U.S. patent application number 14/847545 was filed with the patent office on 2016-07-07 for identification of non-small cell lung carcinoma (nsclc) tumors expressing pdgfr-alpha.
The applicant listed for this patent is Cell Signaling Technology, Inc.. Invention is credited to Katherine Eleanor Crosby, Ailan Guo, Kimberly A Lee, Roberto Polakiewicz, Klarisa Rikova, Quingfu Zeng.
Application Number | 20160193208 14/847545 |
Document ID | / |
Family ID | 37604774 |
Filed Date | 2016-07-07 |
United States Patent
Application |
20160193208 |
Kind Code |
A1 |
Rikova; Klarisa ; et
al. |
July 7, 2016 |
Identification of Non-Small Cell Lung Carcinoma (NSCLC) Tumors
Expressing PDGFR-alpha
Abstract
The invention discloses a previously unidentified subset of
mammalian non-small cell lung carcinomas (NSCLC) in which
platelet-derived growth factor receptor alpha (PDGFR.alpha.) is
expressed and is driving the disease, and provides methods for
identifying a mammalian NSCLC tumor that belongs to a subset of
NSCLC tumors in which PDGFR.alpha. is expressed, and for
identifying a NSCLC tumor that is likely to respond to a
PDGFR.alpha.-inhibiting therapeutic. The invention also provides
methods for inhibiting the progression of a mammalian NSCLC tumor
in which PDGFR.alpha. is expressed, and for determining whether a
compound inhibits the progression of a PDGFR.alpha.-expressing
mammalian NSCLC tumor.
Inventors: |
Rikova; Klarisa; (Reading,
MA) ; Polakiewicz; Roberto; (Lexington, MA) ;
Guo; Ailan; (Lexington, MA) ; Crosby; Katherine
Eleanor; (Middleton, MA) ; Zeng; Quingfu;
(Hamilton, MA) ; Lee; Kimberly A; (Boston,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Cell Signaling Technology, Inc. |
Danvers |
MA |
US |
|
|
Family ID: |
37604774 |
Appl. No.: |
14/847545 |
Filed: |
September 8, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13780982 |
Feb 28, 2013 |
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14847545 |
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12982490 |
Dec 30, 2010 |
8466160 |
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13780982 |
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11174051 |
Jul 1, 2005 |
7932044 |
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12982490 |
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Current U.S.
Class: |
514/252.18 ;
435/7.23 |
Current CPC
Class: |
A61K 31/506 20130101;
G01N 33/6893 20130101; G01N 2333/71 20130101; G01N 33/57423
20130101 |
International
Class: |
A61K 31/506 20060101
A61K031/506; G01N 33/574 20060101 G01N033/574 |
Claims
1. A method for identifying a mammalian non-small cell lung
carcinoma (NSCLC) tumor that belongs to a subset of NSCLC tumors in
which platelet-derived growth factor receptor alpha (PDGFR.alpha.)
is expressed, said method comprising the step of determining
whether PDGFR.alpha. is expressed in a biological sample comprising
cells from a NSCLC tumor using at least one PDGFR.alpha. specific
reagent, wherein expression of PDGFR.alpha. in said biological
sample identifies said NSCLC tumor as belonging to a subset of
NSCLC tumors in which PDGFR.alpha. is expressed.
2. The method of claim 1, wherein said mammal is a human.
3. The method of claim 1, wherein identifying said NSCLC tumor as
belonging to a subset of NSCLC tumors in which PDGFR.alpha. is
expressed Identifies said NSCLC tumor as being likely to respond to
a composition comprising at least one PDGFR.alpha.-inhibiting
therapeutic.
4. The method of claim 3, wherein said PDGFR.alpha.-inhibiting
therapeutic comprises a small molecule inhibitor of
PDGFR.alpha..
5. The method of claim 4, wherein said small molecule inhibitor of
PDGFR.alpha. is Imatinib mesylate (STI-571).
6. The method of claim 4 wherein said small molecule inhibitor of
PDGFR.alpha. is an Imatinib mesylate (STI-571) analogue.
7. The method of claim 6, wherein said Imatinib mesylate (STI-571)
analogue is selected from the group consisting of BAY 43-93006,
XL-999 and SU11248.
8. The method of claim 1, wherein said biological sample comprises
cells obtained from a tumor biopsy, a tumor fine needle aspirate,
or a pleural effusion.
9. The method of claim 1, wherein said PDGFR.alpha.-specific
reagent comprises a PDGFR.alpha.-specific antibody.
10. The method of claim 9, wherein said PDGFR.alpha.-specific
antibody is a phosphorylation site-specific antibody.
11. The method of claim 1, wherein said PDGFR.alpha.-specific
reagent comprises a heavy-isotope labeled peptide (AQUA peptide)
that corresponds to a PDGFR.alpha. peptide sequence.
12. The method of claim 11, wherein said AQUA peptide is a peptide
having a sequence selected from the group consisting of SEQ ID NOs:
3-8.
13. The method of claim 1, wherein said method is implemented in a
flow-cytometry (FC), immuno-histochemistry (IHC), or
immuno-fluorescence (IF) assay format.
14. A method for determining whether a compound inhibits the
progression of a mammalian NSCLC tumor belonging to a subset of
NSCLC tumors in which PDGFR.alpha. is expressed, said method
comprising the step of determining whether said compound inhibits
the expression and/or activity of PDGFR.alpha. in said NSCLC
tumor.
15. The method of claim 14, wherein inhibition of expression and/or
activity of PDGFR.alpha. is determined by examining a biological
sample comprising cells from said NSCLC tumor.
16. The method of claim 14, wherein inhibition of expression and/or
activity of PDGFR.alpha. is determined using at least one
PDGFR.alpha.-specific reagent.
17. The method of claim 16, wherein said PDGFR.alpha.-specific
reagent comprises a phosphorylation-site specific antibody.
18. A method for inhibiting the progression of a mammalian NSCLC
tumor belonging to a subset of NSCLC tumors in which PDGFR.alpha.
is expressed, said method comprising the step of inhibiting the
expression and/or activity of PDGFR.alpha. in said tumor.
19. The method of claim 18, wherein expression and/or activity of
PDGFR.alpha. is inhibited with composition comprising Imatinib
mesylate (STI-571) and/or an analogue of Imatinib mesylate
(STI-571).
20. The method of claim 19, wherein said analogue is selected from
the group consisting of BAY 43-93006, XL-999 and SU11248.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
13/780,982, filed on Feb. 28, 2013 (pending), which is a
continuation of U.S. Ser. No. 12/982,490, filed on Dec. 30, 2010
(now issued as U.S. Pat. No. 8,466,160), which is a divisional
application of U.S. Ser. No. 11/174,051, filed on Jul. 1, 2005 (now
issued as U.S. Pat. No. 7,932,044), the entire contents of each of
which are hereby incorporated by reference.
FIELD OF THE INVENTION
[0002] The invention relates generally to cancer and antibodies,
and the use of antibodies in characterizing cancer.
BACKGROUND OF THE INVENTION
[0003] Many cancers are characterized by disruptions in cellular
signaling pathways that lead to aberrant control of cellular
processes, or to uncontrolled growth and proliferation of cells.
These disruptions are often caused by changes in the
phosphorylation state, and thus the activity of, particular
signaling proteins. Among these cancers is non-small cell lung
carcinoma (NSCLC). NSCLC is the leading cause of cancer death in
the United States, and accounts for about 87% of all lung cancers.
There are about 151,000 new cases of NSCLC in the United States
annually, and it is estimated that over 120,000 patients will die
annually from the disease in the United States alone. See "Cancer
Facts and Figures 2003," American Cancer Society. NSCLC, which
comprises three distinct subtypes, is often only detected after it
has metastasized, and thus the mortality rate is 75% within two
years of diagnosis.
[0004] NSCLC, like most cancers, involves defects in signal
transduction pathways. Receptor tyrosine kinases (RTKs) play a
pivotal role in these signaling pathways, transmitting
extracellular molecular signals into the cytoplasm and/or nucleus
of a cell. Among such RTKs are the receptors for polypeptide growth
factors such as epidermal growth factor (EGF), insulin,
platelet-derived growth factor (PDGF), neurotrophins (i.e., NGF),
and fibroblast growth factor (FGF). Phosphorylation of such RTKs
activates their cytoplasmic domain kinase function, which in turns
activates downstream signaling molecules. Thus, RTKs are key
mediators of cellular signaling as well as oncogenesis resulting
from over-expression and activation of such RTKs and their
substrates. Due to their pivotal role in normal and aberrant
signaling, RTKs are the subject of increasing focus as potential
drug targets for the treatment of certain types of cancer. For
example, Herceptin.RTM., an inhibitor of HER2/neu, is currently an
approved therapy for a certain subset of breast cancer. Iressa.TM.
(ZD1839) and Tarceva.TM. (OSI-774), both small-molecule inhibitors
of EGFR, have been approved for the treatment of NSCLC.
[0005] Platelet-derived growth factor (PDGF) and its receptors
(PDGFRs) are a family of RTKs that play an important role in the
regulation of normal cell growth and differentiation. PDGFRs are
involved in a variety of pathological processes, including
atherosclerosis, neoplasia, tissue repair, and inflammation (see,
e.g. Ross et al., Cell 46: 155-169 (1986); Ross et al., Adv. Exp
Med. Biol. 234: 9-21 (1988)). PDGFRs, which consist of two isoforms
(alpha (a) and beta ((3)), are membrane protein-tyrosine kinases
that, upon binding to PDGF, become activated and, via recruitment
of SH2 domain-containing effector molecules, initiate distinct or
overlapping signaling cascades that coordinate cellular
responses.
[0006] Expression of a constitutively active PDGFR leads to
cellular transformation (see Gazit et al., Cell 39: 89-97 (1984))
and suggests that, in normal cells, PDGFR activity must be tightly
regulated to oppose continuous activation of its downstream
effectors. PDGFR beta, for example, is known to be over-expressed
in a large number of tumors, and PDGF treatment causes
transformation and malignant tumors in a variety of experimental
systems (reviewed in Heldin et al., Physiol. Rev. 79(4): 1283-1316
(1999)). It has therefore been proposed that over-expression or
constitutive activation of the PDGF receptors plays a role in the
origin or tumorigenesis of certain cancer cells. It has been
reported that PDGFR is activated by a fusion to the transcription
factor TEL (see Ide et al., PNAS 99(22): 14404-14409 (2002)) in a
subset of patients with chronic myelomonocytic leukemia (CML).
PDGFR activation has also been implicated in growth of certain
solid tumors, such as glioblastoma (see, e.g. Vassbotn et al., J.
Cell. Physiol. 158: 381-389 (1994)).
[0007] Accordingly, inhibition of PDGFR and its downstream pathway
has become an area of increasing focus for drug development.
Specific inhibitors of PDGFR, such as the small-molecule drug
Gleevec (STI-571; Imatinib mesylate), have recently been developed
and are in clinical trials for treatment of certain cancers,
including prostate and ovarian cancers. It has been shown that
Gleevec.RTM. induces durable responses in patients with chronic
myelo-proliferative diseases associated with activation of PDGFR
(see Apperley et al., N. Engl. J. Med 347(7): 481-7 (2002)).
However, while PDGFR expression has been linked to the progression
of a few cancers, such as CML and glioblastoma, this association
has not been made in many other types of cancers. Similarly,
although certain signaling defects underlying progression of NSCLC
have been identified (including EGFR over-expression), the precise
molecular mechanisms driving this disease are not completely
understood.
[0008] One study reported the apparent expression of PDGFR alpha
(a) in nearly 100% of human lung cancer tumors examined, and
reported the growth inhibition of a lung cancer cell line, A549, by
Gleevec.RTM., an effect that was surmised to be mediated via PDGFR
inhibition (see Zhang et al., Mol. Cancer 2(1): 1-10 (2003)). The
report, however, was inconclusive since the antibody employed in
the study was later shown (by the present inventors) to be
non-specific, and cross-reacts with a variety of proteins other
than PDGFR.alpha.; thus it is unclear which protein(s) was/were
actually being detected in the Zhang study. Moreover, PDGFR.alpha.
is not detectable in the A549 cell line employed in that
study--which is consistent with the present inventors' inability to
reproduce the growth inhibition of this cell line by
Gleevec.RTM.--further evidencing that the observation reported in
Zhang was either erroneous or was mediated by some mechanism other
than expression and inhibition of PDGFR.alpha..
[0009] Since the new generation of targeted therapeutics against
RTKs like PDGFR and EGFR are highly specific, there is a continuing
and imperative need to identify the particular tumors that are, in
fact, being driven by the RTK being targeted by these drugs, since
such tumors are most likely to respond to the inhibitor. It is now
widely accepted that most types of cancer have distinct subsets of
tumors, which are being driven by different signaling pathways. For
example, two distinct subsets of breast cancer are known to exist,
one driven by Her2/Neu signaling and the other by EGFR signaling,
but only the former is responsive to the Her2-targeted therapeutic
Herceptin.RTM.. It is likely that most types of cancer, including
those in which an RTK has already been identified (and targeted) as
a driver of the disease, will in fact have multiple subtypes being
driven by other, presently unknown RTKs and pathways. Indeed, the
modest response rates thus far observed in clinical trials of
several highly specific targeted therapeutics (including those
against EGFR and PDGFR) evidence that many of the cancers being
treated may, in fact, comprise subgroups being driven by
alternative RTKs and pathways that are not being adequately
targeted.
[0010] Accordingly, there is a continuing and pressing need to
identify the particular signaling molecules, including RTKs, whose
expression and/or activation is, in fact, driving a certain type of
cancer (or a subset of that cancer). Identification of such
signaling molecules will enable the development of new and improved
diagnostic and/or prognostic assays to help ensure a particular
patient gets a targeted therapeutic most likely to be effective
against their disease, as well as providing novel drug targets for
treatment of these cancers. Some cancers, like NSCLC, are often not
detected until after the disease has already metastasized, making
prompt and effective treatment paramount. Therefore, the ability to
identify subgroups of cancers that are being driven by
presently-untargeted RTKs and signaling pathways would greatly
assist in developing alternative and more beneficial therapeutic
strategies, and to avoiding prescribing ineffective therapies to
patients who are not likely to respond to them.
SUMMARY OF THE INVENTION
[0011] In accordance with the invention, a previously unknown
subset of mammalian non-small cell lung carcinoma (NSCLC) tumors in
which platelet-derived growth factor receptor alpha (PDGFR.alpha.)
is expressed, and driving the disease, has now been identified. The
ability to identify NSCLC tumors in which PDGFR.alpha. is expressed
and is driving the disease enables the identification of NSCLC
tumors that are likely to respond to inhibitors of PDGFR.alpha.,
such as Imatinib mesylate (STI-571; Gleevec.RTM.). The invention
thus provides methods for identifying a mammalian NSCLC tumor that
belongs to a subset of NSCLC tumors in which PDGFR.alpha. is
expressed, and for identifying a NSCLC tumor that is likely to
respond to a PDGFR.alpha.-inhibiting therapeutic. The invention
also provides methods for determining whether a compound inhibits
progression of a NSCLC tumor expressing PDGFR.alpha., and for
inhibiting the progression of a mammalian NSCLC tumor in which
PDGFR.alpha. is expressed by inhibiting the activity of
PDGFR.alpha..
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0013] FIG. 1--is the amino acid sequence (1-letter code) of human
PDGFR alpha (a) (SEQ ID NO: 1) (SwissProt Accession No.
P16234).
[0014] FIG. 2--is a graphic presentation of human PDGFR.alpha.
kinase with the known/reported tyrosine phosphorylation sites
labeled.
[0015] FIG. 3--is the DNA sequence encoding human PDGFR.alpha.
(Accession No. NM 006206).
[0016] FIG. 4--consists of a Western blot analysis of extracts from
human NSCLC cell lines using various antibodies made against
PDGFR.alpha., demonstrating that some commercially available
antibodies are not in fact specific for PDGFR.alpha..
[0017] FIG. 5--consists of two Western blot analyses of extracts
from three different cell lines using different anti-PDGFR.alpha.
antibodies, demonstrating that some commercially available
antibodies falsely detect PDGFR.alpha. in A549 cells.
[0018] FIG. 6--is a Western blot analysis of extracts from two
NSCLC cell lines induced with PDGFaa using antibodies made against
PDGFR.alpha., phosphoPDGFR.alpha. and for the downstream kinase,
phospho-AKT, demonstrating that the H1703 cell line expresses
PDGFR.alpha. that may be activated by PDGFaa while the A549 cell
line does not express the receptor and is not responsive to
PDGFaa.
[0019] FIG. 7--is an IHC analysis of H1703 xenograft samples probed
with two antibodies made against PDGFR.alpha.. The results
demonstrate that one of the commercial antibodies detects
non-specific staining in the xenografts, consistent with the
Western results on the cell lines.
[0020] FIG. 8--presents the effects of Gleevec.RTM. treatment on
cell growth and cell apoptosis in NSCLC cell lines. Panel A
presents growth curves for four cell lines with increasing
concentrations of Gleevec.RTM. demonstrating that the H1703 cell
line is sensitive to Gleevec.RTM.. Panel B presents Western blot
results demonstrating that Gleevec.RTM. induces apoptosis in the
H1703 cell line as shown by the cleavage of PARP. Panel C is a bar
graph showing that apoptosis is induced in H1703 cells by
administration of Gleevec.RTM., as determined by presence of
cleaved Caspase-3 using flow cytometry.
[0021] FIG. 9--is a Western blot analysis of extracts from H1703
cells treated with EGF, Gleevec.RTM. and Iressa.RTM.. The results
demonstrate that the cell line has PDGFR.alpha. constitutively
activated leading to AKT activation and that this activation may be
inhibited by Gleevec.RTM. but not by Iressa.RTM..
[0022] FIG. 10--depicts the inhibition of PDGFR.alpha.-expressing
NSCLC tumor xenografts, in mice, by Gleevec.RTM.. Panel A is a
graph showing reduction in tumor volume in mice treated with this
PDGFR.alpha. inhibitor. Panel B is a Western blot analysis of tumor
cell extracts from the mouse xenografts demonstrating that exposure
of the xenograft to Gleevec.RTM. correlates with loss of
PDGFR.alpha. phosphorylation, and no change in total AKT
levels.
[0023] FIG. 11--is an immunohistochemical (IHC) analysis of cells
from mouse NSCLC tumor xenografts (expressing PDGFR.alpha.) either
treated (panel B) or untreated (panel A) with the
PDGFR.alpha.-inhibitor Gleevec.RTM. (STI-571) demonstrating that
exposure to Gleevec results in a significant decrease in
phosphorylation of PDGFR.alpha. and AKT while the total level of
the receptor does not change.
DETAILED DESCRIPTION OF THE INVENTION
[0024] In accordance with the invention, a previously unknown
subset of mammalian non-small cell lung carcinoma (NSCLC) tumors in
which platelet-derived growth factor receptor alpha (PDGFR.alpha.)
is expressed and is driving the disease has presently been
identified. Epidermal growth factor receptor (EGFR)
expression/activation is known to occur in many NSCLC tumors (see
Neal et al., supra.; Sainsbury et al., supra.) and the receptor is
presently a therapeutic target for the treatment of NSCLC. However,
although activation of PDGFR is known to drive a subset of certain
cancers, e.g. prostrate cancer, the association of PDGFR.alpha.
expression in a subset of NSCLC tumors has not previously been
conclusively reported.
[0025] A previous study (Zhang et al., supra.) reported the
apparent expression of PDGFR.alpha. in nearly 100% of human lung
cancer tumors examined. This study also reported that Gleevec.RTM.
