U.S. patent application number 13/237833 was filed with the patent office on 2012-09-27 for stratification of cancer patients for susceptibility to therapy with ptk2 inhibitors.
This patent application is currently assigned to BOEHRINGER INGELHEIM INTERNATIONAL GMBH. Invention is credited to Guenther ADOLF, Pilar GARIN-CHESA, Ulrich HIRT.
Application Number | 20120244141 13/237833 |
Document ID | / |
Family ID | 43085773 |
Filed Date | 2012-09-27 |
United States Patent
Application |
20120244141 |
Kind Code |
A1 |
ADOLF; Guenther ; et
al. |
September 27, 2012 |
Stratification of cancer patients for susceptibility to therapy
with PTK2 inhibitors
Abstract
A method for determining whether a cancer patient is susceptible
to treatment with a protein tyrosine kinase 2 (PTK2) inhibitor,
comprising detecting the expression of the E-cadherin protein in a
cancer sample of said cancer patient, wherein an E-cadherin protein
immunoreactivity score (IRS) of 0-2 indicates that the cancer
patient is susceptible to treatment with a PTK2 inhibitor. The
invention further encompasses treatment of a patient with a protein
tyrosine kinase 2 (PTK2) inhibitor if it has been so determined
that said patient is susceptible to such treatment.
Inventors: |
ADOLF; Guenther; (Vienna,
AT) ; GARIN-CHESA; Pilar; (Vienna, AT) ; HIRT;
Ulrich; (Vienna, AT) |
Assignee: |
BOEHRINGER INGELHEIM INTERNATIONAL
GMBH
Ingelheim am Rhein
DE
|
Family ID: |
43085773 |
Appl. No.: |
13/237833 |
Filed: |
September 20, 2011 |
Current U.S.
Class: |
424/130.1 ;
435/7.1; 514/44A |
Current CPC
Class: |
C12Q 1/6886 20130101;
G01N 33/57492 20130101; G01N 2333/705 20130101; G01N 33/57484
20130101; G01N 2333/91205 20130101; G01N 2800/52 20130101; A61P
35/00 20180101; G01N 33/57496 20130101 |
Class at
Publication: |
424/130.1 ;
435/7.1; 514/44.A |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 35/00 20060101 A61P035/00; A61K 31/713 20060101
A61K031/713; A61K 31/7105 20060101 A61K031/7105; G01N 21/76
20060101 G01N021/76; G01N 33/574 20060101 G01N033/574 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 28, 2010 |
EP |
10 180 981.2 |
Claims
1. A method of treating a cancer in a cancer patient, said method
comprising the following steps: (a) determining whether said
patient is susceptible to treatment with a protein tyrosine kinase
2 (PTK2) inhibitor by detecting the amount of expression of the
E-cadherin protein in a cancer sample taken from said patient,
wherein an E-cadherin protein immunoreactivity score (IRS) of 0-2
indicates that the patient is susceptible to treatment with a PTK2
inhibitor; and (b) treating said patient with a protein tyrosine
kinase 2 (PTK2) inhibitor if it has been determined that said
patient is susceptible to treatment with a protein tyrosine kinase
2 (PTK2) inhibitor.
2. The method of claim 1, wherein said cancer patient is a
mammal.
3. The method of claim 2, wherein said mammal is a human.
4. The method of claim 1, wherein said cancer is a carcinoma.
5. The method of claim 1, wherein said cancer is selected from the
group consisting of carcinoma of the duodenum, colon, rectum and
anus; carcinoma of the pancreas; carcinoma of the urinary bladder;
lung tumours (small-cell lung cancer (SCLC), non-small-cell lung
cancer (NSCLC) such as for example squamous cell carcinomas,
adenocarcinomas (acinary, papillary, bronchiolo-alveolar) and
large-cell bronchial carcinoma (giant cell carcinoma, clear-cell
carcinoma)); breast cancer such as ductal, lobular, mucinous or
tubular carcinoma, ovarian cancer (ovarian carcinoma--mucinous or
serous cystoadenocarcinoma, endometriod carcinoma, and clear cell
tumour); head and neck tumours; liver cell carcinoma
(hepatocellular carcinoma (HCC); kidney cancer such as for example
(clear cell renal cell carcinoma, papillary renal cell carcinoma,
chromophobe renal cell carcinoma and collecting duct carcinoma);
prostate cancer; and cancer of the vulva.
6. The method of claim 1, wherein said detection of the expression
of the E-cadherin protein in a cancer sample of a cancer patient is
conducted by way of an immunohistochemistry (IHC) method.
7. The method of claim 6, wherein said IHC method employs a primary
antibody which is specific for E-cadherin and a secondary antibody
which specifically reacts with the primary antibody.
8. A method of screening for a therapeutically effective PTK2
inhibitor comprising the following steps: (a) providing cancer
cells or a cancer cell line which are characterized by an
E-cadherin protein immunoreactivity score of 2, 1, or 0 (1 being
preferred and 0 being even more preferred) (b) contacting the
cancer cell or the cancer cell line of (a) with a PTK2 inhibitor;
and (c) evaluating whether the PTK2 inhibitor negatively affects
the cancer cell/cancer cell lines.
Description
[0001] The present invention relates to a method for determining
whether a cancer patient is susceptible to treatment with a protein
tyrosine kinase 2 (PTK2) inhibitor, comprising detecting the
expression of the E-cadherin protein in a cancer sample of said
cancer patient, wherein an E-cadherin protein immunoreactivity
score (IRS) of 0-2 indicates that the cancer patient is susceptible
to treatment with a PTK2 inhibitor. Said detection of the
expression of the E-cadherin protein in a cancer sample of a cancer
patient is preferably conducted by way of an immunohistochemistry
(IHC) method. Said IHC method preferably employs a primary antibody
which is specific for E-cadherin and a secondary antibody which
specifically reacts with the primary antibody. The present
invention also relates to a method of treating a cancer patient
whose cancer is characterized by an E-cadherin protein
immunoreactivity score (IRS) of 0-2, comprising administering to
the patient a therapeutically effective amount of a PTK2 inhibitor.
In a further aspect, the present invention relates to a PTK2
inhibitor for use in the treatment of a cancer patient whose cancer
is characterized by an E-cadherin protein immunoreactivity score
(IRS) of 0-2. The present invention also provides a method of
screening for a therapeutically effective PTK2 inhibitor comprising
the steps of (a) providing cancer cells or a cancer cell line which
are characterized by an E-cadherin protein immunoreactivity score
of 2, 1, or 0 (1 being preferred and 0 being even more preferred);
(b) contacting the cancer cell or the cancer cell line of (a) with
a PTK2 inhibitor; and (c) evaluating whether the PTK2 inhibitor
negatively affects the cancer cell/cancer cell lines. In a further
aspect, the present invention relates to a method for stratifying
cancer patients that are susceptible to treatment with a PTK2
inhibitor, comprising determining the E-cadherin IRS score in a
cancer sample of said patient, wherein an E-cadherin protein
immunoreactivity score (IRS) of 0-2 (i.e. 2, 1, or 0) indicates
that the cancer patient is susceptible to treatment with a PTK2
inhibitor. The present invention also relates to a pharmaceutical
package comprising a PTK2 inhibitor, and (a) instructions and/or an
imprint indicating that said PTK2 inhibitor is to be used for the
treatment of patients which suffer from a cancer which is
characterized by an E-cadherin protein immunoreactivity score of 2,
1, or 0 (1 being preferred and 0 being more preferred); and/or (b)
instructions and/or an imprint indicating that said patient is to
be stratified by a method of the present invention; and/or (c)
means to carry out a method as defined herein.
[0002] Protein tyrosine kinase 2 (PTK2), also known as focal
adhesion kinase 1 (FAK1) is a non-receptor tyrosine kinase that is
predominantly localized in focal adhesions. PTK2 serves as a linker
between extracellular signals transmitted through integrins and
growth factor receptors and signal transducers inside the cells.
Activated PTK2 appears to be involved in the regulation of cell
survival, proliferation and motility. Therefore, inhibition of PTK2
may inhibit cancer growth and the formation of metastases. PTK2
inhibitors have been previously described and several compounds are
currently under investigation in early clinical trials.
[0003] PTK2 kinase inhibitors show efficacy in a variety of
experimental models of cancer, in particular in human cancer
xenograft models in immunodeficient mice. However, their efficacy
varies widely among different cancer models: whereas cancer
regression or complete inhibition of growth can be achieved in some
models, treatment of other cancer types results in partial
inhibition of growth and some cancers are not affected at all.
Oncogenic mutations or gene amplifications have been described for
a number of genes, e.g. EGFR, HER2 or BRAF. Their presence
determines the sensitivity of a given cancer to treatment with the
corresponding kinase inhibitors, and eligibility of patients for
therapy with such inhibitors can easily be determined by analysis
of the cancer DNA sequence or gene copy number. For the PTK2 gene,
however, no mutations or amplifications have been described so far
in human cancers, or in the preclinical model tumors that are
sensitive to PTK2 inhibition.
[0004] Therefore, the identification of predictive biomarkers for
selection of patients most likely to benefit from therapy with PTK2
inhibitors is urgently required. No such predictive biomarkers or a
genetic signature associated with therapeutic benefit are currently
available.
[0005] Thus, the technical problem underlying the present invention
is to provide means and methods for selecting susceptible cancer
patients and/or cancer types for the treatment with PTK2
inhibitors.
[0006] The present invention addresses this need and provides the
cellular markers and methods which will allow the selection of
cancer patients susceptible to treatment with PTK2 inhibitors.
[0007] In our pre-clinical studies using xenograft models of human
cancers, we have found surprisingly that the expression level of
E-cadherin protein in cancer cells, which can be evaluated for
example with immunohistochemistry (IHC) methods, can be used to
predict sensitivity to PTK2 inhibitors. In view of that, we propose
to examine the E-cadherin expression level prior to the
administration of PTK2 inhibitors in order to determine whether a
cancer/cancer patient is susceptible to treatment with a PTK2
inhibitor or not. Further embodiments of the present invention are
characterized and described herein and also reflected in the
claims.
[0008] It must be noted that as used herein, the singular forms
"a", "an", and "the", include plural references unless the context
clearly indicates otherwise. Thus, for example, reference to "a
reagent" includes one or more of such different reagents and
reference to "the method" includes reference to equivalent steps
and methods known to those of ordinary skill in the art that could
be modified or substituted for the methods described herein. Unless
otherwise indicated, the term "at least" preceding a series of
elements is to be understood to refer to every element in the
series. "At least one" includes for example, one, two, three, four,
or five or even more.
[0009] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
present invention. Throughout this specification and the claims
which follow, unless the context requires otherwise, the word
"comprise", and variations such as "comprises" and "comprising",
will be understood to imply the inclusion of a stated integer or
step or group of integers or steps but not the exclusion of any
other integer or step or group of integer or step.
[0010] Several documents are cited throughout the text of this
specification. Each of the documents cited herein (including all
patents, patent applications, scientific publications,
manufacturer's specifications, instructions, etc.), whether supra
or infra, are hereby incorporated by reference in their entirety.
Nothing herein is to be construed as an admission that the
invention is not entitled to antedate such disclosure by virtue of
prior invention.
[0011] In a first aspect, the present invention relates to a method
for determining whether a cancer and the respective cancer patient
is susceptible to treatment with a protein tyrosine kinase 2 (PTK2)
inhibitor, comprising detecting the expression of the E-cadherin
protein in a cancer sample (obtained) from said cancer or cancer
patient, wherein an E-cadherin protein immunoreactivity score (IRS)
0-2, preferably of 0-1, more preferably an IRS score of 1 and even
more preferably an IRS score of 0, indicates that the cancer and
the respective cancer patient is susceptible to treatment with a
PTK2 inhibitor.
[0012] An IRS score (or IRS) of "0-2" means an IRS of 0, 1, or 2.
Likewise, an IRS score of "0-1" means an IRS score of 0 or 1.
Methods allowing the skilled person to evaluate said IRS score in a
given cancer/cancer sample are explained herein elsewhere. The term
"IRS" as used herein denotes the immunoreactivity score of
E-cadherin as disclosed herein.
[0013] In a preferred embodiment, said detection is carried out by
way of an immunohistochemistry method (IHC).
