U.S. patent application number 14/962875 was filed with the patent office on 2016-06-09 for biological markers predictive of anti-cancer response to epidermal growth factor receptor kinase inhibitors.
The applicant listed for this patent is OSI Pharmaceuticals, LLC. Invention is credited to Lukas A. AMLER, David A. EBERHARD, Graeme GRIFFIN, John D. HALEY, Robert L. YAUCH.
Application Number | 20160158235 14/962875 |
Document ID | / |
Family ID | 36586095 |
Filed Date | 2016-06-09 |
United States Patent
Application |
20160158235 |
Kind Code |
A1 |
HALEY; John D. ; et
al. |
June 9, 2016 |
BIOLOGICAL MARKERS PREDICTIVE OF ANTI-CANCER RESPONSE TO EPIDERMAL
GROWTH FACTOR RECEPTOR KINASE INHIBITORS
Abstract
The present invention provides diagnostic and prognostic methods
for predicting the effectiveness of treatment of a cancer patient
with an EGFR kinase inhibitor. Methods are provided for predicting
the sensitivity of tumor cell growth to inhibition by an EGFR
kinase inhibitor, comprising assessing whether the tumor cell has
undergone an epithelial to mesenchymal transition (EMT), by
determining the expression level of epithelial and/or mesenchymal
biomarkers, wherein tumor cells that have undergone an EMT are
substantially less sensitive to inhibition by EGFR kinase
inhibitors. Improved methods for treating cancer patients with EGFR
kinase inhibitors that incorporate the above methodology are also
provided. Additionally, methods are provided for the identification
of new biomarkers that are predictive of responsiveness of tumors
to EGFR kinase inhibitors. Furthermore, methods for the
identification of agents that restore the sensitivity of tumor
cells that have undergone EMT to inhibition by EGFR kinase
inhibitors are also provided.
Inventors: |
HALEY; John D.;
(Farmingdale, NY) ; GRIFFIN; Graeme; (Farmingdale,
NY) ; AMLER; Lukas A.; (San Francisco, CA) ;
EBERHARD; David A.; (San Francisco, CA) ; YAUCH;
Robert L.; (San Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
OSI Pharmaceuticals, LLC |
Melville |
NY |
US |
|
|
Family ID: |
36586095 |
Appl. No.: |
14/962875 |
Filed: |
December 8, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13374132 |
Dec 12, 2011 |
9244058 |
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14962875 |
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11377530 |
Mar 16, 2006 |
8093011 |
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13374132 |
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60671821 |
Apr 15, 2005 |
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60662545 |
Mar 16, 2005 |
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Current U.S.
Class: |
514/266.4 |
Current CPC
Class: |
G01N 33/5011 20130101;
G01N 33/57407 20130101; A61K 31/517 20130101; G01N 2800/52
20130101; A61P 35/00 20180101; A61P 43/00 20180101; A61P 35/04
20180101; G01N 33/57484 20130101 |
International
Class: |
A61K 31/517 20060101
A61K031/517 |
Claims
1.-21. (canceled)
22. A method of treating tumors or tumor metastases comprising
administering to a patient in need thereof a therapeutically
effective amount of one or more EGFR kinase inhibitors, wherein
said patient's tumor cells have not undergone an
epithelial-mesenchymal transition.
23. The method of claim 22, wherein said tumor cells express one or
more of 1) a high level of one or more epithelial biomarkers; 2) a
low or undetectable level of one or more mesenchymal biomarkers;
and 3) a high ratio of one or more epithelial biomarkers to one or
more mesenchymal biomarkers, sufficient to indicate said tumor
cells have not undergone an epithelial-mesenchymal transition.
24. The method of claim 23, wherein said epithelial biomarker is
Brk, .gamma.-catenin, .alpha.1-catenin, .alpha.2-catenin,
.alpha.3-catenin, keratin 8, keratin 18, connexin 31, plakophilin
3, stratafin 1, laminin alpha-5, or ST14.
25. The method of claim 23, wherein said mesenchymal biomarker is
vimentin, fibronectin, fibrillin-2, collagen alpha-2(IV), collagen
alpha-2(V), LOXL1, nidogen, C11orf9, tenascin, N-cadherin, and
embryonal EDB.sup.+ fibronectin, tubulin alpha-3, or
epimorphin.
26. The method of claim 22, wherein said patient s human.
27. The method of claim 22, wherein said tumor is lung cancer,
pancreatic cancer, head and neck cancer, gastric cancer, breast
cancer, colon cancer, or ovarian cancer.
28. The method of claim 22, wherein said EGFR kinase inhibitor is
erlotinib or a pharmaceutically acceptable salt thereof.
29. The method of claim 22, wherein said patient is administered as
least one other active agent.
30. A method of treating tumors or tumor metastases comprising
administering to a patient in need thereof a therapeutically
effective amount of one or more EGFR kinase inhibitors, wherein
said patient's tumor cells have been determined not to have
undergone an epithelial-mesenchymal transition.
31. The method of claim 30, wherein said determination is made by
assessing if said tumor cells express one or more of 1) a high
level of one or more epithelial biomarkers; 2) a low or
undetectable level of one or more mesenchymal biomarkers; and 3) a
high ratio of one or more epithelial biomarkers to one or more
mesenchymal biomarkers.
32. The method of claim 31, wherein said epithelial biomarker is
Brk, .gamma.-catenin, .alpha.1-catenin, .alpha.2-catenin,
.alpha.3-catenin, keratin 8, keratin 18, connexin 31, plakophilin
3, stratafin 1, laminin alpha-5, or ST14.
33. The method of claim 31, wherein said mesenchymal biomarker is
vimentin, fibronectin, fibrillin-1, fibrillin-2, collagen
alpha-2(IV), collagen alpha-2(V), LOXL1, nidogen, C11orf9,
tenascin, N-cadherin, and embryonal EDB.sup.+ fibronectin, tubulin
alpha-3, or epimorphin.
34. The method of claim 30, wherein said patient is human.
35. The method of claim 30, wherein said tumor is lung cancer,
pancreatic cancer, head and neck cancer, gastric cancer, breast
cancer, colon cancer, or ovarian cancer.
36. The method of claim 30, wherein said EGFR kinase inhibitor is
erlotinib or a pharmaceutically acceptable salt thereof.
37. The method of claim 30, wherein said patient is administered as
least one other active agent.
38. A method of treating tumors or tumor metastases comprising
administering to a human patient in need thereof a therapeutically
effective amount of one or more EGFR kinase inhibitors, wherein
said human patient's tumor cells have been determined not to have
undergone an epithelial-mesenchymal transition by assessing if said
tumor cells express one or more of 1) a high level of one or more
epithelial biomarkers; 2) a low or undetectable level of one or
more mesenchymal biomarkers; and 3) a high ratio of one or more
epithelial biomarkers to one or more mesenchymal biomarkers,
wherein said epithelial biomarker is Brk, .gamma.-catenin,
.alpha.1-catenin, .alpha.2-catenin, .alpha.3-catenin, keratin 8,
keratin 18, connexin 31, plakophilin 3, stratafin 1, laminin
alpha-5, or ST14, wherein said mesenchymal biomarker is vimentin,
fibronectin, fibrillin-1, fibrillin-2, collagen alpha-2(IV),
collagen alpha-2(V), LOXL1, nidogen, C11orf9, tenascin, N-cadherin,
and embryonal EDB.sup.+ fibronectin, tubulin alpha-3, or
epimorphin, and wherein said tumor is lung cancer, pancreatic
cancer, head and neck cancer, gastric cancer, breast cancer, colon
cancer, or ovarian cancer.
39. The method of claim 38, wherein said EGFR kinase inhibitor is
erlotinib or a pharmaceutically acceptable salt thereof.
40. The method of claim 38, wherein said patient is administered as
least one other active agent.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
application Ser. No. 13/374,132, filed Dec. 12, 2011, now allowed;
which is a divisional application of U.S. application Ser. No.
11/377,530, filed Mar. 16, 2006, issued as U.S. Pat. No. 8,093,011;
which claims the benefit of U.S. Provisional Application No.
60/671,821, filed Apr. 15, 2005, and U.S. Provisional Application
No. 60/662,545, filed Mar. 16, 2005; all of which are herein
incorporated by reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] The present invention is directed to methods for diagnosing
and treating cancer patients. In particular, the present invention
is directed to methods for determining which patients will most
benefit from treatment with an epidermal growth factor receptor
(EGFR) kinase inhibitor.
[0003] Cancer is a generic name for a wide range of cellular
malignancies characterized by unregulated growth, lack of
differentiation, and the ability to invade local tissues and
metastasize. These neoplastic malignancies affect, with various
degrees of prevalence, every tissue and organ in the body.
[0004] A multitude of therapeutic agents have been developed over
the past few decades for the treatment of various types of cancer.
The most commonly used types of anticancer agents include:
DNA-alkylating agents (e.g., cyclophosphamide, ifosfamide),
antimetabolites (e.g., methotrexate, a folate antagonist, and
5-fluorouracil, a pyrimidine antagonist), microtubule disrupters
(e.g., vincristine, vinblastine, paclitaxel), DNA intercalators
(e.g., doxorubicin, daunomycin, cisplatin), and hormone therapy
(e.g., tamoxifen, flutamide).
[0005] The epidermal growth factor receptor (EGFR) family comprises
four closely related receptors (HER1/EGFR, HER2, HER3 and HER4)
involved in cellular responses such as differentiation and
proliferation. Over-expression of the EGFR kinase, or its ligand
TGF-alpha, is frequently associated with many cancers, including
breast, lung, colorectal, ovarian, renal cell, bladder, head and
neck cancers, glioblastomas, and astrocytomas, and is believed to
contribute to the malignant growth of these tumors. A specific
deletion-mutation in the EGFR gene (EGFRvIII) has also been found
to increase cellular tumorigenicity. Activation of EGFR stimulated
signaling pathways promote multiple processes that are potentially
cancer-promoting, e.g. proliferation, angiogenesis, cell motility
and invasion, decreased apoptosis and induction of drug resistance.
Increased HER1/EGFR expression is frequently linked to advanced
disease, metastases and poor prognosis. For example, in NSCLC and
gastric cancer, increased HER1/EGFR expression has been shown to
correlate with a high metastatic rate, poor tumor differentiation
and increased tumor proliferation.
[0006] Mutations which activate the receptor's intrinsic protein
tyrosine kinase activity and/or increase downstream signaling have
been observed in NSCLC and glioblastoma. However the role of
mutations as a principle mechanism in conferring sensitivity to EGF
receptor inhibitors, for example erlotinib (TARCEVA.TM.) or
gefitinib (IRESSA.TM.), has been controversial. Recently, a mutant
form of the full length EGF receptor has been reported to predict
responsiveness to the EGF receptor tyrosine kinase inhibitor
gefitinib (Paez, J. G. et al. (2004) Science 304:1497-1500; Lynch,
T. J. et al. (2004) N. Engl. J. Med. 350:2129-2139). Cell culture
studies have shown that cell lines which express the mutant form of
the EGF receptor (i.e. H3255) were more sensitive to growth
inhibition by the EGF receptor tyrosine kinase inhibitor gefitinib,
and that much higher concentrations of gefitinib was required to
inhibit the tumor cell lines expressing wild type EGF receptor.
These observations suggests that specific mutant forms of the EGF
receptor may reflect a greater sensitivity to EGF receptor
inhibitors, but do not identify a completely non-responsive
phenotype.
[0007] The development for use as anti-tumor agents of compounds
that directly inhibit the kinase activity of the EGFR, as well as
antibodies that reduce EGFR kinase activity by blocking EGFR
activation, are areas of intense research effort (de Bono J. S. and
Rowinsky, E. K. (2002) Trends in Mol. Medicine 8:S19-S26; Dancey,
J. and Sausville, E. A. (2003) Nature Rev. Drug Discovery
2:92-313). Several studies have demonstrated, disclosed, or
suggested that some EGFR kinase inhibitors might improve tumor cell
or neoplasia killing when used in combination with certain other
anti-cancer or chemotherapeutic agents or treatments (e.g. Herbst,
R. S. et al. (2001) Expert Opin. Biol. Ther. 1:719-732; Solomon, B.
et al (2003) Int. J. Radiat. Oncol. Biol. Phys. 55:713-723;
Krishnan, S. et al. (2003) Frontiers in Bioscience 8, e1-13;
Grunwald, V. and Hidalgo, M. (2003) J. Nat. Cancer Inst.
95:851-867; Seymour L. (2003) Current Opin. Investig. Drugs
4(6):658-666; Khalil, M. Y. et al. (2003) Expert Rev. Anticancer
Ther. 3:367-380; Bulgaru, A. M. et al. (2003) Expert Rev.
Anticancer Ther. 3:269-279; Dancey, J. and Sausville, E. A. (2003)
Nature Rev. Drug Discovery 2:92-313; Ciardiello, F. et al. (2000)
Clin. Cancer Res. 6:2053-2063; and Patent Publication No: US
2003/0157104).
[0008] Erlotinib (e.g. erlotinib HCl, also known as TARCEVA.TM. or
OSI-774) is an orally available inhibitor of EGFR kinase. In vitro,
erlotinib has demonstrated substantial inhibitory activity against
EGFR kinase in a number of human tumor cell lines, including
colorectal and breast cancer (Moyer J. D. et al. (1997) Cancer Res.
57:4838), and preclinical evaluation has demonstrated activity
against a number of EGFR-expressing human tumor xenografts
(Pollack, V. A. et al (1999) J. Pharmacol. Exp. Ther. 291:739).
More recently, erlotinib has demonstrated promising activity in
phase I and II trials in a number of indications, including head
and neck cancer (Soulieres, D., et al. (2004) J. Clin. Oncol.
22:77), NSCLC (Perez-Soler R, et al. (2001) Proc. Am. Soc. Clin.
Oncol. 20:310a, abstract 1235), CRC (Oza, M., et al. (2003) Proc.
Am. Soc. Clin. Oncol. 22:196a, abstract 785) and MBC (Winer, E., et
al. (2002) Breast Cancer Res. Treat. 76:5115a, abstract 445). In a
phase III trial, erlotinib monotherapy significantly prolonged
survival, delayed disease progression and delayed worsening of lung
cancer-related symptoms in patients with advanced,
treatment-refractory NSCLC (Shepherd, F. et al. (2004) J. Clin.
Oncology, 22:14S (July 15 Supplement), Abstract 7022). While most
of the clinical trial data for erlotinib relate to its use in
NSCLC, preliminary results from phase I/II studies have
demonstrated promising activity for erlotinib and
capecitabine/erlotinib combination therapy in patients with wide
range of human solid tumor types, including CRC (Oza, M., et al.
(2003) Proc. Am. Soc. Clin. Oncol. 22:196a, abstract 785) and MBC
(Jones, R. J., et al. (2003) Proc. Am. Soc. Clin. Oncol. 22:45a,
abstract 180). In November 2004 the U.S. Food and Drug
Administration (FDA) approved TARCEVA.TM. for the treatment of
patients with locally advanced or metastatic non-small cell lung
cancer (NSCLC) after failure of at least one prior chemotherapy
regimen. TARCEVA.TM. is the only drug in the epidermal growth
factor receptor (EGFR) class to demonstrate in a Phase III clinical
trial an increase in survival in advanced NSCLC patients.
[0009] An anti-neoplastic drug would ideally kill cancer cells
selectively, with a wide therapeutic index relative to its toxicity
towards non-malignant cells. It would also retain its efficacy
against malignant cells, even after prolonged exposure to the drug.
Unfortunately, none of the current chemotherapies possess such an
ideal profile. Instead, most possess very narrow therapeutic
indexes. Furthermore, cancerous cells exposed to slightly
sub-lethal concentrations of a chemotherapeutic agent will very
often develop resistance to such an agent, and quite often
cross-resistance to several other antineoplastic agents as well.
Additionally, for any given cancer type one frequently cannot
predict which patient is likely to respond to a particular
treatment, even with newer gene-targeted therapies, such as EGFR
kinase inhibitors, thus necessitating considerable trial and error,
often at considerable risk and discomfort to the patient, in order
to find the most effective therapy.
[0010] Thus, there is a need for more efficacious treatment for
neoplasia and other proliferative disorders, and for more effective
means for determining which tumors will respond to which treatment.
Strategies for enhancing the therapeutic efficacy of existing drugs
have involved changes in the schedule for their administration, and
also their use in combination with other anticancer or biochemical
modulating agents. Combination therapy is well known as a method
that can result in greater efficacy and diminished side effects
relative to the use of the therapeutically relevant dose of each
agent alone. In some cases, the efficacy of the drug combination is
additive (the efficacy of the combination is approximately equal to
the sum of the effects of each drug alone), but in other cases the
effect is synergistic (the efficacy of the combination is greater
than the sum of the effects of each drug given alone).
[0011] Target-specific therapeutic approaches, such as erlotinib,
are generally associated with reduced toxicity compared with
conventional cytotoxic agents, and therefore lend themselves to use
in combination regimens. Promising results have been observed in
phase MI studies of erlotinib in combination with bevacizumab
(Mininberg, E. D., et al. (2003) Proc. Am. Soc. Clin. Oncol.
22:627a, abstract 2521) and gemcitabine (Dragovich, T., (2003)
Proc. Am. Soc. Clin. Oncol. 22:223a, abstract 895). Recent data in
NSCLC phase III trials have shown that first-line erlotinib or
gefitinib in combination with standard chemotherapy did not improve
survival (Gatzemeier, U., (2004) Proc. Am. Soc. Clin. Oncol. 23:617
(Abstract 7010); Herbst, R. S., (2004) Proc. Am. Soc. Clin. Oncol.
23:617 (Abstract 7011); Giaccone, G., et al. (2004) J. Clin. Oncol.
22:777; Herbst, R., et al. (2004) J. Clin. Oncol. 22:785). However,
pancreatic cancer phase III trials have shown that first-line
erlotinib in combination with gemcitabine did improve survival (OSI
Pharmaceuticals/Genentech/Roche Pharmaceuticals Press Release,
9/20/04).
[0012] Several groups have investigated potential biomarkers to
predict a patient's response to EGFR inhibitors (see for example,
PCT publications: WO 2004/063709, WO 2005/017493, WO 2004/111273,
WO 2004/071572, WO 2005/117553 and WO 2005/070020; and US published
patent applications: US 2005/0019785, and US 2004/0132097).
However, no diagnostic or prognostic tests have yet emerged that
can guide practicing physicians in the treatment of their patients
with EGFR kinase inhibitors.
[0013] During most cancer metastases, an important change occurs in
a tumor cell known as the epithelial-mesenchymal transition (EMT)
(Thiery, J. P. (2002) Nat. Rev. Cancer 2:442-454; Savagner, P.
(2001) Bioessays 23:912-923; Kang Y. and Massague, J. (2004) Cell
118:277-279; Julien-Grille, S., et al. Cancer Research
63:2172-2178; Bates, R. C. et al. (2003) Current Biology
13:1721-1727; Lu Z., et al. (2003) Cancer Cell. 4(6):499-515)).
Epithelial cells, which are bound together tightly and exhibit
polarity, give rise to mesenchymal cells, which are held together
more loosely, exhibit a loss of polarity, and have the ability to
travel. These mesenchymal cells can spread into tissues surrounding
the original tumor, as well as separate from the tumor, invade
blood and lymph vessels, and travel to new locations where they
divide and form additional tumors. EMT does not occur in healthy
cells except during embryogenesis. Under normal circumstances
TGF-.beta. acts as a growth inhibitor. However it is believed that
during cancer metastasis, TGF-.beta. begins to promote EMT.
[0014] Thus, there remains a critical need for improved methods for
determining the best mode of treatment for any given cancer patient
and for the incorporation of such determinations into more
effective treatment regimens for cancer patients, whether such
inhibitors are used as single agents or combined with other
anti-cancer agents.
SUMMARY OF THE INVENTION
[0015] The present invention provides diagnostic and prognostic
methods for predicting the effectiveness of treatment of a cancer
patient with an EGFR kinase inhibitor. Based on the surprising
discovery that the sensitivity of tumor cell growth to inhibition
by EGFR kinase inhibitors is dependent on whether such tumor cells
have undergone an EMT, methods have been devised for determining
epithelial and/or mesenchymal biomarkers to predict the sensitivity
of tumor cells to EGFR kinase inhibitors.
[0016] Accordingly, the present invention provides a method of
predicting the sensitivity of tumor cell growth to inhibition by an
EGFR kinase inhibitor, comprising: assessing the level of an
epithelial biomarker expressed by a tumor cell; and predicting the
sensitivity of tumor cell growth to inhibition by an EGFR kinase
inhibitor, wherein high expression levels of tumor cell epithelial
biomarkers correlate with high sensitivity to inhibition by EGFR
kinase inhibitors.
[0017] The present invention also provides a method of predicting
the sensitivity of tumor cell growth to inhibition by an EGFR
kinase inhibitor, comprising: assessing the level of a mesenchymal
biomarker expressed by a tumor cell; and predicting the sensitivity
of tumor cell growth to inhibition by an EGFR kinase inhibitor,
wherein high expression levels of tumor cell mesenchymal biomarkers
correlate with low sensitivity to inhibition by EGFR kinase
inhibitors.
[0018] Improved methods for treating cancer patients with EGFR
kinase inhibitors that incorporate the above methodology are also
provided. Thus, the present invention further provides a method for
treating tumors or tumor metastases in a patient, comprising the
steps of diagnosing a patient's likely responsiveness to an EGFR
kinase inhibitor by assessing whether the tumor cells have
undergone an epithelial-mesenchymal transition, and administering
to said patient a therapeutically effective amount of an EGFR
kinase inhibitor.
[0019] Additionally, methods are provided for the identification of
new epithelial or mesenchymal biomarkers that are predictive of
responsiveness of tumors to EGFR kinase inhibitors.
[0020] Thus, for example, the present invention further provides a
method of identifying an epithelial biomarker that is diagnostic
for more effective treatment of a neoplastic condition with an EGFR
kinase inhibitor, comprising: measuring the level of a candidate
epithelial biomarker in neoplastic cell-containing samples from
patients with a neoplastic condition, and identifying a correlation
between the level of said candidate epithelial biomarker in the
sample from the patient with the effectiveness of treatment of the
neoplastic condition with an EGFR kinase inhibitor, wherein a
correlation of high levels of the epithelial biomarker with more
effective treatment of the neoplastic condition with an EGFR kinase
inhibitor indicates that said epithelial biomarker is diagnostic
for more effective treatment of the neoplastic condition with an
EGFR kinase inhibitor.
[0021] The present invention further provides a method of
identifying a mesenchymal biomarker that is diagnostic for less
effective treatment of a neoplastic condition with an EGFR kinase
inhibitor, comprising: (a) measuring the level of a candidate
mesenchymal biomarker in neoplastic cell-containing samples from
patients with a neoplastic condition, and (b) identifying a
correlation between the level of said candidate mesenchymal
biomarker in the sample from the patient with the effectiveness of
treatment of the neoplastic condition with an EGFR kinase
inhibitor, wherein a correlation of high levels of the mesenchymal
biomarker with less effective treatment of the neoplastic condition
with an EGFR kinase inhibitor indicates that said mesenchymal
biomarker is diagnostic for less effective treatment of the
neoplastic condition with an EGFR kinase inhibitor.
