WO1998024481A2

WO1998024481A2 - A radiolabelled ligand for selective introduction into the nucleus of a cancer cell

Info

Publication number: WO1998024481A2
Application number: PCT/CA1997/000909
Authority: WO
Inventors: Raymond M. Reilly
Original assignee: Individual
Current assignee: Individual
Priority date: 1996-12-02
Filing date: 1997-12-01
Publication date: 1998-06-11
Anticipated expiration: 1999-06-02
Also published as: CA2273946A1; WO1998024481A3; AU5113198A

Abstract

The invention is a radiolabelled ligand for introducing an Auger electron emitting radionuclide into the nucleus of a cancer cell. The ligand binds selectively to a cell surface receptor that is unique to a cancer cell or which is overexpressed on a cancer cell. An Auger electron emitting radionuclide is bonded to the ligand either directly or by means of a chelator. The radiolabelled ligand is internalized by the cell upon binding to the receptor, and a sufficient portion of the ligands so internalized are transported to the nucleus to provide a lethal dose of radiation to it.

Description

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 . 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 shortlived 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 ^1:L1In 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 ¹²⁵I and 8 electrons for ^llxIn. 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 α-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 ¹²⁵I and ^11:lIn have sufficient range (8-14 μm) to deposit energy in the nucleus from decays on the cell membrane, the subcellular range of most Auger electrons requires that the radionuclide be internalized to exert its full effect. The highest radiotoxicity is observed for the thymidine analog ¹²⁵I-iododeoxyuridine (¹²⁵IUdR) 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 ¹²⁵I was also radiotoxic to V79 cells but 80-fold less potent than ^12SIUdR. ¹²⁵IUdR 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, ¹²⁵I 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 ^12SI . The use of ¹²⁵I as 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 ^In-DTPA-hEGF.

Figure 2 is a chromatogram showing the radiochemical purity of ^11:LIn-DTPA-hEGF.

Figure 3 is a graph showing total binding, non-specific binding and specific binding of ^llxIn-DTPA-hEGF to MDA-MB-468 breast cancer cells. Figure 4 is a graph showing the kinetics of binding and retention of ^11:LIn-DTPA-hEGF and ¹²⁵I-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 ⁿⁱj_{n raα}:i₀p armaceuticals.

Figure 6 is a graph showing the radiotoxicity of internalized ^1:L1In-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:LIn is radiotoxic to cells when it is internalized. Studies using ^11:LIn-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) . ^1:L1In-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 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 overexpressed 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 <10⁴ 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 10⁴ - 3 X 10⁵ receptors/cell) likely reflecting its role in the elimination of EGF from the blood (39-41) .

Utilizing the Auger electron emitter ^1:L1In, an initial study to illustrate the utility of the invention was carried out using ^1:L1In-DTPA-hEGF. This radiolabelled ligand was constructed using human epidermal growth 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 o;-amino group using the acid anhydride, followed by chelation of ^11:LIn using an appropriate salt .

Assuming that ¹¹¹In-DTPA-hEGF containing a single ^llxIn atom is bound to 5% of the 10⁶ receptors on the cell membrane, 90% 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 rads considered necessary to sterilize deposits of breast cancer cells (43, 44).

Human epidermal growth factor (hEGF) has been conjugated with DTPA for labelling with ^llxIn and it has been demonstrated that ^11:LIn-DTPA-hEGF exhibits an affinity identical to ¹⁵I-hEGF for binding to EGFR on MDA-MB-468 breast cancer cells (Ka of 7 X 10⁸ L/mol) . ^11:LIn-DTPA-hEGF detected different levels of EGFR on breast cancer cell lines in vitro. ^1:L1In-DTPA-hEGF detected 1.3 X 10⁶ EGFR/cell on MDA-MB-468 human breast cancer cells, 2.9 X 10⁴ EGFR/cell on its subclone SI (EGFR down-regulated) , or 1.5 X 10⁴ EGFR/cell on MCF-7 breast cancer cells.

Fluoreεcein 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 ^11:1In-DTPA-hEGF with MDA-MB-468 cells have been examined. ^1:L1In-DTPA-hEGF bound rapidly to the cells, was internalized and in contrast to ^12SI-hEGF remained in the cell. The proportion of ^11:1In-DTPA-hEGF internalized increased from 70% at 0.25 hours to >95% at 24 hours.

The radiotoxicity of internalized ^1:L1In in breast cancer cells has been confirmed by labelling MDA-MB-468 cells with ^xllIn-oxine. A dose-related growth inhibition was observed with the number of cells recovered decreasing by 93% at <7 pCi of ^11:LIn/cell (Fig. 5) . Radiotoxicity was not observed for ^11:LIn-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 ^11:IIn to a specific activity of 100-400 mCi/mg by incubation with ^llxIn chloride mixed 1:1

(v/v) with 1 M sodium acetate buffer pH 6 for 30 minutes. ^llxIn-DTPA-hEGF was purified from excess ^llxIn 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 R_f values for ^11:1ln-DTPA-hEGF and free ^11:LIn are 0.0 and 1.0 respectively.