(STI-571), a small molecule inhibitor with activity against PDGFR,
could inhibit the progression of a lung cancer cell line, A549, an
effect that was thus surmised by the authors to be mediated via
inhibition of PDGFR.alpha. in these cells. However, as presently
shown (see Example 2), the possible antibodies employed in the
study (obtained from Santa Cruz Biotechnology) are not specific for
PDGFR.alpha. and in fact cross-react with a variety of other
proteins. Accordingly, it is unclear which protein(s) was/were
actually detected in the Zhang study and whether PDGFR.alpha. was
expressed in the examined cells at all. Indeed, as presently shown
(see Example 2), the A549 cell line employed in the Zhang study
does not, in fact, express detectable PDGFR.alpha.. The lack of
PDGFR.alpha. expression in this cell line is consistent with the
present finding that Gleevec.RTM. does not, in fact, inhibit growth
of this cell line, further evidencing that the observation reported
in Zhang was either erroneous or was mediated by some mechanism
other than PDGFR.alpha. inhibition.
[0026] The present discovery is surprising since a link between
PDGFR.alpha. expression and a subset of NSCLC tumors has not, until
now, been conclusively established, despite significant therapeutic
development activity for this cancer. The identification of a
distinct subset of NSCLC tumors in which PDGFR.alpha. is expressed
has important implications for the management and treatment of this
prevalent disease. NSCLC is the leading cause of cancer death in
the United States, and is often difficult to diagnose until after
it has metastasized, increasing the difficulty of effectively
treating or curing this disease. The mortality rate of NSCLC is
therefore 75% within two years of diagnosis. See American Cancer
Society, supra. Although targeted EGFR-inhibitors are presently
approved for the treatment of NSCLC, it is likely that this therapy
will be ineffective against the subgroup of patients having tumors
in which PDGFR.alpha. (rather than or in addition to EGFR) is
expressed and driving the disease, in whole or in part.
[0027] The present discovery that a subset of NSCLC tumors is being
driven by the expression of PDGFR.alpha. enables important new
methods for accurately identifying mammalian NSCLC tumors in which
PDGFR.alpha. is expressing, as these tumors are likely to respond
to PDGFR.alpha.-inhibiting therapeutics, such as Imatinib mesylate
(STI-571; Gleevec.RTM.). The ability to identify such tumors as
early as possible will greatly assist in clinically determining
which therapeutic, or combination of therapeutics, will be most
appropriate for a particular NSCLC tumor, thus helping to avoid
prescription of EGFR-inhibitors in cases where such inhibitors are
likely to be partially or wholly ineffective (i.e. where receptors
other than the one targeted are driving the disease, in whole or in
part, in the tumor). Therefore, the invention provides, in part,
methods for identifying a mammalian NSCLC tumor that belongs to a
subset of NSCLC tumors in which PDGFR.alpha. is expressed. The
identification of such a tumor identifies the tumor as being likely
to respond to a composition comprising one or more
PDGFR.alpha.-inhibiting therapeutics, such as Gleevec.RTM..
[0028] The invention also provides a method for determining whether
a compound inhibits the progression of a mammalian NSCLC tumor
belonging to a subset of NSCLC tumors in which PDGFR.alpha. is
expressed, by determining whether the compound inhibits the
expression or activity of PDGFR.alpha. in such NSCLC tumor. Further
provided by the invention is a method for inhibiting the
progression of a mammalian NSCLC tumor in which PDGFR.alpha. is
expressed by inhibiting the expression or activity of
PDGFR.alpha..
[0029] The further aspects, advantages, and embodiments of the
invention are described in more detail below. All references cited
herein are hereby incorporated by reference.
A. PDGFR.alpha.-Expressing Subset of NSCLC Tumors.
[0030] A distinct subset of human NSCLC tumors in which
PDGFR.alpha. is expressed and driving the disease was identified,
surprisingly, during examination of global phosphorylated peptide
profiles in extracts from known human NSCLC tumor cell lines
including four cells lines; the A549, H441, H1373 and H1703 cell
line. The phosphorylation profiles of these cell lines were
elucidated using a recently described technique for the isolation
and mass spectrometric characterization of modified peptides from
complex mixtures (see U.S. Patent Publication No. 20030044848, Rush
et al., "Immunoaffinity Isolation of Modified Peptides from Complex
Mixtures" (the "IAP" technique), as further described in Example 1
herein. Application of the IAP technique using a
phosphotyrosine-specific antibody (CELL SIGNALING TECHNOLOGY, INC.,
Beverly, Mass., 2003/04 Cat. #9411), identified that the H1703 cell
expressed PDGFR.alpha., in contrast to the other cell lines, which
lacked PDGFR.alpha. but often expressed EGFR (Table 1, Example 1
lists the PDGFR.alpha. phosphosites only observed in the H1703 cell
line). This novel finding indicated the existence of a previously
unidentified subset of NSCLC tumors in which PDGFR.alpha. was
expressed, and that this subset of tumors would likely survive
despite clinically targeting only the EGFR pathway.
[0031] This initial finding was then confirmed by
immunohistochemical (NC) analysis of a tissue microarray comprising
tumor biopsy tissue samples from 304 different human NSCLC
patients, as further described in Example 3 below. Seventeen (17)
out of 305 tumors (or 6% of all tumors examined) belonged to the
PDGFR.alpha.-expressing subset, indicating that incidence of this
subset of NSCLC tumors is rare (see Table 2 in Example 3). Within
the PDGFR.alpha.-expressing subset of NSCLC tumors, adenocarcinomas
and bronchioloalveolar carcinomas account for 76% (11 out of 17) of
the tumors, and PDGFR.alpha.-expressing NSCLC tumors occur more
often in women (65%, 11 out of 17) than in men.
[0032] The low frequency of PDGFR.alpha.-expressing NSCLC tumors
(in the large patient sample population examined) disclosed herein
starkly contrasts with the nearly 100% frequency reported in Zhang
et al., supra., using a small (33 sample) patient population. This
contrast indicates that the earlier report in Zhang was either
erroneous, or resulted from the use of an antibody that was not in
fact PDGFR.alpha.-specific, but rather binds multiple other
proteins (see Example 2 below). Indeed, as presently shown (in
Example 2), the A549 NSCLC cell line utilized in Zhang does not
appreciably express PDGFR.alpha. and the growth of this cell line
is not inhibited by the PDGFR.alpha.-inhibiting compound,
Gleevec.RTM. (STI571). See FIG. 10.
[0033] Inhibition of PDGFR activation and downstream signaling was
then demonstrated on the H1703 cell line (see FIG. 9). In the same
figure, results indicate that the constitutive cellular signal
transduction in these cells is not affected by Iressa.TM. or EGFR
inhibition. These results suggest that Gleevec treatment may
inhibit tumor growth in tumors that are driven by PDGFR.alpha..
This hypothesis was tested using mouse xenografts derived from the
H1703 cell line. Indeed, inhibition of PDGFR.alpha. activity in
vivo was shown by treating mouse xenografts harboring
PDGFR.alpha.-expressing human NSCLC tumors (H1703) with a small
molecule targeted inhibitor of PDGFR.alpha., Gleevec.RTM. (STI-571)
as shown in FIGS. 9 and 10.
[0034] The ability to selectively identify NSCLC tumors that belong
to a subset of NSCLC tumors in which PDGFR.alpha. is expressed and
driving the disease (in whole or in part) enables important new
methods for accurately identifying such tumors for diagnostic
purposes, as well as obtaining information useful in determining
whether such a tumor is likely to respond to a
PDGFR.alpha.-inhibiting therapeutic composition, or likely to be
partially or wholly non-responsive to an EGFR inhibitor when
administered as a single agent for the treatment of NSCLC.
[0035] Accordingly, in one embodiment, the invention provides a
method for identifying a mammalian non-small cell lung carcinoma
(NSCLC) tumor that belongs to a subset of NSCLC tumors in which
platelet-derived growth factor receptor alpha (PDGFR.alpha.) is
expressed, said method comprising the step of determining whether
PDGFR.alpha. is expressed in a biological sample comprising cells
from a NSCLC tumor using at least one PDGFR.alpha.-specific
reagent, wherein expression of PDGFR.alpha. in said biological
sample identifies said NSCLC tumor as belonging to a subset of
NSCLC tumors in which PDGFR.alpha. is expressed.
[0036] Biological samples useful in the practice of the present
invention are described in further detail in section B below. In
one preferred embodiment, the mammalian NSCLC tumor is a human
tumor, while in other preferred embodiments the mammal is a dog, a
cat, or a horse. In other preferred embodiments, the biological
sample comprises cells (or lysates of cells) obtained from a tumor
biopsy, a tumor fine needle aspirate, or a pleural effusion. In
another preferred embodiment, identifying the NSCLC tumor as
belonging to a subset of NSCLC tumors in which PDGFR.alpha. is
expressed identifies the NSCLC tumor as being likely to respond to
a composition comprising at least on PDGFR.alpha.-inhibiting
therapeutic. PDGFR.alpha.-inhibiting therapeutics useful in the
practice of the present invention is described in further detail in
section F below. In one preferred embodiment, the
PDGFR.alpha.-inhibiting therapeutic comprises a small molecule
inhibitor of PDGFR.alpha.. In other preferred embodiments, the
small molecule inhibitor of PDGFR.alpha. is Imatinib mesylate
(STI-571; Gleevec.RTM.) or its analogues, while in another
preferred embodiment, the small molecule inhibitor of PDGFR.alpha.
is selected from the group consisting of BAY 43-93006, XL-999 and
SU11248.
[0037] PDGFR.alpha.-specific reagents useful in the practice of the
methods of the invention are described in further detail in section
C below. In one preferred embodiment, the PDGFR.alpha.-specific
reagent comprises a PDGFR.alpha.-specific antibody. Such antibody
may, in one preferred embodiment, be a phosphorylation
site-specific antibody. In another preferred embodiment, the
PDGFR.alpha.-specific reagent comprises a heavy-isotope labeled
phosphopeptide (AQUA peptide) corresponding to a PDGFR.alpha.
peptide sequence (which may correspond to a phosphorylation site
within PDGFR.alpha.).
[0038] The method of the invention described above may also
optionally comprise the step of determining the level of activated
or expressed epidermal growth factor receptor (EGFR) in said
biological sample. Profiling both PDGFR.alpha.
expression/activation and EGFR expression/activation in a given
NSCLC tumor can provide valuable information on which pathway, or
pathways, is/are driving the disease, and which therapeutic regime
is therefore likely to be of most benefit.
[0039] The ability to identify a mammalian NSCLC tumor belonging to
a subset of NSCLC tumors in which PDGFR.alpha. is activated can
provide clinical information that is valuable to assessing whether
a patient's tumor is likely to respond to a particular therapeutic
(for example, an EGFR inhibitor or a PDGFR.alpha. inhibitor).
Accordingly, in one preferred embodiment of the above-described
method, identifying a NSCLC tumor as belonging to a subset of NSCLC
tumors in which PDGFR.alpha. is activated identifies the tumor as
likely to respond to a composition comprising at least one
PDGFR.alpha.-inhibiting therapeutic. PDGFR.alpha.-inhibiting
therapeutics useful in the practice of the methods of the invention
is described in further detail in section E below.
[0040] PDGFR.alpha.-specific reagents, including antibodies and
AQUA peptides, useful in the practice of the methods of the
invention are described in further detail in section C below. In
one preferred embodiment, the PDGFR.alpha.-specific reagent is an
antibody, and in one preferred embodiment the antibody is a
phosphorylation site-specific antibody that specifically binds
PDGFR.alpha. only when phosphorylated. In another preferred
embodiment, the PDGFR.alpha.-specific reagent is a heavy-isotope
labeled phosphopeptide (AQUA peptide) corresponding to a
PDGFR.alpha. peptide sequence.
[0041] The newly identified, distinct subset of mammalian NSCLC
tumors in which PDGFR.alpha. is expressed and driving the disease
(in whole or in part)--as opposed to the subset of tumors in which
only EGFR is activated and driving the cancer--also has important
implications for the treatment of NSCLC. The progression of NSCLC
tumors belonging to the subset in which PDGFR.alpha. is driving the
disease may be inhibited or stopped by inhibiting the expression
and/or activity of PDGFR.alpha. (as opposed to targeting only EGFR,
which is likely to be wholly or partially ineffective against this
subset of tumors).
[0042] Accordingly, the invention also provides, in part, a method
for inhibiting the progression of a mammalian NSCLC tumor belonging
to a subset of NSCLC tumors in which PDGFR.alpha. is expressed,
said method comprising the step of inhibiting the activity and/or
expression of PDGFR.alpha. in the NSCLC tumor. In a preferred
embodiment, the activity of PDGFR.alpha. is inhibited by contacting
the tumor with a composition comprising at least one
PDGFR.alpha.-inhibiting therapeutic. Compositions and
PDGFR.alpha.-inhibiting compounds suitable for practice of the
method of the invention are described in further detail in section
E below. In one preferred embodiment, the PDGFR.alpha.-inhibiting
therapeutic comprises a small molecule inhibitor of PDGFR.alpha.,
and in some preferred embodiments, the small molecule inhibitor of
PDGFR.alpha. is Imatinib mesylate (STI-571, Gleevec.RTM.) or its
analogues. In other preferred embodiments, the small molecule
inhibitor of PDGFR.alpha. is selected from the group consisting of
BAY43-9006, XL-999 and SU11248. The NSCLC tumor may be contacted
with a therapeutically effective amount of such
PDGFR.alpha.-inhibiting therapeutic, in accordance with standard
dosing and administration approaches.
[0043] The invention also provides, in part, a method for
determining whether a compound inhibits the progression of a
mammalian NSCLC tumor belonging to a subset of NSCLC tumors in
which PDGFR.alpha. is expressed, the method comprising the step of
determining whether the compound inhibits the expression and/or
activity of PDGFR.alpha. in said NSCLC tumor. In one preferred
embodiment, inhibition of activity of PDGFR.alpha. is determined by
examining a biological sample comprising cells from said NSCLC
tumor. In another preferred embodiment, inhibition of activity of
PDGFR.alpha. is determined using at least one PDGFR.alpha.
activation state-specific reagent, and in one preferred embodiment,
the activation-state specific reagent is a phosphorylation-site
specific antibody. The compound may, for example, be a kinase
inhibitor, such as a small molecule or antibody inhibitor.
PDGFR.alpha.-inhibiting compounds are discussed in further detail
in section E below. Patient biological samples may be taken before
and after treatment with the inhibitor and then analyzed, using
methods described below in section D, for the biological effect of
the inhibitor on PDGFR.alpha. phosphorylation or the
phosphorylation of downstream proteins. Such a pharmacodynamic
assay may be useful in determining the biologically active dose of
the drug which may be preferable to a maximal tolerable dose. Such
information would also be useful in submissions for drug approval
by demonstrating the mechanism of drug action.
DEFINITIONS
[0044] As used throughout this specification, the following terms
have the meanings indicated:
[0045] "cells from a NSCLC tumor" means whole cells or extracts of
cells from a NSCLC tumor or neoplasm.
[0046] "expression" or "expressed" with respect to PDGFR.alpha. in
a biological sample means significantly expressed as compared to
control sample in which PDGFR.alpha. is not significantly
expressed.
[0047] "PDGFR.alpha.-specific reagent" means any detectable
reagent, chemical or biological, which can specifically react with,
bind to, detect, and/or quantify PDGFR.alpha. in a biological
sample, and which does not substantially react with PDGFR beta
(.beta.) or other RTKs or kinases, as compared to the reagent's
reactivity to PDGFR.alpha..
[0048] "PDGFR.alpha.-inhibiting therapeutic" means any composition
comprising one or more compounds, chemical or biological, which
inhibits, either directly or indirectly, the expression and/or
activity of PDGFR.alpha. in vivo.
B. Biological Samples
[0049] Biological samples useful in the practice of the methods of
the invention may be obtained from any mammal in which a NSCLC
tumor is present or developing. In one embodiment, the mammal is a
human, and the human may be a candidate for a
PDGFR.alpha.-inhibiting therapeutic, for the treatment of NSCLC.
The human candidate may be a patient currently being treated with,
or considered for treatment with, an EGFR inhibitor, such as
Tarceva.TM. or Iressa.TM.. In another embodiment, the mammal is
large animal, such as a horse or cow, while in other embodiments,
the mammal is a small animal, such as a dog or cat, all of which
are known to develop NSCLC.
[0050] Any biological sample comprising cells (or extracts of
cells) from a mammalian NSCLC tumor is suitable for use in the
methods of the invention. In one embodiment, the biological sample
comprises cells obtained from a tumor biopsy. The biopsy may be
obtained, according to standard clinical techniques, from primary
NSCLC tumors occurring in the lung of a mammal, or by secondary
NSCLC tumors that have metastasized in other tissues. In another
embodiment, the biological sample comprises cells obtained from a
fine needle aspirate taken from a NSCLC tumor, and techniques for
obtaining such aspirates are well known in the art (see Cristallini
et al., Acta Cytol. 36(3): 416-22 (1992)).
[0051] In still another preferred embodiment, the biological sample
comprises cells obtained from a NSCLC pleural effusion. Pleural
effusions (liquid that forms outside the lung in the thoracic
cavity and which contains cancerous cells) are known to form in
many patients with advanced NSCLC, and the presence of such
effusion is predictive of a poor outcome and short survival time.
See Mott et al., Chest 119: 317-318 (2001). Effective and prompt
treatment is therefore particularly critical in such cases.
Standard techniques for obtaining pleural effusion samples have
been described and are well known in the art (see Sahn Clin Chest
Med. 3(2): 443-52 (1982)). Circulating NSCLC cells may also be
obtained from serum using tumor markers, cytokeratin protein
markers or other methods of negative selection as described (see Ma
et al. Anticancer Res. 23(1A): 49-62 (2003)).
[0052] A biological sample may comprise cells from a NSCLC tumor in
which PDGFR.alpha. is expressed and activated but EGFR is not.
Alternatively, the sample may comprise cells from a NSCLC tumor in
which both PDGFR.alpha. and EGFR.alpha. are expressed and
activated, or in which EGFR.alpha. is expressed and activated but
PDGFR.alpha. is not.
[0053] Cellular extracts of the foregoing biological samples may be
prepared, either crude or partially (or entirely) purified, in
accordance with standard techniques, and used in the methods of the
invention. Alternatively, biological samples comprising whole cells
may be utilized in preferred assay formats such as
immunohistochemistry (IHC), flow cytometry (FC), and
immunofluorescence (IF), as further described in section D below.
Such whole-cell assays are advantageous in that they minimize
manipulation of the tumor cell sample and thus reduce the risks of
altering the in vivo signaling/activation state of the cells and/or
introducing artifact signals. Whole cell assays are also
advantageous because they characterize expression and signaling
only in tumor cells, rather than a mixture of tumor and normal
cells.
[0054] In practicing the disclosed method for determining whether a
compound inhibits progression of a NSCLC tumor in which
PDGFR.alpha. is expressed, biological samples comprising cells from
mammalian xenografts may also be advantageously employed. Preferred
xenografts are small mammals, such as mice, harboring human NSCLC
tumors that express PDGFR.alpha.. Xenografts harboring human NSCLC
tumors are well known in the art (see Kal, Cancer Treat Res. 72:
155-69 (1995)) and the production of mammalian xenografts harboring
human tumors is well described (see Winograd et al., In Vivo. 1(1):
1-13 (1987)).
[0055] In assessing PDGFR.alpha. expression in a biological sample
comprising cells from a mammalian NSCLC tumor, a control sample
representing the background in vivo activation of PDGFR.alpha. may
desirably be employed for comparative purposes. Ideally, the
control sample comprises cells from a NSCLC tumor that is
representative of the subset of NSCLC tumors in which PDGFR.alpha.
is not expressed. Comparing the level of expressed PDGFR.alpha. in
control sample versus the test biological sample thus identifies
whether PDGFR.alpha. is expressed. Alternatively, since
PDGFR.alpha. is not expressed in the majority of NSCLC tumors (that
do not belong to the presently disclosed subset of tumors), any
tissue that similarly does not express PDGFR.alpha. may be employed
as a control.
