Note: Descriptions are shown in the official language in which they were submitted.
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A RADIOLABELLED LIGAND FOR SELECTIVELY INTRODUCING
AN AUGER ELECTRON EMITTING RADIONUCLIDE INTO THE
NUCLEUS OF A CANCER CELL
The invention is a radiolabelled ligand for
selectively introducing an Auger electron emitting
radionuclide into the nucleus of a cancer cell. The
invention allows for the selective killing of cancer
cells using Auger electrons while not affecting normal
cells to which the fusion protein is not bound and
internalized.
BACKGROUND OF THE INVENTION
A strategy for treatment of cancer is to identify
cell surface markers which are unique to the cancer or
which are overexpressed as compared to normal cells so
that a therapeutic agent can be targeted to such markers
of cancer cells. It is known, for example, that certain
cancers possess an overexpression of oncogene-encoded
growth factor receptors on their cell surfaces. Growth
factors are internalized after binding to their receptors
and in certain cases are translocated to the cell
nucleus.
A growth factor receptor which is frequently
overexpressed in breast cancer is the epidermal growth
factor receptor (EGFR). Thus, the polypeptide ligand,
epidermal growth factor (EGF), may constitute the basis
for constructing a polypeptide labelled with an Auger
electron emitting radionuclide for use in treatment of
this type of cancer. The invention provides a
radiolabelled ligand having the requisite characteristics
for a cancer therapeutic composition, and as such,
overcomes various shortcomings of prior art targeting
vehicles employing Auger emitting radionuclides for
cancer treatment.
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The present invention will be described in relation
to its application to breast cancer in which EGFR is
overexpressed. The skilled person will appreciate that
the invention has a broader scope than the specific
embodiments described herein.
Breast cancer accounts for about 180,000 new cases
and 45,000 deaths in North America yearly. Locally
confined breast cancer can be treated by surgery and
radiation, but metastatic disease requires systemic
therapy. Responses to systemic therapy can be short-
lived due to the development of drug resistance or
down-regulation of estrogen receptors. In this regard,
radiopharmaceuticals of the invention constitute a new
addition to the catalog of agents useful for the systemic
treatment of this and other cancers.
The radionuclides 125I and 111In decay by electron
capture where a proton is converted to a neutron by
capturing an electron from either a D, L or M shell. The
excess energy released causes a cascade of electrons from
higher shells to fill vacancies in lower shells with
release of 10-20 low energy electrons per decay for 1251
and 8 electrons for 111In. This cascade of electrons was
named after Pierre Auger who first reported it in 1925.
Auger electrons have a very limited range in tissue of <1
cell diameter, but their high rate of energy deposition
over very short distances causes ionization tracks
comparable or exceeding those of high linear energy
transfer radiation such as a-particles.
For the treatment of cancer, chromosomal DNA is the
radiosensitive target in the cell for Auger electrons.
Although the K-shell Auger electrons of 1251 and 111In have
sufficient range (8-14 ~,m) to deposit energy in the
nucleus from decays on the cell membrane, the subcellular
range of most Auger electrons requires that the
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radionuclide be internalized to exert its full effect.
The highest radiotoxicity is observed for the thymidine
analog 12SI_iododeoxyuridine (lzSIUdR) which is
incorporated into DNA (1). Chan et al. (2) showed that
<3 pCi of 125IUdR/cell reduced the survival of Chinese
hamster V79 fibroblasts to 1%. The survival curve had no
shoulder, characteristic of high linear energy transfer
radiation. Kassis (3) showed that rhodamine-123 (a
mitochondrial dye) labelled with lzSI was also radiotoxic
to V79 cells but 8o-fold less potent than lasIUdR. lzSIUdR
has limitations as a radiotherapeutic agent due to its
lack of specificity, targeting cells only in S-phase and
being subject to extensive deiodination by the liver (1).
Nevertheless, 125IUdR is being studied for treatment of
bladder cancer (4), gliomas (5) and hepatic metastases
(6, 7) where normal tissue uptake can be minimized by
local administration.
