U.S. patent application number 14/580019 was filed with the patent office on 2015-06-25 for circulating tumor cell assay.
The applicant listed for this patent is PFIZER INC., VERIDEX LLC. Invention is credited to David Chianese, Mark Carle Connelly, Antonio Gualberto, Carrie L. Melvin, Maria Luisa Paccagnella, Madeline I. Repollet, Leonardus Wendelinus Mathias Marie Terstappen.
Application Number | 20150177252 14/580019 |
Document ID | / |
Family ID | 38476880 |
Filed Date | 2015-06-25 |
United States Patent
Application |
20150177252 |
Kind Code |
A1 |
Gualberto; Antonio ; et
al. |
June 25, 2015 |
CIRCULATING TUMOR CELL ASSAY
Abstract
Methods for the detection, enumeration and analysis of
circulating tumor cells expressing insulin-like growth factor-1
receptors (IGF-1R) are disclosed. These methods are useful for
cancer screening and staging, development of treatment regimens,
and for monitoring for treatment responses, cancer recurrence or
the like. Test kits that facilitate the detection, enumeration and
analysis of such circulating tumor cells are also provided.
Inventors: |
Gualberto; Antonio; (East
Greenwich, RI) ; Paccagnella; Maria Luisa; (Noank,
CT) ; Melvin; Carrie L.; (Ledyard, CT) ;
Repollet; Madeline I.; (Fort Washington, PA) ;
Chianese; David; (Ardmore, PA) ; Connelly; Mark
Carle; (Doylestown, PA) ; Terstappen; Leonardus
Wendelinus Mathias Marie; (Huntingdon Valley, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
VERIDEX LLC
PFIZER INC. |
RARITAN
NEW YORK |
NJ
NY |
US
US |
|
|
Family ID: |
38476880 |
Appl. No.: |
14/580019 |
Filed: |
December 22, 2014 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12303243 |
Jul 29, 2009 |
8940493 |
|
|
PCT/IB07/01483 |
May 29, 2007 |
|
|
|
14580019 |
|
|
|
|
60810811 |
Jun 2, 2006 |
|
|
|
Current U.S.
Class: |
435/7.23 |
Current CPC
Class: |
G01N 33/57496 20130101;
G01N 2800/52 20130101; G01N 33/5023 20130101; G01N 30/02 20130101;
G01N 33/574 20130101; G01N 33/5064 20130101; G01N 2333/71 20130101;
G01N 2458/00 20130101; G01N 2333/91205 20130101; A61P 35/00
20180101; G01N 33/57492 20130101; G01N 2333/4742 20130101; G01N
2333/4745 20130101; G01N 30/02 20130101; B01D 15/3804 20130101 |
International
Class: |
G01N 33/574 20060101
G01N033/574 |
Claims
1. A method for predicting efficacy of IGF-1R antagonist therapy in
a patient, comprising the steps of: a) preparing a sample wherein a
blood sample from the patient is mixed with a ligand that reacts
specifically with tumor cells, to the substantial exclusion of
other sample components, so as to obtain a cell population enriched
for tumor cells; b) contacting the enriched cell population with an
anti-cytokeratin antibody; c) contacting the enriched cell
population with an antibody to insulin-like growth factor receptors
(IGF-1R); and d) determining the presence of cells bound by both
the anti-cytokeratin antibody and the anti-IGF-1R antibody; wherein
the presence of cells bound by both antibodies is predictive of
efficacy of IGF-1R antagonist therapy in the patient.
2. The method of claim 1, wherein the determining step comprises
determining the number of cells bound by both antibodies.
3. The method of claim 1, further comprising the step of adding to
the sample a cell specific dye to allow exclusion of residual
non-nucleated cells and cell debris from analysis.
4. The method of claim 1, wherein the determining step is performed
using at least one process selected from the group consisting of
multiparameter flow cytometry, immunofluorescent microscopy, laser
scanning cytometry, bright field base image analysis, capillary
volumetry, spectral imaging analysis, manual cell analysis,
CELLSPOTTER analysis, and automated cell analysis.
5. The method of claim 1, wherein the sample is an immunomagnetic
sample comprising the blood sample mixed with magnetic particles
coupled to the ligand, and further comprising the step of
subjecting the immunomagnetic sample to a magnetic field, such that
the immunomagnetic sample becomes an enriched tumor cell
suspension.
6. The method of claim 5, wherein the magnetic particles are
colloidal.
7. A method for monitoring efficacy of an IGF-1R antagonist
therapy-in a patient, comprising the steps of: a) preparing a first
sample wherein a first blood sample from the patient is mixed with
a ligand that reacts specifically with tumor cells, to the
substantial exclusion of other sample components, so as to obtain a
cell population enriched for tumor cells; b) contacting the
enriched cell population with an anti-cytokeratin antibody; c)
contacting the enriched cell population with an antibody to
insulin-like growth factor receptors (IGF-1R); d) determining the
presence and number of cells bound by both the anti-cytokeratin
antibody and the anti-IGF-1R antibody; e) preparing a second sample
from a second blood sample from the patient, after administration
of an IGF-1R antagonist therapy, wherein the second blood sample is
mixed with the ligand that reacts specifically with tumor cells,
and performing steps b)-d) on the second sample; and f) comparing
the number of cells bound by both the anti-cytokeratin antibody and
the anti-IGF-1R antibody in the first sample to the number of cells
bound by both antibodies in the second sample; wherein a lower
number of cells bound by both antibodies in the second sample is
indicative of efficacy of the IGF-1R antagonist therapy in the
patient.
8. The method of claim 7, wherein the determining steps are
performed using at least one process selected from the group
consisting of multiparameter flow cytometry, immunofluorescent
microscopy, laser scanning cytometry, bright field base image
analysis, capillary volumetry, spectral imaging analysis, manual
cell analysis, CELLSPOTTER analysis, and automated cell
analysis.
9. The method of claim 7, wherein the sample is an immunomagnetic
sample comprising the blood sample mixed with magnetic particles
coupled to the ligand, and further comprising the step of
subjecting the immunomagnetic sample to a magnetic field, such that
the immunomagnetic sample becomes an enriched tumor cell
suspension.
10. The method of claim 9, wherein the magnetic particles are
colloidal.
11. The method of claim 1, wherein the IGF-1R antagonist therapy
comprises an anti-IGF-1R antibody.
12. The method of claim 7, wherein the IGF-1R antagonist therapy
comprises an anti-IGF-1R antibody.
13. The method of claim 1, wherein the anti-cytokeratin antibody is
labeled with fluorescein isothiocyanate (FITC).
14. The method of claim 1, wherein the antibody to IGF-1R is
labeled with phycoerythrin (PE).
15. The method of claim 1, wherein the ligand is an antibody to
epithelial cell adhesion molecule (EpCAM).
