EP0614377A1

EP0614377A1 - Method for diagnosing and treating cancer

Info

Publication number: EP0614377A1
Application number: EP93900522A
Authority: EP
Inventors: Frederick C. Leung; Darrell R. Fisher; Michael R. Thompson; Scott D. Harvey
Original assignee: Battelle Memorial Institute Inc
Current assignee: Battelle Memorial Institute Inc
Priority date: 1991-11-14
Filing date: 1992-11-16
Publication date: 1994-09-14
Also published as: CA2123588A1; WO1993009816A1; AU3177893A; JPH07501332A

Abstract

La présente invention décrit des conjugués de facteurs de croissance et de radionucléides alphaémetteurs appropriés pour détecter et traiter le cancer. On décrit également des procédés de traitement du cancer utilisant des conjugués de facteurs de croissance et d'iode non radioactif, des conjugués de facteurs de croissance et d'un oxyanion d'un métal, et des conjugués d'un facteur de croissance et d'un isotope radioactif.The present invention describes conjugates of growth factors and alpha emitting radionuclides suitable for detecting and treating cancer. Also disclosed are methods of treating cancer using conjugates of growth factors and non-radioactive iodine, conjugates of growth factors and a metal oxyanion, and conjugates of growth factor and d 'a radioactive isotope.

Description

METHOD FOR DIAGNOSING AND TREATING CANCER

5 Statement of Government Interest

This invention was made with government support under contract DE-A606-76RLO 1830, awarded by the U.S. Department of Energy. The government has certain rights in the invention.

10 Technical Field

The present invention relates generally to methods for diagnosing and treating cancer.

Background of the Invention

15 Cancer accounts for one-fifth of the total mortality in the United

States, and is the second leading cause of death after cardiovascular diseases and stroke. The three leading types of tumors found in man are lung, prostate, and colorectal cancer, and the three leading types of tumors found in women are breast, lung, and colorectal cancer. Common therapeutic approaches for the

20 treatment of cancer generally involve the surgical removal of solid tumors, followed by chemotherapy and/or radiotherapy. One disadvantage of this general approach, however, is that most chemotherapeutic or radiotherapeutic agents are not tumor-cell specific, thus damaging normal tissue during the course of treatment.

25 Various methods have been utilized in order to more effectively direct or target therapeutic agents to tumor cells. For example, many tumor cells have an increased number of certain cell surface antigens as compared to normal cells. This difference between tumor and normal cells may be exploited in order to more effectively target therapeutic agents to tumor cells. More specifically,

30 targeting agents such as monoclonal antibodies may be used to specifically target and bind to the tumor cells, resulting in the localization and internalization of the ^' therapeutic agents. For example, monoclonal antibodies such as the anti-gpl6() antibody for human lung cancer {see Sugiyama et al., "Selective Growth Inhibition of Human Lung Cancer Cell Lines Bearing a Surface Glycoprotein gp lόO by ¹²⁵I-

35 Labeled Anti-gpl60 Monoclonal Antibody," Cancer Res. 48:2168-2113, 1988), a "TNT-l" monoclonal antibody for human cervical carcinoma (see Chen et al, 'Tumor Necrosis Treatment of ME- 180 Human Cervical Carcinoma Model with -^I-Labeled TNT-1 Monoclonal Antibody," Department of Pathology, University of Southern California School of Medicine, Los Angeles, California), and antibodies against the epidermal growth factor receptor for KB carcinoma (see Aboud-Pirak et al., "Efficacy of Antibodies to Epidermal Growth Factor Receptor Against KB Carcinoma In Vitro and in Nude Mice," J. National Cancer Institute ■50(20):1605-1611, 1988) have been used to specifically localize tumor cells. Monoclonal antibodies, however, are disadvantageous because they are typically developed in mouse systems, and injection of such antibodies into humans results in the generation of an extensive immune response against the antibody itself, thus limiting its effectiveness in killing tumor cells.

In order to kill tumor cells, targeting agents have been coupled to various chemotherapeutic agents including, among others, ricin, abrin, diptheria toxin, cholera toxin, gelonin, Pseudomonas toxin, Shigella toxin, and Pokeweed antiviral toxin (see U.S. Patent No. 4,545,985; see also Jansen et al., "Immunotoxins: Hybrid Molecules Combining High Specificity and Potent Cytotoxicity," Immunological Review 62:185-216, 1982; see also Thorpe and Ross, The Preparation and Cytotoxic Properties of Antibody-Toxin Conjugates," Immunological Review 62:119-158). Similarly, various radiotherapeutic agents have also been utilized to kill tumor cells including, for example, the beta emitters ¹³¹I, ⁶⁷Cu, ¹⁸⁶Re, and ⁹⁰Y. Beta emitters, however, are disadvantageous because of their low specific activity, low linear energy transfer, low dose rates (allowing for cell repair of radiation damage), damage to surrounding normal tissues, and in some cases the lack of an associated imageable photon (e.g., yttrium-90).

The present invention overcomes the disadvantages discussed above, and further provides other related advantages.

Summary of the Invention

The present invention provides reagents and methods for detecting and treating cancer. Within one aspect of the present invention, a conjugate of a growth factor and an alpha-emitting radionuclide is provided, the growth factor being capable of specifically binding to a defined population of cancer cells. Within various embodiments, the growth factor is coupled to the alpha-emitting radionuclide by a linker, such as a short polycarbon compound, to separate the alpha-emitting radionuclide from the growth factor. Preferred linkers may be selected from the group consisting of disulfides, dicarboxylic acids, and multi- carbon chain linkers (polycarbons). A particularly preferred linker is hexamethylene diamine. This linker may be coupled to a portion of the growth factor selected from the group consisting of the N-terminus and the C-terminus. In addition, within other embodiments of the invention, the alpha-emitting radionuclide is bound to a sequestering agent, such as, for example, a macrocyclic complexing agent. Preferred macrocyclic complexing agents include crown ethers such as a 21-crown-7 or an 18-crown-6 ether. Within another aspect of the present invention, a pharmaceutical composition is provided comprising a conjugate of a growth factor and an alpha-emitting radionuclide, and a pharmaceutically acceptable carrier or diluent, the growth factor being capable of specifically binding to a defined population of cancer cells. Within various embodiments of the present invention, the alpha- emitting radionuclide is selected from the group consisting of lead-212/bismuth- 212, bismuth-213/polonium-213, bismuth-212m, bismuth-212, polonium-206, polonium-210, astatine-211, radium-223, radium-224, and actinium-225.

Within another aspect of the present invention, a conjugate of a growth factor and non-radioactive iodine is provided, the growth factor being capable of specifically binding to a defined population of cancer cells. Pharmaceutical compositions are also provided, comprising a conjugate of a growth factor and non-radioactive iodine, and a pharmaceutically acceptable carrier or diluent, the growth factor being capable of specifically binding to a defined population of cancer cells.

