the level
of erbB-3 activation) or measuring the level of DNA synthesis for
the cells in the triggered sample and in the control sample (an
increase in the level of DNA synthesis for the cells correlates
with an increase in the level of erbB-3 activation).
[0026] Similarly, the presence of an erbB-3 blocking or inhibiting
ligand in a source containing a potential erbB-3 ligand can be
detected by a) contacting a first sample of a cell line that
expresses erbB-3 protein with the source containing a potential
erbB-3 ligand for a time and under conditions sufficient to allow
erbB-3 ligand contained in the source to bind to erbB-3 protein to
form a blocked sample, wherein the cell line expresses erbB-3
protein having high level intrinsic tyrosine phosphorylation; b)
contacting a second sample of the cell line with a control medium
for the time and under the conditions as given in step a) to form a
control sample; c) determining the level of erbB-3 activation in
the blocked sample and in the control sample; and d) comparing the
level of erbB-3 activation in the blocked sample with the level of
erbB-3 activation in the control sample, wherein a decrease in
activation in the blocked sample over the control sample indicates
the presence of an erbB-3 blocking ligand in the source containing
a potential erbB-3 ligand. Alternatively, chimaric receptors, as
shown in FIG. 11, can be utilized to screen for erbB-3 blocking
ligands.
[0027] In addition, the concentration of various ligands can be
utilized to affect the erbB-3 activity. For example, a ligand which
promotes erbB-3 activity at low concentrations can be administered
or promoted to high concentrations which can inhibit erbB-3
activity.
[0028] This invention additionally provides a method of decreasing
a biochemical or biological activity mediated by the erbB-3
receptor, comprising blocking the binding of an erbB-3 activating
ligand with the erbB-3 receptor. The blocking can be accomplished
by an antibody reactive with the ligand binding domain of the
erbB-3 receptor or by an erbB-3 blocking ligand. Furthermore, a
method of promoting a biochemical or biological activity mediated
by the erbB-3 receptor, comprising contacting an erbB-3 activating
ligand with the erbB-3 receptor is provided.
[0029] This invention also provides a method of detecting the
overexpression of erbB-3 in a sample from a subject. The method
comprises detecting the amount of erbB-3 in the sample and
comparing the amount in the sample to the amount in an equivalent
sample having normal expression, the presence of erbB-3 in a
greater amount indicating overexpression of erbB-3. By "greater
amount" is meant a statistically significant amount. Such amount
depends on the conditions utilized and can readily be determined
given the teachings set forth herein. Generally, a two-fold or
greater increase would be predictive of overexpression. erbB-3 can
be detected, for example, by detecting mRNA utilizing Northern
hybridization, RNA dot blot, RNA slot blot, or in situ
hybridization. erbB-3 can also be detected at the protein level
utilizing, for example, Western blots, immunoprecipitation,
immunohistochemistry, ELISA, and radioimmunoassay. Once
overexpression is detected, the overexpression of erbB-3 can be
correlated to a tumor. Such correlation can be used to diagnose a
tumor or monitor the progression of a previously diagnosed
tumor.
[0030] Also provided is a method of detecting the activation of
erbB-3 in a test sample from a subject, comprising detecting the
presence of phosphorylation of erbB-3, the presence of
phosphorylation of erbB-3 indicating the presence of erbB-3
activation in the sample. This method can further comprise
comparing the amount of erbB-3 phosphorylation in the test sample
to the amount of erbB-3 phosphorylation in a sample from a normal
subject and correlating an increase in phosphorylation in the test
sample, with the presence of a neoplastic condition in the subject.
Such correlation can be used to diagnose a tumor or monitor the
progression of a previously diagnosed tumor.
[0031] This invention further comprises a purified antibody
specific for the human erbB-3 polypeptide having the amino acid
sequence defined in FIG. 4 (SEQ ID NO: 4) or the mature
gp180.sup.erbB-3 protein or a unique portion thereof, such as those
polypeptides given by SEQ ID NOS: 5-9. In this embodiment of the
invention, the antibodies are monoclonal or polyclonal in origin,
and are generated using erbB-3 receptor-related polypeptides or
peptides from natural, recombinant or synthetic chemistry sources.
The term "specific" refers to an erbB-3 antibody capable of binding
or otherwise associating nonrandomly with an antigen of erbB-3 such
that it does not cross react substantially with other-antigens.
These antibodies specifically bind to an erbB-3 protein which
includes the sequence of such polypeptide. In other words, these
antibodies bind substantially only to erbB-3 receptor proteins and
not to erbB (EGFR) or erbB-2 proteins. Also, preferred antibodies
of this invention bind to an erbB-3 protein when that protein is in
its native (biologically active) conformation. For instance, MAb
E-31 has been shown to detect the native erbB-3 protein.
[0032] Fragments of antibodies of this invention, such as Fab or
F(ab)' fragments, which retain antigen binding activity and can be
prepared by methods well known in the art, also fall within the
scope of the present invention. Further, this invention comprises a
pharmaceutical composition of the antibodies of this invention, or
an active fragment thereof, which can be prepared using materials
and methods for preparing pharmaceutical compositions for
administration of polypeptides that are well known in the art and
can be adapted readily for administration of the present antibodies
without undue experimentation.
[0033] These antibodies and active fragments thereof, can be used,
for example, for specific detection or purification of the novel
erbB-3 receptor. Such antibodies could also be used in various
methods known in the art for targeting therapeutic drugs, including
cytotoxic agents, to tissues with high levels of erbB-3 receptors,
for- example, in the treatment of appropriate tumors with
conjugates of such antibodies and cell killing agents. Accordingly,
the present invention further relates to a method for targeting a
therapeutic drug to cells having high levels of erbB-3 receptors,
comprising the steps of i) conjugating an antibody specific for an
erbB-3 receptor, or an active fragment of that antibody, to the
therapeutic drug; and ii) administering the resulting conjugate to
an individual with cells having high levels of erbB-3 receptors in
an effective amount and by an effective route such that the
antibody is able to bind to the erbB-3 receptors on those
cells.
[0034] The antibody of this invention is exemplified by rabbit
antisera containing antibodies which specifically bind to erbB-3
protein. Such receptor specific antisera are raised to synthetic
peptides representing a unique portion of the erbB-3 amino acid
sequence, having six or more amino acids in sequences which are
sufficient to provide a binding site for an antibody specific for
this portion of the erbB-3 polypeptide. Further, this unique
portion of an erbB-3 amino acid sequence, of course, includes
sequences not present in an erbB or an erbB-2 amino acid sequence,
as predicted by the respective cDNA sequences. The erbB-3 specific
anti-peptide antibody of the present invention is exemplified by an
anti-peptide antibody in polyclonal rabbit antiserum raised against
any of the synthetic peptides given in SEQ ID NOS: 5-9, which are
derived from the predicted sequence of the erbB-3 polypeptide. The
specific detection of erbB-3 polypeptide with antiserum raised
against the peptide given in SEQ ID NO: 5 is illustrated in
mammalian cells transformed with an expression vector carrying a
human erbB-3 cDNA (see FIG. 7). The antibody of this invention is
further exemplified by erbB-3-specific monoclonal antibodies, such
as the monoclonal antibody MAb E3-1, which was raised against the
recombinantly expressed protein and is capable of detecting the
native erbB-3 protein. MAb E3-1 specifically immunoprecipitated the
mature 180 kDa erbB-3 protein from LTR-erbB-3 transfectants (FIG.
9A) and did not exhibit cross-reactivity with the EGFR or erbB-2
proteins.
[0035] Antibodies to peptides are prepared by chemically
synthesizing the peptides, conjugating them to a carrier protein,
and injecting the conjugated peptides into rabbits with complete
Freund's adjuvant, according to standard methods of peptide
immunization. For example, the peptide is synthesized by standard
methods (Merrifield, R. B., 1963, J. Amer. Soc, 85:2149) on a solid
phase synthesizer. The crude peptide is purified by HPLC and
conjugated to the carrier, keyhole limpet hemocyanin or bovine
thyroglobulin, for example, by coupling the amino terminal cysteine
to the carrier through a maleimido linkage according to well-known
methods (e.g., Lerner R. A. et al., 1981, Pros Nat. Acadi Sci. USA,
78:3403). In one standard method of peptide immunology, rabbits are
immunized with 100 .mu.g of the erbB-3 peptide-carrier conjugate (1
mg/ml) in an equal volume of complete Freund's adjuvant and then
boosted at 10-14 day intervals with 100 .mu.g of conjugated peptide
in incomplete Freund's adjuvant. Additional boosts with similar
doses at 10-14 day intervals are continued until anti-peptide
antibody titer, as determined, for example, by routine ELISA
assays, reaches a plateau.
[0036] The antibody can be labeled with a detectable moiety or
attached to a solid support by methods known in the art to
facilitate detection of an antibody/antigen complex. Such a
detectable moiety will allow visual detection of a precipitate or a
color change, visual detection by microscopy, or automated
detection by spectrometry or radiometric measurement or the like.
Examples of detectable moieties include fluorescein and rhodamine
(for fluorescence microscopy), horseradish peroxidase (for either
light microscopy or electron microscopy and biochemical detection),
biotin-strepavidin (for light or electron microscopy) and alkaline
phosphatase (for biochemical detection by color change). The
detection methods and moieties used can be selected, for example,
from the list above or other suitable examples by the standard
criteria applied to such selections (Harlow and Lane, Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y., 1988).
[0037] Thus, by following the teachings of the present disclosure,
including application of generally known immunological methods
cited herein, one of ordinary skill in the art is able to obtain
erbB-3-specific antibodies and use them in a variety of
immunological assays, for example, for diagnostic detection of
unusually high or low expression in normal or tumor tissues. Thus,
the present invention also relates to a bioassay for detecting an
erbB-3 antigen in a biological sample comprising the steps of: i)
contacting that sample with an antibody of the present invention
specific for an erbB-3 polypeptide, under conditions such that a
specific complex of that antibody and that antigen can be, formed;
and ii) determining the amount of that antibody present in the form
of those complexes.
[0038] The present invention may be understood more readily by
reference to the following detailed description of specific
embodiments and the Examples and Figures included therein.
DESCRIPTION OF THE FIGURES
[0039] FIG. 1. Detection of v-erbB-related DNA fragments in DNAs
from normal human thymus (lane 1), human mammary tumor lines
MDA-MB468 (lane 2), and SK-BR-3 (lane 3). Hybridization was
conducted at reduced (panel A) or intermediate (panel B) stringency
conditions. The arrow denotes a novel 9 kilobase pair (kbp)
erbB-related restriction fragment distinct from those of the EGFR
gene (erbB) and erbB-2;
[0040] FIG. 2. Genomic and cDNA cloning of erbB-3. The region of
(v-erbB) homology within the genomic 9 kbp SacI insert of
.lambda.E3-1 was subcloned into the plasmid pUC (pE3-1) and
subjected to nucleotide sequence analysis. The three predicted
exons are depicted as solid boxes. erbB-3 cDNA clones were isolated
from oligo dT-primed libraries of mRNAs from normal human placenta
(shaded bars) and the breast tumor cell line MCF-7 (open bar). The
entire nucleotide sequence was determined for both strands on
erbB-3 complementary DNA from normal human placenta and upstream of
the 5' XhoI site on pE3-16. The coding sequence is shown as a solid
bar and splice junctions of the three characterized genomic exons
are indicated by vertical white lines. Solid lines in the cDNA map
represent untranslated sequences. Restriction sites: A=AccI,
Av=AvaI, B=BamHI, Bg=EglII, E=EcoRI, H=HindIII, K=KpnI, M=MstII,
P=PstI, S=SacI, Sm=SmaI, Sp=SpeI;
[0041] FIG. 3. Nucleotide sequence of the region of v-erbB homology
in the human erbB-3 gene derived from human genomic DNA clone E3-1,
in the 1.5 kbp region from the EcoRI to the PstI sites. This region
contains three open reading frames bordered by splice junction
consensus sequences (underlined). The predicted amino acid
sequences of the three exons are shown in three letter code below
the relevant DNA sequences;
[0042] FIG. 4. Comparison of the predicted amino acid sequence of
the erbB-3 polypeptide with other receptor-like tyrosine kinases.
The amino acid sequence is shown in single letter code and is
numbered on the right. The putative extracellular domain (light
shading) extends between the predicted signal sequence (solid box)
at the amino-terminus and a single hydrophobic transmembrane region
(solid box) within the polypeptide. The two cysteine clusters (Cys)
in the extracellular domain and the predicted tyrosine kinase
domain (TK) within the cytoplasmic portion of the polypeptide are
outlined by dark shading. The putative ATP-binding site at the
amino-terminus of the TK domain is circled. Potential
autophosphorylation sites within the carboxyl-terminal domain
(COOH) are indicated by asterisks. Potential N-linked glycosylation
sites (.fwdarw.) are marked above the amino acid sequence. The
percentage of amino acid homology of erbB-3 in individual domains
with erbB-2, EGFR, met, eph, insulin receptor (IR), and fms is
listed below. Less than 16% identity is denoted by (-);
[0043] FIG. 5. Assignment of the genomic locus of erbB-3 was
assigned to human chromosomal locus 12q13. A total of 142 grains
were localized on the 400-band ideogram. As depicted in the
diagram, specific labeling of chromosome 12 was observed, where 38
out of 51 grains were localized to band q13;
[0044] FIG. 6. Elevated erbB-3 transcript levels in human mammary
tumor cell lines. A Northern blot containing 10 .mu.g total
cellular RNA from AB589 mammary epithelial cells (lane 1), as well
as mammary tumor cell lines MDA-MB415 (lane 2) and MDA-MB453 (lane
3) was hybridized with an erbB-3 cDNA probe (panel A). Following
signal decay the same blot was rehybridized with a human
.beta.-actin cDNA probe (Gunning, P., Ponte, P., Okayama, H.,
Engel, J., Blau, H. & Kedes, L, 1983, Mol. Cell Biol.