[0022] Furthermore, methods for the identification of agents that
restore the sensitivity of tumor cells that have undergone EMT to
inhibition by EGFR kinase inhibitors are also provided. Thus, for
example, the present invention provides a method for the
identification of an agent that enhances sensitivity of the growth
of a tumor cell to an EGFR kinase inhibitor, said tumor cell having
being characterized as one that has previously undergone an
epithelial-mesenchymal transition, comprising contacting a sample
of said tumor cells with an EGFR kinase inhibitor, contacting an
identical sample of said tumor cells with an EGFR kinase inhibitor
in the presence of a test agent, comparing the EGFR kinase
inhibitor-mediated growth inhibition in the presence and absence of
the test agent, and determining whether the test agent is an agent
that enhances sensitivity of the growth of the tumor cell to an
EGFR kinase inhibitor.
BRIEF DESCRIPTION OF THE FIGURES
[0023] The file of this patent contains at least one drawing
executed in color. Copies of this patent with color drawing(s) will
be provided by the Patent and Trademark Office upon request and
payment of the necessary fee.
[0024] FIG. 1: In vivo activity of erlotinib against NSCLC
xenografts.
[0025] FIG. 2A: Proteomic profiling of NSCLC lines, sensitive or
relatively insensitive to EGFR kinase inhibition in vitro, showed
markedly increased LCMS/MS detection of vimentin and fibronectin
peptides in cell lines relatively insensitive to erlotinib. FIG.
2B: NSCLC lines sensitive to EGF receptor inhibition express
elevated levels of E-cadherin, with trends observed for .gamma.-
and .alpha.-catenins. E-cadherin immunoblots were performed with
two distinct antibodies with similar results (data not shown).
NSCLC lines relatively insensitive to growth inhibition by
erlotinib expressed the mesenchymal proteins vimentin and/or
fibronectin. No relationship between total EGF receptor protein
expression and sensitivity was observed, though all lines tested
expressed detectable EGF receptor. FIG. 2C: Confocal microscopy of
NSCLC lines sensitive to growth inhibition by erlotinib, H292 and
H441, showing membrane expression of E-cadherin, but not in the
cell lines Calu6 and H1703 that are relatively insensitive to
erlotinib. Conversely, the relatively insensitive lines Calu6 and
H1703 expressed intermediate filament staining for vimentin, while
the erlotinib sensitive lines H292 and H441 did not.
[0026] FIG. 3: NSCLC lines were grown as subcutaneous xenografts in
SCID mice to a volume of .about.500 mm.sup.3, excised and flash
frozen in liquid nitrogen (4 animals per cell line). Tumor tissue
was pulverized while frozen, subjected to detergent lysis and SDS-P
AGE as described and immunoblots probed with antibodies to
E-cadherin, .gamma.-catenin, Brk, fibronectin, vimentin, and GAPDH.
Consistent with in vitro results, E-cadherin expression was
restricted to erlotinib sensitive lines and fibronectin to
relatively insensitive lines.
[0027] FIG. 4: Immunoblot showing higher Brk expression levels in
NSCLC cell lines that are most sensitive to EGFR kinase
inhibition.
[0028] FIG. 5A: Pancreatic cell lines sensitive to EGF receptor
inhibition express elevated levels of the epithelial cell junction
proteins E-cadherin and .gamma.-catenin. The mesenchymal marker
vimentin was most abundant in the insensitive PANC1 cells. FIG. 5B:
Confocal microscopy of a pancreatic cell line sensitive to growth
inhibition by erlotinib, BxPC3, showing membrane expression of
E-cadherin, but not in the cell line MiaPaca2, that is relatively
insensitive to erlotinib. Conversely, the relatively insensitive
line MiaPaca2 expressed intermediate filament staining for
vimentin, while the erlotinib sensitive line BxPC3 did not.
[0029] FIG. 6A: Kaplan-Meier curve illustrating time to disease
progression (TTP) is longer for patients receiving erlotinib in
combination with chemotherapy compared to patients receiving
chemotherapy only whose tumors with E-cadherin staining intensity
of >=2. FIG. 6B: Kaplan-Meier curve illustrating time to disease
progression (TIP) is not extended for patients having tumor
E-cadherin staining intensity of <=1 who are treated with
erlotinib in combination with chemotherapy compared to patients
receiving chemotherapy alone.
DETAILED DESCRIPTION OF THE INVENTION
[0030] The term "cancer" in an animal refers to the presence of
cells possessing characteristics typical of cancer-causing cells,
such as uncontrolled proliferation, immortality, metastatic
potential, rapid growth and proliferation rate, and certain
characteristic morphological features. Often, cancer cells will be
in the form of a tumor, but such cells may exist alone within an
animal, or may circulate in the blood stream as independent cells,
such as leukemic cells.
[0031] "Abnormal cell growth", as used herein, unless otherwise
indicated, refers to cell growth that is independent of normal
regulatory mechanisms (e.g., loss of contact inhibition). This
includes the abnormal growth of: (1) tumor cells (tumors) that
proliferate by expressing a mutated tyrosine kinase or
overexpression of a receptor tyrosine kinase; (2) benign and
malignant cells of other proliferative diseases in which aberrant
tyrosine kinase activation occurs; (4) any tumors that proliferate
by receptor tyrosine kinases; (5) any tumors that proliferate by
aberrant serine/threonine kinase activation; and (6) benign and
malignant cells of other proliferative diseases in which aberrant
serine/threonine kinase activation occurs.
[0032] The term "treating" as used herein, unless otherwise
indicated, means reversing, alleviating, inhibiting the progress
of, or preventing, either partially or completely, the growth of
tumors, tumor metastases, or other cancer-causing or neoplastic
cells in a patient. The term "treatment" as used herein, unless
otherwise indicated, refers to the act of treating.
[0033] The phrase "a method of treating" or its equivalent, when
applied to, for example, cancer refers to a procedure or course of
action that is designed to reduce or eliminate the number of cancer
cells in an animal, or to alleviate the symptoms of a cancer. "A
method of treating" cancer or another proliferative disorder does
not necessarily mean that the cancer cells or other disorder will,
in fact, be eliminated, that the number of cells or disorder will,
in fact, be reduced, or that the symptoms of a cancer or other
disorder will, in fact, be alleviated. Often, a method of treating
cancer will be performed even with a low likelihood of success, but
which, given the medical history and estimated survival expectancy
of an animal, is nevertheless deemed an overall beneficial course
of action.
[0034] The term "therapeutically effective agent" means a
composition that will elicit the biological or medical response of
a tissue, system, animal or human that is being sought by the
researcher, veterinarian, medical doctor or other clinician.
[0035] The term "therapeutically effective amount" or "effective
amount" means the amount of the subject compound or combination
that will elicit the biological or medical response of a tissue,
system, animal or human that is being sought by the researcher,
veterinarian, medical doctor or other clinician.
[0036] The data presented in the Examples herein below demonstrate
that tumor cells, such as NSCLC or pancreatic cancer cells,
containing wild type EGFR, grown either in cell culture or in vivo,
show a range of sensitivities to inhibition by EGFR kinase
inhibitors, dependent on whether they have undergone an epithelial
to mesenchymal transition (EMT). Prior to EMT, tumor cells are very
sensitive to inhibition by EGFR kinase inhibitors such as erlotinib
HCl (TARCEVA.TM.), whereas tumor cells which have undergone an EMT
are substantially less sensitive to inhibition by such compounds.
The data indicates that the EMT may be a "general biological
switch" that determines the level of sensitivity of tumors to EGFR
kinase inhibitors. It is demonstrated that the level of sensitivity
of tumors to EGFR kinase inhibitors can be assessed by determining
the level of biomarkers expressed by a tumor cell that are
characteristic for cells either prior to or subsequent to an EMT
event. For example, high levels of tumor cell expression of
epithelial biomarkers such as E-cadherin, indicative of a cell that
has not yet undergone an EMT, correlate with high sensitivity to
EGFR kinase inhibitors. Conversely, high levels of tumor cell
expression of mesenchymal biomarkers such as vimentin or
fibronectin, indicative of a cell that has undergone an EMT,
correlate with low sensitivity to EGFR kinase inhibitors. Thus,
these observations can form the basis of valuable new diagnostic
methods for predicting the effects of EGFR kinase inhibitors on
tumor growth, and give oncologists an additional tool to assist
them in choosing the most appropriate treatment for their
patients.
[0037] Accordingly, the present invention provides a method of
predicting the sensitivity of tumor cell growth to inhibition by an
EGFR kinase inhibitor, comprising: assessing the level of an
epithelial biomarker expressed by a tumor cell; and predicting the
sensitivity of tumor cell growth to inhibition by an EGFR kinase
inhibitor, wherein high expression levels of tumor cell epithelial
biomarkers correlate with high sensitivity to inhibition by EGFR
kinase inhibitors. Preferred examples of epithelial biomarkers
include E-cadherin and Brk (i.e. PTK-6) (see Table 1). Additional
examples of epithelial biomarkers that can be utilized in the
method of this invention include .gamma.-catenin (i.e. junction
plakoglobin), .alpha.-catenin (i e .alpha.1, .alpha.2, or .alpha.3
catenin), keratin 8, keratin 18, connexin 31, plakophilin 3,
stratafin 1, laminin alpha-5 and ST14 (see Table 1).
[0038] The present invention also provides a method of predicting
the sensitivity of tumor cell growth to inhibition by an EGFR
kinase inhibitor, comprising: assessing the level of a mesenchymal
biomarker expressed by a tumor cell; and predicting the sensitivity
of tumor cell growth to inhibition by an EGFR kinase inhibitor,
wherein high expression levels of tumor cell mesenchymal biomarkers
correlate with low sensitivity to inhibition by EGFR kinase
inhibitors. Preferred examples of mesenchymal biomarkers include
vimentin and fibronectin (see Table 1). Additional examples of
mesenchymal biomarkers that can be utilized in the method of this
invention include fibrillin-1, fibrillin-2, collagen alpha-2(IV),
collagen alpha-2(V), LOXL1, nidogen, C11orf9, tenascin, N-cadherin,
and embryonal EDB.sup.+ fibronectin, tubulin alpha-3 and epimorphin
(see Table 1).
[0039] In the practice of this invention, with preferred epithelial
biomarkers, the level of expression in tumor cells that are
sensitive to EGFR kinase inhibitors will generally be at such a
high level that the biomarker will be very readily detectable,
using for example a specific anti-biomarker antibody for detection.
With preferred epithelial biomarkers, the level of expression in
tumor cells that are relatively insensitive to EGFR kinase
inhibitors will generally be at such a low level that the biomarker
will be barely detectable, if at all, using similar procedures
(e.g. in the data presented in the Examples herein below, compare
E-cadherin levels between sensitive and relatively insensitive
tumor cells in FIGS. 2B, 3 and 5).
[0040] However, for other less preferred epithelial biomarkers, the
level of biomarker expression in tumor cells that are relatively
insensitive to EGFR kinase inhibitors may be readily detectable,
but nevertheless will be at a substantially lower level of
expression than in tumor cells that are sensitive to EGFR kinase
inhibitors (e.g., in the data presented in the Examples herein
below, compare .alpha.-catenin levels for the relatively
insensitive tumor cells H1703 or SW1573 with the sensitive tumor
cells H441, H358, H322 and H292 in FIG. 2B).
[0041] Similarly, in the practice of this invention, with preferred
mesenchymal biomarkers, the level of expression in tumor cells that
are relatively insensitive to EGFR kinase inhibitors will generally
be at such a high level that the biomarker will be very readily
detectable, using for example a specific anti-biomarker antibody
for detection. With preferred mesenchymal biomarkers, the level of
expression in tumor cells that are relatively sensitive to EGFR
kinase inhibitors will generally be at such a low level that the
biomarker will be barely detectable, if at all, using similar
procedures (e.g. in the data presented in the Examples herein
below, compare fibronectin or vimentin levels between sensitive and
relatively insensitive tumor cells in FIGS. 2B, 3 and 5).
[0042] Also, for other less preferred mesenchymal biomarkers, the
level of biomarker expression in tumor cells that are relatively
sensitive to EGFR kinase inhibitors may be readily detectable, but
nevertheless will be at a substantially lower level of expression
than in tumor cells that are relatively insensitive to EGFR kinase
inhibitors.
[0043] For any given epithelial or mesenchymal biomarker, the range
of expression level between tumor cells that are relatively
insensitive to EGFR kinase inhibitors and those that are sensitive,
can readily be assessed by one of skill in the art, for example by
testing on a panel of tumor cells as described herein (e.g. FIG.
2B), or by testing in tumor biopsies from patients whose tumors
display a range of sensitivities to an EGFR kinase inhibitor (e.g.
TARCEVATm).
[0044] In the context of this invention, for a relatively small
percentage of tumor cells that are relatively insensitive to EGFR
kinase inhibitors, the methods described above for predicting the
sensitivity of tumor cell growth to inhibition by an EGFR kinase
inhibitor, comprising assessing the level of an epithelial or
mesenchymal biomarker expressed by a tumor cell, in circumstances
where only a single biomarker level is assessed, may falsely
predict that tumor cell growth is sensitive to inhibition by an
EGFR kinase inhibitor. For example, in the data presented in the
Examples herein below, the levels of the epithelial biomarkers
.gamma.-catenin and .alpha.-catenin in H460 tumor cells, or the
mesenchymal biomarker fibronectin in H1703 cells, falsely predict
high sensitivity to EGFR kinase inhibitors (see FIG. 2B). Thus,
based on such false predictions, a physician may be lead to treat a
small number of patients with EGFR kinase inhibitors, and the tumor
may not be sensitive to the inhibitor. However, for the vast
majority of tumor cells (e.g. at least 90%, from the data presented
in the Examples herein below), assessment of a single biomarker
expression level would be expected to provide an accurate
prediction of level of sensitivity to EGFR kinase inhibitors.
[0045] Furthermore, most importantly in the context of this
invention, no tumor cells that are sensitive to EGFR kinase
inhibitors have been found that when tested by the above methods
(where only a single biomarker level is assessed) give a false
prediction that tumor cell growth will be insensitive to inhibition
by an EGFR kinase inhibitor. Thus, utilizing the testing methods
described herein should never lead a physician to withhold
treatment with an EGFR kinase inhibitor in cases where the patient
may benefit from such treatment.
[0046] In addition, one of skill in the medical arts, particularly
pertaining to the application of diagnostic tests and treatment
with therapeutics, will recognize that biological systems are
somewhat variable and not always entirely predictable, and thus
many good diagnostic tests or therapeutics are occasionally
ineffective. Thus, it is ultimately up to the judgement of the
attending physician to determine the most appropriate course of
treatment for an individual patient, based upon test results,
patient condition and history, and his own experience. There may
even be occasions, for example, when a physician will choose to
treat a patient with an EGFR kinase inhibitor even when a tumor is
not predicted to be particularly sensitive to EGFR kinase
inhibitors, based on data from diagnostic tests or from other
criteria, particularly if all or most of the other obvious
treatment options have failed, or if some synergy is anticipated
when given with another treatment. The fact that the EGFR kinase
inhibitors as a class of drugs are relatively well tolerated
compared to many other anti-cancer drugs, such as more traditional
chemotherapy or cytotoxic agents used in the treatment of cancer,
makes this a more viable option.
[0047] Preferred examples of suitable epithelial biomarkers for use
in this invention, such as E-cadherin, do not lead to any false
predictions when used in the methods described above (where only a
single biomarker level is assessed).
[0048] Furthermore, this invention also provides additional methods
wherein simultaneous assessment of the expression level in tumor
cells of more than one biomarker level is utilized. In preferred
embodiments of these methods (described below) there is no level of
false prediction, as is the case for some of the methods described
above where a single biomarker expression level is assessed.
[0049] Accordingly, the present invention provides a method of
predicting the sensitivity of tumor cell growth to inhibition by an
EGFR kinase inhibitor, comprising: assessing the level of one or
more (or a panel of) epithelial biomarkers expressed by a tumor
cell; and predicting the sensitivity of tumor cell growth to
inhibition by an EGFR kinase inhibitor, wherein simultaneous high
expression levels of all of the tumor cell epithelial biomarkers
correlates with high sensitivity to inhibition by EGFR kinase
inhibitors. In one preferred embodiment of this method the
epithelial biomarkers comprise E-cadherin and Brk, wherein
simultaneous high expression level of the two tumor cell epithelial
biomarkers correlates with high sensitivity to inhibition by EGFR
kinase inhibitor. In another preferred embodiment of this method
the epithelial biomarkers comprise E-cadherin and .gamma.-catenin,
wherein simultaneous high expression level of the two tumor cell
epithelial biomarkers correlates with high sensitivity to
inhibition by EGFR kinase inhibitor. Note that in the two latter
preferred embodiments a high expression level of both biomarkers is
required to indicate high sensitivity.
[0050] The present invention also provides a method of predicting
the sensitivity of tumor cell growth to inhibition by an EGFR
kinase inhibitor, comprising: assessing the level of one or more
(or a panel of) mesenchymal biomarkers expressed by a tumor cell;
and predicting the sensitivity of tumor cell growth to inhibition
by an EGFR kinase inhibitor, wherein simultaneous low or
undetectable expression levels of all of the tumor cell mesenchymal
biomarkers correlates with high sensitivity to inhibition by EGFR
kinase inhibitors. In one preferred embodiment of this method the
mesenchymal biomarkers comprise vimentin and fibronectin, wherein
simultaneous low or undetectable expression level of the two tumor
cell mesenchymal biomarkers correlates with high sensitivity to
inhibition by EGFR kinase inhibitor. Note that in the latter
preferred embodiment a low or undetectable expression of both
biomarkers is required to indicate high sensitivity.
[0051] The present invention also provides a method of predicting
the sensitivity of tumor cell growth to inhibition by an EGFR
kinase inhibitor, comprising: assessing the level of an epithelial
biomarker expressed by a tumor cell; assessing the level of a
mesenchymal biomarker expressed by a tumor cell; and predicting the
sensitivity of tumor cell growth to inhibition by an EGFR kinase
inhibitor, wherein a high ratio of epithelial to mesenchymal
biomarker expression levels correlates with high sensitivity to
inhibition by EGFR kinase inhibitors. In one preferred embodiment
of this method the epithelial biomarker comprises E-cadherin and
the mesenchymal biomarker comprises fibronectin. In another
preferred embodiment of this method the epithelial biomarker
comprises Brk and the mesenchymal biomarker comprises fibronectin.
In another preferred embodiment of this method the epithelial
biomarker comprises E-cadherin and the mesenchymal biomarker
comprises vimentin. In another preferred embodiment of this method
the epithelial biomarker comprises .gamma.-catenin and the
mesenchymal biomarker comprises fibronectin.
[0052] The present invention also provides a method of predicting
the sensitivity of tumor growth to inhibition by an EGFR kinase
inhibitor, comprising: assessing the level of one or more (or a
panel of) epithelial biomarkers expressed by cells of the tumor;
and predicting the sensitivity of tumor growth to inhibition by an
EGFR kinase inhibitor, wherein simultaneous high expression levels
of all of the tumor cell epithelial biomarkers correlates with high
sensitivity to inhibition by EGFR kinase inhibitors. In one
preferred embodiment of this method the epithelial biomarkers
comprise E-cadherin and Brk, wherein simultaneous high expression
level of the two tumor cell epithelial biomarkers correlates with
high sensitivity to inhibition by EGFR kinase inhibitor. In another
preferred embodiment of this method the epithelial biomarkers
comprise E-cadherin and .gamma.-catenin, wherein simultaneous high
expression level of the two tumor cell epithelial biomarkers
correlates with high sensitivity to inhibition by EGFR kinase
inhibitor. Note that in the two latter preferred embodiments a high
expression level of both biomarkers is required to indicate high
sensitivity.
[0053] The present invention also provides a method of predicting
the sensitivity of tumor growth to inhibition by an EGFR kinase
inhibitor, comprising: assessing the level of one or more (or a
panel of) mesenchymal biomarkers expressed by cells of the tumor;
and predicting the sensitivity of tumor growth to inhibition by an
EGFR kinase inhibitor, wherein simultaneous low or undetectable
expression levels of all of the tumor cell mesenchymal biomarkers
correlates with high sensitivity to inhibition by EGFR kinase
inhibitors. In one preferred embodiment of this method the
mesenchymal biomarkers comprise vimentin and fibronectin, wherein
simultaneous low or undetectable expression level of the two tumor
cell mesenchymal biomarkers correlates with high sensitivity to
inhibition by EGFR kinase inhibitor. Note that in the latter
preferred embodiment a low or undetectable expression of both
biomarkers is required to indicate high sensitivity.
[0054] The present invention also provides a method of predicting
the sensitivity of tumor growth to inhibition by an EGFR kinase
inhibitor, comprising: assessing the level of an epithelial
biomarker expressed by cells of the tumor; assessing the level of a
mesenchymal biomarker expressed by cells of the tumor; and
predicting the sensitivity of tumor growth to inhibition by an EGFR
kinase inhibitor, wherein a high ratio of epithelial to mesenchymal
biomarker expression levels correlates with high sensitivity to
inhibition by EGFR kinase inhibitors. In one preferred embodiment
of this method the epithelial biomarker comprises E-cadherin and
the mesenchymal biomarker comprises fibronectin. In another
preferred embodiment of this method the epithelial biomarker
comprises Brk and the mesenchymal biomarker comprises fibronectin.
In another preferred embodiment of this method the epithelial
biomarker comprises E-cadherin and the mesenchymal biomarker
comprises vimentin. In another preferred embodiment of this method
the epithelial biomarker comprises .gamma.-catenin and the
mesenchymal biomarker comprises fibronectin.
[0055] The present invention also provides a method of predicting
whether a cancer patient is afflicted with a tumor that will
respond effectively to treatment with an EGFR kinase inhibitor,
comprising: assessing the level of one or more (or a panel of)
epithelial biomarkers expressed by cells of the tumor; and
predicting if the tumor will respond effectively to treatment with
an EGFR kinase inhibitor, wherein simultaneous high expression
levels of all of the tumor cell epithelial biomarkers correlates
with a tumor that will respond effectively to treatment with an
EGFR kinase inhibitor. In one preferred embodiment of this method
the epithelial biomarkers comprise E-cadherin and Brk, wherein
simultaneous high expression level of the two tumor cell epithelial
biomarkers correlates with a tumor that will respond effectively to
treatment with an EGFR kinase inhibitor. In another preferred
embodiment of this method the epithelial biomarkers comprise
E-cadherin and .gamma.-catenin, wherein simultaneous high
expression level of the two tumor cell epithelial biomarkers
correlates with a tumor that will respond effectively to treatment
with an EGFR kinase inhibitor. Note that in the two latter
preferred embodiments a high expression level of both biomarkers is
required to indicate a tumor that will respond effectively to
treatment with an EGFR kinase inhibitor.
[0056] The present invention also provides a method of predicting
whether a cancer patient is afflicted with a tumor that will
respond effectively to treatment with an EGFR kinase inhibitor,
comprising: assessing the level of one or more (or a panel of)
mesenchymal biomarkers expressed by cells of the tumor; and
predicting if the tumor will respond effectively to treatment with
an EGFR kinase inhibitor, wherein simultaneous low or undetectable
expression levels of all of the tumor cell mesenchymal biomarkers
correlates with a tumor that will respond effectively to treatment
with an EGFR kinase inhibitor. In one preferred embodiment of this
method the mesenchymal biomarkers comprise vimentin and
fibronectin, wherein simultaneous low or undetectable expression
level of the two tumor cell mesenchymal biomarkers correlates with
a tumor that will respond effectively to treatment with an EGFR
kinase inhibitor. Note that in the latter preferred embodiment a
low or undetectable expression of both biomarkers is required to
indicate a tumor that will respond effectively to treatment with an
EGFR kinase inhibitor.