Within other aspects of the invention, a method for treating cancer in warm-blooded animals is provided, comprising administering to a warm¬ blooded ammal an effective amount of a conjugate of a growth factor and an alpha-emitting radionuclide, a conjugate of a growth factor and non-radioactive iodine, a conjugate of a growth factor and yttrium-90, or a conjugate of a growth factor and an oxyanion of a metal, the growth factor being capable of specifically binding to a defined population of cancer cells. Within particularly preferred embodiments of the invention, the above-described method further comprises, prior to the step of administering an effective amount of a conjugate, administering an unlabeled growth factor capable of specifically binding to the defined population of cancer cells, in an amount sufficient to mask growth factor receptors in healthy tissues of the animal.

Within yet another aspect of the present invention, a method for detecting cancer is provided, comprising the steps of (a) administering to a warm- blooded animal an effective amount of a conjugate of a growth factor and an alpha-emitting radionuclide, the growth factor being capable of specifically binding to a defined population of cancer cells; and (b) detecting the presence and location of the conjugate within the warm-blooded animal and therefrom determining the presence of cancer.

Within another aspect of the present invention, a method for detecting the presence of cancer in warm-blooded animals is provided, comprising the steps of (a) administering to the animal an unlabeled growth factor capable of specifically binding to a defined population of cancer cells, in an amount sufficient to mask growth factor receptor sites in healthy tissues of the animal, (b) administering to the animal an effective amount of a conjugate of the growth factor and a radioactive isotope which emits gamma radiation, and (c) detecting the presence and location of the conjugate within the warm-blooded animal and therefrom determining the presence of cancer. Within one embodiment, the radioactive isotope is selected from the group consisting of rhenium-186, technetium-99m, iodine- 131, selenium-75, iodine-123, iodine-125, iodine- 124, indium-Ill, copper-67, radium-223, gold-198, yttrium-90, chromium-51, iron-52, copper-64, gallium-67, gallium-66, gallium-72, galIium-68, zircoπium-89, ruthenium-97, lead-203, rhodium-105, rhenium-188, gold-199, astatine-211, bromine-76, bromine-77, fluorine-18, bismuth-206, mercury-197, and mercury-203. Within yet another aspect of the present invention, a method for diagnosing and treating cancer in warm-blooded animals is provided, comprising the steps of (a) administering to the animal an unlabeled growth factor capable of specifically binding to a defined population of cancer cells, in an amount sufficient to mask growth factor receptor sites in healthy tissues of the animal, (b) administering to the animal an effective amount of conjugate of the growth factor and a radioactive isotope which emits gamma radiation, (c) detecting the presence and location of the conjugate within the warm-blooded animal and therefrom determining the presence of the cancer, and (d) administering an effective amount of a second conjugate of a growth factor and a radioactive isotope or non- radioactive iodine, such that the cancer is treated.

Within another aspect of the present invention, a method for diagnosing and treating cancer in warm-blooded animals is provided, comprising the steps of (a) administering to the animal an unlabeled growth factor capable of specifically binding to a defined population of cancer cells, in an amount sufficient to mask growth factor receptor sites in healthy tissues of the animal, (b) administering to the animal an effective amount of a first conjugate of a growth factor and a radioactive isotope which emits gamma radiation, (c) detecting the presence and location of the conjugate within the warm-blooded animal and therefrom determining the presence of the cancer, and (d) administering an effective amount of a second conjugate of a growth factor and a cytotoxic metal ion, such that the cancer is treated. Within one embodiment, the cytotoxic agent is an oxyanion of a metal selected from the group consisting of manganese, technetium, rhenium, chromium, molybdenum, tungsten, vanadium, and tellurium. Within another embodiment, the cytotoxic agent is an alpha particle emitting radioactive isotope selected from the group consisting of lead-212/bismuth-212, bismuth-213/polonium-213, bismuth-212m, bismuth-212, polonium-206, radium- 224, and actinium-225.

Within yet other embodiments of the above invention, the growth factor is selected from the group consisting of epidermal growth factor, transforming growth factor - alpha, fibroblast growth factors, insulin-like growth factor I and II, and nerve growth factor.

These and other aspects of the present invention will become evident upon reference to the following drawings and detailed description.

Brief Description of the Drawings

FIGURE 1 is a table listing various radioactive nuclides which emit alpha-particle radiation.

FIGURE 2 schematically illustrates a decay series starting with Cm-243. Radium-223 is a member of this decay series.

FIGURE 3 is a graph which illustrates the iodination profile of ^{1J 1}I to epidermal growth factor.

FIGURE 4 is a graph which compares cells treated with ³ I, ¹³¹I- epidermal growth factor, and epidermal growth factor alone. FIGURE 5 is a graph which illustrates the effects of various concentrations of -^--I-epideπnal growth factor on A431 cells.

FIGURE 6 is a graph which illustrates the effects of various concentrations of ¹³¹I-epidermal growth factor on L cells.

FIGURE 7 is a graph which illustrates the effects of various concentrations of non-radioactive iodine-epidermal growth factor on A431 cells.

FIGURE 8 is a graph which illustrates the effects of various concentrations of non-radioactive iodine-epidermal growth factor on L cells.

Detailed Description of the Invention As noted above, the present invention provides reagents for detecting and treating cancer. These reagents generally comprise a conjugate of a growth factor and an alpha-emitting radionuclide, a conjugate of a growth factor and non-radioactive iodine, or any of a number of growth factor conjugates which are described in more detail below, the growth factor being chosen such that it is capable of specifically binding to a defined population of cancer cells.

Many growth factors known to one of ordinary skill in the art may be utilized within the present invention. Representative examples include platelet derived growth factors, transforming growth factor-beta, iπterleukins (Le., IL-1, IL-2, IL-3, IL- , IL-5, IL-6, IL-7, IL-8, or IL-9), granulocyte-macrophage colony stimulating factor (GMCSF), erythropoietin, tumor necrosis factor, endothelial cell growth factor, platelet basic proteins, capillary endothelial cell growth factor, cartilage-derived growth factor, chondrosarcoma-derived growth factor, retina-derived growth factor, hepatoma derived growth factor, bombesin, and parathyroid hormone. Particularly preferred growth factors include epidermal growth factor, transforming growth factor - alpha, fibroblast growth factors, insulin like growth factor I and II, and nerve growth factor. The growth factor should be selected such that it is capable of specifically binding to a defined population of cancer cells which include, for example, preneoplastic cells, premetastatic cells, and tumor cells (both benign and malignant). As will be understood by one of ordinary skill in the art, a defined population of cancer cells may generally be differentiated from normal cells based upon the greater number of growth factor receptors on the cell surface. Consequently, within the context of the present invention a growth factor may be defined to be "specifically binding" to a defined population of cancer cells if this population of cells has greater than approximately two times the number of growth factor receptors on its surface as compared to normal cells, and preferably greater than five to ten times the number of growth factor receptors. In addition, this difference in the number of growth factor receptors on cancer cells, as compared to normal cells, may be exploited in order to more specifically target growth factor conjugates. In particular, the number of growth factor receptors on cells in healthy tissue may be determined, and compared to the number of growth factor receptors on cancer cells. As described in more detail below, unlabeled growth factor capable of specifically binding to a defined population of cancer cells may then be administered in an amount sufficient to mask growth factor receptor sites on the normal cells of healthy tissues, in order to mask the less abundant growth factor receptors on normal cells (if present) prior to the addition of a conjugated growth factor.