[0014] It will be understood that the cancer sample is preferably
obtained from a cancer patient which, however, does not mean that
the step of obtaining said cancer sample from said patient is
necessarily included in the scope of the present invention.
[0015] "E-cadherin" or "E-cadherin protein", also known as CD324,
LCAM or ECAD belongs to the "cadherins" (calcium-dependent adhesion
molecules) which are a class of type-1 transmembrane proteins. They
play important roles in cell adhesion, ensuring that cells within
tissues are bound together. Cadherins are dependent on calcium
(Ca.sup.2+) ions to function, hence their name. The E-cadherin
protein, encoded by the CDH1 gene, is composed of five
extracellular cadherin repeats, a transmembrane region, and a
highly conserved cytoplasmic tail and can be found in all
epithelial tissues. The cytoplasmatic domain is bound to the actin
cytoskeleton via intracellular attachment proteins, the catenins.
The actin cytoskeleton forms a transcellular network that mediates
the structural integrity of the cells and its polarity and is
important for epithelial cell morphogenesis.
[0016] E-cadherin thus serves as a biomarker for epithelial cells
and most epithelial-derived cancers. Recent immunohistochemical
analyses have indicated that decreased membrane expression of
E-cadherin on cancer cells is associated with adverse prognostic
features and lower overall survival in patients with epithelial
cancers (Saito T, et al; Cancer, 2003; 97:1002-9). It thus appears
that E-cadherin is not only a biomarker for epithelial cells as
such but may also serve as a prognostic marker for carcinoma
progression.
[0017] The decreased protein expression or lack of expression of
E-cadherin on the membrane of epithelial cancer cells was found in
our preclinical studies to correlate, most to our surprise, as well
with sensitivity of the respective cancer cells to PTK2 inhibition,
translating into significant cancer growth inhibition and cancer
regression in animal models of human cancer. Therefore, the
E-cadherin expression level may serve as a biomarker for the
selection of patients for treatment with PTK2 inhibitors. These
PTK2 inhibitors are well-known to the skilled artisan and are also
described in great detail herein below. It could be demonstrated by
the present inventors that cancers with low E-cadherin expression,
e.g. an E-cadherin score of 0-2, preferably of 0-1, more preferably
an IRS score of 1 and even more preferably of 0 (said score being
explained in detail herein), will more likely be susceptible to
treatment with a PTK2 inhibitor than cancers with high E-cadherin
expression.
[0018] The "membrane of the cancer cells" means the cell membrane
which separates the exterior of a cancer cell from the interior of
the cancer cell. E-cadherin is regularly expressed on the cell
membrane of epithelial cells ensuring that cells within tissues are
attached to each other to maintain tissue integrity.
[0019] The term "susceptible to treatment with a PTK2 inhibitor"
when used herein means that a PTK2 inhibitor may potentially have a
therapeutic effect in a patient to whom a PTK2 inhibitor is and/or
will be administered. Said term when used herein is equivalent to
the term "sensitive to treatment with a PTK2 inhibitor" or
"responsive to treatment with a PTK2 inhibitor".
[0020] By "therapeutic effect" or "therapeutically effective" is
meant that a PTK2 inhibitor may produce the therapeutic effect for
which it is administered. Preferably, a therapeutic effect includes
the reduction, stabilization or inhibition of progression of a
cancer-associated symptom, such as cancer size, number of
metastases or other symptoms which are caused by/associated with
the presence and/or progression of a cancer. The response includes
a complete response, a partial response, a stable disease (without
progression or relapse), and/or a response with a later relapse of
the patient. Preferably, as described herein the PTK2 inhibitor may
affect that cancer cells will undergo cell death thereby,
ameliorating and/or treating a cancer of a patient provided that
said cancer cells express the PTK2 protein. The therapeutic effect
of the respective methods or method steps of the present invention
may be detectable by all established methods and approaches which
will indicate a therapeutic effect. Alternatively, it is also
envisaged that cancer markers in the serum of the patient (if
present) are detected in order to diagnose whether or not the
therapeutic approach is effective. The skilled person is aware of
numerous other ways which will enable him or her to observe a
therapeutic effect of a PTK2 inhibitor.
[0021] It is envisaged that a cancer sample of a patient who may be
treated with a PTK2 inhibitor is to be obtained prior to the
treatment, during the treatment and/or after the treatment with
said PTK2 inhibitor. Preferably, the sample is obtained prior to
the treatment in order to determine, in accordance with the means
and methods of the present invention, whether or not a cancer
patient may be susceptible to the treatment with a PTK2 inhibitor,
whether or not a patient may respond favorably to the treatment
with a PTK2 inhibitor, or whether or not a patient may benefit from
the treatment with a PTK2 inhibitor.
[0022] The term "potentially" when used in the context of a
therapeutic effect means that a PTK2 inhibitor--though such an
inhibitor is deemed to have a therapeutic effect based on the
outcome of the methods of the present invention--does not
necessarily have to be therapeutically effective. This is so
because--self-explanatory as it is--the methods of the present
invention cannot provide a 100% safe prediction whether or not a
patient may be susceptible to a PTK2 inhibitor, since, apart from
the expression of the E-cadherin protein, individual factors such
as age, body weight, general health, sex, diet, drug interaction
and the like may have an influence as to whether or not a patient
will be susceptible to a PTK2 inhibitor.
[0023] "Treat" or "treatment" as used herein, means to reduce,
stabilize, or inhibit progression of a symptom, such as cancer
size, number of metastases or other symptoms which are caused
by/associated with the presence and/or progression of a cancer.
[0024] The term "cancer", as used herein, refers to malignant cell
growth and proliferation, including all pre-cancerous and cancerous
cells and tissues. Cancers are sometimes also denoted herein as
malignant cancers or neoplasias or tumors. Invasive malignant
cancers of transformed epithelial cells, i.e. carcinomas, are
preferred in the embodiments of the present invention. It is
therefore envisaged that, in the embodiments of the present
invention, said cancer sample essentially consists of/comprises
malignant epithelial cancer cells.
[0025] The cancers described herein may be metastatic (i.e. the
cancer metastasizes) or non-metastatic.
[0026] It will be understood that a cancer to be treated with a
PTK2 inhibitor in accordance with the embodiments of the present
invention, expresses the PTK2 protein. As already discussed
hereinbefore, the present invention relates in essence to a method
for determining whether a cancer patient is susceptible to
treatment with a PTK2 inhibitor. It goes without saying that the
cancer patients to be treated are therefore patients with PTK2
polypeptide expressing cancer cells. A "PTK2 polypeptide-expressing
cancer" is a cancer comprising cells that have a PTK2 polypeptide
present. A "PTK2 polypeptide-expressing cancer" optionally produces
sufficient levels of PTK2 polypeptide in cells thereof, such that a
PTK2 inhibitor can interact with it. The PTK2 polypeptide may be
determined in various ways, i.e. the skilled person is well aware
how to test whether a cancer/cancer cell is PTK2-positive or not.
It will be understood that the evaluation of the PTK2 polypeptide
in the cancer cells is not mandatory, i.e. the embodiments of the
present invention do not necessarily include this step. Presently,
it is assumed that almost all relevant epithelial cancers express
PTK2. Even so, and as mentioned before, the efficacy of PTK2 kinase
inhibitors varies widely among different cancer models: whereas
cancer regression or complete inhibition of growth can be achieved
in some models, treatment of other cancer types results in partial
inhibition of growth and some cancers are not affected at all.
Thus, PTK2 expression as such is obviously not predictive for the
susceptibility of a cancer to treatment with PTK-2 inhibitors. This
gap has been closed by the present invention which provides, for
the first time, a suitable biomarker which allows the
identification of cancers/cancer patients which are susceptible to
treatment with a PTK2-inhibitor: the mentioned biomarker is the
E-cadherin expression as explained throughout the
specification.
[0027] In the context of the present invention the term "cancer
patient" (sometimes also denoted as "patient" or "subject") means a
subject having a cancer described herein (including a subject
diagnosed to suffer from a cancer) but also includes a subject
during an adjuvant therapy, for example after the resection of the
primary cancer.
[0028] Preferably, said subject is a mammalian, such as a human, a
horse, a camel, a dog, a cat, a pig, a cow, a goat or a fowl. A
human subject is most preferred. The compositions, compounds, uses
and methods of the present invention are thus applicable to both
human therapy and veterinary applications.
[0029] A "tissue sample" (sometimes also denoted as "cancer sample"
or "sample of the cancer" or the like) is derived or obtained from
a subject (the cancer patient) and may be obtained via biopsy such
as needle biopsy, surgical biopsy, bone marrow biopsy etc. A cancer
sample includes a specimen of a cancer, parts of a cancer, cancer
cells derived from a cancer (including cancer cell lines which may
be derived from a cancer and which are grown in cell culture) and
also the cancer mass as a whole, but also cancer cell lines as
such, and cells and/or tissue which are/is derived from a subject
and which are/is suspected of being cancerous or which are/is
suspected of comprising cancerous cells. It is thus envisaged that
the cancer sample may also comprise non-cancerous cells. For
example cancer cells and/or (micro) metastases are frequently
surrounded by healthy, i.e. non-cancerous tissue, i.e. the cancer
cells could then form a subset of cells within the healthy tissue.
A cancer sample thereby could comprise a subset of healthy
(non-cancerous) cells and a subset of cancerous cells. The term
"sample" is interchangeable with "specimen".
[0030] A non-limiting exemplary list of cancers which can be
treated with PTK2 inhibitors includes, but is not limited to, one
or more of the following: intestinal cancer including carcinomas of
the duodenum, colon, rectum, and anus; carcinoma of the pancreas
(e.g. pancreatic adenocarcinoma); carcinoma of the bladder; lung
tumours (small-cell lung cancer (SCLC), non-small-cell lung cancer
(NSCLC) such as for example squamous cell carcinomas,
adenocarcinomas (acinary, papillary, bronchiolo-alveolar) and
large-cell bronchial carcinoma (giant cell carcinoma, clear-cell
carcinoma); breast cancer such as ductal, lobular, mucinous or
tubular carcinoma, Paget's carcinoma; uterine cancer (corpus
carcinoma or endometrial carcinoma); CUP syndrome (Cancer of
Unknown Primary); ovarian cancer (ovarian carcinoma--mucinous or
serous cystoadeno-carcinoma, endometrioid carcinomas, clear cell
tumour, Brenner's tumour); gall bladder cancer; bile duct cancer
such as for example Klatskin tumour; testicular cancer (germinal or
non-germinal germ cell tumours); laryngeal cancer such as for
example supra-glottal, glottal and subglottal tumours of the vocal
cords; head and neck tumours (HNO tumours) such as for example
tumours of the lips, and oral cavity (carcinoma of the lips,
tongue, oral cavity), nasopharyngeal carcinoma (tumours of the
nose, lymphoepithelioma), pharyngeal carcinoma, oropharyngeal
carcinomas, carcinomas of the tonsils (tonsil malignoma) and (base
of the) tongue, hypopharyngeal carcinoma, laryngeal carcinoma
(cancer of the larynx), tumours of the paranasal sinuses and nasal
cavity, tumours of the salivary glands and ears; eyelid tumours
(basalioma or adenocarcinoma of the eyelid apparatus); liver cell
carcinoma (hepatocellular carcinoma (HCC); stomach cancer
(papillary, tubular or mucinous adenocarcinoma, adenosquamous,
squamous or undifferentiated carcinoma; kidney cancer such as for
example clear cell renal cell carcinoma, papillary renal cell
carcinoma, chromophobe renal cell carcinoma and collecting duct
carcinoma; oesophageal cancer; penile cancer; prostate cancer (e.g.
hormone refractory prostate cancer); vaginal cancer or vaginal
carcinoma; thyroid carcinomas such as for example papillary,
follicular, medullary or anaplastic thyroid carcinoma; cancer of
the urethra (carcinoma of the urethra, urothelial carcinoma) and
cancer of the vulva.