[0057] The present invention also provides a method of predicting
whether a cancer patient is afflicted with a tumor that will
respond effectively to treatment with an EGFR kinase inhibitor,
comprising: assessing the level of an epithelial biomarker
expressed by cells of the tumor; assessing the level of a
mesenchymal biomarker expressed by cells of the tumor; and
predicting if the tumor will respond effectively to treatment with
an EGFR kinase inhibitor, wherein a high ratio of epithelial to
mesenchymal biomarker expression levels correlates with a tumor
that will respond effectively to treatment with an EGFR kinase
inhibitor. In one preferred embodiment of this method the
epithelial biomarker comprises E-cadherin and the mesenchymal
biomarker comprises fibronectin. In another preferred embodiment of
this method the epithelial biomarker comprises Brk and the
mesenchymal biomarker comprises fibronectin. In another preferred
embodiment of this method the epithelial biomarker comprises
E-cadherin and the mesenchymal biomarker comprises vimentin. In
another preferred embodiment of this method the epithelial
biomarker comprises .gamma.-catenin and the mesenchymal biomarker
comprises fibronectin.
[0058] In the context of the methods of this invention, biomarkers
expressed by a tumor cell can include molecular and cellular
markers that indicate the transition state of the tumor cell. In a
preferred embodiment the biomarker is an individual marker protein,
or its encoding mRNA, characteristic of the particular transition
state of the tumor, i.e. a tumor exhibiting epithelial or
mesenchymal characteristics. In an alternative embodiment, in
certain circumstances the biomarker may be a characteristic
morphological pattern produced in the tumor cell by cellular
macromolecules that is characteristic of either an epithelial or
mesenchymal condition.
TABLE-US-00001 TABLE 1 Molecular Biomarker Gene Identification
Human Biomarker NCBI GeneID.sup.1 NCBI RefSeq.sup.2 E-cadherin 999
NP_004351 Brk 5753 NP_005966 .gamma.-catenin 3728 NP_002221
.alpha.1-catenin 1495 NP_001894 .alpha.2-catenin 1496 NP_004380
.alpha.3-catenin 29119 NP_037398 keratin 8 3856 NP_002264 keratin
18 3875 NP_000215 connexin 31 2707 NP_076872 plakophilin 3 11187
NP_009114 stratifin 1 2810 NP_006133 laminin alpha-5 3911 NP_005551
ST14 19143 NP_035306 vimentin 7431 NP_003371 fibronectin 1 2335
NP_002017 fibrillin-1 2200 NP_000129 fibrillin-2 2201 NP_001990
collagen alpha2(IV) 1284 NP_001837 collagen alpha2(V) 1290
NP_000384 LOXL1 4016 NP_005567 nidogen 4811 NP_002499 C11orf9 745
NP_037411 tenascin 3371 NP_002151 N-cadherin 1000 NP_001783 tubulin
alpha-3 7846 NP_006009 epimorphin 2054 NP_919337 .sup.1The NCBI
GenelD number is a unique identifier of the biomarker gene from the
NCBI Entrez Gene database record (National Center for Biotechnology
Information (NCBI), U.S. National Library of Medicine, 8600
Rockville Pike, Building 38A, Bethesda, MD 20894; Internet address
http://www.ncbi.nlm.nih.gov/). .sup.2The NCBI RefSeq (Reference
Sequence) is an example of a sequence expressed by the biomarker
gene.
[0059] Table 1 lists the genes coding for examples of molecular
biomarkers that can be used in the practice of the methods of the
invention described herein. The molecular biomarkers can include
any product expressed by these genes, including variants thereof,
e.g. expressed mRNA or protein, splice variants, co- and
post-translationally modified proteins, polymorphic variants etc.
In one embodiment the biomarker is the embryonal EDB.sup.+
fibronectin, a splice variant expressed by the fibronectin 1 gene
(Kilian, O. et al. (2004) Bone 35(6):1334-1345). A possible
advantage of determining this fetal form of fibronectin is that one
could readily distinguish mesenchymal-like tumors from surrounding
stromal tissue. In an additional embodiment the biomarker can be an
animal homologue of the human gene product (e.g. from dog, mouse,
rat, rabbit, cat, monkey, ape, etc.).
[0060] In the methods described herein the tumor cell will
typically be from a patient diagnosed with cancer, a precancerous
condition, or another form of abnormal cell growth, and in need of
treatment. The cancer may be lung cancer (e.g. non-small cell lung
cancer (NSCLC)), pancreatic cancer, head and neck cancer, gastric
cancer, breast cancer, colon cancer, ovarian cancer, or any of a
variety of other cancers described herein below. The cancer is
preferably one known to be potentially treatable with an EGFR
kinase inhibitor.
[0061] In the methods of this invention, biomarker expression level
can be assessed relative to a control molecule whose expression
level remains constant throughout EMT, or when comparing tumor
cells expressing either epithelial or mesenchymal transition states
as indicated by molecular biomarkers (e.g. a "housekeeping" gene,
such as GAPDH, .beta.-actin, tubulin, or the like). Biomarker
expression level can also be assessed relative to the other type of
tumor cell biomarker (i.e. epithelial compared to mesenchymal), or
to the biomarker level in non-tumor cells of the same tissue, or
another cell or tissue source used as an assay reference.
[0062] In the methods of this invention, the level of an epithelial
or mesenchymal biomarker expressed by a tumor cell can be assessed
by using any of the standard bioassay procedures known in the art
for determination of the level of expression of a gene, including
for example ELISA, RIA, immunopreciptation, immunoblotting,
immunofluorescence microscopy, RT-PCR, in situ hybridization, cDNA
microarray, or the like, as described in more detail below.
[0063] In the methods of this invention, the expression level of a
tumor cell epithelial or mesenchymal biomarker is preferably
assessed by assaying a tumor biopsy. However, in an alternative
embodiment, expression level of the tumor cell biomarker can be
assessed in bodily fluids or excretions containing detectable
levels of biomarkers originating from the tumor or tumor cells.
Bodily fluids or excretions useful in the present invention include
blood, urine, saliva, stool, pleural fluid, lymphatic fluid,
sputum, ascites, prostatic fluid, cerebrospinal fluid (CSF), or any
other bodily secretion or derivative thereof. By blood it is meant
to include whole blood, plasma, serum or any derivative of blood.
Assessment of tumor epithelial or mesenchymal biomarkers in such
bodily fluids or excretions can sometimes be preferred in
circumstances where an invasive sampling method is inappropriate or
inconvenient.
[0064] In the methods of this invention, the tumor cell can be a
lung cancer tumor cell (e.g. non-small cell lung cancer (NSCLC)), a
pancreatic cancer tumor cell, a breast cancer tumor cell, a head
and neck cancer tumor cell, a gastric cancer tumor cell, a colon
cancer tumor cell, an ovarian cancer tumor cell, or a tumor cell
from any of a variety of other cancers as described herein below.
The tumor cell is preferably of a type known to or expected to
express EGFR kinase, as do all tumor cells from solid tumors. The
EGFR kinase can be wild type or a mutant form.
[0065] In the methods of this invention, the EGFR kinase inhibitor
can be any EGFR kinase inhibitor as described herein below, but is
preferably
6,7-bis(2-methoxyethoxy)-4-quinazolin-4-yl]-(3-ethynylphenyl)amine
(also known as erlotinib, OSI-774, or TARCEVA.TM. (erlotinib HCl),
including pharmacologically acceptable salts or polymorphs
thereof.
[0066] The following methods represent additional specific
embodiments of the method of the invention.
[0067] The present invention provides a method of predicting the
sensitivity of tumor growth to inhibition by an EGFR kinase
inhibitor, comprising: assessing the level of an epithelial
biomarker expressed by cells of the tumor; and predicting the
sensitivity of tumor growth to inhibition by an EGFR kinase
inhibitor, wherein high expression levels of tumor cell epithelial
biomarkers correlate with high sensitivity of tumor growth to
inhibition by EGFR kinase inhibitors.
[0068] The present invention provides a method of predicting the
sensitivity of tumor growth to inhibition by an EGFR kinase
inhibitor, comprising: assessing the level of a mesenchymal
biomarker expressed by cells of the tumor; and predicting the
sensitivity of tumor growth to inhibition by an EGFR kinase
inhibitor, wherein high expression levels of tumor cell mesenchymal
biomarkers correlate with low sensitivity of tumor growth to
inhibition by EGFR kinase inhibitors.
[0069] The present invention provides a method of predicting
whether a cancer patient is afflicted with a tumor that will
respond effectively to treatment with an EGFR kinase inhibitor,
comprising: assessing the level of an epithelial biomarker
expressed by cells of the tumor; and predicting if the tumor will
respond effectively to treatment with an EGFR kinase inhibitor,
wherein high expression levels of tumor cell epithelial biomarkers
correlate with a tumor that will respond effectively to treatment
with an EGFR kinase inhibitor.
[0070] In the methods of this invention, the tumor can be a lung
cancer tumor (e.g. non-small cell lung cancer (NSCLC)), a
pancreatic cancer tumor, a breast cancer tumor, a head and neck
cancer tumor, a gastric cancer tumor, a colon cancer tumor, an
ovarian cancer tumor, or a tumor from any of a variety of other
cancers as described herein below. The tumor is preferably of a
type whose cells are known to or expected to express EGFR kinase,
as do all solid tumors. The EGFR kinase can be wild type or a
mutant form.
[0071] The present invention provides a method of predicting
whether a cancer patient is afflicted with a tumor that will
respond effectively to treatment with an EGFR kinase inhibitor,
comprising: assessing the level of a mesenchymal biomarker
expressed by cells of the tumor; and predicting if the tumor will
respond effectively to treatment with an EGFR kinase inhibitor,
wherein high expression levels of tumor cell mesenchymal biomarkers
correlate with a tumor that will respond less effectively to
treatment with an EGFR kinase inhibitor.
[0072] The present invention provides a method of predicting the
sensitivity of tumor cell growth to inhibition by an EGFR kinase
inhibitor comprising: determining the tumor cell level of at least
one epithelial biomarker polypeptide; determining the tumor cell
level of at least one control polypeptide; comparing the tumor cell
level of at least one epithelial biomarker polypeptide to the tumor
cell level of at least one control polypeptide; wherein a high
ratio of tumor cell biomarker polypeptide to tumor cell control
polypeptide indicates a high predicted sensitivity of tumor cell
growth to inhibition by an EGFR kinase inhibitor. For this method,
examples of useful epithelial biomarker polypeptides include
E-cadherin, .gamma.-catenin, keratin 8, keratin 18, connexin 31,
plakophilin 3, stratafin 1, laminin alpha-5, ST14 and Brk.
[0073] The present invention provides a method of predicting the
sensitivity of tumor cell growth to inhibition by an EGFR kinase
inhibitor comprising: determining the tumor cell level of at least
one epithelial biomarker polynucleotide that encodes an
polypeptide; determining the tumor cell level of at least one
control polynucleotide; comparing the tumor cell level of at least
one epithelial biomarker polynucleotide that encodes a polypeptide
to the tumor cell level of at least one control polynucleotide;
wherein a high ratio of tumor cell biomarker polynucleotide to
tumor cell control polynucleotide indicates a high predicted
sensitivity of tumor cell growth to inhibition by an EGFR kinase
inhibitor. For this method examples of polypeptides encoded by the
epithelial biomarker polynucleotide include E-cadherin,
.gamma.-catenin, keratin 8, keratin 18, connexin 31, plakophilin 3,
stratafin 1, laminin alpha-5, ST14 and Brk.
[0074] The present invention provides a method of predicting the
sensitivity of tumor cell growth to inhibition by an EGFR kinase
inhibitor comprising: determining the tumor cell level of at least
one mesenchymal biomarker polypeptide; determining the tumor cell
level of at least one control polypeptide; comparing the tumor cell
level of at least one mesenchymal biomarker polypeptide to the
tumor cell level of at least one control polypeptide; wherein a low
ratio of tumor cell biomarker polypeptide to tumor cell control
polypeptide indicates a high predicted sensitivity of tumor cell
growth to inhibition by an EGFR kinase inhibitor. For this method,
examples of useful mesenchymal biomarker polypeptides include
vimentin and fibronectin.
[0075] The present invention provides a method of predicting the
sensitivity of tumor cell growth to inhibition by an EGFR kinase
inhibitor comprising: determining the tumor cell level of at least
one mesenchymal biomarker polynucleotide that encodes an
polypeptide; determining the tumor cell level of at least one
control polynucleotide; comparing the tumor cell level of at least
one mesenchymal biomarker polynucleotide that encodes an
polypeptide to the tumor cell level of at least one control
polynucleotide; wherein a low ratio of tumor cell biomarker
polynucleotide to tumor cell control polynucleotide indicates a
high predicted sensitivity of tumor cell growth to inhibition by an
EGFR kinase inhibitor. For this method, examples of useful
polypeptides encoded by the biomarker polynucleotide include
vimentin and fibronectin.
[0076] The present invention provides a method of predicting the
sensitivity of tumor cell growth to inhibition by an EGFR kinase
inhibitor comprising: determining the tumor cell level of at least
one epithelial biomarker polypeptide; determining a non-tumor cell
level of at least one epithelial biomarker polypeptide; comparing
the tumor cell level of at least one epithelial biomarker
polypeptide to the non-tumor cell level of at least one epithelial
biomarker polypeptide; wherein a high ratio of tumor cell biomarker
polypeptide to non-tumor cell biomarker polypeptide indicates a
high predicted sensitivity of tumor cell growth to inhibition by an
EGFR kinase inhibitor. For this method, examples of useful
epithelial biomarker polypeptide include E-cadherin,
.gamma.-catenin, keratin 8, keratin 18, connexin 31, plakophilin 3,
stratafin 1, laminin alpha-5, ST14 and Brk.
[0077] The present invention provides a method of predicting the
sensitivity of tumor cell growth to inhibition by an EGFR kinase
inhibitor comprising: determining the tumor cell level of at least
one epithelial biomarker polynucleotide that encodes an
polypeptide; determining a non-tumor cell level of at least one
epithelial biomarker polynucleotide that encodes an polypeptide;
comparing the tumor cell level of at least one epithelial biomarker
polynucleotide that encodes an polypeptide to the non-tumor cell
level of at least one epithelial biomarker polynucleotide that
encodes an polypeptide; wherein a high ratio of tumor cell
biomarker polynucleotide to non-tumor cell biomarker polynucleotide
indicates a high predicted sensitivity of tumor cell growth to
inhibition by an EGFR kinase inhibitor. For this method, examples
of useful polypeptides encoded by the epithelial biomarker
polynucleotide include E-cadherin, .gamma.-catenin, keratin 8,
keratin 18, connexin 31, plakophilin 3, stratafin 1, laminin
alpha-5, ST14 and Brk.
[0078] The present invention provides a method of predicting the
sensitivity of tumor cell growth to inhibition by an EGFR kinase
inhibitor comprising: determining the tumor cell level of at least
one mesenchymal biomarker polypeptide; determining a non-tumor cell
level of at least one mesenchymal biomarker polypeptide; comparing
the tumor cell level of at least one mesenchymal biomarker
polypeptide to the non-tumor cell level of at least one mesenchymal
biomarker polypeptide; wherein a low ratio of tumor cell biomarker
polypeptide to non-tumor cell biomarker polypeptide indicates a
high predicted sensitivity of tumor cell growth to inhibition by an
EGFR kinase inhibitor. For this method, examples of useful
mesenchymal biomarker polypeptides include vimentin and
fibronectin.
[0079] The present invention provides a method of predicting the
sensitivity of tumor cell growth to inhibition by an EGFR kinase
inhibitor comprising: determining the tumor cell level of at least
one mesenchymal biomarker polynucleotide that encodes an
polypeptide; determining a non-tumor cell level of at least one
mesenchymal biomarker polynucleotide that encodes an polypeptide;
comparing the tumor cell level of at least one mesenchymal
biomarker polynucleotide that encodes an polypeptide to the
non-tumor cell level of at least one mesenchymal biomarker
polynucleotide that encodes an polypeptide; wherein a low ratio of
tumor cell biomarker polynucleotide to non-tumor cell biomarker
polynucleotide indicates a high predicted sensitivity of tumor cell
growth to inhibition by an EGFR kinase inhibitor. For this method,
examples of useful polypeptides encoded by the biomarker
polynucleotide include vimentin and fibronectin.
[0080] The present invention provides a method of predicting the
sensitivity of tumor cell growth to inhibition by an EGFR kinase
inhibitor comprising: determining the tumor cell level of at least
one epithelial biomarker polypeptide; determining the tumor cell
level of at least one mesenchymal biomarker polypeptide; comparing
the level of at least one epithelial biomarker polypeptide to the
level of at least one mesenchymal biomarker polypeptide; wherein a
high ratio of epithelial biomarker polypeptide to mesenchymal
biomarker polypeptide indicates a high predicted sensitivity of
tumor cell growth to inhibition by an EGFR kinase inhibitor. For
this method, examples of useful epithelial biomarker polypeptides
include E-cadherin, catenin, keratin 8, keratin 18, connexin 31,
plakophilin 3, stratafin 1, laminin alpha-5, ST14 and Brk. For this
method, examples of useful mesenchymal biomarker polypeptides
include vimentin and fibronectin.
[0081] The present invention provides a method of predicting the
sensitivity of tumor cell growth to inhibition by an EGFR kinase
inhibitor comprising: determining the tumor cell level of at least
one epithelial biomarker polynucleotide that encodes a polypeptide;
determining the tumor cell level of at least one mesenchymal
biomarker polynucleotide that encodes a polypeptide; (c) comparing
the level of at least one epithelial biomarker polynucleotide to
the level of at least one mesenchymal biomarker polynucleotide;
wherein a high ratio of epithelial biomarker polynucleotide to
mesenchymal biomarker polynucleotide indicates a predicted high
sensitivity of tumor cell growth to inhibition by an EGFR kinase
inhibitor. For this method, examples of useful polypeptides encoded
by the epithelial biomarker polynucleotide include E-cadherin,
.gamma.-catenin, keratin 8, keratin 18, connexin 31, plakophilin 3,
stratafin 1, laminin alpha-5, ST14 and Brk. For this method,
examples of useful polypeptides encoded by the mesenchymal
biomarker polynucleotide include vimentin and fibronectin.
[0082] The present invention provides a method of assessing whether
a cancer patient is afflicted with a cancer that will respond
effectively to treatment with an EGFR kinase inhibitor, the method
comprising comparing: the level of expression of a mesenchymal
biomarker in a patient sample; and the normal level of expression
of the biomarker in a control non-cancer sample, wherein a
significant increase in the level of expression of the mesenchymal
biomarker in the patient sample over the normal level is an
indication that the patient is afflicted with a cancer which is
less likely to respond effectively to treatment with an EGFR kinase
inhibitor. For this method, examples of useful mesenchymal
biomarkers include vimentin and fibronectin, and nucleic acids
encoding for these proteins.
[0083] The present invention provides a method of assessing whether
a cancer patient is afflicted with a cancer that will respond
effectively to treatment with an EGFR kinase inhibitor, the method
comprising comparing: the level of expression of an epithelial
biomarker in a patient sample; and the normal level of expression
of the biomarker in a control non-cancer sample, wherein a
significant decrease in the level of expression of the epithelial
biomarker in the patient sample over the normal level is an
indication that the patient is afflicted with a cancer which is
less likely to respond effectively to treatment with an EGFR kinase
inhibitor. For this method, examples of useful epithelial
biomarkers include E-cadherin, .gamma.-catenin, keratin 8, keratin
18, connexin 31, plakophilin 3, stratafin 1, laminin alpha-5, ST14
and Brk, and nucleic acids encoding for these proteins.
[0084] The present invention provides a method of assessing whether
a cancer patient is afflicted with a cancer that will respond
effectively to treatment with an EGFR kinase inhibitor, the method
comprising comparing: the level of expression of an epithelial
biomarker in a patient sample; and the level of expression of a
mesenchymal biomarker in a patient sample, wherein a high ratio of
the level of expression of the epithelial biomarker to the level of
expression of the mesenchymal biomarker is an indication that the
patient is afflicted with a cancer which is likely to respond
effectively to treatment with an EGFR kinase inhibitor. For this
method, examples of useful epithelial biomarkers include
E-cadherin, .gamma.-catenin, keratin 8, keratin 18, connexin 31,
plakophilin 3, stratafin 1, laminin alpha-5, ST14 and Brk, and
nucleic acids encoding for these proteins. For this method,
examples of useful mesenchymal biomarkers include vimentin and
fibronectin, and nucleic acids encoding for these proteins.
[0085] In any of the above methods referring to a patient sample,
an example of such a sample can be a tumor biopsy.
[0086] The present invention provides a method of determining
whether in a human subject a tumor will be responsive to treatment
with an EGFR kinase inhibitor, comprising: (a) collecting a sample
of a bodily substance containing human nucleic acid or protein,
said nucleic acid or protein having originated from cells of the
human subject, (b) determining quantitatively or
semi-quantitatively in the sample a level of expression for one or
more epithelial cell biomarker proteins or one or more epithelial
cell biomarker protein-specific mRNAs; and (c) comparing the
expression level in (b) to the level of biomarker expression in a
normal control, or to the level of a control polypeptide or nucleic
acid in the sample, wherein reduced expression of one or more
epithelial cell biomarker proteins or one or more epithelial cell
biomarker protein-specific mRNAs, with respect to the control
level, indicates the presence in the human subject of a tumor which
is less likely to respond effectively to treatment with an EGFR
kinase inhibitor.
[0087] The present invention provides a method of determining
whether in a human subject a tumor will be responsive to treatment
with an EGFR kinase inhibitor, comprising: (a) collecting a sample
of a bodily substance containing human nucleic acid or protein,
said nucleic acid or protein having originated from cells of the
human subject, (b) determining quantitatively or
semi-quantitatively in the sample a level of expression for one or
more mesenchymal cell biomarker proteins or one or more mesenchymal
cell biomarker protein-specific mRNAs; and (c) comparing the
expression level in (b) to the level of biomarker expression in a
normal control, or to the level of a control polypeptide or nucleic
acid in the sample, wherein increased expression of one or more
mesenchymal cell biomarker proteins or one or more mesenchymal cell
biomarker protein-specific mRNAs, with respect to the control
level, indicates the presence in the human subject of a tumor which
is less likely to respond effectively to treatment with an EGFR
kinase inhibitor.
[0088] The present invention provides a method of determining the
likelihood that a patient with a tumor will show relatively long
survival benefit from therapy with an EGFR kinase inhibitor,
comprising determining the level of one or more epithelial
biomarkers in the cells of the tumor, comparing said level with the
level of epithelial biomarker expression in a non-tumor control, or
to the level of a control polypeptide or nucleic acid in the tumor
sample, and determining whether the cells of the tumor contain a
relatively high level of one or more epithelial biomarkers, a high
level being indicative that a patient with a tumor will show
relatively long survival benefit from therapy with an EGFR kinase
inhibitor.
[0089] The present invention provides a method of determining the
likelihood that a patient with a tumor will show relatively long
survival benefit from therapy with an EGFR kinase inhibitor,
comprising determining the level of one or more mesenchymal
biomarkers in the cells of the tumor, comparing said level with the
level of mesenchymal biomarker expression in a non-tumor control,
or to the level of a control polypeptide or nucleic acid in the
tumor sample, and determining whether the cells of the tumor
contain a relatively low level of one or more mesenchymal
biomarkers, a low level being indicative that a patient with a
tumor will show relatively long survival benefit from therapy with
an EGFR kinase inhibitor.
