U.S. patent application number 10/100217 was filed with the patent office on 2003-09-25 for methods of detection and treatment of breast cancer.
This patent application is currently assigned to Beth Israel Deaconess Medical Center. Invention is credited to Avraham, Hava, Groopman, Jerome E..
Application Number | 20030181404 10/100217 |
Document ID | / |
Family ID | 26711902 |
Filed Date | 2003-09-25 |
United States Patent
Application |
20030181404 |
Kind Code |
A1 |
Avraham, Hava ; et
al. |
September 25, 2003 |
Methods of detection and treatment of breast cancer
Abstract
Novel methods of detecting and treating breast cancer are
described.
Inventors: |
Avraham, Hava; (Brookline,
MA) ; Groopman, Jerome E.; (Brookline, MA) |
Correspondence
Address: |
HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
530 VIRGINIA ROAD
P.O. BOX 9133
CONCORD
MA
01742-9133
US
|
Assignee: |
Beth Israel Deaconess Medical
Center
Boston
MA
|
Family ID: |
26711902 |
Appl. No.: |
10/100217 |
Filed: |
March 14, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10100217 |
Mar 14, 2002 |
|
|
|
09315928 |
May 20, 1999 |
|
|
|
6368796 |
|
|
|
|
09315928 |
May 20, 1999 |
|
|
|
08876882 |
Jun 16, 1997 |
|
|
|
5981201 |
|
|
|
|
60035228 |
Jan 8, 1997 |
|
|
|
Current U.S.
Class: |
514/44R ;
424/450; 424/94.5; 435/6.14 |
Current CPC
Class: |
C07K 14/71 20130101;
A61K 38/45 20130101; C12N 2799/023 20130101; G01N 33/573 20130101;
C12N 9/1205 20130101; G01N 33/57415 20130101; C12N 2799/026
20130101; A61K 48/00 20130101 |
Class at
Publication: |
514/44 ;
424/94.5; 424/450; 435/6 |
International
Class: |
A61K 048/00; A61K
038/51; C12Q 001/68; A61K 009/127 |
Goverment Interests
[0002] This invention described herein was supported in whole or
part by the National Institutes of Health Grant No. HL51456-02. The
United States Government has certain rights in this invention.
Claims
What is claimed is:
1. A method of inhibiting breast cancer cell growth comprising
supplying Csk Homologous Kinase to breast cells whereby breast
cancer cell growth is inhibited.
2. The method of claim 1, wherein a nucleic acid sequence encoding
Csk Homologous Kinase, or a biologically active fragment, analog or
derivative thereof, is supplied to the breast cells, wherein the
nucleic acid sequence encoding Csk Homologous Kinase or a
biologically active fragment, analog or derivation thereof, is
expressed in the breast cell.
3. The method of claim 2 wherein Csk Homologous Kinase is supplied
to the cell by a nucleic acid construct containing a nucleic acid
sequence encoding Csk Homologous Kinase or a biologically active
fragment, analog or derivative thereof, wherein the construct is
introduced into the breast cell and Csk Homologous Kinase is
expressed.
4. The method of claim 1, wherein Csk Homologous Kinase protein or
a biologically active Csk Homologous Kinase peptide is supplied to
breast cells.
5. The method of claim 4, wherein the Csk Homologous Kinase protein
or peptide is supplied to the cell by introducing into the cell a
liposome containing the Csk Homologous Kinase or Csk Homologous
Kinase peptide.
6. The method of claim 5, wherein the liposome is contained in a
topical formulation and is introduced to the breast cells by
topical application.
7. A Csk Homologous Kinase derivative produced by altering native
or variant Csk Homologous Kinase DNA within the Csk Homologous
Kinase SH2 domain to optimize its association with ErbB-2.
8. The derivative of claim 7, wherein the mutation is a
substitution of one or more amino acids between amino acid 127 and
150 of SEQ ID NO: 1.
9. The derivative of claim 7, wherein the mutation is in an amino
acid residue conserved between csk and c-src, but not conserved
between csk and Csk Homologous Kinase.
10. The derivative of claim 9, wherein the mutation is a
substitution of amino acid 129 of SEQ ID NO: 1.
11. The derivative of claim 9, wherein the mutation is a
substitution from said residue to the corresponding residue
conserved in csk and c-src.
12. A derivative of Csk Homologous Kinase comprising a mutation
which is a substitution of one or more amino acids between amino
acid 127 and 150 of SEQ ID NO: 1.
13. The derivative of claim 12, wherein the mutation is in an amino
acid residue conserved between csk and c-src, but not conserved
between csk and Csk Homologous Kinase.
14. The derivative of claim 12, wherein the mutation is a
substitution of amino acid 129 of SEQ ID NO: 1.
15. The derivative of claim 12, wherein the mutation is a
substitution from said residue to the corresponding residue
conserved in csk and c-src.
16. The derivative of claim 12, wherein the mutation is in an amino
acid residue conserved between csk, c-src and Csk Homologous
Kinase.
17. The derivative of claim 12 wherein the mutation is a
substitution of amino acid 147 of SEQ ID NO: 1
18. A Csk Homologous Kinase derivative produced by altering native
or variant Csk Homologous Kinase DNA within the Csk Homologous
Kinase SH2 domain to inhibit its association with ErbB-2.
19. A CHK analog that mimics the binding of the derivative of claim
7.
20. A CHK analog that mimics the binding of the derivative of claim
12.
21. A pharmaceutical composition that contains an effective amount
of the CHK derivative of claim 7.
22. A pharmaceutical composition that contains an effective amount
of the CHK derivative of claim 12.
23. A method of identifying a biologically active CHK analog with
greater biological activity than native CHK, comprising: a.
contacting ErbB-2 with the CHK analog to be tested for biological
activity, under conditions sufficient for binding of ErbB-2 by the
CHK analog; b. contacting ErbB-2 with native CHK, under conditions
sufficient for binding of ErbB-2 by the native CHK; and c.
comparing the binding of ErbB-2 by the CHK analog with the binding
of ErB-2 by the native CHK.
24. The method of claim 23, wherein the CHK analog is the analog of
claim 19.
25. The method of claim 23, wherein the CHK analog is an analog of
claim 20.
26. The CHK analog identified by the method of claim 23.
27. A method of identifying a biologically active CHK derivative
with greater biological activity than native CHK, comprising: a.
contacting ErbB-2 with the CHK derivative to be tested for
biological activity, under conditions sufficient for binding of
ErbB-2 by the CHK derivative; b. contacting ErbB-2 with native CHK,
under conditions sufficient for binding of ErbB-2 by the native
CHK; and c. comparing the binding of ErbB-2 by the CHK derivative
with the binding of ErB-2 by the native CHK.
28. The method of claim 27, wherein the CHK derivative is the
derivative of claim 7.
29. The method of claim 27, wherein the CHK derivative is the
derivative of claim 12.
30. The CHK derivative identified by the method of claim 27.
31. A CHK analog that binds to ErbB-2 and enhances the activity of
CHK.
32. A pharmaceutical composition that comprises an effective amount
of a CHK analog, wherein the CHK analog binds to ErbB-2 and
enhances the activity of CHK.
Description
RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. Ser. No.:
09/315,928, filed May 20, 1999, which is a Divisional of U.S. Ser.
No.: 08/876,882, filed Jun. 16, 1997, which claims priority to
pending Provisional Application No. 60/035,228, filed Jan. 8, 1997,
the entire teachings of which are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0003] Breast cancer is the second leading cause of cancer death
among women in the United States and is the leading cause of death
among women aged 30-70. (Abeloff, M. D., Curr. Opin. Oncol.,
8:447-448 (1996)). The inheritance of germ-line mutations in
autosomal dominant susceptibility genes appears to be responsible
for 5-10% of all breast cancer cases (Fitzgerald, M. G., et al.,
New Engl. J. Med., 334:143-149 (1996)), and up to 36% of the cases
diagnosed before age 30. BRCA1 was the first isolated breast cancer
susceptibility gene (Langston, A. A., et al., New Engl. J. Med.,
334:137-142 (1996); Couch, F. J. and Weber, B. L., Hum. Mutat.,
8:8-18 (1996)) and mutations in BRCA1 alone account for
approximately 45% of the families with high incidence of breast and
ovarian cancer (Chen, Y. M., et al., Science, 272:125-126 (1996);
Sully, R., et al., Science, 272:123-126 (1996)). In addition, a
second breast cancer susceptibility gene, BRCA2, has been isolated
recently (Wooster, R., et al., Nature, 378:789-792 (1995);
Tavtigian, S. V., et al., Nat. Genet., 12:333-337 (1996)).
[0004] However, the majority of breast carcinomas appear to be
sporadic and have a complex accumulation of molecular and cellular
abnormalities that constitute the malignant phenotype. A number of
somatic gene alterations, such as loss of expression of specific
tumor suppressor genes, have been found to occur in primary human
breast tumors (Borg, A., et al., Cancer Res., 52:2991-2994 (1992);
Eeles, R. A., et al., Cancer Surveys, 25:101-124 (1995)).
Additionally, there is considerable evidence that genetic
alterations in growth factor signaling pathways can contribute to
human breast malignancies. In this regard, activation of different
proto-oncogenes has been found in primary breast tumor (Berns, E.
M., et al., Cancer Res., 52:1036-1039 (1992); Borg, A., et al.,
Brit. J. Cancer, 63:136-142 (1991); Gullick, W. J., et al., Brit.
J. Cancer, 63:434-438 (1991)). Thus, there is considerable
importance in identifying, at a molecular level, factors that
contribute to the progression from normal growth towards
malignancy.
SUMMARY OF THE INVENTION
[0005] The present invention relates to the demonstration that a
cytoplasmic protein tyrosine kinase, Csk Homologous Kinase or CHK,
is expressed in human breast cancer, but not in adjacent normal
breast tissue. Specifically, the present invention relates to
methods of detecting the presence of cancer in mammalian breast
tissue by the detection of the protein tyrosine kinase CHK, or the
detection of nucleic acids encoding the CHK in mammalian tissue,
specifically breast tissue. The detection of CHK in breast tissue
is indicative of cancer.
[0006] The presence of CHK in breast tissue can be determined by
detecting the expression of CHK protein, or a protein fragment, in
breast tissue samples obtained from the mammal. For example, biopsy
tissue can be obtained from the mammal, fixed in a suitable medium
and contacted with anti-CHK antibodies, for example rabbit
anti-CHK, which specifically bind to the CHK protein if it is
present in the tissue sample. The anti-CHK antibody can itself be
detectably labeled, or a detectably labeled second antibody, for
example, peroxidase-conjugated mouse anti-rabbit antibody, can be
used.
[0007] The presence of CHK in breast tissue can also be detected
using an immunoblot (e.g., Northern blot) assay. For example,
tissue can be obtained from the mammal and a cell lysate prepared
which contains proteins released from the tissue cells. The lysate
proteins can be separated by electrophetic means, such as by size
by SDS polyacrylamide gel electrophoresis, and contacted with
anti-CHK antibody which specifically binds to CHK if it is present
in the lysate. Again, the anti-CHK antibody can be detectably
labeled, or a detectably labeled second antibody can be used.