The use of targeted radiopharmaceuticals for the
delivery of Auger electron emitters to cancer cells has
been recognized to be desirable (8). To date, 1251
labelled targeting vehicles such as monoclonal antibodies
(9, 10), oligonucleotides complementary to certain genes
(11), or estradiol (12-14) have been investigated. All
of these radiopharmaceuticals reported to date, however,
suffer from various shortcomings as therapeutic agents.
Nucleotides cannot be specifically targeted in vivo.
Monoclonal antibodies have so far been disappointing as
therapeutic agents in a large part because they are
themselves immunogenic. Radiolabelled estradiol may be
useful in treatment of some breast cancers where there is
an overexpression of the estradiol receptor, but the
action of estradiol depends on a passive diffusion of the
compound across the cell membrane to the receptor which
is located inside the cell. This need for passive
diffusion across the cell membrane essentially limits the
use of Auger electron emitters to 125I. The use of 1251 as
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the radionuclide source of Auger electrons has some
disadvantages, as discussed below.
The present invention focuses on the hitherto
unexplored utilization of overexpressed or uniquely
expressed oncogene-encoded cell surface receptors to'
provide a means for targeting cancer cells for the
delivery of Auger emitting radionuclides. In this
regard, growth factor receptors present particularly
favorable candidates for targeting (15, 16?. The growth
factors, which are polypeptides, are internalized after
binding to their cell surface receptors, and in certain
cases are translocated to the cell nucleus (17).
The invention provides a radiolabelled ligand which
selectively binds a cell surface receptor for introducing
an Auger electron emitting radionuclide into a cancer
cell. The ligand binds a cell surface receptor that is
unique to a cancer cell or that is overexpressed on a
cancer cell. An Auger electron emitting radionuclide is
bonded to the ligand by, for example, a chelator. The
radiolabelled ligand is internalized by the cancer cell
after binding to the cell surface receptor in the same
fashion as is the naked ligand itself.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a chromatogram showing the purification
of 111In-DTPA-hEGF .
Figure 2 is a chromatogram showing the radiochemical
purity of 111In_DTPA-hEGF.
Figure 3 is a graph showing total binding,
non-specific binding and specific binding of
111In-DTPA-hEGF to MDA-MB-468 breast cancer cells.
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Figure 4 is a graph showing the kinetics of binding
and retention of llln-DTPA-hEGF and lzSI-hEGF to
MDA-MB-468 breast cancer cells.
Figure 5 is a graph showing the inhibition of the
growth of MDA-MB-468 breast cancer cells by internalizing
mlln radiopharmaceuticals.
Figure 6 is a graph showing the radiotoxicity of
internalized 111In-hEGF for MDA-MB-468 breast cancer cells
using a colony forming assay.
Figures 7 A-C, are fluorescent micrographs showing
the rapid binding and internalization of fluorescein-hEGF
with MDA-MB-468 breast cancer cells.
DETAILED DESCRIPTION OF THE INVENTION
There is a growing body of evidence that indicates
11'In is radiotoxic to cells when it is internalized.
Studies using 111In-oxine, which is a lipophilic complex
that internalizes nonspecifically into cells, show
toxicity due to chromosomal damage in lymphocytes (18),
hematopoietic stem cells (19), fibroblasts (20, 21),
sperm heads (22) , and tumour cells (23, 24) . 111In-oxine
cannot be used as a radiotherapeutic agent because of its
lack of specificity, but the present invention overcomes
this problem by providing a vehicle for preferentially .
delivering an Auger electron emitting radionuclide to a
cancer cell.
Many cancer types exhibit an overexpression of at
least one cell surface receptor as compared to normal
cells. There is considerable ongoing effort to identify
such cell surface markers as well as markers which may be
unique to cancer cells. One well documented cell surface
receptor which is overexpressed in a variety of cancers
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is the epidermal growth factor receptor (EGFR). Breast
cancer cells express up to 100-fold higher levels of EGFR
than do normal epithelial tissues (25). Overexpression
of EGFR has also been reported in colon cancer and
squamous cell carcinoma. Other growth factor receptors
which may be averexpressed in cancer cells include nerve
growth factor and platelet-derived growth factor
receptors.