16. The method of claim 7, wherein the anti-cytokeratin antibody is
labeled with fluorescein isothiocyanate (FITC).
17. The method of claim 7, wherein the antibody to IGF-1R is
labeled with phycoerythrin (PE).
18. The method of claim 7, wherein the ligand is an antibody to
epithelial cell adhesion molecule (EpCAM).
19. A test kit for screening a patient sample for the presence of
circulating tumor cells expressing IGF-1R, comprising: a) coated
magnetic nanoparticles comprising a magnetic core material, a
protein base coating material, and an antibody that binds
specifically to a characteristic determinant of the tumor cells,
the antibody being coupled, directly or indirectly, to said base
coating material; b) a cell specific dye for excluding sample
components other than the tumor cells from analysis; and c) at
least one detectably labeled agent having binding affinity for
IGF-1R.
20. The test kit of claim 19, further comprising a device for the
detection of circulating tumor cells expressing IGF-1R.
Description
BACKGROUND OF THE INVENTION
[0001] This application is a continuation application of U.S.
patent application Ser. No. 12/303,243, filed Jul. 29, 2009, which
is the National Stage of International Application No.
PCT/IB2007/001483, filed May 29, 2007, which claims the benefit of
U.S. Provisional Application No. 60/810,811, filed Jun. 2, 2006,
all of which are incorporated herein by reference in their
entirety.
[0002] The present invention relates to the fields of oncology and
diagnostic testing, and more particularly to methods for cancer
screening and for predicting and monitoring chemotherapy treatment
responses, cancer recurrence or the like.
[0003] Insulin-like growth factor (IGF-1) is a 7.5 kD polypeptide
that circulates in plasma in high concentrations and is detectable
in most tissues. IGF-1, which is structurally similar to insulin,
stimulates cell differentiation and cell proliferation, and is
required by most mammalian cell types for sustained proliferation.
These cell types include, among others, human diploid fibroblasts,
epithelial cells, smooth muscle cells, T lymphocytes, neural cells,
myeloid cells, chondrocytes, osteoblasts, and bone marrow stem
cells.
[0004] The first step in the transduction pathway leading to
IGF-1-stimulated cellular proliferation or differentiation is
binding of IGF-1 or IGF-2 (or insulin at supraphysiological
concentrations) to the IGF-1 receptor (IGF-1R). The IGF-1R belongs
to the family of tyrosine kinase growth factor receptors (Ullrich
et al., Cell 61: 203-212, 1990), and is structurally similar to the
insulin receptor (Ullrich et al., EMBO J. 5: 2503-2512, 1986).
[0005] Epidemiological studies suggest that high end normal levels
of IGF-1 increase the risk of cancers such as lung, breast,
prostate and colorectal, compared to individuals with IGF-1 levels
at the low end of normal. Further, there is considerable evidence
for a role for IGF-1 and/or IGF-1R in the maintenance of tumor
cells in vitro and in vivo. IGF-1R levels are elevated in tumors of
lung (Kaiser et al., J. Cancer Res. Clin. Oncol. 119: 665-668,
1993; Moody et al., Life Sciences 52: 1161-1173, 1993; Macauley et
al., Cancer Res., 50: 2511-2517, 1990), breast (Pollak et al.,
Cancer Lett. 38: 223-230, 1987; Foekens et al., Cancer Res. 49:
7002-7009, 1989; Arteaqa et al., J. Clin. Invest. 84: 1418-1423,
1989), prostate and colon (Remaole-Bennet et al., J. Clin.
Endocrinol. Metab. 75: 609-616, 1992; Guo et al., Gastroenterol.
102: 1101-1108, 1992). Deregulated expression of IGF-1 in prostate
epithelium leads to neoplasia in transgenic mice (DiGiovanni et
al., Proc. Nat'l. Acad. Sci. USA 97: 3455-3460, 2000). In addition,
IGF-1 appears to be an autocrine stimulator of human gliomas
(Sandberg-Nordqvist et al., Cancer Res. 53 (11): 2475-78, 1993),
while IGF-1 has been shown to stimulate the growth of fibrosarcomas
that overexpress IGF-1R (Butler et al., Cancer Res. 58: 3021-3027,
1998). For a review of the role IGF-1/IGF-1R interaction plays in
the growth of a variety of human tumors, see Macaulay, Br. J.
Cancer, 65: 311-20, 1992.
[0006] Using antisense expression vectors or antisense
oligonucleotides to the IGF-1R RNA, it has been shown that
interference with IGF-1R leads to inhibition of IGF-1-mediated cell
growth (see, e.g., Wraight et al., Nat. Biotech. 18: 521-526,
2000). Growth can also be inhibited using peptide analogues of
IGF-1 (Pietrzkowski et al., Cell Growth & Duff. 3: 199-205,
1992; Pietrzkowski et al., Mol. Cell. Biol. 12: 3883-3889, 1992),
or a vector expressing an antisense RNA to the IGF-1 RNA (Trojan et
al., Science 259: 94-97, 1992). In addition, antibodies to IGF-1R
(Arteaga et al., Breast Canc. Res. Treatm. 22: 101-106, 1992; and
Kalebic et al., Cancer Res. 54: 5531-34, 1994), and dominant
negative mutants of IGF-1R (Prager et al., Proc. Nat'l Acad. Sci.
USA 91: 2181-85, 1994; Li et al., J. Biol. Chem. 269: 32558-2564,
1994; Jiang et al., Oncogene 18: 6071-6077, 1999), can reverse the
transformed phenotype, inhibit tumorigenesis, and induce loss of
the metastatic phenotype.
[0007] IGF-1 is also important in the regulation of apoptosis.
Apoptosis, which is programmed cell death, is involved in a wide
variety of developmental processes, including immune and nervous
system maturation. In addition to its role in development,
apoptosis also has been implicated as an important cellular
safeguard against tumorigenesis (Williams, Cell 65: 1097-1098,
1991; Lane, Nature 362: 786-787, 1993). Suppression of the
apoptotic program may contribute to the development and progression
of malignancies.
[0008] IGF-1 protects from apoptosis by cytokine withdrawal in
IL-3-dependent hematopoietic cells (Rodriguez-Tarduchy, G. et al.,
J. Immunol. 149: 535-540, 1992), and from serum withdrawal in
Rat-1/mycER cells (Harrington, E. et al., EMBO J. 13:3286-3295,
1994). The demonstration that c-myc driven fibroblasts are
dependent on IGF-1 for their survival suggests that there is an
important role for the IGF-1 receptor in the maintenance of tumor
cells by specifically inhibiting apoptosis, a role distinct from
the proliferative effects of IGF-1 or IGF-1R.