Alternatively, CHK protein present in a cell lysate can be detected
by enzyme linked immunosorbent assay (ELISA), radioimmunoassay
(RIA) or other immunoassays.
[0008] The presence of CHK in breast tissue can also be determined
by detecting the presence of a nucleic acid sequence encoding all,
or a portion, of the CHK protein. The nucleic acid can be DNA or
RNA. For example, genomic DNA, cDNA or RNA can be obtained from a
sample of breast tissue and contacted with a polynucleotide (a
nucleic acid probe) that forms a stable hybrid with the nucleic
acid sequence encoding CHK. The probe can be detectably labeled.
The DNA or RNA obtained from the mammal can be amplified prior to
assay, for example using the polymerase chain reaction (PCR) or the
ligase chain reaction (LCR), using specific nucleic acid primers.
Primers useful to amplify the CHK nucleic acid specifically
hybridize to the CHK nucleic acid or to nucleic acid sequence that
flanks the target CHK nucleic acid sequence region.
[0009] Overexpression of the receptor tyrosine kinase, ErbB-2 (also
termed neu/HER-2) has been associated with the development of
breast cancer. (Slamon, D. J., et al., Science, 244:707-712 (1989);
Olsson, H., et al., J. Natl. Cancer Instit., 83:1483-1487 (1991)).
A common pathway linking the activation mechanisms in ErbB-2
amplification in breast cancer is increased tyrosine kinase
activity which leads to cellular transformation. The abundance of
ErbB-2 receptors and their ligands (e.g., heregulin or HGR) in
breast cancer points to a functional role in the pathogenesis of
this malignancy. As demonstrated herein, CHK specifically interacts
with activated ErbB-2 upon HGR stimulation and results described
herein suggest that CHK functions as a negative regulator of ErbB-2
mediated mitogenic signaling.
[0010] Accordingly, the present invention also encompasses methods
of inhibiting breast cancer cell growth (also referred to herein as
neoplastic cell growth), specifically ErbB-2 mediated neoplastic
cell growth, by supplying CHK to cancer cells. For example, CHK
protein, peptide or a biologically active fragment thereof, or a
CHK analog or derivative, can be supplied to mammalian breast
tissue which is abnormal, e.g., neoplastic, or at risk of becoming
abnormal. The CHK protein can be supplied to the target breast
tissue by introducing into target cells a liposome preparation that
contains CHK. Specifically encompassed by this invention is the
topical application of such liposomes in a cream or ointment.
[0011] Alternatively, CHK can be supplied to the target tissue by
introducing a nucleic acid sequence encoding CHK, or a biologically
active fragment, analog, or derivative of CHK which is then
expressed in the breast tissue.
[0012] As described herein, for the first time, Csk-homologous
Kinase has been identified as playing an important role in
signaling in neoplastic breast tissue and as functioning as a
negative regulator of ErbB-2. As a result of this work, novel
methods of detecting and inhibiting breast cancer are now
available.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIGS. 1A-D depict autoradiographs showing the association of
the CHK-SH2 domain with ErbB-2 upon stimulation with HRG. Lysates
were precipitated with the CHK-SH2 GST fusion protein and
immunoblotted with monoclonal antiphosphotyrosine antibody (PY20),
(A), or with polyclonal anti-ErbB-2 antibodies (B).
Immunoprecipitations of the same lystates were performed using 3E8
monoclonal anti-ErbB-2 antibody and blotted with PY20 (C) or with
anti-ErbB-2 antibodies (D). Molecular size markers are indicated on
the left (kDa).
[0014] FIGS. 2A-B show the nucleotide (SEQ ID NO: 1) and deduced
amino acid sequence (SEQ ID NO: 2) of two overlapping matk cDNA
clones representing the full-length cDNA. Nucleotide numbers are
shown on the left. The putative initiation codon at nucleotide
position 263 is shown in bold type.
[0015] FIG. 3 shows the nucleotide sequence (SEQ ID NO: 3) and
deduced amino acid sequence (SEQ ID NO: 4) of a CHK fragment.
Specific primer positions are indicated.
[0016] FIG. 4 is a graphic representation showing the results of an
in vitro kinase assay of CHK and Csk immunoprecipitates.
DETAILED DESCRIPTION OF THE INVENTION
[0017] The family of protein tyrosine kinases (PTKs) includes
oncogenes and growth factor receptors, several of which have been
linked to the pathogenesis and progression of certain cancers
(Bishop, J. M., Genes. Dev., 9:1309-1315 (1995) Cancer, W. G., et
al., Breast Can. Res. & Treat., 35:105-1114 (1995)). Increasing
evidence indicates that the c-src proto-oncogene may play an
important role in breast cancer. Human breast cancers often show
much higher levels of src protein kinase activity than normal
adjacent epithelium (Hennipman, A., et al., Cancer Research,
49:516-521 (1989), Ottenhoff-Kalff, A. E., et al, Cancer Research,
52:4773-4778 (1992)). Indeed, about 70% of the elevated total
tyrosine kinase activity found in primary breast cancers can be
attributed to increased src activity. Involvement of pp60c-src with
two major signaling pathways in human breast cancer has been
demonstrated. In human breast carcinoma cell lines, the SH2 domain
of src binds to activated epidermal growth factor (EGF-R) and
p185.sup.ErbB-2, a receptor tyrosine kinase (Luttrell, D. K., et
al., Proc. Natl. Acad. Sci. USA, 91:83-87 (1994)).
[0018] Overexpression of the receptor tyrosine kinase ErbB-2 (also
termed neu/HER-2) has been also associated with the development of
breast cancer (Salmon, D. J., et al., Science, 244:707-712 (1989),
Williams, T. M., et al., Pathobiology, 59:45-52 (1991)). A common
pathway linking the activation mechanisms in ErbB-2 amplification
in breast cancer is increased tyrosine kinase activity which leads
to cellular transformation (Olsson, H., et al., J. Natl. Cancer.
Inst., 83:1483-1487 (1991)).
[0019] Four members of the ErbB (HER) family are presently know:
p170.sup.ErbB-1 (epidermal growth factor receptor, EGR-R),
p185.sup.ErbB-2, p180.sup.ErbB-3 and p180.sup.ErbB-4. In
particular, the overexpression of the p185.sup.ErbB-2 correlates
with a poor clinical prognosis in breast cancer (Beerli, R. R., et
al., Mol. Cell. Biol., 15:6496-6505 (1995), Holmes, W. E., et al.,
Science, 256:1205-1210 (1992), Wen, D., et al., Cell, 69:559-572
(1992)). The overall amino acid homology within this receptor
family ranges from 40-50%, and all the family members are
characterized by two cysteine-rich regions in the extracellular
domain, a single transmembrane region and a large cytoplasmic
domain that exhibits tyrosine kinase activity (Wen, D., et al.,
Cell, 69:559-572 (1992)).
[0020] Several ligands that bind to and stimulate the kinase
activity of the ErbB family members have been identified and are
classified as EGF-like ligands. EGF, HB-EGF, amphiregulin,
betacellulin, epiregulin and transforming growth factor-.alpha.
(TGF-.alpha.) are the ligands for the EGF-R (ErbB-1) (Cohen, B. D.,
et al., J. Biol. Chem., 271:4813-4818 (1996), Johnson, G. R., et
al., J. Biol. Chem., 268:2924-2931 (1993)). Heregulin (HRG) and its
rat homologue, neu differentiation factor (NDF), are a subfamily of
neruegulins which are EGF-like ligands that bind to and activate
both ErbB-3 and ErbB-4. Although none of these factors binds
directly to the ErbB-2, both EGF and HRG induce its tyrosine
phosphorylation, presumably by ligand-driven heterodimerization and
cross-phosphorylation. Interestingly, ErbB-2, by heterodimerizing
with the EGF-R and ErbB-3, confers high affinity binding sites for
EGF and HRG, respectively (Beerli, R. R., et al., Mol. Cell. Biol.,
15:6469-6505 (1995), Marchionni, M. A., et al., Nature, 362:312-318
(1993)).
[0021] Recently, a cytoplasmic tyrosine kinase, CHK (Csk Homologous
Kinase), previously referred to as MATK (Megakaryocyte Associate
Tyrosine Kinase), has been identified. The CHK protein, primarily
expressed in hematopoietic cells and in human brain has an apparent
molecular weight of 58 kD, and shares 50% homology with the human
Csk (c-terminal src kinase). Like Csk, CHK contains SH3, SH2 and
tyrosine kinase domains, and lacks the src family N-terminal
myristylation and autophosphorylation sites. CHK was found to
phosphorylate the inhibitory carboxyl-terminal conserved tyrosine
of several src-related enzymes in vitro, including Lck, Fyn and
c-src, and to reduce the elevated phosphotyrosine levels of src
family kinases in Csk-deficient fibroblasts.
[0022] As described herein, for the first time, the interaction of
CHK with ErbB-2 upon the activation of breast cancer cells by HRG
has been demonstrated. This interaction occurred via the SH2 domain
of CHK and was specific to the activated ErbB-2 receptor upon HRG
stimulation. Also described herein for the first time, is the
demonstration that CHK is expressed in human breast cancer cells
but not in adjacent normal breast tissue cells.
[0023] The present invention relates to methods of detecting and
treating breast cancer in mammals wherein the methods encompass the
detection or use of CHK protein, or nucleic acids encoding CHK. As
defined herein, the term CHK protein encompasses the full-length
CHK protein as described in Bennett, B. D., et al., J. Biol. Chem.,
269::1068-10741 (1995), the teachings of which are hereby
incorporated by reference, and also biologically active CHK
fragments, derivatives analogs, variants and mutants.
[0024] The term "biologically active" CHK fragments, derivatives
analogs, variants and mutants is defined herein as the activity
encompassing the specific association of CHK with the intracellular
domain of ErbB-2, or chimeric ErbB-2 molecules, such as EGF-ErbB-2
molecules. As described herein, this association is mediated by the
SH2 domain of CHK. Association of CHK with ErbB-2 can be
demonstrated, for example, using immunoprecipation experiments as
described in the Examples. Because CHK is a tyrosine kinase,
biological activity is also defined herein as the ability of CHK to
phosphorylate tyrosine, specifically the phosphorylation of the
carboxyl-terminal tyrosine of src-related kinases, thereby
repressing their activity. Several src-related kinases include Lck,
Fyn and c-src. Assays that demonstrate the phosphorylation ability
of CHK include immune complex kinase reactions and the ability to
phosphorylate kinases in yeast co-expression systems as described
in (Avraham, S., et al., J. Biol. Chem., 270:1833-1842 (1995),
Chow, L. M., et al., Oncogene, 9:3371-3374 (1994), Klags, S., et
al., Proc. Natl. Acad. Sci. USA, 19:2597-2601 (1994) and Davidson,
D., et al., J. Bil. Chem., 272:1355-1362 (1997)), the teachings of
which are hereby incorporated by reference. Other methods of
measuring kinase activity are known to those of skill in the
art.