The invention will be described in relation to the
utilization of EGFR overexpression as a means for
targeting cancer cells. Several investigators have
reported that a proportion of internalized EGF molecules
are translocated to the cell nucleus (17, 26, 27).
Overexpression of the EGFR occurs in up to 60% of breast
cancers and is inversely correlated with estrogen
receptor expression (28-36). EGFR expression in breast
cancer biopsies has ranged from 1-1200 fmol/mg membrane
protein (approx. 600-700,000 receptors/cell) with
overexpression considered to be >10 fmol/mg (37).
Elevated EGFR expression is associated with a high
relapse rate and poor long term survival. -
Normal epithelial cells express <10' receptors/cell.
For the normal breast cell line HBL-100, 8000 EGFR/cell
has been reported (29). The expression of EGFR in breast
cancer cell lines has a reported range of 800 EGFR/cell
for MCF-7 cells to 106 EGFR/cell for MDA-MB-468 cells
(25, 29, 38). The liver is the only normal tissue
exhibiting moderate high levels of EGFR (8 X 10' - 3 X
105 receptors/cell) likely reflecting its role in the
elimination of EGF from the blood (39-41).
Utilizing the Auger electron emitter 111In, an
initial study to illustrate the utility of the invention
was carried out using 111In_DTPA-hEGF. This radiolabelled
ligand was constructed using human epidermal growth
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factor which is commercially available. The chelator
diethylenetriamine pentaacetic acid (DPTA) was bonded to
the E-amino groups of the two free lysine residues (K28
and K48) or to the N-terminal a-amino group using the
acid anhydride, followed by chelation of 111In using an
appropriate salt.
Assuming that 11'In-DTPA-hEGF containing a single llln
atom is bound to 50 of the 106 receptors on the cell
membrane, 900 of the bound ligand is internalized, and 5%
is translocated to the nucleus; a dose projection of
15000 rads to the cell would be expected using cellular
microdosimetry models (42) with 13000 rads being
delivered to the nucleus. This dosage comfortably
exceeds the 6000 cads considered necessary to sterilize
deposits of breast cancer cells (43, 44).
Human epidermal growth factor (hEGF) has been
conjugated with DTPA for labelling with 111In and it has
been demonstrated that lllln_DTPA-hEGF exhibits an
affinity identical to 125I-hEGF for binding to EGFR on
MDA-MB-468 breast cancer cells (Ka of 7 X 10a L/mol).
111In-DTPA-hEGF detected different levels of EGFR on
breast cancer cell lines in vitro. 111In-DTPA-hEGF
detected 1.3 X 106 EGFR/cell on MDA-MB-468 human breast
cancer cells, 2.9 X 10' EGFR/cell on its subclone S1
(EGFR down-regulated), or 1.5 X 10' EGFR/cell on MCF-7
breast cancer cells.
Fluorescein has also been conjugated with hEGF
(fsc-hEGF). Fluorescence microscopy of MDA-MB-468 breast
cancer cells incubated with fsc-hEGF for 1 hour showed
membrane staining (Fig. 7A), but at 5 hours, there was
almost complete internalization into cytoplasmic vesicles
with some nuclear localization (Fig. 7B). The kinetics
of binding and internalization of 121In-DTPA-hEGF with
MDA-MB-468 cells have been examined. 111In-DTPA-hEGF bound
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rapidly to the cells, was internalized and in contrast to
125I_hEGF remained in the cell. The proportion of
111In_DTPA-hEGF internalized increased from 70o at 0.25
hours to >95% at 24 hours.
The radiotoxicity of internalized 111In in breast
cancer cells has been confirmed by labelling MDA-MB-458
cells with 111In-oxine. A dose-related growth inhibition
was observed with the number of cells recovered
decreasing by 93% at <7 pCi of 111In/cell (Fig. 5).