The number of growth factor receptors on a cell may be readily determined based upon the ability of the cell to bind to the growth factor receptor's substrate. For example, assays such as radioreceptor binding assays which determine the quantity of receptor substrate that binds to a cell over the course of time may be readily utilized to determine the number and type of cell surface receptors (see, for example, Ladda et a ., Anal. Biochem. 93:286-294, 1979). Briefly, utilizing radiolabeled growth factor and membrane preparations isolated from both normal and tumor cells, one can readily determine both growth factor receptor number and affinity by a standard competitive binding assay followed by a Scatchard plot analysis (see Scatchard, Anal. N.Y. Acad. Sci. 51:660-612, 1949).

In order to determine which growth factor conjugate would be the most effective therapeutical ly or diagnostically, within one embodiment the cells of interest (e.g., tumor cells) are removed from the patient. The removal of cells may typically be accomplished through surgical procedures, although many other methods may also be utilized, dependent of course on the type of tumor and its location. Once the tumor cells have been removed, they may be maintained in an in vitro culture using conventional media (see, for example, "Media Formulations," ATCC Cell Lines & Hybridomas, 1988). The number and type of receptors may then be readily determined using methods described above; and a growth factor conjugate selected on the basis of its ability to specifically bind to the tumor cells. Additionally, the therapeutic (or diagnostic) effectiveness of the growth factor conjugate upon tumor cells may be readily determined by in vitro assays. A representative assay is described below in Examples 1 and 2.

Alternatively, within another embodiment growth factor conjugates may be utilized for therapeutic or diagnostic purposes based only upon the known characteristics of certain tumors. For example, certain types of tumors such as human epidermal carcinomas are already well defined, and have been shown to possess abnormally high numbers of epidermal growth factor receptors (see Berger et al., "Epidermal Growth Factor Receptors in Lung Tumors," J. Pathology 152:297-307, 1987; Dotzlaw et al., "Epidermal Growth Factor Gene Expression in Human Breast Biopsy Samples: Relationship to Estrogen and Progesterone Receptor Gene Expression," Cancer Res. 50:4204-4208, 1990; Maddy et al., "Epidermal Growth Factor Receptors in Human Prostate Cancer: Correlation with Histological Differentiation of the Tumor," Br. J. Cancer 60:41-44, 1989; Liberman et al., "Expression of Epidermal Growth Factor Receptors in Human Brain Tumors," Cancer Res. 44:573-160, 1984; Neal et al., "Epidermal Growth Factor Receptors in Human Bladder Cancer: Comparisons of Invasive and Superficial Tumors," Lancet 7:366-368, 1985; and Moorghen et al., "Epidermal Growth Factor Receptors in Colorectal Carcinoma," Anticancer Res. 70:605-612, 1990). Thus, an epidermal growth factor conjugate may be readily applied to an epidermal carcinoma without the need to first determine which growth factor to use.

Similarly, Interleukin-2 receptors are expressed by abnormal T cells in patients with certain lymphoid malignancies or autoimmune disorders, but not by resting cells. For example, HTLV-I associated adult T-cell leukemia cells constitutively produce large numbers of IL-2 Tac receptors (see Waldmann, Cancer Surveys 8(4):&91-903, 1989, see also Waldmann, J. Nad. Cane. Inst. S7(12):914-923, 1989). Once a malignancy has been classified as an HTLV-I associated adult T-cell leukemia, an IL-2 growth factor conjugate may be utilized therapeutically without the need to further classify the malignancy as discussed above.

Similarly, a combination of growth factor conjugates may be utilized based upon the known distribution of tumor types in a given disease. For example, if 80% of human lung tumors express growth factor receptor type A, 15% of human lung tumors express growth factor receptor type B, and the remaining 5% of human lung tumors express growth factor type C; a conjugate may be prepared for the treatment of lung cancer comprising a combination of growth factors conjugates A, B, and C. Within one aspect of the present invention the growth factor is conjugated to an alpha-emitting radionuclide. Alpha-emitting radionuclides are particularly preferred because they have short range (35-70 μ through solid tissue and 35-700 μm through lung tissue), and are extremely efficient in killing cells. On the average, only about 1 to 3 alpha particle emissions must penetrate through the nucleus of a cell to kill the cell. If the three-dimensional geometry of cells is considered, about 25 alpha-particle emissions are needed per single cell to achieve complete cell killing in a tumor mass with uniform labeling of the cell surface by a radiolabeled protein (see 4th Int. Radiopharmaceutical Dosimetry Symposium, CONF-851113, pp 26-36, 1985). Many alpha-emitting radionuclides are well known in the art, and may be utilized within the present invention. A representative list is presented in Figure 1. Preferred alpha-emitting radionuclides include lead-212/bismuth-212, bismuth-213/poIonium-213, bismuth- 212m, bismuth-212, polonium-206, polonium-210, astatiπe-211. radium-223, radium-224, and actinium-225. Particularly preferred alpha-emitters are radium-223 (half- life = 11.4 days) and actinium-225 (half-life = 10.0 days). Radium-223 is a member of the natural uranium-235 decay series (see Figure 2). It exists naturally in all soils containing uranium and daughter products, but may be found at higher concentrations in uranium mill tailing piles. It may be removed by chemical separation from tailing sands by recovering its predecessor actinium-227.

Briefly, actinium-227 decays naturally to Th-227, which decays naturally to Ra-223 (see Figure 2). Radium-223 may be separated chemically from both Ac-227 and Th-227 by, for example, passing a saline solution over an ion- exchange resin containing the parent radionuclides. The purified salt radium-223 may thus be eluted from the column (see Pilger, UCRL-3877, 1957, University of California Radiation Laboratory, Berkeley, California; Mϋller, "Praparative Arbeiten uber Ac-227 und seine Folgeprodukte, Sonderdruck aus Radiochimica Ada 9:181-186, 1968; and Atcher et al., "A Radionuclide Generator for the Production of Pb-211 and its Daughters," J. Radioanal. Nucl. Chem. (Letters) 255(3):215-221, 1989). Radium-223 may also similarly be separated from enriched U-235 stockpiles in which natural radioactive decay has allowed the build-up of Ac-227.