[0090] The present invention provides a method for determining for
a patient with a tumor the likelihood that said patient will show
relatively long survival benefit from therapy with an EGFR kinase
inhibitor, comprising: determining the level of one or more
epithelial biomarkers in the cells of the tumor, comparing said
level with the level of epithelial biomarker expression in a
non-tumor control, or to the level of a control polypeptide or
nucleic acid in the tumor sample, and determining whether the cells
of the tumor contain a relatively high level of one or more
epithelial biomarkers; determining the level of one or more
mesenchymal biomarkers in the cells of the tumor, comparing said
level with the level of mesenchymal biomarker expression in a
non-tumor control, or to the level of a control polypeptide or
nucleic acid in the tumor sample, and determining whether the cells
of the tumor contain a relatively low level of one or more
mesenchymal biomarkers, wherein a high level of one or more
epithelial biomarkers and a low level of one or more mesenchymal
biomarkers is indicative that a patient with a tumor will show
relatively long survival benefit from therapy with an EGFR kinase
inhibitor.
[0091] The present invention provides a method of determining a
prognosis for survival for a patient with a neoplastic condition in
response to therapy with an EGFR kinase inhibitor, comprising:
measuring the level of an epithelial biomarker associated with
neoplastic cells, and comparing said level of epithelial biomarker
to a non-neoplastic epithelial biomarker reference level, or to the
level of a control polypeptide or nucleic acid associated with the
neoplastic cells, wherein a decreased level of epithelial biomarker
associated with the neoplastic cells correlates with decreased
survival of said patient.
[0092] The present invention provides a method of determining a
prognosis for survival for a patient with a neoplastic condition in
response to therapy with an EGFR kinase inhibitor, comprising:
measuring the level of an mesenchymal biomarker associated with
neoplastic cells, and comparing said level of mesenchymal biomarker
to a non-neoplastic mesenchymal biomarker reference level, or to
the level of a control polypeptide or nucleic acid associated with
the neoplastic cells, wherein an increased level of mesenchymal
biomarker associated with the neoplastic cells correlates with
decreased survival of said patient.
[0093] For assessment of tumor cell epithelial or mesenchymal
biomarker expression, patient samples containing tumor cells, or
proteins or nucleic acids produced by these tumor cells, may be
used in the methods of the present invention. In these embodiments,
the level of expression of the biomarker can be assessed by
assessing the amount (e.g. absolute amount or concentration) of the
marker in a tumor cell sample, e.g., a tumor biopsy obtained from a
patient, or other patient sample containing material derived from
the tumor (e.g. blood, serum, urine, or other bodily fluids or
excretions as described herein above). The cell sample can, of
course, be subjected to a variety of well-known post-collection
preparative and storage techniques (e.g., nucleic acid and/or
protein extraction, fixation, storage, freezing, ultrafiltration,
concentration, evaporation, centrifugation, etc.) prior to
assessing the amount of the marker in the sample. Likewise, tumor
biopsies may also be subjected to post-collection preparative and
storage techniques, e.g., fixation.
[0094] In the methods of the invention, one can detect expression
of biomarker proteins having at least one portion which is
displayed on the surface of tumor cells which express it. It is a
simple matter for the skilled artisan to determine whether a marker
protein, or a portion thereof, is exposed on the cell surface. For
example, immunological methods may be used to detect such proteins
on whole cells, or well known computer-based sequence analysis
methods may be used to predict the presence of at least one
extracellular domain (i.e. including both secreted proteins and
proteins having at least one cell-surface domain). Expression of a
marker protein having at least one portion which is displayed on
the surface of a cell which expresses it may be detected without
necessarily lysing the tumor cell (e.g. using a labeled antibody
which binds specifically with a cell-surface domain of the
protein).
[0095] Expression of a biomarkers described in this invention may
be assessed by any of a wide variety of well known methods for
detecting expression of a transcribed nucleic acid or protein.
Non-limiting examples of such methods include immunological methods
for detection of secreted, cell-surface, cytoplasmic, or nuclear
proteins, protein purification methods, protein function or
activity assays, nucleic acid hybridization methods, nucleic acid
reverse transcription methods, and nucleic acid amplification
methods.
[0096] In one embodiment, expression of a biomarker is assessed
using an antibody (e.g. a radio-labeled, chromophore-labeled,
fluorophore-labeled, or enzyme-labeled antibody), an antibody
derivative (e.g. an antibody conjugated with a substrate or with
the protein or ligand of a protein-ligand pair {e.g.
biotin-streptavidin}), or an antibody fragment (e.g. a single-chain
antibody, an isolated antibody hypervariable domain, etc.) which
binds specifically with a biomarker protein or fragment thereof,
including a biomarker protein which has undergone either all or a
portion of post-translational modifications to which it is normally
subjected in the tumor cell (e.g. glycosylation, phosphorylation,
methylation etc.).
[0097] In another embodiment, expression of a biomarker is assessed
by preparing mRNA/cDNA (i.e. a transcribed polynucleotide) from
cells in a patient sample, and by hybridizing the mRNA/cDNA with a
reference polynucleotide which is a complement of a biomarker
nucleic acid, or a fragment thereof cDNA can, optionally, be
amplified using any of a variety of polymerase chain reaction
methods prior to hybridization with the reference polynucleotide.
Expression of one or more biomarkers can likewise be detected using
quantitative PCR to assess the level of expression of the
biomarker(s). Alternatively, any of the many known methods of
detecting mutations or variants (e.g. single nucleotide
polymorphisms, deletions, etc.) of a biomarker of the invention may
be used to detect occurrence of a biomarker in a patient.
[0098] In a related embodiment, a mixture of transcribed
polynucleotides obtained from the sample is contacted with a
substrate having fixed thereto a polynucleotide complementary to or
homologous with at least a portion (e.g. at least 7, 10, 15, 20,
25, 30, 40, 50, 100, 500, or more nucleotide residues) of a
biomarker nucleic acid. If polynucleotides complementary to or
homologous with are differentially detectable on the substrate
(e.g. detectable using different chromophores or fluorophores, or
fixed to different selected positions), then the levels of
expression of a plurality of biomarkers can be assessed
simultaneously using a single substrate (e.g. a "gene chip"
microarray of polynucleotides fixed at selected positions). When a
method of assessing biomarker expression is used which involves
hybridization of one nucleic acid with another, it is preferred
that the hybridization be performed under stringent hybridization
conditions.
[0099] When a plurality of biomarkers of the invention are used in
the methods of the invention, the level of expression of each
biomarker in a patient sample can be compared with the normal level
of expression of each of the plurality of biomarkers in
non-cancerous samples of the same type, either in a single reaction
mixture (i.e. using reagents, such as different fluorescent probes,
for each biomarker) or in individual reaction mixtures
corresponding to one or more of the biomarkers.
[0100] The level of expression of a biomarker in normal (i.e.
non-cancerous) human tissue can be assessed in a variety of ways.
In one embodiment, this normal level of expression is assessed by
assessing the level of expression of the biomarker in a portion of
cells which appears to be non-cancerous, and then comparing this
normal level of expression with the level of expression in a
portion of the tumor cells. Alternately, and particularly as
further information becomes available as a result of routine
performance of the methods described herein, population-average
values for normal expression of the biomarkers of the invention may
be used. In other embodiments, the `normal` level of expression of
a biomarker may be determined by assessing expression of the
biomarker in a patient sample obtained from a non-cancer-afflicted
patient, from a patient sample obtained from a patient before the
suspected onset of cancer in the patient, from archived patient
samples, and the like.
[0101] An exemplary method for detecting the presence or absence of
a biomarker protein or nucleic acid in a biological sample involves
obtaining a biological sample (e.g. a tumor-associated body fluid)
from a test subject and contacting the biological sample with a
compound or an agent capable of detecting the polypeptide or
nucleic acid (e.g., mRNA, genomic DNA, or cDNA). The detection
methods of the invention can thus be used to detect mRNA, protein,
cDNA, or genomic DNA, for example, in a biological sample in vitro
as well as in vivo. For example, in vitro techniques for detection
of mRNA include Northern hybridizations and in situ hybridizations.
In vitro techniques for detection of a biomarker protein include
enzyme linked immunosorbent assays (ELISAs), Western blots,
immunoprecipitations and immunofluorescence. In vitro techniques
for detection of genomic DNA include Southern hybridizations. In
vivo techniques for detection of mRNA include polymerase chain
reaction (PCR), Northern hybridizations and in situ hybridizations.
Furthermore, in vivo techniques for detection of a biomarker
protein include introducing into a subject a labeled antibody
directed against the protein or fragment thereof. For example, the
antibody can be labeled with a radioactive marker whose presence
and location in a subject can be detected by standard imaging
techniques.
[0102] A general principle of such diagnostic and prognostic assays
involves preparing a sample or reaction mixture that may contain a
biomarker, and a probe, under appropriate conditions and for a time
sufficient to allow the biomarker and probe to interact and bind,
thus forming a complex that can be removed and/or detected in the
reaction mixture. These assays can be conducted in a variety of
ways.
[0103] For example, one method to conduct such an assay would
involve anchoring the biomarker or probe onto a solid phase
support, also referred to as a substrate, and detecting target
biomarker/probe complexes anchored on the solid phase at the end of
the reaction. In one embodiment of such a method, a sample from a
subject, which is to be assayed for presence and/or concentration
of biomarker, can be anchored onto a carrier or solid phase
support. In another embodiment, the reverse situation is possible,
in which the probe can be anchored to a solid phase and a sample
from a subject can be allowed to react as an unanchored component
of the assay.
[0104] There are many established methods for anchoring assay
components to a solid phase. These include, without limitation,
biomarker or probe molecules which are immobilized through
conjugation of biotin and streptavidin. Such biotinylated assay
components can be prepared from biotin-NHS (N-hydroxy-succinimide)
using techniques known in the art (e.g., biotinylation kit, Pierce
Chemicals, Rockford, Ill.), and immobilized in the wells of
streptavidin-coated 96 well plates (Pierce Chemical). In certain
embodiments, the surfaces with immobilized assay components can be
prepared in advance and stored.
[0105] Other suitable carriers or solid phase supports for such
assays include any material capable of binding the class of
molecule to which the biomarker or probe belongs. Well-known
supports or carriers include, but are not limited to, glass,
polystyrene, nylon, polypropylene, nylon, polyethylene, dextran,
amylases, natural and modified celluloses, polyacrylamides,
gabbros, and magnetite.
[0106] In order to conduct assays with the above mentioned
approaches, the non-immobilized component is added to the solid
phase upon which the second component is anchored. After the
reaction is complete, uncomplexed components may be removed (e.g.,
by washing) under conditions such that any complexes formed will
remain immobilized upon the solid phase. The detection of
biomarker/probe complexes anchored to the solid phase can be
accomplished in a number of methods outlined herein.
[0107] In one embodiment, the probe, when it is the unanchored
assay component, can be labeled for the purpose of detection and
readout of the assay, either directly or indirectly, with
detectable labels discussed herein and which are well-known to one
skilled in the art.
[0108] It is also possible to directly detect biomarker/probe
complex formation without further manipulation or labeling of
either component (biomarker or probe), for example by utilizing the
technique of fluorescence energy transfer (i.e. FET, see for
example, Lakowicz et al., U.S. Pat. No. 5,631,169; Stavrianopoulos,
et al., U.S. Pat. No. 4,868,103). A fluorophore label on the first,
`donor` molecule is selected such that, upon excitation with
incident light of appropriate wavelength, its emitted fluorescent
energy will be absorbed by a fluorescent label on a second
`acceptor` molecule, which in turn is able to fluoresce due to the
absorbed energy. Alternately, the `donor` protein molecule may
simply utilize the natural fluorescent energy of tryptophan
residues. Labels are chosen that emit different wavelengths of
light, such that the `acceptor` molecule label may be
differentiated from that of the `donor`. Since the efficiency of
energy transfer between the labels is related to the distance
separating the molecules, spatial relationships between the
molecules can be assessed. In a situation in which binding occurs
between the molecules, the fluorescent emission of the `acceptor`
molecule label in the assay should be maximal. An FET binding event
can be conveniently measured through standard fluorometric
detection means well known in the art (e.g., using a
fluorimeter).
[0109] In another embodiment, determination of the ability of a
probe to recognize a biomarker can be accomplished without labeling
either assay component (probe or biomarker) by utilizing a
technology such as real-time Biomolecular Interaction Analysis
(BIA) (see, e.g., Sjolander, S. and Urbaniczky, C., 1991, Anal.
Chem. 63:2338-2345 and Szabo et al., 1995, Curr. Opin. Struct.
Biol. 5:699-705). As used herein, "BIA" or "surface plasmon
resonance" is a technology for studying biospecific interactions in
real time, without labeling any of the interactants (e.g.,
BIAcore). Changes in the mass at the binding surface (indicative of
a binding event) result in alterations of the refractive index of
light near the surface (the optical phenomenon of surface plasmon
resonance (SPR)), resulting in a detectable signal which can be
used as an indication of real-time reactions between biological
molecules.
[0110] Alternatively, in another embodiment, analogous diagnostic
and prognostic assays can be conducted with biomarker and probe as
solutes in a liquid phase. In such an assay, the complexed
biomarker and probe are separated from uncomplexed components by
any of a number of standard techniques, including but not limited
to: differential centrifugation, chromatography, electrophoresis
and immunoprecipitation. In differential centrifugation,
biomarker/probe complexes may be separated from uncomplexed assay
components through a series of centrifugal steps, due to the
different sedimentation equilibria of complexes based on their
different sizes and densities (see, for example, Rivas, G., and
Minton, A. P., 1993, Trends Biochem Sci. 18(8):284-7). Standard
chromatographic techniques may also be utilized to separate
complexed molecules from uncomplexed ones. For example, gel
filtration chromatography separates molecules based on size, and
through the utilization of an appropriate gel filtration resin in a
column format, for example, the relatively larger complex may be
separated from the relatively smaller uncomplexed components.
Similarly, the relatively different charge properties of the
biomarker/probe complex as compared to the uncomplexed components
may be exploited to differentiate the complex from uncomplexed
components, for example through the utilization of ion-exchange
chromatography resins. Such resins and chromatographic techniques
are well known to one skilled in the art (see, e.g., Heegaard, N.
H., 1998, J. Mol. Recognit. Winter 11(1-6):141-8; Hage, D. S., and
Tweed, S. A. J. Chromatogr B Biomed Sci Appl 1997 Oct. 10;
699(1-2):499-525). Gel electrophoresis may also be employed to
separate complexed assay components from unbound components (see,
e.g., Ausubel et al., ed., Current Protocols in Molecular Biology,
John Wiley & Sons, New York, 1987-1999). In this technique,
protein or nucleic acid complexes are separated based on size or
charge, for example. In order to maintain the binding interaction
during the electrophoretic process, non-denaturing gel matrix
materials and conditions in the absence of reducing agent are
typically preferred. Appropriate conditions to the particular assay
and components thereof will be well known to one skilled in the
art.
[0111] In a particular embodiment, the level of biomarker mRNA can
be determined both by in situ and by in vitro formats in a
biological sample using methods known in the art. The term
"biological sample" is intended to include tissues, cells,
biological fluids and isolates thereof, isolated from a subject, as
well as tissues, cells and fluids present within a subject. Many
expression detection methods use isolated RNA. For in vitro
methods, any RNA isolation technique that does not select against
the isolation of mRNA can be utilized for the purification of RNA
from tumor cells (see, e.g., Ausubel et al., ed., Current Protocols
in Molecular Biology, John Wiley & Sons, New York 1987-1999).
Additionally, large numbers of tissue samples can readily be
processed using techniques well known to those of skill in the art,
such as, for example, the single-step RNA isolation process of
Chomczynski (1989, U.S. Pat. No. 4,843,155).
[0112] The isolated mRNA can be used in hybridization or
amplification assays that include, but are not limited to, Southern
or Northern analyses, polymerase chain reaction analyses and probe
arrays. One preferred diagnostic method for the detection of mRNA
levels involves contacting the isolated mRNA with a nucleic acid
molecule (probe) that can hybridize to the mRNA encoded by the gene
being detected. The nucleic acid probe can be, for example, a
full-length cDNA, or a portion thereof, such as an oligonucleotide
of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length
and sufficient to specifically hybridize under stringent conditions
to a mRNA or genomic DNA encoding a biomarker of the present
invention. Other suitable probes for use in the diagnostic assays
of the invention are described herein. Hybridization of an mRNA
with the probe indicates that the biomarker in question is being
expressed.
[0113] In one format, the mRNA is immobilized on a solid surface
and contacted with a probe, for example by running the isolated
mRNA on an agarose gel and transferring the mRNA from the gel to a
membrane, such as nitrocellulose. In an alternative format, the
probe(s) are immobilized on a solid surface and the mRNA is
contacted with the probe(s), for example, in an Affymetrix gene
chip array. A skilled artisan can readily adapt known mRNA
detection methods for use in detecting the level of mRNA encoded by
the biomarkers of the present invention.
[0114] An alternative method for determining the level of mRNA
biomarker in a sample involves the process of nucleic acid
amplification, e.g., by RT-PCR (the experimental embodiment set
forth in Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain
reaction (Barany, 1991, Proc. Natl. Acad. Sci. USA, 88:189-193),
self sustained sequence replication (Guatelli et al., 1990, Proc.
Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification
system (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA
86:1173-1177), Q-Beta Replicase (Lizardi et al., 1988,
Bio/Technology 6:1197), rolling circle replication (Lizardi et al.,
U.S. Pat. No. 5,854,033) or any other nucleic acid amplification
method, followed by the detection of the amplified molecules using
techniques well known to those of skill in the art. These detection
schemes are especially useful for the detection of nucleic acid
molecules if such molecules are present in very low numbers. As
used herein, amplification primers are defined as being a pair of
nucleic acid molecules that can anneal to 5' or 3' regions of a
gene (plus and minus strands, respectively, or vice-versa) and
contain a short region in between. In general, amplification
primers are from about 10 to 30 nucleotides in length and flank a
region from about 50 to 200 nucleotides in length. Under
appropriate conditions and with appropriate reagents, such primers
permit the amplification of a nucleic acid molecule comprising the
nucleotide sequence flanked by the primers.
[0115] For in situ methods, mRNA does not need to be isolated from
the tumor cells prior to detection. In such methods, a cell or
tissue sample is prepared/processed using known histological
methods. The sample is then immobilized on a support, typically a
glass slide, and then contacted with a probe that can hybridize to
mRNA that encodes the biomarker.
[0116] As an alternative to making determinations based on the
absolute expression level of the biomarker, determinations may be
based on the normalized expression level of the biomarker.
Expression levels are normalized by correcting the absolute
expression level of a biomarker by comparing its expression to the
expression of a gene that is not a biomarker, e.g., a housekeeping
gene that is constitutively expressed. Suitable genes for
normalization include housekeeping genes such as the actin gene, or
epithelial cell-specific genes. This normalization allows the
comparison of the expression level in one sample, e.g., a patient
sample, to another sample, e.g., a non-tumor sample, or between
samples from different sources.
[0117] Alternatively, the expression level can be provided as a
relative expression level. To determine a relative expression level
of a biomarker (e.g. a mesenchymal biomarker), the level of
expression of the biomarker is determined for 10 or more samples of
normal versus cancer cell isolates, preferably 50 or more samples,
prior to the determination of the expression level for the sample
in question. The mean expression level of each of the genes assayed
in the larger number of samples is determined and this is used as a
baseline expression level for the biomarker. The expression level
of the biomarker determined for the test sample (absolute level of
expression) is then divided by the mean expression value obtained
for that biomarker. This provides a relative expression level.
[0118] In another embodiment of the present invention, a biomarker
protein is detected. A preferred agent for detecting biomarker
protein of the invention is an antibody capable of binding to such
a protein or a fragment thereof, preferably an antibody with a
detectable label. Antibodies can be polyclonal, or more preferably,
monoclonal. An intact antibody, or a fragment or derivative thereof
(e.g., Fab or F(ab').sub.2) can be used. The term "labeled", with
regard to the probe or antibody, is intended to encompass direct
labeling of the probe or antibody by coupling (i.e., physically
linking) a detectable substance to the probe or antibody, as well
as indirect labeling of the probe or antibody by reactivity with
another reagent that is directly labeled. Examples of indirect
labeling include detection of a primary antibody using a
fluorescently labeled secondary antibody and end-labeling of a DNA
probe with biotin such that it can be detected with fluorescently
labeled streptavidin.
[0119] Proteins from tumor cells can be isolated using techniques
that are well known to those of skill in the art. The protein
isolation methods employed can, for example, be such as those
described in Harlow and Lane (Harlow and Lane, 1988, Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.).
[0120] A variety of formats can be employed to determine whether a
sample contains a protein that binds to a given antibody. Examples
of such formats include, but are not limited to, enzyme immunoassay
(EIA), radioimmunoassay (RIA), Western blot analysis and enzyme
linked immunoabsorbant assay (ELISA). A skilled artisan can readily
adapt known protein/antibody detection methods for use in
determining whether tumor cells express a biomarker of the present
invention.
[0121] In one format, antibodies, or antibody fragments or
derivatives, can be used in methods such as Western blots or
immunofluorescence techniques to detect the expressed proteins. In
such uses, it is generally preferable to immobilize either the
antibody or proteins on a solid support. Suitable solid phase
supports or carriers include any support capable of binding an
antigen or an antibody. Well-known supports or carriers include
glass, polystyrene, polypropylene, polyethylene, dextran, nylon,
amylases, natural and modified celluloses, polyacrylamides,
gabbros, and magnetite.
[0122] One skilled in the art will know many other suitable
carriers for binding antibody or antigen, and will be able to adapt
such support for use with the present invention. For example,
protein isolated from tumor cells can be run on a polyacrylamide
gel electrophoresis and immobilized onto a solid phase support such
as nitrocellulose. The support can then be washed with suitable
buffers followed by treatment with the detectably labeled antibody.
The solid phase support can then be washed with the buffer a second
time to remove unbound antibody. The amount of bound label on the
solid support can then be detected by conventional means.
[0123] For ELISA assays, specific binding pairs can be of the
immune or non-immune type. Immune specific binding pairs are
exemplified by antigen-antibody systems or hapten/anti-hapten
systems. There can be mentioned fluorescein/anti-fluorescein,
dinitrophenyl/anti-dinitrophenyl, biotin/anti-biotin,
peptide/anti-peptide and the like. The antibody member of the
specific binding pair can be produced by customary methods familiar
to those skilled in the art. Such methods involve immunizing an
animal with the antigen member of the specific binding pair. If the
antigen member of the specific binding pair is not immunogenic,
e.g., a hapten, it can be covalently coupled to a carrier protein
to render it immunogenic. Non-immune binding pairs include systems
wherein the two components share a natural affinity for each other
but are not antibodies. Exemplary non-immune pairs are
biotin-streptavidin, intrinsic factor-vitamin B.sub.12, folic
acid-folate binding protein and the like.
[0124] A variety of methods are available to covalently label
antibodies with members of specific binding pairs. Methods are
selected based upon the nature of the member of the specific
binding pair, the type of linkage desired, and the tolerance of the
antibody to various conjugation chemistries. Biotin can be
covalently coupled to antibodies by utilizing commercially
available active derivatives. Some of these are
biotin-N-hydroxy-succinimide which binds to amine groups on
proteins; biotin hydrazide which binds to carbohydrate moieties,
aldehydes and carboxyl groups via a carbodiimide coupling; and
biotin maleimide and iodoacetyl biotin which bind to sulfhydryl
groups. Fluorescein can be coupled to protein amine groups using
fluorescein isothiocyanate. Dinitrophenyl groups can be coupled to
protein amine groups using 2,4-dinitrobenzene sulfate or
2,4-dinitrofluorobenzene. Other standard methods of conjugation can
be employed to couple monoclonal antibodies to a member of a
specific binding pair including dialdehyde, carbodiimide coupling,
homofunctional crosslinking, and heterobifunctional crosslinking.