Radiotoxicity was not observed for 121In-DTPA, which does
not enter the cell.
Methods
Radiolabelling of Human Epidermal Growth Factor
Lyophilized human epidermal growth factor (hEGF) was
dissolved in 50 mM sodium bicarbonate in 150 mM sodium
chloride buffer pH 7.5 to a concentration of 10 mg/mL.
hEGF was then reacted with the bicyclic anhydride of
diethylenetriamine-pentaacetic acid (DTPA) at a molar
ratio (DTPA:hEGF) of 5:1 at room temperature for 30
minutes. The DTPA-conjugated hEGF was purified from
excess DTPA by size-exclusion chromatography on a P-2
mini-column (BioRad) eluted with 50 mM sodium bicarbonate
in 150 mM sodium chloride buffer pH 7.5. The absorbance
of fractions was measured at 280 nm. Purified DTPA-hEGF
was radiolabelled with 111In to a specific activity of
100-400 mCi/mg by incubation with 111In chloride mixed 1:1
(v/v) with 1 M sodium acetate buffer pH 6 for 30 minutes.
111In-DTPA-hEGF was purified from excess 111In by
size-exclusion chromatography on a P-2 mini-column
(BioRad) eluted with 50 mM sodium bicarbonate in 150 mM
sodium chloride buffer pH 7.5. Radiochemical purity was
confirmed by instant thin layer chromatography in 100 mM
sodium citrate pH 5. The Rf values for 111In-DTPA-hEGF
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and free 11'In are 0.0 and 1.0 respectively.
Measurement of Binding to bmA-MB-468 Breast Cancer Cells
111In-DTPA-hEGF (0.25-80 ng) was incubated with 1.5 X
106 MDA-MB-468 cells in 1 mL of 0.1% human serum albumin
in 35 mm multiwell culture dishes at 37°C for 30 minutes.
The cells were transferred to a centrifuge tube and
centrifuged. The cell pellet was separated from the
supernatant and counted in a g-scintillation counter to
determine bound (B) and free (F) radioactivity.
Non-specific binding was determined by conducting the
assay in 100 nM hEGF5l. The affinity constant (Ka) and
number of receptors/cell were determined from Scatchard
plots of B/F versus B. The kinetics of binding was
determined by incubating 1 ng of 111In-DTPA-hEGF or
125I_hEGF with 3 X 106 MDA-MB-468 breast cancer cells at
37°C and determining the proportion of radioactivity
bound to the cells at various times up to 24 hours. The
internalized fraction was measured by determining the
proportion of radioactivity which could not be displaced
from the cell surface by 100 nM hEGF.
Growth Inhibition Assay of 111In-DTPA-hEGF Against
bmA-MB-468 Cells
MDA-MB-468 breast cancer cells expressing
approximately 106 epidermal growth factor receptors/cell
were incubated with l~~In-DTPA-hEGF, unlabelled hEGF or
111In-oxine) centrifuged to remove free ligand, then
assayed and seeded (106 cells/dish) into 35 mm culture
dishes. Growth medium was added and the cells were
cultured at 37°C/5o COZ for 4 days. The cells were then
recovered by trypsinization and counted in a
hemocytometer. Control dishes contained cells cultured
in growth medium containing 111In-DTPA or growth medium
alone.
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Cytotoxicity Assay of 111Ia-DTPA-hEGF Against bmA-D2B-468
Cells
MDA-MB-468 breast cancer cells expressing
approximately 106 epidermal growth factor receptors/cell
were incubated with increasing amounts 111In-DTPA-hEGF or
111In_oxine, centrifuged to remove free ligand, assayed
and then seeded into 50 mm culture dishes. The number of
cells seeded was varied from 3 X 10' to 3 X 106 cells to
obtain approximately 300 viable colonies/dish taking into
account the plating efficiency and the expected level of
cytotoxicity. Control dishes contained MDA-MB-468 breast
cancer cells which were incubated with normal saline.