[0031] Carcinoma of the duodenum, colon, rectum and anus; carcinoma
of the pancreas (e.g. pancreatic adenocarcinoma); carcinoma of the
urinary bladder; lung tumours (small-cell lung cancer (SCLC),
non-small-cell lung cancer (NSCLC) such as for example squamous
cell carcinomas, adenocarcinomas (acinary, paillary,
bronchiolo-alveolar) and large-cell bronchial carcinoma (giant cell
carcinoma, clear-cell carcinoma); breast cancer such as ductal,
lobular, mucinous or tubular carcinoma, ovarian cancer (ovarian
carcinoma--mucinous or serous cystoadenocarcinoma, endometriod
carcinoma, and clear cell tumour); head and neck tumours; liver
cell carcinoma (hepatocellular carcinoma (HCC); kidney cancer such
as for example (clear cell renal cell carcinoma, papillary renal
cell carcinoma, chromophobe renal cell carcinoma and collecting
duct carcinoma); prostate cancer (e.g. hormone refractory prostate
cancer); and cancer of the vulva, are of great importance and
therefore preferred.
[0032] Protein tyrosine kinase 2 (PTK2), also known as FAK, FADK,
FAK1, pp 125FAK, EC 2.7.10.2) is a cytoplasmic protein tyrosine
kinase which is found concentrated in the focal adhesions that form
at the cell membrane of cells growing in the presence of
extracellular matrix constituents. The encoded protein is a member
of the FAK subfamily of protein tyrosine kinases but lacks
significant sequence similarity to kinases from other subfamilies.
PTK2 has three functional domains: (1) a focal adhesion targeting
(FAT) domain, which is important for localization of FAK to focal
adhesions and for binding integrin-associated proteins such as
paxillin and talin; (2) a catalytic domain with tyrosine kinase
activity; and (3) a N-terminal domain, important for the
interaction with integrins and growth factor receptors (Parsons, J.
T. (2003). Focal adhesion kinase: The first ten years. J. Cell Sci.
116, 1409-1416). PTK2 has multiple phosphorylation sites that are
required for binding to adaptor proteins containing SH2 domains. An
important phosphorylation site is Tyr397, which appears to be
important for the interaction of PTK2 with downstream signaling
molecules such as Rho kinase.
[0033] Convincing evidence suggests that PTK2 plays an essential
role in cell-matrix signal transduction pathways (Clark and Brugge
1995, Science 268: 233-239) and its aberrant activation is
associated with an increase in the metastatic potential of cancers
(Owens et al. 1995, Cancer Research 55: 2752-2755). PTK2 was
originally identified as a 125 kDa protein highly
tyrosine-phosphorylated in cells transformed by v-Src. PTK2 was
subsequently found to be a tyrosine kinase that localizes to focal
adhesions, which are contact points between cultured cells and
their underlying substratum and sites of intense tyrosine
phosphorylation. PTK2 is phosphorylated and, thus, activated in
response to extracellular matrix (ECM)-binding to integrins.
Recently, studies have demonstrated that an increase in PTK2 mRNA
levels is accompanied by a more invasive behaviour of cancers and
attenuation of the expression of PTK2 (through the use of antisense
oligonucleotides) induces apoptosis in cancer cells (Xu et al.
1996, Cell Growth and Diff. 7: 413-418). In addition to being
expressed in most tissue types, PTK2 is found at elevated levels in
most human cancers, particularly in highly invasive and metastatic
cancers. Even so, and as mentioned before, the efficacy of PTK2
kinase inhibitors varies widely among different cancer models.
Thus, PTK2 expression as such is obviously not predictive for the
susceptibility of a cancer to treatment with PTK-2 inhibitors. This
gap has been closed by the present invention which provides, for
the first time, a suitable biomarker which allows the
identification of cancers/cancer patients which are susceptible to
treatment with a PTK2-inhibitor: the mentioned biomarker is the
E-cadherin expression as explained throughout the
specification.
[0034] The term "PTK2 inhibitor" defines in the context of the
present invention a compound or a plurality of compounds which
interact(s) with PTK2 (preferably the human PTK2) such that the
kinase activity is reduced. Assays which are suitable to detect
such inhibitors are explained in more detail herein below. The term
"plurality of compounds" is to be understood as a plurality of
substances which may or may not be identical. The plurality of
compounds may preferably act additively or synergistically. Said
compound or plurality of compounds may be chemically synthesized or
microbiologically produced and/or comprised in, for example,
samples, e.g., cell extracts from, e.g., plants, animals or
microorganisms.
[0035] The term "reduced PTK2 kinase activity" or "reducing the
PTK2 kinase activity" as used herein defines the reduction of the
kinase activity of PTK2, preferably to at least about the same
level as compared to a normal/natural state of a comparable
control-cell/subject. In this context, the term "normal/natural
state of a comparable control-cell/subject" means the PTK2 kinase
activity in a control-cell which is preferably of the same nature
as the test-cell (e.g. both cells are epithelial cells) but which
is derived from a different source. "A different source" includes
e.g. a cell/tissue sample obtained from a healthy subject,
preferably from a subject who does not suffer from a carcinoma or a
cell/tissue sample obtained from a distinct part of the same
subject wherein said distinct part appears to be free from
associated symptoms of a carcinoma. However, even in cases where
the PTK2 inhibitor will not reduce the kinase activity of PTK2 to
about the normal/natural state of a comparable control-cell/subject
but actually reduces the PTK2 kinase activity when compared to the
PTK2 kinase activity before the addition of said inhibitor, it will
be appreciated that said inhibitor has a beneficial effect.
[0036] Accordingly, it is envisaged that a PTK2 inhibitor at least
reduces the kinase activity of PTK2 about 5%, 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90% or even 100% when compared to the PTK2
kinase activity that is achieved without the addition of said
inhibitor. Suitable test systems to measure the PTK2 kinase
activity are disclosed herein. Accordingly, it is preferred that
the inhibitors of the present invention reduce the kinase activity
of PTK2 about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or
even 100%, for example under conditions which are similar or
identical to the test system disclosed herein (for example the PTK2
enzyme test).
PTK2 Enzyme Test
[0037] This test uses active PTK2 enzyme (Invitrogen Code PV3832)
and poly-Glu-Tyr (4:1, Sigma P-0275) as the kinase substrate. The
kinase activity is detected by means of the phosphorylation of the
substrate in a DELFIA.TM. assay. The phosphorylated substrate is
detected with the europium-labelled phosphotyrosine antibody PT66
(Perkin Elmer, No.: AD0040). In order to determine
concentration-activity curves with PTK2 inhibitors the compounds
are serially diluted in 10% DMSO/H.sub.2O and 10 .mu.L of each
dilution are dispensed per well in a 96-well microtitre plate
(clear U-shaped base plate, Greiner No. 650101) (the inhibitors are
tested in duplicates) and mixed with 10 .mu.L/well of PTK2 kinase
(0.01 .mu.g/well). PTK2 kinase is diluted accordingly beforehand
with kinase dilution buffer (20 mM TRIS/HCl pH 7.5, 0.1 mM EDTA,
0.1 mM EGTA, 0.286 mM sodium orthovanadate, 10% glycerol with the
addition of freshly prepared BSA (fraction V 1 mg/mL) and DTT (1
mM)). The test compound and the PTK2 kinase are pre-incubated for 1
h at RT and shaken at 500 rpm. Then 20 .mu.L ATP Mix (30 mM
TRIS/HCl pH 7.5, 0.02% Brij, 0.2 mM sodium orthovanadate, 10 mM
magnesium acetate, 0.1 mM EGTA, 1.times. Phosphatase Inhibitor
Cocktail 1 (Sigma, No.: P2850), 50 .mu.M ATP (Sigma, No.: A3377; 15
mM stock solution)) are added. The reaction is started by the
addition of 10 .mu.L/well of poly (Glu, Tyr) substrate (25
.mu.g/well poly (Glu, Tyr), 0.05 .mu.g/well biotinylated poly (Glu,
Tyr) dissolved in 250 mM TRIS/HCl pH 7.5, 9 mM DTT)--the final
concentration of DMSO is 2%. After 1 h kinase reaction (the plates
are shaken at 500 rpm), the reaction is stopped by the addition of
12 .mu.L/well of 100 mM EDTA, pH 8. And shaken for a further 5 min
at RT (500 U/min).
[0038] 55 .mu.L of the reaction mixture are transferred into a
streptavidin plate (Strepta Well High Bind (transparent, 96-well)
made by Roche, No.: 11989685001) and incubated for 1 h at RT
(shaking at 500 rpm). Then the microtitre plate is washed three
times with 200 .mu.L/well D-PBS (Invitrogen, No.: 14190). 100 .mu.L
of 1:2000 diluted DELFIA Eu-NI Anti-Phosphotyrosine PT66 antibody
(Perkin Elmer, No.: AD0040, 1:2000 diluted in DELFIA test buffer
(Perkin Elmer, No.: 1244-111)) is then added and it is incubated
for 1 h at RT (shaking at 500 rpm). Then the plate is washed three
times with 200 .mu.L/well DELFIA washing buffer (Perkin Elmer, No.:
1244-114), 200 .mu.L/well strengthening solution (Perkin Elmer,
No.: 1244-105) is added and the whole is incubated for 10 min at RT
(shaking at 300 rpm).
[0039] The time-delayed europium fluorescence is then measured in a
microtitre plate reader (Victor, Perkin Elmer). The positive
control consists of wells that contain solvent (2% DMSO in test
buffer) and display uninhibited kinase activity. Wells that contain
test buffer instead of enzyme act as a control for the background
kinase activity. The IC.sub.50 values are determined from
concentration-activity analyses by iterative calculation using a
sigmoidal curve analysis algorithm (FIFTY, based on GraphPAD Prism
Version 3.03) with a variable Hill coefficient.
[0040] Further assays which might be used to identify PTK2
inhibitors are well-known to the skilled person and include inter
alia a PTK2 soft agar assay or a Phospho-PTK2 (pY397) Assay. Both
assays are explained in detail herein below.
PTK2 Soft-Agar Assay
[0041] This cellular test is used to determine the influence of
PTK2 inhibitors on the growth of PC-3 prostate carcinoma cells in
soft agar (`anchorage-independent growth`). After an incubation
time of two weeks the cell vitality is demonstrated by Alamar Blue
(resazurin) staining. PC-3 cells (ATCC CRL-1435) are grown in cell
culture flasks (175 cm.sup.2) with F12 Kaighn's Medium (Gibco, No.:
21127) which has been supplemented with 10% foetal calf serum
(Invitrogen, No.: 16000-044). The cultures are incubated in the
incubator at 37.degree. C. and 5% CO.sub.2 and are run twice a
week. The test is carried out in microtitre plates (Greiner, No.:
655 185) and consists of a lower layer made up of 90 .mu.L of
medium with 1.2% agarose (Invitrogen, 4% agarose gel 1.times.
liquid 40 mL, No.: 18300-012), followed by a cell layer in 60 .mu.L
medium and 0.3% agarose and finally a top layer comprising 30 .mu.L
medium which contains the test compounds (without the addition of
agarose). To prepare the lower layer, 4% agarose are decocted with
10.times.D-PBS (Gibco, No.: 14200) and H.sub.2O and thus prediluted
on 3% agarose in 1.times.D-PBS. The latter is adjusted with culture
medium (F12 Kaighn's/10% FCS) and FCS to a final dilution of 1.2%
agarose in F12 Kaighn's Medium with 10% FCS. Each well of a
microtitre plate is supplied with 90 .mu.L of the suspension for
the lower layer and cooled to RT for 1 h. For the cell layer, PC-3
cells are detached using trypsin (Gibco, 0.05%; No.: 25300),
counted and seeded in 60 .mu.L F12 Kaighn's (10% FCS) with the
addition of 0.3% agarose (37.degree. C.). After cooling to RT for 1
h the test compounds (30 .mu.L from serial dilutions) are added for
quadruple measurements. The concentration of the test compounds
usually covers a test range of between 10 .mu.M and 0.3 nM. The
compounds (stock solution: 10 mM in 100% DMSO) are prediluted in
F12 Kaighn's Medium+6% DMSO, to obtain a final concentration of 1%
DMSO. The cells are incubated at 37.degree. C. and 5% CO.sub.2 in a
steam-saturated atmosphere for 14 days. The metabolic activity of
living cells is then demonstrated with the dye Alamar Blue (AbD
Serotec, No.: BUFO 12B). To do this, 18 .mu.L/well of an Alamar
Blue suspension are added and the whole is incubated for approx. 8
h in the incubator at 37.degree. C. The positive control consists
of empty wells that are filled with a mixture of 18 .mu.L of Alamar
Blue reduced by autoclaving and 180 .mu.L of F12 Kaighn's Medium
(10% FCS). The fluorescence intensity is determined by means of a
fluorescence spectrometer (SpectraMAX GeminiXS, Molecular Devices).