An alternative method of producing radium-223 for medical applications is to start with natural radium-226. Within this method, radium-226 is first irradiated in a nuclear reactor to produce radium-227. The radium-227 then beta decays to actinium-227. For example, 1.0 curies of radium-226 is irradiated for about 120 days in a hydride assembly (such as the Fast Flux Test Facility, Richland, Washington). This assembly produces neutrons of epithermal energy, optimum for conversion of Ra-226 to Ra-227, which then beta-decays to Ac-227. Other nuclear reactors may, however, also be used to activate Ra-226 to Ra-227. This procedure produces about 9.5 curies of actinium-227. Radium-223 may then be chemically separated from actinium-227 and thorium-227 utilizing methods described above.

The growth factor may be conjugated to the alpha-emitting radionuclide by various methods, although it is particularly preferred to bind the alpha-emitting radionuclide to a sequestering agent, for example by positioning the alpha-emitting radionuclide within the sequestering agent, which is in turn coupled by a linker to the growth factor. A variety of diverse organic macrocyclic complexing agents may be used to sequester the alpha-emitting radionuclide including, among others, the following groups: ( 1) spherands, (2) cryptaspherands, (3) cryptands, (4) hemispherands, (5) corrands (modified crown ethers), and (6) podands (acyclic hosts) (see Cram, Science 240:760-67, 1988). In general, these macrocyclic ring compounds are large, somewhat spherical organic compounds which resemble cage structures, and have the ability to hold a heavy radionuclide as a ligand holds a metal ion. The sequestering agent should be selected such that it has both a high affinity and specificity for the alpha-emitting radionuclide as well as a low intrinsic mammalian toxicity. High specificity is essential to avoid displacement by other divalent cations (Mg⁺² and Ca⁺²) that are prevalent in physiological fluids. Additionally, the compound should either contain a functional group, or have chemistry which is compatible with the introduction of an appropriate functional group, to allow attachment to the linker.

The affinity of the sequestering agent for the alpha-emitting radionuclide is defined by the system energetics as described by Cram (supra). More specifically, as inferred by X-ray crystallographic data of complexed and non-complexed crown ethers, it is believed that the solution conformations of non- complexed ethers lack well-defined cavities with the associated convergently aligned binding sites. During the process of complexation, the crown ether undergoes desolvation and reordering of structure, a process which requires energy. If the sequestering agent presents a rigid prestructured and desolvated cavity to the ion (as is the case for spherands), the energy normally consumed by desolvation and reorganization is reflected in a larger binding constant for the ion. Based on this fundamental principle of reorganization, Cram lists the affinity of hosts for their most complimentary guests as: spherands > cryptaspherands > cryptands > hemispherands > corrands > podands. The difference in binding affinity between spherands and podands is dramatic, for example, the binding constant of a lithium sequestering spherand was found to be 10¹² higher than its corresponding open-chain podaπd (see Cram, supra). Thus, although many different sequestering agents may be utilized within the context of the present invention, spherands which are designed and synthesized specifically to sequester radium-223 are particularly preferred.

Particularly preferred sequestering agents include 18-crown-6 or 21- crown-7 ethers, including for example modified crown ethers such as dicyclohexano-21-crown-7 (Case and McDowell, Radioact. Radiochem. 7:58, 1990; McDowell et al, Solvent Extr. Ion Exch. 7:377, 1989; for other crown ethers or macrocyclic polyethers, see Pedersen, Science 247:536-540, 1988, U.S. Patent No. 4,943,375, Eia et al., Heterocycles J2(4):711-722, 1991; Wai and Du, Anal Chem 62(21):2412-14, 1990; Tang and Wai, Analyst (London) 774(4):451-453, 1989). Briefly, Ra²⁺ is bound by the etherate oxygen network comprising the interior cavity of the spherical crown-ether molecule. This binding is believed to be pH dependent: Rar complexes with a combination of a proton and smaller Group IA ions for the binding site within the crown cavity. These crown ethers may additionally be modified with polarizable functional groups (similar to changes made with closo- and mdo-carboamyl species used in boron-neutron capture therapy), resulting in compounds with greater solubility in aqueous media (see generally, Mizusawa et al., Inorg. C em. 24:1911, 1985). Such changes improve retention of biological specificity after conjugation, and improve the conjugate loading capability of the biological agent. These modifications may be accomplished in tandem with the synthesis of the above-noted crown ethers under appropriate conditions for mild conjugation to the biological delivery system.

Additional crown ethers suitable for use within the present invention may be synthesized, or purchased from various sources including, among others, Aldrich Chemical Co. (Milwaukee, Wis.), Fluka Chemical Corp. (Ronkonkoma, N.Y.), and Nisso Research Chemicals, (Iwai Co. Ltd., Tokyo, Japan). Sequestration of the alpha-emitting radionuclide may be achieved by mixing the sequestering agent with a salt of the alpha-emitting radionuclide which has been dissolved in solvent. The particular solvent chosen depends of course on the solubility of the sequestering agent and alpha-emitting radionuclide. For example, Cram and co-workers prepared the sodium complex of a spherand simply by adding excess salt dissolved in acetonitrile to a methylene chloride solution of the spherand (see Cram and Lein, / A . Chem. Soc. 707:3657-3668, 1985).

The ability of the crown ether to sequester or complex with the alpha-emitting radionuclide may be readily determined (see Cox et al., "Rates and Equilibria of Alkaline-Earth-Metal Complexes with Diaza Crown Ethers in Methanol," Inorg. Chem., 27:4018-4021, 1988; see also Mohite and Khopkar, "Separation of Barium From Alkaline Earths and Associated Elements by Extraction with Dibenzo-18-crown-6 From a Picrate Medium," Analytica Chimica Ada, 206:363-367, 1988). Briefly, separation of the complexed and free radionuclide can be accomplished by partitioning between an organic solvent (such as chloroform) and water. The complexed radionuclide will partition into the organic phase, whereas the free radionuclide will reside exclusively in the aqueous phase. Alternatively, a variety of chromatographic techniques such as High Performance Liquid Chromatography (HPLC) or Reverse-Phase High Performance Liquid Chromatography (RP-HPLC) may be utilized to separate sequestered radionuclide from the free cation. Once isolated, verification of the molecular architecture may be accomplished. Briefly, the mode of cation binding can take two forms: ( 1) through external association (i.e., anion/cation pairing without bond formation), or (2) via coordination of the cation to the crown-ether oxygen network. Specificity and strong binding, which are preferred for the present applications, are dependent on the latter type of association. Single crystal X-ray diffraction techniques may be used to unambiguously assign the type of interaction for the solid materials, and ¹⁷0, ¹³C and *H-NMR may be used to determine the structures of target materials in solution.