3:787-795);
[0045] FIG. 7. Expression of a human erbB-3 polypeptide in cells
transformed by a cDNA segment as detected by an erbB-3-specific
anti-peptide antiserum. Cellular lysates (100 .mu.g of each sample)
were electrophoresed and transferred to nitrocellulose membranes
for analysis by Western blotting. Panel A. Detection of erbB-3
polypeptide with the antiserum. Panel B. Preincubation of the
antiserum with homologous peptide. Antibody blocking indicates
binding specificity. Lane 1: Selected cultures of NIH3T3 cells
transfected with 1 .mu.g LTRerbB-3 expression vector. Lane 2:
control NIH3T3 cells;
[0046] FIG. 8. Characterization of gp180.sup.erbB-3 recombinantly
expressed in NIH/3T3 cells. A: Immunoblot analysis of transfectants
with erbB-3 peptide antisera MK4 and MK5 and peptide competition.
B: Immunoprecipitation with MK5 antiserum of LTR-rbB-3
transfectants metabolically labeled for 2 h in the presence or
absence of glycosylation inhibitor tunicamycin (1 .mu.g/ml). C:
Pulse-chase analysis: LTR-erbB-3 transfectants were pulse labeled
for 15 min with 0.5 mCi each of [.sup.35S methionine and [.sup.35S]
cysteine and immediately lysed (0) or chased with 100 .mu.g/ml each
of unlabeled methionine and cysteine for the indicated time
periods. 1.times.10.sup.7 TCA-precipitable counts were
immunoprecipitated from total lysates using MK5 antiserum;
[0047] FIG. 9. Immunolocalization of gp180.sup.erbB-3 on the
surface of LTR-erbB3 cells. A: Immunoprecipitation analysis of
metabolically labeled LTR-erbB-3 transfectants with monoclonal
antibody E3-1 and a non-immune control. B: Indirect
immunofluorescence: Formalin-fixed LTR-erbB-3 transfectants were
incubated with MAb E3-1 (upper left) or non-immune IgG (lower left)
and stained with a fluorescein-conjugated secondary antibody
(100.times. original magnification). Indirect immunofluorescence
with MAb E3-1 of native LTR-erbB-3 cells (right panel; 1000.times.
original magnification);
[0048] FIG. 10. Autophosphorylation in vitro and chronic tyrosine
phosphorylation in vivo of gp180.sup.erbB-3. LTRerbB-3 or control
lystaes were immunoprecipitated with erbB-3 monoclonal antibody
(E3-1) or non-immune IgG (NI). Parallel immunoprecipitates were
subjected either to immunoblot analysis with MK4 antiserum (A) or
to an immunocomplex kinase assay in the presence of
[.sup.32P]-.gamma.ATP (B). Tyrosine Phosphorylation in vivo was
assayed by immunoprecipitation with monoclonal anti-P-Tyr
antibodies followed by immunoblotting with MK4 antiserum (C);
[0049] FIG. 11. EGF-dependent tyrosine phosphorylation of an
EGFR/erbB-3 chimeric receptor, gp180.sup.EGFR/erbB-3. Serum-starved
LTR-erbB-3, LTR-EGFR/erbB-3, and LTR-EGFR transfectants were
triggered with 100 ng/ml EGF. Similar amounts of gp180.sup.erbB-3,
gp180.sup.EGFR/B-3, and EGFR were immunoprecipitated with erbB-3
(E3-1) or EGFR (AB-1) monoclonal antibodies followed by immunoblot
analysis with anti-P-Tyr antibodies or peptide antisera; and
[0050] FIG. 12. Activation of gp180.sup.erbB-3 signaling function
in human breast tumor cells. The erbB-3 protein was
immunoprecipitated with MAb E3-1 from 1 mg total protein lysate and
subjected to immunoblot analysis with erbB-3 peptide antiserum or
phosphotyrosine antibodies as indicated.
DESCRIPTION OF SPECIFIC EMBODIMENTS
[0051] As used herein, the terms "polypeptide", "protein", "gene
product", "antigen", "receptor", "receptor protein" and the like,
when used in reference to erbB-3, encompass the erbB-3 amino acid
functional sequence as given in SEQ ID NO: 4, the mature erbB-3
glycoprotein, gp180.sup.erbB-3, and these entities modified by
other post-translational modifications, such as glycosylation or
tyrosine phosphorylation. However, as is common in the art, the
term "erbB-3 polypeptide" typically refers to the sequence as given
in SEQ ID NO: 4 while the remaining terms typically refer to
gp180.sup.erbB-3.
[0052] The identification of a third member of the erbB-EGF
receptor, family of membrane spanning tyrosine kinases and the
cloning of its full length coding sequence is described the
Examples herein. The presence of apparent structural domains
resembling those of the EGF receptor suggests the existence of an
extracellular binding site for a ligand. The structural relatedness
of the extracellular domain of the erbB-3 receptor with that of the
EGF receptor indicates that one or more of an increasing number of
EGF-like ligands (Shoyab, M., Plowman, G. D., McDonald, V. L.
Bradley, J. G. & Todaro, G. J., 1989, Science 243:1074-1076)
interacts with the erbB-3 product. Accordingly, the erbB-3 gene is
expected to play important roles in both normal and neoplastic
processes, as is known for the EGFR and erbB-2 genes.
[0053] Despite extensive collinear homology with the EGF receptor
and erbB-2, distinct regions within the predicted erbB-3 coding
sequence revealed relatively higher degrees of divergence. For
example, its carboxyl terminal domain failed to exhibit significant
collinear identity scores with either erbB-2 or EGFR. The
divergence at the carboxyl terminus also accounts for minor size
differences among the three polypeptides of erbB-3, erbB-2, and
EGFR, which possess estimated molecular weights of 148 kilodaltons
(kd), 138 kd, and 131 kd, respectively. Within the tyrosine kinase
domain, which represents the most conserved region of the predicted
erbB-3 protein, a short stretch of 29 amino acids closer to the
carboxyl terminus than the ATP binding site differed from regions
of the predicted erbB-2 and EGFR coding sequence in 28 and 25
positions, respectively. Such regions of higher divergence in their
cytoplasmic domains are likely to confer different functional
specificity to these closely related receptor-like molecules. Thus,
mutations or other alterations in expression of the erbB-3 gene are
likely- to cause cancers or genetic disorders different from those
associated with such defects in the erbB and erbB-2 genes.
[0054] Chromosomal mapping localized erbB-3 to human chromosome 12
at the q11-13 locus, whereas the related EGFR and erbB-2 genes are
located at chromosomal sites-7p12-13 and 17p12-21.3, respectively.
Thus, each gene appears to be localized to a region containing a
different homeobox and a different collagen chain gene locus.
Keratin type I and type II genes also map to regions of 12 and 17,
respectively, consistent with the different localizations of erbB-3
and erbB-2, respectively. Thus, the DNA segments of the present
invention represent novel probes to aid in genetic mapping of any
heritable diseases which are associated with chromosomal
aberrations in the vicinity of the 12q11-13 locus.
[0055] There is evidence for autocrine as well as paracrine
effectors of normal cell proliferation. The former are factors that
are produced by the same cells upon which they stimulate cell
proliferation, whereas the latter factors are secreted by cells
other than those that arc affected by those factors. However, the
inherent transforming potential of autocrine growth factors
suggests that growth factors most commonly act on their target cell
populations by a paracrine route. The present survey of erbB-3 gene
expression indicates its normal expression in cells of epithelial
and neuroectodermal derivation. Comparative analysis of the three
erbB receptor-like genes in different cell types of epidermal
tissue revealed that keratinocytes expressed all three genes. In
contrast, melanocytes and stromal fibroblasts specifically lacked
EGFR and erbB-3 transcripts, respectively. Thus, melanocytes and
stromal fibroblasts may be sources of paracrine growth factors for
EGFR and erbB-3 products, respectively, that are expressed by the
other cell types residing in close-proximity in epidermal
tissues.
[0056] Given that both erbB and erbB-2 have been causally
implicated in human malignancy, the present findings (Example 5)
that the erbB-3 transcript is overexpressed in a significant
fraction of human mammary tumor cell lines indicates that this new
member of the EGFR receptor family also plays an important role in
some human malignancies.
[0057] Characterization of the human erbB-3 gene product,
gp180.sup.erbB-3, shows that it is a transmembrane glycoprotein
exhibiting properties characteristic of a receptor-like tyrosine
kinase. The recombinant human erbB-3 protein shared identical
electrophoretic mobility with the natural erbB-3 product expressed
in human breast tumor cell lines. Moreover, both recombinant and
endogenously expressed gp180.sup.erbB-3 were recognized by
different antibodies directed against distinct epitopes, such as
monoclonal (i.e., Mab E3-1) and peptide antibodies directed against
epitopes in the extracellular and carboxyl-terminal domains. The
145 kDa erbB-3 polypeptide precursor conformed with predicted
erbB-3 encoded protein following cleavage of its signal sequence.
Finally, demonstration of its inherent signaling properties
established functional integrity of recombinantly expressed
gp180.sup.erbB-3.
[0058] Although the erbB-3 tyrosine kinase domain shares greater
than 60% amino acid identity with the EGFR and erbB-2 proteins,
single amino acid substitutions differences in highly conserved
residues shared among known tyrosine kinases raised a question as
to whether erbB-3 harbors intrinsic catalytic activity involved in
signal propagation instead of signal attenuation as has been
postulated for certain receptor tyrosine kinase-like molecules
(Chou et al., Proc. Natl. Acad. Sci. USA 88:4897 (1991)). Most
notably, codon 834 within the tyrosine kinase domain predicts
asparagine in erbB-3, while aspartate is present at this position
in essentially all known protein kinases. Moreover, substitution of
asparagine for aspartate in this position abolishes c-kit and v-fps
tyrosine kinase activity. The present characterization of the
erbB-3 cytoplasmic domain demonstrates not only its catalytic
function but also the ability to transduce a mitogenic signal as
well gp180.sup.erbB-3 demonstrated autokinase activity in vitro
and, in some cell lines, tyrosine phosphorylation in vivo.
Moreover, EGF-dependent activation of gp180.sup.EGFR/erbB-3 was
associated both with mitogenic signaling and in vivo tyrosine
phosphorylation of the chimeric receptor. All of these findings
imply that the erbB-3 protein represents a biologically active
membrane spanning receptor capable of transducing a mitogenic
signal in a ligand-dependent manner. Thus, the erbB-3 gene encodes
a membrane spanning molecule possessing all the properties of a
functional growth receptor.
[0059] Constitutive activation of erbB-3 catalytic activity was
demonstrated in LTR-erbB-3 transfectants. These results raise the
possibility that NIH/3T3 cells may express an erbB-3 ligand. If so,
this putative ligand would unlikely interact with the EGFR, since
overexpression of the latter in NIH/3T3 cells is not associated
with its chronic tyrosine phosphorylation in the absence of
exogenous EGF. This invention further established that EGF neither
enhanced in vivo tyrosine phosphorylation of gp180.sup.erbB-3 nor
elicited a mitogenic response in LTR-rbB-3 cells. Additional
ligands of the EGF family, including TGF.alpha., amphiregulin, and
HB-EGF, have also failed to stimulate gp180.sup.erbB-3 tyrosine
phosphorylation or DNA synthesis in LTR-erbB-3 cells. While a low
affinity interaction of known EGF-related ligands for
gp180.sup.erbB-3 cannot be excluded, these findings indicated that
erbB-3 and EGFR proteins possess distinct ligand specificities. The
ability to trigger the erbB-3 catalytic domain in the EGFR/erbB-3
chimeric molecule should make it possible to more readily identify
its substrates as well as to compare them with those of its closely
related family members.
[0060] Based upon this invention's demonstration that the erbB-3
protein is both catalytically active and can elicit a proliferative
response in NIH/3T3 cells, the instant findings of its chronic
activation in some human breast tumor cells suggest its
contribution to the malignant phenotype in such tumors. Analogous
evidence has implicated overexpression associated with gene
amplification of both EGFR and erbB-2 in a variety of tumors as
well. In such tumors, there is precedence for activation of
receptor kinase activity by mechanisms involving autocrine loops as
well as genetic alterations affecting regulatory or coding
sequences.
[0061] Both EGFR and erbB-2 genes have been implicated as oncogenes
based upon demonstration of their overexpression and constitutive
activation in various human tumors. The results of this invention
argue strongly that the most recently identified family member,
erbB-3, is activated in some human breast tumors. Overexpression of
the erbB-3 protein did not invariably correlate with its chronic
tyrosine phosphorylation. Hence, erbB-3 activation may involve
autocrine stimulation or subtle genetic alterations. In addition to
breast tumors, expression of the erbB-3 transcript has been
observed in a wide range of human carcinomas, including colon,
lung, kidney, pancreas, and skin. These findings prompt the search
for evidence of erbB-3 activation as an oncogene in these other
common human cancers.
EXAMPLE 1
[0062] Identification of a Human DNA Fragment Related to the erbB-3
Proto-Oncogene Family
[0063] In an effort to detect novel erbB-related genes, human
genomic DNA was cleaved with a variety of restriction endonucleases
and subjected to Southern blot analysis with v-erbB as a probe.
Normal mammary epithelial cells AB589 (Walen, K H. & Stampfer,
M. R., 1989, Cancer. Genet. Cytogenet. 37:249-261) and immortalized
keratinocytes RHEK have been described previously (Rhim; J. S.,
Jay, G., Arnstein, P., Price, F. M., Sanford, K. K. & Aaronson,
S. A., 1985, Science 227:1250-52). Normal human epidermal
melanocytes (NHEM) and keratinocytes (NHEK) were obtained from
Clonetics. Sources for human embryo fibroblasts (Rubin, J. S.,
Osada, H., Finch, P. W., Taylor, W. G., Rudikoff, S., &
Aaronson, S. A., 1989, Proc Nat Acad Sci. USA 86:802-806) or
mammary tumor cell lines SK-BR-3, MDA-MB468, MDA-MB453, and
MDA-MB415 (Kraus, M. H., Popescu, N. C., Amsbaugh, S. C. &
King, C. R., 1987, EMBO. J. 6:605-610) have been described. For
nucleic acid RNA hybridization, DNA and RNA were transferred to
nitrocellulose membranes as previously described (Kraus, M. H., et
al., 1987, supra). High stringency hybridization was conducted in
50% formamide and 5.times.SSC at 42.degree. C. Filters were washed
at 50.degree. C. in 0.1.times.SSC. Reduced stringency hybridization
of DNA was carried out in 30% formamide followed by washes in
0.6.times.SSC, while intermediate stringency was achieved by
hybridization in 40% formamide and washing in 0.25.times.SSC. For
the specific results depicted in FIG. 1, DNAs were restricted with
SacI and hybridized with probe specific for an oncogenic viral form
of the erbB gene, v-erbB, spanning from the upstream BamHI site to
the EcoRI site in the avian erythroblastosis proviral DNA
(Vennstrom, B., Fanshier, L, Moscovici, C. & Bishop, J. M.,
1980, J. Virol. 36:575-585).