[0056] The methods described above will have valuable diagnostic
utility for mammalian NSCLC tumors, and treatment decisions
pertaining to the same. For example, biological samples may be
obtained from a subject that has not been previously diagnosed as
having NSCLC, nor has yet undergone treatment for such cancer, and
the method is employed to diagnostically identify a NSCLC tumor in
such subject as belonging to a subset of NSCLC tumors in which
PDGFR.alpha. is expressed. Alternatively, a biological sample may
be obtained from a subject that has been diagnosed as having NSCLC
and has been receiving therapy, such as EGFR inhibitor therapy
(e.g. Tarceva.TM. Iressa.TM.) for treatment of such cancer, and the
method of the invention is employed to identify whether the
subject's NSCLC tumor belongs to a subset of NSCLC that is likely
to respond to such therapy and/or whether alternative or additional
PDGFR.alpha. inhibiting therapy is desirable or warranted. The
methods of the invention may also be employed to monitor the
progression or inhibition of a PDGFR.alpha.-expressing NSCLC tumor
following treatment of a subject with a composition comprising a
PDGFR.alpha.-inhibiting therapeutic or combination of
therapeutics.
[0057] Such diagnostic assay may be carried out subsequent to or
prior to preliminary evaluation or surgical surveillance
procedures. The identification method of the invention may be
advantageously employed as a diagnostic to identify NSCLC patients
having tumors driven by PDGFR.alpha., which patients would be most
likely to respond to therapeutics targeted at inhibiting
PDGFR.alpha. activity, such as STI-571 (Gleevec.RTM.) or its
analogues. The ability to select such patients would also be useful
in the clinical evaluation of efficacy of future PDGFR.alpha.
targeted therapeutics as well as in the future prescription of such
drugs to NSCLC patients.
C. PDGFR.alpha.-Specific Reagents
[0058] PDGFR.alpha.-activation state-specific reagents useful in
the practice of the disclosed methods include, among others,
PDGFR.alpha.-specific antibodies and AQUA peptides (heavy-isotope
labeled peptides) corresponding to, and suitable for detection and
quantification of, PDGFR.alpha. expression in a biological sample.
A PDGFR.alpha.-specific reagent is any reagent, biological or
chemical, capable of specifically binding to, detecting and/or
quantifying the presence/level of expressed PDGFR.alpha. in a
biological sample. The term includes, but is not limited to, the
preferred antibody and AQUA peptide reagents discussed below, and
equivalent reagents are within the scope of the present
invention.
[0059] Antibodies.
[0060] Antibodies suitable for use in practice of the methods of
the invention include a PDGFR.alpha.-specific antibody and a
PDGFR.alpha. phosphorylation site-specific antibody. A
PDGFR.alpha.-specific antibody is an isolated antibody or
antibodies that specifically bind(s) the PDGFR alpha (a) protein
(e.g. human, see SEQ ID NO: 1) regardless of phosphorylation state,
but including phosphorylated forms of the protein. A PDGFR.alpha.
phosphorylation site-specific antibody is an isolated antibody or
antibodies that specifically bind(s) PDGFR alpha (a) protein only
when phosphorylated at a particular tyrosine, serine, or threonine
residue, and does not substantially bind the unphosphorylated form
of the protein, or the protein when phosphorylated at a different
site than that for which the antibody is specific.
[0061] Human PDGFR.alpha.-specific, and phosphorylation
site-specific, antibodies may also bind to highly homologous and
equivalent epitopic peptide sequences in other mammalian species,
for example murine or rabbit PDGFR.alpha., and vice versa.
Antibodies useful in practicing the methods of the invention
include (a) monoclonal antibodies, (b) purified polyclonal
antibodies that specifically bind to the target protein (e.g. a
phosphorylated form of PDGFR.alpha.), (c) antibodies as described
in (a)-(c) above that bind equivalent and highly homologous
epitopes or phosphorylation sites in other non-human species (e.g.
mouse, rat), and (d) fragments of (a)-(c) above that bind to the
antigen (or more preferably the epitope) bound by the exemplary
antibodies disclosed herein.
[0062] The term "antibody" or "antibodies" as used herein refers to
all types of immunoglobulins, including IgG, IgM, IgA, IgD, and
IgE. The antibodies may be monoclonal or polyclonal and may be of
any species of origin, including (for example) mouse, rat, rabbit,
horse, or human, or may be chimeric antibodies. See, e.g., M.
Walker et al., Molec. Immunol. 26: 403-11 (1989); Morrision et al.,
Proc. Natl. Acad. Sci. 81: 6851 (1984); Neuberger et al., Nature
312: 604 (1984)). The antibodies may be recombinant monoclonal
antibodies produced according to the methods disclosed in U.S. Pat.
No. 4,474,893 (Reading) or U.S. Pat. No. 4,816,567 (Cabilly et al.)
The antibodies may also be chemically constructed specific
antibodies made according to the method disclosed in U.S. Pat. No.
4,676,980 (Segel et al.).
[0063] PDGFR.alpha.-specific antibodies are commercially available
(see Cell Signaling Technology, 2005 Catalogue, #3164, and Santa
Cruz Biotechnology, 2005 Catalogue, #338). Certain preferred
embodiments of the methods of the invention employ a
phosphorylation site-specific antibody that specifically binds
PDGFR.alpha. only when phosphorylated at a tyrosine known to be
relevant to protein activity, for example tyrosines 720 and 754 in
the human PDGFR.alpha. protein sequence (SEQ ID NO: 1). Some or all
of these phosphorylation-site specific antibodies are commercially
available (see Cell Signaling Technology 2005 Catalogue, #2992, and
Santa Cruz Biotechnology, 2005 Catalogue, #12911). The production
and use of PDGFR.alpha.-specific antibodies has been described.
See, e.g. U.S. Pat. No. 6,660,488, Dec. 9, 2003, Matsui et al.
[0064] The preferred epitopic site of a PDGFR.alpha.-specific
antibody of the invention is a peptide fragment consisting
essentially of about 11 to 17 amino acids of the human PDGFR.alpha.
protein sequence (SEQ ID NO: 1). For PDGFR.alpha. phosphorylation
site-specific antibodies, the epitope comprises the particular
phosphorylated residue (tyrosine, serine, or threonine), with about
5 to 9 amino acids positioned on each side of it (for example,
residues 746-762 of SEQ ID NO: 1, comprising the phosphotyrosine at
position 754). It will be appreciated that antibodies that
specifically binding shorter or longer peptides/epitopes within
PDGFR.alpha. are within the scope of the present invention. The
amino acid sequence of human PDGFR.alpha. has been published (see
FIG. 1 (SEQ ID NO: 1), as are the sequences of PDGFR.alpha. from
other species.
[0065] The invention is not limited to use of antibodies, but
includes equivalent molecules, such as protein binding domains or
nucleic acid aptamers, which bind, in a fusion-protein specific
manner, to essentially the same epitope to which a PDGFR.alpha.
antibody useful in the methods of the invention binds. See, e.g.,
Neuberger et al., Nature 312: 604 (1984). Such equivalent
non-antibody reagents may be suitably employed in the methods of
the invention further described below.
[0066] Polyclonal antibodies useful in practicing the methods of
the invention may be produced according to standard techniques by
immunizing a suitable animal (e.g., rabbit, goat, etc.) with an
antigen encompassing a desired epitope of PDGFR.alpha., collecting
immune serum from the animal, and separating the polyclonal
antibodies from the immune serum, and purifying polyclonal
antibodies having the desired specificity, in accordance with known
procedures. The antigen may be a synthetic peptide antigen
comprising the desired epitopic sequence, selected and constructed
in accordance with well-known techniques. See, e.g., ANTIBODIES: A
LABORATORY MANUAL, Chapter 5, p. 75-76, Harlow & Lane Eds.,
Cold Spring Harbor Laboratory (1988); Czernik, Methods In
Enzymology, 201: 264-283 (1991); Merrifield, J. Am. Chem. Soc. 85:
21-49 (1962)). Polyclonal antibodies produced as described herein
may be screened and isolated as further described below.
[0067] Monoclonal antibodies may also be beneficially employed in
the methods of the invention, and may be produced in hybridoma cell
lines according to the well-known technique of Kohler and Milstein.
Nature 265: 495-97 (1975); Kohler and Milstein, Eur. J. Immunol. 6:
511 (1976); see also, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,
Ausubel et al. Eds. (1989). Monoclonal antibodies so produced are
highly specific, and improve the selectivity and specificity of
assay methods provided by the invention. For example, a solution
containing the appropriate antigen (e.g. a synthetic peptide
comprising a phosphorylation site within PDGFR.alpha.) may be
injected into a mouse and, after a sufficient time (in keeping with
conventional techniques), the mouse sacrificed and spleen cells
obtained. The spleen cells are then immortalized by fusing them
with myeloma cells, typically in the presence of polyethylene
glycol, to produce hybridoma cells. Rabbit fusion hybridomas, for
example, may be produced as described in U.S. Pat. No. 5,675,063,
C. Knight, Issued Oct. 7, 1997. The hybridoma cells are then grown
in a suitable selection media, such as
hypoxanthine-aminopterin-thymidine (HAT), and the supernatant
screened for monoclonal antibodies having the desired specificity,
as described below. The secreted antibody may be recovered from
tissue culture supernatant by conventional methods such as
precipitation, ion exchange or affinity chromatography, or the
like.
[0068] Monoclonal Fab fragments may also be produced in Escherichia
coli by recombinant techniques known to those skilled in the art.
See, e.g., W. Huse, Science 246: 1275-81 (1989); Mullinax et al.,
Proc. Nat'/Acad. Sci. 87: 8095 (1990). If monoclonal antibodies of
one isotype are preferred for a particular application, particular
isotypes can be prepared directly, by selecting from the initial
fusion, or prepared secondarily, from a parental hybridoma
secreting a monoclonal antibody of different isotype by using the
sib selection technique to isolate class-switch variants
(Steplewski, et al., Proc. Nat'l. Acad. Sci., 82: 8653 (1985);
Spira et al., J. Immunol. Methods, 74: 307 (1984)). The antigen
combining site of the monoclonal antibody can be cloned by PCR and
single-chain antibodies produced as phage-displayed recombinant
antibodies or soluble antibodies in E. coli (see, e.g., ANTIBODY
ENGINEERING PROTOCOLS, 1995, Humana Press, Sudhir Paul
editor.).
[0069] Antibodies useful in the methods of the invention, whether
polyclonal or monoclonal, may be screened for epitope and
phosphorylation-state specificity according to standard techniques.
See, e.g. Czemik et al., Methods in Enzymology, 201: 264-283
(1991). For example, the antibodies may be screened against a
peptide library by ELISA to ensure specificity for both the desired
antigen and, if desired, for reactivity only with the
phosphorylated form of the antigen. The antibodies may also be
tested by Western blotting against cell preparations containing
target protein to confirm reactivity with the only the desired
target and to ensure no appreciable binding to other isoforms of
PDGFR.
[0070] PDGFR.alpha.-specific, and phosphorylation-specific,
antibodies useful in the methods of the invention may exhibit some
limited cross-reactivity with non-PDGFR.alpha. epitopes. This is
not unexpected as most antibodies exhibit some degree of
cross-reactivity, and anti-peptide antibodies will often
cross-react with epitopes having high homology or identity to the
immunizing peptide. See, e.g., Czemik, supra. Cross-reactivity with
non-PDGFR.alpha. proteins is readily characterized by Western
blotting alongside markers of known molecular weight. Amino acid
sequences of cross-reacting proteins may be examined to identify
sites highly homologous or identical to the PDGFR.alpha. sequence
to which the antibody binds. Undesirable cross-reactivity can be
removed by negative selection using antibody purification on
peptide columns.
[0071] PDGFR.alpha.-specific antibodies useful in practicing the
methods of the invention are ideally specific for human
PDGFR.alpha., but are not limited only to binding the human
species, per se. The invention includes the use of antibodies that
also bind conserved and highly homologous or identical epitopes in
other mammalian species (e.g. mouse, rat, monkey). Highly
homologous or identical sequences in other species can readily be
identified by standard sequence comparisons, such as using BLAST,
with the human PDGFR.alpha. sequence disclosed herein (SEQ ID NO:
1).
[0072] Antibodies employed in the methods of the invention may be
further characterized by, and validated for, use in a particular
assay format, for example FC, IHC, and/or ICC. The use of
antibodies against PDGFR.alpha. in such methods is further
described in section D below. Antibodies may also be advantageously
conjugated to fluorescent dyes (e.g. Alexa488, PE), or labels such
as quantum dots, for use in multi-parametric analyses along with
other signal transduction (phospho-AKT, phospho-Erk 1/2) and/or
cell marker (cytokeratin) antibodies, as further described in
section D below.
[0073] In practicing the methods of the invention, the expression
and/or activity of EGFR in a given NSCLC tumor may also be
advantageously examined using an EGFR-specific antibody and/or an
EGFR phosphorylation site-specific antibody. EGFR-specific and
phosphorylation-site specific antibodies are commercially available
(see CELL SIGNALING TECHNOLOGY, INC., Beverly Mass., 2003/04
Catalogue, #'s 2231, 2232, and 2234-2237; and Santa Cruz
Biotechnology, 2005 Catalogue, #03). Such antibodies may also be
produced according to standard methods, as described above. The
amino acid sequence of human EGFR is published (see accession
#NP-005219), as are the sequences of EGFR from other species.
Detection of EGFR expression and/or activation, along with
PDGFR.alpha. expression, in an NSCLC tumor can provide information
on whether PDGFR.alpha. alone is driving the tumor, or whether EGFR
is also activated and driving the tumor. Such information is
clinically useful in assessing whether targeting either, or both,
receptors is likely to be most beneficial in inhibiting progression
of the NSCLC tumor, and in selecting an appropriate therapeutic or
combination thereof.
[0074] It will be understood that more than one antibody may be
used in the practice of the above-described methods. For example,
one or more PDGFR.alpha.-specific antibodies together with one or
more antibodies specific for another kinase, receptor, or kinase
substrate that is suspected of being, or potentially is, activated
in a NSCLC tumor may be simultaneously employed to detect the
activity of such other signaling molecules in a biological sample
comprising cells from such NSCLC tumor.
[0075] Heavy-Isotope Labeled Peptides (AQUA Peptides).
[0076] PDGFR.alpha.-activation state-specific reagents useful in
the practice of the disclosed method may also comprise
heavy-isotope labeled peptides suitable for the absolute
quantification of expressed PDGFR.alpha. (preferably phosphorylated
at a disclosed site) in a biological sample. The production and use
of AQUA peptides for the absolute quantification of proteins (AQUA)
in complex mixtures has been described. See WO/03016861, "Absolute
Quantification of Proteins and Modified Forms Thereof by Multistage
Mass Spectrometry," Gygi et al. and also Gerber et al. Proc. Natl.
Acad. Sci. U.S.A. 100: 6940-5 (2003) (the teachings of which are
hereby incorporated herein by reference, in their entirety).
[0077] The AQUA methodology employs the introduction of a known
quantity of at least one heavy-isotope labeled peptide standard
(which has a unique signature detectable by LC-SRM chromatography)
into a digested biological sample in order to determine, by
comparison to the peptide standard, the absolute quantity of a
peptide with the same sequence and protein modification in the
biological sample. Briefly, the AQUA methodology has two stages:
peptide internal standard selection and validation and method
development; and implementation using validated peptide internal
standards to detect and quantify a target protein in sample. The
method is a powerful technique for detecting and quantifying a
given peptide/protein within a complex biological mixture, such as
a cell lysate, and may be employed, e.g., to quantify change in
protein phosphorylation as a result of drug treatment, or to
quantify differences in the level of a protein in different
biological states.
[0078] Generally, to develop a suitable internal standard, a
particular peptide (or modified peptide) within a target protein
sequence is chosen based on its amino acid sequence and the
particular protease to be used to digest. The peptide is then
generated by solid-phase peptide synthesis such that one residue is
replaced with that same residue containing stable isotopes
(.sup.13C, .sup.15N). The result is a peptide that is chemically
identical to its native counterpart formed by proteolysis, but is
easily distinguishable by MS via a 7-Da mass shift. The newly
synthesized AQUA internal standard peptide is then evaluated by
LC-MS/MS. This process provides qualitative information about
peptide retention by reverse-phase chromatography, ionization
efficiency, and fragmentation via collision-induced dissociation.
Informative and abundant fragment ions for sets of native and
internal standard peptides are chosen and then specifically
monitored in rapid succession as a function of chromatographic
retention to form a selected reaction monitoring (LC-SRM) method
based on the unique profile of the peptide standard.
[0079] The second stage of the AQUA strategy is its implementation
to measure the amount of a protein or modified protein from complex
mixtures. Whole cell lysates are typically fractionated by SDS-PAGE
gel electrophoresis, and regions of the gel consistent with protein
migration are excised. This process is followed by in-gel
proteolysis in the presence of the AQUA peptides and LC-SRM
analysis. (See Gerber et al. supra.) AQUA peptides are spiked in to
the complex peptide mixture obtained by digestion of the whole cell
lysate with a proteolytic enzyme and subjected to immunoaffinity
purification as described above. The retention time and
fragmentation pattern of the native peptide formed by digestion
(e.g. trypsinization) is identical to that of the AQUA internal
standard peptide determined previously; thus, LC-MS/MS analysis
using an SRM experiment results in the highly specific and
sensitive measurement of both internal standard and analyte
directly from extremely complex peptide mixtures.
[0080] Since an absolute amount of the AQUA peptide is added (e.g.
250 fmol), the ratio of the areas under the curve can be used to
determine the precise expression levels of a protein or
phosphorylated form of a protein in the original cell lysate. In
addition, the internal standard is present during in-gel digestion
as native peptides are formed, such that peptide extraction
efficiency from gel pieces, absolute losses during sample handling
(including vacuum centrifugation), and variability during
introduction into the LC-MS system do not affect the determined
ratio of native and AQUA peptide abundances.
[0081] An AQUA peptide standard is developed for a known
phosphorylation site sequence previously identified by the
IAP-LC-MS/MS method within in a target protein. One AQUA peptide
incorporating the phosphorylated form of the particular residue
within the site may be developed, and a second AQUA peptide
incorporating the non-phosphorylated form of the residue developed.
In this way, the two standards may be used to detect and quantify
both the phosphorylated and non-phosphorylated forms of the site in
a biological sample.
[0082] Peptide internal standards may also be generated by
examining the primary amino acid sequence of a protein and
determining the boundaries of peptides produced by protease
cleavage. Alternatively, a protein may actually be digested with a
protease and a particular peptide fragment produced can then
sequenced. Suitable proteases include, but are not limited to,
serine proteases (e.g. trypsin, hepsin), metallo proteases (e.g.
PUMP1), chymotrypsin, cathepsin, pepsin, thermolysin,
carboxypeptidases, etc.
[0083] A peptide sequence within a target protein is selected
according to one or more criteria to optimize the use of the
peptide as an internal standard. Preferably, the size of the
peptide is selected to minimize the chances that the peptide
sequence will be repeated elsewhere in other non-target proteins.
Thus, a peptide is preferably at least about 6 amino acids. The
size of the peptide is also optimized to maximize ionization
frequency. Thus, peptides longer than about 20 amino acids are not
preferred. The preferred ranged is about 7 to 15 amino acids. A
peptide sequence is also selected that is not likely to be
chemically reactive during mass spectrometry, thus sequences
comprising cysteine, tryptophan, or methionine are avoided.
[0084] A peptide sequence that does not include a modified region
of the target region may be selected so that the peptide internal
standard can be used to determine the quantity of all forms of the
protein. Alternatively, a peptide internal standard encompassing a
modified amino acid may be desirable to detect and quantify only
the modified form of the target protein. Peptide standards for both
modified and unmodified regions can be used together, to determine
the extent of a modification in a particular sample (i.e. to
determine what fraction of the total amount of protein is
represented by the modified form). For example, peptide standards
for both the phosphorylated and unphosphorylated form of a protein
known to be phosphorylated at a particular site can be used to
quantify the amount of phosphorylated form in a sample.