[0025] Another biological activity of CHK is the antigenic property
of inducing a specific immunological response as determined using
well-known laboratory techniques. For example, a biologically
active CHK can induce an immunological response which produces
antibodies specific for CHK (anti-CHK antibodies).
[0026] To be "functionally" or "biologically active" a CHK protein
fragment, analog, mutant or derivative typically shares substantial
sequence (amino acid or nucleic acid) identity (e.g., at least
about 65%, typically at least about 80% and most typically about
90-95%) with the corresponding sequences of endogenous, or
naturally occurring, CHK and possesses one or more of the functions
of endogenous CHK thereof. For example, a biologically active CHK
fragment typically shares sequence homology with endogenous CHK
protein in the domains, e.g., tyrosine kinase domain, or SH2
domain, important for biological activity.
[0027] CHK of the present invention is understood to specifically
include CHK proteins having amino acid sequences analogous to the
sequence of the endogenous CHK. Such proteins are defined herein as
CHK analogs. An "analog" is defined herein to mean an amino acid
sequence with sufficient identity to the amino acid sequence of
endogenous CHK protein to possess the biological activity of the
protein. For example, an analog of a polypeptide can be introduced
with "silent" changes in the amino acid sequence wherein one or
more amino acid residues differ form the amino acid sequence of
CHK, yet possess e.g., kinase activity or associates with ErbB-2.
Examples of such differences include additions, deletions or
substitutions of residues. Also encompassed by the present
invention are proteins that exhibit greater or lesser biological
activity of CHK protein.
[0028] The present invention also encompasses biologically active
fragments of CHK protein. Such fragments can include only a part of
the full-length amino acid sequence of CHK yet possess iological
activity. As used herein, a "biologically active fragment" means a
fragment that can exert a biological or physical effect of the
full-length protein, or has a biological characteristic, e.g.,
antigenicity, of the full-length protein. The antigenicity of a
peptide fragment can be determined, for example, as described in
Geysen, et al., WO 84/03564, the teachings of which are herein
incorporated by reference. Such activities and characteristics are
described above. Such fragments can be produced by amino and
carboxyl terminal deletions as well as internal deletions. Also
included are active fragments of the protein as obtained by
enzymatic digestion. Such peptide fragments can be tested for
biological activity as described herein.
[0029] "Derivatives" and "variants" of CHK are CHK proteins that
have been modified. They include CHK proteins that have been
modified by alterations in their amino acid sequence. They also
include truncated and hybrid forms of CHK. "Truncated" forms are
shorter versions of CHK, typically modified so as to remove the
C-terminal regions which effect binding or secretion. "Hybrid" or
"chimeric" forms are CHK proteins that are composed of one or more
CHK proteins combined with one or more other proteins, such as
another kinase.
[0030] Variants can be produced using methods discussed below. The
CHK gene can be mutated in vitro or in vivo using techniques well
known to those of skill in the art, for example, site-specific
mutagenesis and oligonucleotide mutagenesis. Manipulations of the
CHK protein sequence can be made at the protein level as well. Any
of numerous chemical modifications can be carried out by known
techniques including, but not limited to, specific chemical
cleavage by cyanogen bromide, trypsin and papain. It can also be
structurally modified or denatured, for example, by heat or by
being immobilized on a solid surface.
[0031] The amino acid sequences of the CHK proteins of the present
invention can be altered to optimize CHK association with ErbB-2 by
methods known in the art by introducing appropriate nucleotide
changes into native or variant DNA encoding the CHK, or by in vitro
synthesis of the desired CHK. Alterations can be created outside or
within the CHK SH2 domain.
[0032] In general, mutations can be conservative or
non-conservative amino acid substitutions, amino acid insertions or
amino acid deletions. The mutations can be at or near (within 5 or
10 amino acids) the SH2 binding domain. More preferably, DNA
encoding an CHK amino acid sequence variant is prepared by
site-directed mutagenesis of DNA that encodes a variant or a
nonvariant version of CHK. Site-directed (site-specific)
mutagenesis allows the production of CHK variants through the use
of specific oligonucleotide sequences that encode the DNA sequence
of the desired mutation, as well as a sufficient number of adjacent
nucleotides, to provide a primer sequence of sufficient size and
sequence complexity to form a stable duplex on both sides of the
deletion junction being traversed.
[0033] Typically, a primer of about 20 to 25 nucleotides in length
is preferred, with about 5 to 10 residues on both sides of the
junction of the sequence being altered. In general, the techniques
of site-specific mutagenesis are well known in the art, as
exemplified by publications such as Edelman et al., DNA 2, 183
(1983). The site-specific mutagenesis technique typically employs a
phage vector that exists in both a single-stranded and
double-stranded form. Typical vectors useful in site-directed
mutagenesis include vectors such as the M13 phage, for example, as
disclosed by Messing et al., Third Cleveland Symposium on
Macromolecules and Recombinant DNA, A. Walton, ed., Elsevier,
Amsterdam (1981). This and other phage vectors are commercially
available and their use is well-known to those skilled in the art.
A versatile and efficient procedure for the construction of
oligonucleotide directed site-specific mutations in DNA fragments
using M13-derived vectors was published by Zoller, M. J. and Smith,
M., Nucleic Acids Res. 10:6487-6500 (1982)). Also, plasmid vectors
that contain a single-stranded phage origin of replication can be
employed to obtain single-stranded DNA. Veira et al., Meth.
Enzymol. 153:3 (1987). Alternatively, nucleotide substitutions can
be introduced by synthesizing the appropriate DNA fragment in
vitro, and amplifying it by PCR procedures known in the art.
[0034] In general, site-specific mutagenesis can be performed by
first obtaining a single-stranded vector that includes within its
sequence a DNA sequence that encodes the relevant protein. An
oligonucleotide primer bearing the desired mutated sequence is
prepared, generally synthetically, for example, by the method of
Crea et al., Proc. Natl. Acad. Sci. USA 75, 5765 (1978). This
primer can then be annealed with the single-stranded protein
sequence-containing vector, and subjected to DNA-polymerizing
enzymes such as E. coli polymerase I Klenow fragment, to complete
the synthesis of the mutation-bearing strand. Thus, a heteroduplex
is formed wherein one strand encodes the original non-mutated
sequence and the second strand bears the desired mutation. This
heteroduplex vector can then be used to transform appropriate host
cells such as JM 101 cells, and clones can be selected that include
recombinant vectors bearing the mutated sequence arrangement.
Thereafter, the mutated region can be removed and placed in an
appropriate expression vector for protein production.
[0035] The PCR technique can also be used in creating amino acid
sequence variants of an CHK. When small amounts of template DNA are
used as starting material in a PCR, primers that differ slightly in
sequence from the corresponding region in a template DNA can be
used to generate relatively large quantities of a specific DNA
fragment that differs from the template sequence only at the
positions where the primers differ from the template. For
introduction of a mutation into a plasmid DNA, one of the primers
can be designed to overlap the position of the mutation and to
contain the mutation; the sequence of the other primer is
preferably identical to a stretch of sequence of the opposite
strand of the plasmid, but this sequence can be located anywhere
along the plasmid DNA. It is preferred, however, that the sequence
of the second primer is located within 500 nucleotides from that of
the first, such that in the end the entire amplified region of DNA
bounded by the primers can be easily sequenced. PCR amplification
using a primer pair like the one just described results in a
population of DNA fragments that differ at the position of the
mutation specified by the primer, and possibly at other positions,
as template copying is somewhat error-prone.
[0036] If the ratio of template to product material is extremely
low, the vast majority of product DNA fragments incorporate the
desired mutation(s). This product can be used to replace the
corresponding region in the plasmid that served as PCR template
using standard DNA technology. Mutations at separate positions can
be introduced simultaneously by either using a mutant second primer
or performing a second PCR with different mutant primers and
ligating the two resulting PCR fragments simultaneously to the
vector fragment in a three (or more) part ligation.
[0037] Another method for preparing variants, cassette mutagenesis,
is based on the technique described by Wells et al. Gene 34, 315
(1985). The starting material can be the plasmid (or vector)
comprising the CHK DNA to be mutated. The codon(s) within the CHK
to be mutated are identified. There must be unique restriction
endonuclease sites on each side of the identified mutation site(s).
If such restriction sites do not exist, they can be generated using
the above-described oligonucleotide-mediated mutagenesis method to
introduce them at appropriate locations in the CHK DNA. After the
restriction sites have been introduced into the plasmid, the
plasmid is cut at these sites to linearize it. A double stranded
oligonucleotide encoding the sequence of the DNA between the
restriction sites but containing the desired mutation(s) is
synthesized using standard procedures. The two strands are
synthesized separately and then hybridized together using standard
techniques. This double-stranded oligonucleotide is referred to as
the cassette. This cassette is designed to have 3' and 5' ends that
are compatible with the ends of the linearized plasmid, such that
it can be directly ligated to the plasmid. This plasmid now
contains the mutated CHK DNA sequence, that can be expressed to
produce CHK with altered binding activity.
[0038] Specifically encompassed by the present invention are
methods of detecting the presence or absence of CHK protein in
mammalian cells wherein detection of the presence of (e.g., the
expression of) CHK is indicative of breast cancer. A biological
sample to be tested for the presence or absence of CHK protein is
obtained from the mammal. Typically, the sample is breast tissue or
tissue adjacent to the breast. The tissue sample can include lymph
nodes. The sample is typically obtained by biopsy techniques well
known to those of skill in the art.
[0039] CHK protein expression can be detected in a tissue sample by
immunohistochemical techniques as described herein. For example,
the tissue can be imbedded in paraffin or frozen and sectioned in
to thin slices, typically mounted on microscope slides. The tissue
is contacted with an anti-CHK antibody under conditions suitable
for the anti-CHK antibody to specifically bind to CHK present in
the tissue sample as described herein. The anti-CHK antibodies can
be monoclonal or polyclonal. The antibody can be detectably
labeled, for example, with a flourescent dye. Alternatively, a
second antibody that is detectably labeled can be used. For
example, if the first antibody is a mouse anti-CHK antibody, a
second antibody can be detectably labeled rabbit anti-mouse.
Techniques for producing, purifying and labeling antibodies are
well-known to those of skill in the art.
[0040] Expression of CHK protein can also be detected by Western
blot (immunoblot) analysis using anti-CHK antibodies as described
herein. Additionally, the expression of CHK protein can be detected
by immunoprecipitation using anti-CHK antibodies, also as described
herein. Additional techniques suitable for use to detect the
presence of CHK protein includes e.g., immunofluorescence staining,
confocal staining and ELISA when using soluble lysates. Such
techniques are also well known to those of skill in the art.