The excitation wavelength is 530 nm, the emission wavelength is 590
nm.
[0042] The EC.sub.50 values are determined from
concentration-activity analyses by iterative calculation using a
sigmoidal curve analysis algorithm (FIFTY, based on GraphPAD Prism
Version 3.03) with a variable Hill coefficient.
Phospho-PTK2 (pY397) Assay
[0043] This cellular test is used to determine the influence of
PTK2 inhibitors on the state of the PTK2 phosphorylation at
tyrosine 397 (pY397).
[0044] PC-3 cells (prostate carcinoma, ATCC CRL-1435) are grown in
cell culture flasks (175 cm.sup.2) with F 12 Kaighn's Medium
(Gibco, No.: 21127) with the addition of 10% foetal calf serum
(Invitrogen, No.: 16000-044). The cultures are incubated in the
incubator at 37.degree. C. and 5% CO.sub.2 and run twice a
week.
[0045] For the test, 2.times.104 cells pro well/90 .mu.L medium are
plated out in 96-well microtitre plates (Costar, No.: 3598) and
incubated overnight in the incubator at 37.degree. C. and 5%
CO.sub.2. The test compounds (10 .mu.L from serial dilution) are
added the next day. The concentration of the test compounds usually
covers a range of 50 .mu.M and 0.8 nM. The test compounds (stock
solution: 10 mM in 100% DMSO) are diluted in medium/medium 10% DMSO
such that the final concentration is 1% DMSO. The cells are then
incubated in the incubator at 37.degree. C. and 5% CO.sub.2 for 2
h. Then the culture supernatant is removed and the cells are fixed
with 150 .mu.L 4% formaldehyde in D-PBS for 20 min at RT. The cell
lawn is washed five times with 200 .mu.L 0.1% Triton X-100 in D-PBS
for 5 min in each case and then incubated for 90 min with blocking
buffer (5% skimmed milk powder (Maresi Fixmilch) in TBST (25 mM
Tris/HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20). The blocking buffer
is replaced by 50 .mu.L of the first antibody anti-phospho PTK2
[pY397] rabbit monoclonal (Invitrogen/Biosource, No.: 44-625G),
which is diluted 1:200 in blocking buffer. For control purposes,
alternatively a PTK2 [total] antibody (clone 4.47 mouse monoclonal,
Upstate, No.: 05-537), diluted 1:400 in blocking buffer is used.
This incubation is carried out at 4.degree. C. overnight. Then the
cell lawn is washed five times with 100 .mu.L of 0.1% Tween in
D-PBS for 5 min in each case and 50 .mu.L/well of second antibody
are added. In order to detect bound phospho-PTK2 [pY397] antibody a
goat-anti-rabbit antibody is used which is coupled with horseradish
peroxidase (Dako, No.: P0448; 1:500 dilution in blocking buffer).
In order to detect bound PTK2 [total]-antibodies a
rabbit-anti-mouse antibody is used, which is also coupled with
horseradish peroxidase (Dako, No.: PO161; 1:1000 dilution in
blocking buffer). This incubation is carried out for 1 h at RT with
gentle shaking. The cell lawn is then again washed five times with
100 .mu.L of 0.1% Tween in D-PBS for 5 min in each case. Peroxidase
staining is carried out by adding 100 .mu.L staining solution (1:1
mixture of TMB peroxidase substrate (KPL, No.: 50-76-02) and
peroxidase solution B (H.sub.2O.sub.2) (KPL, No.: 50-65-02). The
development of the stain takes place for 10-30 min in the dark. The
reaction is stopped by the addition of 100 .mu.L/well of a 1 M
phosphoric acid solution. The absorption is determined
photometrically at 450 nm with an absorption measuring device
(VICTOR PerkinElmer). The inhibition of the anti-phospho PTK2
[pY397] immune staining is used to determine EC50 values. The
staining with anti-PTK2 [total]-antibodies is for control purposes
and should remain constant under the influence of inhibitor. The
EC50 values are determined from concentration-activity analyses by
iterative calculation with the aid of a sigmoidal curve analysis
algorithm (FIFTY, based on GraphPAD Prism Version 3.03) with a
variable Hill coefficient.
[0046] Compounds which effect a reduction of the amount of active
PTK2 in cells, in tissues comprising said cells or subjects
comprising said tissues or cells are likewise envisaged as PTK2
inhibitors and comprise, for example, aptamers, antibodies or
functional fragments thereof which are able to bind to and thereby
to inhibit PTK2; antisense oligonucleotides, iRNA, miRNA or siRNA
which specifically bind to the nucleotides sequences encoding PTK2
and thereby reduce the amount of active PTK2 in a cell or a tissue.
Such antibodies and interfering nucleic acid sequences are
well-known to the skilled person; plenty of them are even
commercially available.
[0047] Examples of PTK2 inhibitors which are envisaged in the
context of the present invention are the compounds which are
exemplified in WO 2010/106097, WO 2010/136559, WO 2011/039344, WO
2007/063384, WO 2010/058032, WO 2010/058030, WO 2010/055117, WO
2009/07153, WO 2005/1110245, WO 2008/129380, WO 2007/072158, WO
2005/111022, WO 2005/111023, WO 2005/111016, WO 2004/056807, WO
2009/071535, WO 2004/056786, WO 2010/062578, EP 2047849, WO
2008/115369, WO 2009/024332, WO 2008/129380, WO 2007/072158, WO
2004/056807, WO 2006/021457, WO 2010/062578, WO 2009/105498, WO
2004/030620, US 2008/167368, and/or WO 2009/153589 although the
invention is in no way limited thereto. The aforementioned
documents are included herein in their entirety by way of reference
thereto.
[0048] In the context of the present invention, the term "PTK2
inhibitor" is interchangeable with the term "PTK2 antagonist" or
the like.
[0049] PTK2 inhibitors show efficacy in a variety of experimental
models of cancer, in particular in human cancer xenograft models in
immunodeficient mice. However, their efficacy varies widely among
different cancer models: whereas cancer regression or complete
inhibition of growth can be achieved in some models, treatment of
other cancer types results in partial inhibition of growth and some
cancers are not affected at all. The present invention is based in
essence on the surprising finding that the level of expression of
E-cadherin protein in cancer cells from epithelial cancers can be
used to predict the sensitivity of the respective cancers to PTK2
inhibitors when scored by way of the scoring system as established
by the present invention. In view of that, it is envisaged to
detect the expression of E-cadherin protein in a cancer sample from
a patient, wherein an E-cadherin protein immunoreactivity score
(IRS) of 0-2, preferably of 0-1, more preferably an IRS score of 1
and even more preferably of 0, indicates that the cancer/cancer
patient is susceptible to treatment with a PTK2 inhibitor.
[0050] Said IRS score is preferably evaluated (detected) by way of
an IHC method, although other immunological methods are not
excluded.
[0051] An "IHC method" means the detection of targets (e.g.
antigens) in tissue sections by the use of binding domains as
specific reagents through target-binding domain interactions that
are visualized by a label. It is envisaged that said tissue
sections (which are for example about 2-5 .mu.m in thickness) are
taken from a tissue sample that has been preferably embedded, for
example in paraffin. IHC protocols describing details of the
methods of detection and the sources of the E-cadherin antibodies
are described below. The IHC method used in the examples was the
indirect avidin-biotin complex (ABC) immunoperoxidase method with
DAB as a substrate for the reaction. Further IHC methods are
explained in more detail herein below.
[0052] The term "binding domain" characterizes in connection with
the present invention a domain of a polypeptide which specifically
recognizes E-cadherin. The term "specifically recognizing
E-cadherin" or "specific for E-cadherin", means in accordance with
the present invention that the binding domain, e.g. an antibody, is
capable of specifically interacting with and/or binding to
E-cadherin. As used herein, the term "binds" in connection with the
interaction between E-cadherin and a binding domain indicates that
the binding domain associates with (e.g., interacts with or
complexes with) E-cadherin to a statistically significant degree as
compared to association with proteins generally (i.e., non-specific
binding). Thus, the term "binding domain" is also understood to
refer to a domain that has a statistically significant association
or binding with E-cadherin.
[0053] A preferred example of a binding domain in line with the
present invention is or comprises an epitope binding domain,
preferably an antibody, more preferably a monoclonal antibody or an
antigen binding fragment thereof.
[0054] The term "antibody" refers to a monoclonal or a polyclonal
antibody (see Harlow and Lane, "Antibodies, A Laboratory Manual",
CSH Press, Cold Spring Harbor, USA, 1988) which binds to a target,
or a derivative of said antibody which retains or essentially
retains its binding specificity. Preferred derivatives of such
antibodies are chimeric antibodies comprising, for example, a mouse
or rat variable region and a human constant region. The term
"antibody" also comprises bifunctional (bispecific) antibodies and
antibody constructs, like single-chain Fvs (scFv) or
antibody-fusion proteins. The term "scFv fragment" (single-chain Fv
fragment) is well understood in the art and preferred due to its
small size and the possibility to produce such fragments
recombinantly. Said antibody or antibody binding portion is for
example a rat, mouse, camel, goat, sheep, chicken, horse, or human
antibody or a humanized antibody. The term "humanized antibody"
means, in accordance with the present invention, an antibody of
non-human origin, where at least one complementarity determining
region (CDR) in the variable regions such as the CDR3 and
preferably all 6 CDRs have been replaced by CDRs of an antibody of
human origin having a desired specificity. Optionally, the
non-human constant region(s) of the antibody has/have been replaced
by (a) constant region(s) of a human antibody. Methods for the
production of humanized antibodies are described in, e.g., EP-A10
239 400 and WO90/07861. The term antibody or functional fragment
thereof also includes heavy chain antibodies and the variable
domains thereof, which are mentioned in WO 94/04678, WO 96/34103
and WO 97/49805, WO 04/062551, WO 04/041863, WO 04/041865, WO
04/041862 and WO 04/041867; as well as domain antibodies or
"dAb's", which are based on or derived from the heavy chain
variable domain (VH) or the light chain variable domain (VL) of
traditional 4 chain antibody molecules (see, e.g., Ward et al. 1989
Nature 341, 544-546).
[0055] The term "antigen binding fragment" as used herein refers to
fragments of the antibodies as specified herein which retain or
essentially retain the binding specificity of the antibodies like,
separated light and heavy chains, Fab, Fab/c, Fv, Fab', F(ab')2. An
antigen-binding fragment may comprise a light chain variable region
(VL) and a heavy chain variable region (VR) of an antibody;
however, it does not have to comprise both. Fd fragments, for
example, have two VH regions and often retain antigen-binding
function of the intact antigen-binding fragment.
[0056] The following exemplary antibodies can be employed in the
embodiments of the present invention. The invention is however not
limited to these specific antibodies: [0057] Mouse monoclonal
antibody [HECD-1] to extracellular domain of E-cadherin (Abcam
Ab1416). [0058] Mouse monoclonal antibody to E-cadherin (Dako
M3612) recognizes the 120 kD mature form and 82 kD fragment of
E-cadherin [0059] Mouse Anti-E-cadherin monoclonal antibody
(Invitrogen 18-0223); reacts with the cytoplasmic domain of human
E-cadherin. [0060] Mouse monoclonal antibody to E-cadherin
(Sigma-Aldrich WH0000999M1) [0061] Rabbit polyclonal antibody to
E-cadherin (Cell Signalling 4065)
[0062] The term "epitope binding domain" includes, besides
antibodies or antigen binding fragments thereof (sometimes also
denoted as "functional fragments"), other binding entities which
bind to (specifically bind to) a proteinaceous target such as
E-cadherin. The term "epitope binding domain" includes, for
example, a domain antibody (dAb), for example a human, camelid or
shark immunoglobulin single variable domain or it may be a domain
which is a derivative of a scaffold selected from the group
consisting of CTLA-4 (Evibody); lipocalin; Protein A derived
molecules such as Z-domain of Protein A (Affibody, SpA), A-domain
(Avimer/Maxibody); Heat shock proteins such as GroEl and GroES;
transferrin (trans-body); ankyrin repeat protein (DARPin); peptide
aptamer; C-type lectin domain (Tetranectin); human
.gamma.-crystallin and human ubiquitin (affilins); PDZ domains;
scorpion toxin kunitz type domains of human protease inhibitors;
and fibronectin (adnectin); which has been subjected to protein
engineering in order to obtain binding to a ligand other than the
natural ligand. CTLA-4 (Cytotoxic T Lymphocyte-associated Antigen
4) is a CD28 family receptor expressed on mainly CD4+ T-cells. Its
extracellular domain has a variable domain-like Ig fold. Loops
corresponding to CDRs of antibodies can be substituted with
heterologous sequence to confer different binding properties.