[0085] The peptide is labeled using one or more labeled amino acids
(i.e. the label is an actual part of the peptide) or less
preferably, labels may be attached after synthesis according to
standard methods. Preferably, the label is a mass-altering label
selected based on the following considerations: The mass should be
unique to shift fragments masses produced by MS analysis to regions
of the spectrum with low background; the ion mass signature
component is the portion of the labeling moiety that preferably
exhibits a unique ion mass signature in MS analysis; the sum of the
masses of the constituent atoms of the label is preferably uniquely
different than the fragments of all the possible amino acids. As a
result, the labeled amino acids and peptides are readily
distinguished from unlabeled ones by the ion/mass pattern in the
resulting mass spectrum. Preferably, the ion mass signature
component imparts a mass to a protein fragment that does not match
the residue mass for any of the 20 natural amino acids.
[0086] The label should be robust under the fragmentation
conditions of MS and not undergo unfavorable fragmentation.
Labeling chemistry should be efficient under a range of conditions,
particularly denaturing conditions, and the labeled tag preferably
remains soluble in the MS buffer system of choice. The label
preferably does not suppress the ionization efficiency of the
protein and is not chemically reactive. The label may contain a
mixture of two or more isotopically distinct species to generate a
unique mass spectrometric pattern at each labeled fragment
position. Stable isotopes, such as .sup.2H, .sup.13C, .sup.15N,
.sup.17O, .sup.18O, or .sup.34S, are among preferred labels. Pairs
of peptide internal standards that incorporate a different isotope
label may also be prepared. Preferred amino acid residues into
which a heavy isotope label may be incorporated include leucine,
proline, valine, and phenylalanine.
[0087] Peptide internal standards are characterized according to
their mass-to-charge (m/z) ratio, and preferably, also according to
their retention time on a chromatographic column (e.g. an HPLC
column). Internal standards that co-elute with unlabeled peptides
of identical sequence are selected as optimal internal standards.
The internal standard is then analyzed by fragmenting the peptide
by any suitable means, for example by collision-induced
dissociation (CID) using, e.g., argon or helium as a collision gas.
The fragments are then analyzed, for example by multi-stage mass
spectrometry (MS.sup.n) to obtain a fragment ion spectrum, to
obtain a peptide fragmentation signature. Preferably, peptide
fragments have significant differences in m/z ratios to enable
peaks corresponding to each fragment to be well separated, and a
signature is that is unique for the target peptide is obtained. If
a suitable fragment signature is not obtained at the first stage,
additional stages of MS are performed until a unique signature is
obtained.
[0088] Fragment ions in the MS/MS and MS.sup.3 spectra are
typically highly specific for the peptide of interest, and, in
conjunction with LC methods, allow a highly selective means of
detecting and quantifying a target peptide/protein in a complex
protein mixture, such as a cell lysate, containing many thousands
or tens of thousands of proteins. Any biological sample potentially
containing a target protein/peptide of interest may be assayed.
Crude or partially purified cell extracts are preferably employed.
Generally, the sample has at least 0.01 mg of protein, typically a
concentration of 0.1-10 mg/mL, and may be adjusted to a desired
buffer concentration and pH.
[0089] A known amount of a labeled peptide internal standard,
preferably about 10 femtomoles, corresponding to a target protein
to be detected/quantified is then added to a biological sample,
such as a cell lysate. The spiked sample is then digested with one
or more protease(s) for a suitable time period to allow digestion.
A separation is then performed (e.g. by HPLC, reverse-phase HPLC,
capillary electrophoresis, ion exchange chromatography, etc.) to
isolate the labeled internal standard and its corresponding target
peptide from other peptides in the sample. Microcapillary LC is a
preferred method.
[0090] Each isolated peptide is then examined by monitoring of a
selected reaction in the MS. This involves using the prior
knowledge gained by the characterization of the peptide internal
standard and then requiring the MS to continuously monitor a
specific ion in the MS/MS or MS.sup.n spectrum for both the peptide
of interest and the internal standard. After elution, the area
under the curve (AUC) for both peptide standard and target peptide
peaks are calculated. The ratio of the two areas provides the
absolute quantification that can be normalized for the number of
cells used in the analysis and the protein's molecular weight, to
provide the precise number of copies of the protein per cell.
Further details of the AQUA methodology are described in Gygi et
al., and Gerber et al. supra.
[0091] AQUA internal peptide standards (heavy-isotope labeled
peptides) may desirably be produced, as described above, to detect
any quantify any phosphorylation site with PDGFR.alpha. relevant to
activity of this RTK. For example, an AQUA phosphopeptide may be
prepared that corresponds to any of the following preferred
PDGFR.alpha. tyrosine phosphorylation sites: tyrosine 572, tyrosine
742, tyrosine 762, tyrosine 768, tyrosine 849, or tyrosine 1018 (in
the human PDGFR.alpha. protein sequence (SEQ ID NO: 1); see also
Table 1). Peptide standards for a given phosphorylation site (e.g.
the tyrosine 572 site in human PDGFR.alpha.) may be produced for
both the phosphorylated and non-phosphorylated forms of the site,
and such standards employed in the AQUA methodology to detect and
quantify both forms of such phosphorylation site in a biological
sample.
[0092] The six phosphorylation site peptide sequences identified in
Table 1 (see Example 1) (SEQ ID NOs: 3-8) are particularly well
suited for development of corresponding AQUA peptides, since the
IAP method by which they were identified (see Part A above, and
Example 1) inherently confirmed that such peptides are in fact
produced by enzymatic digestion (trypsinization) and are in fact
suitably fractionated/ionized in MS/MS. For example, the peptide
sequence QADTTQyVPMLER (SEQ ID NO: 4; see Table 1), which
encompasses phosphorylatable tyrosine 742 (human PDGFR.alpha.
sequence) may desirably be selected for development of AQUA
peptides for quantifying phosphorylated (Y742) PDGFR.alpha. in a
biological sample. Heavy-isotope labeled equivalents of any of
these preferred peptides (both in phosphorylated and
unphosphorylated form) can be readily synthesized and their unique
MS and LC-SRM signature determined, so that the peptides are
validated as AQUA peptides and ready for use in quantification
experiments.
[0093] It will be appreciated that larger AQUA peptides comprising
a PDGFR.alpha. phosphorylation site sequence (and additional
residues downstream or upstream of it) may also be constructed.
Similarly, a smaller AQUA peptide comprising less than all of the
residues of such phosphorylation site sequence (but still
comprising the phosphorylatable tyrosine residue of interest) may
alternatively be constructed. Such larger or shorter AQUA peptides
are within the scope of the present invention, and the selection
and production of preferred AQUA peptides, whether to quantify
total PDGFR.alpha. or phosphorylated PDGFR.alpha., may be carried
out as described above (see Gygi et al., Gerber et al.,
supra.).
D. Assay Formats
[0094] Immunoassays useful in the practice of the methods of the
invention may be homogenous immunoassays or heterogeneous
immunoassays. In a homogeneous assay the immunological reaction
usually involves a PDGFR.alpha.-specific reagent (e.g. a
PDGFR.alpha.-specific antibody), a labeled analyte, and the
biological sample of interest. The signal arising from the label is
modified, directly or indirectly, upon the binding of the antibody
to the labeled analyte. Both the immunological reaction and
detection of the extent thereof are carried out in a homogeneous
solution. Immunochemical labels that may be employed include free
radicals, radio-isotopes, fluorescent dyes, enzymes,
bacteriophages, coenzymes, and so forth. Semi-conductor nanocrystal
labels, or "quantum dots", may also be advantageously employed, and
their preparation and use has been well described. See generally,
K. Barovsky, Nanotech. Law & Bus. 1(2): Article 14 (2004) and
patents cited therein.
[0095] In a heterogeneous assay approach, the reagents are usually
the biological sample, a PDGFR.alpha.-specific reagent (e.g., an
antibody), and suitable means for producing a detectable signal.
Biological samples as described above in section B may be used. The
antibody is generally immobilized on a support, such as a bead,
plate or slide, and contacted with the sample suspected of
containing the antigen in a liquid phase. The support is then
separated from the liquid phase and either the support phase or the
liquid phase is examined for a detectable signal employing means
for producing such signal. The signal is related to the presence of
the analyte in the biological sample. Means for producing a
detectable signal include the use of radioactive labels,
fluorescent labels, enzyme labels, quantum dots, and so forth. For
example, if the antigen to be detected contains a second binding
site, an antibody which binds to that site can be conjugated to a
detectable group and added to the liquid phase reaction solution
before the separation step. The presence of the detectable group on
the solid support indicates the presence of the antigen in the test
sample. Examples of suitable immunoassays are the radioimmunoassay,
immunofluorescence methods, enzyme-linked immunoassays, and the
like.
[0096] Immunoassay formats and variations thereof, which may be
useful for carrying out the methods disclosed herein, are well
known in the art. See generally E. Maggio, Enzyme-Immunoassay,
(1980) (CRC Press, Inc., Boca Raton, Fla.); see also, e.g., U.S.
Pat. No. 4,727,022 (Skold et al., "Methods for Modulating
Ligand-Receptor Interactions and their Application"); U.S. Pat. No.
4,659,678 (Forrest et al., "Immunoassay of Antigens"); U.S. Pat.
No. 4,376,110 (David et al., "Immunometric Assays Using Monoclonal
Antibodies"). Conditions suitable for the formation of
reagent-antibody complexes are well known to those of skill in the
art. See id. PDGFR.alpha.-specific or phosphorylation site-specific
monoclonal antibodies may be used in a "two-site" or "sandwich"
assay, with a single hybridoma cell line serving as a source for
both the labeled monoclonal antibody and the bound monoclonal
antibody. Such assays are described in U.S. Pat. No. 4,376,110. The
concentration of detectable reagent should be sufficient such that
the binding of PDGFR.alpha. is detectable compared to
background.
[0097] Antibodies useful in the practice of the methods disclosed
herein may be conjugated to a solid support suitable for a
diagnostic assay (e.g., beads, plates, slides or wells formed from
materials such as latex or polystyrene) in accordance with known
techniques, such as precipitation. Antibodies or other PDGFR.alpha.
binding reagents may likewise be conjugated to detectable groups
such as radiolabels (e.g., .sup.35S, .sup.125I, .sup.131I) enzyme
labels (e.g., horseradish peroxidase, alkaline phosphatase), and
fluorescent labels (e.g., fluorescein) in accordance with known
techniques.
[0098] Cell-based assays, such flow cytometry (FC),
immuno-histochemistry (IHC), or immunofluorescence (IF) are
particularly desirable in practicing the methods of the invention,
since such assay formats are clinically-suitable, allow the
detection of PDGFR.alpha. activation in vivo, and avoid the risk of
artifact changes in activity resulting from manipulating cells
obtained from an NSCLC tumor in order to obtain extracts.
Accordingly, in some preferred embodiment, the methods of the
invention are implemented in a flow-cytometry (FC),
immunohistochemistry (IHC), or immunofluorescence (IF) assay
format.
[0099] Flow cytometry (FC) may be employed to determine the
activation status of PDGFR.alpha. in a mammalian NSCLC tumor
before, during, and after treatment with a drug targeted at
inhibiting PDGFR.alpha. kinase activity. For example, tumor cells
from a fine needle aspirate may be analyzed by flow cytometry for
PDGFR.alpha. expression and/or activation, as well as for markers
identifying lung cancer cell types, etc., if so desired. Flow
cytometry may be carried out according to standard methods. See,
e.g. Chow et al., Cytometry (Communications in Clinical Cytometry)
46: 72-78 (2001). Briefly and by way of example, the following
protocol for cytometric analysis may be employed: fixation of the
cells with 2% paraformaldehyde for 10 minutes at 37.degree. C.
followed by permeabilization in 90% methanol for 30 minutes on ice.
Cells may then be stained with the primary PDGFR.alpha.-specific
antibody, washed and labeled with a fluorescent-labeled secondary
antibody. The cells would then be analyzed on a flow cytometer
(e.g. a Beckman Coulter FC500) according to the specific protocols
of the instrument used. Such an analysis would identify the level
of expressed PDGFR.alpha. protein in the tumor. Similar analysis
after treatment of the tumor with a PDGFR.alpha.-inhibiting
therapeutic would reveal the responsiveness of a
PDGFR.alpha.-expressing tumor to the targeted inhibitor or
PDGFR.alpha. kinase.
[0100] Immunohistochemical (IHC) staining may be also employed to
determine the expression and/or activation status of PDGFR.alpha.
in a mammalian NSCLC tumor before, during, and after treatment with
a drug targeted at inhibiting PDGFR.alpha. activity. IHC may be
carried out according to well-known techniques. See, e.g.,
ANTIBODIES: A LABORATORY MANUAL, Chapter 10, Harlow & Lane
Eds., Cold Spring Harbor Laboratory (1988). Briefly, and by way of
example, paraffin-embedded tissue (e.g. tumor tissue from a biopsy)
is prepared for immunohistochemical staining by deparaffinizing
tissue sections with xylene followed by ethanol; hydrating in water
then PBS; unmasking antigen by heating slide in sodium citrate
buffer; incubating sections in hydrogen peroxide; blocking in
blocking solution; incubating slide in primary
anti-PDGFR.alpha.antibody and secondary antibody; and finally
detecting using ABC avidin/biotin metho according to manufacturer's
instructions.
[0101] Immunofluorescence (IF) assays may be also employed to
determine the expression and/or activation status of PDGFR.alpha.
in a mammalian NSCLC tumor before, during, and after treatment with
a drug targeted at inhibiting PDGFR.alpha. kinase activity. IF may
be carried out according to well-known techniques. See, e.g., J. M.
polak and S. Van Noorden (1997) INTRODUCTION TO
IMMUNOCYTOCHEMISTRY, 2nd Ed.; ROYAL MICROSCOPY SOCIETY MICROSCOPY
HANDBOOK 37, BioScientific/Springer-Verlag. Briefly, and by way of
example, patient samples may be fixed in paraformaldehyde followed
by methanol, blocked with a blocking solution such as horse serum,
incubated with the primary antibody against PDGFR.alpha. followed
by a secondary antibody labeled with a fluorescent dye such as
Alexa 488 and analyzed with an epifluorescent microscope.
Antibodies employed in the above-described assays may be
advantageously conjugated to fluorescent dyes (e.g. Alexa488, PE),
or other labels, such as quantum dots, for use in multi-parametric
analyses along with other signal transduction (EGFR, phospho-AKT,
phospho-Erk 1/2) and/or cell marker (cytokeratin) antibodies.
[0102] Similarly, AQUA peptides for the detection/quantification of
expressed PDGFR.alpha. in a biological sample comprising cells from
a NSCLC tumor may be prepared and used in standard AQUA assays, as
described in detail in section C above. Accordingly, in some
preferred embodiments of the methods of the invention, the
PDGFR.alpha.-specific reagent comprises a heavy isotope labeled
phosphopeptide (AQUA peptide) corresponding to a PDGFR.alpha.
peptide sequence (e.g., a phosphorylation site), as described above
in section C.
[0103] PDGFR.alpha.-specific reagents useful in practicing the
methods of the invention may also be mRNA, oligonucleotide or DNA
probes that can directly hybridize to, and detect, PDGFR.alpha.
expression transcripts in a biological sample. Briefly, and by way
of example, formalin-fixed, paraffin-embedded patient samples may
be probed with a fluorescein-labeled RNA probe followed by washes
with formamide, SSC and PBS and analysis with a fluorescent
microscope.
E. PDGFR.alpha.-Inhibiting Therapeutics.
[0104] In accordance with the present invention, it has now been
shown that the progression of a distinct subset of mammalian NSCLC
tumors in which PDGFR.alpha. is expressed may be inhibited, in
vivo, by inhibiting the activity of PDGFR.alpha. in such tumors.
PDGFR.alpha. activity in this newly identified and distinct subset
of NSCLC tumors may be inhibited by contacting the tumor with a
PDGFR.alpha. inhibiting therapeutic, such as a small-molecule
PDGFR.alpha. inhibitor like Imatinib mesylate (STI-571;
Gleevec.RTM.). As further described in Example 5 herein, growth
inhibition of PDGFR.alpha.-expressing NSCLC tumors can be
accomplished by inhibiting this RTK, using an exemplary
PDGFR.alpha.-inhibiting therapeutic, Gleevec.RTM.. Accordingly, the
invention provides, in part, a method for inhibiting the
progression of a PDGFR.alpha.-expressing mammalian NSCLC tumor by
inhibiting the expression and/or activity of PDGFR.alpha. in the
tumor.
[0105] A PDGFR-inhibiting therapeutic may be any composition
comprising at least one compound, biological or chemical, which
inhibits, directly or indirectly, the expression and/or activity of
PDGFR.alpha. in vivo, including the exemplary classes of compounds
described below. Such compounds include therapeutics that act
directly on PDGFR.alpha. itself, or on proteins or molecules that
modify the activity of PDGFR.alpha., or that act indirectly by
inhibiting the expression of PDGFR.alpha.. Such compositions also
include compositions comprising only a single PDGFR.alpha.
inhibiting compound, as well as compositions comprising multiple
therapeutics (including those against other RTKs), which may also
include a non-specific therapeutic agent like a chemotherapeutic
agent or general transcription inhibitor.
Small-Molecule Inhibitors.
[0106] In some preferred embodiments, a PDGFR.alpha.-inhibiting
therapeutic useful in the practice of the methods of the invention
is a targeted, small molecule inhibitor, such as Gleevec.RTM.
(STI-571), and its analogues. As presently shown (see Example 5),
administration of Gleevec.RTM. to mice harboring human NSCLC
xenografts selectively inhibited the progression of the disease in
those mice with PDGFR.alpha.-expressing tumors. Gleevec.RTM., which
specifically binds to and blocks the ATP-binding site of
PDGFR.alpha. (as well as Bcr-Abl kinase), thereby preventing
phosphorylation and activation of this enzyme, is commercially
available and its properties are well known. The
PDGFR.alpha.-specific inhibitory properties of Gleevec.RTM. have
been described. See, e.g. Martinelli et al., Haematologica 89(2):
236-7 (2004). Other preferred small-molecule inhibitors of PDGFR
include BAY 43-93006, XL-999 and 5U11248. These compounds are under
clinical investigation and their PDGFR.alpha.-specific inhibitory
properties have been described. See Wilhelm et al., Cancer Res.
64(19): 7099-109 (2004) and Mendel et al., Clin Cancer Res. 9(1):
327-37 (2003).
[0107] Small molecule targeted inhibitors are a class of molecules
that typically inhibit the activity of their target enzyme by
specifically, and often irreversibly, binding to the catalytic site
of the enzyme, and/or binding to an ATP-binding cleft or other
binding site within the enzyme that prevents the enzyme from
adopting a conformation necessary for its activity. Small molecule
inhibitors may be rationally designed using X-ray crystallographic
or computer modeling of PDGFR.alpha. three-dimensional structure,
or may found by high throughput screening of compound libraries for
inhibition of PDGFR.alpha.. Such methods are well known in the art,
and have been described. Specificity of PDGFR.alpha. inhibition may
be confirmed, for example, by examining the ability of such
compound to inhibit PDGFR.alpha. activity, but not other kinase
activity, in a panel of kinases, and/or by examining the inhibition
of PDGFR.alpha. activity in a biological sample comprising NSCLC
tumor cells, as described above. Such screening methods are further
described below.
[0108] Other small molecules with PDGFR.alpha.-inhibitory
properties, such as quinoline and quinoxaline compounds, and
1,3-diazine compounds, have been described. See, e.g. U.S. Pat.
Nos. 6,821,962; 6,696,434; 6,169,088. Methods for identifying
antagonists of PDGFR.alpha. have also been described. See, e.g.
U.S. Pat. No. 6,566,075, May 20, 2003, Escobedo et al.
Antibody Inhibitors.