[0064] Under reduced stringency hybridization, four SacI
restriction fragments were detected. Two were identified as EGFR
gene fragments by their amplification in the mammary tumor cell
line MDA-MB468 (FIG. 1A, lane 1,2) known to contain EGFR gene
amplification and one as an erbB-2 specific gene fragment due to
its increased signal intensity in another mammary tumor cell line,
SK-BR-3, known to have erbB-2 amplified (FIG. 1A, lane 1,3).
However, a single 9 kbp SacI fragment exhibited equal signal
intensities in DNAs from normal human thymus, SK-BR-3 and a line
with high levels of EGFR, A431 (FIG. 1A). When the hybridization
stringency was raised by 7.degree. C., this fragment did not
hybridize, whereas EGFR and erbB-2 specific restriction fragments
were still detected with v-erbB as a probe (FIG. 1B). Taken
together, these findings suggested the specific detection of a
novel v-erbB-related DNA sequence within the 9 kbp SacI
fragment.
EXAMPLE 2
[0065] Cloning of the Human DNA Fragment Related to erbB
[0066] For further characterization, a normal human genomic library
was prepared from SacI cleaved thymus DNA enriched for 8 to 12 kbp
fragments. For convenience, bacteriophage .lambda.sep-lac5 was
obtained from L. Prestidge and D. Hogness (Stanford University);
many other cloning vectors derived from phage .lambda. or other
genomes can be used for cloning this DNA fragment according to
standard recombinant DNA methods that are well known in the art.
Purified phage DNA was subjected to cos-end ligation, restriction
with SacI, and fractionation in a continuous 10-40% sucrose
gradient. A genomic library was prepared by ligating SacI
restriction fragments of normal human thymus DNA in the molecular
weight range of 8 kbp to 12 kbp (isolated by sucrose gradient
sedimentation) with the purified phage arms. Ten recombinant clones
detected by v-erbB under reduced stringency conditions did not
hybridize with human EGFR or erbB-2 cDNA probes at high stringency.
As shown in the restriction map of a representative clone with 9
kbp insert, the region of v-erbB homology was localized by
hybridization analysis to a 1.5 kbp segment spanning from the EcoRI
to the downstream PstI site.
[0067] The nucleotide sequence of a portion of a clone of the novel
human genomic DNA fragment related to erbB was determined for both
DNA strands by the dideoxy chain termination method (Sanger, F.,
Nicklen, S. & Coulson, A. R., 1977, Proc. Nat. Acad Sci. USA.
74:5463-67) using supercoiled plasmid DNA as template. The
nucleotide sequence was assembled and translated using
IntelliGenetics software. Amino acid sequence comparison was
performed with the alignment program by Pearson and Lipman
(Pearson, W. R. & Lipman, D. J., 1988, supra) as implemented on
the computers of the NCI Advanced Scientific Computing Laboratory.
Hydrophobic and hydrophilic regions in the predicted protein were
identified according to Kyte and Doolittle (Kyte, J. &
Doolittle, R. F., 1982, J Mol. Biol. 157:105-132). Nucleotide
sequence analysis revealed that the region of v-erbB homology in
the 1.5 kbp segment from the EcoRI to the PstI contained three open
reading frames bordered by splice junction consensus sequences
(FIG. 2). Computerized comparisons of the predicted amino acid
sequence of these three open reading frames with other known
proteins revealed the highest identity scores of 64% to 67% to
three regions which are contiguous in the tyrosine kinasc domains
of v-erbB, as well as human EGFR and erbB-2 proteins. Furthermore,
all splice junctions of the three characterized exons in the new
gene were conserved with erbB-2. Amino acid sequence homology to
other known tyrosine kinases was significantly lower, ranging from
39% to 46%.
[0068] A single 6.2 kb specific mRNA was identified by Northern
blot analysis of human epithelial cells using the 150 bp SpeI-AccI
exon-containing fragment as probe (FIG. 2). Under the stringent
hybridization conditions employed, this robe detected neither the 5
kb erbB-2 mRNA nor the 6 kb and 10 kb EGIFR mRNAs. All of these
findings suggested that the present work has identified a new
functional member of the erbB proto-oncogene family, which
tentatively has been designated as erbB-3.
EXAMPLE 3
[0069] Cloning and Characterization of cDNAs for the mRNA of the
Human erbB-3 Gene
[0070] In an effort to characterize the entire erbB-3 coding
sequence, overlapping cDNA clones were isolated from oligo
dT-primed cDNA libraries from sources with known erbB-3 expression,
utilizing gene-specific genomic exons or cDNA fragments as probes.
In brief, an oligo dT-primed human placenta cDNA library in
.lambda.gt11 was obtained from Clontech. MCF-7 cDNA was prepared by
first strand synthesis from 5 .mu.g poly A.sup.+ RNA using an oligo
dT containing linker-primer and Mo-MuLV reverse transcriptase,
followed by second strand synthesis with DNA polymerase I, RNaseH,
and subsequent T4 DNA polymerase treatment. Double-stranded cDNA
was directionally cloned into the SfiI site of .lambda.pCEV9 using
specific linker-adapter oligonucleotides (Miki, T., Matusi, T.,
Heidaran, M. A. & Aaronson, S. A., 1989, Gene 83:137-146; see
also, U.S. application Ser. No. 07/386,053 of Miki et al., filed
Jul. 28, 1989). Following plaque purification, phage DNA inserts
were subcloned into pUC-based plasmid vectors for further
characterization. The clones were initially characterized by
restriction analysis and hybridization to the mRNA, and were
subsequently subjected to nucleotide sequence analysis. Clones
designated pE3-6, pE3-8, pE-9, and pE3-11 carrying inserts with
molecular weights ranging from 1.3 kbp to 4.3 kbp were isolated
from a human placenta library, whereas the pE3-16 clone containing
a 5 kbp insert was obtained by screening the MCF-7 cDNA library
with the upstream most coding sequence of pE3-11 as a probe. The
clones pE3-8, pE3-9, pE3-11, and pE3-16 contained identical 3' ends
terminating in a poly A stretch (FIG. 2).
[0071] The complete coding sequence of erbB-3 was contained within
a single long open reading frame of 4080 nuclcotides extending from
position 46 to an in-frame termination codon at position 4126. The
most upstream ATG codon at position 100 was the likely initiation
codon, as it was preceded by an in-frame stop codon at nucleotide
position 43 and fulfilled the criteria of Kozak for an authentic
initiation codon. The open reading frame comprised 1342 codons
predicting a 148 kd polypeptide. Downstream from the termination
codon, multiple stop codons were present in all frames. As shown in
SEQ ID NO: 4, the deduced amino acid sequence of the erbB-3
polypeptide predicted a transmembrane receptor tyrosine kinase most
closely related to EGFR and erbB-2. A hydrophobic signal sequence
of erbB-3 was predicted to comprise the 19 amino-terminal amino
acid residues. Cleavage of this signal sequence between glycine at
position 19 and serine at position 20 would generate a processed
polypeptidc of 1323 amino acids with an estimated molecular weight
of 145 kd. A single hydrophobic membrane spanning domain
encompassing 21 amino acids was identified within the coding
sequence separating an extracellular domain of 624 amino acids from
a cytoplasmic domain comprising 678 amino acids (SEQ ID NO: 4).
[0072] The putative erbB-3 ligand-binding domain was 43% and 45%
identical in amino acid residues with the predicted erbB-2 and EGFR
protein, respectively. Within the extracellular domain, all 50
cysteine residues of the processed erbB-3 polypeptide were
conserved and similarly spaced when compared to the EGFR and
erbB-2. Forty-seven cysteine residues were organized in two
clusters containing 22 and 25 cysteines respectively, a structural
hallmark of this tyrosine kinase receptor subfamily (see, for
example, Yamamoto, T., Ikawa, S., Akiyama, T., Semba, K, Nomura,
N., Miyajima, N., Saito, T. and Toyoshima, K., 1986, Nature
319:230-234). Ten potential N-linked glycosylation sites were
localized within the erbB-3 extracellular domain. In comparison
with the EGFR and erbB-2 proteins, five and two of these
glycosylation sites were conserved, respectively. Among these, the
site proximal to the transmembrane domain was conserved among all
three proteins (SEQ ID NO: 4).
[0073] Within the cytoplasmic domain, a core of 277 amino acids
from position 702 through 978 revealed the most extensive homology
with the tyrosine kinase domains of EGFR and erbB-2. In this region
60% or 62% of amino acid, residues were identical and 90% or 89%
were conserved, respectively. This stretch of amino acid homology
coincides with the minimal catalytic domain of tyrosine kinases
(Hanks, S. K., Quinn, A. M. & Hunter, T., 1988, Science
241:42-52).
[0074] There was significantly lower homology with other tyrosine
kinases (FIG. 4). The consensus sequence for an ATP-binding site
(GxGxxG, Hanks, S. K. et al., 1988, supra) was identified at amino
acid positions 716 through 721. This sequence as well as a lysine
residue located 21 amino acid residues further toward the carboxyl
terminus was conserved between the three erbB-related receptors.
Taken together these findings defined the region between amino acid
position 702 and 978 as the putative catalytic domain of the erbB-3
protein (SEQ ID NO: 4).
[0075] The most divergent region of erbB-3 compared to either EGFR
or erbB-2 was its carboxyl terminus comprising 364 amino acids.
This region showed a high degree of hydrophilicity and the frequent
occurrence of proline and tyrosine residues. Among these tyrosine
residues, those at positions 1197, 1199, and 1262 matched closest
with the consensus sequence for putative phosphorylation sites. The
peptide sequence YEYMN (SEQ ID NO: 12), encompassing tyrosine 1197
and 1199, was repeated at positions 1260-1264 and was at both
locations surrounded by charged residues, providing an environment
of high local hydrophilicity. These observations render tyrosines
1197, 1199 and 1262 likely candidates for autophosphorylation sites
of the erbB-3 protein.
EXAMPLE 4
[0076] Chromosomal Mapping of the Human erbB-3 Gene.
[0077] The chromosomal location of the erbB-3 gene was determined
by in situ hybridization (Popescu, N. C., King, C. R. & Kraus,
M. H., 1989, Genomics 4:362-366) with a .sup.3H-labeled plasmid
containing the amino-terminal erbB-3 coding sequence. A total of
110 human chromosome spreads was examined prior and subsequent to G
banding for identification of individual chromosomes. A total of
142 grains was localized on a 400-band ideogram. Specific labeling
of chromosome 12 was observed, where 38 out of 51 grains were
localized to band q13 (FIG. 5). Thus, the genomic locus of erbB-3
was assigned to 12q13. In this region of chromosome 12, several
genes have previously been mapped including the melanoma-associated
antigen ME491, histone genes and the gene for lactalbumin. In
addition, two proto-oncogenes, int-1 and gli are located in close
proximity to erbB-3.
EXAMPLE 5
[0078] erbB-3 Expression in Normal and Malignant Human Cells
[0079] To investigate its pattern of expression, a number of human
tissues were surveyed for the erbB-3 transcript. The 6.2 kb erbB-3
specific mRNA was observed in term placenta, postnatal skin,
stomach, lung, kidney, and brain, while it was not detectable in
skin fibroblasts, skeletal muscle or lymphoid cells. Among the
fetal tissues analyzed, the erbB-3 transcript was expressed in
liver, kidney, and brain, but not in fetal heart or embryonic lung
fibroblasts. These observations indicate the preferential
expression of erbB-3 in epithelial tissues and brain.
[0080] ErbB-3 expression was also investigated in individual cell
populations derived from normal human epithelial tissues including
keratinocytes, glandular epithelial cells, melanocytes, and
fibroblasts. For comparison levels of EGFR and erbB-2 transcripts
were analyzed. As shown in Table 1, erbB-3 mRNA levels were
relatively high in keratinocytes, comparable with those of erbB2
and EGFR in these cells. Lower, but similar expression levels of
each transcript were detected in cells derived from glandular
epithelium. These findings are consistent with growth regulatory
roles of all three receptor-like molecules in squamous and
glandular epithelium. Whereas erbB-2 and EGFR transcripts were also
readily observed in normal fibroblasts, the same cells lacked
detectable erbB-3 mRNA In contrast, normal human melanocytes, which
expressed both erbB-3 and erbB-2 at levels comparable with human
keratinocytes, lacked detectable EGFR transcripts. Thus, the
expression patterns of these receptor-like molecules were different
in specialized cell populations derived from epidermal tissues.
1TABLE 1 Normal expression pattern of human erbB gene family
members Cell Source of Transcripts Gene Relative RNA levels
Embryonic fibroblast (M426) erbB-3 - erbB-2 + EGF-R + Skin
fibroblast (501T) erbB-3 - erbB-2 + EGF-R + Immortal keratinocyte
(RHEK) erbB-3 ++ erbB-2 ++ EGF-R ++ Primary keratinocyte (NHEK)
erbB-3 + erbB-2 + EGF-R ++ Glandular epithelium (AB589) erbB-3 (+)
erbB-2 (+) EGF-R (+) Melanocyte (NHEM) erbB-3 ++ erbB-2 ++ EGF-R -
Replicate Northern blots were hybridized with equal amounts (in
cpm) of probes of similar specific activities for erbB-3, erbB-2,
and EGFR, respectively. Relative signal intensities were estimated:
- not detectable, (+) weakly positive, + positive, ++ strongly
positive.
