US00606883OA United States Patent (19) 11 Patent Number: 6,068,830 Diamandis et al. (45) Date of Patent: May 30, 2000

54) LOCALIZATION AND THERAPY OF FOREIGN PATENT DOCUMENTS NON-PROSTATIC ENDOCRINE CANCER 0217577 4/1987 European Pat. Off.. WITH AGENTS DIRECTED AGAINST 0453082 10/1991 European Pat. Off.. PROSTATE SPECIFIC ANTIGEN WO 92/O1936 2/1992 European Pat. Off.. WO 93/O1831 2/1993 European Pat. Off.. 75 Inventors: Eleftherios P. Diamandis, Toronto; Russell Redshaw, Nepean, both of OTHER PUBLICATIONS Canada Clinical BioChemistry vol. 27, No. 2, (Yu, He et al), pp. 73 Assignee: Nordion International Inc., Canada 75-79, dated Apr. 27, 1994. Database Biosis BioSciences Information Service, AN 21 Appl. No.: 08/569,206 94:393008 & Journal of Clinical Laboratory Analysis, vol. 8, No. 4, (Yu, He et al), pp. 251-253, dated 1994. 22 PCT Filed: Jul. 14, 1994 Bas. Appl. Histochem, Vol. 33, No. 1, (Papotti, M. et al), 86 PCT No.: PCT/CA94/00392 Pavia pp. 25–29 dated 1989. S371 Date: Apr. 11, 1996 Primary Examiner Yvonne Eyler S 102(e) Date: Apr. 11, 1996 Attorney, Agent, or Firm-Banner & Witcoff, Ltd. 87 PCT Pub. No.: WO95/02424 57 ABSTRACT It was discovered that prostate-specific antigen is produced PCT Pub. Date:Jan. 26, 1995 by non-proStatic endocrine cancers. It was further discov 30 Foreign Application Priority Data ered that non-prostatic endocrine cancers with recep tors can be stimulated with to cause them to produce Jul. 14, 1993 GB United Kingdom ...... 93.14623 PSA either initially or at increased levels. This invention 51) Int. Cl." ...... A61K 51/00; A61K 49/00; relates to the imaging of non-prostatic endocrine cancers by G01N 31/00; G01N 33/48 labelled biological binding units which bind to prostate 52 U.S. Cl...... 424/9.34; 424/1.49; 424/9.323; Specific antigen in an imaging procedure, Such as, radioim 435/723; 436/64 aging or magnetic resonance imaging. Further, the PSA 58 Field of Search ...... 435/7.23; 424/1.49, binding units may be constructed to deliver a toxic agent, 424/9.34, 9.323; 436/64 Such as a radioisotope, toxin or a drug to provide endocrine cancer therapy. Another aspect of the invention is passive 56) References Cited immunotherapy against endocrine cancers by treatment with PSA-binding units. U.S. PATENT DOCUMENTS 5,162,504 11/1992 Horoszewicz ...... 530/388.2 7 Claims, 4 Drawing Sheets U.S. Patent May 30, 2000 Sheet 1 of 4 6,068,830

6 A Free PSA 2 45000 E 5 CS d S 4 35000 g 35 25000

1 15000 SD O o O 1 O 20 30 40 50 60 5000 Fraction Number F. G. A.

B Free PSA 45OOO ACT-PSA t 35OOO

25000 l i 15000 adtw. 5OOO 1O 20 30 40 50 60 Fraction Number F. G. B. U.S. Patent May 30, 2000 Sheet 2 of 4 6,068,830

PSA

4.4 U.S. Patent May 30, 2000 Sheet 3 of 4 6,068,830

1O 1.

S 1 O) 2 P CC { ... 1

O1 -11 -10 -9 -8 -7 -6 Log(Steroid Concentration,mol/L) F. G. 3. U.S. Patent May 30, 2000 Sheet 4 of 4 6,068,830