[0109] PDGFR.alpha.-inhibiting therapeutics useful in the methods
of the invention may also be targeted antibodies that specifically
bind to critical catalytic or binding sites or domains required for
PDGFR.alpha. activity, and inhibit the kinase by blocking access of
substrates or secondary molecules to PDGFR.alpha. and/or preventing
the enzyme from adopting a conformation necessary for its activity.
The production, screening, and therapeutic use of humanized
target-specific antibodies has been well-described. See Merluzzi et
al., Adv Clin Path. 4(2): 77-85 (2000). Commercial technologies and
systems, such as Morphosys, Inc.'s Human Combinatorial Antibody
Library (HuCAL.RTM.), for the high-throughput generation and
screening of humanized target-specific inhibiting antibodies are
available.
[0110] The production of various anti-receptor kinase targeted
antibodies and their use to inhibit activity of the targeted
receptor has been described. See, e.g. U.S. Patent Publication No.
20040202655, "Antibodies to IGF-I Receptor for the Treatment of
Cancers," Oct. 14, 2004, Morton et al.; U.S. Patent Publication No.
20040086503, "Human anti-Epidermal Growth Factor Receptor
Single-Chain Antibodies," Apr. 15, 2004, Raisch et al.; U.S. Patent
Publication No. 20040033543, "Treatment of Renal Carcinoma Using
Antibodies Against the EGFr," Feb. 19, 2004, Schwab et. al.
Standardized methods for producing, and using, receptor tyrosine
kinase activity-inhibiting antibodies are known in the art. See,
e.g., European Patent No. EP1423428, "Antibodies that Block
Receptor Tyrosine Kinase Activation, Methods of Screening for and
Uses Thereof," Jun. 2, 2004, Borges et al.
[0111] Phage display approaches may also be employed to generate
PDGFR.alpha. antibody inhibitors, and protocols for bacteriophage
library construction and selection of recombinant antibodies are
provided in the well-known reference text CURRENT PROTOCOLS IN
IMMUNOLOGY, Colligan et al. (Eds.), John Wiley & Sons, Inc.
(1992-2000), Chapter 17, Section 17.1. See also U.S. Pat. No.
6,319,690, Nov. 20, 2001, Little et al.; U.S. Pat. No. 6,300,064,
Oct. 9, 2001, Knappik et al.; U.S. Pat. No. 5,840,479, Nov. 24,
1998, Little et al.; U.S. Patent Publication No. 20030219839, Nov.
27, 2003, Bowdish et al.
[0112] A library of antibody fragments displayed on the surface of
bacteriophages may be produced (see, e.g. U.S. Pat. No. 6,300,064,
Oct. 9, 2001, Knappik et al.) and screened for binding to a soluble
dimeric form of a receptor protein tyrosine kinase. An antibody
fragment that binds to the soluble dimeric form of the RTK used for
screening is identified as a candidate molecule for blocking
constitutive activation of the target RTK in a cell. See European
Patent No. EP1423428, Borges et al., supra.
[0113] PDGFR.alpha.-binding targeted antibodies identified in
screening of antibody libraries as describe above may then be
further screened for their ability to block the activity of
PDGFR.alpha., both in vitro kinase assay and in vivo in cell lines
and/or tumors. PDGFR.alpha. inhibition may be confirmed, for
example, by examining the ability of such antibody therapeutic to
inhibit PDGFR.alpha. activity, but not other kinase activity, in a
panel of kinases, and/or by examining the inhibition of
PDGFR.alpha. activity in a biological sample comprising NSCLC tumor
cells, as described above. Methods for screening such compounds for
PDGFR.alpha. inhibition are further described above.
Indirect Inhibitors.
[0114] PDGFR.alpha.-inhibiting compounds useful in the practice of
the disclosed methods may also be compounds that indirectly inhibit
PDGFR.alpha. activity by inhibiting the activity of proteins or
molecules other than PDGFR.alpha. itself. Such inhibiting
therapeutics may be targeted inhibitors that modulate the activity
of key regulatory kinases that phosphorylate or de-phosphorylate
(and hence activate or deactivate) PDGFR.alpha. itself. As with
other receptor tyrosine kinases, PDGFR.alpha. regulates downstream
signaling through a network of adaptor proteins and downstream
kinases. As a result, induction of cell growth and survival by
PDGFR.alpha. activity may be inhibited by targeting these
interacting or downstream proteins. Drugs currently in development
that could be used in this manner include AKT inhibitors (RX-0201)
and mTOR inhibitors (rapamycin and its analogs such as CC1-779,
Rapamune and RAD001).
[0115] PDGFR.alpha. activity may also be indirectly inhibited by
using a compound that inhibits the binding of an activating
molecule, such as the platelet-derived growth factor (PDGF) A or B,
necessary for PDGFR.alpha. to adopt its active conformation. For
example, the production and use of anti-PDGF antibodies has been
described. See U.S. Patent Publication No. 20030219839, "Anti-PDGF
Antibodies and Methods for Producing Engineered Antibodies,"
Bowdish et al. Inhibition of PDGF binding to PDGFR.alpha. directly
down-regulates PDGFR.alpha. activity.
[0116] Indirect inhibitors of PDGFR.alpha. activity may be
rationally designed using X-ray crystallographic or computer
modeling of PDGFR.alpha. three dimensional structure, or may found
by high throughput screening of compound libraries for inhibition
of key upstream regulatory enzymes and/or necessary binding
molecules, which results in inhibition of PDGFR.alpha.. Such
approaches are well known in the art, and have been described.
PDGFR.alpha. inhibition by such therapeutics may be confirmed, for
example, by examining the ability of the compound to inhibit
PDGFR.alpha. activity, but not other kinase activity, in a panel of
kinases, and/or by examining the inhibition of PDGFR.alpha.
activity in a biological sample comprising NSCLC tumor cells, as
described above. Methods for identifying compounds that inhibit
PDGFR.alpha. activity in NSCLC tumors are further described
below.
Anti-Sense and/or Transcription Inhibitors.
[0117] PDGFR.alpha.-inhibiting therapeutics may also comprise
anti-sense and/or transcription inhibiting compounds that inhibit
PDGFR.alpha. activity by blocking transcription of the gene
encoding PDGFR.alpha.. The inhibition of various receptor kinases,
including VEGFR, EGFR, and IGFR, and FGFR, by antisense
therapeutics for the treatment of cancer has been described. See,
e.g., U.S. Pat. Nos. 6,734,017; 6,710,174, 6,617,162; 6,340,674;
5,783,683; 5,610,288.
[0118] Antisense oligonucleotides may be designed, constructed, and
employed as therapeutic agents against target genes in accordance
with known techniques. See, e.g. Cohen, J., Trends in Pharmacol.
Sci. 10(11): 435-437 (1989); Marcus-Sekura, Anal. Biochem. 172:
289-295 (1988); Weintraub, H., Sci. AM pp. 40-46 (1990); Van Der
Krol et al., BioTechniques 6(10): 958-976 (1988); Skorski et al.,
Proc. Natl. Acad. Sci. USA (1994) 91: 4504-4508 Inhibition of human
carcinoma growth in vivo using an antisense RNA inhibitor of EGFR
has recently been described. See U.S. Patent Publication No.
20040047847, "Inhibition of Human Squamous Cell Carcinoma Growth In
vivo by Epidermal Growth Factor Receptor Antisense RNA Transcribed
from a Pol III Promoter," Mar. 11, 2004, He et al. Similarly, a
PDGFR.alpha.-inhibiting therapeutic comprising at least one
antisense oligonucleotide against a mammalian PDGFR.alpha. gene may
be prepared according to methods described above. Pharmaceutical
compositions comprising PDGFR.alpha.-inhibiting antisense compounds
may be prepared and administered as further described below.
Small Interfering RNA.
[0119] Small interfering RNA molecule (siRNA) compositions, which
inhibit translation, and hence activity, of PDGFR.alpha. through
the process of RNA interference, may also be desirably employed in
the methods of the invention.
[0120] RNA interference, and the selective silencing of target
protein expression by introduction of exogenous small
double-stranded RNA molecules comprising sequence complimentary to
mRNA encoding the target protein, has been well described. See,
e.g. U.S. Patent Publication No. 20040038921, "Composition and
Method for Inhibiting Expression of a Target Gene," Feb. 26, 2004,
Kreutzer et al.; U.S. Patent Publication No. 20020086356, "RNA
Sequence-Specific Mediators of RNA Interference," Jun. 12, 2003,
Tuschl et al.; U.S. Patent Publication 20040229266, "RNA
Interference Mediating Small RNA Molecules," Nov. 18, 2004, Tuschl
et. al.
[0121] Double-stranded RNA molecules (dsRNA) have been shown to
block gene expression in a highly conserved regulatory mechanism
known as RNA interference (RNAi). Briefly, the RNAse III Dicer
processes dsRNA into small interfering RNAs (siRNA) of
approximately 22 nucleotides, which serve as guide sequences to
induce target-specific mRNA cleavage by an RNA-induced silencing
complex RISC (see Hammond et al., Nature (2000) 404: 293-296).
[0122] RNAi involves a catalytic-type reaction whereby new siRNAs
are generated through successive cleavage of longer dsRNA. Thus,
unlike antisense, RNAi degrades target RNA in a non-stoichiometric
manner. When administered to a cell or organism, exogenous dsRNA
has been shown to direct the sequence-specific degradation of
endogenous messenger RNA (mRNA) through RNAi.
[0123] A wide variety of target-specific siRNA products, including
vectors and systems for their expression and use in mammalian
cells, are now commercially available. See, e.g. Promega, Inc.
(www.promega.com); Dharmacon, Inc. (www.dharmacon.com). Detailed
technical manuals on the design, construction, and use of dsRNA for
RNAi are available. See, e.g. Dharmacon's "RNAi Technical Reference
& Application Guide"; Promega's "RNAi: A Guide to Gene
Silencing." PDGFR.alpha.-inhibiting siRNA products are also
commercially available, and may be suitably employed in the method
of the invention. See, e.g. Dharmacon, Inc., Lafayette, Colo. (Cat
Nos. M-003162-03, MU-003162-03, D-003162-07 thru -10 (siGENOME.TM.
SMARTselection and SMARTpool.RTM. siRNAs).
[0124] It has recently been established that small dsRNA less than
49 nucleotides in length, and preferably 19-25 nucleotides,
comprising at least one sequence that is substantially identical to
part of a target mRNA sequence, and which dsRNA optimally has at
least one overhang of 1-4 nucleotides at an end, are most effective
in mediating RNAi in mammals. See U.S. Patent Publication No.
20040038921, Kreutzer et al., supra; U.S. Patent Publication No.
20040229266, Tuschl et al., supra. The construction of such dsRNA,
and their use in pharmaceutical preparations to silence expression
of a target protein, in vivo, are described in detail in such
publications.
[0125] If the sequence of the gene to be targeted in a mammal is
known, 21-23 nt RNAs, for example, can be produced and tested for
their ability to mediate RNAi in a mammalian cell, such as a human
or other primate cell. Those 21-23 nt RNA molecules shown to
mediate RNAi can be tested, if desired, in an appropriate animal
model to further assess their in vivo effectiveness. Target sites
that are known, for example target sites determined to be effective
target sites based on studies with other nucleic acid molecules,
for example ribozymes or antisense, or those targets known to be
associated with a disease or condition such as those sites
containing mutations or deletions, can be used to design siRNA
molecules targeting those sites as well.
[0126] Alternatively, the sequences of effective dsRNA can be
rationally designed/predicted screening the target mRNA of interest
for target sites, for example by using a computer folding
algorithm. The target sequence can be parsed in silico into a list
of all fragments or subsequences of a particular length, for
example 23 nucleotide fragments, using a custom Perl script or
commercial sequence analysis programs such as Oligo, MacVector, or
the GCG Wisconsin Package.
[0127] Various parameters can be used to determine which sites are
the most suitable target sites within the target RNA sequence.
These parameters include but are not limited to secondary or
tertiary RNA structure, the nucleotide base composition of the
target sequence, the degree of homology between various regions of
the target sequence, or the relative position of the target
sequence within the RNA transcript. Based on these determinations,
any number of target sites within the RNA transcript can be chosen
to screen siRNA molecules for efficacy, for example by using in
vitro RNA cleavage assays, cell culture, or animal models. See,
e.g., U.S. Patent Publication No. 20030170891, Sep. 11, 2003,
McSwiggen J. An algorithm for identifying and selecting RNAi target
sites has also recently been described. See U.S. Patent Publication
No. 20040236517, "Selection of Target Sites for Antisense Attack of
RNA," Nov. 25, 2004, Drlica et al.
[0128] Commonly used gene transfer techniques include calcium
phosphate, DEAE-dextran, electroporation and microinjection and
viral methods (Graham et al. (1973) Virol. 52: 456; McCutchan et
al., (1968), J. Natl. Cancer Inst. 41: 351; Chu et al. (1987),
Nucl. Acids Res. 15: 1311; Fraley et al. (1980), J. Biol. Chem.
255: 10431; Capecchi (1980), Cell 22: 479). DNA may also be
introduced into cells using cationic liposomes (Feigner et al.
(1987), Proc. Natl. Acad. Sci USA 84: 7413). Commercially available
cationic lipid formulations include Tfx 50 (Promega) or
Lipofectamin 200 (Life Technologies). Alternatively, viral vectors
may be employed to deliver dsRNA to a cell and mediate RNAi. See
U.S Patent Publication No. 20040023390, "siRNA-mediated Gene
Silencing with Viral Vectors," Feb. 4, 2004, Davidson et al.
[0129] Transfection and vector/expression systems for RNAi in
mammalian cells are commercially available and have been well
described. See, e.g. Dharmacon, Inc., DharmaFECT.TM. system;
Promega, Inc., siSTRIKE.TM. U6 Hairpin system; see also Gou et al.
(2003) FEBS. 548, 113-118; Sui, G. et al. A DNA vector-based RNAi
technology to suppress gene expression in mammalian cells (2002)
Proc. Natl. Acad. Sci. 99, 5515-5520; Yu et al. (2002) Proc. Natl.
Acad. Sci. 99, 6047-6052; Paul, C. et al. (2002) Nature
Biotechnology 19, 505-508; McManus et al. (2002) RNA 8,
842-850.
[0130] siRNA interference in a mammal using prepared dsRNA
molecules may then be effected by administering a pharmaceutical
preparation comprising the dsRNA to the mammal. The pharmaceutical
composition is administered in a dosage sufficient to inhibit
expression of the target gene. dsRNA can typically be administered
at a dosage of less than 5 mg dsRNA per kilogram body weight per
day, and is sufficient to inhibit or completely suppress expression
of the target gene. In general a suitable dose of dsRNA will be in
the range of 0.01 to 2.5 milligrams per kilogram body weight of the
recipient per day, preferably in the range of 0.1 to 200 micrograms
per kilogram body weight per day, more preferably in the range of
0.1 to 100 micrograms per kilogram body weight per day, even more
preferably in the range of 1.0 to 50 micrograms per kilogram body
weight per day, and most preferably in the range of 1.0 to 25
micrograms per kilogram body weight per day. A pharmaceutical
composition comprising the dsRNA is administered once daily, or in
multiple sub-doses, for example, using sustained release
formulations well known in the art. The preparation and
administration of such pharmaceutical compositions may be carried
out accordingly to standard techniques, as further described
below.
[0131] Such dsRNA may then be used to inhibit PDGFR.alpha.
expression and activity in a NSCLC tumor, by preparing a
pharmaceutical preparation comprising a therapeutically-effective
amount of such dsRNA, as described above, and administering the
preparation to a human subject having a PDGFR.alpha.-activated
NSCLC tumor, for example, via direct injection to the tumor. The
similar inhibition of other receptor tyrosine kinases, such as
VEGFR and EGFR using siRNA inhibitors has recently been described.
See U.S. Patent Publication No. 20040209832, Oct. 21, 2004,
McSwiggen et al.; U.S. Patent Publication No. 20030170891, Sep. 11,
2003, McSwiggen; U.S. Patent Publication No. 20040175703, Sep. 9,
2004, Kreutzer et al.
[0132] Therapeutic Compositions; Administration.
[0133] PDGFR.alpha.-Inhibiting Therapeutic Compositions Useful in
the Practice of the methods of the invention may be administered to
a mammal by any means known in the art including, but not limited
to oral or peritoneal routes, including intravenous, intramuscular,
intraperitoneal, subcutaneous, transdermal, airway (aerosol),
rectal, vaginal and topical (including buccal and sublingual)
administration.
[0134] For oral administration, a PDGFR.alpha.-inhibiting
therapeutic will generally be provided in the form of tablets or
capsules, as a powder or granules, or as an aqueous solution or
suspension. Tablets for oral use may include the active ingredients
mixed with pharmaceutically acceptable excipients such as inert
diluents, disintegrating agents, binding agents, lubricating
agents, sweetening agents, flavoring agents, coloring agents and
preservatives. Suitable inert diluents include sodium and calcium
carbonate, sodium and calcium phosphate, and lactose, while corn
starch and alginic acid are suitable disintegrating agents. Binding
agents may include starch and gelatin, while the lubricating agent,
if present, will generally be magnesium stearate, stearic acid or
talc. If desired, the tablets may be coated with a material such as
glyceryl monostearate or glyceryl distearate, to delay absorption
in the gastrointestinal tract.
[0135] Capsules for oral use include hard gelatin capsules in which
the active ingredient is mixed with a solid diluent, and soft
gelatin capsules wherein the active ingredients is mixed with water
or an oil such as peanut oil, liquid paraffin or olive oil. For
intramuscular, intraperitoneal, subcutaneous and intravenous use,
the pharmaceutical compositions of the invention will generally be
provided in sterile aqueous solutions or suspensions, buffered to
an appropriate pH and isotonicity. Suitable aqueous vehicles
include Ringer's solution and isotonic sodium chloride. The carrier
may consists exclusively of an aqueous buffer ("exclusively" means
no auxiliary agents or encapsulating substances are present which
might affect or mediate uptake of the PDGFR.alpha.-inhibiting
therapeutic). Such substances include, for example, micellar
structures, such as liposomes or capsids, as described below.
Aqueous suspensions may include suspending agents such as cellulose
derivatives, sodium alginate, polyvinylpyrrolidone and gum
tragacanth, and a wetting agent such as lecithin. Suitable
preservatives for aqueous suspensions include ethyl and n-propyl
p-hydroxybenzoate.
[0136] PDGFR.alpha.-inhibiting therapeutic compositions may also
include encapsulated formulations to protect the therapeutic (e.g.
a dsRNA compound) against rapid elimination from the body, such as
a controlled release formulation, including implants and
microencapsulated delivery systems. Biodegradable, biocompatible
polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No. 4,522,811;
PCT publication WO 91/06309; and European patent publication
EP-A-43075. An encapsulated formulation may comprise a viral coat
protein. The viral coat protein may be derived from or associated
with a virus, such as a polyoma virus, or it may be partially or
entirely artificial. For example, the coat protein may be a Virus
Protein 1 and/or Virus Protein 2 of the polyoma virus, or a
derivative thereof.
[0137] PDGFR.alpha.-inhibiting compositions can also comprise a
delivery vehicle, including liposomes, for administration to a
subject, carriers and diluents and their salts, and/or can be
present in pharmaceutically acceptable formulations. For example,
methods for the delivery of nucleic acid molecules are described in
Akhtar et al., 1992, Trends Cell Bio., 2, 139; DELIVERY STRATEGIES
FOR ANTISENSE OLIGONUCLEOTIDE THERAPEUTICS, ed. Akbtar, 1995,
Maurer et al., 1999, Mol. Membr. Biol., 16, 129-140; Hofland and
Huang, 1999, Handb. Exp. Pharmacol., 137, 165-192; and Lee et al.,
2000, ACS Symp. Ser., 752, 184-192. Beigelman et al., U.S. Pat. No.