Carbodiimide coupling is an effective method of coupling carboxyl
groups on one substance to amine groups on another. Carbodiimide
coupling is facilitated by using the commercially available reagent
1-ethyl-3-(dimethyl-aminopropyl)-carbodiimide (EDAC).
[0125] Homobifunctional crosslinkers, including the bifunctional
imidoesters and bifunctional N-hydroxysuccinimide esters, are
commercially available and are employed for coupling amine groups
on one substance to amine groups on another. Heterobifunctional
crosslinkers are reagents which possess different functional
groups. The most common commercially available heterobifunctional
crosslinkers have an amine reactive N-hydroxysuccinimide ester as
one functional group, and a sulfhydryl reactive group as the second
functional group. The most common sulfhydryl reactive groups are
maleimides, pyridyl disulfides and active halogens. One of the
functional groups can be a photoactive aryl nitrene, which upon
irradiation reacts with a variety of groups.
[0126] The detectably-labeled antibody or detectably-labeled member
of the specific binding pair is prepared by coupling to a reporter,
which can be a radioactive isotope, enzyme, fluorogenic,
chemiluminescent or electrochemical materials. Two commonly used
radioactive isotopes are .sup.125I and .sup.3H. Standard
radioactive isotopic labeling procedures include the chloramine T,
lactoperoxidase and Bolton-Hunter methods for .sup.125I and
reductive methylation for .sup.3H. The term "detectably-labeled"
refers to a molecule labeled in such a way that it can be readily
detected by the intrinsic enzymic activity of the label or by the
binding to the label of another component, which can itself be
readily detected.
[0127] Enzymes suitable for use in this invention include, but are
not limited to, horseradish peroxidase, alkaline phosphatase,
.beta.-galactosidase, glucose oxidase, luciferases, including
firefly and renilla, .beta.-lactamase, urease, green fluorescent
protein (GFP) and lysozyme. Enzyme labeling is facilitated by using
dialdehyde, carbodiimide coupling, homobifunctional crosslinkers
and heterobifunctional crosslinkers as described above for coupling
an antibody with a member of a specific binding pair.
[0128] The labeling method chosen depends on the functional groups
available on the enzyme and the material to be labeled, and the
tolerance of both to the conjugation conditions. The labeling
method used in the present invention can be one of, but not limited
to, any conventional methods currently employed including those
described by Engvall and Pearlmann, Immunochemistry 8, 871 (1971),
Avrameas and Ternynck, Immunochemistry 8, 1175 (1975), Ishikawa et
al., J. Immunoassay 4(3):209-327 (1983) and Jablonski, Anal.
Biochem. 148:199 (1985).
[0129] Labeling can be accomplished by indirect methods such as
using spacers or other members of specific binding pairs. An
example of this is the detection of a biotinylated antibody with
unlabeled streptavidin and biotinylated enzyme, with streptavidin
and biotinylated enzyme being added either sequentially or
simultaneously. Thus, according to the present invention, the
antibody used to detect can be detectably-labeled directly with a
reporter or indirectly with a first member of a specific binding
pair. When the antibody is coupled to a first member of a specific
binding pair, then detection is effected by reacting the
antibody-first member of a specific binding complex with the second
member of the binding pair that is labeled or unlabeled as
mentioned above.
[0130] Moreover, the unlabeled detector antibody can be detected by
reacting the unlabeled antibody with a labeled antibody specific
for the unlabeled antibody. In this instance "detectably-labeled"
as used above is taken to mean containing an epitope by which an
antibody specific for the unlabeled antibody can bind. Such an
anti-antibody can be labeled directly or indirectly using any of
the approaches discussed above. For example, the anti-antibody can
be coupled to biotin which is detected by reacting with the
streptavidin-horseradish peroxidase system discussed above.
[0131] In one embodiment of this invention biotin is utilized. The
biotinylated antibody is in turn reacted with
streptavidin-horseradish peroxidase complex. Orthophenylenediamine,
4-chloro-naphthol, tetramethylbenzidine (TMB), ABTS, BTS or ASA can
be used to effect chromogenic detection.
[0132] In one immunoassay format for practicing this invention, a
forward sandwich assay is used in which the capture reagent has
been immobilized, using conventional techniques, on the surface of
a support. Suitable supports used in assays include synthetic
polymer supports, such as polypropylene, polystyrene, substituted
polystyrene, e.g. aminated or carboxylated polystyrene,
polyacrylamides, polyamides, polyvinylchloride, glass beads,
agarose, or nitrocellulose.
[0133] The invention also encompasses kits for detecting the
presence of a biomarker protein or nucleic acid in a biological
sample. Such kits can be used to determine if a subject is
suffering from or is at increased risk of developing a tumor that
is less susceptible to inhibition by EGFR kinase inhibitors. For
example, the kit can comprise a labeled compound or agent capable
of detecting a biomarker protein or nucleic acid in a biological
sample and means for determining the amount of the protein or mRNA
in the sample (e.g., an antibody which binds the protein or a
fragment thereof, or an oligonucleotide probe which binds to DNA or
mRNA encoding the protein). Kits can also include instructions for
interpreting the results obtained using the kit.
[0134] For antibody-based kits, the kit can comprise, for example:
(1) a first antibody (e.g., attached to a solid support) which
binds to a biomarker protein; and, optionally, (2) a second,
different antibody which binds to either the protein or the first
antibody and is conjugated to a detectable label.
[0135] For oligonucleotide-based kits, the kit can comprise, for
example: (1) an oligonucleotide, e.g., a detectably labeled
oligonucleotide, which hybridizes to a nucleic acid sequence
encoding a biomarker protein or (2) a pair of primers useful for
amplifying a biomarker nucleic acid molecule. The kit can also
comprise, e.g., a buffering agent, a preservative, or a protein
stabilizing agent. The kit can further comprise components
necessary for detecting the detectable label (e.g., an enzyme or a
substrate). The kit can also contain a control sample or a series
of control samples which can be assayed and compared to the test
sample. Each component of the kit can be enclosed within an
individual container and all of the various containers can be
within a single package, along with instructions for interpreting
the results of the assays performed using the kit.
[0136] The present invention further provides a method for treating
tumors or tumor metastases in a patient, comprising the steps of
diagnosing a patient's likely responsiveness to an EGFR kinase
inhibitor by assessing whether the tumor cells have undergone an
epithelial-mesenchymal transition, by for example any of the
methods described herein for determining the expression level of
tumor cell epithelial and/or mesenchymal biomarkers, and
administering to said patient a therapeutically effective amount of
an EGFR kinase inhibitor. For this method, an example of a
preferred EGFR kinase inhibitor would be erlotinib, including
pharmacologically acceptable salts or polymorphs thereof. In this
method one or more additional anti-cancer agents or treatments can
be co-administered simultaneously or sequentially with the EGFR
kinase inhibitor, as judged to be appropriate by the administering
physician given the prediction of the likely responsiveness of the
patient to an EGFR kinase inhibitor, in combination with any
additional circumstances pertaining to the individual patient.
[0137] It will be appreciated by one of skill in the medical arts
that the exact manner of administering to said patient of a
therapeutically effective amount of an EGFR kinase inhibitor
following a diagnosis of a patient's likely responsiveness to an
EGFR kinase inhibitor will be at the discretion of the attending
physician. The mode of administration, including dosage,
combination with other anti-cancer agents, timing and frequency of
administration, and the like, may be affected by the diagnosis of a
patient's likely responsiveness to an EGFR kinase inhibitor, as
well as the patient's condition and history. Thus, even patients
diagnosed with tumors predicted to be relatively insensitive to
EGFR kinase inhibitors may still benefit from treatment with such
inhibitors, particularly in combination with other anti-cancer
agents, or agents that may alter a tumor's sensitivity to EGFR
kinase inhibitors.
[0138] The present invention further provides a method for treating
tumors or tumor metastases in a patient, comprising the steps of
diagnosing a patient's likely responsiveness to an EGFR kinase
inhibitor by assessing whether the tumor cells have undergone an
epithelial-mesenchymal transition, by for example any of the
methods described herein for determining the expression level of
tumor cell epithelial and/or mesenchymal biomarkers, identifying
the patient as one who is likely to demonstrate an effective
response to treatment with an EGFR kinase inhibitor, and
administering to said patient a therapeutically effective amount of
an EGFR kinase inhibitor.
[0139] The present invention further provides a method for treating
tumors or tumor metastases in a patient, comprising the steps of
diagnosing a patient's likely responsiveness to an EGFR kinase
inhibitor by assessing whether the tumor cells have undergone an
epithelial-mesenchymal transition, by for example any of the
methods described herein for determining the expression level of
tumor cell epithelial and/or mesenchymal biomarkers, identifying
the patient as one who is less likely or not likely to demonstrate
an effective response to treatment with an EGFR kinase inhibitor,
and treating said patient with an anti-cancer therapy other than an
EGFR kinase inhibitor.
[0140] The present invention further provides a method of
identifying an epithelial biomarker whose expression level is
predictive of the sensitivity of tumor cell growth to inhibition by
an EGFR kinase inhibitor, comprising: (a) measuring the expression
level of a candidate epithelial biomarker in a panel of tumor cells
that displays a range of sensitivities to an EGFR kinase inhibitor,
and (b) identifying a correlation between the expression level of
said candidate epithelial biomarker in the tumor cells and the
sensitivity of tumor cell growth to inhibition by the EGFR kinase
inhibitor, wherein a correlation of high levels of the epithelial
biomarker with high sensitivity of tumor cell growth to inhibition
by the EGFR kinase inhibitor indicates that the expression level of
said epithelial biomarker is predictive of the sensitivity of tumor
cell growth to inhibition by an EGFR kinase inhibitor. In one
embodiment of this method the panel of tumor cells is a panel of
tumor cell lines. In an alternative embodiment the panel of tumor
cells is a panel of primary tumor cells, prepared from tumor
samples derived from patients or experimental animal models. In an
additional embodiment the panel of tumor cells is a panel of tumor
cell lines in mouse xenografts, wherein tumor cell growth can for
example be determined by monitoring a molecular marker of growth or
a gross measurement of tumor growth, e.g. tumor dimensions or
weight.
[0141] The present invention further provides a method of
identifying a mesenchymal biomarker whose expression level is
predictive of the sensitivity of tumor cell growth to inhibition by
an EGFR kinase inhibitor, comprising: (a) measuring the expression
level of a candidate mesenchymal biomarker in a panel of tumor
cells that displays a range of sensitivities to an EGFR kinase
inhibitor, and (b) identifying a correlation between the expression
level of said candidate mesenchymal biomarker in the tumor cells
and the sensitivity of tumor cell growth to inhibition by the EGFR
kinase inhibitor, wherein a correlation of high levels of the
mesenchymal biomarker with low sensitivity of tumor cell growth to
inhibition by the EGFR kinase inhibitor indicates that the
expression level of said mesenchymal biomarker is predictive of the
lack of sensitivity of tumor cell growth to inhibition by an EGFR
kinase inhibitor. In one embodiment of this method the panel of
tumor cells is a panel of tumor cell lines. In an alternative
embodiment the panel of tumor cells is a panel of primary tumor
cells, prepared from tumor samples derived from patients or
experimental animal models. In an additional embodiment the panel
of tumor cells is a panel of tumor cell lines in mouse xenografts,
wherein tumor cell growth can for example be determined by
monitoring a molecular marker of growth or a gross measurement of
tumor growth, e.g. tumor dimensions or weight.
[0142] The present invention further provides a method of
identifying an epithelial biomarker that is diagnostic for more
effective treatment of a neoplastic condition with an EGFR kinase
inhibitor, comprising: (a) measuring the level of a candidate
epithelial biomarker in neoplastic cell-containing samples from
patients with a neoplastic condition, and (b) identifying a
correlation between the level of said candidate epithelial
biomarker in the sample from the patient with the effectiveness of
treatment of the neoplastic condition with an EGFR kinase
inhibitor, wherein a correlation of high levels of the epithelial
biomarker with more effective treatment of the neoplastic condition
with an EGFR kinase inhibitor indicates that said epithelial
biomarker is diagnostic for more effective treatment of the
neoplastic condition with an EGFR kinase inhibitor.
[0143] The present invention further provides a method of
identifying a mesenchymal biomarker that is diagnostic for less
effective treatment of a neoplastic condition with an EGFR kinase
inhibitor, comprising: (a) measuring the level of a candidate
mesenchymal biomarker in neoplastic cell-containing samples from
patients with a neoplastic condition, and (b) identifying a
correlation between the level of said candidate mesenchymal
biomarker in the sample from the patient with the effectiveness of
treatment of the neoplastic condition with an EGFR kinase
inhibitor, wherein a correlation of high levels of the mesenchymal
biomarker with less effective treatment of the neoplastic condition
with an EGFR kinase inhibitor indicates that said mesenchymal
biomarker is diagnostic for less effective treatment of the
neoplastic condition with an EGFR kinase inhibitor.
[0144] The effectiveness of treatment in the preceding methods can
for example be determined by measuring the decrease in size of
tumors present in the patients with the neoplastic condition, or by
assaying a molecular determinant of the degree of proliferation of
the tumor cells.
[0145] The present invention provides a method of identifying an
epithelial biomarker that is diagnostic for increased survival of a
patient with a neoplastic condition when treated with an EGFR
kinase inhibitor, comprising: (a) measuring the level of the
candidate epithelial biomarker in neoplastic cell-containing
samples from patients with a neoplastic condition, and (b)
identifying a correlation between the level of said candidate
epithelial biomarker in the sample from the patient with the
survival of that patient when treated with an EGFR kinase
inhibitor, wherein the correlation of an epithelial biomarker with
survival in said patients indicates said epithelial biomarker is
diagnostic for increased survival of a patient with said neoplastic
condition when treated with an EGFR kinase inhibitor.
[0146] The present invention provides a method of identifying a
mesenchymal biomarker that is diagnostic for decreased survival of
a patient with a neoplastic condition when treated with an EGFR
kinase inhibitor, comprising: (a) measuring the level of the
candidate mesenchymal biomarker in neoplastic cell-containing
samples from patients with a neoplastic condition, and (b)
identifying an inverse correlation between the level of said
candidate mesenchymal biomarker in the sample from the patient with
the survival of that patient when treated with an EGFR kinase
inhibitor, wherein the inverse correlation of a mesenchymal
biomarker with survival in said patients indicates said mesenchymal
biomarker is diagnostic for decreased survival of a patient with
said neoplastic condition when treated with an EGFR kinase
inhibitor.
[0147] The present invention provides a method for the
identification of an agent that enhances sensitivity of the growth
of a tumor cell to an EGFR kinase inhibitor, said tumor cell having
being characterized as one that has previously undergone an
epithelial-mesenchymal transition, comprising contacting a sample
of said tumor cells with an EGFR kinase inhibitor, contacting an
identical sample of said tumor cells with an EGFR kinase inhibitor
in the presence of a test agent, comparing the EGFR kinase
inhibitor-mediated growth inhibition in the presence and absence of
the test agent, and determining whether the test agent is an agent
that enhances sensitivity of the growth of the tumor cell to an
EGFR kinase inhibitor. For this method, an example of a preferred
EGFR kinase inhibitor would be erlotinib, including
pharmacologically acceptable salts or polymorphs thereof. In one
embodiment of this method the sample of tumor cells can be cells in
vitro, such as a tumor cell line or a primary tumor cell culture.
In an alternative embodiment the sample of tumor cells can be cells
in vivo, such as tumor cells in a mouse xenograft. In the latter
embodiment, tumor cell growth can for example be determined by
monitoring a molecular marker of growth or a gross measurement of
tumor growth, e.g. tumor dimensions or weight.
[0148] Suitable test agents which can be tested in the preceding
method include combinatorial libraries, defined chemical entities,
peptide and peptide mimetics, oligonucleotides and natural product
libraries, such as display (e.g. phage display libraries) and
antibody products. Test agents may be used in an initial screen of,
for example, 10 substances per reaction, and the substances of
these batches which show inhibition or activation tested
individually. Test agents may be used at a concentration of from 1
nM to 1000 .mu.M, preferably from 1 .mu.M to 100 .mu.M, more
preferably from 1 .mu.M to 10 .mu.M.
[0149] Agents which enhances sensitivity of the growth of a tumor
cell to an EGFR kinase inhibitor which have been identified by the
preceding methods can be used in the treatment of patients with
cancers which are predicted to be less responsive to inhibition by
EGFR kinase inhibitors (including lung cancer, pancreatic cancer,
or any of the other cancer types described herein), and are an
additional embodiment of this invention. Thus the present invention
further provides a composition of matter comprising such an agent,
which may be formulated and administered by any of the methods
known in the art, including those described herein in relation to
EGFR kinase inhibitors. Such agents that enhances sensitivity of
the growth of a tumor cell to an EGFR kinase inhibitor may for
example be agents that induce a mesenchymal to epithelial
transition (MET), or that inhibit a specific cellular activity
responsible for reduced sensitivity to EGFR kinase inhibitors, or
induce a specific cellular activity that enhances sensitivity to
EGFR kinase inhibitors. Examples of suitable agents include
antagonists of EMT inducing agents, TGF-beta antagonists or
TGF-beta receptor antagonists (for example: anti-TGF-beta and
anti-TGF-beta receptor antibodies,
4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole
(SB 203580);
4-[4-(3,4-Methylenedioxyphenyl)-5-(2-pyridyl)-1H-imidazol-2-yl]-benzamide
(SB431542); and similarly or more active analogues or homologues of
such compounds), inhibitors of FAK, ILK, SRC, FYN or YES
protein-tyrosine kinases, and calpain inhibitors.
[0150] The present invention further provides a method of treating
tumors or tumor metastases in a patient, comprising administering
to the patient a therapeutically effective amount of an EGFR kinase
inhibitor and in addition, simultaneously or sequentially, one or
more antagonists of an EMT inducing agent. In a preferred
embodiment said tumor is first determined to have epithelial
phenotype by the presence one or more epithelial biomarkers. In a
particular embodiment, said EMT inducing agent is an anti-TGF-beta
antibody, an anti-TGF-beta receptor antibody,
4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole
(SB 203580); or
4-[4-(3,4-Methylenedioxyphenyl)-5-(2-pyridyl)-1H-imidazol-2-yl]-benzamide
(SB431542). In a particular embodiment, said EGFR antagonist is
erlotinib.
[0151] The present invention further provides the preceding methods
for treating tumors or tumor metastases in a patient, comprising
administering to the patient a therapeutically effective amount of
an EGFR kinase inhibitor and in addition, simultaneously or
sequentially, one or more other cytotoxic, chemotherapeutic or
anti-cancer agents, or compounds that enhance the effects of such
agents.
[0152] In the context of this invention, additional other
cytotoxic, chemotherapeutic or anti-cancer agents, or compounds
that enhance the effects of such agents, include, for example:
alkylating agents or agents with an alkylating action, such as
cyclophosphamide (CTX; e.g. CYTOXAN.RTM.), chlorambucil (CHL; e.g.
LEUKERAN.RTM.), cisplatin (CisP; e.g. PLATINOL.RTM.) busulfan (e.g.
MYLERAN.RTM.), melphalan, carmustine (BCNU), streptozotocin,
triethylenemelamine (TEM), mitomycin C, and the like;
anti-metabolites, such as methotrexate (MTX), etoposide (VP16; e.g.
VEPESID.RTM.), 6-mercaptopurine (6MP), 6-thiocguanine (6TG),
cytarabine (Ara-C), 5-fluorouracil (5-FU), capecitabine (e.g.
XELODA.RTM.), dacarbazine (DTIC), and the like; antibiotics, such
as actinomycin D, doxorubicin (DXR; e.g. ADRIAMYCIN.RTM.),
daunorubicin (daunomycin), bleomycin, mithramycin and the like;
alkaloids, such as vinca alkaloids such as vincristine (VCR),
vinblastine, and the like; and other antitumor agents, such as
paclitaxel (e.g. TAXOL.RTM.) and pactitaxel derivatives, the
cytostatic agents, glucocorticoids such as dexamethasone (DEX; e.g.
DECADRON.RTM.) and corticosteroids such as prednisone, nucleoside
enzyme inhibitors such as hydroxyurea, amino acid depleting enzymes
such as asparaginase, leucovorin and other folic acid derivatives,
and similar, diverse antitumor agents. The following agents may
also be used as additional agents: amifostine (e.g. ETHYOL.RTM.),
dactinomycin, mechlorethamine (nitrogen mustard), streptozocin,
cyclophosphamide, lomustine (CCNU), doxorubicin lipo (e.g.
DOXIL.RTM.), gemcitabine (e.g. GEMZAR.RTM.), daunorubicin lipo
(e.g. DAUNOXOME.RTM.), procarbazine, mitomycin, docetaxel (e.g.
TAXOTERE.RTM.), aldesleukin, carboplatin, oxaliplatin, cladribine,
camptothecin, CPT 11 (irinotecan), 10-hydroxy 7-ethyl-camptothecin
(SN38), floxuridine, fludarabine, ifosfamide, idarubicin, mesna,
interferon beta, interferon alpha, mitoxantrone, topotecan,
leuprolide, megestrol, melphalan, mercaptopurine, plicamycin,
mitotane, pegaspargase, pentostatin, pipobroman, plicamycin,
tamoxifen, teniposide, testolactone, thioguanine, thiotepa, uracil
mustard, vinorelbine, chlorambucil.
[0153] The present invention further provides the preceding methods
for treating tumors or tumor metastases in a patient, comprising
administering to the patient a therapeutically effective amount of
an EGFR kinase inhibitor and in addition, simultaneously or
sequentially, one or more anti-hormonal agents. As used herein, the
term "anti-hormonal agent" includes natural or synthetic organic or
peptidic compounds that act to regulate or inhibit hormone action
on tumors.
[0154] Antihormonal agents include, for example: steroid receptor
antagonists, anti-estrogens such as tamoxifen, raloxifene,
aromatase inhibiting 4(5)-imidazoles, other aromatase inhibitors,
42-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone,
and toremifene (e.g. FARESTON.RTM.); anti-androgens such as
flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and
pharmaceutically acceptable salts, acids or derivatives of any of
the above; agonists and/or antagonists of glycoprotein hormones
such as follicle stimulating hormone (FSH), thyroid stimulating
hormone (TSH), and luteinizing hormone (LH) and LHRH (leuteinizing
hormone-releasing hormone); the LHRH agonist goserelin acetate,
commercially available as ZOLADEX.RTM. (AstraZeneca); the LHRH
antagonist D-alaninamide
N-acetyl-3-(2-naphthalenyl)-D-alanyl-4-chloro-D-phenylalanyl-3-(3-pyridin-
yl)-D-alanyl-L-seryl-N6-(3-pyridinylcarbonyl)-L-lysyl-N6-(3-pyridinylcarbo-
nyl)-D-lysyl-L-leucyl-N6-(1-methylethyl)-L-lysyl-L-proline (e.g
ANTIDE.RTM., Ares-Serono); the LHRH antagonist ganirelix acetate;
the steroidal anti-androgens cyproterone acetate (CPA) and
megestrol acetate, commercially available as MEGACE.RTM.
(Bristol-Myers Oncology); the nonsteroidal anti-androgen flutamide
(2-methyl-N-[4,20-nitro-3-(trifluoromethyl)phenylpropanamide),
commercially available as EULEXIN.RTM. (Schering Corp.); the
non-steroidal anti-androgen nilutamide,
(5,5-dimethyl-3-[4-nitro-3-(trifluoromethyl-4'-nitrophenyl)-4,4-dimethyl--
imidazolidine-dione); and antagonists for other non-permissive
receptors, such as antagonists for RAR, RXR, TR, VDR, and the
like.