[0009] The protective effects of IGF-1 on apoptosis are dependent
upon having IGF-1R present on cells to interact with IGF-1
(Resnicoff et al., Cancer Res. 55: 3739-41, 1995). Support for an
anti-apoptotic function of IGF-1R in the maintenance of tumor cells
was also provided by a study using antisense olignucleotides to the
IGF-1R that identified a quantitative relationship between IGF-1R
levels, the extent of apoptosis and the tumorigenic potential of a
rat syngeneic tumor (Rescinoff et al., Cancer Res. 55: 3739-3741,
1995). It has been found that overexpressed IGF-1R protects tumor
cells in vitro from etoposide-induced apoptosis (Sell et al.,
Cancer Res. 55: 303-06, 1995) and, even more dramatically, that a
decrease in IGF-1R levels below wild type levels caused massive
apoptosis of tumor cells in vivo (Resnicoff et al., Cancer Res. 55:
2463-69, 1995).
[0010] Some studies suggest that expression levels of IGF-1R
correlate with clinical outcome. In tumor models, IGF-1R modulates
cell proliferation, survival and metastasis and induces resistance
to targeted therapies. Inhibition of IGF-1R significantly increases
the activity of cytotoxic agents (Cohen, B. e al., Clin. Cancer
Res. 11(5): 2063-73). Inhibition of IGF-1R signaling thus appears
to be a promising strategy for the development of novel cancer
therapies.
[0011] Malignant tumors of epithelial tissues are the most common
form of cancer and are responsible for the majority of
cancer-related deaths. Because of progress in the surgical
treatment of these tumors, mortality is linked increasingly to
early metastasis and recurrence, which is often occult at the time
of primary diagnosis (Racila et al., Proc. Nat'l Acad. Sci. USA
95:4589-94, 1998; Pantel et al., J. Nat'l Cancer Inst. 91(13):
1113-24, 1999). For example, the remote anatomical location of some
organs makes it unlikely that tumors in those organs will be
detected before they have invaded neighboring structures and grown
to larger than 1 cm. Even with respect to breast cancers, 12-37% of
small tumors of breast cancer (<1 cm) detected by mammography
already have metastasized at diagnosis (Chadha M. et al., Cancer
73(2): 350-3, 1994.
[0012] Circulating tumor cells ("CTCs") are cells of epithelial
origin that are present in the circulation of patients with
different solid malignancies. They are derived from clones of the
primary tumor and are malignant. (See Fehm et al., Clin. Cancer
Res. 8: 2073-84, 2002.) Evidence has accumulated in the literature
showing that CTCs can be considered an independent diagnostic for
cancer progression of carcinomas (Beitsch & Clifford, Am. J.
Surg. 180(6): 446-49, 2000 (breast); Feezor et al., Ann. OncoL
Surg. 9(10): 944-53, 2002 (colorectal); Ghossein et al., Diagn.
Mol. PathoL 8(4): 165-75, 1999 (melanoma, prostate, thyroid);
Glaves, Br. J. Cancer 48: 665-73, 1983 (lung); Matsunami et al.,
Ann. Surg. Oncol. 10(2): 171-5, 2003 (gastric); Racila et al.,
1998; Pantel et al., 1999).
[0013] Detection and enumeration of circulating tumor cells is
important for patient care for a number of reasons. They may be
detectable before the primary tumor, thus allowing early stage
diagnosis. They decrease in response to therapy, so the ability to
enumerate CTCs allow one to monitor the effectiveness of a give
therapeutic regimen. They can be used as a tool to monitor for
recurrence in patients with no measurable disease in the adjuvant
setting. For example, CTC were found to be present in 36% of breast
cancer patients 8-22 years after mastectomy, apparently from
micrometastases (deposits of single tumor cells or very small
clusters of neoplastic cells). Meng et al., Clin. Can. Res. 10(24):
8152-62, 2004.
[0014] In addition, CTCs may be used to predict progression-free
survival (PFS) and overall survival (OS), as the presence/number of
circulating tumor cells in patients with metastatic carcinoma has
been shown to be correlated with both PFS and OS. See e.g.,
Cristofanilli et al., J. Clin. Oncol. 23(1): 1420-1430, 2005;
Cristofanilli et al., N. Engl. J. Med. 351(8): 781-791, 2004.
[0015] However, there remains a need for rapid and reliable assays
that are more sensitive than mere detection of CTCs.
SUMMARY OF THE INVENTION
[0016] The present invention is directed to a method for predicting
efficacy of IGF-1R antagonist therapy in a patient, comprising the
steps of: a) obtaining a biological specimen from the patient; b)
preparing a sample wherein the biological specimen is mixed with a
ligand that reacts specifically with tumor cells, to the
substantial exclusion of other sample components; c) contacting the
sample with at least one reagent that specifically binds epithelial
cells; d) contacting the sample with an agent having binding
affinity for insulin-like growth factor receptors (IGF-1R) on
cells; and e) analyzing the sample to determine the presence of
tumor cells expressing IGF-1R, the presence of tumor cells
expressing IGF-1R in the sample being predictive of efficacy of
IGF-1R antagonist therapy in the patient.
[0017] The present invention is also directed to a method for
monitoring efficacy of an IGF-1R antagonist therapy in a patient,
comprising the steps of: a) obtaining a first biological specimen
from the patient; b) preparing a first sample wherein the first
biological specimen is mixed with a ligand that reacts specifically
with tumor cells, to the substantial exclusion of other sample
components; c) contacting the first sample with at least one
reagent that specifically binds epithelial cells; d) contacting the
first sample with an agent having binding affinity for insulin-like
growth factor receptors (IGF-1R) on cells; e) analyzing the first
sample to determine the presence and number of tumor cells
expressing IGF-1R; f) administering to the patient an IGF-1R
antagonist therapy; g) after administering the IGF-1R antagonist
therapy, obtaining a second biological specimen from the patient;
h) preparing a second sample from the second biological specimen,
wherein the second biological specimen is mixed with the ligand
that reacts specifically with tumor cells, and performing steps
c)-e) on the second sample; and i) comparing the number of tumor
cells expressing IGF-1R in the first sample to the number of tumor
cells expressing IGF-1R in the second sample, with a lower number
in the second sample being indicative of effectiveness of the
IGF-1R antagonist therapy in the patient.
[0018] In a preferred embodiment, the IGF-1R antagonist therapy is
an anti-IGF-1R antibody.
[0019] The present invention is further directed to a kit for
screening a patient sample for the presence of circulating tumor
cells expressing IGF-1R, comprising: a) coated magnetic
nanoparticles comprising a magnetic core material, a protein base
coating material, and an antibody that binds specifically to a
characteristic determinant of the tumor cells, the antibody being
coupled, directly or indirectly, to said base coating material; b)
a cell specific dye for excluding sample components other than the
tumor cells from analysis; and c) at least one detectably labeled
agent having binding affinity for IGF-1R.