6,395,713 and Sullivan et al., PCT WO 94/02595 further describe the
general methods for delivery of nucleic acid molecules. These
protocols can be utilized for the delivery of virtually any nucleic
acid molecule.
[0138] PDGFR.alpha.-inhibiting therapeutics can be administered to
a mammalian tumor by a variety of methods known to those of skill
in the art, including, but not restricted to, encapsulation in
liposomes, by iontophoresis, or by incorporation into other
vehicles, such as hydrogels, cyclodextrins, biodegradable
nanocapsules, and bioadhesive microspheres, or by proteinaceous
vectors (O'Hare and Normand, International PCT Publication No. WO
00/53722). Alternatively, the therapeutic/vehicle combination is
locally delivered by direct injection or by use of an infusion
pump. Direct injection of the composition, whether subcutaneous,
intramuscular, or intradermal, can take place using standard needle
and syringe methodologies, or by needle-free technologies such as
those described in Conry et al., 1999, Clin. Cancer Res., 5,
2330-2337 and Barry et al., International PCT Publication No. WO
99/31262.
[0139] Pharmaceutically acceptable formulations of
PDGFR.alpha.-inhibitory therapeutics include salts of the above
described compounds, e.g., acid addition salts, for example, salts
of hydrochloric, hydrobromic, acetic acid, and benzene sulfonic
acid. A pharmacological composition or formulation refers to a
composition or formulation in a form suitable for administration,
e.g., systemic administration, into a cell or patient, including
for example a human. Suitable forms, in part, depend upon the use
or the route of entry, for example oral, transdermal, or by
injection. Such forms should not prevent the composition or
formulation from reaching a target cell. For example,
pharmacological compositions injected into the blood stream should
be soluble. Other factors are known in the art, and include
considerations such as toxicity and forms that prevent the
composition or formulation from exerting its effect.
[0140] Administration routes that lead to systemic absorption (i.e.
systemic absorption or accumulation of drugs in the blood stream
followed by distribution throughout the entire body), are desirable
and include, without limitation: intravenous, subcutaneous,
intraperitoneal, inhalation, oral, intrapulmonary and
intramuscular. Each of these administration routes exposes the
PDGFR.alpha. inhibiting therapeutic to an accessible diseased
tissue or tumor. The rate of entry of a drug into the circulation
has been shown to be a function of molecular weight or size. The
use of a liposome or other drug carrier comprising the compounds of
the instant invention can potentially localize the drug, for
example, in certain tissue types, such as the tissues of the
reticular endothelial system (RES). A liposome formulation that can
facilitate the association of drug with the surface of cells, such
as, lymphocytes and macrophages is also useful. This approach can
provide enhanced delivery of the drug to target cells by taking
advantage of the specificity of macrophage and lymphocyte immune
recognition of abnormal cells, such as cancer cells.
[0141] By "pharmaceutically acceptable formulation" is meant, a
composition or formulation that allows for the effective
distribution of the nucleic acid molecules of the instant invention
in the physical location most suitable for their desired activity.
Nonlimiting examples of agents suitable for formulation with the
nucleic acid molecules of the instant invention include:
P-glycoprotein inhibitors (such as Pluronic P85), which can enhance
entry of drugs into the CNS (Jolliet-Riant and Tillement, 1999,
Fundam. Clin. Pharmacol., 13, 16-26); biodegradable polymers, such
as poly (DL-lactide-coglycolide) microspheres for sustained release
delivery after intracerebral implantation (Emerich et al, 1999,
Cell Transplant, 8, 47-58) (Alkermes, Inc. Cambridge, Mass.); and
loaded nanoparticles, such as those made of polybutylcyanoacrylate,
which can deliver drugs across the blood brain barrier and can
alter neuronal uptake mechanisms (Prog Neuro-psychopharmacol Biol
Psychiatry, 23, 941-949, 1999). Other non-limiting examples of
delivery strategies for the PDGFR.alpha.-inhibiting compounds
useful in the method of the invention include material described in
Boado et al., 1998, J. Pharm. Sci., 87, 1308-1315; Tyler et al.,
1999, FEBS Lett., 421, 280-284; Pardridge et al., 1995, PNAS USA.,
92, 5592-5596; Boado, 1995, Adv. Drug Delivery Rev., 15, 73-107;
Aldrian-Herrada et al., 1998, Nucleic Acids Res., 26, 4910-4916;
and Tyler et al., 1999, PNAS USA., 96, 7053-7058.
[0142] Therapeutic compositions comprising surface-modified
liposomes containing poly (ethylene glycol) lipids (PEG-modified,
or long-circulating liposomes or stealth liposomes) may also be
suitably employed in the methods of the invention. These
formulations offer a method for increasing the accumulation of
drugs in target tissues. This class of drug carriers resists
opsonization and elimination by the mononuclear phagocytic system
(MPS or RES), thereby enabling longer blood circulation times and
enhanced tissue exposure for the encapsulated drug (Lasic et al.
Chem. Rev. 1995, 95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull.
1995, 43, 1005-1011). Such liposomes have been shown to accumulate
selectively in tumors, presumably by extravasation and capture in
the neovascularized target tissues (Lasic et al., Science 1995,
267, 1275-1276; Oku et al., 1995, Biochim. Biophys. Acta, 1238,
86-90). The long-circulating liposomes enhance the pharmacokinetics
and pharmacodynamics of DNA and RNA, particularly compared to
conventional cationic liposomes which are known to accumulate in
tissues of the MPS (Liu et al., J. Biol. Chem. 1995, 42,
24864-24870; Choi et al., International PCT Publication No. WO
96/10391; Ansell et al., International PCT Publication No. WO
96/10390; Holland et al., International PCT Publication No. WO
96/10392). Long-circulating liposomes are also likely to protect
drugs from nuclease degradation to a greater extent compared to
cationic liposomes, based on their ability to avoid accumulation in
metabolically aggressive MPS tissues such as the liver and
spleen.
[0143] Therapeutic compositions may include a pharmaceutically
effective amount of the desired compounds in a pharmaceutically
acceptable carrier or diluent. Acceptable carriers or diluents for
therapeutic use are well known in the pharmaceutical art, and are
described, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES,
Mack Publishing Co. (A. R. Gennaro edit. 1985). For example,
preservatives, stabilizers, dyes and flavoring agents can be
provided. These include sodium benzoate, sorbic acid and esters of
p-hydroxybenzoic acid. In addition, antioxidants and suspending
agents can be used.
[0144] A pharmaceutically effective dose is that dose required to
prevent, inhibit the occurrence, or treat (alleviate a symptom to
some extent, preferably all of the symptoms) of a disease state.
The pharmaceutically effective dose depends on the type of disease,
the composition used, the route of administration, the type of
mammal being treated, the physical characteristics of the specific
mammal under consideration, concurrent medication, and other
factors that those skilled in the medical arts will recognize.
Generally, an amount between 0.1 mg/kg and 100 mg/kg body
weight/day of active ingredients is administered dependent upon
potency of the negatively charged polymer.
[0145] Dosage levels of the order of from about 0.1 mg to about 140
mg per kilogram of body weight per day are useful in the treatment
of the above-indicated conditions (about 0.5 mg to about 7 g per
patient per day). The amount of active ingredient that can be
combined with the carrier materials to produce a single dosage form
varies depending upon the host treated and the particular mode of
administration. Dosage unit forms generally contain between from
about 1 mg to about 500 mg of an active ingredient. It is
understood that the specific dose level for any particular patient
depends upon a variety of factors including the activity of the
specific compound employed, the age, body weight, general health,
sex, diet, time of administration, route of administration, and
rate of excretion, drug combination and the severity of the
particular disease undergoing therapy.
[0146] For administration to non-human animals, the composition can
also be added to the animal feed or drinking water. It can be
convenient to formulate the animal feed and drinking water
compositions so that the animal takes in a therapeutically
appropriate quantity of the composition along with its diet. It can
also be convenient to present the composition as a premix for
addition to the feed or drinking water.
[0147] A PDGFR.alpha.-inhibiting therapeutic useful in the practice
of the invention may comprise a single compound as described above,
or a combination of multiple compounds, whether in the same class
of inhibitor (i.e. antibody inhibitor), or in different classes
(i.e antibody inhibitors and small-molecule inhibitors). Such
combination of compounds may increase the overall therapeutic
effect in inhibiting the progression of a PDGFR.alpha.-expressing
NSCLC tumor in the mammal. For example, the therapeutic composition
may a small molecule inhibitor, such as STI-571 (Gleevec.RTM.
alone, or in combination with other Gleevec.RTM. analogues
targeting PDGFR.alpha. activity and/or small molecule inhibitors of
EGFR, such as Tarceva.TM. or Iressa.TM.. The therapeutic
composition may also comprise one or more non-specific
chemotherapeutic agent in addition to one or more targeted
inhibitors. Such combinations have recently been shown to provide a
synergistic tumor killing effect in many cancers. The effectiveness
of such combinations in inhibiting PDGFR.alpha. activity and NSCLC
tumor growth in vivo can be assessed as described below.
Identification of PDGFR.alpha.-Inhibiting Compounds.
[0148] The invention also provides, in part, a method for
determining whether a compound inhibits the progression of a
mammalian NSCLC tumor belonging to a subset of NSCLC tumors in
which PDGFR.alpha. is activated, by determining whether the
compound inhibits the activity of PDGFR.alpha. in the NSCLC tumor.
In one preferred embodiment, inhibition of activity of PDGFR.alpha.
is determined by examining a biological sample comprising cells
from the NSCLC tumor. In another preferred embodiment, inhibition
of activity of PDGFR.alpha. is determined using at least one
PDGFR.alpha. activation state-specific reagent, and in one
preferred embodiment, the activation-state specific reagent is a
phosphorylation-site specific antibody.
[0149] The tested compound may be any type of therapeutic or
composition as described above. Methods for assessing the efficacy
of a compound, both in vitro and in vivo, are well established and
known in the art. For example, a composition may be tested for
ability to inhibit PDGFR.alpha. in vitro using a cell or cell
extract in which PDGFR.alpha. is activated. A panel of compounds
may be employed to test the specificity of the compound for
PDGFR.alpha. (as opposed to other targets, such as EGFR or PDGFR
beta).
[0150] A compound found to be an effective inhibitor of
PDGFR.alpha. activity in vitro may then be examined for its ability
to inhibit NSCLC tumor growth, in vivo, using, for example,
mammalian xenografts harboring human PDGFR.alpha. expressing NSCLC
tumors. In this procedure, cell lines known to be driven by
PDGFR.alpha. are placed subcutaneously in the mouse. The cells then
grow into a tumor mass that may be visually monitored. The mouse
may then be treated with the drug. The effect of the drug treatment
on tumor size may be externally observed. The mouse is then
sacrificed and the tumor removed for analysis by IHC and Western
blot. In this way, the effects of the drug may be observed in a
biological setting most closely resembling a patient. The drug's
ability to alter signaling in the tumor cells or surrounding
stromal cells may be determined by analysis with
phosphorylation-specific antibodies. The drug's effectiveness in
inducing cell death or inhibition of cell proliferation may also be
observed by analysis with apoptosis specific markers such as
cleaved caspase 3 and cleaved PARP.
[0151] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD50 (the dose
lethal to 50% of the population) and the ED50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD50/ED50. Compounds which exhibit
high therapeutic indices are preferred.
[0152] The following Examples are provided only to further
illustrate the invention, and are not intended to limit its scope,
except as provided in the claims appended hereto. The present
invention encompasses modifications and variations of the methods
taught herein which would be obvious to one of ordinary skill in
the art.
Example 1
Identification of PDGFR.alpha.-Expression in a NSCLC Cell Line by
Global Phosphopeptide Profiling
[0153] The global phosphorylation profiles of four human NSCLC cell
lines, A549, H441, H1373, and H1703, were examined using a recently
described and powerful technique for the isolation and mass
spectrometric characterization of modified peptides from complex
mixtures (the "IAP" technique, see Rush et al., supra). The IAP
technique was performed using a phosphotyrosine-specific antibody
(CELL SIGNALING TECHNOLOGY, INC., Beverly, Mass., 2003/04 Cat.
#9411) to isolate, and subsequently characterize,
phosphotyrosine-containing peptides from extracts of the NSCLC cell
lines.
[0154] Tryptic phosphotyrosine-containing peptides were purified
and analyzed from extracts of each of the cell lines mentioned
above, as follows. Cells were cultured in DMEM medium or RPMI 1640
medium supplemented with 10% fetal bovine serum and
penicillin/streptomycin. Cells were harvested by low speed
centrifugation. After complete aspiration of medium, cells were
resuspended in 1 mL lysis buffer per 1.25.times.10.sup.8 cells (20
mM HEPES pH 8.0, 9 M urea, 1 mM sodium vanadate, supplemented or
not with 2.5 mM sodium pyro-phosphate, 1 mM
.beta.-glycerol-phosphate) and sonicated.
[0155] Sonicated cell lysates were cleared by centrifugation at
20,000.times.g, and proteins were reduced with DTT at a final
concentration of 4.1 mM and alkylated with iodoacetamide at 8.3 mM.
For digestion with trypsin, protein extracts were diluted in 20 mM
HEPES pH 8.0 to a final concentration of 2 M urea and soluble
TLCK-trypsin (Worthington) was added at 10-20 .mu.g/mL. Digestion
was performed for 1-2 days at room temperature.
[0156] Trifluoroacetic acid (TFA) was added to protein digests to a
final concentration of 1%, precipitate was removed by
centrifugation, and digests were loaded onto Sep-Pak C.sub.18
columns (Waters) equilibrated with 0.1% TFA. A column volume of
0.7-1.0 ml was used per 2.times.10.sup.8 cells. Columns were washed
with 15 volumes of 0.1% TFA, followed by 4 volumes of 5%
acetonitrile (MeCN) in 0.1.degree./0 TFA. Peptide fraction I was
obtained by eluting columns with 2 volumes each of 8, 12, and 15%
MeCN in 0.1% TFA and combining the eluates. Fractions II and III
were a combination of eluates after eluting columns with 18, 22,
25% MeCN in 0.1% TFA and with 30, 35, 40% MeCN in 0.1% TFA,
respectively. All peptide fractions were lyophilized.
[0157] Peptides from each fraction corresponding to
2.times.10.sup.8 cells were dissolved in 1 ml of IAP buffer (20 mM
Tris/HCl or 50 mM MOPS pH 7.2, 10 mM sodium phosphate, 50 mM NaCl)
and insoluble matter (mainly in peptide fractions III) was removed
by centrifugation. IAP was performed on each peptide fraction
separately. The phosphotyrosine monoclonal antibody P-Tyr-100 (Cell
Signaling Technology, Inc., catalog number 9411) was coupled at 4
mg/ml beads to protein G (Roche), respectively. Immobilized
antibody (15 .mu.l, 60 .mu.g) was added as 1:1 slurry in TAP buffer
to 1 ml of each peptide fraction, and the mixture was incubated
overnight at 4.degree. C. with gentle rotation. The immobilized
antibody beads were washed three times with 1 ml TAP buffer and
twice with 1 ml water, all at 4.degree. C. Peptides were eluted
from beads by incubation with 75 .mu.l of 0.1.degree./0 TFA at room
temperature for 10 minutes.
[0158] Alternatively, one single peptide fraction was obtained from
Sep-Pak C18 columns by elution with 2 volumes each of 10%, 15%,
20%, 25%, 30.degree. A), 35.degree.A) and 40.degree. A)
acetonitrile in 0.1% TFA and combination of all eluates. IAP on
this peptide fraction was performed as follows: After
lyophilization, peptide was dissolved in 1.4 ml IAP buffer (MOPS pH
7.2, 10 mM sodium phosphate, 50 mM NaCl) and insoluble matter was
removed by centrifugation. Immobilized antibody (40 .mu.l, 160
.mu.g) was added as 1:1 slurry in IAP buffer, and the mixture was
incubated overnight at 4.degree. C. with gentle shaking. The
immobilized antibody beads were washed three times with 1 ml IAP
buffer and twice with 1 ml water, all at 4.degree. C. Peptides were
eluted from beads by incubation with 55 .mu.l of 0.15% TFA at room
temperature for 10 min (eluate 1), followed by a wash of the beads
(eluate 2) with 45 .mu.l of 0.15% TFA. Both eluates were
combined.
[0159] Analysis by LC-MS/MS Mass Spectrometry.
[0160] 40 .mu.l or more of IAP eluate were purified by 0.2 .mu.l
StageTips or ZipTips. Peptides were eluted from the microcolumns
with 1 .mu.l of 40% MeCN, 0.1% TFA (fractions I and II) or 1 .mu.l
of 60% MeCN, 0.1% TFA (fraction III) into 7.6 .mu.l of 0.4% acetic
acid/0.005% heptafluorobutyric acid. For single fraction analysis,
1 .mu.l of 60% MeCN, 0.1% TFA, was used for elution from the
microcolumns. This sample was loaded onto a 10 cm.times.75 .mu.m
PicoFrit capillary column (New Objective) packed with Magic C18 AQ
reversed-phase resin (Michrom Bioresources) using a Famos
autosampler with an inert sample injection valve (Dionex). The
column was then developed with a 45-min linear gradient of
acetonitrile delivered at 200 nl/min (Ultimate, Dionex), and tandem
mass spectra were collected in a data-dependent manner with an LCQ
Deca XP Plus ion trap mass spectrometer.
[0161] Database Analysis & Assignments.
[0162] MS/MS spectra were evaluated using TurboSequest in the
Sequest Browser package (v. 27, rev. 12) supplied as part of
BioWorks 3.0 (ThermoFinnigan). Individual MS/MS spectra were
extracted from the raw data file using the Sequest Browser program
CreateDta, with the following settings: bottom MW, 700; top MW,
4,500; minimum number of ions, 20; minimum TIC, 4.times.10.sup.5;
and precursor charge state, unspecified. Spectra were extracted
from the beginning of the raw data file before sample injection to
the end of the eluting gradient. The lonQuest and VuDta programs
were not used to further select MS/MS spectra for Sequest analysis.
MS/MS spectra were evaluated with the following TurboSequest
parameters: peptide mass tolerance, 2.5; fragment ion tolerance,
0.0; maximum number of differential amino acids per modification,
4; mass type parent, average; mass type fragment, average; maximum
number of internal cleavage sites, 10; neutral losses of water and
ammonia from bandy ions were considered in the correlation
analysis. Proteolytic enzyme was specified except for spectra
collected from elastase digests.
[0163] Searches were performed against the NCBI human protein
database (either as released on Apr. 29, 2003 and containing 37,490
protein sequences or as released on Feb. 23, 2004 and containing
27,175 protein sequences). Cysteine carboxamidomethylation was
specified as a static modification, and phosphorylation was allowed
as a variable modification on serine, threonine, and tyrosine
residues or on tyrosine residues alone. It was determined that
restricting phosphorylation to tyrosine residues had little effect
on the number of phosphorylation sites assigned.
[0164] In proteomics research, it is desirable to validate protein
identifications based solely on the observation of a single peptide
in one experimental result, in order to indicate that the protein
is, in fact, present in a sample. This has led to the development
of statistical methods for validating peptide assignments, which
are not yet universally accepted, and guidelines for the
publication of protein and peptide identification results (see Carr
et al., Mol. Cell Proteomics 3: 531-533 (2004)), which were
followed in this Example. However, because the immunoaffinity
strategy separates phosphorylated peptides from unphosphorylated
peptides, observing just one phosphopeptide from a protein is a
common result, since many phosphorylated proteins have only one
tyrosinephosphorylated site.
[0165] For this reason, it is appropriate to use additional
criteria to validate phosphopeptide assignments. Assignments are
likely to be correct if any of these additional criteria are met:
(i) the same sequence is assigned to co-eluting ions with different
charge states, since the MS/MS spectrum changes markedly with
charge state; (ii) the site is found in more than one peptide
sequence context due to sequence overlaps from incomplete
proteolysis or use of proteases other than trypsin; (iii) the site
is found in more than one peptide sequence context due to
homologous but not identical protein isoforms; (iv) the site is
found in more than one peptide sequence context due to homologous
but not identical proteins among species; and (v) sites validated
by MS/MS analysis of synthetic phosphopeptides corresponding to
assigned sequences, since the ion trap mass spectrometer produces
highly reproducible MS/MS spectra. The last criterion is routinely
employed to confirm novel site assignments of particular
interest.