[0155] The use of the cytotoxic and other anticancer agents
described above in chemotherapeutic regimens is generally well
characterized in the cancer therapy arts, and their use herein
falls under the same considerations for monitoring tolerance and
effectiveness and for controlling administration routes and
dosages, with some adjustments. For example, the actual dosages of
the cytotoxic agents may vary depending upon the patient's cultured
cell response determined by using histoculture methods. Generally,
the dosage will be reduced compared to the amount used in the
absence of additional other agents.
[0156] Typical dosages of an effective cytotoxic agent can be in
the ranges recommended by the manufacturer, and where indicated by
in vitro responses or responses in animal models, can be reduced by
up to about one order of magnitude concentration or amount. Thus,
the actual dosage will depend upon the judgment of the physician,
the condition of the patient, and the effectiveness of the
therapeutic method based on the in vitro responsiveness of the
primary cultured malignant cells or histocultured tissue sample, or
the responses observed in the appropriate animal models.
[0157] The present invention further provides the preceding methods
for treating tumors or tumor metastases in a patient, comprising
administering to the patient a therapeutically effective amount of
an EGFR kinase inhibitor and in addition, simultaneously or
sequentially, one or more angiogenesis inhibitors.
[0158] Anti-angiogenic agents include, for example: VEGFR
inhibitors, such as SU-5416 and SU-6668 (Sugen Inc. of South San
Francisco, Calif., USA), or as described in, for example
International Application Nos. WO 99/24440, WO 99/62890, WO
95/21613, WO 99/61422, WO 98/50356, WO 99/10349, WO 97/32856, WO
97/22596, WO 98/54093, WO 98/02438, WO 99/16755, and WO 98/02437,
and U.S. Pat. Nos. 5,883,113, 5,886,020, 5,792,783, 5,834,504 and
6,235,764; VEGF inhibitors such as IM862 (Cytran Inc. of Kirkland,
Wash., USA); angiozyme, a synthetic ribozyme from Ribozyme
(Boulder, Colo.) and Chiron (Emeryville, Calif.); and antibodies to
VEGF, such as bevacizumab (e.g. AVASTIN.TM., Genentech, South San
Francisco, Calif.), a recombinant humanized antibody to VEGF;
integrin receptor antagonists and integrin antagonists, such as to
.alpha..sub.v.beta..sub.3, .alpha..sub.v.beta..sub.5 and
.alpha..sub.v.beta..sub.6 integrins, and subtypes thereof, e.g.
cilengitide (EMD 121974), or the anti-integrin antibodies, such as
for example .alpha..sub.v.beta..sub.3 specific humanized antibodies
(e.g. VITAXIN.RTM.); factors such as IFN-alpha (U.S. Pat. Nos.
41,530,901, 4,503,035, and 5,231,176); angiostatin and plasminogen
fragments (e.g. kringle 1-4, kringle 5, kringle 1-3 (O'Reilly, M.
S. et al. (1994) Cell 79:315-328; Cao et al. (1996) J. Biol. Chem.
271: 29461-29467; Cao et al. (1997) J. Biol. Chem.
272:22924-22928); endostatin (O'Reilly, M. S. et al. (1997) Cell
88:277; and International Patent Publication No. WO 97/15666);
thrombospondin (TSP-1; Frazier, (1991) Curr. Opin. Cell Biol.
3:792); platelet factor 4 (PF4); plasminogen activator/urokinase
inhibitors; urokinase receptor antagonists; heparinases; fumagillin
analogs such as TNP-4701; suramin and suramin analogs; angiostatic
steroids; bFGF antagonists; flk-1 and flt-1 antagonists;
anti-angiogenesis agents such as MMP-2 (matrix-metalloproteinase 2)
inhibitors and MMP-9 (matrix-metalloproteinase 9) inhibitors.
Examples of useful matrix metalloproteinase inhibitors are
described in International Patent Publication Nos. WO 96/33172, WO
96/27583, WO 98/07697, WO 98/03516, WO 98/34918, WO 98/34915, WO
98/33768, WO 98/30566, WO 90/05719, WO 99/52910, WO 99/52889, WO
99/29667, and WO 99/07675, European Patent Publication Nos.
818,442, 780,386, 1,004,578, 606,046, and 931,788; Great Britain
Patent Publication No. 9912961, and U.S. Pat. Nos. 5,863,949 and
5,861,510. Preferred MMP-2 and MMP-9 inhibitors are those that have
little or no activity inhibiting MMP-1. More preferred, are those
that selectively inhibit MMP-2 and/or MMP-9 relative to the other
matrix-metalloproteinases (i.e. MMP-1, MMP-3, MMP-4, MMP-5, MMP-6,
MMP-7, MMP-8, MMP-10, MMP-11, MMP-12, and MMP-13).
[0159] The present invention further provides the preceding methods
for treating tumors or tumor metastases in a patient, comprising
administering to the patient a therapeutically effective amount of
an EGFR kinase inhibitor and in addition, simultaneously or
sequentially, one or more tumor cell pro-apoptotic or
apoptosis-stimulating agents.
[0160] The present invention further provides the preceding methods
for treating tumors or tumor metastases in a patient, comprising
administering to the patient a therapeutically effective amount of
an EGFR kinase inhibitor and in addition, simultaneously or
sequentially, one or more signal transduction inhibitors.
[0161] Signal transduction inhibitors include, for example: erbB2
receptor inhibitors, such as organic molecules, or antibodies that
bind to the erbB2 receptor, for example, trastuzumab (e.g.
HERCEPTIN.RTM.); inhibitors of other protein tyrosine-kinases, e.g.
imitinib (e.g. GLEEVEC.RTM.); ras inhibitors; raf inhibitors (e.g.
BAY 43-9006, Onyx Pharmaceuticals/Bayer Pharmaceuticals); MEK
inhibitors; mTOR inhibitors; cyclin dependent kinase inhibitors;
protein kinase C inhibitors; and PDK-1 inhibitors (see Dancey, J.
and Sausville, E. A. (2003) Nature Rev. Drug Discovery 2:92-313,
for a description of several examples of such inhibitors, and their
use in clinical trials for the treatment of cancer).
[0162] ErbB2 receptor inhibitors include, for example: ErbB2
receptor inhibitors, such as GW-282974 (Glaxo Wellcome plc),
monoclonal antibodies such as AR-209 (Aronex Pharmaceuticals Inc.
of The Woodlands, Tex., USA) and 2B-1 (Chiron), and erbB2
inhibitors such as those described in International Publication
Nos. WO 98/02434, WO 99/35146, WO 99/35132, WO 98/02437, WO
97/13760, and WO 95/19970, and U.S. Pat. Nos. 5,587,458, 5,877,305,
6,465,449 and 6,541,481.
[0163] The present invention further provides the preceding methods
for treating tumors or tumor metastases in a patient, comprising
administering to the patient a therapeutically effective amount of
an EGFR kinase inhibitor and in addition, simultaneously or
sequentially, an anti-HER2 antibody or an immunotherapeutically
active fragment thereof.
[0164] The present invention further provides the preceding methods
for treating tumors or tumor metastases in a patient, comprising
administering to the patient a therapeutically effective amount of
an EGFR kinase inhibitor and in addition, simultaneously or
sequentially, one or more additional anti-proliferative agents.
[0165] Additional antiproliferative agents include, for example:
Inhibitors of the enzyme farnesyl protein transferase and
inhibitors of the receptor tyrosine kinase PDGFR, including the
compounds disclosed and claimed in U.S. Pat. Nos. 6,080,769,
6,194,438, 6,258,824, 6,586,447, 6,071,935, 6,495,564, 6,150,377,
6,596,735 and 6,479,513, and International Patent Publication WO
01/40217.
[0166] The present invention further provides the preceding methods
for treating tumors or tumor metastases in a patient, comprising
administering to the patient a therapeutically effective amount of
an EGFR kinase inhibitor and in addition, simultaneously or
sequentially, a COX II (cyclooxygenase II) inhibitor. Examples of
useful COX-II inhibitors include alecoxib (e.g. CELEBREX.TM.),
valdecoxib, and rofecoxib.
[0167] The present invention further provides the preceding methods
for treating tumors or tumor metastases in a patient, comprising
administering to the patient a therapeutically effective amount of
an EGFR kinase inhibitor and in addition, simultaneously or
sequentially, treatment with radiation or a
radiopharmaceutical.
[0168] The source of radiation can be either external or internal
to the patient being treated. When the source is external to the
patient, the therapy is known as external beam radiation therapy
(EBRT). When the source of radiation is internal to the patient,
the treatment is called brachytherapy (BT). Radioactive atoms for
use in the context of this invention can be selected from the group
including, but not limited to, radium, cesium-137, iridium-192,
americium-241, gold-198, cobalt-57, copper-67, technetium-99,
iodine-123, iodine-131, and indium-111. Where the EGFR kinase
inhibitor according to this invention is an antibody, it is also
possible to label the antibody with such radioactive isotopes.
[0169] Radiation therapy is a standard treatment for controlling
unresectable or inoperable tumors and/or tumor metastases. Improved
results have been seen when radiation therapy has been combined
with chemotherapy. Radiation therapy is based on the principle that
high-dose radiation delivered to a target area will result in the
death of reproductive cells in both tumor and normal tissues. The
radiation dosage regimen is generally defined in terms of radiation
absorbed dose (Gy), time and fractionation, and must be carefully
defined by the oncologist. The amount of radiation a patient
receives will depend on various considerations, but the two most
important are the location of the tumor in relation to other
critical structures or organs of the body, and the extent to which
the tumor has spread. A typical course of treatment for a patient
undergoing radiation therapy will be a treatment schedule over a 1
to 6 week period, with a total dose of between 10 and 80 Gy
administered to the patient in a single daily fraction of about 1.8
to 2.0 Gy, 5 days a week. In a preferred embodiment of this
invention there is synergy when tumors in human patients are
treated with the combination treatment of the invention and
radiation. In other words, the inhibition of tumor growth by means
of the agents comprising the combination of the invention is
enhanced when combined with radiation, optionally with additional
chemotherapeutic or anticancer agents. Parameters of adjuvant
radiation therapies are, for example, contained in International
Patent Publication WO 99/60023.
[0170] The present invention further provides the preceding methods
for treating tumors or tumor metastases in a patient, comprising
administering to the patient a therapeutically effective amount of
an EGFR kinase inhibitor and in addition, simultaneously or
sequentially, treatment with one or more agents capable of
enhancing antitumor immune responses.
[0171] Agents capable of enhancing antitumor immune responses
include, for example: CTLA4 (cytotoxic lymphocyte antigen 4)
antibodies (e.g. MDX-CTLA4), and other agents capable of blocking
CTLA4. Specific CTLA4 antibodies that can be used in the present
invention include those described in U.S. Pat. No. 6,682,736.
[0172] In the context of this invention, an "effective amount" of
an agent or therapy is as defined above. A "sub-therapeutic amount"
of an agent or therapy is an amount less than the effective amount
for that agent or therapy, but when combined with an effective or
sub-therapeutic amount of another agent or therapy can produce a
result desired by the physician, due to, for example, synergy in
the resulting efficacious effects, or reduced side effects.
[0173] As used herein, the term "patient" preferably refers to a
human in need of treatment with an EGFR kinase inhibitor for any
purpose, and more preferably a human in need of such a treatment to
treat cancer, or a precancerous condition or lesion. However, the
term "patient" can also refer to non-human animals, preferably
mammals such as dogs, cats, horses, cows, pigs, sheep and non-human
primates, among others, that are in need of treatment with an EGFR
kinase inhibitor.
[0174] In a preferred embodiment, the patient is a human in need of
treatment for cancer, a precancerous condition or lesion, or other
forms of abnormal cell growth. The cancer is preferably any cancer
treatable, either partially or completely, by administration of an
EGFR kinase inhibitor. The cancer may be, for example, lung cancer,
non small cell lung (NSCL) cancer, bronchioloalviolar cell lung
cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the
head or neck, cutaneous or intraocular melanoma, uterine cancer,
ovarian cancer, rectal cancer, cancer of the anal region, stomach
cancer, gastric cancer, colon cancer, breast cancer, uterine
cancer, carcinoma of the fallopian tubes, carcinoma of the
endometrium, carcinoma of the cervix, carcinoma of the vagina,
carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus,
cancer of the small intestine, cancer of the endocrine system,
cancer of the thyroid gland, cancer of the parathyroid gland,
cancer of the adrenal gland, sarcoma of soft tissue, cancer of the
urethra, cancer of the penis, prostate cancer, cancer of the
bladder, cancer of the kidney or ureter, renal cell carcinoma,
carcinoma of the renal pelvis, mesothelioma, hepatocellular cancer,
biliary cancer, chronic or acute leukemia, lymphocytic lymphomas,
neoplasms of the central nervous system (CNS), spinal axis tumors,
brain stem glioma, glioblastoma multiforme, astrocytomas,
schwannomas, ependymomas, medulloblastomas, meningiomas, squamous
cell carcinomas, pituitary adenomas, including refractory versions
of any of the above cancers, or a combination of one or more of the
above cancers. The precancerous condition or lesion includes, for
example, the group consisting of oral leukoplakia, actinic
keratosis (solar keratosis), precancerous polyps of the colon or
rectum, gastric epithelial dysplasia, adenomatous dysplasia,
hereditary nonpolyposis colon cancer syndrome (HNPCC), Barrett's
esophagus, bladder dysplasia, and precancerous cervical
conditions.
[0175] For purposes of the present invention, "co-administration
of" and "co-administering" an EGFR kinase inhibitor with an
additional anti-cancer agent (both components referred to
hereinafter as the "two active agents") refer to any administration
of the two active agents, either separately or together, where the
two active agents are administered as part of an appropriate dose
regimen designed to obtain the benefit of the combination therapy.
Thus, the two active agents can be administered either as part of
the same pharmaceutical composition or in separate pharmaceutical
compositions. The additional agent can be administered prior to, at
the same time as, or subsequent to administration of the EGFR
kinase inhibitor, or in some combination thereof. Where the EGFR
kinase inhibitor is administered to the patient at repeated
intervals, e.g., during a standard course of treatment, the
additional agent can be administered prior to, at the same time as,
or subsequent to, each administration of the EGFR kinase inhibitor,
or some combination thereof, or at different intervals in relation
to the EGFR kinase inhibitor treatment, or in a single dose prior
to, at any time during, or subsequent to the course of treatment
with the EGFR kinase inhibitor.
[0176] The EGFR kinase inhibitor will typically be administered to
the patient in a dose regimen that provides for the most effective
treatment of the cancer (from both efficacy and safety
perspectives) for which the patient is being treated, as known in
the art, and as disclosed, e.g. in International Patent Publication
No. WO 01734574. In conducting the treatment method of the present
invention, the EGFR kinase inhibitor can be administered in any
effective manner known in the art, such as by oral, topical,
intravenous, intra-peritoneal, intramuscular, intra-articular,
subcutaneous, intranasal, intra-ocular, vaginal, rectal, or
intradermal routes, depending upon the type of cancer being
treated, the type of EGFR kinase inhibitor being used (for example,
small molecule, antibody, RNAi, ribozyme or antisense construct),
and the medical judgement of the prescribing physician as based,
e.g., on the results of published clinical studies.
[0177] The amount of EGFR kinase inhibitor administered and the
timing of EGFR kinase inhibitor administration will depend on the
type (species, gender, age, weight, etc.) and condition of the
patient being treated, the severity of the disease or condition
being treated, and on the route of administration. For example,
small molecule EGFR kinase inhibitors can be administered to a
patient in doses ranging from 0.001 to 100 mg/kg of body weight per
day or per week in single or divided doses, or by continuous
infusion (see for example, International Patent Publication No. WO
01/34574). In particular, erlotinib HCl can be administered to a
patient in doses ranging from 5-200 mg per day, or 100-1600 mg per
week, in single or divided doses, or by continuous infusion. A
preferred dose is 150 mg/day. Antibody-based EGFR kinase
inhibitors, or antisense, RNAi or ribozyme constructs, can be
administered to a patient in doses ranging from 0.1 to 100 mg/kg of
body weight per day or per week in single or divided doses, or by
continuous infusion. In some instances, dosage levels below the
lower limit of the aforesaid range may be more than adequate, while
in other cases still larger doses may be employed without causing
any harmful side effect, provided that such larger doses are first
divided into several small doses for administration throughout the
day.
[0178] The EGFR kinase inhibitors and other additional agents can
be administered either separately or together by the same or
different routes, and in a wide variety of different dosage forms.
For example, the EGFR kinase inhibitor is preferably administered
orally or parenterally. Where the EGFR kinase inhibitor is
erlotinib HCl (TARCEVATm), oral administration is preferable. Both
the EGFR kinase inhibitor and other additional agents can be
administered in single or multiple doses.
[0179] The EGFR kinase inhibitor can be administered with various
pharmaceutically acceptable inert carriers in the form of tablets,
capsules, lozenges, troches, hard candies, powders, sprays, creams,
salves, suppositories, jellies, gels, pastes, lotions, ointments,
elixirs, syrups, and the like. Administration of such dosage forms
can be carried out in single or multiple doses. Carriers include
solid diluents or fillers, sterile aqueous media and various
non-toxic organic solvents, etc. Oral pharmaceutical compositions
can be suitably sweetened and/or flavored.
[0180] The EGFR kinase inhibitor can be combined together with
various pharmaceutically acceptable inert carriers in the form of
sprays, creams, salves, suppositories, jellies, gels, pastes,
lotions, ointments, and the like. Administration of such dosage
forms can be carried out in single or multiple doses. Carriers
include solid diluents or fillers, sterile aqueous media, and
various non-toxic organic solvents, etc.
[0181] All formulations comprising proteinaceous EGFR kinase
inhibitors should be selected so as to avoid denaturation and/or
degradation and loss of biological activity of the inhibitor.
[0182] Methods of preparing pharmaceutical compositions comprising
an EGFR kinase inhibitor are known in the art, and are described,
e.g. in International Patent Publication No. WO 01/34574. In view
of the teaching of the present invention, methods of preparing
pharmaceutical compositions comprising an EGFR kinase inhibitor
will be apparent from the above-cited publications and from other
known references, such as Remington's Pharmaceutical Sciences, Mack
Publishing Company, Easton, Pa., 18.sup.th edition (1990).
[0183] For oral administration of EGFR kinase inhibitors, tablets
containing one or both of the active agents are combined with any
of various excipients such as, for example, micro-crystalline
cellulose, sodium citrate, calcium carbonate, dicalcium phosphate
and glycine, along with various disintegrants such as starch (and
preferably corn, potato or tapioca starch), alginic acid and
certain complex silicates, together with granulation binders like
polyvinyl pyrrolidone, sucrose, gelatin and acacia. Additionally,
lubricating agents such as magnesium stearate, sodium lauryl
sulfate and talc are often very useful for tableting purposes.
Solid compositions of a similar type may also be employed as
fillers in gelatin capsules; preferred materials in this connection
also include lactose or milk sugar as well as high molecular weight
polyethylene glycols. When aqueous suspensions and/or elixirs are
desired for oral administration, the EGFR kinase inhibitor may be
combined with various sweetening or flavoring agents, coloring
matter or dyes, and, if so desired, emulsifying and/or suspending
agents as well, together with such diluents as water, ethanol,
propylene glycol, glycerin and various like combinations
thereof.
[0184] For parenteral administration of either or both of the
active agents, solutions in either sesame or peanut oil or in
aqueous propylene glycol may be employed, as well as sterile
aqueous solutions comprising the active agent or a corresponding
water-soluble salt thereof. Such sterile aqueous solutions are
preferably suitably buffered, and are also preferably rendered
isotonic, e.g., with sufficient saline or glucose. These particular
aqueous solutions are especially suitable for intravenous,
intramuscular, subcutaneous and intraperitoneal injection purposes.
The oily solutions are suitable for intra-articular, intramuscular
and subcutaneous injection purposes. The preparation of all these
solutions under sterile conditions is readily accomplished by
standard pharmaceutical techniques well known to those skilled in
the art. Any parenteral formulation selected for administration of
proteinaceous EGFR kinase inhibitors should be selected so as to
avoid denaturation and loss of biological activity of the
inhibitor.
[0185] Additionally, it is possible to topically administer either
or both of the active agents, by way of, for example, creams,
lotions, jellies, gels, pastes, ointments, salves and the like, in
accordance with standard pharmaceutical practice. For example, a
topical formulation comprising an EGFR kinase inhibitor in about
0.1% (w/v) to about 5% (w/v) concentration can be prepared.
[0186] For veterinary purposes, the active agents can be
administered separately or together to animals using any of the
forms and by any of the routes described above. In a preferred
embodiment, the EGFR kinase inhibitor is administered in the form
of a capsule, bolus, tablet, liquid drench, by injection or as an
implant. As an alternative, the EGFR kinase inhibitor can be
administered with the animal feedstuff, and for this purpose a
concentrated feed additive or premix may be prepared for a normal
animal feed. Such formulations are prepared in a conventional
manner in accordance with standard veterinary practice.
[0187] As used herein, the term "EGFR kinase inhibitor" refers to
any EGFR kinase inhibitor that is currently known in the art or
that will be identified in the future, and includes any chemical
entity that, upon administration to a patient, results in
inhibition of a biological activity associated with activation of
the EGF receptor in the patient, including any of the downstream
biological effects otherwise resulting from the binding to EGFR of
its natural ligand. Such EGFR kinase inhibitors include any agent
that can block EGFR activation or any of the downstream biological
effects of EGFR activation that are relevant to treating cancer in
a patient. Such an inhibitor can act by binding directly to the
intracellular domain of the receptor and inhibiting its kinase
activity. Alternatively, such an inhibitor can act by occupying the
ligand binding site or a portion thereof of the EGF receptor,
thereby making the receptor inaccessible to its natural ligand so
that its normal biological activity is prevented or reduced.
Alternatively, such an inhibitor can act by modulating the
dimerization of EGFR polypeptides, or interaction of EGFR
polypeptide with other proteins, or enhance ubiquitination and
endocytotic degradation of EGFR. EGFR kinase inhibitors include but
are not limited to low molecular weight inhibitors, antibodies or
antibody fragments, antisense constructs, small inhibitory RNAs
(i.e. RNA interference by dsRNA; RNAi), and ribozymes. In a
preferred embodiment, the EGFR kinase inhibitor is a small organic
molecule or an antibody that binds specifically to the human
EGFR.
[0188] EGFR kinase inhibitors that include, for example quinazoline
EGFR kinase inhibitors, pyrido-pyrimidine EGFR kinase inhibitors,
pyrimido-pyrimidine EGFR kinase inhibitors, pyrrolo-pyrimidine EGFR
kinase inhibitors, pyrazolo-pyrimidine EGFR kinase inhibitors,
phenylamino-pyrimidine EGFR kinase inhibitors, oxindole EGFR kinase
inhibitors, indolocarbazole EGFR kinase inhibitors, phthalazine
EGFR kinase inhibitors, isoflavone EGFR kinase inhibitors,
quinalone EGFR kinase inhibitors, and tyrphostin EGFR kinase
inhibitors, such as those described in the following patent
publications, and all pharmaceutically acceptable salts and
solvates of said EGFR kinase inhibitors: International Patent
Publication Nos. WO 96/33980, WO 96/30347, WO 97/30034, WO
97/30044, WO 97/38994, WO 97/49688, WO 98/02434, WO 97/38983, WO
95/19774, WO 95/19970, WO 97/13771, WO 98/02437, WO 98/02438, WO
97/32881, WO 98/33798, WO 97/32880, WO 97/3288, WO 97/02266, WO
97/27199, WO 98/07726, WO 97/34895, WO 96/31510, WO 98/14449, WO
98/14450, WO 98/14451, WO 95/09847, WO 97/19065, WO 98/17662, WO
99/35146, WO 99/35132, WO 99/07701, and WO 92/20642; European
Patent Application Nos. EP 520722, EP 566226, EP 787772, EP 837063,
and EP 682027; U.S. Pat. Nos. 5,747,498, 5,789,427, 5,650,415, and
5,656,643; and German Patent Application No. DE 19629652.