Measurement of Binding to MDA-MB-468 Breast Cancer Cells

^In-DTPA-hEGF (0.25-80 ng) was incubated with 1.5 X 10^s 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 hEGF51. 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 ^1:L1In-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 ^1X1In-DTPA-hEGF Against MDA-MB-468 Cells

MDA-MB-468 breast cancer cells expressing approximately 10⁶ epidermal growth factor receptors/cell were incubated with ^In-DTPA-hEGF, unlabelled hEGF or ^1:L1In-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/5% C0₂ for 4 days. The cells were then recovered by trypsinization and counted in a hemocytometer . Control dishes contained cells cultured in growth medium containing ^X11ln-DTPA or growth medium alone . Cytotoxicity Assay of ^lxlIn-DTPA-hEGF Against MDA-MB-468 Cells

MDA-MB-468 breast cancer cells expressing approximately 10⁶ epidermal growth factor receptors/cell were incubated with increasing amounts ^1:L1In-DTPA-hEGF or ^11:LIn-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 10⁴ 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/5% C0₂ for 14 days. The growth medium was removed and the colonies were stained with methylene blue (1% 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 ^1:11In-DTPA-hEGF or ^1:11In-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 MDA-MB-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 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 Growth Factor

Human epidermal growth factor (hEGF) was conjugated with approximately 1 mole of DTPA/mole of hEGF. DTPA-hEGF was radiolabelled with ^llxIn 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 ^1:llIn-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 Binding to MDA-MB-468 Breast Cancer Cells

The affinity constant for binding of ^11:LIn-DTPA-hEGF to MDA-MB-468 cells was 7 X 10^s 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 ^1:L1In-DTPA-hEGF to MDA-MB-468 human breast cancer cells is shown in Fig. 4. ^1:L1In-DTPA-hEGF was rapidly bound by the breast cancer cells and was retained for at least 24 hours. Over a 24 hour period at 37°C, <8% of ^11:LIn radioactivity was lost from the cells in-vitro. The proportion of ^1:L1In-EGF internalized (i.e. not displaceable from the cell surface by 100 nM EGF) increased from 70% at 0.25 hours to >95% at 24 hours.

Growth Inhibition Assay of ^1:ιlIn-DTPA-hEGF Against MDA-MB-468 Cells

^1:11In-hEGF (3.4 pCi/cell) achieved a 63% growth inhibition of the MDA-MB-468 cells compared to the medium control, whereas ^11:LIn-oxine (3.5 pCi/cell) resulted in 89% growth inhibition (Fig. 5) . ^In-DTPA which does not internalize, had no effect on growth. By varying the amount of ^1:L1In-oxine, we observed a dose-related effect with 80% growth inhibition at 0.7 pCi/cell increasing to 93% at 6.9 pCi/cell.

Cytotoxicity Assay of ^X11In-DTPA-hEGF Against MDA-MB-468 Cells

Using a colony-forming assay, the radiotoxicity of internalized ^1:L1In for MDA-MB-468 breast cancer cells was evaluated. ^11:LIn-DTPA-hEGF (8 pCi/cell) resulted in a 99% reduction in cell survival for MDA-MB-468 cells (Fig. 6) . ^11:lIn-oxine was also radiotoxic with greater than 99% 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) . 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, ^{1 E}I-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 ^X11ln 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 ^llxIn should not increase its immunogenicity. Clinical experience with the somatostatin peptide analog, octreotide conjugated with DTPA and labelled with ^1:L1In 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 ^11:LIn has certain advantages as compared to, for example, ¹⁵I. The uptake of the radioligand can be monitored by external imaging because ^11:LIn also emits gamma radiation of sufficient energy to be detected outside the body using a gamma ray camera. In contrast, ^{1 B}I 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 ^11:LIn labelled hEGF has been shown by the data presented to retain ^11:LIn within the cell. Over a 24 hour period at 37°C, <8% of ^1:L1In radioactivity was lost from cells in vitro as compared to 77% loss for cells which internalized a ¹²⁵I labelled molecule. ¹²⁵I labelled peptides and proteins are catabolized in the cell to ¹²⁵I-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, ^11;LIn labelled peptides and proteins are degraded in the cell to the terminal catabolite ^1:L1In-DTPA-lysine which is not exported from the cell. Retention of ^1:L1In 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 ¹²⁵I as the radiolabel; and therefore, suffer from intracellular catabolism and export of free ¹²⁵I from the cell. References

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Claims

CLAIMS :

1. A radiolabelled ligand for introducing an Auger electron emitting radionuclide into the nucleus of a cancer cell, comprising:

a ligand which binds selectively to a cell surface receptor that is unique to a cancer cell or which receptor is overexpressed on a cancer cell; and

an Auger electron emitting radionuclide bonded to the ligand, the radiolabelled ligand being internalized by the cell upon binding to the receptor and a sufficient number of radiolabelled ligands or radiolabelled ligand degradation products so internalized being transported to the nucleus of the cell to provide a lethal dose of radiation to the nucleus .

2. A radiolabelled ligand as claimed in claim 1, wherein the ligand has a chelator bonded to it and the radionuclide is bound to the chelator.

3. A radiolabelled ligand as claimed in claim 1, wherein the ligand is human epidermal growth factor

(hEGF) .

4. A radiolabelled ligand as claimed in claim 2, wherein the radionuclide is ^11:LIn.

5. A radiolabelled ligand as claimed in claim 2, wherein the chelator is diethylenetriamine pentaacetic acid.

6. The radiolabelled ligand ^In-DTPA-hEGF having at least one ^11:LIn-DTPA moiety.

7. A pharmaceutical composition for use in the treatment of cancer comprising a radiolabelled ligand as claimed in claim 1.