CTLA-4 molecules engineered to have different binding specificities
are also known as Evibodies. For further details see Journal of
Immunological Methods 248 (1-2), 31-45 (2001).
[0063] The binding domain of the present invention is either
labeled or unlabeled.
[0064] A label refers in the context of the invention to a compound
or composition detectable by spectroscopic, photochemical,
biochemical, immunochemical, or chemical means.
[0065] The label may be directly or indirectly detectable.
[0066] "Directly" means that the label as such generates the signal
such as a radioactive, chromogenic, or fluorescent signal. Direct
labels include radiolabels, fluorescent label, electron-dense
reagents; etc. The direct label has or generates a measurable
signal that can be used to quantify and/or detect (qualitatively)
the bound binding domain.
[0067] "Indirectly" means that the label is for example bound by
another entity which as such is then detectable (detection entity).
Indirect detection or indirect label involves the binding of a
second directly or indirectly detectable binding domain to the
indirect label. For example, the indirect label of the binding
domain of the invention can be the ligand of a binding partner,
such as biotin, which is a binding partner for streptavidin, or a
nucleotide sequence, which is the binding partner for a
complementary sequence, to which it can specifically hybridize etc.
"Indirect labelling" is therefore characterized in that the primary
binding domain is manipulated such that it can be detected by a
second binding domain (sometimes also denoted detection entity)
which is specific for that manipulation (e.g. a biotin label).
[0068] In a preferred embodiment said binding domain is an antibody
(primary antibody) and said detection entity is a secondary
antibody which specifically reacts with the primary antibody as
such or the label of the primary antibody.
[0069] It is also envisaged that indirect and direct labelling is
mixed. For example, already the direct label is able to generate a
signal (e.g. a fluorescent label like FITC) but the secondary
binding domain, which is also labelled, e.g. with a direct label
binds specifically to that label (anti FITC antibody) binds to the
label of the primary signal and, thereby, may increase the
detectable signal. Such means and methods are well-known to the
skilled person, in particular to practitioners in the field of
immunochemistry.
[0070] In another embodiment, the primary binding domain (e.g. an
antibody) is not labeled as such but is detected/detectable by way
of a secondary binding domain (detection entity) which binds to the
primary binding domain (for example a primary non-labeled antibody
raised in mouse is detected by a second, labeled antibody, which
was raised in another species and specifically binds to mouse
antibodies (e.g. goat anti-mouse)). The secondary binding domain is
then directly or indirectly labeled. Such antibody detection
sandwiches are well-established and the skilled person will have no
problem to generate/establish or create such systems.
[0071] The "detection" (sometimes also denoted as "determination"
and grammatical variants thereof) which takes place ex vivo is
carried out by standard detection techniques which are well-known
to the skilled person and include, but are not limited to, any kind
of suitable IHC detection techniques, such as light and
fluorescence-based microscopy including near infrared based
microscopy, and confocal microscopy.
[0072] The term "ex vivo", which is interchangeable with "in vitro"
refers to activities conducted in cells in a controlled
environment. The methods of the present invention are conducted ex
vivo.
[0073] As mentioned hereinbefore, immunohistochemistry (IHC) is the
detection of targets (in the context of the present invention e.g.
E-cadherin) and/or subsets of cells presenting said target in
tissue sections by the use of binding domains which are either
directly labeled (direct IHC) or indirectly labeled (indirect IHC),
which binding domains react with their target through specific
target-binding domain interactions. In the context of the present
invention, said target is E-cadherin.
[0074] These interactions are then visualized by the mentioned
label. There are mainly two strategies used for the
immunohistochemical (IHC) detection of antigens in tissue, the
direct method and the indirect method. The direct method of IHC
uses one directly labelled binding domain, which binds directly to
the target being stained for. The direct method is thus a one-step
staining method, and involves e.g. a labelled antibody (e.g. FITC
conjugated antiserum) reacting directly with the antigen in tissue
sections. This technique utilizes only one antibody and the
procedure is therefore simple and rapid. The indirect method of IHC
uses one binding domain against E-cadherin, and a second, labelled,
binding domain against the first binding domain. A second binding
domain is for example an antibody raised against the IgG of the
animal species in which the primary antibody has been raised.
[0075] It is envisaged that in some embodiments of the methods of
the present invention, said IHC method is characterized by the
following steps: [0076] (a) optionally providing a cancer sample
(or, alternatively, optionally providing a container comprising a
cancer sample); [0077] (b) fixation of said cancer sample; [0078]
(c) optionally embedding the cancer sample in paraffin; [0079] (c)
incubation of the fixed cancer sample with an E-cadherin specific
binding domain; [0080] (d) directly or indirectly detecting the
binding domain and thereby the E-cadherin expression on the cancer
cells.
[0081] "Fixing" or "fixation" means a fixation procedure which is
suitable to prepare the cancer sample for a subsequent IHC
detection procedure. A "fixation" is particularly carried out in
order to ensure the preservation of tissue architecture and cell
morphology. Suitable fixation conditions are well-known and also
disclosed herein. Alternatively, it is also envisaged that the
tissue/subset is preserved by way of deep-freezing (e.g. in liquid
nitrogen).
[0082] All the above pre-treatment steps/measures are within the
scope of the term "fixation", i.e. fixation specifically includes
fixation with fixing agents like formaldehyde, paraformaldehyde;
and/or deep-freezing of the tissue sample/subset of cells, and/or
optionally also the embedding of the tissue/subset of cells in
paraffin or similar agents.
[0083] Means and methods to put the different IHC protocols into
practice are well-known to the skilled person. See for example the
respective protocols in the literature or in the internet (for
example www.ihcworld.com).
[0084] The most common fixative used for immunohistochemistry is
paraformaldehyde, which is frequently used in diverse buffers
containing about 1 to 5% paraformaldehyde. Specific buffers which
are based on paraformaldehyde are exemplified in the following:
[0085] a) 4% paraformaldehyde in 0.1 M phosphate buffer [0086] b)
2% paraformaldehyde with 0.2% picric acid in 0.1 M phosphate buffer
[0087] c) PLP (paraformaldehyde, lysine, paraformaldehyde)
fixative: e.g. 4% paraform-aldehyde, 0.2% periodate and 1.2% lysine
in 0.1 M phosphate buffer [0088] d) 4% paraformaldehyde with 0.05%
glutaraldehyde.
[0089] These buffers are not intended to limit the invention but
simply illustrate specific conditions which are normally applied to
achieve a sufficient fixation of tissue. The standard fixation time
is about 5, 10, 15, 20, 30 min to overnight. The so-treated tissue
is frequently subject to a subsequent paraffin embedding protocol,
followed by the incubation in organic solvents like for example
xylene and ethanol treatment. The sample is then normally hydrated
by placing it in 95%, 70%, 50%, 30% alcohol (e.g. ethanol) for
several minutes each. There is, however, no standard protocol for
IHC, i.e. the protocol will vary depending on the tissue, the
binding domains used etc. All this is known to the skilled person
and routinely handled without further ado. Specific protocols are
disclosed for example in the internet (searchable with the string
"IHC protocols" in a search machine like Google etc.). Some
antigens will not even survive moderate amounts of aldehyde
fixation. Under this condition, tissues are often fresh frozen in
liquid nitrogen and cut with a cryostat. The sections are kept
frozen at -20.degree. C. or lower until fixation with cold acetone
or alcohol.
[0090] Once a "signal" is obtained, proving that signal truly
reflects the distribution of the target is still a matter of some
difficulty. The simplest negative control is the absence of
expression in tissues in which the RNA for E-cadherin is known not
to be expressed. An alternative negative control is the elimination
of the signal by pre-incubating the binding domain with an excess
of the peptide or protein with which it was raised.
[0091] It is also envisaged that the IHC methods of the present
invention are combined with other techniques which are applicable
on tissue sections, such as in situ hybridization techniques (e.g.
fluorescent in situ hybridization), in order to verify or detect
further cancer associated signals and or other intracellular
signals of cancer cells.
[0092] Methods, for example IHC methods, which can be employed in
the context of the present invention are disclosed herein and
exemplified in the appended examples.
[0093] The gist of the present invention is in essence the
surprising finding that the level of expression of E-cadherin
protein in cancer cells can be used to predict the sensitivity of
the respective cancers to PTK2 inhibitors. In view of that, it is
envisaged to detect the expression of E-cadherin protein in a
cancer/in a cancer sample from a patient, wherein an E-cadherin
protein immunoreactivity score (IRS) of 0-2, preferably an IRS
score of 0-1, more preferably an IRS score of 1 and even more
preferably an IRS score of 0 indicates that the cancer/cancer
patient is susceptible to treatment with a PTK2 inhibitor.
[0094] The IRS score of the present invention was established as
follows. We have assessed the number of membrane-bound E-cadherin
positive cancer cells using paraform-aldehyde-fixed and
paraffin-embedded tissue samples. The cancers used were derived
from xenograft models of human cancer cell lines grown in nude mice
(BomTac:NMRI-Foxn1nu), including pancreatic cancer cell lines
MiaPaCa-2 and BxPC-3; prostate cancer cells PC-3 and ovarian cancer
cells TOV-21G.
[0095] The entire section from each sample was analyzed and an
average of the percentage of E-cadherin positive cancer cells
determined as the number of E-cadherin positive cells over the
total number of cancer cells in all the histologic fields examined.
Only membrane E-cadherin immunoreactivity in cancer cells was
considered positive for scoring purposes. The person skilled in the
art of pathology understands which cells are relevant under the
conditions present when performing the method and may determine the
fraction of positive cells based on his/her general knowledge and
the teachings of the present disclosure.
[0096] The scoring as proposed here is semi-quantitative; the
protein expression levels are recorded as 0, 1, 2, 3 or 4 with 0
being substantially no detectable protein expression (less than 1%)
and 4 being the highest detected protein expression (>60%).
[0097] As positive control, tissue sections from normal colonic
mucosa and/or colorectal cancer samples known to express E-cadherin
could be included. The connective tissue present in any given
tissue (normal and tumor) could serve as a negative control in
these assays.
[0098] The mentioned "control", "positive control" or "control
sample" is preferably a sample (cell or tissue) which allows a
comparison with the test sample, for example because both samples
are mainly composed of the same cell type (e.g. both consist of
epithelial cells) or both samples are derived from the same tissue
yet from a different source. "A different source" includes e.g. a
cell/tissue sample obtained from a healthy subject, preferably from
a subject who does not suffer from a cancer or a cell/tissue sample
obtained from a distinct part of the same subject wherein said
distinct part appears to be free from associated symptoms of a
cancer.
[0099] The number of E-cadherin positive cancer cells was scored as
follows:
[0100] 0 (less than 1%), 1 (about 1-10%), 2 (about 11-30%), 3
(about 31-60%), 4 (>60%). "Positive cells" thereby means cancer
cells showing E-cadherin expression as explained herein above. The
scoring mentioned herein applies to all embodiments of the present
invention. Preferably, an E-cadherin protein immunoreactivity score
(IRS) of IRS-0 is characterized by an average of less than 1%
membrane E-cadherin positive cancer cells over the total number of
cancer cells in one or more histologic field(s) examined; IRS-1 is
characterized by an average of about 1-10% membrane E-cadherin
positive cancer cells over the total number of cancer cells in one
or more histologic field(s) examined; IRS-2 is characterized by an
average of about 11-30% membrane E-cadherin positive cancer cells
over the total number of cancer cells in one or more histologic
field(s) examined; IRS-3 is characterized by an average of about
31-60% membrane E-cadherin positive cancer cells over the total
number of cancer cells in one or more histologic field(s) examined;
and IRS-4 is characterized by an average of more than 60% membrane
E-cadherin positive cancer cells over the total number of cancer
cells in one or more histologic field(s) examined. The mentioned
scoring is, in a preferred embodiment, conducted via IHC using
paraformaldehyde-fixed and paraffin-embedded tissue samples.