[0020] With the foregoing and other objects, advantages and
features of the invention that will become hereinafter apparent,
the nature of the invention may be more clearly understood by
reference to the following detailed description of the invention,
the figures, and to the appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 is an overlay of histograms of the
IGF-1R-phycoerythrin staining of various cell lines.
[0022] FIG. 2 is a collection of fluorescent microscope images of
labeled MCF-7 breast cancer cells (Panel A) and T-24 bladder cells
(Panel B).
[0023] FIG. 3 is a collection of fluorescent microscope images of
potential circulating tumor cells.
[0024] FIGS. 4A-D are graphic representations of the number of
total circulating tumor cells and IGF-1R-positive circulating tumor
cells of four patients treated with an anti-IGF-1R antibody.
[0025] FIGS. 5A-D are graphic representations of the number of
total circulating tumor cells and IGF-1R-positive circulating tumor
cells of four patients treated with an anti-IGF-1R antibody in
combination with docetaxel.
[0026] FIGS. 6A-D are graphic representations of the number of
total circulating tumor cells and IGF-1R-positive circulating tumor
cells of four patients treated with an anti-IGF-1R antibody in
combination with paclitaxel and carboplatin.
DETAILED DESCRIPTION OF THE INVENTION
[0027] Unless otherwise defined herein, scientific and technical
terms used in connection with the present invention shall have the
meanings that are commonly understood by those of ordinary skill in
the art. Further, unless otherwise required by context, singular
terms shall include pluralities and plural terms shall include the
singular.
[0028] Each year in the United States, more than 1 million new
cases of cancer are diagnosed; approximately one out of every five
deaths in this country is caused by cancer or complications
associated with its treatment. Considerable efforts are continually
directed at improving treatment and diagnosis of this disease. Most
cancer patients are not killed by their primary tumor; rather they
succumb to metastases: multiple widespread tumor colonies
established by malignant cells that detach themselves from the
original tumor and travel through the body, often to distant sites.
Unfortunately, metastatic colonies are frequently more difficult to
detect and eliminate than the primary tumor and it is often
impossible to treat all of them successfully. The ability of
malignant cells to metastasize remains one of the major obstacles
to the treatment of cancer and may be accelerated by the IGF-1
receptor. See, e.g., Bahr et al., Growth Factors 23:1-14, 2005.
[0029] Based on the complexity of cancer and cancer metastasis and
the frustration in treating cancer patients over the years, many
attempts have been made to develop diagnostic tests to guide
treatment and monitor the effects of such treatment on metastasis
or relapse. Such tests presumably could also be used for cancer
screening, replacing relatively crude tests such as mammography for
breast tumors or digital rectal exams for prostate cancers.
[0030] In view of the knowledge of the relationship of IGF-1 and
IGF-1R to certain cancers, studies were undertaken to evaluate the
effect of anti-IGF-1R antibodies on the number of circulating tumor
cells and their IGF-1R expression, as well as clinical efficacy of
the antibodies. An assay to detect and enumerate circulating tumor
cells expressing IGF-1R (IGF-1R-positive CTCs) has now been
developed that is useful in the diagnosis and treatment of cancer,
and superior to prior art methods involving CTCs. The assay may
contribute to a better understanding of the biological functions of
the IGF-1 receptor. For example, while it has been postulated that
elevated IGF-1R levels are needed to trigger the tumor cell
invasive/metastatic phenotype, the relationship between levels of
IGF-1R expression and metastatic potential has not yet been
completely elucidated. It has now been found that patients with
high IGF-1R-positive CTC counts appeared to have more aggressive
tumors as evidenced by rapid disease progression. A potential
relationship between increased IGF-1R expression and metastatic
potential could thus be the basis for the detection of
IGF-1R-positive CTCs as a predictor of poor outcome and/or
therapeutic intervention.
[0031] Initially, the CTC-IGF-1R assay methods of the present
invention are useful for the early detection of a tumor, or to
confirm a diagnosis. They also may be used for assessing
prognosis.
[0032] The methods of the present invention are also useful for
treatment planning. It has been found that patients with
IGF-1R-positive circulating tumor cells prior to treatment are more
likely to respond to IGF-1R antagonist therapy that those who do
not. By screening patients for IGF-1R-positive antibodies prior to
the initiation of any treatment, it is possible to pre-select a
population most likely to respond to IGF-1R antagonist therapy and
plan a treatment regimen accordingly. Potential uses of the
CTC-IGF-1R assays as biomarkers of anti-IGF-1R antibodies could
include identification of an optimal biological dose, dose and
treatment regimen selection and determination of treatment
duration.
[0033] It has been found that there is a good correlation between
changes in the level of IGF-1R-positive CTCs in the blood with
chemotherapy and clinical status. In view of this correlation, it
is also possible to assess the patient's response to treatment, or
disease progression using the assay methods of the present
invention. Measurement of IGF-1 receptors on circulating tumor
cells provides a real pharmacodynamic (PD) endpoint. Once treatment
has started, measurement of IGF-1R on tumor cells can be used to
determine if maximum inhibition of the target has been reached
without reaching maximum tolerated dose (MTD). The development of
resistance to a given treatment can also be monitored. An
additional benefit of the present invention is that drug effect can
be determined more frequently by measuring CTC than by traditional
means.
[0034] Finally, the methods of the present invention may also be
used to detect recurrence of a tumor, even in the absence of
clinical symptoms.
[0035] The methods of the present invention may be used in
conjunction with the diagnosis and/or treatment of
non-hematological malignancies, including cancers of the breast,
prostate, ovaries, lung and colon, particularly non-small cell lung
cancer (NSCLC) and hormone-refractory prostate cancer (HRPC).
[0036] Screening of patients with multiple tumor types indicates
that CTCs and IGF-1R-positive CTCs are frequently detected in HRPC
patients. The identification of these cells in HRPC may have
prognostic or therapeutic implications. In fact, in a previous
study, the presence of CTCs was found to be the most significant
parameter predictive of survival in HRPC patients (Moreno et al.,
Urology 65: 713-718, 2005). Multiple studies have established a
role for IGF-1R in the development of prostate cancer, and in vitro
data suggest that increased IGF-1R expression and/or activity are
associated with progression to the hormone refractory phenotype
(Hellawell et al., Cancer Res., 62: 2942-2950, 2002; Chott et al.,
Am. J. Pathol. 155: 1271-1279, 1999). In one study described
herein, HRPC patients that were considered IGF-1R positive (i.e.,
at least one IGF-1R-positive CTC detected) had a median level of
serum PSA at enrollment higher than those patients with no
detectable CTCs. Furthermore, PSA levels and CTC and
IGF-1R-positive CTC counts changed in parallel during treatment
response or disease progression. It has previously been shown that
patients with progressive metastatic HRPC had CTC counts
significantly higher than those in the earlier disease group, and a
drop in CTC counts one week after initial treatment with docetaxel
was reported in two patients (Moreno et al., 2005, supra). Both
patients, however, progressed, exhibiting a rise in CTC counts and
PSA levels despite additional doses of docetaxel. The studies
described herein suggest that only sustained decreases in CTC
counts are associated with response to therapy in HRPC. Thus, CTC
counts may provide prognostic information independent to that of
PSA level. Importantly, preclinical data indicate that changes in
PSA in response to anti-IGF-1R treatment reflect changes in
prostate tumor growth (Wu et al., Clin. Cancer Res. 11: 3065-3074,
2005).