As noted above, within one embodiment of the present invention the alpha-emitting radionuclide is positioned within a sequestering agent which is i turn coupled by a linker to preferably either the amino ("N") or carboxy ("C") terminus of the growth factor. The linker serves to place an inert "spacer" between the biologically active growth factor and the alpha-emitting radionuclide containing complex. This space minimizes steric interactions that may interfere with the growth factor's affinity towards its target. The optimum length of the spacer arm is primarily dependent on the affinity of the growth factor for its target receptor. The higher this affinity, the smaller the relative importance of stearic repulsion between the sequestering agent and the target receptors. A virtually limitless number of linkers may be selected which are suitable for use within the present invention, although presently preferred linkers include disulfides, dicarboxylic acids, polycarbon chains, and modified polycarbon chains. Preferred linkers include hydrocarbon chains which range in length from 4 to 18 carbon atoms. Particularly preferred linkers have at least she methylene units such as hexamethylene diamine.

The linker may be attached to any of a number of extraanular functionalities on the sequestering agent, although carboxy and amino functionalities are particularly preferred. Within one aspect of the invention, if the extraanular fuπctionalization is a carboxy group, then a first synthetic step could involve reaction of the sequestering agent with hexamethylene diamine. Subsequent reaction with the C-terminus of the growth factor would complete synthesis of the conjugate. Alternatively, as noted above, the linker may be coupled to other aspects of the growth factor such as the N-terminus. Within this embodiment, after reaction with hexamethylene diamine the sequestering agent may be reacted with succinic anhydride. Subsequent coupling of the linker to the growth factor may then be accomplished through the N-terminus of the growth factor.

Alternatively, within another aspect of the present invention, the sequestering agent may contain an amino functionality. In these cases, a dicarboxylic acid linker (for example, octanedioic acid) may be utilized to couple the sequestering agent to the N-terminus of the growth factor. On the other hand. if the sequestering agent is reacted with ethylene diamine after condensation with the dicarboxylic acid, linkage to the growth factor may be accomplished through the C-terminus.

Within one embodiment of the present invention, in order to allow the covalent attachment of the sequestering agent to the linker an appropriate functionality is inserted into the sequestering agent. For example, a bromine atom may be incorporated into the appropriate position of an aromatic constituent during synthesis of the macrocyclic compound (see Skowronska-Ptasinska et al., J. Org. Chem 53:5484-91, 1988). Sequential treatment of this compound with n- butyllithium and CO2 yields the carboxy analog:

Sequestering

Agent

It should be noted, however, that synthetic reactions leading to these types of sequestering agents may produce very low yields.

Since the growth factor is likely to be the limiting reactant, the next step within this embodiment of the invention is the reaction between the functionalized sequestering agent and the linker:

If the sequestering agent is not immobilized on a rigid support the following by-product may also be produced:

Thus, chromatographic purification of the reaction mixture to isolate the desired product may be necessary before proceeding. Briefly, standard semi-preparative chromatographic separations based upon, for example, RP- HPLC or HPLC purification, may be utilized to purify the target compounds from the synthetic mixtures. Products may be detected either by refractive index or by the more sensitive technique of ultraviolet adsorption detection. Within one embodiment, a chromophoric benzene moiety is incorporated into the sequestering agent to facilitate detection during chromatographic purification.

The final reaction within this embodiment involves a similar reaction between the sequestering agent-linker (organic soluble) and the carboxy terminus of the growth factor (water soluble) as summarized below:

H (CH₂)₆NHOC-Growth Factor

Solubility incompatibilities may be overcome by use of a 50:50 dimethylformamide:water solvent system (see generally Cooper, The Tools of Biochemistry, Wiley, New York, pp. 234-255, 1977; Cuatrecasas, "Protein Purification by Affinity Chromatography on Polyacrylamide Beads," J Biol. Chem. 245:3059, 1970; and Cuatrecasas, "Affinity Chromatography of Macromolecules," in Advances in Enzymology, A. Meister (ed.), Wiley, New York, p.29, 1972). Within another aspect of the present invention, the growth factor is conjugated to non-radioactive iodine. Briefly, non-radioactive iodine may be obtained from many commercial sources, including, for example, Sigma Chemical Co. (St. Louis, Mo.). Various methods which are typically used to label proteins with radioactive iodine may also be utilized to conjugate non-radioactive iodine to the growth factor. For example, iodide (normally supplied as Nal) may be oxidized to form I2, which then attacks tyrosyl and histidyl side chains. Representative methods utilizing this technique include the Chloramine T method (Hunter and Greenwood, Nature 794:495-496, 1962), the Iodogen method (see Fraker and Speck, Biochem. Biophys. Res. Commun. 80:849-857, 1978), and the lactoperoxidase method (see Hubbard and Cohn, J. Cell Biol. 55:290-405, 1972). Alternatively, an iodinated reagent containing a reactive coupling group may be bound to the protein (see Bolton and Hunter, Biochem. J. 733:529-539, 1973).

Within other aspects of the invention, numerous additional growth factor conjugates are provided. Within one embodiment, these growth factor conjugates comprise a growth factor, and a radioactive isotope which emits gamma radiation. Representative examples of such radioactive isotopes include rhenium-186, technetium-99m, iodine-131, selenium-75, iodine-123, iodine-125, iodine-124, indium-I l l, copper-67, radium-223, gold-198, yttrium-90, chromium- 51, iron-52, copper-64, gallium-67, gallium-66, gallium-72, gallium-68, zirconium- 89, ruthenium-97, lead-203, rhodium- 105, rhenium-188, gold- 199, astatine-211, bromine-76, bromine-77, fluorine-18, bismuth-206, mercury-197, and mercury-203. Within other embodiments of the invention, the growth factor conjugate comprises a growth factor and a cytotoxic agent. Representative examples of cytotoxic agents include (in addition to the various alpha and gamma emitters discussed above) oxyanions of a metal selected from the group consisting of manganese, technetium, rhenium, chromium, molybdenum, tungsten, vanadium, and tellurium.

Conjugated growth factors of the present invention may additionally be purified utilizing a variety of techniques, including among others, column chromatography, HPLC, and RP-HPLC.

Conjugates of the present invention may be utilized in various ways. For example, they may be employed in in vitro assays as described below in order to kill specific cells. Additionally, as noted above, the conjugates of the present invention may be utilized for the treatment and detection of cancer in warm¬ blooded animals. Many warm-blooded animals may be treated and diagnosed for

SUBSTITUTE SHEET cancer, including for example, mice, rats, sheep, cows, pigs, monkeys, and humans. Briefly, as noted above, within one aspect of the present invention a method for treating cancer in warm-blooded animals is provided, comprising the step of administering to the animal an effective amount of a conjugate of a growth factor and an alpha-emitting radionuclide, the growth factor conjugate being capable of specifically binding to a defined population of cancer cells. Within one embodiment, the alpha-emitting radionuclide is selected from the group consisting of lead 212/bismuth-212, bismuth-213/polonium-213, bismuth-212m, bismuth-212, polonium-206, polonium-223, radium-224, and actinium-225. Within another aspect of the present invention, a method for treating cancer in warm-blooded animals is provided, comprising administering to the animal an effective amount of a conjugate of a growth factor and yttrium-90, the growth factor conjugate being capable of specifically binding to a defined population of cancer cells. Within yet another aspect of the present invention, a method for treating cancer in warm-blooded animals is provided, comprising the step of administering to the animal an effective amount of a conjugate of a growth factor and an oxyanion of a metal selected from the group consisting of manganese, technetium, rhenium, chromium, molybdenum, tungsten, vanadium, and tellurium, the growth factor conjugate being capable of specifically binding to a defined population of cancer cells.