& ------& & S.&

6,068,830 1 2 LOCALIZATION AND THERAPY OF NON Direct approaches to radionuclide imaging include radio PROSTATIC ENDOCRINE CANCER WITH labelled chemotherapeutic agents, simple ionic Substances, AGENTS DIRECTED AGAINST PROSTATE metabolite imaging, immunologic and receptor imaging. SPECIFIC ANTIGEN The use of radiolabelled chemotherapeutic agents, Such as bleomycin, have not demonstrated clinical value (14). ''Ga FIELD OF THE INVENTION citrate is the most commonly used simple ionic tracer for This invention relates to the localization and therapy of tumor imaging, however it localizes in other pathologies and non-prostatic endocrine cancers by agents that have been is non specific (15, 16, 17, 18). Metabolite imaging carried constructed to target prostate specific antigen (PSA). out with positron emitting radionuclides such as F fluorodeoxyglucose, 'C-methionine and 'C-thymidine BACKGROUND OF THE INVENTION provides tumor information that has been shown Cancer of the breast is the most common cause of cancer to be clinically valuable for disease staging (19, 20, 21). death in middle aged women in Europe and North America The receptor imaging of breast cancer has been attempted and both its incidence and mortality are on the increase by Several approaches. Spicer et al. (22) and Hochberg (23) (1-5). The predominant indications for breast tumor imag 15 and others (24-29) have developed radiolabelled estradiols ing are: detecting the presence of tumor, localizing Sites of and have been able to demonstrate imaging in disease, and following the effects of therapy (6). Trends in receptor positive breast cancers. It has been postulated that Scintigraphic imaging have been towards developing imag a therapeutic response could result with Auger electrons ing pharmaceuticals to provide quantitative information on from 'I or 'I radiolabelled estradiols (30,31), or from B the pathophysiological characteristics of a tumor, Such as its emitting radioisotopes such as Re conjugated to proges anaplasticity, or likely response to a given therapy (7). For terone (32). A problem with receptor based imaging is the example, to determine Via Scintigraphic imaging how a interference that antagonists, Such as patients breast cancer will respond to the administration of a growth Suppressor, Such as Somatostatin, or the estrogen , may have in the clinical environment. receptor antagonist tamoxifen (8). Diamandis in PCT Appli 25 It is known that proteins, Such as antibodies, can be cation CA 94/00267 has shown that the presence of PSA in developed against Specific antigens that are either produced breast tumors, as measured by in vitro methods, has prog or associated with tumors, can be used to localize tumors. nostic value. Thus, imaging of these tumors may not only U.S. Pat. No. 3,927,193 to Hansen et al. (33) discloses a reveal occult disease, but may also provide clinically valu process whereby antibodies to carcinoembryonic antigen able pathophysiological information. (CEA) and labelled with 'I and "I were used to image the location of tumors present in hamsters. From this work it Tumor imaging is commonly carried out using a gamma was proposed that the location of a tumor in a human could emitting radionuclide conjugate and a Scintillation gamma be determined by in Vivo administration of a parenteral camera, or with a positron emitting radiopharmaceutical and Solution containing an antibody-radioisotope conjugate fol a positron or PET camera, or with a magnetopharmaceutical lowed by imaging by a gamma camera. Goldenberg et al. and a magnetic resonance imaging device. The Scintillation 35 camera, also known as an Anger camera, consists of a reported Success in clinical trials of tumor detection and detector head, and a display console. The Anger camera head localization by Scintillation Scanning of patients that is composed of Sodium iodide crystals that absorb gamma received radiolabelled antibodies to CEA (34). rays and emits the absorbed energy as flashes of light Based on the original work of Milstein and Kohler (35), Scintillations that are proportional in brightness to the energy 40 monoclonal antibodies have been developed against a vari absorbed. In a gamma camera the Sodium iodide crystals are ety of tumour antigens such as CA 19.9, CA 125, melanoma coupled to photomultiplier tubes that convert light pulses asSociated antigens, TAG 72, C. fetal protein, ferritin, into electronic pulses. These voltages are translated via a choriogonadotropin, prostatic acid , and PSA for computing circuit to a cathode ray tube. The data from the radioimmunoimaging and therapy. camera head may be in either analog or digital form that can 45 Several investigators have reported on the development of be stored in a computer and can reconstruct the data to monoclonal antibodies against epitopes of various malignant provide an image. Single-photon emission computed tomog prostate cell components (36, 37, 38, 39, 40). Moveover, raphy (SPECT) imaging involves the use of a gamma PSA was purified and well characterized and found to have Scintillation camera where multiple images, typically a molecular weight in the range of 34,000 (41). PSA is used encompassing 