[0037] It has also been found that the proportion of responders to
combined anti-IGF-1R antibody and docetaxel therapy was higher in
those patients with detectable IGF-1R-positive CTCs at study entry
than in those in which these cells were not detected. Furthermore,
late responses, which are not necessarily associated with clinical
benefit (see, e.g., Petrylak et al., J. Nat'l Cancer Inst. 98:
516-521, 2006), were seen in IGF-1R-CTC negative patients. These
data are suggestive of a potential use of IGF-1R CTC enumeration
for the identification of HRPC patients that could benefit from
anti-IGF-1R therapy.
[0038] The methods of the present invention may be used in the
planning and/or monitoring of treatment with various
chemotherapeutic compounds that inhibit IGF-1R signaling.
Particularly preferred are anti-IGF-1R antibodies such as those
described in U.S. Pat. No. 7,037,498 and U.S. Patent Application
Publication No. 2005/0069539. Other preferred anti-IGF-1R
antibodies include F-50035 and MK-0646 (Pierre Fabre/Merck); 19D12
(Schering-Plough); R1507 (Roche/Genmab); EM-164/AVE-1642
(Immunogen/Sanofi-Aventis); IMC-A12 (ImClone Systems); AMG479
(Amgen); as well as antibodies described in International Patent
Application No. WO2006/069202; U.S. Patent Application Publication
No. 2005/0147612; U.S. Patent Application Publication No.
2005/0084906; U.S. Patent Application Publication No. 2005/0249730;
U.S. Patent Application Publication No. 2004/0018191; U.S. Patent
Application Publication No. 2005/0136063; U.S. Patent Application
Publication No. 2003/0235582; U.S. Patent Application Publication
No. 2004/0265307; U.S. Patent Application Publication No.
2004/0228859; U.S. Patent Application Publication No. 2005/0008642;
European Patent Application No. 1622942; U.S. Patent Application
Publication No. 2003/0165502; and U.S. Patent Publication No.
2005/0048050.
[0039] Other classes of molecules suitable for use with the present
invention include peptide aptamers that specifically bind to
IGF-1R, antisense oligonucleotide IGF-1R modulators; and small
molecule IGF-1R inhibitors. Preferred small molecule IGF-1R
inhibitors include OSI-906 (OSI Pharmaceuticals); AEW-541
(Novartis); BMS-536924 and BMS-554417 (Bristol-Myers Squibb);
INSM-18 (Insmed); AG-1024 (Pfizer); XL228 (Exelixis),
picropodophyllin, and those disclosed in International Patent
Application Nos. WO2004/043962 and WO2004/054996.
[0040] As set forth below in greater detail, the methods of the
present invention involve the selective removal of cells having
certain antigenic reactive sites from a patient sample. Methods and
apparatuses for such selective removal are well known to one of
skill in the art. See, e.g., U.S. Pat. Nos. 4,551,435; 4,795,698;
4,925,788; 5,108,933; and 5,200,084; and U.S. Patent Application
Publication No. 2004/0157271. In a preferred embodiment, the cells
of interest are immunomagnetically isolated from the patient sample
using ferrofluids. Ferrofluids contain tiny magnetic particles in a
colloidal suspension whose flow can be controlled by magnets or
magnetic fields.
[0041] In order that this invention may be better understood, the
following examples are set forth. These examples are for purposes
of illustration only and are not to be construed as limiting the
scope of the invention in any manner.
EXAMPLES
[0042] In the examples set forth herein, blood samples were taken
at multiple geographic locations from human subjects into CELLSAVE
Preservative Tubes (Immunicon, Huntingdon Valley, Pa.), evacuated
10 mL blood draw tubes containing a cell preservative to preserve
cell morphology and cells surface antigen expression. Samples were
maintained at room temperature and processed as described above
within 72 hours of blood collection.
[0043] Patient response to treatment was assessed radiologically
using the Response Evaluation Criteria in Solid Tumors (RECIST)
(see Therasse et al., J. Nat'l Cancer Inst. 92: 205-216, 2000) or,
in HRPC patients, using prostate specific antigen working group
(PSAWG) criteria (see Bubley et al., J. Clin. Oncol. 17:3461-3467,
1999).
Example 1
Development of IGF-1R Circulating Tumor Cell Assay
[0044] Cell Culture and Cell Spiking:
[0045] The breast cancer cell line MCF-7, the prostate cell line
PC3-9, the bladder cell line T-24 and the hematopoeitic cell line
CEM were cultured in flasks containing RPMI-1640 cell culture
medium supplemented with 10% FCS and subsequently harvested using
trypsin. The cell suspensions were only used when their viability
as assessed by trypan blue exclusion exceeded 90%. To determine the
actual cell number, a 50 .mu.L aliquot of the cells was
permeabilized and fluorescently labeled by adding 200 .mu.l of PBS
containing 0.05% saponin and 10 .mu.l anti-cytokeratin monoclonal
antibody conjugated to phycoerythrin (PE) at a final concentration
of 0.5 .mu.g/ml. After a 15 minute incubation at room temp, 200
.mu.l of buffer and 20 .mu.l of fluorescent beads (Beckman-Coulter,
Inc., Miami, Fla.) containing approximately 20,000 total beads were
added. Duplicate tubes containing beads only were run on a flow
cytometer (FACSCalibur, BD Biosciences, San Jose, Calif.) until
100% of the sample was aspirated. This provided an accurate
estimate of the number of beads present in 20 .mu.l. The
experimental tubes were then tested in triplicate on the flow
cytometer until 10,000 beads were counted in each tube. Using the
known number of beads per unit volume, the concentration of cells
was determined. For IGF-1R detection spiked cell numbers were
estimated to be between 130 and 220 in 7.5 mL of blood.