[0041] Detection of the presence or absence of CHK can also be
accomplished by the detection of the presence or absence of nucleic
acids, either DNA or RNA, encoding the CHK protein in a biological
sample. The biological sample, e.g., breast tissue, can be prepared
in a manner that renders the nucleic acid encoding CHK available
for hybridization with a nucleic acid probe that specifically
hybridizes with a nucleic acid sequence that encodes all, or a
portion, of CHK. For example, Northern blot analysis, or Southern
blot analysis can be used to detect the presence of CHK RNA or DNA
in a biological sample. These techniques are well-known to those of
skill in the art. (See e.g., Sambrook et al. MOLECULAR CLONING: A
LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., (1989) or Ausubel et al., CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, J. Wiley and Sons, New York, N.Y. (1992)). For
example, the standard Southern blot methods includes extracting
genomic DNA from the sample, and digesting the genomic DNA with
suitable restriction enzymes to obtain DNA fragments. The DNA
fragments are then separated by electrophoretic means on e.g.,
agarose gels and transferred to nylon membranes which are exposed
to detectably-labeled probes under conditions sufficient for the
probes to specifically hybridize to nucleic acids encoding CHK.
Detection can be accomplished by, e.g., autoradiography,
spectrometry or fluorometry.
[0042] Nucleic acid probes useful in the present invention comprise
at least about 15 nucleotides, typically about 21 to 45 nucleotides
and most typically about 100 nucleotides. This number of
nucleotides typically provides the minimal length required of a
probe that would specifically hybridize to a CHK-encoding sequence.
The probes are of a specificity and sufficient length to form
stable hybrid duplexes with the target sequence under stringent
conditions. As used herein, stringent conditions are defined as
conditions under which specific hybrid duplexes will be stable and
maintained and under which non-specific hybrid duplexes will be not
be stable (e.g., stable during wash conditions while non-specific
hybrid duplexes will be (eluted during wash conditions). Probes and
conditions useful in the present invention are described in WO
93/15201, entitled "Novel Protein Tyrosine Kinases", the teachings
of which are herein incorporated in their entirety by reference
(Also see FIG. 3, SEQ ID NO: 3, which is a nucleic acid sequence
encoding a CHK peptide). Techniques for identifying probes and
conditions of stringency (e.g., moderate or high) are also
well-known to those of skill in the art and e.g., are described in
Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989) or
Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, J. Wiley
and Sons, New York, N.Y. (1992).
[0043] It is important to note that the nucleotide sequences of
probes that are useful in the present invention need not be fully
complementary to the target sequence. Probes need only be
substantially complementary. As defined herein, substantially
complementary means that the probe sequence is sufficiently similar
in sequence identity to the target sequence that the probe
specifically hybridizes with the target sequence under specified
conditions. For example, non-complementary bases can be
interspersed within the probe sequence, or the probe can be longer
or shorter than the target sequence, provided that the probe still
specifically hybridizes with the target sequence.
[0044] Detection of hybrid duplexes is typically accomplished by
the use of detectably labeled probes. Such labels and methods of
labeling probes are well-known to those of skill in the art. For
example, labels can be radiolabels, chemiluminescent labels,
fluorescent labels, biotin, enzymes or other labels known to those
of skill in the art. Alternatively, the probe can be unlabeled but
detectable by subsequent binding or hybridization to a second,
detectably labeled molecule.
[0045] Detection of nucleic acids encoding CHK can also be
accomplished by amplification techniques which directly amplify the
target nucleic acid present in a sample, for example, by polymerase
chain reaction (PCR) (See e.g., Saiki, et al., Science,
230:1350-1353 (1986) or ligase chain reaction (LCR) (See e.g.,
Weiss, R., Science, 254:1292-1293 (1991)). Such amplification
techniques can also be used a preliminary steps for detection
techniques described above. For example, conditions and specific
primers suitable for use in a PCR method to amplify nucleic acids
encoding CHK are described herein.
[0046] In situ hybridization analysis on tissue samples can also be
used to detect the presence or absence of CHK in a biological
sample. Such techniques are also well-known to those of skill in
the art. (See for example, Sure Site II System.TM. hybridization
kit by NoVagen.)
[0047] The present invention also encompasses the use of the CHK
proteins and nucleic acids encoding these proteins as a basis of
rational drug design to produce biologically active CHK analogs
that have substantially comparable, or lesser or greater biological
activity of CHK. Also encompassed are the use of the CHK proteins
to identify small molecules which interact with CHK and, thus, can
act as agonists, antagonists or inhibitors of CHK activity.
[0048] A further embodiment encompassed by the present invention
includes methods of inhibiting neoplastic (tumor) cell growth by
supplying CHK to cells. Specifically encompassed by the present
invention are methods that inhibit ErbB-2 mediated-breast cancer
cell growth. Cells that are in need of CHK and are supplied with,
or receive CHK protein, are referred to herein as target or
recipient cells. The recipient cells are either substantially
deficient in CHK (e.g., fail to produce an amount of CHK sufficient
to suppress neoplastic growth, or hyperplasia, which is abnormal
growth) or produce adequate amounts of CHK, but the CHK produced is
functionally abnormal (e.g., the CHK lacks biological activity to
suppress neoplastic growth). As defined herein, the term "inhibit"
means either to completely suppress or prevent neoplastic cell
growth or to substantially, or significantly decrease neoplastic or
hypeplastic cell growth. Inhibition or decrease of cancer cell
growth, or hyperplasia, can be measured as described herein, e.g.,
by comparing growth of breast tumor cells that have been supplied
with CHK to breast tumor cells that have not been supplied with
CHK, and by other methods well-known to those of skill in the
art.
[0049] CHK protein, peptide or a biologically active fragment
thereof, or a CHK analog or derivative, can be supplied to
mammalian breast tissue that manifests neoplastic cell growth, or
is at risk of producing neoplastic cell growth e.g., hyperplastic
tissue. CHK can be supplied to (e.g., introduced into) the target
recipient cells by methods well-known to those of skill in the art.
For example, CHK can be introduced into recipient cells by
injection of a pharmaceutical composition that contains an
effective amount of CHK in a physiologically compatible solution,
or by a liposome preparation that contains an effective amount of
CHK. Specifically encompassed by this invention is the topical
application of liposomes in a cream or ointment which contain an
effective amount of CHK. An effective amount of CHK is defined
herein as an amount of CHK which inhibits neoplastic or
hyperplastic cell growth, specifically ErbB-2 mediated breast
cancer cell growth.
[0050] Suitable physiologically acceptable carriers include but are
not limited to water, salt solutions, alcohols, gum arabic,
vegetable oil, benzyl alcohols, polyethylene glycols, gelatine,
carbohydrates such as lactose, amylose or starch, magnesium
stearate, talc, silicic acid. Viscous paraffin, perfume oil, fatty
acid esters, hydroxymethylcellulose, polyvinyl pyrrolidone, etc.
The pharmaceutical preparations can be sterilized and if desired,
mixed with auxiliary agents, e.g., lubricants, preservatives,
stabilizers, wetting agents, emulsifiers, salts for influencing
osmotic pressure, buffers and the like which do not deleteriously
react with the active compounds. They can also be combined, where
desired, with other active agents, e.g., enzyme inhibitors, to
further reduce metabolic degradation.
[0051] For topical application, there are employed as nonsprayable
forms, viscous semi-solid or solid forms comprising a carrier
compatible with topical application and having a dynamic viscosity
preferably greater than water. Suitable formulations include but
are not limited to solutions, suspensions, emulsions, creams,
foams, ointments, powders, liniments, salves, aerosols, etc., which
are, if desired, sterilized or mixed with auxiliary agents, e.g.,
preservatives, stabilizers, wetting agents, buffers or salts for
influencing osmotic pressure, etc. For topical application, also
suitable are sprayable aerosol preparations wherein the active
ingredient, preferably in combination with a solid or liquid inert
carrier material, is packaged in a squeeze bottle or in admixture
with a pressurized volatile, normally gaseous propellant, e.g.,
pressurized air. Also included are transdermal patches as
discussed, for example, in WO 93/07870, the teachings of which are
incorporated by reference.
[0052] Alternatively, CHK can be supplied to the recipient cells by
introducing a nucleic acid sequence encoding CHK, or a biologically
active fragment, analog, or derivative of CHK which is then
expressed in the recipient cells. Methods of introducing nucleic
acids encoding specific proteins such as CHk are well-known to
those of skill in the art. For example, expression vectors can be
designed and produced that contain a nucleic acid insert which
encodes CHK or a biologically active fragment of CHK. Methods to
construct these expression vectors are well-known to those of skill
in the art. For example, described herein is an expression vector
comprising vaccinia virus useful for expressing a DNA insert
encoding CHK. In addition to vaccinia virus, other virus or plasmid
vectors, such as retroviruses, or plasmid vectors can be used to
introduce nucleic acids encoding CHK into recipient cells.
Additionally naked DNA can be injected into recipient cells, or
methods such as elctroporation, co-precipitation or a "gene gun"
can be used to deliver the DNA to the recipient cells.
[0053] Other techniques using naked plasmids or DNA, and cloned
genes encapsidated in targets liposomes or in erythrocyte ghosts,
can be used to introduce the receptor into the host (Friedman, T.,
Science, 244:1275-1281 (1990); Rabinovich, N. R., et al., Science,
265:1401-1404 (1994)). The construction of expression vectors and
the transfer of vectors and nucleic acids into various host cells
can be accomplished using genetic engineering techniques, or by
using commercially available kits as described in Sambrook, J., et
al. Molec. Cloning, Cold Spring Harbor Press (1989) or Ausubel, F.
M., et al. Current Protocols in Molecular Biology, Greene
Publishing Assoc. and Wiley-Interscience (1989) the teachings of
which are hereby incorporated, in their entirety, by reference.
[0054] Cells from a patient's tumor can be analyzed by the
diagnostic methods described above to determine the presence of CHK
or to determine the biological activity of CHK present in their
cells. A vector as described herein, containing a nucleic acid
encoding CHK and operably linked to expression control elements
required for the expression of a protein in the recipient cells, is
then introduced into the patient, either at the site of the tumor
or by intravenous or other parenteral injection in order to reach
any tumor cells that may have metastasized to other sites. The
introduction may be repeated as necessary in order to achieve the
desired effect of inhibiting neoplastic growth. A description of
techniques that may be used to specifically target breast cells is
described in EP 0 699 754 A1, the teachings of which are herein
incorporated by reference.
[0055] Thus, as a result of the work described herein, novel
methods of detecting and inhibiting breast cancer, or hyperplastic
growth that may result in cancer, are now available.
[0056] The following examples more specifically illustrate the
invention and are not intended to be limiting in any way.
EXAMPLE 1
Expression of CHK in Human Breast Cancer Tissue
[0057] Materials:
[0058] Recombinant heregulin (rHRG(1, 177-244), rabbit polyclonal
anti-ErbB-2 antibodies, and 3E8-monoclonal anti-ErbB-2 antibodies,
were obtained from Genentech, Inc. (San Francisco, Calif.), Levi,
A. D., Bunge, R. P., Lofgren, J. A., Meima, L., Hefti, F.,
Nikolics, K., and Sliwkowski, M. X., J. Neurosci., 15, 1329-1340
(1995)). EGF and IL-6 were purchased from Collaborative Biomedical
Products (Bedford, Mass.) and from R & D Systems (Minneapolis,
Minn.) respectively. Monoclonal anti-phosphotyrosine antibody
(PY20) conjugated to horse radish peroxidase (HRP) was obtained
from Zymed, Inc. (San Francisco, Calif.). Polyclonal antibodies for
EGF-R, ErbB-3, ErbB-4 and polyclonal anti-CHK (anti-LSK) antibodies
were obtained from Santa Cruz Biotechnology (Santa Cruz, Calif.).