[0081] To search for evidence of erbB-3 involvement in the
neoplastic process, erbB-3 mRNA levels in a series of human tumor
cell lines were surveyed. The erbB-3 transcript was detected in 36
of 38 carcinomas of 2 of 12 sarcomas while 7 tumor cell lines of
hematopoictic origin lacked measurable erbB-3 mRNA. Markedly
elevated levels of a normal-sized transcript were observed in 6 out
of 17 tumor cell lines derived from human mammary carcinomas. By
Southern blot analysis, neither gross gene rearrangement nor
amplification was detected in the cell lines. FIG. 6A shows the
results of Northern blot analysis with control AB589 nonmalignant
human mammary epithelial cells (lane 1) and two representative
human mammary tumor lines, MDA-MB415 (lane 2) and MDA-MB453 (lane
3). Hybridization of the same filter with human .beta.-actin probe
(FIG. 6B) verified actual levels of mRNA in each lane.
Densitometric scanning indicated that the erbB-3 transcript in each
tumor cell line was elevated more than 100 fold above that of the
control cell line. Thus, overexpression of this new member of the
erbB family, as in the case of the EGFR and erbB-2 genes, is likely
to play an important role in some human malignancies.
EXAMPLE 6
[0082] Further Characterization of the Normal erbB-3 Gene
Product
[0083] The pZIPneo expression vector (Cepko et al, Cell 37:1053
(1984)) was modified by introduction of a unique Sal I cloning
site. Following deletion of the Sal I site in the tetracyclin
resistance gene, the synthetic oligonucleotides
5'-GATCTCGAGTCGAC-3' (SEQ ID NO: 10) and 5'-GATCGTCGACTCGA-3' (SEQ
ID NO: 11) were annealed and ligated into the single Bam HI site to
generate pZIPneo.sub.Sal. The erbB-3 open reading frame including 7
nucleotides upstream of the initiation codon and the termination
codon (nucleotides 93-4128) was Tinkered with Sal I ends, employing
the polymerase chain reaction (PCR) and cloned into
pZIPneo.sub.sal(LTR-rbB). Sense orientation and integrity of the
open reading frame were confirmed by restriction analysis as well
as nucleotide sequence analysis of cloning boundaries and
PCR-amplified regions.
[0084] For structural and functional characterization of the erbB-3
gene product, the complete erbB-3 open reading-frame was inserted
as given above into the modified ZIPneo vector, placing the cDNA
under the transcriptional control of the Moloney murine leukemia
virus long-terminal-repeat sequence (LTR-erbB-3). NIH/3T3
fibroblasts were transfected with LTR-erbB-3 or LTR-neo control DNA
and cultured in the presence or absence of the selective drug G418.
Under conditions in which efficient drug resistance
(6.times.10.sup.3 colonies/pmol) was conferred by LTR-erbB-3, no
transformed foci were detectable. In contrast, LTRerbB-2 or
EGF-triggered LTR-EGFR induced i morphological transformation of
NIH-3T3 cells with efficiencies of around 1.2.times.10.sup.4/pmol
and 2.3.times.10.sup.2/pmol, respectively.
[0085] To test for expression of the erbB-3 protein, polyclonal
rabbit antisera, including MK4 and MK5, were developed against
synthetic peptides. MK4 and MK5 were raised against peptides that
encompass the residues given in SEQ ID NO: 5 and SEQ ID NO: 6,
respectively, which are within the carboxyl terminus of the
predicted erbB-3 product. For immunization, peptides were coupled
to thyroglobulin using glutaraldehyde. Immunoblot analysis of
lysates from marker-selected LTR-neo and LTR-erbB-3 transfectants
revealed a major 180 kDa band only in LTR-erbB-3 cells. This band
was independently recognized by both antisera (FIG. 8A). There was
no cross-reactivity of either antiserum with the related EGFR or
erbB-2 proteins overexpressed in NIH/3T3 cells. Immunoreactivity of
either antiserum with the 180 kDa band in LTR-erbB-3 transfectants
was competed by the antigenic peptide, while MK4 reactivity with a
faint 125 kDa band was not affected by preincubation with peptide
(FIG. 8A). These results established specificity of erbB-3 protein
detection by both polyclonal antisera. By comparison, the 180 kDa
erbB-3 protein migrated distinctly slower than the 170 kDa EGFR and
slightly faster than the 185 kDa erbB-2 protein.
[0086] To characterize processing of the erbB-3 protein, we
performed immunoprecipitation experiments with MK5 antiserum.
Metabolic labeling of LTR-erbB-3 transfectants in the presence or
absence of tunicamycin demonstrated that the 145 kDa erbB-3 core
polypeptide is modified by N-linked glycosylation (FIG. 8B).
Pulse-chase analysis further indicated cotranslational processing,
resulting in a predominant 170 kDa precursor protein in addition to
faint erbB-3 specific bands of 150 kDa and 160 kDa, following 15
min of pulse-labeling (FIG. 8C). The mature 180 kDa erbB-3 protein
appeared after 0.5 h of chase, and the majority was converted into
gp180.sup.erbB-3 by 1 h. By analysis of further time points, we
estimate an approximate half-life of 2-3h (FIG. 8C). Thus, in
NIH/3T3 cells, gp180.sup.erbB-3 exhibits an apparently faster
turn-over than the EGFR, for which a biosynthesis time of 3h and
approximate half-life of 3-6 h has been reported.
[0087] For immunolocalization of the erbB-3 protein,
erbB-3-specific monoclonal antibodies, including MAb E3-1, were
raised against the recombinantly expressed protein. BALB/c mice
were immunized with live LTR-erbB-3 cells. Somatic cell hybrids
were prepared by fusion of immune splenocytes with murine
non-secreting myeloma cells NS-1. Hybridoma supernatants were
screened for differential immunoreactivity with LTR-erbB-3 but not
LTR-neo transfectants by enzyme-linked immunosorbent assay (ELISA)
using both live cells or cell extracts as antigen source. Positive
hybridoma cell lines were cloned twice by limiting dilution and
further characterized by immunoprecipitation and immunofluorescencc
analysis. One monoclonal antibody, MAb E3-1 (IgG2a isotype),
specifically immunoprecipitated gp180.sup.erbB-3 from LTR-erbB-3
transfectants (FIG. 9A) and did not exhibit cross-reactivity with
the EGFR or erbB-2 proteins overexpressed in an NIH/3T3 cell
background. Immunofluorescence analysis using a labeled second
antibody revealed heterogeneous membrane immunostaining of
formalin-fixed LTR-erbB-3 cells using MAb E3-1, but not with a
non-specific immunoglobulin of matching isotype (FIG. 9B). MAb
E3-1-specific membrane fluorescence of native LTR-erbB-3 cells
(FIG. 9B) indicated that gp180.sup.erbB-3 was expressed at the cell
surface, as expected for a membrane-anchored protein.
[0088] To investigate its function, we next analyzed the erbB-3
protein for in vitro kinase activity. LTRerbB-3 and control LTR-neo
cell lysates were first immunoprecipitated with E3-1 followed by
immunoblot analysis with MK4 antiserum (FIG. 10A). When the same
immunoprecipitates were incubated in autokinase buffer containing
[.sup.32P]-.gamma.ATP, a predominant 180 kDa phosphoprotein was
labeled only in immunoprecipitates containing the erbB-3 protein
(FIG. 10B). These findings indicated that gp180.sup.erbB-3
possessed intrinsic protein kinase activity. To assess its
enzymatic activity in vivo, LTR-rbB-3 lysates were subjected to
immunoprecipitation with phosphotyrosine-specific monoclonal
antibodies (anti-P-Tyr) followed by immunoblotting with MK4
antiserum. As shown in FIG. 10C, the erbB-3 protein was recovered
from anti-P-Tyr immunoprecipitates, and immunodetection was
competed either by phenyl phosphate in the immunoprecipitation or
the erbB-3 peptide in Western blot analysis. These findings
indicated that recombinant gp.sub.180.sup.erbB-3 expressed in
NIH/3T3 cells was chronically phosphorylated on tyrosine
residues.
[0089] The protein lysates were prepared in Staph A buffer
containing the protease inhibitors phenylmethyl sulfonyl fluoride
(1 mM) and aprotinin (10 .mu.g/ml; Boehringer Mannheim). For the
analysis of phospho-tyrosine proteins, the phosphatase inhibitors
sodium orthovanadate (2 mM) and sodium pyrophosphate (10 mM) were
added. Immunoblot analysis using peptide antisera was essentially
conducted as previously reported. For the detection of
phosphotyrosine proteins, membranes were blocked in PBS containing
5% BSA and immunostained with a mixture of monoclonal anti-P-Tyr
antibodies (PY20 and PY69; ICN) diluted 1:500 in PBS containing 1%
BSA. Filters were washed with PBS containing 0.05% Tween20.
Immunoprecipitation was conducted using gammabind G agarose
(Pharmacia) to collect the immunocomplexes. The beads were coupled
with goat anti-mouse-IgG second antibody (Boehringer Mannheim) in
immunoprecipitations using erbB-3 or EGFR monoclonal antibodies.
For in vitro kinase assays, 4 mg total lysates were precleared with
gammabind G agarose. Following immunoprecipitation, washed
immunocomplexes were equilibrated in autokinase buffer containing
40 mM Hepes 7.5, 10 mM MgCl, and 0.05% Triton. The immunocomplexes
were subsequently divided for immunoblot analysis or immunocomplex
kinase assay, respectively. Autokinase reactions were carried out
in 40 .mu.l autokinase buffer containing 20 Ci .gamma.ATP (3000
ci/mmol) at 25.degree. for 10 min and terminated by addition of SDS
containing sample buffer.
EXAMPLE 7
[0090] EGF-Dependent Mitogenic Signaling by an EGFR/erb B-3
Chimeric Receptor
[0091] To explore erbB-3 signaling, a chimeric receptor,
LTR-EGFR/erbB-3, containing the ligand-binding domain of the
closely related EGF receptor (aa 1-682) and the intracellular
portion of erbB-3 (aa 681-1342) was engineered. Linearized
expression constructs (0.01-10 .mu.g/plate) were transfected into
NIH/3T3 cells by calcium phosphate precipitation using 40 .mu.g of
calf thymus DNA as carrier. Mass cultures expressing the
recombinant proteins were obtained by selection with 750 .mu.g/ml
G418. Selected LTR-EGFR/erbB-3 transfectants were enriched for
expression of the chimeric protein by preparative FACS sorting
using EGFR monoclonal antibody AB-1 (Oncogene Sciences).
[0092] Transfection of NIH/3T3 cells with this construct did not
result in detectable focus formation either in the presence or
absence of EGF. To quantitate expression of the chimeric receptor,
selected mass cultures were analyzed for EGF-binding in comparison
to NIH/3T3 cells overexpressing the EGFR (LTR-EGFR). Scatchard
analysis established around 5.7.times.10.sup.5 EGF binding
sites/cell for the LTR-EGFR/erbB-3 transfectant as compared to
2.5.times.10.sup.6 binding sites/cell for LTR-EGFR transfectant.
The LTR-EGFR/erbB-3 transfectant exhibited two populations of
binding sites with affinities of 0.11 nM and 5 nM, respectively.
The high-affinity sites were in the minority (2.3.times.10.sup.4),
and there were 5.5.times.10.sup.5 low-affinity binding sites.
Similar results were obtained with the wild-type EGFR in LTR-EGFR
transfectants, which displayed 1.1.times.10.sup.5 high affinity
(0.13 nM) and 2.4.times.10.sup.6 low affinity receptors (7 nM).
[0093] To investigate EGF responsiveness of erbB-3 enzymatic
activity, the in vivo tyrosine phosphorylation of the chimeric
receptor in the presence or absence of EGF was compared. This
protein, as well as the EGFR and erbB-3 proteins from independent
transfectants were enriched by immunoprecipitation and subjected to
immunoblot analysis with either anti-P-Tyr or the appropriate
specific antiserum. As shown in FIG. 11, the steady state level of
tyrosine phosphorylated gp180.sup.erbB-3 in NIH/3T3 cells was not
altered upon EGF exposure (lane 1,2). The chimeric EGFR/erbB-3
receptor, which was expressed as a 180 kDa protein,
gp180.sup.EGFR/erbB-3 displayed low, but detectable level of
tyrosine phosphorylation in serum-free medium (lane 3). However,
EGF triggering of the chimera resulted in a substantial increase in
tyrosine phosphorylation, demonstrating EGF-dependent activation of
erbB-3 catalytic function (lane 4). The wild-type EGFR showed
somewhat higher level of EGF-dependent tyrosine phosphorylation
under the same conditions (lane 6). Of note, the relative level of
gp180.sup.erbB-3 tyrosine phosphorylation was comparable to that of
EGF-activated chimeric receptor expressed at a similar protein
level, indicating constitutive activation of erbB-3 catalytic
properties in LTR-erbB-3 transfectants.
[0094] Whether the erbB-3 catalytic domain was capable of
transducing a mitogenic signal was then assessed. When the
LTR-EGFR/erbB-3 transfectant was exposed to increasing EGF
concentrations, there was a dose-dependent stimulation of DNA
synthesis similar to that observed with EGFR overexpressing NIH-3T3
cells. Under the same conditions, neither LTR-neo nor LTR-erbB-3
transfectants showed a significant increase in DNA synthesis even
at high EGF concentrations, consistent with previous observations.
It should be noted that basal-levels of DNA synthesis of the
LTR-erbB-3 transfectant were 2-3 fold above those of the other
transfectants, findings that were reproducible with several
independent selected mass cultures.
[0095] The biological effects of activated erbB-3 catalytic
function were assessed by testing the transfectants for
anchorage-independent growth. To test anchorage-independent growth,
cell suspensions were seeded at 10-fold serial dilutions in
semisolid agarose medium containing growth medium and 0.45%
seaplaque agarose (FMC Corp.). Visible colonies comprising >100
cells were scored at 14 days. EGF was added at a concentration of
20 ng/ml. Human mammary tumor cell lines were obtained from the
American Type Culture Collection and propagated in Dulbecco's
modified Eagle medium containing 10% fetal calf serum.