[0046] Isolation and Enumeration of CTCs:
[0047] Samples for the isolation of cells from blood were prepared
and analyzed using the CELLTRACKS system (Immunicon, Huntingdon
Valley, Pa.), which consists of a CELLTRACKS AUTOPREP system, a
reagent kit and the CELLSPOTTER Analyzer. The CELLTRACKS AUTOPREP
system is an automated sample preparation system for rare cell
detection. The reagent kit consists of ferrofluids coated with
antibodies to immunomagnetically enrich cells, a cocktail of
fluorescently conjugated antibodies (antibodies conjugated to PE
and allophycocyanin (APC) to label epithelial cells and leukocytes
respectively), a nuclear dye, and buffers to wash, permeabilize and
resuspend the cells. For the detection of carcinoma cells, 7.5 mL
of blood is mixed with ferrofluids coated with antibodies directed
against the tumor-associated antigen EpCAM (epithelial cell
adhesion molecule, or epithelial cell surface antigen). After
immunomagnetic enrichment, fluorescein isothyocyanate
(FITC)-labeled antibodies recognizing cytokeratins 4, 5, 6, 8, 10,
13, 18 and 19, APC-labeled antibodies recognizing leukocyte antigen
CD45, PE-labeled antibodies recognizing IGF-1R, and the nucleic
acid dye 4',6-diamidino-2-phenylindole (DAPI) were added in
conjunction with a permeabilization buffer to fluorescently label
the immunomagnetically-labeled cells. After incubation on the
system, the magnetic separation was repeated and excess staining
reagents were aspirated. In the final processing step, the cells
were resuspended in the MAGNEST Cell Presentation Device
(Immunicon, Huntingdon Valley, Pa.). This device consists of a
chamber and two magnets that orient the immunomagnetically-labeled
cells for analysis using a fluorescent microscope.
[0048] The MAGNEST was placed on the CELLSPOTTER Analyzer, a four
color semi-automated fluorescent microscope. Image frames covering
the entire surface of the cartridge for each of the four
fluorescent filter cubes were captured. The captured images
containing objects that met predetermined criteria were
automatically presented in a web-enabled browser from which final
selection of cells was made by the operator. The criteria for an
object to be defined as a CTC included round to oval morphology, a
visible nucleus (DAPI positive), positive staining for cytokeratin,
and lack of expression of CD45 (as determined by negative CD45-APC
staining). Results of cell enumeration were always expressed as the
number of cells per 7.5 mL of blood.
[0049] Selection of IGF-1R antibodies and detection of IGF-1R on
tumor cell lines: The anti-IGF-1R antibodies 1H7 (PE conjugate; BD
Biosciences, San Jose, Calif.) and 33255.111 (R&D Systems,
Minneapolis, Minn.) were titrated on cells from the breast cancer
cell line MCF-7 cells. Cross blocking experiments demonstrated no
inhibition suggesting that these antibodies bind to different and
noncompetitive epitopes of the IGF-1R. Cross blocking experiments
with CP-751,871 human anti-IGF-1R antibody (Pfizer Inc.; see U.S.
Pat. No. 7,037,498) showed that antibody 33255.111 was completely
blocked from binding cells by essentially equimolar amounts of
CP-751,871. The 1H7 antibody, in contrast, showed no inhibition of
binding in the presence of CP-751,871. These data demonstrate that
33255.111 and CP-751,871 bind to either the same or related
epitopes. Since the binding of the 1H7 antibody to IGF-1R is not
blocked in the presence of CP-751,871, 1H7 was selected for further
evaluation.
[0050] The antigen density on the hematopoeitic cell line CEM, the
prostate cancer cell line PC3-9, the bladder cell line T-24 and the
MCF-7 breast cancer cell line was assessed by staining with the
PE-labeled anti-IGF-1R antibody 1H7 followed by flow cytometric
analysis. FIG. 1 shows an overlay of histograms of the IGF-1R-PE
staining of the cell lines. Staining of CEM cells was similar to
that of the controls and IGF-1R density was thus below the
detection limit. IGF-1R-PE staining of the PC3-9 cells could be
resolved from the background and T24 and MCF-7 cells clearly had a
brighter staining. Estimation of the antigen density was obtained
by qualibration of the flow cytometer with beads with a known
number of PE molecules. IGF-1R density on the PC3-9 cells was
approximately 10,000 IGF-1R antigens, on T-24 cells approximately
50,000 IGF-1R antigens and on MCF-7 cells approximately 1,000,000
IGF-1R antigens.
[0051] IGF-1R Assay Characterization:
[0052] The standard CTC assay using the CELLTRACKS system uses PE
for detection of cytokeratin present on cells of epithelial origin,
APC for detection of CD45 present on cells of hematopoeitic origin,
and FITC for detection of analyte specific reagents on OTCs defined
as cytokeratin-positive, CD45-negative nucleated cells. The current
limitation of detection of the CELLSPOTTER Analyzers for detection
of antigens with FITC-labeled antibodies is approximately 100,000
antigens per cell. To increase this sensitivity, the CTC assay was
reconfigured to lower the detection limit for IGF-1R detection.
Cytokeratin expressed at high densities on epithelial cells was
labeled with FITC, which permitted the use of the PE-labeled
anti-IGF-1R antibodies. In separate experiments 130 to 200 PC3-9,
T-24 or MCF-7 cells were spiked into 7.5 mL of blood and prepared
with the newly configured staining reagents. After preparation the
samples were scanned on the CELLSPOTTER Analyzer. The analyzer was
reconfigured such that cytokeratin FITC-positive, nucleated
DAPI-positive events were presented to the user as CTC
candidates.
[0053] In Panel A of FIG. 2 a typical example of MCF-7 cells
recovered from 7.5 mL of blood is shown. The top row shows a
cluster of 3 MCF-7 cells that clearly expressed the IGF-1R
receptor, the checkmark next to the composite image indicates that
the operator classified the cell as a CTC and the checkmark next to
the image showing the IGF-1R staining indicated that the operator
classified this CTC as one that expressed IGF-1R. All cells shown
in Panel A clearly expressed the IGF-1R receptor. In blood from
sixteen healthy individuals spiked with MCF-7 cells 80.6% (SD7.7)
of the recovered MCF-7 cells were classified as OTCs that expressed
IGF-1R. In Panel B of FIG. 2 a typical example of T-24 cells
recovered from 7.5 mL of blood is shown. Expression of IGF-1R of
the four T-24 cells was clearly dimmer as compared to the IGF-1R
staining of MCF-7 cells and the operator only classified the bottom
two cells as OTCs that expressed the IGF-1R receptor. In blood from
six healthy individuals spiked with T-24 cells, 13.6% (SD3.9) of
the recovered MCF-7 cells were classified as OTCs that expressed
IGF-1R. In blood from six healthy individuals spiked with PC3-9
cells, 3.8% (SD6.0) of the recovered MCF-7 cells were classified as
OTCs that expressed IGF-1R. These data provided guidance for the
IGF-1R antigen density on OTCs of patients with metastatic
carcinomas that is needed for it to be detected by this assay.