Anti-GST monoclonal antibodies were purchased from Pharmacia
Biotech, Inc. (Piscataway, N.J.). GST fusion proteins containing
the NH2-SH2 domain of p85 P13 kinase and SH2-SH2-SH3 domains of
PLC-1 were obtained from Santa Cruz Biotechnology. The primers for
the polymerase chain reaction (PCR) were synthesized by an
automated DNA Synthesizer (Applied Biosystems, Model 394). Reagents
for electrophoresis were obtained from BioRad (Hercules, Calif.).
ECL reagents were purchased from Amersham Corp. (Arlington Heights,
Ill.). All other reagents were purchased from Sigma (St. Louis,
Mo.).
[0059] Experimental Procedures
[0060] Immunohistochemical staining was performed on
paraffin-embedded 5 mm-thick tissue sections of human breast
cancer. Sections were deparaffinized in xylene and then incubated
in decreasing concentrations of ethyl alcohol. After several rinses
in water, the slides were incubated in methanol/hydrogen peroxide
(1:4), briefly rinsed in water and then in PBS (pH 7.6). Subsequent
immunohistochemical staining was performed using a 1:100 dilution
in PBS of rabbit anti-CHK antisera (1 hr incubation) followed by
the addition of the secondary antibodies, peroxidase-conjugated
rabbit anti-mouse IgG (Sigma) at 50 .mu.g/ml in PBS.
[0061] Analyses of CHK expression in human breast cancer tissues at
different stages were performed using immunohistochemistry on
paraffin sections. Results (Table 1) revealed that CHK is expressed
in the majority of breast cancers, but was not detected in normal
adjacent tissue.
1TABLE 1 CHK Expression in Primary Breast Cancer Tissues BREAST
CANCER PATIENTS NO. PATIENTS (+) FOR CHK Stage I 32/41 Stage II
29/35 Stage III 4/4 Unknown Stage 5/6 Normal Breast 0/3
Fibroadenoma 0/6
[0062] Immunohistochemical staining was performed on paraffin
embedded sections of infiltrating ductal carcinoma using anti-CHK
antibodies.
EXAMPLE 2
CHK is Associated With Activated ErbB-2 Upon Stimulation With
HRG
[0063] Experiments were performed using the T47D breast cancer cell
line and the GST-fusion protein containing the SH2 domain of CHK
(CHK-SH2). T47D cells express the ErbB family receptors and the CHK
protein as observed by immunohistochemistry. The T47D human breast
cancer cell line was obtained from ATCC (American Type Culture
Collection, Rockville, Md.). T47D cells were grown in RPMI-1640
medium (GIBCO/BRL, Life Technologies, Gaithersburg, Md.)
supplemented with 10% fetal bovine serum (FBS) and 3.5 (g/ml
insulin (Sigma). Prior to stimulation with HRG, cells were starved
overnight in media containing 1% FBS and then for 4 hr in
serum-free medium. The starved cells were then stimulated with HRG
(10 nM) for the indicated time points (FIGS. 1A-D). Cells were
lysed, and the supernatants were incubated with the purified
CHK-SH2 fusion protein (FIGS. 1A, 1B) or with the 3E8 monoclonal
antibody to ErbB-2 (FIGS. 1C, 1D). The co-precipitated proteins
were analyzed on 7% SDS-PAGE, and immunoblotted with PY20 (FIGS.
1A, 1C).
[0064] As shown in FIG. 1A, a tyrosine-phosphorylated 185 kD
protein was associated with CHK-SH2 within 2 min of the HRG
stimulation. The association of the 185 KD with CHK-SH2 was maximal
at 2-8 min after HRG stimulation and then gradually decreased. In
order to determine whether the 185 kD protein was ErbB-2, the blot
was deprobed and reblotted with polyclonal anti-ErbB-2 antibody. As
shown in FIG. 1B, the 185 kD protein was confirmed to be the ErbB-2
protein. These results indicated that the CHK protein can interact
with the HRG-activated ErbB-2 receptor.
[0065] When lysates from HRG-treated cells were immunoprecipitated
with the 3E8 monoclonal anti-ErbB-2 antibody, the pattern of the
phosphorylated ErbB-2 was different from that of the ErbB-2
precipitated with the SH2 domain of CHK (compare FIG. 1C with FIG.
1A). Blotting of the same samples with the polyclonal anti-ErbB-2
antibody (FIG. 1D) confirmed these observations.
[0066] CHK-SH2 fusion proteins also precipitated other as of yet
unidentified tyrosine-phosphorylated proteins as shown in FIG. 1A.
However, these phosphorylated proteins were also precipitated from
the unstimulated cells and their phosphorylation pattern did not
appear to change over the time course of these studies.
EXAMPLE 3
The Association of CHK With ErbB-2 is Specific for HRG
Stimulation
[0067] In order to determine whether the observed association of
CHK with ErbB-2 was receptor-specific and stimulus-specific,
experiments were performed to analyze whether CHK could associate
with either the EGF-R or IL-6 receptors which are both known to be
expressed in T47D cells. The association of CHK-SH2 with ErbB-2 in
lysates from HRG, EGF and IL-6 stimulated cells was compared. T47D
cells were serum starved as described above and then activated
either with HRG (10(nM) for 8 min or with (EGF (100 ng/ml) or IL-6
(100 ng/ml) for 5 min. The experimental time points and the
concentrations of EGF and IL-6 were optimized in initial kinetic
studies. The stimulated cells were lysed and precipitated with the
CHK-SH2 fusion protein as described above. The precipitates were
then analyzed on SDS-PAGE and immunoblotted with PY20 antibodies or
with polyclonal anti-ErbB-2 antibodies. Only HRG stimulation
induced the association of ErbB-2 with the purified CHK-SH2 fusion
protein. EGF or IL-6 stimulation failed to induce CHK-SH2
association either to ErbB-2, or to the EGF-receptor or the IL-6
receptor.
[0068] The association of ErbB-2 with other SH2 domain-containing
signaling molecules such as p85 of P13-kinase, PLC-1 or Shc was
also examined. The SH2-SH2-SH3 domain of PLC-1 was found to be
associated with the HRG-activated ErbB-2 as well as with Shc. The
SH2 domain of P13-kinase precipitated ErbB-2, probably as a result
of the ErbB-2 heterodimerization with ErbB-3. Taken together, these
results indicate that ErbB-2 associates with all three signaling
molecules in HRG-activated T47D cells.
[0069] Experiments were also performed to show that the SH3 domain
of CHK is not involved in the interaction between CHK and
ErbB-2.
[0070] The potential involvement of other domains of CHK in the
interaction with ErbB-2 was also examined. GST-fusion proteins
containing the SH3 domain of CHK (CHK-SH3), the N-terminal domain
plus SH3 domain (NH2-SH3), the SH3 and SH2 domains of CHK
(SH3-SH2), the SH2 domain of CHK as well as the GST protein alone
were prepared as follows.
[0071] Generation of Flag-CHK Construct in pCDNA3 Vector:
[0072] The CHK cDNA (1.6 kb, (SEQ ID NO: 1) was cloned into EcoRI
sites in the pCDNA3 neo vector. The nucleotide sequence for the
Flag epitope (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) (SEQ ID NO: 5) was
introduced to the 5' end of the ORF (open reading frame) of the CHK
cDNA sequence by PCR, using 1.6 kb CHK cDNA as a template. The 5'
sense primer included a BamH I restriction site, ATG initiation
codon, the Flag sequence and CHK sequences from nucleotides #269 to
#295 of SEQ ID NO: 1, FIG. 2 (Bennett, B. D., et al. (1994) J.
Biol. Chem. 269, 1068-1074), the teachings of which are herein
incorporated, in their entirety, by reference). The 3'-antisense
primer was composed of CHK sequences from nucleotides #510 to #481
of SEQ ID NO: 1. The PCR product was double digested with BamHI and
BstEII (New England Bio Labs, Beverly, Mass.), gel-purified and
then cloned into BamHI, and BstEII sites in the pDNA3 neo-Flag-CHK.
The construct was analyzed by restriction mapping and nucleotide
sequencing.
[0073] Transfection:
[0074] Transfection of MCF-7 cells was performed using the
Lipofectamine.TM. (Gibco/BRL) according to the manufacturer's
protocol. The transfected cells were selected in 1.2 mg/ml G418
(Sigma). Positive transfectants were chosen based on their
immunoreactivity on Western blots probed with polyclonal anti-CHK
and monoclonal anti-Flag (M5) antibodies (Eastman Kodak Company,
New Haven, Conn.).
[0075] Construction and Purification of GST-Fusion Proteins of
CHK:
[0076] To express the NH2-SH3 and SH3-SH2 domains of CHK as
GST-fusion proteins, the corresponding DNA sequences were amplified
by PCR with sense and antisense primers of CHK cDNA which contained
BamHI and EcoRI restriction sites. For the NH2-SH3 construct, we
used the sense primer from nucleotides #4 to #27 SEQ ID NO: 1 and
the antisense primer from nucleotides #343 to #321 of SEQ ID NO: 1.
For the SH3-SH2 construct, we the sequence from nucleotides #127 to
#150 of SEQ ID NO: 1 was used as the sense primer and from
nucleotides #657 to #634 SEQ ID NO: 1 as the antisense primer. The
DNA fragments obtained from PCR were restriction digested with
BamHI and EcoRI and ligated into the pGEX-2T vector (Pharmacia).
The sequence and orientation were confirmed by sequencing both
strands. Construction of the GST-fusion proteins of CHK-SH2 and
CHK-SH3 were performed as described in Jhun, B. H., Rivnay, B.,
Price, D., and Avraham, H. (1995) J. Biol. Chem. 270,
9661-9666.
[0077] GST-fusion proteins were produced by the induction of
transformed bacteria using 10 mM isopropyl-(-thiogalactopyranoside
(IPTG), and purified on a large scale by affinity chromatography on
glutathione-sepharose beads (Pharmacia) according to the
manufacturer's protocol.
[0078] HRG-stimulated T47D cell lysates were incubated with the
different GST-fusion proteins, analyzed by SDS-PAGE, and
immunoblotted either with PY20, rabbit anti-ErbB-2 antibody or with
anti-GST antibody. Neither the SH3 domain of the CHK protein nor
the NH2-SH3 domain precipitated ErbB-2. Binding to ErbB-2 was
detected only in the presence of the CHK-SH2 and CHK-SH3-SH2 fusion
proteins. As expected, no binding was detected when the same
lysates were incubated with the GST protein alone. The amounts of
the different fusion proteins loaded on the gel were comparable.
These results confirm that CHK can interact with the HRG-stimulated
ErbB-2 in a specific manner via its SH2 domain.