[0096] As shown in Table 2, LTR-neo transfectants failed to exhibit
significant soft agar growth in the presence or absence of EGF. In
contrast, EGF induced soft agar colony formation with both
LTR-EGFR/erbB-3 and LTR-EGFR transfectants. The latter showed a
larger colony number (Table 2) as well as colony size (data not
shown). By comparison, the LTR-erbB-3 transfectant displayed
EGF-independent colony formation with an efficiency similar to that
of EGF-activated LTR-EGFR/erbB-3 transfectant (Table 2). All of
these findings establish that ligand activation of a chimeric
EGFR/erbB-3 receptor causes mitogenic signaling in NIH/3T3 cells
and suggest that chronic tyrosine phosphorylation of erbB-3 in
LTR-erbB-3 transfectants is associated with constitutive signaling
in these cells.
2TABLE 2 Anchorage-independent growth of NIH/3T3 transfectants #
colonies*/10.sup.4 cells NIH3T3 transfectants -EGF +EGF LTR-neo
1(.+-.1) 4(.+-.2) LTR-EGFR 2(.+-.2) 206(.+-.49) LTR-EGFR/erbB-3
7(.+-.3) 88(.+-.16) LTR-erbB-3 97(.+-.20) 94(.+-.29) *mean
(.+-.standard error) of 3 independent assays
EXAMPLE 8
[0097] Evidence for Activated erbB-3 Signaling Function in Human
Breast Tumor Cells
[0098] The availability of erbB-3 specific antibodies made it
possible to explore expression and activity of gp180.sup.erbB-3 in
human tumor cells. Based upon our previous evidence for erbB-3 mRNA
overexpression in certain breast cancer cell lines, we measured
erbB-3 protein levels and tyrosine phosphorylation in such tumor
lines, using the procedures given above. Following
immunoprecipitation with Mab E3-1, immunoblot analysis with MK4
antiserum revealed the natural human erbB-3 product as a 180 kDa
protein. The levels of erbB-3 protein varied markedly along the
tumor lines analyzed, with highest expression in BT483, MDA-M1453,
MDA-MB134, MDA-MB361, SK-BR-3, and MDA-MB468 (FIG. 12). The lowest
levels were observed in BT20 and MDA-MB175 cell lines (FIG. 12),
comparable with that expressed by nonmalignant 184B5 mammary
epithelial cells (data not shown). Thus, erbB-3 protein expression
varied by at least 20-30 fold among the lines tested, consistent
with results of transcript analysis (data not shown).
[0099] Immunoblot analysis of the same immunoprecipitates with
anti-P-Tyr antibodies revealed that tyrosine phosphorylation of the
native erbB-3 product was undetectable in 8 of the tumor cell
lines, including MDA-MB134, MDA-MB361, and MDA-MB468, which
harbored increased erbB-3 levels. In contrast, erbB-3 protein
expressed by 4 cell lines, including MDA-MB453, BT474, MDA-MB175,
and SK-BR-3, demonstrated readily detectable chronic tyrosine
phosphorylation (FIG. 12, lanes 5, 7, 9 and 11). In MDA-MB175,
there was no significantly elevated level of erbB-3 protein. Thus,
in 4 out of 12 breast tumor cells lines, the gp180.sup.erbB-3
signaling function was activated at steady state. Whether chronic
erbB-3 phosphorylation involves autocrine stimulation or subtle
structural alterations, these findings provide evidence for
constitutive gp180.sup.erbB-3 activation in these human breast
tumor cells.
EXAMPLE 9
[0100] Identification, Purification and Characterization of erbB-3
Ligands
[0101] As shown in FIG. 12, the gp.sub.180.sup.erbB-3 that is
overexpressed in some human breast tumor cell lines can be either
functionally activated or not, depending on the cell line. Further,
in some other human breast tumor cell lines, the erbB-3 polypeptide
is not overexpressed and, again, can be either activated or not
activated. These differences, and the common property of growth
factor receptor-like tyrosine kinases to rapidly autophosphorylate
on tyrosine residues in response to ligand triggering, can be
exploited to identify, isolate and characterize ligands, preferably
specific ligands, that can activate or down-regulate erbB-3. The
term "erbB-3 ligand" refers to a molecule that binds to the erbB-3
protein, particularly to the extracellular domain of the erbB-3
protein, and can activate ("erbB-3 activating ligand") or
down-regulate ("erbB-3 blocking ligand") the biochemical and/or
biological activity of the erbB-3 protein. Depending on the
concentration of the ligand, a ligand can both activate and
down-regulate activity.
[0102] A source containing a potential erbB-3 ligand, such as
conditioned medium, body fluid extracts, cell extracts, tissue
extracts or the like, with or without agents which can modify
erbB-3 activity, can be screened for the presence of such a ligand
by the ability of the solution, in the case of an activating
ligand, to enhance erbB-3 phosphorylation. With respect to
screening for an erbB-3 activating ligand, cells from a cell line
whose expressed erbB-3 protein contains nonexistent or low level
intrinsic tyrosine phosphorylation can be contacted with potential
ligand sources or control medium for a time and under conditions
sufficient to allow binding of an erbB-3 ligand, if present, to
bind to erbB-3. Typically, if an erbB-3 ligand is present, binding
will occur within a short time. Thus, the cells are exposed to
potential ligand sources or control medium for, preferably, no
longer than 30 minutes, most preferably, 10 minutes or less.
Appropriate conditions to allow binding of the ligand can be
determined by one skilled in the art, such as physiological
conditions at 37.degree. C. or the conditions given in FIG. 5 of
Holmes et al., Science 256:1205 (1992). erbB-3 modifying agents, if
administered, can be present in a concentration between 10.sup.-2
pM and 10.sup.5 pM. The cell line employed can overexpress erbB-3,
such as the mammary tumor cell lines MDA-M1415, MDA-MB134,
MDA-MB468, BT483, MDA-MB361, MCF-7 and ZR-75-1, which express an
increased amount of the erbB-3 protein with low level intrinsic
tyrosine phosphorylation compared to protein amounts and activation
levels for corresponding nonmalignant cells. One such potential
activating ligand source can be derived from cell lines that not
only overexpress erbB-3 but also exhibit high level intrinsic
tyrosine phosphorylation, such as MDA-MB453, SK-BR-3, and BT474. In
addition, any normal cell which does not overexpress erbB-3 can be
utilized, e.g., fibroblasts.
[0103] Similarly, with respect to screening for an erbB-3
inhibitory down-regulating ligand, a cell line whose expressed
erbB-3 protein contains high level intrinsic tyrosine
phosphorylation can be exposed to potential ligand sources or
control medium for a time and under conditions sufficient to allow
binding of an erbB-3 ligand, if present, to bind to erbB-3. The
cell line employed preferably expresses activated erbB-3, such as
the mammary tumor cell lines MDA-MB453, SK-BR-3 and BT474. In
addition, an activating ligand at higher concentration can be
down-regulating. Such activity can be routinely screened given the
teaching herein.
[0104] The triggering or blocking of erbB-3 activation can be
detected by comparing the level of erbB-3 tyrosine phosphorylation
in the cell line after exposure to the potential ligand source with
the normal level, e.g., the level obtained after exposure to the
control medium. For example, in a negative control the cells can be
in serum free medium and for activating ligand the conditioned
medium is from cell lines with increased erbB-3 that don't have
phosphorylation. For instance, to measure erbB-3 specific tyrosine
phosphorylation, potentially triggered (or blocked) cells and the
control cells are lysed. Using procedures such as those discussed
above, the erbB-3 protein is immunoprecipitated with an erbB-3
specific antibody, preferably a monoclonal such as MAb E3-1. The
immunoprecipitates arc divided and subjected to immunoblot analysis
with either antiphosphotyrosine or erbB-3 antibodies. The presence
of an erbB-3 activating or blocking ligand can be monitored by a
relative increase or decrease, respectively, of phosphotyrosine
levels in comparison to the untriggered control. Any increase can
be significant, especially a two-fold or greater increase. This
ligand-detection system can be used repeatedly throughout the
ligand purification procedures so as to monitor protein
purification of the erbB-3 ligand to homogeneity.
[0105] Alternatively, following exposure of the cell lines to the
potential ligand source as discussed above, detection of an erbB-3
activating ligand or blocking i ligand can be accomplished by
measurement of cell growth and/or mitogenic signals resulting from
the activation or inhibition of erbB-3 catalytic activity, using,
for example, the procedures given in Example 7 above. An increase
in colony number or colony size and/or a dose-dependent increase of
DNA synthesis for the cells exposed to the potential ligand
relative to those exposed to the control medium correlates with the
presence of an activating ligand in the potential ligand source.
Conversely, respective decreases correlate with the presence of a
blocking ligand in the potential ligand source.
[0106] Following the isolation and purification of the erbB-3
ligand, the identity of the ligand can be determined by protein
identification methods known in the art, such as amino acid
sequencing. Further, the erbB-3 ligand can be molecularly
characterized. For instance, similar to the procedures outlined in
Holmes et al., Science 256:1205 (1992), the nucleic acid sequence
that corresponds to the ligand's amino acid sequence, or a partial