Within another aspect of the present invention, a method for treating cancer in warm-blooded animals is provided, comprising the step of administering to the animal an effective amount of a conjugate of a growth factor and non-radioactive iodine, the growth factor conjugate being capable of specifically binding to a defined population of cancer cells.

Within particularly preferred embodiments of the invention, the present invention provides, prior to the step of administering an effective amount of a conjugate as described above, administering an unlabeled growth factor capable of specifically binding to the defined population of cancer cells, in an amount sufficient to mask growth factor receptors in healthy tissues of the animal. Briefly, in order to concentrate the radioisotope preferentially in cancer cells and avoid excessive damage to normal cells, administration of the conjugated growth factor is preceded by the step of administering a "cold" or unlabeled growth factor capable of binding to growth factor receptors in both normal and cancer cells, thereby reducing the number of receptor sites on normal cells available for binding and thus minimizing radiation damage to normal cells. Masking of growth factor receptors may be accomplished in methods for both treating and diagnosing cancer, as described herein.

Within one aspect of the present invention, a method for detecting cancer is provided, comprising the steps of (a) administering to a warm-blooded animal an effective amount of a conjugate of a growth factor and an alpha- emitting radionuclide, the growth factor being capable of specifically binding to a defined population of cancer cells, and (b) detecting the presence of the conjugate within the warm-blooded animal, and therefrom determining the presence of cancer. Briefly, conjugates or pharmaceutical compositions as described above may be administered in an effective amount as determined by experimental trials. The presence of the conjugate may be detected by any suitable nuclear medicine radiation camera which detects the requisite particle emissions (e.g., alpha or gamma). In the case of alpha emitters, a Nuclear Medicine Anger camera fitted with a collimator for the Tc"^^m energy window is particularly preferred. Within another aspect of the present invention, a method for detecting the presence of cancer in warm-blooded animals is provided comprising the steps of (a) administering to the warm-blooded animal an effective amount of a conjugate of a growth factor and an alpha-emitting radionuclide, the growth factor conjugate being capable of specifically binding to a defined population of cancer cells, and (b) detecting the presence and location of the conjugate within the warm-blooded animal and therefrom determining the presence of cancer.

Within yet another aspect of the present invention a method for detecting the presence of cancer in warm-blooded animals is provided, comprising the steps of (a) administering to the animal an unlabeled growth factor capable of specifically binding to a defined population of cancer cells, in an amount sufficient to mask growth factor receptor sites in healthy tissues of the animal, (b) administering to the animal an effective amount of a conjugate of the growth factor and a radioactive isotope which emits gamma radiation, and (c) detecting the presence and location of the conjugate within the warm-blooded animal and therefrom determining the presence of cancer. Within various embodiments of the present invention, the radioactive isotope is selected from the group consisting of rhenium- 186, technetium-99m, iodine-131, selenium-75, iodine-123, iodine-125, iodine-124, indium- 111, copper-67, radium-223, gold- 198, yttrium-90, chromium- 51, iron-52, copper-64, gallium-67, gallium-66, gallium-72, gallium-68, zirconium- 89, ruthenium-97, lead-203, rhodium-105, rhenium-188, gold-199, astatine-211, bromine-76, bromine-77, fluorine-18, bismuth-206, mercury-197, and mercury-203. Within another aspect of the present invention, a method for diagnosing and treating cancer in warm-blooded animals is provided, comprising (a) administering to the animal an unlabeled growth factor capable of specifically binding to a defined population of cancer cells, in an amount sufficient to mask growth factor receptor sites in healthy tissues of the animal, (b) administering to the animal an effective amount of conjugate of the growth factor and a radioactive isotope which emits gamma radiation, (c) detecting the presence and location of the conjugate within the warm-blooded animal and therefrom determining the presence of the cancer, and (d) administering an effective amount of a second conjugate of a growth factor and a radioactive isotope or non-radioactive iodine, such that the cancer is treated.

Within yet another aspect of the present invention, a method for diagnosing and treating cancer in warm-blooded animals is provided, comprising the steps of (a) administering to the animal an unlabeled growth factor capable of specifically binding to a defined population of cancer cells, in an amount sufficient to mask growth factor receptor sites in healthy tissues of the animal, (b) administering to the animal an effective amount of a first conjugate of a growth factor and a radioactive isotope which emits gamma radiation, (c) detecting the presence and location of the conjugate within the warm-blooded animal and therefrom determining the presence of the cancer, and (d) administering an effective amount of a second conjugate of a growth factor and a cytotoxic metal ion, such that the cancer is treated.

Within various embodiments of the invention, the cytotoxic agent is an oxyanion of a metal selected from the group consisting of manganese, technetium, rhenium, chromium, molybdenum, tungsten, vanadium, and tellurium.

Within yet other embodiments of the invention, the cytotoxic agent is an alpha particle emitting radioactive isotope selected from the group consisting of lead-

212 bismuth-212, bismuth-213/poIonium-213, bismuth-212m, bismuth-212, polonium-206, radium-224, and actinium-225. Within a further embodiment of the invention, pharmaceutical compositions are provided. Briefly, representative examples of pharmaceutical compositions include a conjugate of a growth factor and an alpha-emitting radionuclide, or a conjugate of a growth factor and non-radioactive iodine, or any of the other growth factor conjugates discussed above, along with a pharmaceutically acceptable carrier or diluent. Suitable pharmaceutically acceptable carriers or diluents include neutral buffered saline or saline.

Additionally, the pharmaceutical composition may contain other constituents, including for example buffers, carbohydrates such as glucose, sucrose, or dextrose, preservatives, as well as other stabilizers or excipients. Although appropriate dosages may be determined by experimental trials, about 5x10^**" to 5x10* ^■* conjugate complexes/70kg of adult weight may be administered assuming a 1:1 ratio of growth factor to the alpha-emitter or non-radioactive iodine. Nevertheless, the amount and frequency of administration will depend of course on many factors such as the condition of the patient, the nature and severity of the disease, as well as the type of cancer being treated. In addition, as discussed above, it is generally preferable to first mask growth factor receptors with unlabeled growth factor, in order to minimize damage to normal healthy tissues.

The following examples are offered by way of illustration, and not by way of limitation.