EXAMPLE 4
In Vivo Association of Intact CHK With ErbB-2
[0079] The MCF-7 human breast cancer cell line was obtained from
ATCC (American Type Culture Collection, Rockville, Md.). The MCF-7
cells were grown in MEM (GIBCO) supplemented with 10% FBS, 5
.mu.g/ml insulin (Sigma), 1 mM non-essential amino acids and 1 mM
sodium pyruvate. Prior to stimulation, cells were starved overnight
in media containing 1% FBS and then for 4 hr in serum-free
medium.
[0080] To further confirm the association of ErbB-2 with CHK, the
CHK protein was overexpressed in MCF-7 breast cancer cells. CHK
expression in MCF-7 cells was detected only by PCR analysis.
Expression of the ErbB receptor family in MCF-7 cells was similar
to that observed in T47D cells. Stable transfections were performed
using the Flag-CHK pCDNA3 neo construct as described above. The
transfected cells were analyzed for CHK expression by Western blot
using anti-Flag and anti-CHK antibodies and also by
immunofluorescence using confocal microscopy. MCF-7 cells
transfected with Flag-CHK pCDNA3 neo (Flag-CHK), MCF-7 cells
transfected with the pCDNA3 neo vector alone, or untransfected
MCF-7 control cells, were stimulated with HRG and then lysed.
[0081] Immunoprecipitation studies were performed as follows.
Approximately 5.times.10.sup.6 cells/plate were starved overnight
in media containing 1% FBS, followed by additional starvation in
serum-free medium for 4 hr at 37.degree. C. The starved cells were
then stimulated with 10 nM HRG for 8 min or with 100 ng/ml EGF or
100 ng/ml IL-6 for 5 min at room temperature. The stimulation was
terminated by the addition of an ice-cold lysis buffer (0.1% SDS,
1% Triton X-100, in Tris-buffered saline containing 10% glycerol, 1
mM EDTA, 0.5 mM Na3VO4, 0.2 mM phenylmethylsulfonyl fluoride, 1
.mu.g/ml aprotinin, and 10 mM leupeptin). Lysates were pre-cleared
by centrifugation (14,000 rpm, 15 min) and then incubated for 90
min at 4.degree. C. with 10 .mu.g of GST-fusion proteins coupled to
glutathione-sepharose beads. The beads were washed three times with
the lysis buffer. For the immunoprecipitation experiments,
polyclonal anti-CHK antibody, monoclonal anti-ErbB-2 antibody, 3E8
(10 .mu.g/ml), polyclonal anti-ErbB-3 antibody (10 .mu.g/ml) or
polyclonal anti-ErbB-4 antibody (10 .mu.g/ml) were used. SDS-sample
buffer was added to the samples and analyzed on 7% polyacrylamide
SDS-PAGE. Proteins were transferred onto nitrocellulose or
Immobilon-PTM (Millipore, Inc., Bedford, Mass.) membranes. Bound
proteins were immunoblotted with anti-phosphotyrosine antibody
(PY20), polyclonal anti-ErbB-2 antibody, or polyclonal anti-CHK,
EGF-R, ErbB-3 or ErbB-4 antibodies. The blots were developed using
the enhanced chemiluminescence (ECL) system (Amersham). Blots were
stripped for 30 min at 55.degree. C. in stripping buffer (100 mM
2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.7), according to
the manufacturer's protocol (Amersham).
[0082] The 185 KD tyrosine-phosphorylated protein was
immunoprecipitated with anti-Flag antibodies or anti-CHK antibodies
only in HRG-stimulated Flag-CHK transfected cell lysates, but not
in the untransfected MCF-7 cell lysates or the MCF-7 cell lysates
transfected with the pcDNA3-neo Flag vector alone. Blotting with
the anti-ErbB-2 antibody confirmed that the co-precipitated 185 KD
protein was indeed the ErbB-2. Analysis of the total lysates from
the same experiment revealed that the ErbB-2 was
tyrosine-phosphorylated as a result of the HRG stimulation in the
Flag-CHK cells as well as in the MCF-7 untransfected cells. The
expression of ErbB-2 appeared to be equal in both the Flag-CHK and
MCF-7 cells. Taken together, these in vitro and in vivo data
indicate that the HRG-stimulated ErbB-2 associates with CHK through
the SH2 domain.
EXAMPLE 5
Involvement of Other ErbB-2 Family Members in the Interaction With
CHK
[0083] To further investigate the possible involvement of other
members of the ErbB family in the observed interaction between CHK
and ErbB-2, co-immunoprecipitation experiments using MCF-7 cells
transfected with Flag-CHK were performed. Flag-CHK transfected
cells were stimulated with HRG, and then lysed and
immunoprecipitated with anti-CHK antibody as described above. The
immunocomplexes were separated by SDS-PAGE and immunoblotted with
anti-ErbB-2 antibody or with anti-ErbB-3 antibody. The results
indicated that anti-CHK antibody immunoprecipitated the
HRG-activated ErbB-2. In contrast, no detectable ErbB-3 was found.
However, the possibility that very low amounts of ErbB-3 were
present in the precipitates as a result of the heterodimerization
with the ErbB-2 receptor upon HRG stimulation cannot be
excluded.
[0084] It was also investigated whether ErbB-4 interacted with CHK
under these conditions, however, findings indicated that ErbB-4 was
not involved in the ErbB-2-CHK association.
[0085] In order to confirm the presence and phosphorylation of the
ErbB-3 as well as the heterodimerization of ErbB-3 with ErbB-2 in
the Flag-CHK transfected cells, lysates from HRG-stimulated
Flag-CHK cells were immunoprecipitated with anti-ErbB-3 antibodies
or with anti-ErbB-2 antibodies. Both ErbB-3 and ErbB-2 were
tyrosine-phosphorylated upon HRG stimulation and the formation of
ErbB-2-ErbB-3 heterodimers was demonstrated by the presence of
ErbB-2 in the precipitates of the anti-ErbB-3 antibodies. However,
under these conditions, ErbB-3 was not detected in the samples
immunoprecipitated with anti-ErbB-2 antibody. Taken together, these
observations indicate that upon HRG stimulation, heterodimerization
of ErbB-3 with ErbB-2 receptors occurred in the transfected cells,
suggesting that the ErbB signaling in these cells is not
altered.
[0086] To determine whether EGF-R (ErbB-1) might be involved in
ErbB-2-CHK interactions, Flag-CHK MCF-7 transfected cells were
serum-starved and then stimulated with HRG (10 nM) or with EGF (100
ng/ml). The lysates were immunoprecipitated with anti-CHK
antibodies and analyzed by SDS-PAGE. Only the
tyrosine-phosphorylated ErbB-2 protein was immunoprecipitated with
anti-CHK-antibodies in the HRG-stimulated lysates. No
tyrosine-phosphorylated proteins were detected in the
immunoprecipitates with anti-CHK antibodies from the EGF-stimulated
cells. Reprobing of this blot with anti-ErbB-2 or with anti-EGF-R
antibodies confirmed that neither of these receptors were present
in the CHK immunoprecipitates.
[0087] As a control, immunoprecipitations with anti-EGF-R
antibodies of the EGF-stimulated Flag-CHK cell lysates as well as
of lysates from untransfected MCF-7 cells were performed. The EGF-R
and the ErbB-2 proteins were present in the immunoprecipitates from
the EGF-stimulated cells as a result of the EGF-ErbB-2
heterodimerization. Probing of the same blot with anti-ErbB-2 or
anti-EGF-R antibodies confirm this observation.
[0088] These analyses indicate that CHK associates via its SH2
domain with HRG-stimulated ErbB-2. This association is specific to
HRG-stimulated ErbB-2 and does not appear to prominently involve
other ErbB family members.
EXAMPLE 6
Functional Association of CHK to the NEC (Val.sup.664) and TEC
(Glu.sup.664) EGF-ErbB-2 Hybrid Receptors
[0089] ErbB-2 functions as a co-receptor for growth-regulatory
molecules, including neuregulins. Replacement of the extracellular
domain of ErbB-2 by the ligand biding domain of the receptor for
EGF allows heterologous stimulation of the ErbB-2, which has been
successfully exploited in signal transduction studies (Ben-Levy,
R., et al., EMBO J., 13;3302-3311 (1994). The transforming protein
of ErbB-2, which contains a Glutamine residue (Glu.sup.664) instead
of a Valine (VAL.sup.664) residue, is a constitutively active
receptor permanently coupled to signaling pathways. To confirm that
the association of CHK with ErbB-2 is mediated by the intracellular
domain of ErbB-2 and not by other members of the ErbB-2 family,
chimeric proteins that include the extracellular domain of the EGF
receptor and the transmembrane and cytoplasmic domains of the
ErbB-2, termined NEC (Val.sup.664), or the point-mutated
cytoplasmic domain of ErbB-2 (Glu.sup.664) termined TEC, (kindly
obtained from Dr. Y. Yarden (Department of Chemical Immunology, the
Weizmann Institute of Science, Rehovot, Israel); Peles, E., et al.,
J. Biol. Chem., 267:12266-12274 (1992)) were used in this study. It
is important to note that ErbB-2 does not directly bind to any of
the EGF-like ligands. However, EGF and HRG induce the tyrosine
phosphorylation of ErbB-2, presumably by ligand-driven
heterodimerization and transphosphorylation. NIH3T3 cells were
stably transfected with the chimeric plasmid EGF-TEC-ErbB-2 or with
the chimeric plasmid EGF-NEC-ErbB-2. TEC and NEC cells
(4.times.10.sup.6 cells/plate) were serum-starved and then
unstimulated or stimulated with 100 ng EGF at room temperature for
5 minutes. The lysates were divided to two parts: one half of the
lysates were precipitated with the CHK-SH2 GST-fusion protein (10
.mu.g) for 90 minutes at 4.degree. C. (A-II, B-II). After washing,
the precipitates were separated by 7% SDS-PAGE and immunoblotted
with monoclonal anti-phosphotyrosine antibody (PY20), or with
polyclonal anti-EGF-R antibodies. The other half of the lysates was
immunoprecipitated using monoclonal antibodies for EGF-R for 16 h
at 4.degree. C. The washed precipitates were run on 7% SDS-PAGE and
blotted with PY20 or with anti-EGF-R antibodies.
[0090] CHK association with both EGF-stimulated and unstimulated
NEC and TEC was analyzed. Upon EGF Stimulation, CHK was found to
associate via its SH2 domain with NEC, while its association with
TEC was constitutive and not dependent on EGF stimulation. These
results indicate that the CHK-SH2 domain specifically associates
with the intracellular domain of ErbB-2.