amino acid sequence corresponding to a portion of the ligand, can
be used to design degenerate oligonucleotide probes corresponding
to the amino acid sequence or partial sequence. These degenerate
oligonucleotides can be used to screen a cDNA library and generate
a clone that encodes the precursor of the erbB-3 ligand. Following
determination of the coding sequence, related coding sequences can
be discovered by screening other libraries.
[0107] For purposes of completing the background description and
present disclosure, each of the published articles, patents and
patent applications heretofore identified in this specification are
hereby incorporated by reference into the specification.
[0108] The foregoing invention has been described in some detail
for purposes of clarity and understanding. It will also be obvious
that various changes and combinations in form and detail can be
made without departing from the scope of the invention.
3 # SEQUENCE LIS #TING (1) GENERAL INFORMATION: (iii) NUMBER OF
SEQUENCES: 12 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 1542 base #pairs (B) TYPE: nucleic
acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE
TYPE: DNA (genomic) (ix) FEATURE: (A) NAME/KEY: exon (B) LOCATION:
66..221 (ix) FEATURE: (A) NAME/KEY: exon (B) LOCATION: 780..855
(ix) FEATURE: (A) NAME/KEY: exon (B) LOCATION: 1040..1185 (ix)
FEATURE: (A) NAME/KEY: intron (B) LOCATION: 222..779 (ix) FEATURE:
(A) NAME/KEY: intron (B) LOCATION: 856..1039 (ix) FEATURE: (A)
NAME/KEY: CDS (B) LOCATION: join(66..221 #, 780..855, 1040..1185)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: #1: GAATTCCAGA TCTCAGTGAC
TGATTCCCCC AACCTTAAGA ATACTTTCTT CC #CCTATACC 60 TACAG GGA ATG TAC
TAC CTT GAG GAA CAT GGT #ATG GTG CAT AGA AAC 107 Gly Met Tyr Tyr
Leu Glu Glu #His Gly Met Val His Arg Asn 1 # 5 # 10 CTG GCT GCC CGA
AAC GTG CTA CTC AAG TCA CC #C AGT CAG GTT CAG GTG 155 Leu Ala Ala
Arg Asn Val Leu Leu Lys Ser Pr #o Ser Gln Val Gln Val 15 #20 #25
#30 GCA GAT TTT GGT GTG GCT GAC CTG CTG CCT CC #T GAT GAT AAG CAG
CTG 203 Ala Asp Phe Gly Val Ala Asp Leu Leu Pro Pr #o Asp Asp Lys
Gln Leu 35 # 40 # 45 CTA TAC AGT GAG GCC AAG GTGAGGAGAC ACAAAGGGTA
AG #GAGGCGGG 251 Leu Tyr Ser Glu Ala Lys 50 GGTGGAGTGA AGCATGGGGA
TAGGGAGCAG CCAGTGGTCT CTTCCAGAGG CA #AGCAGATG 311 CTTCATGGTA
AGTTCAAGGA GAGAAGGCTG CAGATGCCAG ATATTTTAGT TC #AGAGGGCA 371
ACAAAGAAAA TAATGATCAA GAACTTGGGA CTGGCCGGGC GCGGTGGCTC AC #GCCTGTAA
431 TCCCAACACT TCGGGAGGCC AAGGCGGGTG GATCACAAGG TCAGGAGATC AA
#GACCATCC 491 TGGCTAGCAC GGTGAAACCC CGTCTCTACT AAATATACAA
AAAAAAAAAA AT #TAGCCAGG 551 CGTGGCGGCA TGCATCTGTA CTCCCAGCTA
CTCGGGAGGC TGAGGCAGGA GA #ATGGCGTG 611 AACCCAGGAG GCGGAGCTTG
CAGTGGGCCG AGATCGCACC ACTGCACTCC AG #TCTGGGCG 671 ACAGAGCGAG
ACTCCGTCTC AAAAAAAAAA AAAAAAGAAT TTGGGACTTG GA #AATCCTAA 731
GAAAATTTGT GGAAATAAAC TTGTGATACC TCTATCTTTA ATCCGCAG ACT #CCA ATT
788 # # Thr # Pro Ile # # # 55 AAG TGG ATG GCC CTT GAG AGT ATC CAC
TTT GG #G AAA TAC ACA CAC CAG 836 Lys Trp Met Ala Leu Glu Ser Ile
His Phe Gl #y Lys Tyr Thr His Gln 60 # 65 # 70 AGT GAT GTC TGG AGC
TAT G GTCAGTGCAT CTGGATG #CCC TCTCTACCAT 885 Ser Asp Val Trp Ser
Tyr 75 CACTGGCCCC AGTTTCAAAT TTACCTTTTG AGAGCCCCCT CTTAGAATCT CT
#AAGCACTT 945 CAGATTTTTG TGTTAGATCA GGTTCTGCCT TCCCTTCACT
TCATGCCCAT GT #CTACTATT 1005 TTGCCAGTGA CTAGTCCATG TCTTCCTGCA ACAG
GT GTG ACA #GTT TGG GAG 1056 # # Gly Val Thr Val Trp Glu # # 80 TTG
ATG ACC TTC GGG GCA GAG CCC TAT GCA GG #G CTA CGA TTG GCT GAA 1104
Leu Met Thr Phe Gly Ala Glu Pro Tyr Ala Gl #y Leu Arg Leu Ala Glu
85 # 90 # 95 GTA CCA GAC CTG CTA GAG AAG GGG GAG CGG TT #G GCA CAG
CCC CAG ATC 1152 Val Pro Asp Leu Leu Glu Lys Gly Glu Arg Le #u Ala
Gln Pro Gln Ile 100 1 #05 1 #10 1 #15 TGC ACA ATT GAT GTC TAC ATG
GTG ATG GTC AA #G TGTGAGTTAC CTGCTGAGCC 1205 Cys Thr Ile Asp Val
Tyr Met Val Met Val Ly #s 120 # 125 CAACCATTTT CTCTTTTTTT
CTTTTTTTTT CTTTTTTTTT TTTTTTTGAG AC #AGAGTCTC 1265 ACAATTGTCA
CCCAGGCTGG AGTGCAATGG TGCAATCAAT CTTGGCTCAC TA #CAACCTCC 1325
GCCTCTCGGG TTCAAGAGAT TCTCCTGCTT CAGCTCCGGA GTAGCTGGGA TT #ACAGCGCC
1385 CGCCACACCT GGATAACTGT TACACTTTTA GTAGAGATGG GGTTTCACCA TG
#TTGGCCAG 1445 GCTGGTCTCA AACTCCTGAC CTCAGGTGAT CCGCCTGCCT
CAGCTTCCCA AA #GTGCTGGG 1505 ATTACAGGTG TGAGCCATCA TGCTCGCCTG
ACTGCAG # # 1542 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 126 amino #acids (B) TYPE: amino acid
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE
DESCRIPTION: SEQ ID NO: #2: Gly Met Tyr Tyr Leu Glu Glu His Gly Met
Va #l His Arg Asn Leu Ala 1 5 # 10 # 15 Ala Arg Asn Val Leu Leu Lys
Ser Pro Ser Gl #n Val Gln Val Ala Asp 20 # 25 # 30 Phe Gly Val Ala
Asp Leu Leu Pro Pro Asp As #p Lys Gln Leu Leu Tyr 35 # 40 # 45 Ser
Glu Ala Lys Thr Pro Ile Lys Trp Met Al #a Leu Glu Ser Ile His 50 #
55 # 60 Phe Gly Lys Tyr Thr His Gln Ser Asp Val Tr #p Ser Tyr Gly
Val Thr 65 #70 #75 #80 Val Trp Glu Leu Met Thr Phe Gly Ala Glu Pr
#o Tyr Ala Gly Leu Arg 85 # 90 # 95 Leu Ala Glu Val Pro Asp Leu Leu
Glu Lys Gl #y Glu Arg Leu Ala Gln 100 # 105 # 110 Pro Gln Ile Cys
Thr Ile Asp Val Tyr Met Va #l Met Val Lys 115 # 120 # 125 (2)
INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 4905 base #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE:
(A) NAME/KEY: CDS (B) LOCATION: 100..4125 (xi) SEQUENCE
DESCRIPTION: SEQ ID NO: #3: ACCAATTCGC CAGCGGTTCA GGTGGCTCTT
GCCTCGATGT CCTAGCCTAG GG #GCCCCCGG 60 GCCGGACTTG GCTGGGCTCC
CTTCACCCTC TGCGGAGTC ATG AGG GCG # AAC GAC 114 # # Met Arg Ala Asn
Asp # # 1 # 5 GCT CTG CAG GTG CTG GGC TTG CTT TTC AGC CT #G GCC CGG
GGC TCC GAG 162 Ala Leu Gln Val Leu Gly Leu Leu Phe Ser Le #u Ala
Arg Gly Ser Glu 10 # 15 # 20 GTG GGC AAC TCT CAG GCA GTG TGT CCT
GGG AC #T CTG AAT GGC CTG AGT 210 Val Gly Asn Ser Gln Ala Val Cys
Pro Gly Th #r Leu Asn Gly Leu Ser 25 # 30 # 35 GTG ACC GGC GAT GCT
GAG AAC CAA TAC CAG AC #A CTG TAC AAG CTC TAC 258 Val Thr Gly Asp
Ala Glu Asn Gln Tyr Gln Th #r Leu Tyr Lys Leu Tyr 40 # 45 # 50 GAG
AGG TGT GAG GTG GTG ATG GGG AAC CTT GA #G ATT GTG CTC ACG GGA 306
Glu Arg Cys Glu Val Val Met Gly Asn Leu Gl #u Ile Val Leu Thr Gly
55 # 60 # 65 CAC AAT GCC GAC CTC TCC TTC CTG CAG TGG AT #T CGA GAA
GTG ACA GGC 354 His Asn Ala Asp Leu Ser Phe Leu Gln Trp Il #e Arg
Glu Val Thr Gly 70 #75 #80 #85 TAT GTC CTC GTG GCC ATG AAT GAA TTC
TCT AC #T CTA CCA TTG CCC AAC 402 Tyr Val Leu Val Ala Met Asn Glu
Phe Ser Th #r Leu Pro Leu Pro Asn 90 # 95 # 100 CTC CGC GTG GTG CGA
GGG ACC CAG GTC TAC GA #T GGG AAG TTT GCC ATC 450 Leu Arg Val Val
Arg Gly Thr Gln Val Tyr As #p Gly Lys Phe Ala Ile 105 # 110 # 115
TTC GTC ATG TTG AAC TAT AAC ACC AAC TCC AG #C CAC GCT CTG CGC CAG
498 Phe Val Met Leu Asn Tyr Asn Thr Asn Ser Se #r His Ala Leu Arg
Gln 120 # 125 # 130 CTC CGC TTG ACT CAG CTC ACC GAG ATT CTG TC #A
GGG GGT GTT TAT ATT 546 Leu Arg Leu Thr Gln Leu Thr Glu Ile Leu Se
#r Gly Gly Val Tyr Ile 135 # 140 # 145 GAG AAG AAC GAT AAG CTT TGT
CAC ATG GAC AC #A ATT GAC TGG AGG GAC 594 Glu Lys Asn Asp Lys Leu
Cys His Met Asp Th #r Ile Asp Trp Arg Asp 150 1 #55 1 #60 1 #65 ATC
GTG AGG GAC CGA GAT GCT GAG ATA GTG GT #G AAG GAC AAT GGC AGA 642
Ile Val Arg Asp Arg Asp Ala Glu Ile Val Va #l Lys Asp Asn Gly Arg
170 # 175 # 180 AGC TGT CCC CCC TGT CAT GAG GTT TGC AAG GG #G CGA
TGC TGG GGT CCT 690 Ser Cys Pro Pro Cys His Glu Val Cys Lys Gl #y
Arg Cys Trp Gly Pro 185 # 190 # 195 GGA TCA GAA GAC TGC CAG ACA TTG
ACC AAG AC #C ATC TGT GCT CCT CAG 738 Gly Ser Glu Asp Cys Gln Thr
Leu Thr Lys Th #r Ile Cys Ala Pro Gln 200 # 205 # 210 TGT AAT GGT
CAC TGC TTT GGG CCC AAC CCC AA #C CAG TGC TGC CAT GAT 786 Cys Asn
Gly His Cys Phe Gly Pro Asn Pro As #n Gln Cys Cys His Asp 215 # 220
# 225 GAG TGT GCC GGG GGC TGC TCA GGC CCT CAG GA #C ACA GAC TGC TTT
GCC 834 Glu Cys Ala Gly Gly Cys Ser Gly Pro Gln As #p Thr Asp Cys
Phe Ala 230 2 #35 2 #40 2 #45 TGC CGG CAC TTC AAT GAC AGT GGA GCC
TGT GT #A CCT CGC TGT CCA CAG 882 Cys Arg His Phe Asn Asp Ser Gly
Ala Cys Va #l Pro Arg Cys Pro Gln 250 # 255 # 260 CCT CTT GTC TAC
AAC AAG CTA ACT TTC CAG CT #G GAA CCC AAT CCC CAC 930 Pro Leu Val
Tyr Asn Lys Leu Thr Phe Gln Le #u Glu Pro Asn Pro His 265 # 270 #
275 ACC AAG TAT CAG TAT GGA GGA GTT TGT GTA GC #C AGC TGT CCC CAT
AAC 978 Thr Lys Tyr Gln Tyr Gly Gly Val Cys Val Al #a Ser Cys Pro
His Asn 280 # 285 # 290 TTT GTG GTG GAT CAA ACA TCC TGT GTC AGG GC
#C TGT CCT CCT GAC AAG 1026 Phe Val Val Asp Gln Thr Ser Cys Val Arg
Al #a Cys Pro Pro Asp Lys 295 # 300 # 305 ATG GAA GTA GAT AAA AAT
GGG CTC AAG ATG TG #T GAG CCT TGT GGG GGA 1074 Met Glu Val Asp Lys
Asn Gly Leu Lys Met Cy #s Glu Pro Cys Gly Gly 310 3 #15 3 #20 3 #25
CTA TGT CCC AAA GCC TGT GAG GGA ACA GGC TC #T GGG AGC CGC TTC CAG
1122 Leu Cys Pro Lys Ala Cys Glu Gly Thr Gly Se #r Gly Ser Arg Phe
Gln 330 # 335 # 340 ACT GTG GAC TCG AGC AAC ATT GAT GGA TTT GT #G
AAC TGC ACC AAG ATC 1170 Thr Val Asp Ser Ser Asn Ile Asp Gly Phe Va
#l Asn Cys Thr Lys Ile 345 # 350 # 355 CTG GGC AAC CTG GAC TTT CTG
ATC ACC GGC CT #C AAT GGA GAC CCC TGG 1218 Leu Gly Asn Leu Asp Phe
Leu Ile Thr Gly Le #u Asn Gly Asp Pro Trp 360 # 365 # 370 CAC AAG
ATC CCT GCC CTG GAC CCA GAG AAG CT #C AAT GTC TTC CGG ACA 1266 His
Lys Ile Pro Ala Leu Asp Pro Glu Lys Le #u Asn Val Phe Arg Thr 375 #
380 # 385 GTA CGG GAG ATC ACA GGT TAC CTG AAC ATC CA #G TCC TGG CCG
CCC CAC 1314 Val Arg Glu Ile Thr Gly Tyr Leu Asn Ile Gl #n Ser Trp
Pro Pro His 390 3 #95 4 #00 4 #05 ATG CAC AAC TTC AGT GTT TTT TCC
AAT TTG AC #A ACC ATT GGA GGC AGA 1362 Met His Asn Phe Ser Val Phe
Ser Asn Leu Th #r Thr Ile Gly Gly Arg 410 # 415 # 420 AGC CTC TAC