Additional non-limiting examples of low molecular weight EGFR
kinase inhibitors include any of the EGFR kinase inhibitors
described in Traxler, P., 1998, Exp. Opin. Ther. Patents
8(12):1599-1625.
[0189] Specific preferred examples of low molecular weight EGFR
kinase inhibitors that can be used according to the present
invention include
[6,7-bis(2-methoxyethoxy)-4-quinazolin-4-yl]-(3-ethynylphenyl)amine
(also known as OSI-774, erlotinib, or TARCEVA.TM. (erlotinib HCl);
OSI Pharmaceuticals/Genentech/Roche) (U.S. Pat. No. 5,747,498;
International Patent Publication No. WO 01/34574, and Moyer, J. D.
et al. (1997) Cancer Res. 57:4838-4848); CI-1033 (formerly known as
PD183805; Pfizer) (Sherwood et al., 1999, Proc. Am. Assoc. Cancer
Res. 40:723); PD-158780 (Pfizer); AG-1478 (University of
California); CGP-59326 (Novartis); PKI-166 (Novartis); EKB-569
(Wyeth); GW-2016 (also known as GW-572016 or lapatinib ditosylate;
GSK); and gefitinib (also known as ZD1839 or IRESSA.TM.;
Astrazeneca) (Woodburn et al., 1997, Proc. Am. Assoc. Cancer Res.
38:633). A particularly preferred low molecular weight EGFR kinase
inhibitor that can be used according to the present invention is
[6,7-bis(2-methoxyethoxy)-4-quinazolin-4-yl]-(3-ethynylphenyl)amine
(i.e. erlotinib), its hydrochloride salt (i.e. erlotinib HCl,
TARCEVA.TM.), or other salt forms (e.g. erlotinib mesylate).
[0190] Antibody-based EGFR kinase inhibitors include any anti-EGFR
antibody or antibody fragment that can partially or completely
block EGFR activation by its natural ligand. Non-limiting examples
of antibody-based EGFR kinase inhibitors include those described in
Modjtahedi, H., et al., 1993, Br. J. Cancer 67:247-253; Teramoto,
T., et al., 1996, Cancer 77:639-645; Goldstein et al., 1995, Clin.
Cancer Res. 1:1311-1318; Huang, S. M., et al., 1999, Cancer Res.
15:59(8):1935-40; and Yang, X., et al., 1999, Cancer Res.
59:1236-1243. Thus, the EGFR kinase inhibitor can be the monoclonal
antibody Mab E7.6.3 (Yang, X. D. et al. (1999) Cancer Res.
59:1236-43), or Mab C225 (ATCC Accession No. HB-8508), or an
antibody or antibody fragment having the binding specificity
thereof. Suitable monoclonal antibody EGFR kinase inhibitors
include, but are not limited to, IMC-C225 (also known as cetuximab
or ERBITUX.TM.; Imclone Systems), ABX-EGF (Abgenix), EMD 72000
(Merck KgaA, Darmstadt), RH3 (York Medical Bioscience Inc.), and
MDX-447 (Medarex/Merck KgaA).
[0191] Additional antibody-based EGFR kinase inhibitors can be
raised according to known methods by administering the appropriate
antigen or epitope to a host animal selected, e.g., from pigs,
cows, horses, rabbits, goats, sheep, and mice, among others.
Various adjuvants known in the art can be used to enhance antibody
production.
[0192] Although antibodies useful in practicing the invention can
be polyclonal, monoclonal antibodies are preferred. Monoclonal
antibodies against EGFR can be prepared and isolated using any
technique that provides for the production of antibody molecules by
continuous cell lines in culture. Techniques for production and
isolation include but are not limited to the hybridoma technique
originally described by Kohler and Milstein (Nature, 1975, 256:
495-497); the human B-cell hybridoma technique (Kosbor et al.,
1983, Immunology Today 4:72; Cote et al., 1983, Proc. Nati. Acad.
Sci. USA 80: 2026-2030); and the EBV-hybridoma technique (Cole et
al, 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,
Inc., pp. 77-96).
[0193] Alternatively, techniques described for the production of
single chain antibodies (see, e.g., U.S. Pat. No. 4,946,778) can be
adapted to produce anti-EGFR single chain antibodies.
Antibody-based EGFR kinase inhibitors useful in practicing the
present invention also include anti-EGFR antibody fragments
including but not limited to F(ab').sub.2 fragments, which can be
generated by pepsin digestion of an intact antibody molecule, and
Fab fragments, which can be generated by reducing the disulfide
bridges of the F(ab').sub.2 fragments. Alternatively, Fab and/or
scFv expression libraries can be constructed (see, e.g., Huse et
al., 1989, Science 246: 1275-1281) to allow rapid identification of
fragments having the desired specificity to EGFR.
[0194] Techniques for the production and isolation of monoclonal
antibodies and antibody fragments are well-known in the art, and
are described in Harlow and Lane, 1988, Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory, and in J. W. Goding, 1986,
Monoclonal Antibodies: Principles and Practice, Academic Press,
London. Humanized anti-EGFR antibodies and antibody fragments can
also be prepared according to known techniques such as those
described in Vaughn, T. J. et al., 1998, Nature Biotech. 16:535-539
and references cited therein, and such antibodies or fragments
thereof are also useful in practicing the present invention.
[0195] EGFR kinase inhibitors for use in the present invention can
alternatively be based on antisense oligonucleotide constructs.
Anti-sense oligonucleotides, including anti-sense RNA molecules and
anti-sense DNA molecules, would act to directly block the
translation of EGFR mRNA by binding thereto and thus preventing
protein translation or increasing mRNA degradation, thus decreasing
the level of EGFR kinase protein, and thus activity, in a cell. For
example, antisense oligonucleotides of at least about 15 bases and
complementary to unique regions of the mRNA transcript sequence
encoding EGFR can be synthesized, e.g., by conventional
phosphodiester techniques and administered by e.g., intravenous
injection or infusion. Methods for using antisense techniques for
specifically inhibiting gene expression of genes whose sequence is
known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135;
6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and
5,981,732).
[0196] Small inhibitory RNAs (siRNAs) can also function as EGFR
kinase inhibitors for use in the present invention. EGFR gene
expression can be reduced by contacting the tumor, subject or cell
with a small double stranded RNA (dsRNA), or a vector or construct
causing the production of a small double stranded RNA, such that
expression of EGFR is specifically inhibited (i.e. RNA interference
or RNAi). Methods for selecting an appropriate dsRNA or
dsRNA-encoding vector are well known in the art for genes whose
sequence is known (e.g. see Tuschi, T., et al. (1999) Genes Dev.
13(24):3191-3197; Elbashir, S. M. et al. (2001) Nature 411:494-498;
Hannon, G. J. (2002) Nature 418:244-251; McManus, M. T. and Sharp,
P. A. (2002) Nature Reviews Genetics 3:737-747; Bremmelkamp, T. R.
et al. (2002) Science 296:550-553; U.S. Pat. Nos. 6,573,099 and
6,506,559; and International Patent Publication Nos. WO 01/36646,
WO 99/32619, and WO 01/68836).
[0197] Ribozymes can also function as EGFR kinase inhibitors for
use in the present invention. Ribozymes are enzymatic RNA molecules
capable of catalyzing the specific cleavage of RNA. The mechanism
of ribozyme action involves sequence specific hybridization of the
ribozyme molecule to complementary target RNA, followed by
endonucleolytic cleavage. Engineered hairpin or hammerhead motif
ribozyme molecules that specifically and efficiently catalyze
endonucleolytic cleavage of EGFR mRNA sequences are thereby useful
within the scope of the present invention. Specific ribozyme
cleavage sites within any potential RNA target are initially
identified by scanning the target molecule for ribozyme cleavage
sites, which typically include the following sequences, GUA, GUU,
and GUC. Once identified, short RNA sequences of between about 15
and 20 ribonucleotides corresponding to the region of the target
gene containing the cleavage site can be evaluated for predicted
structural features, such as secondary structure, that can render
the oligonucleotide sequence unsuitable. The suitability of
candidate targets can also be evaluated by testing their
accessibility to hybridization with complementary oligonucleotides,
using, e.g., ribonuclease protection assays.
[0198] Both antisense oligonucleotides and ribozymes useful as EGFR
kinase inhibitors can be prepared by known methods. These include
techniques for chemical synthesis such as, e.g., by solid phase
phosphoramadite chemical synthesis. Alternatively, anti-sense RNA
molecules can be generated by in vitro or in vivo transcription of
DNA sequences encoding the RNA molecule. Such DNA sequences can be
incorporated into a wide variety of vectors that incorporate
suitable RNA polymerase promoters such as the T7 or SP6 polymerase
promoters. Various modifications to the oligonucleotides of the
invention can be introduced as a means of increasing intracellular
stability and half-life. Possible modifications include but are not
limited to the addition of flanking sequences of ribonucleotides or
deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or
the use of phosphorothioate or 2'-O-methyl rather than
phosphodiesterase linkages within the oligonucleotide backbone.
[0199] In the context of the methods of treatment of this
invention, EGFR kinase inhibitors are used as a composition
comprised of a pharmaceutically acceptable carrier and a non-toxic
therapeutically effective amount of an EGFR kinase inhibitor
compound (including pharmaceutically acceptable salts thereof).
[0200] The term "pharmaceutically acceptable salts" refers to salts
prepared from pharmaceutically acceptable non-toxic bases or acids.
When a compound of the present invention is acidic, its
corresponding salt can be conveniently prepared from
pharmaceutically acceptable non-toxic bases, including inorganic
bases and organic bases. Salts derived from such inorganic bases
include aluminum, ammonium, calcium, copper (cupric and cuprous),
ferric, ferrous, lithium, magnesium, manganese (manganic and
manganous), potassium, sodium, zinc and the like salts.
Particularly preferred are the ammonium, calcium, magnesium,
potassium and sodium salts. Salts derived from pharmaceutically
acceptable organic non-toxic bases include salts of primary,
secondary, and tertiary amines, as well as cyclic amines and
substituted amines such as naturally occurring and synthesized
substituted amines. Other pharmaceutically acceptable organic
non-toxic bases from which salts can be formed include ion exchange
resins such as, for example, arginine, betaine, caffeine, choline,
N',N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol,
2-dimethylaminoethanol, ethanolamine, ethylenediamine,
N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine,
histidine, hydrabamine, isopropylamine, lysine, methylglucamine,
morpholine, piperazine, piperidine, polyamine resins, procaine,
purines, theobromine, triethylameine, trimethylamine,
tripropylamine, tromethamine and the like.
[0201] When a compound used in the present invention is basic, its
corresponding salt can be conveniently prepared from
pharmaceutically acceptable non-toxic acids, including inorganic
and organic acids. Such acids include, for example, acetic,
benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic,
fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic,
lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric,
pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric,
p-toluenesulfonic acid and the like. Particularly preferred are
citric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuric and
tartaric acids.
[0202] Pharmaceutical compositions used in the present invention
comprising an EGFR kinase inhibitor compound (including
pharmaceutically acceptable salts thereof) as active ingredient,
can include a pharmaceutically acceptable carrier and optionally
other therapeutic ingredients or adjuvants. Other therapeutic
agents may include those cytotoxic, chemotherapeutic or anti-cancer
agents, or agents which enhance the effects of such agents, as
listed above. The compositions include compositions suitable for
oral, rectal, topical, and parenteral (including subcutaneous,
intramuscular, and intravenous) administration, although the most
suitable route in any given case will depend on the particular
host, and nature and severity of the conditions for which the
active ingredient is being administered. The pharmaceutical
compositions may be conveniently presented in unit dosage form and
prepared by any of the methods well known in the art of
pharmacy.
[0203] In practice, the EGFR kinase inhibitor compounds (including
pharmaceutically acceptable salts thereof) of this invention can be
combined as the active ingredient in intimate admixture with a
pharmaceutical carrier according to conventional pharmaceutical
compounding techniques. The carrier may take a wide variety of
forms depending on the form of preparation desired for
administration, e.g. oral or parenteral (including intravenous).
Thus, the pharmaceutical compositions of the present invention can
be presented as discrete units suitable for oral administration
such as capsules, cachets or tablets each containing a
predetermined amount of the active ingredient. Further, the
compositions can be presented as a powder, as granules, as a
solution, as a suspension in an aqueous liquid, as a non-aqueous
liquid, as an oil-in-water emulsion, or as a water-in-oil liquid
emulsion. In addition to the common dosage forms set out above, an
EGFR kinase inhibitor compound (including pharmaceutically
acceptable salts of each component thereof) may also be
administered by controlled release means and/or delivery devices.
The combination compositions may be prepared by any of the methods
of pharmacy. In general, such methods include a step of bringing
into association the active ingredients with the carrier that
constitutes one or more necessary ingredients. In general, the
compositions are prepared by uniformly and intimately admixing the
active ingredient with liquid carriers or finely divided solid
carriers or both. The product can then be conveniently shaped into
the desired presentation.
[0204] An EGFR kinase inhibitor compound (including
pharmaceutically acceptable salts thereof) used in this invention,
can also be included in pharmaceutical compositions in combination
with one or more other therapeutically active compounds. Other
therapeutically active compounds may include those cytotoxic,
chemotherapeutic or anti-cancer agents, or agents which enhance the
effects of such agents, as listed above.
[0205] Thus in one embodiment of this invention, the pharmaceutical
composition can comprise an EGFR kinase inhibitor compound in
combination with an anticancer agent, wherein said anti-cancer
agent is a member selected from the group consisting of alkylating
drugs, antimetabolites, microtubule inhibitors, podophyllotoxins,
antibiotics, nitrosoureas, hormone therapies, kinase inhibitors,
activators of tumor cell apoptosis, and antiangiogenic agents.
[0206] The pharmaceutical carrier employed can be, for example, a
solid, liquid, or gas. Examples of solid carriers include lactose,
terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium
stearate, and stearic acid. Examples of liquid carriers are sugar
syrup, peanut oil, olive oil, and water. Examples of gaseous
carriers include carbon dioxide and nitrogen.
[0207] In preparing the compositions for oral dosage form, any
convenient pharmaceutical media may be employed. For example,
water, glycols, oils, alcohols, flavoring agents, preservatives,
coloring agents, and the like may be used to form oral liquid
preparations such as suspensions, elixirs and solutions; while
carriers such as starches, sugars, microcrystalline cellulose,
diluents, granulating agents, lubricants, binders, disintegrating
agents, and the like may be used to form oral solid preparations
such as powders, capsules and tablets. Because of their ease of
administration, tablets and capsules are the preferred oral dosage
units whereby solid pharmaceutical carriers are employed.
Optionally, tablets may be coated by standard aqueous or nonaqueous
techniques.
[0208] A tablet containing the composition used for this invention
may be prepared by compression or molding, optionally with one or
more accessory ingredients or adjuvants. Compressed tablets may be
prepared by compressing, in a suitable machine, the active
ingredient in a free-flowing form such as powder or granules,
optionally mixed with a binder, lubricant, inert diluent, surface
active or dispersing agent. Molded tablets may be made by molding
in a suitable machine, a mixture of the powdered compound moistened
with an inert liquid diluent. Each tablet preferably contains from
about 0.05 mg to about 5 g of the active ingredient and each cachet
or capsule preferably contains from about 0.05 mg to about 5 g of
the active ingredient.
[0209] For example, a formulation intended for the oral
administration to humans may contain from about 0.5 mg to about 5 g
of active agent, compounded with an appropriate and convenient
amount of carrier material that may vary from about 5 to about 95
percent of the total composition. Unit dosage forms will generally
contain between from about 1 mg to about 2 g of the active
ingredient, typically 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg,
500 mg, 600 mg, 800 mg, or 1000 mg.
[0210] Pharmaceutical compositions used in the present invention
suitable for parenteral administration may be prepared as solutions
or suspensions of the active compounds in water. A suitable
surfactant can be included such as, for example,
hydroxypropylcellulose. Dispersions can also be prepared in
glycerol, liquid polyethylene glycols, and mixtures thereof in
oils. Further, a preservative can be included to prevent the
detrimental growth of microorganisms.
[0211] Pharmaceutical compositions used in the present invention
suitable for injectable use include sterile aqueous solutions or
dispersions. Furthermore, the compositions can be in the form of
sterile powders for the extemporaneous preparation of such sterile
injectable solutions or dispersions. In all cases, the final
injectable form must be sterile and must be effectively fluid for
easy syringability. The pharmaceutical compositions must be stable
under the conditions of manufacture and storage; thus, preferably
should be preserved against the contaminating action of
microorganisms such as bacteria and fungi. The carrier can be a
solvent or dispersion medium containing, for example, water,
ethanol, polyol (e.g., glycerol, propylene glycol and liquid
polyethylene glycol), vegetable oils, and suitable mixtures
thereof.
[0212] Pharmaceutical compositions for the present invention can be
in a form suitable for topical sue such as, for example, an
aerosol, cream, ointment, lotion, dusting powder, or the like.
Further, the compositions can be in a form suitable for use in
transdermal devices. These formulations may be prepared, utilizing
an EGFR kinase inhibitor compound (including pharmaceutically
acceptable salts thereof), via conventional processing methods. As
an example, a cream or ointment is prepared by admixing hydrophilic
material and water, together with about 5 wt % to about 10 wt % of
the compound, to produce a cream or ointment having a desired
consistency.
[0213] Pharmaceutical compositions for this invention can be in a
form suitable for rectal administration wherein the carrier is a
solid. It is preferable that the mixture forms unit dose
suppositories. Suitable carriers include cocoa butter and other
materials commonly used in the art. The suppositories may be
conveniently formed by first admixing the composition with the
softened or melted carrier(s) followed by chilling and shaping in
molds.
[0214] In addition to the aforementioned carrier ingredients, the
pharmaceutical formulations described above may include, as
appropriate, one or more additional carrier ingredients such as
diluents, buffers, flavoring agents, binders, surface-active
agents, thickeners, lubricants, preservatives (including
anti-oxidants) and the like. Furthermore, other adjuvants can be
included to render the formulation isotonic with the blood of the
intended recipient. Compositions containing an EGFR kinase
inhibitor compound (including pharmaceutically acceptable salts
thereof) may also be prepared in powder or liquid concentrate
form.
[0215] Dosage levels for the compounds used for practicing this
invention will be approximately as described herein, or as
described in the art for these compounds. It is understood,
however, that the specific dose level for any particular patient
will depend upon a variety of factors including the age, body
weight, general health, sex, diet, time of administration, route of
administration, rate of excretion, drug combination and the
severity of the particular disease undergoing therapy.
[0216] Many alternative experimental methods known in the art may
be successfully substituted for those specifically described herein
in the practice of this invention, as for example described in many
of the excellent manuals and textbooks available in the areas of
technology relevant to this invention (e.g. Using Antibodies, A
Laboratory Manual, edited by Harlow, E. and Lane, D., 1999, Cold
Spring Harbor Laboratory Press, (e.g. ISBN 0-87969-544-7); Roe B.
A. et. al. 1996, DNA Isolation and Sequencing (Essential Techniques
Series), John Wiley & Sons. (e.g. ISBN 0-471-97324-0); Methods
in Enzymology: Chimeric Genes and Proteins", 2000, ed. J. Abelson,
M. Simon, S. Emr, J. Thorner. Academic Press; Molecular Cloning: a
Laboratory Manual, 2001, 3.sup.rd Edition, by Joseph Sambrook and
Peter MacCallum, (the former Maniatis Cloning manual) (e.g. ISBN
0-87969-577-3); Current Protocols in Molecular Biology, Ed. Fred M.
Ausubel, et. al. John Wiley & Sons (e.g. ISBN 0-471-50338-X);
Current Protocols in Protein Science, Ed. John E. Coligan, John
Wiley & Sons (e.g. ISBN 0-471-11184-8); and Methods in
Enzymology: Guide to protein Purification, 1990, Vol. 182, Ed.
Deutscher, M. P., Academic Press, Inc. (e.g. ISBN 0-12-213585-7)),
or as described in the many university and commercial websites
devoted to describing experimental methods in molecular
biology.
[0217] This invention will be better understood from the
Experimental Details that follow. However, one skilled in the art
will readily appreciate that the specific methods and results
discussed are merely illustrative of the invention as described
more fully in the claims which follow thereafter, and are not to be
considered in any way limited thereto.
[0218] Experimental Details:
[0219] Introduction
[0220] Inhibitors of EGF receptor function have shown clinical
utility and the definition of key EGF receptor signaling pathways
which describe patient subsets most likely to benefit from therapy
has become an important area of investigation. Mutations which
activate the receptor's intrinsic protein tyrosine kinase activity
and/or increase downstream signaling have been observed in NSCLC
and glioblastoma. However the role of mutations as a principle
mechanism in conferring sensitivity to EGF receptor inhibitors has
been controversial. In vitro and clinical studies have shown
considerable variability between wt EGF receptor cell lines and
tumors in their cellular responses to EGF receptor inhibition,
which in part has been shown to derive from EGF receptor
independent activation of the phosphatidyl inositol 3-kinase
pathway, leading to the continued phosphorylation of the
anti-apoptotic serine-threonine kinase Akt. The molecular
determinants to alternative routes of PI3-kinase activation and
consequent EGF receptor inhibitor insensitivity are an active area
of investigation. For example the insulin-like growth factor-1
receptor (IGF-1 receptor), which strongly activates the PI3-kinase
pathway, has been implicated in cellular resistance to EGF
inhibitors. The roles of cell-cell and cell-adhesion networks,
which can also exert survival signals through the PI3-kinase
pathway in mediating insensitivity to selective EGF receptor
inhibition are less clear and would be postulated to impact cell
sensitivity to EGF receptor blockade. The ability of tumor cells to
maintain growth and survival signals in the absence of adhesion to
extracellular matrix or cell-cell contacts is important not only in
the context of cell migration and metastasis but also in
maintaining cell proliferation and survival in wound-like tumor
environments where extracellular matrix is being remodeled and cell
contact inhibition is diminished. Here we demonstrate that
sensitivity of NSCLC and pancreatic cells to EGF receptor
inhibition is conferred by an E-cadherin epithelial cell phenotype
in which ErbB family member signaling was active. Conversely
insensitivity to EGF receptor inhibition was mediated through an
epithelial-mesenchymal transition (EMT) associated with the
expression of vimentin and/or fibronectin.
[0221] Materials and Methods
[0222] Cell Culture and Preparation of Cell Extracts
[0223] The NSCLC lines with wt EGFR, H292, H358, H322, H441, A549,
Calu6, H460, H1703 and SW1573 were cultured in the appropriate ATCC
recommended supplemented media. Cell extracts were prepared by
detergent lysis ((50 mM Tris-HCl, pH8, 150 mM NaCl, 1% NP-40, 0.5%
NaDeoxycholate, 0.1% SDS) containing protease and phosphatase
inhibitors. The soluble protein concentration was determined by
micro-BSA assay (Pierce, Rockford Ill.).