[0054] IGF-1R Expression on CTCs in Metastatic Carcinomas:
[0055] In 7.5 mL of blood from 139 healthy individuals CTCs were
virtually absent (0 CTCs in 135 and 1 CTC in 4). To test whether or
not IGF-1R indeed could be detected on CTCs in patients with
metastatic carcinomas, blood samples from 50 patients were tested.
In 7.5 mL of blood CTCs were detected in 11 of 50 (22%) patients.
Among these 50 patients 18 had breast cancer and in 28% CTCs were
detected, 13 had colorectal cancer and in 31% CTCs were detected, 3
had prostate cancer and in 33% CTCs were detected, 12 had lung
cancer and in 8% CTCs were detected and in none of the 4 patients
with ovarian cancer CTCs were detected. Examples of CTCs detected
are shown in FIG. 3. Eight CTC candidates are shown in the figure.
Events 1, 4, 5, 7 and 8 were classified as CTCs and only CTCs in
row 1 and 4 were classified as CTCs that expressed IGF-1R. Note
that potential IGF-1R staining can be observed in CTCs in row 5 and
7 but this was not considered sufficient for classification as
IGF-1R positive CTCs. Table 1 shows the number of CTCs detected in
the 11 patients with CTCs, the number of CTCs that expressed
IGF-1R, and the proportion of CTCs that express IGF-1R. In 8 of the
11 (91%) patients CTCs were detected that expressed IGF-1R. The
proportion of CTCs that expressed IGF-R, however, varied
greatly.
TABLE-US-00001 TABLE 1 IGF-1R(+) % IGF-1R(+) CTCs CTCs CTCs Breast
180 47 26 25 11 44 5 1 20 4 1 25 Colorectal 6 1 17 5 0 0 4 0 0 2 0
0 1 1 100 Prostate 16 1 6 Lung 2 1 50
Example 2
IGF-R Expression on CTCs in Phase 1 Dose-Finding Study of
Anti-IGF-1R Antibody
[0056] Study 1 was a dose-finding Phase 1 study designed to define
the safety and tolerability of a fully human anti-IGF-1R antibody
as described in U.S. Pat. No. 7,037,498 in patients with advanced
solid tumors. In this study, the anti-IGF-1R antibody treatment was
given every 21 days (21-day cycle) at doses from 3 to 20 mg/kg. In
order to evaluate the effect of treatment with anti-IGF-1R antibody
on the number of CTCs and CTCs expressing IGF-1R in these patients,
blood samples were drawn at screening, on Day 1 pre-dosing and
study Day 8 of each 21-day treatment cycle. One additional sample
was taken whenever a patient withdrew from the study due to disease
progression. Twenty-six patients provided blood samples for the
enumeration of CTCs during the course of the study.
[0057] Sixteen of the twenty-six patients (61%) had one or more
CTCs at some point during the study (pre-dosing or while on
treatment). In three of the sixteen patients with CTCs detected at
some point during the study, no IGF-1R-positive CTCs were detected.
In two cases only one CTC was detected in 7.5 mL of blood. Levels
of CTCs and IGF-1R-positive CTCs were plotted in relation to time
and several patterns of response to anti-IGF-1R antibody treatment
were observed. Four examples are depicted in FIGS. 4A-D. In Panel
A, CTC counts from a patient treated with a single dose of 6 mg/kg
of anti-IGF-1R antibody is shown. In this patient, the number of
CTCs and IGF-1R-positive CTCs decreased at Day 8 of treatment with
anti-IGF-1R antibody and were no longer detectable at Day 15 to
then reappear before the start of cycle 2. In Panel B, CTC counts
from a patient treated with a single dose of 10 mg/kg of
anti-IGF-1R antibody is shown. After a slight decrease of CTCs
after 8 and 15 days of treatment, the number of CTCs and
IGF-1R-positive CTCs increased by the time that the patient was
taken off study. This patient presented disease progression by CT
scan. In Panel C CTC counts from a patient treated with a single
dose of 20 mg/kg of anti-IGF-1R antibody is shown. CTCs and
IGF-1R-positive numbers were unaffected except for a spike of CTCs
15 days after administration of the dose. Panel D shows CTC counts
from a patient treated with a single dose of 3 mg/kg of anti-IGF-1R
antibody in which almost all detected CTCs expressed IGF-1R. An
increase of CTCs was noted at the time that the patient was taken
off the study. One possible interpretation of these data is that
most circulating tumor cells in these patients express IGF-1R and
that treatment with anti-IGF-1R antibodies appears to block a
survival signal necessary for the preservation of CTCs.
Alternatively, the effect of the anti-IGF-1R antibody may take
place directly at the tumor mass, inhibiting the migration of
CTCs.
[0058] Despite variability, a decrease in the number of CTCs and
IGF-1R-positive CTCs post-dosing was observed as well as a rebound
in the number of circulating cells by the end of the treatment
cycle. Increases in the number of CTCs and IGF-1R-positive CTCs
were also noted at post-study follow-up visits. These data support
a role for CTC and CTC-IGF-1R assays in the monitoring of the
biological and clinical response to anti-IGF-1R antibodies.
Example 3
IGF-1R Expression on CTCs in Study of Anti-IGF-1R Antibody in
Combination with Docetaxel
[0059] Study 2 was a phase 1 b dose-finding study of a fully human
anti-IGF-1R antibody as described in U.S. Pat. No. 7,037,498 in
combination with docetaxel (TAXOTERE) in patients with advanced
solid tumors. Docetaxel and anti-IGF-1R antibody were administered
on Day 1 and Day 22 at doses of 75 mg/m.sup.2 and 0.1-10 mg/kg,
respectively. Twenty-seven patients provided blood samples for the
enumeration of CTCs, including nineteen hormone-refractory prostate
cancer (HRPC) patients. CTC samples were collected at each cycle on
Day 1 pre-dose and on Day 8.
[0060] Nineteen of the twenty-seven patients (70%) had one or more
CTCs at some point during the study. In only one of the nineteen
patients with CTCs detected at some point of the study, no
IGF-1R-positive CTCs were detected. Interestingly, in all 5 CTC
assessments of this patient, CTCs were detected and no
IGF-1R-positive CTCs were detected, suggesting that the tumor site
itself in this patient may not express IGF-1R.
[0061] Decreases in the number of CTCs in response to treatment
were observed in the majority of patients. Levels of CTCs and
IGF-1R-positive CTCs were plotted in relation to time and several
patterns of response to anti-IGF-1R antibody treatment were
observed. Four examples are depicted in FIGS. 5A-D. Panel A depicts
the number of CTCs and IGF-1R-positive CTCs in a hormone refractory
prostate cancer patient treated with 10 mg/kg of anti-IGF-1R
antibody and 75 mg/m.sup.2 of docetaxel (21 day-cycle). Following
treatment, the total number of CTCs decreased reflecting a response
to the combination treatment. PSA values obtained from this patient
confirmed a clinical response to treatment. In addition, in this
patient, approximately 50% of the CTCs were originally
IGF-1R-positive before anti-IGF-1R antibody/docetaxel therapy; the
number of IGF-1R-positive CTCs quickly decreased with treatment.