AAC CGG GGC TTC TCA TTG TTG AT #C ATG AAG AAC TTG AAT 1410 Ser Leu
Tyr Asn Arg Gly Phe Ser Leu Leu Il #e Met Lys Asn Leu Asn 425 # 430
# 435 GTC ACA TCT CTG GGC TTC CGA TCC CTG AAG GA #A ATT AGT GCT GGG
CGT 1458 Val Thr Ser Leu Gly Phe Arg Ser Leu Lys Gl #u Ile Ser Ala
Gly Arg 440 # 445 # 450 ATC TAT ATA AGT GCC AAT AGG CAG CTC TGC TA
#C CAC CAC TCT TTG AAC 1506 Ile Tyr Ile Ser Ala Asn Arg Gln Leu Cys
Ty #r His His Ser Leu Asn 455 # 460 # 465 TGG ACC AAG GTG CTT CGG
GGG CCT ACG GAA GA #G CGA CTA GAC ATC AAG 1554 Trp Thr Lys Val Leu
Arg Gly Pro Thr Glu Gl #u Arg Leu Asp Ile Lys 470 4 #75 4 #80 4 #85
CAT AAT CGG CCG CGC AGA GAC TGC GTG GCA GA #G GGC AAA GTG TGT GAC
1602 His Asn Arg Pro Arg Arg Asp Cys Val Ala Gl #u Gly Lys Val Cys
Asp 490 # 495 # 500 CCA CTG TGC TCC TCT GGG GGA TGC TGG GGC CC #A
GGC CCT GGT CAG TGC 1650 Pro Leu Cys Ser Ser Gly Gly Cys Trp Gly Pr
#o Gly Pro Gly Gln Cys 505 # 510 # 515 TTG TCC TGT CGA AAT TAT AGC
CGA GGA GGT GT #C TGT GTG ACC CAC TGC 1698 Leu Ser Cys Arg Asn Tyr
Ser Arg Gly Gly Va #l Cys Val Thr His Cys 520 # 525 # 530 AAC TTT
CTG AAT GGG GAG CCT CGA GAA TTT GC #C CAT GAG GCC GAA TGC 1746 Asn
Phe Leu Asn Gly Glu Pro Arg Glu Phe Al #a His Glu Ala Glu Cys 535 #
540 # 545 TTC TCC TGC CAC CCG GAA TGC CAA CCC ATG GA #G GGC ACT GCC
ACA TGC 1794 Phe Ser Cys His Pro Glu Cys Gln Pro Met Gl #u Gly Thr
Ala Thr Cys 550 5 #55 5 #60 5 #65 AAT GGC TCG GGC TCT GAT ACT TGT
GCT CAA TG #T GCC CAT TTT CGA GAT 1842 Asn Gly Ser Gly Ser Asp Thr
Cys Ala Gln Cy #s Ala His Phe Arg Asp 570 # 575 # 580 GGG CCC CAC
TGT GTG AGC AGC TGC CCC CAT GG #A GTC CTA GGT GCC AAG 1890 Gly Pro
His Cys Val Ser Ser Cys Pro His Gl #y
Val Leu Gly Ala Lys 585 # 590 # 595 GGC CCA ATC TAC AAG TAC CCA GAT
GTT CAG AA #T GAA TGT CGG CCC TGC 1938 Gly Pro Ile Tyr Lys Tyr Pro
Asp Val Gln As #n Glu Cys Arg Pro Cys 600 # 605 # 610 CAT GAG AAC
TGC ACC CAG GGG TGT AAA GGA CC #A GAG CTT CAA GAC TGT 1986 His Glu
Asn Cys Thr Gln Gly Cys Lys Gly Pr #o Glu Leu Gln Asp Cys 615 # 620
# 625 TTA GGA CAA ACA CTG GTG CTG ATC GGC AAA AC #C CAT CTG ACA ATG
GCT 2034 Leu Gly Gln Thr Leu Val Leu Ile Gly Lys Th #r His Leu Thr
Met Ala 630 6 #35 6 #40 6 #45 TTG ACA GTG ATA GCA GGA TTG GTA GTG
ATT TT #C ATG ATG CTG GGC GGC 2082 Leu Thr Val Ile Ala Gly Leu Val
Val Ile Ph #e Met Met Leu Gly Gly 650 # 655 # 660 ACT TTT CTC TAC
TGG CGT GGG CGC CGG ATT CA #G AAT AAA AGG GCT ATG 2130 Thr Phe Leu
Tyr Trp Arg Gly Arg Arg Ile Gl #n Asn Lys Arg Ala Met 665 # 670 #
675 AGG CGA TAC TTG GAA CGG GGT GAG AGC ATA GA #G CCT CTG GAC CCC
AGT 2178 Arg Arg Tyr Leu Glu Arg Gly Glu Ser Ile Gl #u Pro Leu Asp
Pro Ser 680 # 685 # 690 GAG AAG GCT AAC AAA GTC TTG GCC AGA ATC TT
#C AAA GAG ACA GAG CTA 2226 Glu Lys Ala Asn Lys Val Leu Ala Arg Ile
Ph #e Lys Glu Thr Glu Leu 695 # 700 # 705 AGG AAG CTT AAA GTG CTT
GGC TCG GGT GTC TT #T GGA ACT GTG CAC AAA 2274 Arg Lys Leu Lys Val
Leu Gly Ser Gly Val Ph #e Gly Thr Val His Lys 710 7 #15 7 #20 7 #25
GGA GTG TGG ATC CCT GAG GGT GAA TCA ATC AA #G ATT CCA GTC TGC ATT
2322 Gly Val Trp Ile Pro Glu Gly Glu Ser Ile Ly #s Ile Pro Val Cys
Ile 730 # 735 # 740 AAA GTC ATT GAG GAC AAG AGT GGA CGG CAG AG #T
TTT CAA GCT GTG ACA 2370 Lys Val Ile Glu Asp Lys Ser Gly Arg Gln Se
#r Phe Gln Ala Val Thr 745 # 750 # 755 GAT CAT ATG CTG GCC ATT GGC
AGC CTG GAC CA #T GCC CAC ATT GTA AGG 2418 Asp His Met Leu Ala Ile
Gly Ser Leu Asp Hi #s Ala His Ile Val Arg 760 # 765 # 770 CTG CTG
GGA CTA TGC CCA GGG TCA TCT CTG CA #G CTT GTC ACT CAA TAT 2466 Leu
Leu Gly Leu Cys Pro Gly Ser Ser Leu Gl #n Leu Val Thr Gln Tyr 775 #
780 # 785 TTG CCT CTG GGT TCT CTG CTG GAT CAT GTG AG #A CAA CAC CGG
GGG GCA 2514 Leu Pro Leu Gly Ser Leu Leu Asp His Val Ar #g Gln His
Arg Gly Ala 790 7 #95 8 #00 8 #05 CTG GGG CCA CAG CTG CTG CTC AAC
TGG GGA GT #A CAA ATT GCC AAG GGA 2562 Leu Gly Pro Gln Leu Leu Leu
Asn Trp Gly Va #l Gln Ile Ala Lys Gly 810 # 815 # 820 ATG TAC TAC
CTT GAG GAA CAT GGT ATG GTG CA #T AGA AAC CTG GCT GCC 2610 Met Tyr
Tyr Leu Glu Glu His Gly Met Val Hi #s Arg Asn Leu Ala Ala 825 # 830
# 835 CGA AAC GTG CTA CTC AAG TCA CCC AGT CAG GT #T CAG GTG GCA GAT
TTT 2658 Arg Asn Val Leu Leu Lys Ser Pro Ser Gln Va #l Gln Val Ala
Asp Phe 840 # 845 # 850 GGT GTG GCT GAC CTG CTG CCT CCT GAT GAT AA
#G CAG CTG CTA TAC AGT 2706 Gly Val Ala Asp Leu Leu Pro Pro Asp Asp
Ly #s Gln Leu Leu Tyr Ser 855 # 860 # 865 GAG GCC AAG ACT CCA ATT
AAG TGG ATG GCC CT #T GAG AGT ATC CAC TTT 2754 Glu Ala Lys Thr Pro
Ile Lys Trp Met Ala Le #u Glu Ser Ile His Phe 870 8 #75 8 #80 8 #85
GGG AAA TAC ACA CAC CAG AGT GAT GTC TGG AG #C TAT GGT GTG ACA GTT
2802 Gly Lys Tyr Thr His Gln Ser Asp Val Trp Se #r Tyr Gly Val Thr
Val 890 # 895 # 900 TGG GAG TTG ATG ACC TTC GGG GCA GAG CCC TA #T
GCA GGG CTA CGA TTG 2850 Trp Glu Leu Met Thr Phe Gly Ala Glu Pro Ty
#r Ala Gly Leu Arg Leu 905 # 910 # 915 GCT GAA GTA CCA GAC CTG CTA
GAG AAG GGG GA #G CGG TTG GCA CAG CCC 2898 Ala Glu Val Pro Asp Leu
Leu Glu Lys Gly Gl #u Arg Leu Ala Gln Pro 920 # 925 # 930 CAG ATC
TGC ACA ATT GAT GTC TAC ATG GTG AT #G GTC AAG TGT TGG ATG 2946 Gln
Ile Cys Thr Ile Asp Val Tyr Met Val Me #t Val Lys Cys Trp Met 935 #
940 # 945 ATT GAT GAG AAC ATT CGC CCA ACC TTT AAA GA #A CTA GCC AAT
GAG TTC 2994 Ile Asp Glu Asn Ile Arg Pro Thr Phe Lys Gl #u Leu Ala
Asn Glu Phe 950 9 #55 9 #60 9 #65 ACC AGG ATG GCC CGA GAC CCA CCA
CGG TAT CT #G GTC ATA AAG AGA GAG 3042 Thr Arg Met Ala Arg Asp Pro
Pro Arg Tyr Le #u Val Ile Lys Arg Glu 970 # 975 # 980 AGT GGG CCT
GGA ATA GCC CCT GGG CCA GAG CC #C CAT GGT CTG ACA AAC 3090 Ser Gly
Pro Gly Ile Ala Pro Gly Pro Glu Pr #o His Gly Leu Thr Asn 985 # 990
# 995 AAG AAG CTA GAG GAA GTA GAG CTG GAG CCA GA #A CTA GAC CTA GAC
CTA 3138 Lys Lys Leu Glu Glu Val Glu Leu Glu Pro Gl #u Leu Asp Leu
Asp Leu 1000 # 1005 # 1010 GAC TTG GAA GCA GAG GAG GAC AAC CTG GCA
AC #C ACC ACA CTG GGC TCC 3186 Asp Leu Glu Ala Glu Glu Asp Asn Leu
Ala Th #r Thr Thr Leu Gly Ser 1015 # 1020 # 1025 GCC CTC AGC CTA
CCA GTT GGA ACA CTT AAT CG #G CCA CGT GGG AGC CAG 3234 Ala Leu Ser
Leu Pro Val Gly Thr Leu Asn Ar #g Pro Arg Gly Ser Gln 1030 1035 #
1040 # 1045 AGC CTT TTA AGT CCA TCA TCT GGA TAC ATG CC #C ATG AAC
CAG GGT AAT 3282 Ser Leu Leu Ser Pro Ser Ser Gly Tyr Met Pr #o Met
Asn Gln Gly Asn 1050 # 1055 # 1060 CTT GGG GAG TCT TGC CAG GAG TCT
GCA GTT TC #T GGG AGC AGT GAA CGG 3330 Leu Gly Glu Ser Cys Gln Glu
Ser Ala Val Se #r Gly Ser Ser Glu Arg 1065 # 1070 # 1075 TGC CCC
CGT CCA GTC TCT CTA CAC CCA ATG CC #A CGG GGA TGC CTG GCA 3378 Cys
Pro Arg Pro Val Ser Leu His Pro Met Pr #o Arg Gly Cys Leu Ala 1080
# 1085 # 1090 TCA GAG TCA TCA GAG GGG CAT GTA ACA GGC TC #T GAG GCT
GAG CTC CAG 3426 Ser Glu Ser Ser Glu Gly His Val Thr Gly Se #r Glu
Ala Glu Leu Gln 1095 # 1100 # 1105 GAG AAA GTG TCA ATG TGT AGA AGC
CGG AGC AG #G AGC CGG AGC CCA CGG 3474 Glu Lys Val Ser Met Cys Arg
Ser Arg Ser Ar #g Ser Arg Ser Pro Arg 1110 1115 # 1120 # 1125 CCA
CGC GGA GAT AGC GCC TAC CAT TCC CAG CG #C CAC AGT CTG CTG ACT 3522
Pro Arg Gly Asp Ser Ala Tyr His Ser Gln Ar #g His Ser Leu Leu Thr
1130 # 1135 # 1140 CCT GTT ACC CCA CTC TCC CCA CCC GGG TTA GA #G
GAA GAG GAT GTC AAC 3570 Pro Val Thr Pro Leu Ser Pro Pro Gly Leu Gl
#u Glu Glu Asp Val Asn 1145 # 1150 # 1155 GGT TAT GTC ATG CCA GAT
ACA CAC CTC AAA GG #T ACT CCC TCC TCC CGG 3618 Gly Tyr Val Met Pro
Asp Thr His Leu Lys Gl #y Thr Pro Ser Ser Arg 1160 # 1165 # 1170
GAA GGC ACC CTT TCT TCA GTG GGT CTT AGT TC #T GTC CTG GGT ACT GAA
3666 Glu Gly Thr Leu Ser Ser Val Gly Leu Ser Se #r Val Leu Gly Thr
Glu 1175 # 1180 # 1185 GAA GAA GAT GAA GAT GAG GAG TAT GAA TAC AT
#G AAC CGG AGG AGA AGG 3714 Glu Glu Asp Glu Asp Glu Glu Tyr Glu Tyr
Me #t Asn Arg Arg Arg Arg 1190 1195 # 1200 # 1205 CAC AGT CCA CCT
CAT CCC CCT AGG CCA AGT TC #C CTT GAG GAG CTG GGT 3762 His Ser Pro
Pro His Pro Pro Arg Pro Ser Se #r Leu Glu Glu Leu Gly 1210 # 1215 #
1220 TAT GAG TAC ATG GAT GTG GGG TCA GAC CTC AG #T GCC TCT CTG GGC
AGC 3810 Tyr Glu Tyr Met Asp Val Gly Ser Asp Leu Se #r Ala Ser Leu
Gly Ser 1225 # 1230 # 1235 ACA CAG AGT TGC CCA CTC CAC CCT GTA CCC
AT #C ATG CCC ACT GCA GGC 3858 Thr Gln Ser Cys Pro Leu His Pro Val
Pro Il #e Met Pro Thr Ala Gly 1240 # 1245 # 1250 ACA ACT CCA GAT
GAA GAC TAT GAA TAT ATG AA #T CGG CAA CGA GAT GGA 3906 Thr Thr Pro
Asp Glu Asp Tyr Glu Tyr Met As #n Arg Gln Arg Asp Gly 1255 # 1260 #
1265 GGT GGT CCT GGG GGT GAT TAT GCA GCC ATG GG #G GCC TGC CCA GCA
TCT 3954 Gly Gly Pro Gly Gly Asp Tyr Ala Ala Met Gl #y Ala Cys Pro
Ala Ser 1270 1275 # 1280 # 1285 GAG CAA GGG TAT GAA GAG ATG AGA GCT
TTT CA #G GGG CCT GGA CAT CAG 4002 Glu Gln Gly Tyr Glu Glu Met Arg
Ala Phe Gl #n Gly Pro Gly His Gln 1290 # 1295 # 1300 GCC CCC CAT
GTC CAT TAT GCC CGC CTA AAA AC #T CTA CGT AGC TTA GAG 4050 Ala Pro
His Val His Tyr Ala Arg Leu Lys Th #r Leu Arg Ser Leu Glu 1305 #
1310 # 1315 GCT ACA GAC TCT GCC TTT GAT AAC CCT GAT TA #C TGG CAT
AGC AGG CTT 4098 Ala Thr Asp Ser Ala Phe Asp Asn Pro Asp Ty #r Trp
His Ser Arg Leu 1320 # 1325 # 1330 TTC CCC AAG GCT AAT GCC CAG AGA
ACG TAACTCCTG #C TCCCTGTGGC 4145 Phe Pro Lys Ala Asn Ala Gln Arg
Thr 1335 # 1340 ACTCAGGGAG CATTTAATGG CAGCTAGTGC CTTTAGAGGG
TACCGTCTTC TC #CCTATTCC 4205 CTCTCTCTCC CAGGTCCCAG CCCCTTTTCC
CCAGTCCCAG ACAATTCCAT TC #AATCTTTG 4265 GAGGCTTTTA AACATTTTGA
CACAAAATTC TTATGGTATG TAGCCAGCTG TG #CACTTTCT 4325 TCTCTTTCCC
AACCCCAGGA AAGGTTTTCC TTATTTTGTG TGCTTTCCCA GT #CCCATTCC 4385
TCAGCTTCTT CACAGGCACT CCTGGAGATA TGAAGGATTA CTCTCCATAT CC #CTTCCTCT
4445 CAGGCTCTTG ACTACTTGGA ACTAGGCTCT TATGTGTGCC TTTGTTTCCC AT
#CAGACTGT 4505 CAAGAAGAGG AAAGGGAGGA AACCTAGCAG AGGAAAGTGT