[0166] All spectra and all sequence assignments made by Sequest
were imported into a relational database. Assigned sequences were
accepted or rejected following a conservative, two-step process. In
the first step, a subset of high-scoring sequence assignments was
selected by filtering for XCorr values of at least 1.5 for a charge
state of +1, 2.2 for +2, and 3.3 for +3, allowing a maximum RSp
value of 10. Assignments in this subset were rejected if any of the
following criteria were satisfied: (i) the spectrum contained at
least one major peak (at least 10% as intense as the most intense
ion in the spectrum) that could not be mapped to the assigned
sequence as an a, b, or y ion, as an ion arising from neutral-loss
of water or ammonia from a b or y ion, or as a multiply protonated
ion; (ii) the spectrum did not contain a series of b or y ions
equivalent to at least six uninterrupted residues; or (iii) the
sequence was not observed at least five times in all the studies we
have conducted (except for overlapping sequences due to incomplete
proteolysis or use of proteases other than trypsin). In the second
step, assignments with below-threshold scores were accepted if the
low-scoring spectrum showed a high degree of similarity to a
high-scoring spectrum collected in another study, which simulates a
true reference library-searching strategy. All spectra supporting
the final list of assigned sequences (not shown here) were reviewed
by at least three scientists to establish their credibility.
[0167] The foregoing IAP analysis identified six phospho-tyrosine
sites in PDGFR.alpha. as being present in the H1703 cell line, but
not in the three other NSCLC cell lines examined (see Table 1
below). In contrast, phospho-tyrosine sites in EGFR were identified
in all four cell lines. This result was surprising since the link
between PDGFR.alpha. expression and/or phosphorylation in a subset
of human NSCLC had not previously been established.
TABLE-US-00001 TABLE 1 Phosphorylation Phosphorylated Kinase Site
Sequence Tyrosine SEQ ID NO: PDGFR.alpha. VIESISPDGHEyIYVDPMQLPYDSR
Y572 SEQ ID NO: 3 PDGFR.alpha. QADTTQyVPMLER Y742 SEQ ID NO: 4
PDGFR.alpha. SLyDRPASYK Y762 SEQ ID NO: 5 PDGFR.alpha. SLYDRPASyK
Y768 SEQ ID NO: 6 PDGFR.alpha. DIMHDSNyVSK Y849 SEQ ID NO: 7
PDGFR.alpha. LSADSGylIPLPDIDPVPEEEDLGKR Y1018 SEQ ID NO: 8
Example 2
Western Blot Analysis and IHC of PDGFR.alpha. Expression in NSCLC
Tumor Cell Lines and Xenografts
[0168] The observation that the H1703 NSCLC tumor cell line--but
not the other NSCLC cell lines--expresses PDGFR.alpha. was
confirmed by Western blot analysis of cell extracts using
antibodies specific for PDGFR.alpha. and other receptor tyrosine
kinases (RTKs) and downstream kinases. Antibody specificity for
receptor tyrosine kinases is often difficult to obtain due to many
possible variables including the close homology among the receptors
and the secondary modifications that the receptors undergo.
Therefore, the first step in determining PDGFR.alpha. expression by
Western blot analysis was to identify an antibody that is specific
for this protein.
[0169] FIG. 4 presents the results of an analysis of three cell
lines probed with two antibodies to PDGFR.alpha. and one antibody
to PDGFR.beta.. The U87 cell line is known to express PDGFR.beta.,
the H358 cell line does not express PDGFR and the H118 cell line
strongly expresses both isoforms. The xenograft samples include
both the cell line and the surrounding stomal cells. As a result,
these samples are expected to include both isoforms. The results
demonstrate that the CST (Cell Signaling Technology, Beverly,
Mass.) PDGFR.alpha. and PDGFR.beta. antibodies (Cat. Nos. 3164 and
3169, respectively) correctly detect the appropriate proteins and
do not detect any other proteins as shown by the lack of additional
bands on the Western blot. In contrast, the PDGFR.alpha. antibody
from Santa Cruz Biotechnology (Santa Cruz, Calif.) (Cat. No.
SC-338) detects multiple proteins. Some of the proteins detected
with this antibody are detected as strongly as the bands at the
correct molecular weight (see FIG. 4). While Santa Cruz
Biotechnology offers multiple antibodies to PDGFR.alpha., SC-338 is
the preferred product for IHC and is the product most often
referenced in the literature.
[0170] A previous report (Zhang et al., (2003), supra.) employed a
PDGFR.alpha. antibody (from Santa Cruz Biotechnology, Inc.) in an
attempt to analyze PDGFR.alpha. expression in the A549 cell line.
This Western blot was presently repeated using the SC-338 antibody
from Santa Cruz Biotechnology as well as a CST antibody to
PDGFR.alpha. and a CST antibody to PDGFR.beta. (results are shown
in FIG. 5(a)). The NIH3T3 cell line was included as a positive
control, as it is known that this cell line expresses both isoforms
of PDGFR. The results indicate that the Santa Cruz Biotechnology
antibody detects multiple proteins in the A549 cells, none of which
match the correct molecular weight for PDGFR. The antibody does
detect a protein in the NIH3T3 cells that has the correct molecular
weight. The CST PDGFR.alpha. (#3164) antibody detects a protein
with the correct molecular weight in the NIH3T3 cell line but not
in the A549 cells. Likewise, the CST PDGFR.beta. (#3169) antibody
detects PDGFR.beta. in the NIH3T3 cell line but not in the A549
cell line. These results clearly demonstrate that the Santa Cruz
antibody is not specific for PDGFR.alpha. in the A549 cell line,
and that Westerns with antibodies that are specific for this
protein indicate that the A549 cell line does not express
detectable levels of PDGFR.alpha.. The present results bring into
doubt the conclusions reached by Zhang et al., and given the lack
of specificity of the antibody employed in that study, it is likely
the authors detected something other than PDGFR.alpha.
expression.
[0171] The initial mass spec screen of NSCLC cell lines indicated
that the H1703 cell line expressed PDGFR.alpha. (see Example 1).
The CST PDGFR antibodies that have been shown to be specific were
used in Western blot analysis of this cell line in FIG. 5b. The
A549 cell line was also included in the analysis as well as two
Santa Cruz Biotechnology PDGFR.alpha. antibodies (SC-338 and
SC-431). The results support the mass spec result, indicating that
the H1703 cell line expresses PDGFR.alpha. while the A549 cell line
does not. The results with the Santa Cruz Biotechnology antibodies
are similar although both antibodies show multiple cross-reactive
bands in the A549 cell line.
[0172] As a final determination of PDGFR.alpha. expression, the
cells were stimulated with PDGFaa growth factor. This homo-dimer of
the a form of the growth factor specifically activates PDGFR.alpha.
and not PDGFR.beta.. Therefore, cells that express PDGFR.alpha.
should show phosphorylation of the receptor and activation of
downstream signaling following treatment with this ligand, while
cells that lack the receptor should not show a response. AKT
phosphorylation was used as a marker of downstream signaling.
[0173] The results in FIG. 6 show that in the H1703 cell line,
PDGFaa treatment results in phosphorylation of PDGFR.alpha. and
AKT. PDGFaa treatment of A549 cells does not result in AKT
activation and no PDGFR.alpha. or phospho-PDGFR.alpha. is detected.
These results, along with the results presented above, clearly
demonstrate that the H1703 cell line expresses PDGFR.alpha. while
the A549 cell line does not.
[0174] Finally, the antibody specificity observed by Western blot
analysis may have significant implications for the use of the
antibody in IHC. To test the use of the PDGFR.alpha. antibodies in
IHC, A549 xenografts were formalin fixed and paraffin embedded, and
probed with the antibodies. FIG. 7 presents the IHC results. As
expected from the Western blot results, the Santa Cruz PDGFR.alpha.
antibody, SC-338, gives non-specific staining of the A549 cells
while the CST #3164 antibody only detects PDGFR.alpha. in the
surrounding stromal cells. This staining of the normal mouse stomal
cells is appropriate as these cells are known to express the
receptor.
Example 3
Immunohistochemical Analysis of PDGFR.alpha. Expression in Human
NSCLC Tumor Samples
[0175] The existence of a distinct subset of human NSCLC tumors in
which PDGFR.alpha. is expressed was further confirmed by IHC
analysis of multiple tissue micro-arrays comprising tumor samples
from 304 human NSCLC patients. Tissues were obtained from multiple
sources including commercial as well as public tissue banks. The
classification of the tumors as well as the scoring of the IHC
staining was performed by a trained pathologist. The IHC was done
with the CST PDGFR.alpha.-specific antibody (#3164) that was shown
to be specific by Western blot as well as peptide absorption (data
not shown). The results of the IHC screen are summarized in Table 2
below.
TABLE-US-00002 TABLE 2 PDGFR.alpha. is Expressed in a Small Subset
of Human NSCLC Tumors. IHC Cases score Pathological diagnosis Age
Sex HL001 2+ Adenocarcinoma 40 F HL002 2+ Adenocarcinoma 62 F HL003
1-2+ Adenocarcinoma 52 M HL004 1+ Adenocarcinoma 51 F HL005 1+
Adenocarcinoma 60 F HL006 2+ Adenocarcinoma 50 M HL007 1-2+
Adenocarcinoma 56 F HL008 1-2+ Bronchioloalveolar carcinoma 58 M
HL009 3+ Bronchioloalveolar carcinoma 57 F CST-219 DIV2 HL010 2-3+
Bronchioloalveolar carcinoma 52 F HL011 1-2+ Bronchioloalveolar
carcinoma 54 M HL012 1+ Bronchioloalveolar carcinoma 52 F HL013 1+
Bronchioloalveolar carcinoma 48 F HL014 1+ Squamous cell carcinoma
67 M HL015 1+ mucoeperdoid carcinoma 26 F HL016 1-2+ adenoid
carcinoma 54 F HL017 3+ Sarcomatoid carcinoma 59 M
[0176] As shown in Table 2, out of 304 NSCLC tumor tissue samples
screened, only 17 (6%) showed positive PDGFR.alpha. staining
PDGFR.alpha. expression was seen more frequently in
Bronchioloalveolar carcinomas (6 cases) and Adenocarcinomas (7
cases) (13/17, 76%), and less frequently in Sarcomatoid carcinomas
(1 case) (1/17, 6%). PDGFR.alpha.-expressing NSCLC tumors occur
more frequently in women (11/17, 65%) than in men (6/17, 35%).
These results are very different than the IHC results reported by
Zhang et al. (2003), supra., and reflect the specificity of the CST
PDGFR.alpha. antibody compared to the non-specific Santa Cruz
Biotechnology antibody. Zhang et al. reported PDGFR.alpha.
expression in 27 out of 29 NSCLC samples. This extremely high level
of staining reported in the Zhang study is most likely is due to
the cross-reactivity of the antibody employed in the IHC analysis.
It is noteworthy that, prior to the present disclosure, no other
reports of PDGFR.alpha. expression in NSCLC have been made
following the Zhang et al. paper.
Example 4
Gleevec.RTM. Inhibits Growth of PDGFR.alpha.-Expressing Mammalian
NSCLC Cell Lines
[0177] In order to confirm that PDGFR.alpha. is driving cell growth
and survival in the subset of NSCLC tumors in which this RTK is
expressed, the ability of a PDGFR.alpha.-inhibitor, Gleevec.RTM.,
to inhibit growth of H1703 cells was examined. A standard MTT cell
proliferation assay (see Mosmann, J. Immunol Methods. 65(1-2):
55-63 (1983)) was performed on the H1703, A549, H1373 and K562 cell
lines using a range of Gleevec.RTM. concentrations. The H1373 cell
line was predicted to be insensitive to Gleevec.RTM. as it is
thought to be driven by erbB2 and erbB3 (see Sithanandam,
Carcinogenesis 24(10): 1581-92 (2003)). The K562 cell line is known
to be driven by the BCR/ABL translocation which is inhibited by
Gleevec.RTM.. The results of the assay are presented in FIG. 8(a).
As predicted, the H1373 cell line is insensitive to Gleevec.RTM.
while the K562 cell line is sensitive at concentrations of 0.1
.mu.M. The H1703 cell line was also sensitive to Gleevec.RTM. at
concentrations similar to what was observed with the K562 cell
line. In contrast, the A549 cell line was not affected by
Gleevec.RTM. at concentrations up to 10 .mu.M.
[0178] To confirm the effect of Gleevec.RTM. on the H1703 cell
line, Western blot analysis was performed on the cells following
exposure to a range of Gleevec.RTM. concentrations. FIG. 8(b)
presented the results of this analysis. As shown, increasing
Gleevec.RTM. concentrations result in an increase in cleaved PARP,
an indication that Gleevec.RTM. treatment is resulting in cell
apoptosis. PARP cleavage is one mechanism known to be involved in
cell apoptosis (see Lazebnik et al. Nature 371: 346-347 (1994)).
Cell apoptosis was also analyzed by analyzing caspase 3 cleavage by
flow cytometry of the cells following Gleevec.RTM. treatment for 1,
2 or 3 days. As shown in FIG. 8(c), caspase 3 cleavage is observed
as early as 1 day of treatment and increases as the exposure time
increases. Similar to PARP cleavage, caspase 3 cleavage is a well
known marker of cell apoptosis (see Fernandes-Alnemri et al., J.
Biol. Chem. 269: 30761-30764 (1994)). These results demonstrate
that Gleevec.RTM. treatment of H1703 cells results in growth
inhibition and apoptosis.
Example 5
Gleevec.RTM. Inhibits Signaling in PDGFR.alpha.-Expressing
Mammalian NSCLC Cell Lines
[0179] If Gleevec.RTM. alters the ability of PDGFR.alpha. to drive
cell proliferation and survival in H1703 cells, then it must
interfere with the cellular signaling that occurs downstream of the
receptor. To test this hypothesis, Western blot analysis was
performed on the cells following Gleevec.RTM. treatment as well as
Iressa.TM. treatment and stimulation with EGF. Iressa.TM. is a
targeted EGFR inhibitor. Phosphorylation of the EGFR receptor, ERK
and AKT were determined while total PDGFR.alpha. and ERK1/2 are
included as loading controls. FIG. 9(a) presents the results of
this analysis. In the untreated control cells, AKT and ERK are both
phosphorylated while the EGFR receptor is not. EGF treatment
induces the phosphorylation of EGFR as well as an increase in
phosphorylation of ERK and AKT as would be expected. Treatment with
Iressa.TM. decreases the phosphorylation of EGFR and ERK but not
AKT. Importantly, only treatment of the cells with Gleevec.RTM.
results in the loss of AKT phosphorylation. AKT is thought to be
the primary driver of cell survival (see Franke, Cell 88: 435-437
(1997)).
[0180] Therefore, these results demonstrate that while these cells
express EGFR that may be inhibited by Iressa.TM., the constitutive
activation of AKT is only inhibited through PDGFR.alpha.. FIG. 9(b)
presents a dose response analysis of Gleevec.RTM. on H1703 cells.
The results indicate that Gleevec.RTM. treatment at doses as low as
0.01 .mu.M inhibit PDGFR.alpha. phosphorylation while doses of 0.1
.mu.M greatly inhibit AKT phosphorylation. These results are
consistent with the hypothesis that Gleevec.RTM. is inhibiting
H1703 cell growth and survival through inhibition of PDGFR.alpha.
and AKT signaling.
Example 6
Gleevec.RTM. Inhibits Growth of PDGFR.alpha.-Expressing Mammalian
NSCLC Tumor Xenografts
[0181] In order to further confirm the ability of Gleevec.RTM. to
inhibit cell growth and survival in the subset of NSCLC tumors in
which this RTK is expressed, human tumor xenografts, in vivo, were
examined. In this model, human cell lines are injected into
immune-compromised mice forming xenograft tumors that resemble
human tumors including vascularization and other features found in
human tumors. The mice are then administered the drug in the same
manner as in human patients. Tumor size may be monitored visually
during drug treatment and the tumors may be removed, fixed and
analyzed by standard IHC procedures or lysed and analyzed by
Western blot.
[0182] FIG. 10(a) presents the results of the xenograft experiments
demonstrating that Gleevec.RTM. treatment results in a significant
decrease in tumor size. The average tumor diameter in the 5 control
mice was approximately 190 mm while the average tumor diameter in
the 3 treated mice was only approximately 20 mm. This dramatic
decrease in tumor size in the treated mice is a strong indication
that Gleevec.RTM. treatment in vivo has a therapeutic effect on
tumors that are driven by PDGFR.alpha..
[0183] To further analyze the mechanism behind this reduction in
tumor size, Western blots were performed on the tumor lysate from 4
treated mice compared to one control mouse. The results in FIG.
10(b) show that in these xenografts, Gleevec.RTM. is inhibiting
PDGFR.alpha. phosphorylation (total AKT was included in the Western
as a loading control). These results are consistent with previous
results that suggest that Gleevec.RTM. is reducing tumor size
through PDGFR.alpha. inhibition. The xenograft tumors were also
analyzed by IHC (see FIG. 11) in which control tumors were compared
to Gleevec.RTM. treated tumors. The results of the IHC analysis
again demonstrate that Gleevec.RTM. treatment results in a decrease
in PDGFR.alpha. and AKT phosphorylation. The IHC results suggest
that mammalian tumors, e.g. from a human patient, may be analyzed
by IHC in a similar manner to determine the biological activity of
a PDGFR.alpha. inhibitor.