Growth medium was added and the cells were cultured at
37°C/5o COZ for 14 days. The growth medium was removed
and the colonies were stained with methylene blue (lo in
a 1:1 mixture of ethanol and water) then washed twice
with normal saline. The number of colonies per dish was
counted using a manual colony counter (Manostat Corp.).
The plating efficiency was calculated by dividing the
number of colonies observed by the number of cells seeded
in each dish. The surviving fraction at increasing
amounts of 111In-DTPA-hEGF or llln-oxine was calculated by
dividing the plating efficiency for dishes containing
treated cells with that observed for control dishes.
Fluorescence Microscopy of hEGF Incubated with bmA-~2B-468
Cells
Lyophilized hEGF was dissolved in 100 mM sodium
bicarbonate buffer pH 9 to a concentration of 10 mg/mL.
hEGF was then reacted with fluorescein isothiocyanate
(FITC, Pierce) at a molar ratio of 12:1 (FITC:hEGF) at
room temperature in the dark for 1 hour. The
fluorescein-conjugated hEGF was purified from excess
fluorescein by size-exclusion chromatography on a P-2
mini-column (BioRad) eluted with normal saline. The
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absorbance of fractions was measured at 495 nm. Purified
fluorescein-hEGF was stored in light-resistant
polypropylene tubes at -10°C. For fluorescence
microscopy, glass slides with adherent MDA-MB-468 breast
cancer cells were incubated with 100 nM of
fluorescein-hEGF for 1 hour at 37°C. The slides were
then washed twice with saline, then fixed with 0.8%
glutaraldehyde and cover slips mounted. The slides were
then examined using a fluorescence microscope with an
excitation wavelength of 494 nm and an emission
wavelength of 520 nm.
Results
Radiolabelling of Epidermal Gro~rth Factor
Human epidermal growth factor (hEGF) was conjugated
with approximately 1 mole of DTPA/mole of hEGF.
DTPA-hEGF was radiolabelled with 111In acetate to a
specific activity of 100-400 mCi/mg and purified on a P-2
size-exclusion mini-column. A representative chromatogram
is shown in Fig. 1. The radiochemical purity of
111In-DTPA-hEGF was >99% by instant thin layer
chromatography in 100 mM sodium citrate pH 5. A
representative chromatogram is shown in Fig. 2.
Measurement of Biadiag to ~A-MB-468 Breast Cancer Cells
The affinity constant for binding of l~lln_DTPA-hEGF
to MDA-MB-468 cells was 7 X 108 L/mol and the number of
binding sites was 1.3 X 106. A typical binding curve
showing total binding (TB), non-specific binding (NSB)
and specific binding (SB) is shown in Fig. 3. The
kinetics of binding of l~lln_DTPA-hEGF to MDA-MB-468 human
breast cancer cells is shown in Fig. 4. 111In-DTPA-hEGF
was rapidly bound by the breast cancer cells and was
retained for at least 24 hours. Over a 24 hour period at
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37°C, <80 of 111In radioactivity was lost from the cells
in-vitro. The proportion of 111In_EGF internalized (i.e.
not displaceable from the cell surface by 100 nM EGF)
increased from 70% at 0.25 hours to >95o at 24 hours.
Growth Inhibition Assay of lmln_DTPA-hEGF Against
I~A-MB-468 Cells
l~lln-hEGF (3.4 pCi/cell) achieved a 63% growth
inhibition of the MDA-MB-468 cells compared to the medium
control , whereas 111In-oxine ( 3 . 5 pCi/cell ) resulted in
89% growth inhibition (Fig. 5).~llln-DTPA which does not
internalize, had no effect on growth. By varying the
amount of 111In-oxine, we observed a dose-related effect
with 80% growth inhibition at 0.7 pCi/cell increasing to
93o at 6.9 pCi/cell.