[0224] Protein Identification and Quantitation by LC-MS/NIS Peptide
Sequencing
[0225] Anti-phosphotyrosine immunoaffinity resins were prepared by
covalent coupling to a solid support by standard methods. Freshly
prepared immunoaffinity resins were used for each biological
experiment to maximize binding and reduce carryover. Briefly,
anti-phosphotyrosine antibodies were crosslinked to solid-support
and non-covalently bound IgG removed by low pH elution. Fresh
affinity resins were prepared for each biological experiment to
avoid cross-contamination. Proteins isolated by
anti-phosphotyrosine affinity selection were measured by iTRAQ
labeling of tryptic peptides as previously described (Ross et al,
2004; Haley et al., 2004). Peptide masses and sequence information
were determined by electrospray LC-MS/MS and database searching.
Peptides with confidence levels of >=90% with scores of >=20
were considered, after which spectra were inspected manually.
Peptide expression ratios were converted to log.sub.2 values and
averaged to yield a single protein expression value for each time
point (1, 4 and 24 hours) after erlotinib exposure (1 uM). Proteins
were clustered by temporal log.sub.2 protein expression ratios
using Euclidian hierarchical methods and self-organizing maps.
[0226] Immunoblot Analysis of NSCLC and Pancreatic Cell Line
Extracts
[0227] Protein immunodetection was performed by electrophoretic
transfer of SDS-PAGE separated proteins to nitrocellulose,
incubation with antibody and chemiluminescent second step detection
(PicoWest; Pierce, Rockford, Ill.). The antibodies included:
E-Cadherin (Santa Cruz Biotechnology, Santa Cruz, Calif.; sc21791),
.alpha.-catenin (sc9988), .beta.-catenin (sc7963), .gamma.-catenin
(sc8415) and Brk (sc1188); Vimentin (BD Biosciences, San Jose,
Calif.; BD550513) and Fibronectin (BD610077); GAPDH (AbCam,
Cambridge, UK); Phospho-Akt (Cell Signaling, Beverly, Mass. #9271),
Akt (CS, #9272), Phospho-p44/42 Map kinase.sup.T202/Y204 (Erk1/2;
CS #9101), Phospho-Src family.sup.Y416 (CS #2101),
Phospho-STAT3.sup.Y705 (CS, #9131) and Phospho-S6.sup.S235/236 (CS,
#2211); .beta.-actin (Sigma, Saint Louis, Mo. #A5441). Antibodies
further included: Phospho-Shc (Cell Signaling, #2434, Beverly,
Mass.), Phospho-Paxillin (Cell Signaling, #2541), Phospho-Akt
(Ser473 and Thr308) (Cell Signaling, #9271 and 9275),
Phospho-HER2/ErbB2 (Cell Signaling, #2245), Phospho-Her3 (Tyr1289)
(Cell Signaling #4791), Phospho-p44/42 Map kinase (Cell Signaling,
#9101), Phospho-EGFR (Tyr845) (Cell Signaling, #2231), Phospho-EGFR
(Tyr992) (Cell Signaling, #2235), Phospho-EGFR (Tyr1045) (Cell
Signaling, #2237), EGFR (Cell Signaling, #2232), Phospho-p70 S6
kinase (Cell Signaling, #9205), Phospho-GSK-3alpha/beta (Cell
Signaling, #9331), Phospho-EGFR (Tyr1068) (Cell Signaling, #2236),
Phospho-Src family (Tyr416) (Cell Signaling #2101),
phospho-SAPK/JNK (Thr183/Tyr185)(Cell Signaling #9251),
phospho-STAT3 (Tyr705) (Cell Signaling #9131), ErbB2 (Cell
Signaling #2242); ErbB4 (Cell Signaling 4795), PY20 (Exalpha
Biologicals Inc.), Brk (Santa Cruz Biochemicals).
[0228] In Vitro Pharmacology
[0229] On day 1, NSCLC cells were plated 3-5.times.10.sup.4
cells/well in 96 well plates in their normal serum-containing
media. After 24 h, erlotinib was added to the plates at a 10.times.
concentration in a 10% DMSO/water solution to achieve a final assay
concentration range from 20 .mu.M to 8 nM. Dilutions were made in
3-fold steps. Final DMSO concentrations in each well was constant
and did not exceed 1%. Following erlotinib addition, cells were
replaced in the incubator and left for 72 h. On day 5, Cell-Titer
Glo (Promega) was used to assess the effects on cell viability.
Manufacturers instructions were followed for the assay. Experiments
were conducted in triplicate to at least an n=3. Data was
normalized as a percentage inhibition compared to DMSO only control
wells and concentration-response analysis was performed using Prizm
graphing software.
[0230] In Vivo Pharmacology
[0231] Female CD-1 nu/nu mice (Charles River Laboratories) were
implanted with harvested NSCLC tumor cells in a single subcutaneous
site on the flank of the mice in the axillary region. Tumors were
allowed to grow to 200.+-.50 mm.sup.3, at which time the animals
were sorted into treatment groups of 8 animals per group based on
weight (+1 g body weight) and tattooed on the tail for permanent
identification. Tumor volumes and body weights were determined
twice weekly. The tumor volume was determined by measuring in two
directions with vernier calipers and calculated using the formula:
Tumor volume=(length.times.width.sup.2)/2. The data were plotted as
the % change in mean values of tumor volume and body weight for
each group. The tumor growth inhibition (% TGI) was determined as %
TGI=100(1-W.sub.t-W.sub.c): where W.sub.t is the median tumor
volume of the treated group at time x and W.sub.c is the median
tumor volume of the control group at time x. TARCEVA.TM. was dosed
in a 6% Captisol (CyDex, Inc) in WFI (Water for Injection) solution
and all control animals were dosed with an equal volume of the
vehicle. Tumor growth inhibition studies were dosed by oral gavage
once a day for 14 days. Pharmacodynamic studies were dosed by oral
gavage for 1-3 days with tumors from 4 control and 4 TARCEVA.TM.
treated animals harvested and snap frozen in liquid nitrogen 4
hours after dosing on Days 1, 2 and 3.
[0232] Confocal Microscopy
[0233] Cells grown on glass coverslips for 24 hours were washed and
fixed with 3.7% formaldehyde in PBS followed by permeabilization in
0.5% NP-40. The cells were washed, blocked with 5% BSA and
incubated with primary antibody for 2 hours at room temperature and
with diluted FITC-conjugated secondary antibody for 1 hour. Nuclei
were stained with DAPI (300 nM for 5 min). The confocal images were
captured using a spinning objective confocal microscope at
60.times. magnification.
[0234] Results
TABLE-US-00002 TABLE 2 Growth inhibition of wt EGF receptor tumor
cell lines sensitive or relatively insensitive to erlotinib
expressed as concentration (.mu.M) required for half-maximal
efficacy (EC.sub.50) and maximum inhibition (%) by erlotinib. Tumor
growth inhibition (TGI) is given for day 15 after xenograft
exposure to erlotinib. Cell Max. %TGI EC.sub.50 Classi- line
Inhibition (%) Day 15 Half Maximal fication H292 69 85 0.1
Sensitive H322 80 nd 0.4 Sensitive H358 72 25 0.6 Sensitive H441 55
60 2 Sensitive A549 30 49 5 Intermediate H460 30 6 5 Insensitive
Calu6 46 0 >10 Insensitive H1703 30 nd 7 Insensitive SW1573 25
nd 9 Insensitive
[0235] NSCLC Lines Containing Wt EGF Receptor Display a Range of
Sensitivities to Erlotinib In Vitro
[0236] NSCLC cell lines containing mutations in the catalytic
domain of EGFR displayed hypersensitivity to treatment with the
selective EGFR inhibitors erlotinib and gefitinib. It has been
suggested that only those patients bearing such mutations would
respond and/or show survival benefit from treatment with EGFR
tyrosine kinase inhibitors. However, a randomized placebo
controlled clinical trial conducted with erlotinib indicated that
the survival rate of patients exposed to the drug was well in
excess of the predicted occurrence of such mutations in the patient
population. This suggested that, although mutations were an
indicator of patient response, other factors were undoubtedly
involved in conferring survival benefit.
[0237] Initially the receptor for epidermal growth factor (EGFR)
was sequenced in 14 NSCLC cell lines. Sequence analysis
demonstrated that the EGFR expressed in all of the cell lines of
this study was wild-type with respect to two recently identified
mutations (deletion and point mutations; data not shown). Having
determined that the receptors were wild-type, the sensitivity of
the panel of non-small cell lung cancer cell lines to erlotinib was
assessed using a cell viability assay.
[0238] Analysis of erlotinib sensitivity in a range of human NSCLC
cell lines, which are wild type for EGFR, indicated a wide range of
sensitivity (Table 2; Griffin et al., 2005). We have thus broadly
classified these cell lines into those that are relatively
insensitive (H1703, SW1573, H460 and Calu6), those which show an
intermediate sensitivity (A549) and those which are sensitive
(H441, H358, H322 and H292) to erlotinib-mediated growth inhibition
in vitro and in xenografts. These differences can be correlated in
part to a failure of the relatively insensitive cell lines to show
erlotinib-mediated inhibition of Akt/PKB phosphorylation (Griffin
et al., 2005). A range of sensitivities of the cells to erlotinib
was observed from cells lines ranging from the most sensitive
(H292) through the least sensitive (H460). There were few
correlations between tumor type and erlotinib sensitivity, although
it is interesting to note that both of the bronchioalveolar
carcinoma (BAC) derived cell lines (11358 and H322) showed a level
of sensitivity to EGFR inhibition. Previous reports from clinical
trials have suggested that of the population of NSCLC patients,
those with BAC histologies tended to have a greater treatment
benefit than other NSCLC patients. However, more BAC derived cell
lines should be tested prior to making any conclusions. The data
from the in vitro pharmacology experiments is summarized in Table
2. The concentration response curves were analyzed in two ways.
Firstly in order to define the more traditionally accepted IC50
values (not shown), the curves have been fit in a 0-100% range.
However, since erlotinib and other EGFR inhibitors may be described
as cytostatic rather than cytotoxic, and therefore would therefore
never be expected to achieve complete cell kill, it is questionable
how relevant an IC50 value is. Indeed, even in the most sensitive
lines a maximal efficacy of about 70-80% was the most observed.
Therefore, an EC50 constraining the curves from 0-80% is a more
relevant potency comparison.
[0239] In order to determine the relevance of the in vitro cell
viability assay to in vivo efficacy, a selection of cell lines
ranging from sensitive through insensitive in vitro were tested in
mouse xenograft models. The data from these experiments are shown
in FIG. 1 and Table 2. The correlation between in vitro sensitivity
and in vivo sensitivity to erlotinib was striking. Those cells that
were most sensitive in vitro, were also the most sensitive in vivo,
with the rank order of sensitivities of all cell lines being
identical between the two assays. Such a finding strongly supports
the use of the in vitro assay as an initial guide for assessing
erlotinib sensitivity in xenograft models. The cell lines chosen
were picked for their range of sensitivities based on in vitro and
in vivo activities. Although a somewhat subjective classification,
two sensitive lines (H292 and H358), two intermediate (H441 and
A549) and two insensitive (H460 and Calu-6) were selected. Despite
its low sensitivity in vitro, A549 were classed as an intermediate
cell line due to a low level of response in vivo. The principle aim
of further study was to determine the molecular determinants of
erlotinib sensitivity in these NSCLC cell lines.
[0240] Changes in Epithelial and Mesenchymal Cell Markers Correlate
with Sensitivity of NSCLC Cell Lines to Erlotinib
[0241] Initially differences in protein tyrosine phosphorylation
and complex formation between NSCLC lines sensitive or relatively
insensitive to erlotinib in vitro and in xenograft models were
measured. These experiments involved anti-phosphotyrosine affinity
selection of cell lysates, tryptic digestion and protein
identification based on LC-MS/MS fragment ion spectra. We observed
a striking difference between the erlotinib sensitive and
relatively insensitive NSCLC lines in the abnormal expression
vimentin and or fibronectin (FIG. 2A). Typically vimentin and
fibronectin expression are characteristic of mesenchymal cells and
are only weakly or unexpressed in epithelial cell lineages.
Vimentin expression was primarily found in H1703 and Calu6, while
fibronectin expression was observed in H460 cells. These three
NSCLC lines were relatively insensitive to growth inhibition by
erlotinib in vitro (>10 uM EC.sub.50) and in vivo (at 200 mg/kg
orally qd). Little or no vimentin or fibronectin expression was
found in the the erlotinib sensitive NSCLC lines H292 and H358, the
intermediate line A549 or in the two mutant EGF receptor cell lines
H1650 and H1975.
[0242] Based on the expression of mesenchymal proteins in NSCLC
lines relatively insensitive to erlotinib, we analyzed protein
extracts from the same panel of relatively insensitive and
sensitive NSCLC cell lines for the presence or absence of markers
characteristic of either epithelial or mesenchymal phenotypes (FIG.
2B). Strikingly, E-cadherin was detected in the sensitive cell
lines (H441, H358, H322 and 11292) but was absent in the relatively
insensitive cell lines (H, 1703, SW1573, H460 and Calu6). The
intermediately sensitive cell line A549 showed low but detectable
expression. A similar loss of .gamma.-catenin was observed in cells
relatively insensitive to erlotinib, with the exception of H460.
Therefore, the relatively insensitive cell lines appear to have
lost expression of epithelial cell marker proteins. Next we asked
whether these cell lines expressed the mesenchymal markers
fibronectin and/or vimentin. The relatively insensitive cell lines
clearly expressed either one or both of fibronectin and vimentin
(FIG. 2B), whereas neither protein was detectable in cell lines
sensitive to erlotinib. Interestingly the intermediately sensitive
cell line A549 again showed low but detectable levels of both
proteins. However, confocal microcopy experiments (results not
shown) using immunostaining with antibodies specific for E-cadherin
and vimentin indicated that the A549 cell culture used appears to
be a mixed population of cells since no dual staining of cells was
observed. This could also explain the somewhat variable results
obtained with this cell line, and is consistent with its
intermediate sensitivity to erlotinib.
[0243] The changes in cell-lineage markers were further analyzed in
two relatively insensitive and two sensitive cell lines by confocal
microscopy after immunostaining with antibodies toward E-cadherin
and vimentin (FIG. 2C). No E-cadherin staining could be detected in
either H1703 or Calu6 cells (FIG. 2C, panels 1 and 2), whereas all
of these cells could be stained for vimentin (FIG. 2C, panels 5 and
6). The reverse was true for the sensitive cell lines H441 and
H292, with clear E-cadherin staining on the membrane of these cells
(FIG. 2C, panels 3 and 4) but no visible vimentin staining (FIG.
2C, panels 7 and 8). Taken together these data indicate that NSCLC
cells which were relatively insensitive to growth inhibition by
erlotinib appeared to have undergone transition to a more
mesenchymal cell type and expressed either vimentin or fibronectin.
In contrast cell lines that were sensitive to growth inhibition by
erlotinib maintained an epithelial phenotype and expressed
E-cadherin.
[0244] Erlotinib Sensitivity Correlates with Maintenance of
Epithelial Markers During Tumor Growth In Vivo
[0245] Tumors xenografts derived from NSCLC cell lines grown in
mice displayed a similar degree of erlotinib sensitivity to that
observed for the respective cell line in vitro. We therefore wished
to examine whether the protein markers identified in vitro were
also predictive of erlotinib sensitivity in vivo. Protein extracts
were prepared from 3 independent tumor xenografts grown from H460,
Calu6, A549, H441 and H292 cells. Immunoblotting of extracts
indicated that E-cadherin was not detectably expressed in the
xenografts derived from the H460 and Calu6 cells that are
relatively insensitive to erlotinib, was expressed at low levels in
xenografts derived from the A549 cells of intermediate sensitivity
and expressed at high levels in H441 and H292 cell lines sensitive
to erlotinib (FIG. 3). A similar result was observed on analysis of
.gamma.-catenin levels. In contrast xenograft samples derived from
Calu6 expressed fibronectin and vimentin (Calu6) or fibronectin
alone (H460), a result consistent with that obtained from in vitro
cell cultures (FIG. 2B). H441 and H292 derived xenograft extracts
showed little or no expression of either fibronectin or vimentin.
These in vivo results further support the in vitro data and
indicate that the presence of these protein markers is not an
artifact of cell culture. Further, they support the hypothesis that
erlotinib sensitivity may be restricted to cells with an epithelial
phenotype and that cells which have undergone EMT become less
dependent upon EGFR signaling for cell proliferation and
survival.
[0246] Expression of Brk in NSCLC Cell Lines that are Relatively
Insensitive or Sensitive to EGF Receptor Inhibition
[0247] The results of these experiments led to the working
hypothesis that erlotinib sensitivity is determined by the ability
of the compound to inhibit Akt signaling. Following this hypothesis
the question arises as to what is unique about these cells that
allows the EGFR pathway to so significantly impact cellular Akt
signaling. Recent papers by (REFS) have suggested an interesting
potential link between EGFR and Akt signaling, which may or may not
involve heterodimerization with other Her members such as ErbB3,
involving the non receptor tyrosine kinase Brk (also known as
PTK6). It was of interest therefore to determine whether there may
be any relationship between Brk expression in sensitive and
insensitive erlotinib lines, thus providing a rationale for why
EGFR inhibition is so intricately linked to Akt in sensitive
compared to insensitive. FIG. 4 shows Western blot analysis of a
number of lines from the NSCLC panel, and their respective
expression of Brk protein. Interestingly there is a very good
correlation between Brk levels and erlotinib sensitivity in so far
as high Brk expression equates to higher erlotinib sensitivity and
absence, or lower expression, of Brk tends to characterize
insensitive lines.
[0248] Analysis of EMT Markers is Predictive of Erlotinib
Sensitivity of Pancreatic Cell Lines in Culture
[0249] We next extended these studies to ask whether these
observations would be applicable to other cancer cell types. As
erlotinib has shown efficacy in Phase III combination studies with
gemcitabine in pancreatic cancer, we examined the sensitivity of
pancreatic cell lines to growth inhibition by erlotinib in vitro
and their expression of epithelial and mesenchymal lineage markers.
Consistent with data in NSCLC, pancreatic cell lines sensitive to
erlotinib expressed E-cadherin but not vimentin or fibronectin,
while pancreatic lines that are relatively insensitive to erlotinib
had lost E-cadherin expression and gained vimentin and/or
fibronectin expression (FIG. 5). These results were observed both
by immunoblot (FIG. 5A) and confocal fluorescence microscopy
studies (FIG. 5B).
[0250] Patients with Tumors Expressing High Levels of E-Cadherin
have Greater Time to Disease Progression when Treated with
Erlotinib+Chemotherapy Compared to Chemotherapy Treatment Alone
[0251] Samples from patients who participated in a randomized,
double-blinded phase III clinical trial referred to as Tribute were
analyzed for E-cadherin expression by Immunohistochemistry (IHC).
Tribute studied 1,079 patients at approximately 150 centers in the
United States having histological confirmed NSCLC who had not
received prior chemotherapy comparing erlotinib+chemotherapy
(carboplatin/paclitaxel) with chemotherapy alone. Patients received
paclitaxel (200 mg/m.sup.2 3 hour i.v. infusion) followed by
carboplatin (AUC=6 mg/ml.times.minute infused over 15-30 minutes
using Calvert formula) with or without erlotinib (100 mg/day p.o.
escalated to 150 mg/day for tolerant patients). Tumor samples,
formalin-fixed paraffin-embedded blocks or unstained slides, from
87 patients in the Tribute trial were immunostained to detect
E-cadherin expression. Staining intensity was scored as 0, 1+, 2+
and 3+ with 65 of the 87 samples having >=2 staining intensity
and 22 had <=1 staining intensity.
[0252] Immunohistochemistry for E-cadherin was performed on
formalin-fixed paraffin embedded tissue sections assembled in a
tissue microarray. Following deparaffinization, antigen retrieval
was performed by pretreating with Target Retrieval Solution at 110
degrees C. for 20 min (DakoCytomation, Carpenteria Calif.). The
pretreated sections were then incubated with primary mouse
monoclonal IgG2 antibody against E-cadherin (clone 36, Pharmingen)
at a concentration of 1 microgram/ml for 60 min at ambient
temperature. Primary antibody bound to the sections was detected
using biotinylated horse anti-mouse IgG, and visualized using the
avidin-biotin peroxidase complex technique (Vectastain ABC Elite,
Vector Laboratories) and diaminobenzidine as chromagen.
[0253] It was determined that patients whose tumors stained for
high levels of membrane and cytoplasmic E-cadherin exhibited
significantly longer time to disease progression (TTP) when treated
with the combination of erlotinib and chemotherapy compared to
chemotherapy alone (34.0 weeks v. 19.3 weeks, p=0.0028). The
results are provided in table 2 and are illustrated by the
Kaplan-Meier curve in FIG. 6a. Conversely, patients whose tumors
had low membrane and cytoplasmic E-cadherin expression (staining
intensity of <=1) did not have a significant difference in TTP
for the two treatment groups which is illustrated by the
Kaplan-Meier curve in FIG. 6b.
TABLE-US-00003 TABLE 2 Time to Progression by E-cadherin staining
for erlotinib + chemotherapy and chemotherapy alone treatment
groups Intensity >= 2 Intensity <= 1 Erlotinib + Erlotinib +
Chemo chemo Chemo chemo N 37 28 14 8 Patients 31 (83.8%) 16 (57.1%)
8 (57.1%) 5 (62.5%) who progressed Censored 6 (16.2%) 12 (42.9%) 6
(42.9%) 3 (37.5%) patients Median 19.3 34.0 30.0 19.1 time (wk) 95%
CI (12.9, 25.7) (13.1, 42.1) (19.1, .) (8.6, .) P-Value 0.0028
0.3976 (Logrank) Hazard 0.37 1.63 ratio (HR) 95% (0.19, 0.73)
(0.50, 5.33) CI for HR
CONCLUSION
[0254] The loss of E-cadherin expression and the acquisition of a
more mesenchymal phenotype has been shown to correlate with poor
prognosis in multiple epithelial-derived solid tumors. The loss of
E-cadherin and to a lesser extent .gamma.-catenin and Brk
correlated with cellular and xenograft insensitivity to EGF
receptor inhibition. Conversely the cellular acquisition of
mesenchymal markers, vimentin, fibronectin or fibrillin correlates
with a loss of sensitivity to EGF receptor inhibitors. We clearly
show that a partial or complete epithelial to mesenchymal
transition negatively impacts cellular responses to EGF receptor
inhibitors in vitro and in xenografts and serves a diagnostic for
patients most likely to benefit from EGF receptor kinase inhibitors
and anti-EGF receptor antibody therapies.
ABBREVIATIONS
[0255] EGF, epidermal growth factor; EMT, epithelial to mesenchymal
transition; NSCLC, non-small cell lung carcinoma; HNSCC, head and
neck squamous cell carcinoma; CRC, colorectal cancer; MBC,
metastatic breast cancer; EGFR, epidermal growth factor receptor;
Brk, Breast tumor kinase (also known as protein tyrosine kinase 6
(PTK6)); LC, liquid chromatography; MS, mass spectrometry; IGF-1,
insulin-like growth factor-1; TGF.alpha., transforming growth
factor alpha; HB-EGF, heparin-binding epidermal growth factor; LPA,
lysophosphatidic acid; TGF.alpha., transforming growth factor
alpha; IC.sub.50, half maximal inhibitory concentration; pY,
phosphotyrosine; wt, wild-type; PI3K, phosphatidyl inositol-3
kinase; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase.
INCORPORATION BY REFERENCE
[0256] All patents, published patent applications and other
references disclosed herein are hereby expressly incorporated
herein by reference.
EQUIVALENTS
[0257] Those skilled in the art will recognize, or be able to
ascertain, using no more than routine experimentation, many
equivalents to specific embodiments of the invention described
specifically herein. Such equivalents are intended to be
encompassed in the scope of the following claims.
* * * * *
References