[0101] "One or more" includes in this regard 1, 2, 3, 4, 5, 6, 7,
8, 9 or even more histological fields which are analysed
(preferably per sample), depending on the circumstances. At least
"3" histological fields are preferred.
[0102] When testing the above mentioned xenograft models of human
cancer cell lines grown in nude mice, we could demonstrate that
PTK2 inhibitors where highly efficacious in certain models whereas
the response in others was rather low. The xenograft models were
established as follows: Athymic female BomTac:NMRI-Foxnlnu mice
about six weeks of age were allowed to adjust to the new
environment for at least 3 days before they were used for
experiments. The animals were housed under standardized conditions
in groups of 5 in Macrolon.RTM. type II cages. Standardized diet
(PROVIMI KLIBA) and autoclaved tap water were provided ad libitum.
To establish subcutaneous tumors, cells were harvested by
trypsinization, centrifuged, washed and resuspended in ice-cold
PBS+5% FCS. 100 .mu.L cell suspension containing 5,000,000 cells
was then injected subcutaneously into the right flank of the nude
mice (1 site per mouse). Mice were randomly distributed between the
treatment and the vehicle control group (10-14 days after cell
injection) when tumors were well established and had reached
diameters of 6-9 mm. The tumor diameter was measured three times a
week (Monday, Wednesday and Friday) with a caliper. The volume of
each tumor [in mm.sup.3] was calculated according to the formula
"tumor volume=length*diameter2*.pi./6". To monitor side effects of
treatment, mice were inspected daily for abnormalities and body
weight was determined three times a week (Monday, Wednesday and
Friday). Animals were sacrificed at the end of the study about
three weeks after start of treatment. Animals with necrotic tumors
or tumor sizes exceeding 2000 mm.sup.3 were sacrificed early during
the studies for ethical reasons.
[0103] When testing the above mentioned xenograft models of human
cancer cell lines grown in nude mice, we could demonstrate that
PTK2 inhibitors where highly efficacious in certain models whereas
the response in others was rather low. For example the human
pancreatic adenocarcinoma model derived from the cell line
MiaPaCa-2 was highly responsive to treatment with PTK2 inhibitors
resulting in strong inhibition of cancer growth (i.e. "tumor growth
inhibition" or TGI), indicated by a TGI of 114% and the occurrence
of cancer regressions. Tissue sections were prepared from this
cancer and stained by immunohistochemistry with an antibody against
E-cadherin. Protein expression of E-cadherin was completely absent
in these cancer sections corresponding to an IRS of "0" (<1%
E-cadherin positive cells). TGI is defined herein below.
[0104] In contrast, two further pancreatic adenocarcinoma models
derived from the cell lines BxPC-3 or AsPC-1 showed only weak or no
sensitivity following the treatment with PTK2 inhibitors with TGIs
of 49% (BxPC-3) and 13% (AsPC-1) and the complete absence of cancer
regressions. Immunohistochemistry of tissue sections from these
cancer models with antibodies against E-cadherin demonstrated a
strong expression of E-cadherin in these cancers corresponding to
the IRS of "4" (>60% positive cells) for BxPC-3 and "3" (31-60%
positive cells) for AsPC-1 (see FIG. 2 for further illustration).
It is therefore envisaged that these adenocarcinoma models could
serve as a reference/reference sample for the evaluation and/or
adjustment of the IRS score of the present invention. A BxPC-3
xenograft could be seen as a reference for an IRS score of "4"
while an AsPC-1 xenograft may serve as a reference for IRS score
"3". Likewise "MiaPaCa-2", could serve as a reference for an
E-cadherin IRS of "0". The generation of such xenografts is
described herein.
[0105] The above data show that a minor or absent expression of
E-cadherin corresponds with the sensitivity of preclinical cancer
models to PTK2 inhibitors whereas cancers with an intermediate to
high percentage of E-cadherin expressing cancer cells seem to be
less sensitive or insensitive. In view of that, it could be
established that an E-cadherin protein immunoreactivity score 0-2,
preferably an IRS score of 0-1, and even more preferably an IRS
score of 0 indicates that the cancer/cancer patient is susceptible
to treatment with a PTK2 inhibitor.
[0106] The "tumor growth inhibition" or "TGI" is defined as
follows:
TGI = 100 * ( C d - C 1 ) - ( T d - T 1 ) ( C d - C 1 ) ,
##EQU00001##
wherein "C.sub.1" and "T.sub.1" means median tumor volumes in
control and treatment group at start of the experiment at day 1,
and "C.sub.d" and "T.sub.d" means median tumor volumes in control
and treatment group at end of the experiment at day d.
[0107] In a further embodiment, the present invention relates to a
method of treating a patient with a cancer having an E-cadherin
protein immunoreactivity score 0-2, preferably an IRS score of 0-1,
more preferably an IRS score of 1 and even more preferably an IRS
score of 0, comprising administering to the patient a
therapeutically effective amount of a PTK2 inhibitor.
[0108] It will be understood that within the context of the
embodiments of the present invention, the E-cadherin protein
immunoreactivity score is preferably evaluated by way of an IHC
method as indicated above and exemplified herein. Suitable IHC
methods are described herein although the present invention is not
limited to these specific protocols.
[0109] By "therapeutically effective amount" or "therapeutically
active" is meant a dose of a PTK2 inhibitor that produces the
therapeutic effects for which it is administered. The
therapeutically effective amount of the drug may reduce the number
of cancer cells, reduce the cancer size, inhibit (i.e., slow to
some extent and preferably stop) cancer cell infiltration into
peripheral organs, inhibit (i.e., slow to some extent and
preferably stop) cancer metastasis, inhibit, at least to some
extent, cancer growth, and/or relieve to some extent one or more of
the symptoms associated with the disorder. The exact dose will be
ascertainable by one skilled in the art using known techniques. As
is known in the art, adjustments for age, body weight, general
health, sex, diet, drug interaction and the severity of the
condition may be necessary, and will be ascertainable with routine
experimentation by those skilled in the art.
[0110] It was shown for the first time by the present inventors
that the E-cadherin protein immunoreactivity score of a cancer
sample correlates well with the susceptibility of the respective
cancer to PTK2 inhibitor treatment. Based on these findings, it is
now possible to stratify cancer patients or cancers (or cancer cell
lines) which are susceptible to a treatment with PTK2 inhibitors.
Based on the findings of the present invention, it is now clear
that patients whose cancers show an E-cadherin protein
immunoreactivity score (IRS) of 0, 1 or 2 should preferentially be
treated with PTK2 inhibitors, as the probability of achieving a
therapeutic benefit will be higher for those patients than for
patients with IRS 3 or 4. It is however envisaged that cancers
having an E-cadherin protein immunoreactivity score of 3 or above 3
may also be treated with an PTK2 inhibitor, as the high IRS score
(3-4) although clearly indicating a lower probability of achieving
a therapeutic benefit for the patient with PTK2 inhibitors, does
not necessarily exclude a residual therapeutic effect of these PTK2
inhibitors. Such patients could also be treated with alternative
(alternative to PTK2 inhibitors) or additional anti-cancer
therapies (i.e. therapies which are not based on PTK2 inhibitors).
Additional or alternative anti-cancer therapies include, but are
not limited to therapies which can be selected from antineoplastic
agents, anti-angiogenic agents, chemotherapeutic agents and peptide
cancer therapy agents. The antineoplastic agents can be selected
from antibiotic-type agents, alkylating agents, antimetabolite
agents, hormonal agents, immunological agents, interferon-type
agents, kinase inhibitors, and combinations thereof. Such
pharmaceutically active compound/agent is for example a traditional
small organic chemical molecule or can be macromolecules such as a
proteins, antibodies (including fragments thereof), peptibodies,
DNA, RNA or fragments of such macromolecules.
[0111] The present invention further relates to a method for
stratifying cancer patients that are susceptible to treatment with
a PTK2 inhibitor, comprising determining the E-cadherin IRS score
in a cancer sample of said patient, wherein an E-cadherin protein
immunoreactivity score (IRS) of 0-2 (i.e. 2, 1, or 0) indicates
that the cancer patient is susceptible to treatment with a PTK2
inhibitor. An E-cadherin protein immunoreactivity score (IRS) of
3-4 (i.e. 3, or 4) indicates that the patient is not susceptible to
treatment with a PTK2 inhibitor or at least that the probability of
achieving a therapeutic benefit for the patient with PTK2
inhibitors is rather low.
[0112] The term "stratify" or "stratifying" refers to sorting
patients into those who are more (or less) likely to benefit from
an anti-cancer therapy which is based on a PTK2 inhibitor than
others. The methods of present invention may thus be employed for
stratifying cancer patients with regard to their susceptibility to
treatment with a PTK2 inhibitor. As mentioned, those patients whose
cancers show an E-cadherin protein immunoreactivity scores IRS of
0-2, preferably an IRS score of 0-1, more preferably an IRS score
of 1 and even more preferably an IRS score of 0 are more likely to
benefit from said PTK2 inhibitor based therapy, while those whose
E-cadherin protein immunoreactivity score (IRS) of the cancer is 3
or greater are less likely to benefit from such a therapy.
[0113] Specifically, a "patient who may benefit" from anti-cancer
therapy with a PTK2 inhibitor is a patient in which a PTK2
inhibitor has a higher likelihood to have a therapeutic effect. The
likelihood that (a) cancer and/or a cancer patient may or may not
respond favorably is dependent on the E-cadherin protein
immunoreactivity score on the cell membrane of the respective
cancer cells of said patient, as described herein.
[0114] Correspondingly, a "patient who may not benefit" from
anti-cancer therapy with a PTK2 inhibitor is a patient in which a
PTK2 inhibitor does not have a higher likelihood to have a
therapeutic effect.
[0115] The present invention further relates to the use of a PTK2
inhibitor as defined herein for the preparation of a pharmaceutical
composition for the treatment of a cancer patient whose cancer is
characterized by an E-cadherin protein immunoreactivity score of
0-2, preferably an IRS score of 0-1, and even more preferably an
IRS score of 0.
[0116] Said pharmaceutical composition may further comprise
pharmaceutically acceptable carriers and/or diluents. Examples of
suitable pharmaceutically acceptable carriers and/or diluents are
well known in the art and include phosphate buffered saline
solutions, water, emulsions, such as oil/water emulsions, various
types of wetting agents, sterile solutions etc. Compositions
comprising such carriers can be formulated by well known
conventional methods.
[0117] These pharmaceutical compositions of the present invention
can be administered to a subject at a suitable dose. The dosage
regimen will be determined by the attending physician and clinical
factors. As is well known in the medical arts, dosages for any one
patient depends upon many factors, including the patient's size,
body surface area, age, the particular compound to be administered,
sex, time and route of administration, general health, and other
drugs being administered concurrently. Preparations for parenteral
administration include sterile aqueous or non-aqueous solutions,
suspensions, and emulsions. Examples of non-aqueous solvents are
propylene glycol, polyethylene glycol, vegetable oils such as olive
oil, and injectable organic esters such as ethyl oleate. Aqueous
carriers include water, alcoholic/aqueous solutions, emulsions or
suspensions, including saline and buffered media. Parenteral
vehicles include sodium chloride solution, Ringer's dextrose,
dextrose and sodium chloride, lactated Ringer's, or fixed oils.
Intravenous vehicles include fluid and nutrient replenishers,
electrolyte replenishers (such as those based on Ringer's
dextrose), and the like. Preservatives and other additives may also
be present such as, for example, antimicrobials, anti-oxidants,
chelating agents, and inert gases and the like.