EXAMPLE 7
Generation and Characterization of MCF-7 Cells Stably Transfected
With CHK cDNA
[0091] In order to study the biological function(s) of CHK in human
mammary epithelial cells, two known breast cancer cell lines, MCF-7
and T47D were chosen. Both cell lines, obtained from American Type
Culture (ATCC, Rockville, Md.), are well established in the field
of breast cancer research and used extensively as models
(Gras-Porta, D., et al., Mol. Cell. Biol., 15:1182-1191 (1995)
Azijsen, R. M., et al., Mol. & Cell Biol., 16:2554-2560
(1996)). The expression of CHK in both these cell lines was
analyzed. While T47D cells expressed CHK mRNA and protein as
detected by Northern and Western blot analyses respectively, CHK
expression in MCF-7 cells was detected only by PCR without evidence
for significant levels of protein using immunoprecipitation or
Western blotting. MCF-7 cells stably transfected with CHK cDNA that
expressed CHK mRNA and CHK protein were generated. The MCF10-A cell
line was used as a model for normal breast epithelial cells (Soule,
H. D., et al., Cancer Research, 50:6075-6086 (1990). These cells
lacked expression of CHK, as evaluated by Northern blot, PCR and
Western blot.
[0092] Stable transfections of MCF-7 cells were performed using the
FLAG-CHK-pcDNA3neo construct or the pcDNA3neo vector as a control.
CHK protein can be detected either by CHK specific antibodies or
FLAG monoclonal antibodies. The proliferation rate of MCF-7 cells
transfected with the FLAG-CHK-pcDNA3neo construct overexpressing
CHK protein was significantly reduced (p<0.001) compared to the
untreated MCF-7 cells or to the MCF-7 cells transfected with the
FLAG pcDNA3neo vector alone.
[0093] Confocal microscopy studies in these MCF-7 cells stably
transfected with the FLAG-CHK-pcDNA3neo construct demonstrated that
CHK was localized in the cytosol fraction. However, upon heregulin
stimulation, CHK was translocated to the membrane. Taken together,
these results, with the data on CHK-SH2 associating with ErbB-2,
suggest that CHK is translocated from the cytosol to the membrane
and associates with the ErbB-2 receptor upon ligand
stimulation.
EXAMPLE 8
Tumor Development in Nude Mice
[0094] Initial studies have shown that CHK negatively regulates src
activity and associates with ErbB-2 upon heregulin stimulation.
Therefore, CHK might function as a negative regulator and might act
to inhibit mitogenic signaling by c-src and ErbB-2. Interestingly,
the proliferation rate of the MCF-7/CHK clone was reduced compared
to the control MCF-7/neo clone or untransfected MCF-7 cells.
Therefore, to evaluate the anti-transforming potential of CHK,
tumor development was monitored in nude mice injected with MCF-7,
MCF-7/neo and MCF-7/CHK cells, using standard laboratory
techniques.
[0095] Tumor development in nude mice injected with MCF-7/CHK cells
was significantly reduced (2/15) compared to tumor development in
nude mice injected with control MCF-7 cells (15/15) or MCF-7/neo
cells (12/15). These experiments suggest that overexpression of CHK
can negatively regulate the growth of MCF-7 breast cancer cells in
nude mice.
EXAMPLE 9
CHK Overexpression Affects S-Phase Entry of MCF-7 Cells
[0096] A number of proto-oncogenes have been shown to affect cell
cycle. Protoncogenes involved in the G.sub.0/G.sub.1 transition,
such as myc and ras, are able to cooperate with cyclin D.sub.1 in
transforming cells. pp60src has been directly implicated in cell
cycle regulation as well (Taylor, S. J., et al., Bioassays, 18:9-11
(1996), Roche, S., et al., Science, 269:1567-1569 (1995)). Since it
has been demonstrated that CHK can regulate pp60src, it was
investigated whether the level of CHK expression might modulate
cell cycle kinetics using MCF-7 cells or transfected MCF-7 cells
that overexpressed CHK protein (i.e., MCF-7/CHK). Growth-arrested
postconfluent MCF-7, MCF-7/neo or MCF-7/CHK cells were obtained by
serum depletion for 4 days. Cells were stimulated by 10% serum and
harvested at specific times. These analyses indicate a significant
delay in the entry to S-phase of the CHK transfected MCF07 cells
compared to the controls. These results suggest that overexpression
of CHK might have an effect on cell cycle.
EXAMPLE 10
Expression of CHK Using the Vaccine Virus/T7 RNA Polymerase Hybrid
System and the Baculovious System
[0097] To analyze the interactions of CHK with ErbB-2, pp60src or
other interacting molecules, a recombinant vaccinia virus was
constructed to drive expression of CHK. CHK was inserted into a
PTM-1 vaccinia recombinant plasmid under the control of the T7 RNA
polymerase promoter. Recombinant viruses were selected, amplified
and titered using standard techniques (Elroy-Stein, O., et al.,
Proc. Natl. Acad. Sci. USA, 87:6743-6747 (1990), Ausubel, F. M., et
al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Son
publishers (1992)). To demonstrate that recombinant viruses produce
appropriately immunoreactive proteins, MCF-7 cells were co-infected
with the CHK recombinant vaccinia virus and the T7 polymerase
recombinant virus at 10.times.MOI (multiplicity of infection) of
each virus in 2.5% FCS DMEM. Cell lysates were run on SDS-PAGE and
analyzed by immunoblotting with the anti-CHK antibodies. Expression
of the 60 Kd immunoreactive CHK protein was demonstrated by
immunoblotting with specific antibody. .sup.35S-labeling of MCF-7
cells co-infected with the CHK recombinant vaccinia virus indicated
that CHK is a major protein being synthesized in these cells.
[0098] To characterize the biochemical and functional properties of
CHK, CHK has also been expressed using the baculovirus system. For
baculovirus expression, CHK cDNA was inserted into a pAcHLT-A.TM.
vector (PharMingen), as directed by the manufacturer. Recombinant
CHK baculovirus was used to infect Sf9 insect cells for 72 hours at
5.times.MOI. Cell lysates were run on SDS-PAGE followed by Western
blotting with anti-CHK antibody, or by protein staining of the gel
with Coomassie Blue. Extracts of recombinant CHK baculovirus
derived from infected Sf9 cells were chromatographed on
phosphotyrosien-Affi-gel, DEAE-Sephacel, and Mono S. Purified CHK
was eluted from these columns as described in Flink, N. A., et al.,
J. Cell. Biochem., 55:389-397 (1994).
EXAMPLE 11
CHK Phosphorylation of the C-Terminal src Peptide, Enolase, and
Poly Glu/Tyr
[0099] In order to confirm the pp60src kinase as a substrate for
CHK, immunoprecipitations of CHK and Csk from mouse brain were
carried out. Mouse brain extracts were immunoprecipitated with
either anti-CHK (murine Ctk), anti-Csk (murine), or normal mouse
serum. Washed immunoprecipitates were used to phosphorylate
substrates in the presence of 25 mM MOPS, pH 7.4, 50 .mu.M Na.sub.3
VO.sub.4 5 mM MnCl.sub.2, 0.5 mM DTT, 125 .mu.M .gamma.[.sup.32P]
ATP. Substrates tested were C-terminal src peptide, enolase, and
Poly Glu/Tyr. Reactions were either terminated by the addition of
SDS sample buffer (enolase, Poly Glu/Tyr) and run on SDS-PAGE, or
terminated by pipetting onto P81 paper (src peptide) and washed
extensively in 75 mM phosphoric acid. In vitro kinase assays of CHK
and Csk immunoprecipates showed that both kinases phosphoylated the
C-terminal scr peptide, enolase and poly Glu/Try to similar degrees
(FIG. 4). =Poly Glu/Tyr; .box-solid.=Enolase; =C-terminal src
peptide.
[0100] Equivalents
[0101] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
5 1 1987 DNA Homo sapiens CDS (263)...(1846) 1 ggagcaactc
gctccaagtt gtgcagccgg gaccgcctcg gggtgtgcag ccggctcgcg 60
gaggccctcc tgggggcggg cggggcgcgg ctcgggggcg ccccctgagc agaaaacagg
120 aagaaccagg ctcggtccag tggcacccag ctccctacct cctgtgccag
ccgcctggcc 180 tgtggcaggc cattcccagc gtccccgact gtgaccactt
gctcagtgtg cctctcacct 240 gcctcagttt cctctggggg cg atg gcg ggg cga
ggc tct ctg gtt tcc tgg 292 Met Ala Gly Arg Gly Ser Leu Val Ser Trp
1 5 10 cgg gca ttt cac ggc tgt gat tct gct gag gaa ctt ccc cgg gtg
agc 340 Arg Ala Phe His Gly Cys Asp Ser Ala Glu Glu Leu Pro Arg Val
Ser 15 20 25 ccc cgc ttc ctc cga gcc tgg cac ccc cct ccc gtc tca