EXAMPLES

EXAMPLE 1 EFFECTS OF ¹³¹I RADIOLABELED EGF ON A431 CELLS

A. Preparation of Cells

The human cervical epidermoid carcinoma cell line A431 (available from the American Type Culture Collection or "ATCC," Rockville, Maryland, under accession number CRL 1555) was grown in Dulbecco's Modified Eagle's Medium with 10% fetal bovine serum. The cells were harvested by trypsinization with 0.05% trypsin and counted with trypan blue to obtain the number of live cells. A431 cells have approximately 1-2 x 10⁶ EGF receptors per cell.

B. Radioiodination of Epidermal Growth Factor

One hundred micrograms of murine EGF (GIBCO

Laboratories/Life Technologies, Inc., Grand Island, N.Y.) was radiolabeled by iodination with ¹³¹I (Dupont, Wilmington, Del.) using a modified method of lactoperoxidase procedure as described by Leung et al. (Proc. Soc. Exp. Biol. Med., 796(4):385-9, 1991) which modifies the procedure of Thorell and Johansson (Biochim. Biophys. Acta 257:363 1971). Briefly, H2O2 was added in four or five aliquots at 1-min intervals to a reaction mixture of 10 mCi of ¹³¹I, 100 μg of EGF, and 100 μg of lactoperoxidase. The labeled EGF was then separated from the free L^13lj aI and lactoperoxidase by gel filtration on a Sephacryl S-200 column (1 x 30 cm) that was previously equilibrated with 0.05 M phosphate-buffered saline containing 0.1% bovine serum albumin pH 7.6). Figure 3 shows the iodination profile of the ^I3iI to EGF, demonstrating that >90% of the ^I31I was labeled into the EGF molecule.

C. Cytotoxicity Assay A431 cells were grown in 6 well cultured plates. Two of the wells were exposed to approximately 500 juCi of radiolabeled EGF (¹³¹I-EGF), two of the wells were exposed to free ¹³ -\ similar to the quantity of ³ -EGF, and the other two wells of A431 cells were exposed to unlabeled EGF similar to the quantity of ^{1 1}I-EGF. The A431 cells were exposed to the three different treatments for one hour. Cells were washed twice with PBS buffer, and fed with DMEM and cultured for 5 days. The experiment was repeated in four replicate of 6 well plates. At the end of day 5, cells were harvested and counted. Figure 3 shows that the wells of A431 cells exposed to ¹³¹I-EGF have significantly fewer viable cells as compared to cells exposed to free ¹³¹I or cells exposed to unlabeled EGF. Thus, radiolabeled growth factor can be used as a specific cytotoxic agent for human tumor cells which possess high numbers of the growth factor receptor.

EXAMPLE 2 EFFECTS OF NON-RADIOACΠVE IODINE LABELED EGF ON A431 AND L CELLS.

A. Preparation of Cells

A431 cells and L cells were prepared as described above in Example 1. L cells are a murine fibroblast cell line which is available from the ATCC under accession number CRL 6362. Unlike A431 cells, L cells have less than 1000 EGF receptors per cell. Cells were grown and harvested as described above in Example IA.

B. Iodination of Epidermal Growth Factor

Epidermal Growth Factor was iodinated utilizing a procedure identical to that described in Example IB above, except that non-radioactive iodine (Sigma Chemical Co., St. Louis, Mo.) was utilized in place of radioactive iodine.

C. Cytotoxicity Assay

Cells were prepared and analyzed essentially as described in Example IC. As illustrated in Figures 4 through 7, ¹³ -EGF has a cytotoxic effect on A431 cells (see Figure 4), but not on cells with low numbers of receptors such as L cells (see Figure 6). When experiments were performed with EGF labeled with non-radioactive iodine, there was a surprising cytotoxic effect similar to that of EGF labeled ^{iJ l}ϊ. Furthermore, the cytotoxic effect did not extend to L cells, indicating that the cytoxic effect was mediated by EGF binding to the cell.

EXAMPLE 3 MASKING OF EGF-GROWTH FACTOR RECEPTORS PRIOR TO ADMINISTRATION or -"^■^I-EGF

In order to determine the biodistribution of ^Lj-^Jl EGF, the following experiment was undertaken. Briefly, approximately 1 x 10" A431 cells were ~~

injected subcutaneously into nude mice. The cells were allowed to grow in the mice for one to two weeks, after which the mice were injected either with or without unlabeled EGF, followed by the injection of ¹²³I-EGF. The mice were then sacrificed and the percent of injected dose per gram determined in the blood, tumor, muscle, lung, kidney, spleen, liver, intestine, thyroid, urine and stomach. The results of this experiment are set forth below in Tables I and II.

TABLE I

A-431 With Labeled EGF (1-123) Biodistribution Summar of Percent In ected Dose Per Gram

TABLE II

A-431 With Labeled EGF (1-123) Biodistribution Summar of Tissue to Blood Ratios

prior to labeled EGF; sacrificed at 3 minutes.

Mouse 25-10-1 was blocked with 25 ug native EGF 3 minutes prior to labeled EGF; sacrificed at 10 minutes

Mice 25-14-1 and 50-14-1 were injected with 25 ug and 50 ug native

EGF respectively, 3 minutes prior to labeled EGF; sacrificed at 14 hours. As shown in Table II, administration of unlabeled growth factor sufficient to mask growth factor receptors in normal healthy tissue, results in the more specific targeting of ¹²³I-EGF.

From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for the purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Furthermore, various references have been cited herein which provide additional detail and experimental insight, and are therefore hereby incorporated by reference. Accordingly, the invention is not limited except as by the appended claims.

While the applicant has disclosed the invention in the context of diagnosis and treatment of cancer, it is believed that the invention in its broadest aspect is applicable to the delivery of therapeutic agents to treat other disease conditions; for example, use of growth factor conjugated to a therapeutic agent such as an antibiotic would enhance the efficacy of the therapeutic agent for treating diseases such as rheumatoid arthritis.

Claims

1. A conjugate of a growth factor and an alpha-emitting radionuclide, said growth factor being capable of specifically binding to a defined population of cancer cells.

2. The conjugate of claim 1 wherein said growth factor is coupled to said alpha-emitting radionuclide by a linker.

3. The conjugate of claim 1 wherein said alpha-emitting radionuclide is bound to a sequestering agent.

4. The conjugate of claim 3 wherein the sequestering agent is a macrocyclic complexing agent.

5. The conjugate of claim 3 wherein said sequestering agent is a crown ether.

6. The conjugate of claim 5 wherein said crown ether is selected from the group consisting of 21-crown-7 ethers and 18-crown-6 ethers.

7. The conjugate of claim 2 wherein said linker is a polycarbon compound.

8. The conjugate of claim 2 wherein said linker is selected from the group consisting of disulfides, dicarboxylic acids, and polycarbons.