[0118] Furthermore, the pharmaceutical compositions of the
invention may comprise further agents such as additional
anti-cancer therapies/agents. Additional anti-cancer therapies
include, but are not limited to therapies which can be selected
from antineoplastic agents, anti-angiogenic agents,
chemotherapeutic agents and peptide cancer therapy agents. The
antineoplastic agents can be selected from antibiotic-type agents,
alkylating agents, antimetabolite agents, hormonal agents,
immunological agents, interferon-type agents, kinase inhibitors,
and combinations thereof. Such pharmaceutically active
compound/agent is for example a traditional small organic chemical
molecule or can be macromolecules such as a proteins, antibodies
(including fragments thereof), peptibodies, DNA, RNA or fragments
of such macromolecules. Such therapies are well known to the
skilled person.
[0119] The route of administration of the PTK2 inhibitors described
herein or the pharmaceutical compositions of the present invention
depends on the circumstances and includes (but is not limited to)
oral administration, parenteral administration, e.g.,
intravenously, intramuscularly, intraperitonealy, etc., subcutan
administration, transdermal adminis-tration, inhalative
administration, by suppository etc.
[0120] In a further embodiment, the present invention relates to a
PTK2 inhibitor as defined herein for use in the treatment of a
cancer patient whose cancer is characterized by an E-cadherin
protein immunoreactivity score of 0-2, preferably an IRS score of
0-1, more preferably an IRS score of 1, and even more preferably an
IRS score of 0.
[0121] It is preferred that within the context of the present
invention, the cancer patient is or has been identified
(characterized or stratified) with a method as defined herein,
preferably with the IHC method described herein. Said
identification or stratification method may be carried out prior to
and/or during said treatment with said PTK2 inhibitor.
[0122] The present inventors established that the E-cadherin
protein immunoreactivity score of a cancer sample correlates very
well with the susceptibility of the respective cancer to PTK2
inhibitor treatment. Based on this finding, it is now possible to
select, based on the respective E-cadherin IRS, an appropriate
anti-cancer therapy which is potentially therapeutically effective
for a cancer patient suffering from cancer, and in particular for
cancer patients suffering from a carcinoma.
[0123] Patients and/or cancers whose E-cadherin protein
immunoreactivity score (IRS) is greater then 3 should, based on the
findings of the present invention, be less susceptible to treatment
with PTK2 inhibitors. These patients can additionally or
alternatively be treated with alternative anti-cancer therapies,
although it cannot be excluded that PTK2 inhibitors might still
exert a beneficial effect in these patients. Thus, it is still
possible to employ these compounds even in patients which are
characterized by an E-cadherin IRS score of 3 or above, although it
is expected that the respective PTK2 inhibitors will not be as
effective in these patients as in patients which are characterized
by an E-cadherin IRS of 2, preferably 1 and even more preferably 0.
Accordingly, it is to be understood that by way of the present
invention it is possible to select a more suitable therapy form
depending on the respective E-cadherin IRS score.
[0124] The present invention therefore relates in a further
embodiment to a method for selecting an anti cancer treatment for a
cancer patient comprising the steps of: [0125] (a) determining the
E-cadherin protein immunoreactivity score of a cancer sample of
said patient; and [0126] (b) selecting preferably a PTK2 inhibitor
as anti cancer treatment for said patient if the E-cadherin protein
immunoreactivity score determined in (a) is 2, 1 or 0, (1 being
preferred and 0 being more preferred), or [0127] (c) selecting
preferably an alternative or an additional anti cancer treatment if
the E-cadherin protein immunoreactivity score determined in (a) is
3 or 4.
[0128] "Selecting preferably a PTK2 inhibitor as anti cancer
treatment" means that it is preferred to use a PTK2 inhibitor based
therapy for cancers having an E-cadherin protein immunoreactivity
score of 2, 1 or 0 (1 being preferred and 0 being more preferred).
It is however envisaged that cancers having an E-cadherin protein
immunoreactivity score of 3 or above 3 may also be treated with an
PTK2 inhibitor, as the high IRS score (3-4) although clearly
indicating a lower probability of achieving a therapeutic benefit
for the patient with PTK2 inhibitors, does not necessarily exclude
a residual therapeutic effect of these PTK2 inhibitor. In such
cases (high IRS score of 3 or above), the PTK2 inhibitor is
preferably not used alone.
[0129] Based on the novel findings of the present invention, namely
that a cancer which is characterized by an E-cadherin protein
immunoreactivity score of 2, 1 or 0 (1 being preferred and 0 being
even more preferred) is susceptible to a PTK2 inhibitor, it is also
envisaged to provide a screening method which employs cancer cells
characterized by an E-cadherin protein immunoreactivity score of 2,
1 or 0 (1 being preferred and 0 being more preferred), to screen
for PTK2 inhibitors, as these cancer cells are (more) susceptible
to PTK2 inhibitors and thereby may enable the positive
identification of novel PTK2 inhibitors which, otherwise (i.e. when
employing cancer cells characterized by an E-cadherin protein
immunoreactivity score of 3 or above 3), would have been sorted
out.
[0130] Therefore, in a further embodiment, the present invention
relates to a method of screening for a therapeutically effective
PTK2 inhibitor comprising the following steps: [0131] (a) providing
cancer cells or a cancer cell line which are characterized by an
E-cadherin protein immunoreactivity score of 2, 1, or 0 (1 being
preferred and 0 being even more preferred) [0132] (b) contacting
the cancer cell or the cancer cell line of (a) with a PTK2
inhibitor; and [0133] (c) evaluating whether the PTK2 inhibitor
negatively affects the cancer cell/cancer cell lines.
[0134] The above screening method is preferably a "phenotypical"
screening method which employs phenotypically detectable changes of
the cancer cells in response to the PTK2 inhibitor (these responses
then indicate whether the PTK2 inhibitor is therapeutically
effective or not). The term "phenotypically detectable changes"
means changes which negatively affect the cancer cells. These
negative effects includes cell death such as apoptosis or necrosis,
reduced migration capabilities of the cancer cells/cancer cell
lines and/or inhibition of proliferation of these cells, to name
some. This list is however not exclusive, i.e. the skilled person
is aware of further phenotypical changes of cancer cells which
might indicate that a test compound actually negatively affects the
respective cell. By way of the above screening method it is easier
to identify therapeutically effective PTK2 inhibitors.
[0135] The present invention also relates to a pharmaceutical
package comprising at least one PTK2 inhibitor, and: [0136] (a)
instructions and/or an imprint indicating that said PTK2 inhibitor
is preferably used for the treatment of patients which suffer from
a cancer which is characterized by an E-cadherin protein
immunoreactivity score of 2 or below 2; and/or [0137] (b)
instructions and/or an imprint indicating that said patient is to
be stratified by a method described herein; and/or [0138] (c) means
to carry out a method as defined herein.
[0139] "Means to carry out a method as defined herein" includes
inter alia E-cadherin specific antibodies, positive and/or negative
controls as described herein elsewhere, buffers which may be used
for IHC methods, and/or other means which can be used for the
detection methods of the present invention, such as control
antibodies, secondary antibodies, glass or plastic slides for IHC
etc.
[0140] In a further embodiment, the present invention relates to a
pharmaceutical kit or package comprising a PTK2 inhibitor and
further comprising an E-cadherin antibody which is used for the
prediction of the E-cadherin IRS.
[0141] In another embodiment, the present invention relates to a
diagnostic kit or package comprising an E-cadherin antibody for the
prediction of the E-cadherin IRS and a PTK2 inhibitor which is used
for the treatment of patients which have been determined,
characterized, identified or stratified in accordance with the
methods of the present invention.
[0142] In a further aspect, the present invention relates to a kit,
preferably a diagnostic kit or diagnostic package comprising means
to detect E-cadherin protein expression in accordance with the
means and methods of the present invention and: [0143] (a) package
inserts and/or instructions as to carry out said E-cadherin
detection (scoring); and/or [0144] (b) positive and/or negative
controls which allow the verification of the score.
[0145] The term "package insert and/or instructions" is used to
refer to instructions customarily included in commercial packages
of diagnostic products that contain information about the methods,
usage, storage, handling, and/or warnings concerning the use of
such diagnostic products. "Positive controls" includes cancer cell
or cancer samples expressing E-cadherin, or E-cadherin as such
(protein control) which might be used in standard immunological
methods as described herein. "Negative controls" includes reference
cells or reference samples which do not express E-cadherin on the
protein level. Both, the positive as well as the negative control
could also be replaced by pictures indicating the respective score
and thereby aiding the practitioner. "Means to detect E-cadherin
protein expression" includes inter alia E-cadherin specific
antibodies (i.e. antibodies binding to E-cadherin).
[0146] The present invention relates in a further embodiment to an
E-cadherin antibody for use in the stratification of cancer
patients with regard to their susceptibility to treatment with a
PTK2 inhibitor. The stratification can be conducted in accordance
with the methods of the present invention.
[0147] Likewise, the present invention relates to an E-cadherin
antibody for use in a method for determining whether a cancer
patient is susceptible to treatment with a protein tyrosine kinase
2 (PTK2) inhibitor. Said method comprises the detection of the
expression of the E-cadherin protein in a cancer sample of said
cancer patient, wherein an E-cadherin protein immunoreactivity
score (IRS) of 0-2 indicates that the cancer patient is susceptible
to treatment with a PTK2 inhibitor.
[0148] The present inventors established that the E-cadherin
protein expression as evaluated for example by way of the protein
immunoreactivity score as described herein, correlates very well
with the susceptibility of the respective cancer to PTK2 inhibitor
treatment. It is however assumed that the expression profile of the
E-cadherin mRNA when evaluated in the respective cancer cells is
likewise predictive for the susceptibility of the respective
cancers/cancer patients to PTK2 inhibitor treatment. Thus, it has
to be understood that gist of the present invention extends to the
evaluation of the E-cadherin mRNA as well. Thus, all the
embodiments of the present invention which are disclosed and
described herein equally apply to the measurement of the expression
level of E-cadherin mRNA in cancer cells. It is particularly
assumed that a decreased expression of E-cadherin mRNA or no
expression of E-cadherin mRNA indicates an increased susceptibility
of the respective cancer/cancer patient to treatment with (a) PTK2
inhibitor(s). Methods which allow the skilled person to evaluate
the expression profile of E-cadherin expressing cancer cells are
well-known to the skilled person and include for example Northern
blotting, mRNA profiling techniques including detection techniques
based on nucleic acid arrays, polymerase chain reaction (PCR)
techniques, such as real time quantitative PCR or "Q-PCR" etc. It
will be understood that the present invention is not limited to
these techniques which are merely exemplified to illustrate some of
the techniques which are known to the skilled person. The term
"quantitative PCR", or "Q-PCR", refers to a variety of methods used
to quantify the results of the polymerase chain reaction for
specific nucleic acid sequences. Such methods typically are
categorized as kinetics-based systems, that generally determine or
compare the amplification factor, such as determining the threshold
cycle (C.sub.t), or as co-amplification methods, that generally
compare the amount of product generated from simultaneous
amplification of target and standard templates. Many Q-PCR
techniques comprise reporter probes, intercalating agents, or both.
For example but not limited to TagMan.RTM. probes (Applied
Biosystems), i-probes, molecular beacons, Eclipse probes, scorpion
primers, Lux.TM. primers, FRET primers, ethidium bromide, SYBR.RTM.
Green I (Molecular Probes), and PicoGreen.RTM. (Molecular Probes).
The skilled person is well aware how to measure the expression
level of the respective E-cadherin mRNA.
[0149] This disclosure may best be understood in conjunction with
the accompanying drawings, incorporated herein by references.
Furthermore, a better understanding of the present invention and of
its many advantages will be had from the following examples, given
by way of illustration and are not intended as limiting.
BRIEF DESCRIPTION OF THE DRAWINGS
[0150] FIG. 1: E-Cadherin Expression on Cultured Cells
[0151] Human tumor cells were seeded in chamber slides and stained
with specific antibodies against human E-cadherin. Antibody binding
was detected with a secondary antibody labeled with fluorescent dye
(Alexa 488, green signal). Nuclear DNA was stained with propidium
iodide (PI, red signal). E-cadherin was either absent or expressed
in a small fraction of cells (see the MiaPaca2 and TOV-21G cell
lines). In contrast, BxPC-3 cells displayed an epithelial phenotype
with strong expression of E-cadherin.