gcc agg atg 388 Pro Arg Phe Leu Arg Ala Trp His Pro Pro Pro Val Ser
Ala Arg Met 30 35 40 cca acg agg cgc tgg gcc ccg ggc acc cag tgt
atc acc aaa tgc gag 436 Pro Thr Arg Arg Trp Ala Pro Gly Thr Gln Cys
Ile Thr Lys Cys Glu 45 50 55 cac acc cgc ccc aag cca ggg gag ctg
gcc ttc cgc aag ggc gac gtg 484 His Thr Arg Pro Lys Pro Gly Glu Leu
Ala Phe Arg Lys Gly Asp Val 60 65 70 gtc acc atc ctg gag gcc tgc
gag aac aag agc tgg tac cgc gtc aag 532 Val Thr Ile Leu Glu Ala Cys
Glu Asn Lys Ser Trp Tyr Arg Val Lys 75 80 85 90 cac cac acc agt gga
cag gag ggg ctg ctg gca gct ggg gcg ctg cgg 580 His His Thr Ser Gly
Gln Glu Gly Leu Leu Ala Ala Gly Ala Leu Arg 95 100 105 gac ggg gag
gcc ctc tcc gca gac ccc aag ctc agc ctc atg ccg tgg 628 Asp Gly Glu
Ala Leu Ser Ala Asp Pro Lys Leu Ser Leu Met Pro Trp 110 115 120 ttc
cac ggg aag atc tcg ggc cag gag gct gtc cag cag ctg cag cct 676 Phe
His Gly Lys Ile Ser Gly Gln Glu Ala Val Gln Gln Leu Gln Pro 125 130
135 ccc gag gat ggg ctg ttc ctg gtg cgg gag tcc gcg cgc cac ccc ggc
724 Pro Glu Asp Gly Leu Phe Leu Val Arg Glu Ser Ala Arg His Pro Gly
140 145 150 gac tac gtc ctg tgc gtg agc ttt ggc cgc gac gtc atc cac
tac cgc 772 Asp Tyr Val Leu Cys Val Ser Phe Gly Arg Asp Val Ile His
Tyr Arg 155 160 165 170 gtg ctg cac cgc gac ggc cac ctc aca atc gat
gag gcc gtg ttc ttc 820 Val Leu His Arg Asp Gly His Leu Thr Ile Asp
Glu Ala Val Phe Phe 175 180 185 tgc aac ctc atg gac atg gtg gag cat
tac agc aag gac aag ggc gct 868 Cys Asn Leu Met Asp Met Val Glu His
Tyr Ser Lys Asp Lys Gly Ala 190 195 200 atc tgc acc aag ctg gtg aga
cca aag cgg aaa cac ggg acc aag tcg 916 Ile Cys Thr Lys Leu Val Arg
Pro Lys Arg Lys His Gly Thr Lys Ser 205 210 215 gcc gag gag gag ctg
gcc agg gcg ggc tgg tta ctg aac ctg cag cat 964 Ala Glu Glu Glu Leu
Ala Arg Ala Gly Trp Leu Leu Asn Leu Gln His 220 225 230 ttg aca ttg
gga gca cag atc gga gag gga gag ttt gga gct gtc ctg 1012 Leu Thr
Leu Gly Ala Gln Ile Gly Glu Gly Glu Phe Gly Ala Val Leu 235 240 245
250 cag ggt gag tac ctg ggg caa aag gtg gcc gtg aag aat atc aag tgt
1060 Gln Gly Glu Tyr Leu Gly Gln Lys Val Ala Val Lys Asn Ile Lys
Cys 255 260 265 gat gtg aca gcc cag gcc ttc ctg gac gag acg gcc gtc
atg acg aag 1108 Asp Val Thr Ala Gln Ala Phe Leu Asp Glu Thr Ala
Val Met Thr Lys 270 275 280 atg caa cac gag aac ctg gtg cgt ctc ctg
ggc gtg atc ctg cac cag 1156 Met Gln His Glu Asn Leu Val Arg Leu
Leu Gly Val Ile Leu His Gln 285 290 295 ggg ctg tac att gtc atg gag
cac gtg agc aag ggc aac ctg gtg aac 1204 Gly Leu Tyr Ile Val Met
Glu His Val Ser Lys Gly Asn Leu Val Asn 300 305 310 ttt ctg cgg acc
cgg ggt cga gcc ctc gtg aac acc gct cag ctc ctg 1252 Phe Leu Arg
Thr Arg Gly Arg Ala Leu Val Asn Thr Ala Gln Leu Leu 315 320 325 330
cag ttt tct ctg cac gtg gcc gag ggc atg gag tac ctg gag agc aag
1300 Gln Phe Ser Leu His Val Ala Glu Gly Met Glu Tyr Leu Glu Ser
Lys 335 340 345 aag ctt gtg cac cgc gac ctg gcc gcc cgc aac atc ctg
gtc tca gag 1348 Lys Leu Val His Arg Asp Leu Ala Ala Arg Asn Ile
Leu Val Ser Glu 350 355 360 gac ctg gtg gcc aag gtc agc gac ttt ggc
ctg gcc aaa gcc gag cgg 1396 Asp Leu Val Ala Lys Val Ser Asp Phe
Gly Leu Ala Lys Ala Glu Arg 365 370 375 aag ggg cta gac tca agc cgg
ctg ccc gtc aag tgg acg gcg ccc gag 1444 Lys Gly Leu Asp Ser Ser
Arg Leu Pro Val Lys Trp Thr Ala Pro Glu 380 385 390 gct ctc aaa cac
ggg ttc acc agc aag tcg gat gtc tgg agt ttt ggg 1492 Ala Leu Lys
His Gly Phe Thr Ser Lys Ser Asp Val Trp Ser Phe Gly 395 400 405 410
gtg ctg ctc tgg gag gtc ttc tca tat gga cgg gct ccg tac cct aaa
1540 Val Leu Leu Trp Glu Val Phe Ser Tyr Gly Arg Ala Pro Tyr Pro
Lys 415 420 425 atg tca ctg aaa gag gtg tcg gag gcc gtg gag aag ggg
tac cgc atg 1588 Met Ser Leu Lys Glu Val Ser Glu Ala Val Glu Lys
Gly Tyr Arg Met 430 435 440 gaa ccc ccc gag ggc tgt cca ggc ccc gtg
cac gtc ctc atg agc agc 1636 Glu Pro Pro Glu Gly Cys Pro Gly Pro
Val His Val Leu Met Ser Ser 445 450 455 tgc tgg gag gca gag ccg ccc
gcc ggc cac cct tcc gca aac tgg ccg 1684 Cys Trp Glu Ala Glu Pro
Pro Ala Gly His Pro Ser Ala Asn Trp Pro 460 465 470 aga agc tgg ccc
ggg agc tac gca gtg cag gtg ccc cag cct ccg tct 1732 Arg Ser Trp
Pro Gly Ser Tyr Ala Val Gln Val Pro Gln Pro Pro Ser 475 480 485 490
cag ggc agg acg ccg acg gtc cac ctc gcc ccg aag cca gga gcc ctg
1780 Gln Gly Arg Thr Pro Thr Val His Leu Ala Pro Lys Pro Gly Ala
Leu 495 500 505 acc cca ccc ggt ggc cct tgg ccc cag agg acc gag aga
gtg gag agt 1828 Thr Pro Pro Gly Gly Pro Trp Pro Gln Arg Thr Glu
Arg Val Glu Ser 510 515 520 gcg gcg tgg ggg cac tga ccaggcccaa
ggagggtcca ggcgggcaag 1876 Ala Ala Trp Gly His 525 tcatcctcct
ggtgcccaca gcaggggctg gcccacgtag ggggctctgg gcggcccgtg 1936
gacaccccag acctgcgaag gatgatcgcc cgataaagac ggattctaag g 1987 2 527
PRT Homo sapiens 2 Met Ala Gly Arg Gly Ser Leu Val Ser Trp Arg Ala
Phe His Gly Cys 1 5 10 15 Asp Ser Ala Glu Glu Leu Pro Arg Val Ser
Pro Arg Phe Leu Arg Ala 20 25 30 Trp His Pro Pro Pro Val Ser Ala
Arg Met Pro Thr Arg Arg Trp Ala 35 40 45 Pro Gly Thr Gln Cys Ile
Thr Lys Cys Glu His Thr Arg Pro Lys Pro 50 55 60 Gly Glu Leu Ala
Phe Arg Lys Gly Asp Val Val Thr Ile Leu Glu Ala 65 70 75 80 Cys Glu
Asn Lys Ser Trp Tyr Arg Val Lys His His Thr Ser Gly Gln 85 90 95
Glu Gly Leu Leu Ala Ala Gly Ala Leu Arg Asp Gly Glu Ala Leu Ser 100
105 110 Ala Asp Pro Lys Leu Ser Leu Met Pro Trp Phe His Gly Lys Ile
Ser 115 120 125 Gly Gln Glu Ala Val Gln Gln Leu Gln Pro Pro Glu Asp
Gly Leu Phe 130 135 140 Leu Val Arg Glu Ser Ala Arg His Pro Gly Asp
Tyr Val Leu Cys Val 145 150 155 160 Ser Phe Gly Arg Asp Val Ile His
Tyr Arg Val Leu His Arg Asp Gly 165 170 175 His Leu Thr Ile Asp Glu
Ala Val Phe Phe Cys Asn Leu Met Asp Met 180 185 190 Val Glu His Tyr
Ser Lys Asp Lys Gly Ala Ile Cys Thr Lys Leu Val 195 200 205 Arg Pro
Lys Arg Lys His Gly Thr Lys Ser Ala Glu Glu Glu Leu Ala 210 215 220
Arg Ala Gly Trp Leu Leu Asn Leu Gln His Leu Thr Leu Gly Ala Gln 225
230 235 240 Ile Gly Glu Gly Glu Phe Gly Ala Val Leu Gln Gly Glu Tyr
Leu Gly 245 250 255 Gln Lys Val Ala Val Lys Asn Ile Lys Cys Asp Val
Thr Ala Gln Ala 260 265 270 Phe Leu Asp Glu Thr Ala Val Met Thr Lys
Met Gln His Glu Asn Leu 275 280 285 Val Arg Leu Leu Gly Val Ile Leu
His Gln Gly Leu Tyr Ile Val Met 290 295 300 Glu His Val Ser Lys Gly
Asn Leu Val Asn Phe Leu Arg Thr Arg Gly 305 310 315 320 Arg Ala Leu
Val Asn Thr Ala Gln Leu Leu Gln Phe Ser Leu His Val 325 330 335 Ala
Glu Gly Met Glu Tyr Leu Glu Ser Lys Lys Leu Val His Arg Asp 340 345
350 Leu Ala Ala Arg Asn Ile Leu Val Ser Glu Asp Leu Val Ala Lys Val
355 360 365 Ser Asp Phe Gly Leu Ala Lys Ala Glu Arg Lys Gly Leu Asp
Ser Ser 370 375 380 Arg Leu Pro Val Lys Trp Thr Ala Pro Glu Ala Leu
Lys His Gly Phe 385 390 395 400 Thr Ser Lys Ser Asp Val Trp Ser Phe
Gly Val Leu Leu Trp Glu Val 405 410 415 Phe Ser Tyr Gly Arg Ala Pro
Tyr Pro Lys Met Ser Leu Lys Glu Val 420 425 430 Ser Glu Ala Val Glu
Lys Gly Tyr Arg Met Glu Pro Pro Glu Gly Cys 435 440 445 Pro Gly Pro
Val His Val Leu Met Ser Ser Cys Trp Glu Ala Glu Pro 450 455 460 Pro
Ala Gly His Pro Ser Ala Asn Trp Pro Arg Ser Trp Pro Gly Ser 465 470
475 480 Tyr Ala Val Gln Val Pro Gln Pro Pro Ser Gln Gly Arg Thr Pro
Thr 485 490 495 Val His Leu Ala Pro Lys Pro Gly Ala Leu Thr Pro Pro
Gly Gly Pro 500 505 510 Trp Pro Gln Arg Thr Glu Arg Val Glu Ser Ala
Ala Trp Gly His 515 520 525 3 147 DNA Homo sapiens CDS (1)...(150)
3 gga tcc att cac aga gac cta gca gca cgc aac atc ctg gtc tca gag
48 Gly Ser Ile His Arg Asp Leu Ala Ala Arg Asn Ile Leu Val Ser Glu
1 5 10 15 gac ctg gta acc aag gtc agc gac ttt ggc ctg gcc aaa gcc
gag cgg 96 Asp Leu Val Thr Lys Val Ser Asp Phe Gly Leu Ala Lys Ala
Glu Arg 20 25 30 aag ggg cta gac tca agc cgg ctg ccc gtc aaa tgg
atg gct ccc gaa 144 Lys Gly Leu Asp Ser Ser Arg Leu Pro Val Lys Trp
Met Ala Pro Glu 35 40 45 ttc 147 Phe 4 49 PRT Homo sapiens 4 Gly
Ser Ile His Arg Asp Leu Ala Ala Arg Asn Ile Leu Val Ser Glu 1 5 10
15 Asp Leu Val Thr Lys Val Ser Asp Phe Gly Leu Ala Lys Ala Glu Arg
20 25 30 Lys Gly Leu Asp Ser Ser Arg Leu Pro Val Lys Trp Met Ala
Pro Glu 35 40 45 Phe 5 8 PRT Artificial Sequence Peptide Flag 5 Asp
Tyr Lys Asp Asp Asp Asp Lys 1 5
* * * * *