Sequence CWU 1
1
811089PRTHomo sapiens 1Met Gly Thr Ser His Pro Ala Phe Leu Val Leu
Gly Cys Leu Leu Thr 1 5 10 15 Gly Leu Ser Leu Ile Leu Cys Gln Leu
Ser Leu Pro Ser Ile Leu Pro 20 25 30 Asn Glu Asn Glu Lys Val Val
Gln Leu Asn Ser Ser Phe Ser Leu Arg 35 40 45 Cys Phe Gly Glu Ser
Glu Val Ser Trp Gln Tyr Pro Met Ser Glu Glu 50 55 60 Glu Ser Ser
Asp Val Glu Ile Arg Asn Glu Glu Asn Asn Ser Gly Leu 65 70 75 80 Phe
Val Thr Val Leu Glu Val Ser Ser Ala Ser Ala Ala His Thr Gly 85 90
95 Leu Tyr Thr Cys Tyr Tyr Asn His Thr Gln Thr Glu Glu Asn Glu Leu
100 105 110 Glu Gly Arg His Ile Tyr Ile Tyr Val Pro Asp Pro Asp Val
Ala Phe 115 120 125 Val Pro Leu Gly Met Thr Asp Tyr Leu Val Ile Val
Glu Asp Asp Asp 130 135 140 Ser Ala Ile Ile Pro Cys Arg Thr Thr Asp
Pro Glu Thr Pro Val Thr 145 150 155 160 Leu His Asn Ser Glu Gly Val
Val Pro Ala Ser Tyr Asp Ser Arg Gln 165 170 175 Gly Phe Asn Gly Thr
Phe Thr Val Gly Pro Tyr Ile Cys Glu Ala Thr 180 185 190 Val Lys Gly
Lys Lys Phe Gln Thr Ile Pro Phe Asn Val Tyr Ala Leu 195 200 205 Lys
Ala Thr Ser Glu Leu Asp Leu Glu Met Glu Ala Leu Lys Thr Val 210 215
220 Tyr Lys Ser Gly Glu Thr Ile Val Val Thr Cys Ala Val Phe Asn Asn
225 230 235 240 Glu Val Val Asp Leu Gln Trp Thr Tyr Pro Gly Glu Val
Lys Gly Lys 245 250 255 Gly Ile Thr Met Leu Glu Glu Ile Lys Val Pro
Ser Ile Lys Leu Val 260 265 270 Tyr Thr Leu Thr Val Pro Glu Ala Thr
Val Lys Asp Ser Gly Asp Tyr 275 280 285 Glu Cys Ala Ala Arg Gln Ala
Thr Arg Glu Val Lys Glu Met Lys Lys 290 295 300 Val Thr Ile Ser Val
His Glu Lys Gly Phe Ile Glu Ile Lys Pro Thr 305 310 315 320 Phe Ser
Gln Leu Glu Ala Val Asn Leu His Glu Val Lys His Phe Val 325 330 335
Val Glu Val Arg Ala Tyr Pro Pro Pro Arg Ile Ser Trp Leu Lys Asn 340
345 350 Asn Leu Thr Leu Ile Glu Asn Leu Thr Glu Ile Thr Thr Asp Val
Glu 355 360 365 Lys Ile Gln Glu Ile Arg Tyr Arg Ser Lys Leu Lys Leu
Ile Arg Ala 370 375 380 Lys Glu Glu Asp Ser Gly His Tyr Thr Ile Val
Ala Gln Asn Glu Asp 385 390 395 400 Ala Val Lys Ser Tyr Thr Phe Glu
Leu Leu Thr Gln Val Pro Ser Ser 405 410 415 Ile Leu Asp Leu Val Asp
Asp His His Gly Ser Thr Gly Gly Gln Thr 420 425 430 Val Arg Cys Thr
Ala Glu Gly Thr Pro Leu Pro Asp Ile Glu Trp Met 435 440 445 Ile Cys
Lys Asp Ile Lys Lys Cys Asn Asn Glu Thr Ser Trp Thr Ile 450 455 460
Leu Ala Asn Asn Val Ser Asn Ile Ile Thr Glu Ile His Ser Arg Asp 465
470 475 480 Arg Ser Thr Val Glu Gly Arg Val Thr Phe Ala Lys Val Glu
Glu Thr 485 490 495 Ile Ala Val Arg Cys Leu Ala Lys Asn Leu Leu Gly
Ala Glu Asn Arg 500 505 510 Glu Leu Lys Leu Val Ala Pro Thr Leu Arg
Ser Glu Leu Thr Val Ala 515 520 525 Ala Ala Val Leu Val Leu Leu Val
Ile Val Ile Ile Ser Leu Ile Val 530 535 540 Leu Val Val Ile Trp Lys
Gln Lys Pro Arg Tyr Glu Ile Arg Trp Arg 545 550 555 560 Val Ile Glu
Ser Ile Ser Pro Asp Gly His Glu Tyr Ile Tyr Val Asp 565 570 575 Pro
Met Gln Leu Pro Tyr Asp Ser Arg Trp Glu Phe Pro Arg Asp Gly 580 585
590 Leu Val Leu Gly Arg Val Leu Gly Ser Gly Ala Phe Gly Lys Val Val
595 600 605 Glu Gly Thr Ala Tyr Gly Leu Ser Arg Ser Gln Pro Val Met
Lys Val 610 615 620 Ala Val Lys Met Leu Lys Pro Thr Ala Arg Ser Ser
Glu Lys Gln Ala 625 630 635 640 Leu Met Ser Glu Leu Lys Ile Met Thr
His Leu Gly Pro His Leu Asn 645 650 655 Ile Val Asn Leu Leu Gly Ala
Cys Thr Lys Ser Gly Pro Ile Tyr Ile 660 665 670 Ile Thr Glu Tyr Cys
Phe Tyr Gly Asp Leu Val Asn Tyr Leu His Lys 675 680 685 Asn Arg Asp
Ser Phe Leu Ser His His Pro Glu Lys Pro Lys Lys Glu 690 695 700 Leu
Asp Ile Phe Gly Leu Asn Pro Ala Asp Glu Ser Thr Arg Ser Tyr 705 710
715 720 Val Ile Leu Ser Phe Glu Asn Asn Gly Asp Tyr Met Asp Met Lys
Gln 725 730 735 Ala Asp Thr Thr Gln Tyr Val Pro Met Leu Glu Arg Lys
Glu Val Ser 740 745 750 Lys Tyr Ser Asp Ile Gln Arg Ser Leu Tyr Asp
Arg Pro Ala Ser Tyr 755 760 765 Lys Lys Lys Ser Met Leu Asp Ser Glu
Val Lys Asn Leu Leu Ser Asp 770 775 780 Asp Asn Ser Glu Gly Leu Thr
Leu Leu Asp Leu Leu Ser Phe Thr Tyr 785 790 795 800 Gln Val Ala Arg
Gly Met Glu Phe Leu Ala Ser Lys Asn Cys Val His 805 810 815 Arg Asp
Leu Ala Ala Arg Asn Val Leu Leu Ala Gln Gly Lys Ile Val 820 825 830
Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp Ile Met His Asp Ser Asn 835
840 845 Tyr Val Ser Lys Gly Ser Thr Phe Leu Pro Val Lys Trp Met Ala
Pro 850 855 860 Glu Ser Ile Phe Asp Asn Leu Tyr Thr Thr Leu Ser Asp
Val Trp Ser 865 870 875 880 Tyr Gly Ile Leu Leu Trp Glu Ile Phe Ser
Leu Gly Gly Thr Pro Tyr 885 890 895 Pro Gly Met Met Val Asp Ser Thr
Phe Tyr Asn Lys Ile Lys Ser Gly 900 905 910 Tyr Arg Met Ala Lys Pro
Asp His Ala Thr Ser Glu Val Tyr Glu Ile 915 920 925 Met Val Lys Cys
Trp Asn Ser Glu Pro Glu Lys Arg Pro Ser Phe Tyr 930 935 940 His Leu
Ser Glu Ile Val Glu Asn Leu Leu Pro Gly Gln Tyr Lys Lys 945 950 955
960 Ser Tyr Glu Lys Ile His Leu Asp Phe Leu Lys Ser Asp His Pro Ala
965 970 975 Val Ala Arg Met Arg Val Asp Ser Asp Asn Ala Tyr Ile Gly
Val Thr 980 985 990 Tyr Lys Asn Glu Glu Asp Lys Leu Lys Asp Trp Glu
Gly Gly Leu Asp 995 1000 1005 Glu Gln Arg Leu Ser Ala Asp Ser Gly
Tyr Ile Ile Pro Leu Pro 1010 1015 1020 Asp Ile Asp Pro Val Pro Glu
Glu Glu Asp Leu Gly Lys Arg Asn 1025 1030 1035 Arg His Ser Ser Gln
Thr Ser Glu Glu Ser Ala Ile Glu Thr Gly 1040 1045 1050 Ser Ser Ser
Ser Thr Phe Ile Lys Arg Glu Asp Glu Thr Ile Glu 1055 1060 1065 Asp
Ile Asp Met Met Asp Asp Ile Gly Ile Asp Ser Ser Asp Leu 1070 1075
1080 Val Glu Asp Ser Phe Leu 1085 26633DNAHomo sapiens 2ttctccccgc
cccccagttg ttgtcgaagt ctgggggttg ggactggacc ccctgattgc 60gtaagagcaa
aaagcgaagg cgcaatctgg acactgggag attcggagcg cagggagttt
120gagagaaact tttattttga agagaccaag gttgaggggg ggcttatttc
ctgacagcta 180tttacttaga gcaaatgatt agttttagaa ggatggacta
taacattgaa tcaattacaa 240aacgcggttt ttgagcccat tactgttgga
gctacaggga gagaaacagg aggagactgc 300aagagatcat ttgggaaggc
cgtgggcacg ctctttactc catgtgtggg acattcattg 360cggaataaca
tcggaggaga agtttcccag agctatgggg acttcccatc cggcgttcct
420ggtcttaggc tgtcttctca cagggctgag cctaatcctc tgccagcttt
cattaccctc 480tatccttcca aatgaaaatg aaaaggttgt gcagctgaat
tcatcctttt ctctgagatg 540ctttggggag agtgaagtga gctggcagta
ccccatgtct gaagaagaga gctccgatgt 600ggaaatcaga aatgaagaaa
acaacagcgg cctttttgtg acggtcttgg aagtgagcag 660tgcctcggcg
gcccacacag ggttgtacac ttgctattac aaccacactc agacagaaga
720gaatgagctt gaaggcaggc acatttacat ctatgtgcca gacccagatg
tagcctttgt 780acctctagga atgacggatt atttagtcat cgtggaggat
gatgattctg ccattatacc 840ttgtcgcaca actgatcccg agactcctgt
aaccttacac aacagtgagg gggtggtacc 900tgcctcctac gacagcagac
agggctttaa tgggaccttc actgtagggc cctatatctg 960tgaggccacc
gtcaaaggaa agaagttcca gaccatccca tttaatgttt atgctttaaa
1020agcaacatca gagctggatc tagaaatgga agctcttaaa accgtgtata
agtcagggga 1080aacgattgtg gtcacctgtg ctgtttttaa caatgaggtg
gttgaccttc aatggactta 1140ccctggagaa gtgaaaggca aaggcatcac
aatgctggaa gaaatcaaag tcccatccat 1200caaattggtg tacactttga
cggtccccga ggccacggtg aaagacagtg gagattacga 1260atgtgctgcc
cgccaggcta ccagggaggt caaagaaatg aagaaagtca ctatttctgt
1320ccatgagaaa ggtttcattg aaatcaaacc caccttcagc cagttggaag
ctgtcaacct 1380gcatgaagtc aaacattttg ttgtagaggt gcgggcctac
ccacctccca ggatatcctg 1440gctgaaaaac aatctgactc tgattgaaaa
tctcactgag atcaccactg atgtggaaaa 1500gattcaggaa ataaggtatc
gaagcaaatt aaagctgatc cgtgctaagg aagaagacag 1560tggccattat
actattgtag ctcaaaatga agatgctgtg aagagctata cttttgaact
1620gttaactcaa gttccttcat ccattctgga cttggtcgat gatcaccatg
gctcaactgg 1680gggacagacg gtgaggtgca cagctgaagg cacgccgctt
cctgatattg agtggatgat 1740atgcaaagat attaagaaat gtaataatga
aacttcctgg actattttgg ccaacaatgt 1800ctcaaacatc atcacggaga
tccactcccg agacaggagt accgtggagg gccgtgtgac 1860tttcgccaaa
gtggaggaga ccatcgccgt gcgatgcctg gctaagaatc tccttggagc
1920tgagaaccga gagctgaagc tggtggctcc caccctgcgt tctgaactca
cggtggctgc 1980tgcagtcctg gtgctgttgg tgattgtgat catctcactt
attgtcctgg ttgtcatttg 2040gaaacagaaa ccgaggtatg aaattcgctg
gagggtcatt gaatcaatca gcccggatgg 2100acatgaatat atttatgtgg
acccgatgca gctgccttat gactcaagat gggagtttcc 2160aagagatgga
ctagtgcttg gtcgggtctt ggggtctgga gcgtttggga aggtggttga
2220aggaacagcc tatggattaa gccggtccca acctgtcatg aaagttgcag
tgaagatgct 2280aaaacccacg gccagatcca gtgaaaaaca agctctcatg
tctgaactga agataatgac 2340tcacctgggg ccacatttga acattgtaaa
cttgctggga gcctgcacca agtcaggccc 2400catttacatc atcacagagt
attgcttcta tggagatttg gtcaactatt tgcataagaa 2460tagggatagc
ttcctgagcc accacccaga gaagccaaag aaagagctgg atatctttgg
2520attgaaccct gctgatgaaa gcacacggag ctatgttatt ttatcttttg
aaaacaatgg 2580tgactacatg gacatgaagc aggctgatac tacacagtat
gtccccatgc tagaaaggaa 2640agaggtttct aaatattccg acatccagag
atcactctat gatcgtccag cctcatataa 2700gaagaaatct atgttagact
cagaagtcaa aaacctcctt tcagatgata actcagaagg 2760ccttacttta
ttggatttgt tgagcttcac ctatcaagtt gcccgaggaa tggagttttt
2820ggcttcaaaa aattgtgtcc accgtgatct ggctgctcgc aacgtcctcc
tggcacaagg 2880aaaaattgtg aagatctgtg actttggcct ggccagagac
atcatgcatg attcgaacta 2940tgtgtcgaaa ggcagtacct ttctgcccgt
gaagtggatg gctcctgaga gcatctttga 3000caacctctac accacactga
gtgatgtctg gtcttatggc attctgctct gggagatctt 3060ttcccttggt
ggcacccctt accccggcat gatggtggat tctactttct acaataagat
3120caagagtggg taccggatgg ccaagcctga ccacgctacc agtgaagtct
acgagatcat 3180ggtgaaatgc tggaacagtg agccggagaa gagaccctcc
ttttaccacc tgagtgagat 3240tgtggagaat ctgctgcctg gacaatataa
aaagagttat gaaaaaattc acctggactt 3300cctgaagagt gaccatcctg
ctgtggcacg catgcgtgtg gactcagaca atgcatacat 3360tggtgtcacc
tacaaaaacg aggaagacaa gctgaaggac tgggagggtg gtctggatga
3420gcagagactg agcgctgaca gtggctacat cattcctctg cctgacattg
accctgtccc 3480tgaggaggag gacctgggca agaggaacag acacagctcg
cagacctctg aagagagtgc 3540cattgagacg ggttccagca gttccacctt
catcaagaga gaggacgaga ccattgaaga 3600catcgacatg atggacgaca
tcggcataga ctcttcagac ctggtggaag acagcttcct 3660gtaactggcg
gattcgaggg gttccttcca cttctggggc cacctctgga tcccgttcag
3720aaaaccactt tattgcaatg cggaggttga gaggaggact tggttgatgt
ttaaagagaa 3780gttcccagcc aagggcctcg gggagcgttc taaatatgaa
tgaatgggat attttgaaat 3840gaactttgtc agtgttgcct ctcgcaatgc
ctcagtagca tctcagtggt gtgtgaagtt 3900tggagataga tggataaggg
aataataggc cacagaaggt gaactttgtg cttcaaggac 3960attggtgaga
gtccaacaga cacaatttat actgcgacag aacttcagca ttgtaattat
4020gtaaataact ctaaccaagg ctgtgtttag attgtattaa ctatcttctt
tggacttctg 4080aagagaccac tcaatccatc catgtacttc cctcttgaaa
cctgatgtca gctgctgttg 4140aactttttaa agaagtgcat gaaaaaccat
ttttgaacct taaaaggtac tggtactata 4200gcattttgct atctttttta
gtgttaagag ataaagaata ataattaacc aaccttgttt 4260aatagatttg
ggtcatttag aagcctgaca actcattttc atattgtaat ctatgtttat
4320aatactacta ctgttatcag taatgctaaa tgtgtaataa tgtaacatga
tttccctcca 4380gagaaagcac aatttaaaac aatccttact aagtaggtga
tgagtttgac agtttttgac 4440atttatatta aataacatgt ttctctataa
agtatggtaa tagctttagt gaattaaatt 4500tagttgagca tagagaacaa
agtaaaagta gtgttgtcca ggaagtcaga atttttaact 4560gtactgaata
ggttccccaa tccatcgtat taaaaaacaa ttaactgccc tctgaaataa
4620tgggattaga aacaaacaaa actcttaagt cctaaaagtt ctcaatgtag
aggcataaac 4680ctgtgctgaa cataacttct catgtatatt acccaatgga
aaatataatg atcagcaaaa 4740agactggatt tgcagaagtt tttttttttt
ttcttcatgc ctgatgaaag ctttggcaac 4800cccaatatat gtattttttg
aatctatgaa cctgaaaagg gtcagaagga tgcccagaca 4860tcagcctcct
tctttcaccc cttaccccaa agagaaagag tttgaaactc gagaccataa
4920agatattctt tagtggaggc tggatgtgca ttagcctgga tcctcagttc
tcaaatgtgt 4980gtggcagcca ggatgactag atcctgggtt tccatccttg
agattctgaa gtatgaagtc 5040tgagggaaac cagagtctgt atttttctaa
actccctggc tgttctgatc ggccagtttt 5100cggaaacact gacttaggtt
tcaggaagtt gccatgggaa acaaataatt tgaactttgg 5160aacagggttg
gaattcaacc acgcaggaag cctactattt aaatccttgg cttcaggtta
5220gtgacattta atgccatcta gctagcaatt gcgaccttaa tttaactttc
cagtcttagc 5280tgaggctgag aaagctaaag tttggttttg acaggttttc
caaaagtaaa gatgctactt 5340cccactgtat gggggagatt gaactttccc
cgtctcccgt cttctgcctc ccactccata 5400ccccgccaag gaaaggcatg
tacaaaaatt atgcaattca gtgttccaag tctctgtgta 5460accagctcag
tgttttggtg gaaaaaacat tttaagtttt actgataatt tgaggttaga
5520tgggaggatg aattgtcaca tctatccaca ctgtcaaaca ggttggtgtg
ggttcattgg 5580cattctttgc aatactgctt aattgctgat accatatgaa
tgaaacatgg gctgtgatta 5640ctgcaatcac tgtgctatcg gcagatgatg
ctttggaaga tgcagaagca ataataaagt 5700acttgactac ctactggtgt
aatctcaatg caagccccaa ctttcttatc caactttttc 5760atagtaagtg
cgaagactga gccagattgg ccaattaaaa acgaaaacct gactaggttc
5820tgtagagcca attagacttg aaatacgttt gtgtttctag aatcacagct
caagcattct 5880gtttatcgct cactctccct tgtacagcct tattttgttg
gtgctttgca ttttgatatt 5940gctgtgagcc ttgcatgaca tcatgaggcc
ggatgaaact tctcagtcca gcagtttcca 6000gtcctaacaa atgctcccac
ctgaatttgt atatgactgc atttgtgggt gtgtgtgtgt 6060tttcagcaaa
ttccagattt gtttcctttt ggcctcctgc aaagtctcca gaagaaaatt
6120tgccaatctt tcctactttc tatttttatg atgacaatca aagccggcct
gagaaacact 6180atttgtgact ttttaaacga ttagtgatgt ccttaaaatg
tggtctgcca atctgtacaa 6240aatggtccta tttttgtgaa gagggacata
agataaaatg atgttataca tcaatatgta 6300tatatgtatt tctatataga
cttggagaat actgccaaaa catttatgac aagctgtatc 6360actgccttcg
tttatatttt tttaactgtg ataatcccca caggcacatt aactgttgca
6420cttttgaatg tccaaaattt atattttaga aataataaaa agaaagatac
ttacatgttc 6480ccaaaacaat ggtgtggtga atgtgtgaga aaaactaact
tgatagggtc taccaataca 6540aaatgtatta cgaatgcccc tgttcatgtt
tttgttttaa aacgtgtaaa tgaagatctt 6600tatatttcaa taaatgatat
ataatttaaa gtt 6633325PRTHomo sapiens 3Val Ile Glu Ser Ile Ser Pro
Asp Gly His Glu Tyr Ile Tyr Val Asp 1 5 10 15 Pro Met Gln Leu Pro
Tyr Asp Ser Arg 20 25 413PRTHomo sapiens 4Gln Ala Asp Thr Thr Gln
Tyr Val Pro Met Leu Glu Arg 1 5 10 510PRTHomo sapiens 5Ser Leu Tyr
Asp Arg Pro Ala Ser Tyr Lys 1 5 10 610PRTHomo sapiens 6Ser Leu Tyr
Asp Arg Pro Ala Ser Tyr Lys 1 5 10 711PRTHomo sapiens 7Asp Ile Met
His Asp Ser Asn Tyr Val Ser Lys 1 5 10 826PRTHomo sapiens 8Leu Ser
Ala Asp Ser Gly Tyr Ile Ile Pro Leu Pro Asp Ile Asp Pro 1 5 10 15
Val Pro Glu Glu Glu Asp Leu Gly Lys Arg 20 25
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