Cytotoxicity Assay of 111In-DTPA-hEGF Against MDA-MB-468
Cells
Using a colony-forming assay, the radiotoxicity of
internalized 11'In for MDA-MB-468 breast cancer cells was
evaluated. lIn_DTPA-hEGF (8 pCi/cell) resulted in a 99%
reduction in cell survival for MDA-MB-468 cells (Fig. 6).
111In-oxine was also radiotoxic with greater than 99o cell
killing at <6 pCi/cell.
Fluorescence Microscopy of hEGF Incubated With MDA-MB-468
Cells
Human epidermal growth factor (hEGF) was conjugated
with approximately 1.5 moles of fluorescein/mole of hEGF.
Fluorescence microscopy of MDA-MB-468 human breast cancer
cells incubated with fluorescein-hEGF showed rapid
binding to the cell surface followed by internalization
into cytoplasmic vesicles and then nuclear localization
(Fig. 7 A-C) .
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The skilled person will appreciate the various
advantages of radiolabelled ligands of the invention for
use in cancer therapy. As seen from the foregoing data,
radiolabelled hEGF is rapidly internalized by cancer
cells after binding. In contrast with estradiol, for
example, the internalization process for hEGF involves an
active transport mechanism rather than simple diffusion
across the cell membrane. This active transport mechanism
for hEGF also includes nuclear translocation which allows
for a maximal radiation dose of Auger electrons to be
delivered to the cell's DNA. In contrast, 125I_estradiol
enters the cell by simple diffusion, the estradiol
receptor being located inside the cell. Because
diffusion of estradiol across the cell membrane is
dependent on maintaining its lipid solubility,
modifications to the molecule to allow radiolabelling may
adversely affect the targetability of the molecule.
Thus, conjugation of estradiol with a chelating agent
such as DTPA for labelling the molecule with 111In would
decrease lipid solubility, and significantly impair the
ability of the molecule to diffuse into the cell.
The problem of immunogenicity associated with the
use of monoclonal antibodies in vivo should largely be
avoided by the invention employing a human polypeptide
like hEGF. hEGF being an endogenous polypeptide is not
itself immunogenic in humans, and the conjugation of the
molecule with DTPA for labelling with 111In should not
increase its immunogenicity. Clinical experience with
the somatostatin peptide analog, octreotide conjugated
with DTPA and labelled with 111In has shown very low
immunogenicity in a study conducted on 1000 patients in
Europe.
While the invention may be used in association with
various Auger electron emitting radionuclides, it is felt
that for most applications 111In has certain advantages as
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compared to, for example, 1251. The uptake of the
radioligand can be monitored by external imaging because
l~lln also emits gamma radiation of sufficient energy to
be detected outside the body using a gamma ray camera.
In contrast, 1251 does not emit gamma radiation of
sufficient energy to be detected outside the body.
Obviously, it is important for the Auger electron
emitter to reside within the cell for a sufficient time
to administer a lethal dose of radiation to the nucleus.
The invention comprising l~lIn labelled hEGF has been
shown by the data presented to retain 111In within the
cell. Over a 24 hour period at 37°C, <8a of 111In
radioactivity was lost from cells in vitro as compared to
770 loss for cells which internalized a 125I labelled
molecule. 1251 labelled peptides and proteins are
catabolized in the cell to 1251-iodotyrosine.
Dehalogenase enzymes then cleave the radioiodine from the
iodotyrosine, and the free radioiodine diffuses away from
the tumour. The free radioiodine may then localize in
other normal tissues such as the thyroid, potentially
increasing toxicity to these tissues. In contrast, 111In
labelled peptides and proteins are degraded in the cell
to the terminal catabolite 111In-DTPA-lysine which is not
exported from the cell. Retention of 111In in the cell
maximizes the radiation dose (which is delivered over the
lifetime of the radionuclide? and minimizes normal tissue
toxicity. Previously utilized Auger electron emitting
radiopharmaceuticals such as iododeoxyuridine,
iodoestradiol or internalizing monoclonal antibodies have
used 1251 as the radiolabel; and therefore, suffer from
intracellular catabolism and export of free 1251 from the
cell .
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