AATTTTGGTT TA #TGACTCTT 4565 AACCCCCTAG AAAGACAGAA GCTTAAAATC
TGTGAAGAAA GAGGTTAGGA GT #AGATATTG 4625 ATTACTATCA TAATTCAGCA
CTTAACTATG AGCCAGGCAT CATACTAAAC TT #CACCTACA 4685 TTATCTCACT
TAGTCCTTTA TCATCCTTAA AACAATTCTG TGACATACAT AT #TATCTCAT 4745
TTTACACAAA GGGAAGTCGG GCATGGTGGC TCATGCCTGT AATCTCAGCA CT #TTGGGAGG
4805 CTGAGGCAGA AGGATTACCT GAGGCAAGGA GTTTGAGACC AGCTTAGCCA AC
#ATAGTAAG 4865 ACCCCCATCT CTTTAAAAAA AAAAAAAAAA AAAAAAAAAA # # 4905
(2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 1342 amino #acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:
#4: Met Arg Ala Asn Asp Ala Leu Gln Val Leu Gl #y Leu Leu Phe Ser
Leu 1 5 # 10 # 15 Ala Arg Gly Ser Glu Val Gly Asn Ser Gln Al #a Val
Cys Pro Gly Thr 20 # 25 # 30 Leu Asn Gly Leu Ser Val Thr Gly Asp
Ala Gl #u Asn Gln Tyr Gln Thr 35 # 40 # 45 Leu Tyr Lys Leu Tyr Glu
Arg Cys Glu Val Va #l Met Gly Asn Leu Glu 50 # 55 # 60 Ile Val Leu
Thr Gly His Asn Ala Asp Leu Se #r Phe Leu Gln Trp Ile 65 #70 #75
#80 Arg Glu Val Thr Gly Tyr Val Leu Val Ala Me #t Asn Glu Phe Ser
Thr 85 # 90 # 95 Leu Pro Leu Pro Asn Leu Arg Val Val Arg Gl #y Thr
Gln Val Tyr Asp 100 # 105 # 110 Gly Lys Phe Ala Ile Phe Val Met Leu
Asn Ty #r Asn Thr Asn Ser Ser 115 # 120 # 125 His Ala Leu Arg Gln
Leu Arg Leu Thr Gln Le #u Thr Glu Ile Leu Ser 130 # 135 # 140 Gly
Gly Val Tyr Ile Glu Lys Asn Asp Lys Le #u Cys His Met Asp Thr 145 1
#50 1 #55 1 #60 Ile Asp Trp Arg Asp Ile Val Arg Asp Arg As #p Ala
Glu Ile Val Val 165 # 170 # 175 Lys Asp Asn Gly Arg Ser Cys Pro Pro
Cys Hi #s Glu Val Cys Lys Gly 180 # 185 # 190 Arg Cys Trp Gly Pro
Gly Ser Glu Asp Cys Gl #n Thr Leu Thr Lys Thr 195 # 200 # 205 Ile
Cys Ala Pro Gln Cys Asn Gly His Cys Ph #e Gly Pro Asn Pro Asn 210 #
215 # 220 Gln Cys Cys His Asp Glu Cys Ala Gly Gly Cy #s Ser Gly Pro
Gln Asp 225 2 #30 2 #35 2 #40 Thr Asp Cys Phe Ala Cys Arg His Phe
Asn As #p Ser Gly Ala Cys Val 245 # 250 # 255 Pro Arg Cys Pro Gln
Pro Leu Val Tyr Asn Ly #s Leu Thr Phe Gln Leu 260 # 265 # 270 Glu
Pro Asn Pro His Thr Lys Tyr Gln Tyr Gl #y Gly Val Cys Val Ala 275 #
280 # 285 Ser Cys Pro His Asn Phe Val Val Asp Gln Th #r Ser Cys Val
Arg Ala 290 # 295 # 300 Cys Pro Pro Asp Lys Met Glu Val Asp Lys As
#n Gly Leu Lys Met Cys 305 3 #10 3 #15 3 #20 Glu Pro Cys Gly Gly
Leu Cys Pro Lys Ala Cy #s Glu Gly Thr Gly Ser 325 # 330 # 335 Gly
Ser Arg Phe Gln Thr Val Asp Ser Ser As #n Ile Asp Gly Phe Val 340 #
345 # 350 Asn Cys Thr Lys Ile Leu Gly Asn Leu Asp Ph #e Leu Ile Thr
Gly Leu 355 # 360
# 365 Asn Gly Asp Pro Trp His Lys Ile Pro Ala Le #u Asp Pro Glu Lys
Leu 370 # 375 # 380 Asn Val Phe Arg Thr Val Arg Glu Ile Thr Gl #y
Tyr Leu Asn Ile Gln 385 3 #90 3 #95 4 #00 Ser Trp Pro Pro His Met
His Asn Phe Ser Va #l Phe Ser Asn Leu Thr 405 # 410 # 415 Thr Ile
Gly Gly Arg Ser Leu Tyr Asn Arg Gl #y Phe Ser Leu Leu Ile 420 # 425
# 430 Met Lys Asn Leu Asn Val Thr Ser Leu Gly Ph #e Arg Ser Leu Lys
Glu 435 # 440 # 445 Ile Ser Ala Gly Arg Ile Tyr Ile Ser Ala As #n
Arg Gln Leu Cys Tyr 450 # 455 # 460 His His Ser Leu Asn Trp Thr Lys
Val Leu Ar #g Gly Pro Thr Glu Glu 465 4 #70 4 #75 4 #80 Arg Leu Asp
Ile Lys His Asn Arg Pro Arg Ar #g Asp Cys Val Ala Glu 485 # 490 #
495 Gly Lys Val Cys Asp Pro Leu Cys Ser Ser Gl #y Gly Cys Trp Gly
Pro 500 # 505 # 510 Gly Pro Gly Gln Cys Leu Ser Cys Arg Asn Ty #r
Ser Arg Gly Gly Val 515 # 520 # 525 Cys Val Thr His Cys Asn Phe Leu
Asn Gly Gl #u Pro Arg Glu Phe Ala 530 # 535 # 540 His Glu Ala Glu
Cys Phe Ser Cys His Pro Gl #u Cys Gln Pro Met Glu 545 5 #50 5 #55 5
#60 Gly Thr Ala Thr Cys Asn Gly Ser Gly Ser As #p Thr Cys Ala Gln
Cys 565 # 570 # 575 Ala His Phe Arg Asp Gly Pro His Cys Val Se #r
Ser Cys Pro His Gly 580 # 585 # 590 Val Leu Gly Ala Lys Gly Pro Ile
Tyr Lys Ty #r Pro Asp Val Gln Asn 595 # 600 # 605 Glu Cys Arg Pro
Cys His Glu Asn Cys Thr Gl #n Gly Cys Lys Gly Pro 610 # 615 # 620
Glu Leu Gln Asp Cys Leu Gly Gln Thr Leu Va #l Leu Ile Gly Lys Thr
625 6 #30 6 #35 6 #40 His Leu Thr Met Ala Leu Thr Val Ile Ala Gl #y
Leu Val Val Ile Phe 645 # 650 # 655 Met Met Leu Gly Gly Thr Phe Leu
Tyr Trp Ar #g Gly Arg Arg Ile Gln 660 # 665 # 670 Asn Lys Arg Ala
Met Arg Arg Tyr Leu Glu Ar #g Gly Glu Ser Ile Glu 675 # 680 # 685
Pro Leu Asp Pro Ser Glu Lys Ala Asn Lys Va #l Leu Ala Arg Ile Phe
690 # 695 # 700 Lys Glu Thr Glu Leu Arg Lys Leu Lys Val Le #u Gly
Ser Gly Val Phe 705 7 #10 7 #15 7 #20 Gly Thr Val His Lys Gly Val
Trp Ile Pro Gl #u Gly Glu Ser Ile Lys 725 # 730 # 735 Ile Pro Val
Cys Ile Lys Val Ile Glu Asp Ly #s Ser Gly Arg Gln Ser 740 # 745 #
750 Phe Gln Ala Val Thr Asp His Met Leu Ala Il #e Gly Ser Leu Asp
His 755 # 760 # 765 Ala His Ile Val Arg Leu Leu Gly Leu Cys Pr #o
Gly Ser Ser Leu Gln 770 # 775 # 780 Leu Val Thr Gln Tyr Leu Pro Leu
Gly Ser Le #u Leu Asp His Val Arg 785 7 #90 7 #95 8 #00 Gln His Arg
Gly Ala Leu Gly Pro Gln Leu Le #u Leu Asn Trp Gly Val 805 # 810 #
815 Gln Ile Ala Lys Gly Met Tyr Tyr Leu Glu Gl #u His Gly Met Val
His 820 # 825 # 830 Arg Asn Leu Ala Ala Arg Asn Val Leu Leu Ly #s
Ser Pro Ser Gln Val 835 # 840 # 845 Gln Val Ala Asp Phe Gly Val Ala
Asp Leu Le #u Pro Pro Asp Asp Lys 850 # 855 # 860 Gln Leu Leu Tyr
Ser Glu Ala Lys Thr Pro Il #e Lys Trp Met Ala Leu 865 8 #70 8 #75 8
#80 Glu Ser Ile His Phe Gly Lys Tyr Thr His Gl #n Ser Asp Val Trp
Ser 885 # 890 # 895 Tyr Gly Val Thr Val Trp Glu Leu Met Thr Ph #e
Gly Ala Glu Pro Tyr 900 # 905 # 910 Ala Gly Leu Arg Leu Ala Glu Val
Pro Asp Le #u Leu Glu Lys Gly Glu 915 # 920 # 925 Arg Leu Ala Gln
Pro Gln Ile Cys Thr Ile As #p Val Tyr Met Val Met 930 # 935 # 940
Val Lys Cys Trp Met Ile Asp Glu Asn Ile Ar #g Pro Thr Phe Lys Glu
945 9 #50 9 #55 9 #60 Leu Ala Asn Glu Phe Thr Arg Met Ala Arg As #p
Pro Pro Arg Tyr Leu 965 # 970 # 975 Val Ile Lys Arg Glu Ser Gly Pro
Gly Ile Al #a Pro Gly Pro Glu Pro 980 # 985 # 990 His Gly Leu Thr
Asn Lys Lys Leu Glu Glu Va #l Glu Leu Glu Pro Glu 995 # 1000 # 1005
Leu Asp Leu Asp Leu Asp Leu Glu Ala Glu Gl #u Asp Asn Leu Ala Thr
1010 # 1015 # 1020 Thr Thr Leu Gly Ser Ala Leu Ser Leu Pro Va #l
Gly Thr Leu Asn Arg 1025 1030 # 1035 # 1040 Pro Arg Gly Ser Gln Ser
Leu Leu Ser Pro Se #r Ser Gly Tyr Met Pro 1045 # 1050 # 1055 Met
Asn Gln Gly Asn Leu Gly Glu Ser Cys Gl #n Glu Ser Ala Val Ser 1060
# 1065 # 1070 Gly Ser Ser Glu Arg Cys Pro Arg Pro Val Se #r Leu His
Pro Met Pro 1075 # 1080 # 1085 Arg Gly Cys Leu Ala Ser Glu Ser Ser
Glu Gl #y His Val Thr Gly Ser 1090 # 1095 # 1100 Glu Ala Glu Leu
Gln Glu Lys Val Ser Met Cy #s Arg Ser Arg Ser Arg 1105 1110 # 1115
# 1120 Ser Arg Ser Pro Arg Pro Arg Gly Asp Ser Al #a Tyr His Ser
Gln Arg 1125 # 1130 # 1135 His Ser Leu Leu Thr Pro Val Thr Pro Leu
Se #r Pro Pro Gly Leu Glu 1140 # 1145 # 1150 Glu Glu Asp Val Asn
Gly Tyr Val Met Pro As #p Thr His Leu Lys Gly 1155 # 1160 # 1165
Thr Pro Ser Ser Arg Glu Gly Thr Leu Ser Se #r Val Gly Leu Ser Ser
1170 # 1175 # 1180 Val Leu Gly Thr Glu Glu Glu Asp Glu Asp Gl #u
Glu Tyr Glu Tyr Met 1185 1190 # 1195 # 1200 Asn Arg Arg Arg Arg His
Ser Pro Pro His Pr #o Pro Arg Pro Ser Ser 1205 # 1210 # 1215 Leu
Glu Glu Leu Gly Tyr Glu Tyr Met Asp Va #l Gly Ser Asp Leu Ser 1220
# 1225 # 1230 Ala Ser Leu Gly Ser Thr Gln Ser Cys Pro Le #u His Pro
Val Pro Ile 1235 # 1240 # 1245 Met Pro Thr Ala Gly Thr Thr Pro Asp
Glu As #p Tyr Glu Tyr Met Asn 1250 # 1255 # 1260 Arg Gln Arg Asp
Gly Gly Gly Pro Gly Gly As #p Tyr Ala Ala Met Gly 1265 1270 # 1275
# 1280 Ala Cys Pro Ala Ser Glu Gln Gly Tyr Glu Gl #u Met Arg Ala
Phe Gln 1285 # 1290 # 1295 Gly Pro Gly His Gln Ala Pro His Val His
Ty #r Ala Arg Leu Lys Thr 1300 # 1305 # 1310 Leu Arg Ser Leu Glu
Ala Thr Asp Ser Ala Ph #e Asp Asn Pro Asp Tyr 1315 # 1320 # 1325
Trp His Ser Arg Leu Phe Pro Lys Ala Asn Al #a Gln Arg Thr 1330 #
1335 # 1340 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 15 amino #acids (B) TYPE: amino acid
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE:
internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: #5: Glu Asp Glu Asp
Glu Glu Tyr Glu Tyr Met As #n Arg Arg Arg Arg 1 5 # 10 # 15 (2)
INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 15 amino #acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: #6: Thr Thr Pro Asp Glu Asp Tyr
Glu Tyr Met As #n Arg Gln Arg Asp 1 5 # 10 # 15 (2) INFORMATION FOR
SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino
#acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE
TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE
DESCRIPTION: SEQ ID NO: #7: Thr Glu Glu Arg Leu Asp Ile Lys His Asn
Ar #g Pro Arg Arg Asp 1 5 # 10 # 15 (2) INFORMATION FOR SEQ ID NO:
8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino #acids (B)
TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:
#8: Arg Ser Arg Ser Arg Ser Arg Ser Pro Arg Pr #o Arg Gly Asp Ser 1
5 # 10 # 15 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 15 amino #acids (B) TYPE: amino acid
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE:
internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: #9: Tyr Met Asn Arg
Arg Arg Arg His Ser Pro Pr #o His Pro Pro Arg 1 5 # 10 # 15 (2)
INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 14 base #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: #10: GATCTCGAGT CGAC # # # 14 (2)
INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 14 base #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: #11: GATCGTCGAC TCGA # # # 14 (2)
INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 5 amino #acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: #12: Tyr Glu Tyr Met Asn 1 5
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