Since the biological effect of the anti-IGF-1R antibody is to
induce IGF-1R down-regulation, these data support potential roles
of the CTC and CTC-IGF-1R assays in the monitoring of both the
clinical and biological (biomarker) activity of anti-IGF-1R
antibodies. Panel B depicts a patient with a similar decrease in
the number of CTCs and IGF-1R positive CTCs. Panel C and D depicts
two patients in which the number of CTCs and IGF-1R-positive CTCs
decreased after administration of each dose but also rebounded back
each time. Both patients were treated with low doses of anti-IGF-1R
antibody (0.8 and 3 mg/kg, respectively).
[0062] HRPC patients enrolled in Study 2 who had at least one
detectable IGF-1R-positive CTC at enrollment had higher PSA levels
(n=10, median PSA 475 ng/mL) than those who were IGF-1R-CTC
negative (n=8, median PSA 92 ng/mL). No assessment was possible in
two patients due to missing samples. Despite the apparent higher
tumor burden, patients that were IGF-1R-CTC positive at enrollment
responded by PSA criteria in a higher proportion (6 out of 10) and,
overall, earlier to the combination of docetaxel and anti-IGF-1R
antibody than those who were IGF-1R-CTC negative (2 out of 8), as
shown in Table 2:
TABLE-US-00002 TABLE 2 Treatment HRPC Patients Baseline Baseline
Cycle of Responding to PSA Baseline IGF-1R (+) Initial PSA
Treatment (ng/mL) CTCs CTCs Response 1006 238 2 1 3.sup.rd 1008 471
4 1 12.sup.th 1014 9944 12 5 2.sup.nd 1019 617 44 19 3.sup.rd 1022
1639 6 2 1.sup.st 1025 807 160 37 1.sup.st 1003 13 0 0 6.sup.th
1011 131 0 0 12.sup.th
Of the eight HRPC patients who had no detectable CTCs at study
entry, six did not respond to treatment, and three of them actually
experienced an increase in CTC and IGF-1R-positive CTC counts at
the time of disease progression. Finally, the one HRPC patient
treated with docetaxel and anti-IGF-1R antibodies with detectable
CTCs, but no detectable IGF-1R-positive CTCs, did not respond to
the combination treatment. The decrease in his CTC count was also
short-lived (1 cycle).
[0063] In Table 3, a summary of Patient Best Responses for HRPC
patients in Study 2 is shown. Two of eight (25%) patients with no
detectable IGF-1R-positive CTCs in 7.5 mL before initiation of
therapy showed a partial response to therapy whereas six of eleven
patients (55%) with .gtoreq.1 IGF-1R-positive CTCs in 7.5 mL showed
a partial response. These data strongly support the notion that the
CTC-IGF-1R assay could be employed to identify patients that could
benefit with anti-IGF-1R antibody therapy, as well as to identify
the optimal dose and determine the duration of treatment.
TABLE-US-00003 TABLE 3 IGF-1R-Positive Circulating Tumor Cells and
Patient Best Response in Study 2 Patient IGF-1R(+) CTCs at baseline
(n) Best Response 1001 2 PD 1005 2 SD 1006 1 PR 1008 1 PR 1012 31
PD 1013 4 PD 1014 5 PR 1019 19 PR 1021 4 PD 1022 2 PR 1025 37 PR
total = 6 PR/11patients 1002 0 SD 1003 0 PR 1010 0 PD 1011 0 PR
1015 0 SD 1016 0 PD 1018 0 SD 1020 0 SD total = 2 PR/8 patients PD
= progressive disease; SD = stable disease; PR = partial
response
Example 4
IGF-1R Expression on CTCs in Study of Anti-IGF-1R Antibody in
Combination with Paclitaxel and Carboplatin
[0064] Study 3 was a phase 1 b study of a fully human anti-IGF-1R
antibody as described in U.S. Pat. No. 7,037,498 in combination
with paclitaxel (TAXOL) and carboplatin (PARAPLATIN) in patients
with advanced solid tumors. The doses of paclitaxel, carboplatin
and anti-IGF-1R antibody were 200 mg/m.sup.2, AUC of 6, and 0.05-10
mg/kg, respectively. Forty-one patients provided blood samples for
the enumeration of CTCs. Blood samples were collected at each Cycle
on Day 1 pre-dose and on Day 8.
[0065] Twenty-one of the forty-one patients (51%) had one or more
CTCs at some point during the study, whereas ten of the forty-one
patients (24%) had one or more CTCs before the first dose was
administered. Sixteen of the forty-one patients (39%) had one or
more IGF-1R-positive CTCs at some point during the study, whereas
eight of the forty-one patients (20%) had one or more
IGF-R-positive CTCs before the first dose was administered. One
HRPC patient enrolled in the study had a large number of CTCs at
study entry (71 CTCs and 15 IGF-1R-positive CTCs) that decreased in
response to treatment (21 CTCs and 2 IGF-1R CTCs by treatment Day
21).
[0066] Levels of CTCs and IGF-1R-positive CTCs were plotted in
relation to time and several patterns of response to treatment were
observed. Four examples are depicted in FIGS. 6A-D. Panel A
represents data from a patient that joined the study with a very
low number of CTCs. After 6 cycles of treatment with
paclitaxel/carboplatin and 0.05 mg/kg of the anti-IGF-1R antibody
followed by 2 additional cycles of 3 mg/kg of anti-IGF-1R antibody,
the patient no longer responded to therapy. This lack of clinical
response was associated with a dramatic increase in the number of
CTCs and IGF-1R-positive CTCs. Progression of disease in this
patient was confirmed by CT scan. In Panel B data are presented
from a patient with a similar pattern of rising CTCs and
IGF-1R-positive CTCs during the course of the study. In Panels C
and D two patients are illustrated with CTCs before the initiation
of therapy. The patients received 1.5 and 6 mg/kg of anti-IGF-1R
antibody, respectively. The number of IGF-1R-positive cells appear
to decrease (Panel C) or remain low (Panel D) while the patient was
on anti-IGF-1R treatment. These data further support a potential
role of CTCs and CTCs-IGF-1R assays in the monitoring of the effect
and the development of resistance to anti-IGF-1R antibody
treatments.
[0067] Although certain presently preferred embodiments of the
invention have been described herein, it will be readily apparent
to those skilled in the art to which the invention pertains that
variations and modifications of the described embodiments may be
made without departing from the spirit and scope of the invention.
Accordingly, it is intended that the invention be limited only to
the extent required by the appended claims and the applicable rules
of law.
* * * * *