9. The conjugate of claim 2 wherein said linker is hexamethylene diamine.

10. The conjugate of claim 2 wherein said linker is coupled to a portion of the growth factor selected from the group consisting of the N-terminus and the C-terminus.

11. The conjugate of claim 1 wherein the alpha-emitting radionuclide is selected from the group consisting of lead-212/bismuth-212, bismuth-213/polonium- 213, bismuth-212m, bismuth-212, polonium-206, polonium-210, astatine-211, radium- 223, radium-224, and actinium-225.

12. A conjugate of a growth factor and non-radioactive iodine, said growth factor being capable of specifically binding to a defined population of cancer cells.

13. The conjugate of claims 1 or 12 wherein said growth factor is selected from the group consisting of epidermal growth factor, transforming growth factor - alpha, fibroblast growth factors, insulin like growth factor I and II, and nerve growth factor.

14. A pharmaceutical composition comprising a conjugate of a growth factor and an alpha-emitting radionuclide, and a pharmaceutically acceptable carrier or diluent, said growth factor being capable of specifically binding to a defined population of cancer cells.

15. A pharmaceutical composition comprising a conjugate of a growth factor and non-radioactive iodine, and a pharmaceutically acceptable carrier or diluent, said growth factor being capable of specifically binding to a defined population of cancer cells.

16. The pharmaceutical composition of claims 14 or 15 wherein said growth factor is selected from the group consisting of epidermal growth factor, transforming growth factor - alpha, fibroblast growth factors, insulin like growth factor I and π, and nerve growth factor.

17. A method for treating cancer in warm-blooded animals comprising adn-ii-nistering to said animal an effective amount of a conjugate of a growth factor and an alpha-emitting radionuclide, said growth factor conjugate being capable of specifically binding to a defined population of cancer cells.

18. The method of claim 17, wherein said alpha-emitting radionuclide is selected from the group consisting of lead 212/bismuth-212, bismuth-213/polonium- 213, bismuth-212m, bismuth-212, polonium-206, polonium-223, radium-224, and actinium-225.

19. A method for treating cancer in warm-blooded animals, comprising administering to said animal an effective amount of a conjugate of a growth factor and yttrium-90, said growth factor conjugate being capable of specifically binding to a defined population of cancer cells.

20. A method for treating cancer in warm-blooded animals, comprising administering to said animal an effective amount of a conjugate of a growth factor and an oxyanion of a metal selected from the group consisting of manganese, technetium, rhenium, chromium, molybdenum, tungsten, vanadium, and tellurium, said growth factor conjugate being capable of specifically binding to a defined population of cancer cells.

21. A method for treating cancer in warm-blooded animals comprising administering to said animal an effective amount of a conjugate of a growth factor and non-radioactive iodine, said growth factor conjugate being capable of specifically binding to a defined population of cancer cells.

22. The method of claims 16 - 21, further comprising, prior to the step of administering an effective amount of a conjugate, administering an unlabeled growth factor capable of specifically binding to a defined population of cancer cells, in an amount sufficient to mask growth factor receptors in healthy tissues of said animal.

23. A method for detecting the presence of cancer in warm-blooded animals, comprising:

(a) administering to said animal an effective amount of a conjugate of a growth factor and an alpha-emitting radionuclide, said growth factor conjugate being capable of specifically binding to a defined population of cancer cells; and

(b) detecting the presence and location of the conjugate within the warm-blooded animal and therefrom determining the presence of cancer.

24. A method for detecting the presence of cancer in warm-blooded animals, comprising:

(a) administering to said animal an unlabeled growth factor capable of specifically binding to a defined population of cancer cells, in an amount sufficient to mask growth factor receptor sites in healthy tissues of said animal;

(b) administering to said animal an effective amount of a conjugate of said growth factor and a radioactive isotope which emits gamma radiation; and (c) detecting the presence and location of the conjugate within the warm-blooded animal and therefrom determining the presence of cancer.

25. The method of claim 24 wherein said radioactive isotope is selected from the group consisting of rhenium-186, technetium-99m, iodine-131, selenium-75, iodine-123, iodine-125, iodine- 124, indium-Ill, copper-67, radium-223, gold-198, yttrium-90, chromium-51, iron-52, copper-64, gallium-67, gallium-66, gallium- 72, gallium-68, zirconium-89, ruthenium-97, lead-203, rhodium-105, rhenium-188, gold- 199, astatine-211, bromine-76, bromine-77, fluorine-18, bismuth-206, mercury-197, and mercuιy-203.

26. A method for diagnosing and treating cancer in warm-blooded animals, comprising:

(b) administering to said animal an effective amount of conjugate of said growth factor and a radioactive isotope which emits gamma radiation;

(c) detecting the presence and location of the conjugate within the warm-blooded ammal and therefrom determining the presence of said cancer; and

(d) administering an effective amount of a second conjugate of a growth factor and a radioactive isotope or non-radioactive iodine, such that said cancer is treated.

27. A method for diagnosing and treating cancer in warm-blooded animals, comprising:

(b) administering to said animal an effective amount of a first conjugate of a growth factor and a radioactive isotope which emits gamma radiation;

(c) detecting the presence and location of the conjugate within the warm-blooded animal and therefrom determining the presence of said cancer; and

(d) administering an effective amount of a second conjugate of a growth factor and a cytotoxic agent, such that said cancer is treated.

28. The method of claims 26 - 27 wherein said radioactive isotope is selected from the group consisting of rhenium-186, technetium-99m, iodine-131, selenium-75, iodine-123, iodine-125, iodine-124, indium-Il l, copper-67, radium-223, gold-198, yttrium-90, chromium-51, iron-52, copper-64, gallium-67, gallium-66, gallium- 72, gallium-68, zirconium-89, ruthenium-97, lead-203, rhodium-105, rhenium-188, gold- 199, astatine-211, bromine-76, bromine-77, fluorine-18, bismuth-206, mercury-197, and mercury-203.

29. The method of claim 27 wherein said cytotoxic agent is an oxyanion of a metal selected from the group consisting of manganese, technetium, rhenium, chromium, molybdenum, tungsten, vanadium, and tellurium.

30. The method of claim 27 wherein said cytotoxic agent is an alpha particle emitting radioactive isotope selected from the group consisting of lead- 212/bismuth-212, bismuth-213/polonium-213, bismuth-212m, bismuth-212, polonium- 206, radium-224, and actinium-225.

31. A composition according to claims 1-12 for use as an active therapeutic substance.

32. A composition according to claim 13 for use as an active therapeutic substance.

33. A conjugate of a growth factor and an alpha-emitting radionuclide, said growth factor conjugate being capable of specifically binding to a defined population of cancer cells, for use in a method for treating cancer.

34. A conjugate of a growth factor and non-radioactive iodine, said growth factor conjugate being capable of specifically binding to a defined population of cancer cells, for use in a method for treating cancer.