[0152] FIG. 2: E-Cadherin Expression in Xenograft Cancers
[0153] Five micrometer thick paraffin sections were obtained from
xenograft cancers derived from MiaPaCa-2, As-PC-1 and BxPC3 cells
and stained for E-cadherin using the ABC immunoperoxidase method
followed by hematoxylin counterstaining. Brown staining in the
membrane of tumor cells indicates the presence of the marker
protein (no staining in MiaPaca2; brown staining in As-PC-1 (30%)
and BxPC-3 (>60%)).
EXAMPLES
[0154] The following examples illustrate the invention. These
examples should not be construed as to limit the scope of this
invention. The examples are included for purposes of illustration
and the present invention is limited only by the claims.
Example 1
Sensitivity of human tumor xenografts to PTK2 inhibitors
[0155] The xenograft models were established as follows: Athymic
female BomTac:NMRI-Foxn1nu mice about six weeks of age were allowed
to adjust to the new environment for at least three days before
they were used for experiments. The animals were housed under
standardized conditions in groups of 5 in Macrolon.RTM. type II
cages. Standardized diet (PROVIMI KLIBA) and autoclaved tap water
were provided ad libitum. To establish subcutaneous tumors, cells
were harvested by trypsinization, centrifuged, washed and
resuspended in ice-cold PBS+5% FCS. 100 .mu.L cell suspension
containing 5,000,000 cells was then injected subcutaneously into
the right flank of the nude mice (1 site per mouse). Mice were
randomly distributed between the treatment and the vehicle control
group (10-14 days after cell injection) when tumors were well
established and had reached diameters of 6-9 mm. The tumor diameter
was measured three times a week (Monday, Wednesday and Friday) with
a caliper. The volume of each tumor [in mm.sup.3] was calculated
according to the formula "tumor volume=length*diameter2*.pi./6". To
monitor side effects of treatment, mice were inspected daily for
abnormalities and body weight was determined three times a week
(Monday, Wednesday and Friday). Animals were sacrificed at the end
of the study about three weeks after start of treatment. Animals
with necrotic tumors or tumor sizes exceeding 2000 mm.sup.3 were
sacrificed early during the studies for ethical reasons.
[0156] The sensitivity of human tumor xenografts growing in nude
mice to treatment with PTK2 inhibitors was as follows:
TABLE-US-00001 Significant Cancer type Model TGI vs. controls
Regressions Score Pancreatic MiaPaCa-2 114% yes yes 0 adenoca
AsPc-1 13% no no 3 Prostate ca PC-3 102% yes yes n.a. Ovarian ca
TOV-21G 100% yes yes 0 Pancreatic AC BxPC-3 49% yes no 4 Colon ca
HCT-116 34% no no 4 LoVo 24% no no 4 HT-29 7% no no 4
Example 2
Immunohistochemistry Protocol (Abc Method) for Determining
E-Cadherin Expression in Xenograft Models
[0157] Deparaffinization: [0158] heat slides for 1 h at 65.degree.
C.; [0159] put slides in xylene for 3.times.5 min, then in 100%
EtOH abs., 96% EtOH, 70% EtOH (3.times.20 sec each), then in dest.
water;
[0160] Antigen Retrieval: [0161] put slides in citrate buffer for
20 min in an autoclave at 121.degree. C./1 bar; [0162] allow slides
to cool at RT for 30 min; [0163] wash with PBS;
[0164] Staining: [0165] incubate slides 5 min in 3% H.sub.2O.sub.2
in PBS; [0166] wash with PBS; [0167] add M.O.M. blocking reagent (2
drops=904 of stock in 2500 .mu.L PBS) to the tissues, incubate 60
min at RT; [0168] wash with PBS; [0169] add M.O.M. diluent (600
.mu.L of stock in 7500 .mu.L PBS) to the tissues, incubate 5 min at
RT; [0170] aspirate; [0171] add antibodies (diluted in M.O.M.
diluent) to the tissues, incubate 60 min at RT; [0172] wash with
PBS; [0173] add M.O.M. biotinylated anti-mouse IgG reagent (10
.mu.L stock in 2500 .mu.L M.O.M. diluent) to the tissues, incubate
10 min at RT; [0174] wash with PBS; [0175] add Vectastain ABC Elite
kit (30 min before use: 2 drops A+2500 .mu.L PBS, mix, add 2 drops
B, mix; Vector #PK-6200) to the tissues, incubate 10 min at RT;
[0176] wash with PBS; [0177] slides 4 min in PBS/0.5% Triton X-100;
[0178] stain in DAB solution; [0179] wash with PBS; [0180] put
slides 1 min in dest. water;
[0181] Counterstaining: [0182] put slides 1 min in Haematoxylin
solution; [0183] wash in running water; [0184] put slides 1 sec in
HCl/EtOH; [0185] wash in running water; [0186] put slides 20 sec in
ammonium-water; [0187] wash in running water; [0188] put slides in
70% EtOH, 96% EtOH, 100% EtOH abs. (3 times/20 sec each); [0189]
put slides in xylene for 1 min, 2 min, 2 min;
[0190] Buffers and Reagents:
[0191] Citrate Buffer:
[0192] 21.01 g citric acid monohydrate in 800 mL dest. water
[0193] Adjust pH=6 with 2 M NaOH, then fill with dest. water to
1000 mL
[0194] Tris/EDTA Buffer: 0.01 M Tris/0.001 M EDTA; pH=8
[0195] HCl/EtOH:
[0196] 175 mL EtOH abs.
[0197] 2.5 mL HCl 37%
[0198] 72.5 mL dest. water
[0199] Ammonium water: 250 mL dest. water+10 drops ammonium
solution (32%)
[0200] Haematoxylin: 160 mL Papanicolaou Solution la Harris'
Haematoxylin solution, Merck #1.092.530.500+80 mL dest. water
[0201] (filter before use)!
[0202] DAB solution: 125 mg DAB in 250 mL PBS/0.5% Triton X-100,
filter and add 25 .mu.L 30% H.sub.2O.sub.2 before use
[0203] Mouse anti-E-cadherin (Abcam #ab1416; 1:100)
[0204] M.O.M. kit basic: Vector #BMK-2202
Example 3
E-Cadherin Expression on Cultured Cells
[0205] Human cancer cells were seeded in chamber slides and stained
with specific antibodies against human E-cadherin. Antibody binding
was detected with a secondary antibody labeled with fluorescent dye
(Alexa 488, green signal). Nuclear DNA was stained with propidium
iodide (PI, red signal). E-cadherin was either absent or expressed
in a small fraction of cells (see the TOV-21G and MiaPaca2 cell
lines). In contrast, BxPC-3 cells displayed an epithelial phenotype
with strong membrane expression of E-cadherin in most cells. The
results of this experiment are depicted in FIG. 1.
Example 4
Immunofluorescence Protocol for Determining E-Cadherin Expression
in Cultured Tumor Cells
[0206] Grow the selected cell lines in 4 chamber
tissue-culture-treated glass slides until near confluency; [0207]
Aspirate the tissue culture medium and fix the slides with
acetone/methanol (1:1 v/v) for 10 min at 4.degree. C.; [0208] Allow
the slides to dry at room temperature for 5 min and store at
-80.degree. C. until use;
[0209] Staining: [0210] thaw slides for 10 min at RT; [0211] wash
with PBS; [0212] incubate slides in blocking serum 10% normal goat
serum, 20 min at RT; [0213] aspirate serum and do not wash; [0214]
incubate with the E-cadherin antibody 1:200 in 2% BSA/PBS for 60
min at RT; [0215] wash with PBS; [0216] incubate with secondary
antibody Alexa 488 conjugated goat anti-mouse (1:1000 in PBS) for
45 min at RT; [0217] wash with PBS; [0218] stain for 2 min at RT
with 0.5 .mu.g/mL propidium iodide in PBS; [0219] wash with PBS;
[0220] coverslip with Dako fluorescence mounting medium; store in
the dark at 4.degree. C. until microscopic examination;
[0221] Buffers and Reagents:
[0222] Citrate Buffer:
[0223] 21.01 g citric acid monohydrate in 800 mL distilled
water
[0224] Adjust pH=6 with 2 M NaOH, then fill with distilled water to
1000 mL
[0225] Mouse anti-human E-cadherin (Abcam #ab1416)
[0226] Normal goat serum (Vector Laboratories #S-1000)
[0227] Alexa 488 conjugated goat anti-mouse Invitrogen #
a-11017
[0228] DakoCytomation fluorescence mounting medium # S3023
[0229] Propidium Iodide Sigma # P4170
Example 5
Immunohistochemistry Protocol (ABC Method) for Determining
E-Cadherin Expression in Human Tissue Samples
[0230] Deparaffinization: [0231] heat slides for 1 h at 65.degree.
C.; [0232] put slides in xylene for 3.times.5 min, then in abs.
EtOH, 96% EtOH, 70% EtOH (3.times.20 sec each); [0233] wash in
distilled water;
[0234] Antigen Retrieval: [0235] put slides in citrate buffer for
20 min in an autoclave at 121.degree. C./1 bar [0236] allow slides
to cool at RT for 30 min; [0237] wash with PBS;
[0238] Staining: [0239] incubate slides for 5 min in 3%
H.sub.2O.sub.2 in PBS; [0240] wash with PBS; [0241] incubate in
blocking serum: 10% normal horse serum (Vector Laboratories
#S-2000) in PBS/2% BSA to the tissues, incubate 30 min at RT;
[0242] aspirate serum and do not wash the slides; [0243] add mouse
anti-E-cadherin (Abcam #ab1416; 1:200 in PBS/2% BSA) to the
tissues, incubate 60 min at RT; [0244] wash with PBS; [0245] add
biotinylated horse anti-mouse IgG (Vector Laboratories #BA-2000;
1:200 in PBS) to the tissues, incubate 30 min at RT; [0246] wash
with PBS; [0247] add Vectastain ABC Standard kit (1:100 in PBS;
Vector #PK-4000) to the tissues, incubate 30 min at RT; [0248] wash
with PBS; [0249] put slides 4 min in PBS/0.5% Triton X-100; [0250]
stain in DAB solution; [0251] wash with PBS; [0252] put slides 1
min in distilled water;
[0253] Counterstaininq: [0254] stain 1 min in haematoxylin
solution; [0255] wash in running water; [0256] put slides 1 sec in
HCl/EtOH; [0257] wash in running water; [0258] put slides 20 sec in
ammonium-water; [0259] wash in running water; [0260] dehydrate the
sections in 70% EtOH, 96% EtOH, abs. EtOH (3 times/20 sec each);
[0261] xylene (.times.3) for 1 min, 2 min, 2 min; [0262] coverslip
with Entellan;
[0263] Buffers and Reagents:
[0264] Citrate Buffer:
[0265] 21.01 g citric acid monohydrate in 800 mL distilled
water
[0266] Adjust pH=6 with 2 M NaOH, then fill with distilled water to
1000 mL
[0267] HCl/EtOH:
[0268] 175 mL abs. EtOH
[0269] 2.5 mL HCl 37%
[0270] 72.5 mL distilled water
[0271] Ammonium-Water:
[0272] 250 mL distilled water+10 drops ammonium solution (32%)
[0273] Haematoxylin:
[0274] 160 mL Papanicolaou Solution la Harris' Haematoxylin
solution, Merck #1.092.530.500 80 mL distilled water
[0275] filtrate before use!
[0276] DAB solution:
[0277] 125 mg DAB (Sigma # D5905) in 250 mL PBS/0.5% Triton X-100,
filtrate and add 25 .mu.L 30% H.sub.2O.sub.2 before use
[0278] Normal horse serum (Vector Laboratories #S-2000)
[0279] Mouse anti-human E-cadherin (Abcam #ab1416)
[0280] Biotinylated horse anti-mouse IgG (Vector Laboratories
#BA-2000)
[0281] Vectastain ABC Standard kit (1/100 in PBS; Vector
#PK-4000)
[0282] Entellan (Merk 1.07961.0100)
[0283] It will be clear that the invention may be practiced
otherwise than as particularly described in the foregoing
description and examples. Numerous modifications and variations of
the present invention are possible in light of the above teachings
and, therefore, are within the scope of the appended claims.
[0284] The entire disclosure of each document cited (including
patents, patent applications, journal articles, abstracts,
laboratory manuals, books, or other disclosures) in the Background
of the Invention, detailed Description, and Examples is hereby
incorporated herein by reference.
* * * * *
References