180 or 360, around the body are taken and 50 widely as a tumor marker for in vitro based analyses for the computer issued to reconstruct multiple tomograms in diagnostic and monitoring purposes of prostatic carcinoma. coronal, Sagittal, and transverse projections. In PET imaging U.S. Pat. No. 5,162,504 describes monoclonal antibodies the positron radionuclide collides with an electron causing that have been developed to recognize malignant prostate annihilation of the particles and creating two photons that epithelium. These antibodies were developed as diagnostic travel in 180 opposite directions. The PET system is 55 and prognostic tools for the detection of cancer of the designed to capture opposite sides and register the count at prostate, not as embodies in this invention, for the detection precisely the same time. A computer is used to manipulate of cancers not associated with the prostate. Until the dis the data and then reconstruct a croSS Sectional image from covery reported by Diamandis in International Application this information. PCT CA94/00267, it was thought that PSA only occurred in There are a number of approaches to breast tumor imag 60 men and was only produced by prostate tissue. ing that may be divided into two groups: indirect and direct. To image breast tumors researchers have developed anti Indirect techniques, are generally utilized to locate meta bodies directed against TAG 72, CA-3, CEA, EGF-R, Static disease by recognizing the Secondary effects of tumor LASA-P, and other glycoproteins associated with breast within an organ System. Indirect techniques include, but are cancer (42, 43, 44). Khaw et al. developed the monoclonal not limited to, the use of radiolabelled "Te phosphonates 65 antibody 323/A3 against a 43 Kd membrane associated to locate bone metastases (9,10) and 'Tc radiocolloids in glycoprotein from the MCF-7 tumor cell line that was able Scans and breast lyphoscintigraphy (11, 12, 13). to image tumors as Small as 0.19 grams (45). Rainsbury et 6,068,830 3 4 al. developed the antibody LICRCON-M8 against human allowing Said binding units to incubate in Vivo and bind milk fat globule and were also able to demonstrate imaging PSA associated with the endocrine cancer, and of human breast tumors, of particular note metastases to the detecting presence of Said imaging agents of bound units bone were found (46). localized to Said endocrine cancer. Antibodies have been labelled directly with radioisotopes 5 According to another aspect of the invention, a method for such as 12I, 125I, 131I, F, 186Re, Re, and 99Tc and detecting and locating endocrine cancers as described above, indirectly with chelating complexes. Such as diaminetrim wherein the human Subject is first given a Steroid which ethylenepentaacetic acid using '''In, 'Y, ''"Tc, Re and induces the cancer cells to express the PSA gene. "Re (47, 48). Antibody-mediated radiotherapy may be Such method further comprises the initial Step of injecting carried out using either beta emitting radionuclides Such as a patient with a Steroid which induces the cancer cells to 18.Re, 188Re, 13 I, 90Y, 153Sm, 3P or 10°Pd, or with alpha produce PSA, Said cancer cells having receptors for the particle emitters such as 'At or 'Pb, or with Auger injected Steroid. electron emitters such as 'I or 'I (47, 48). Therapy may According to another aspect of the invention, a method also be attempted with either drug or toxin based conjugates where entities constructed to target PSA as described above for example Adriamycin-immunoconjugates (49) and 15 deliver a toxic agent which is a radioisotope that emits Auger vinblastine-immunoconjugates (50) have been developed. electrons, and/or other Sub-atomic particles, or toxic com An unexpected finding of the clinical usefulness of immu pounds including, but not limited to, diphtheria toxin, ricin noScintigraphy has been the reported complete remission of toxin, adriamycin, chlorambucil, or daunorubicin. 7 out of 10 FIGO IV ovarian cancer patients who under went According to another aspect of the invention, a method of repeated imaging with an OC 125 antibody and had anti passive immunotherapy to endocrine cancer where PSA idiotypic HAMA (51). antibodies, or constructs thereof including, but not limited Magnetic resonance imaging can be carried out using to, chimeric or human antibodies, or their fragments, Single gadolinium and other lanthanides or metals. Such as iron chain antibodies, molecular recognition units, and peptides conjugated to antibody based proteins. Several versions of or entities that mimic peptides are administered parenterally. these antibody based products are believed to be undergoing 25 clinical evaluation and commercial development presently. BRIEF DESCRIPTION OF THE DRAWINGS To improve the Specific activity and Safety of the immu noconjugate directed towards tumor markers Several FIG. 1 is high performance liquid chromatography approaches have been taken ranging from the use of anti (HPLC) with a gel filtration column. Each HPLC fraction body fragments to genetic engineering of recombinant pro (0.5 mL) was analyzed with an assay that measures free and duced humanized antibody constructs to Synthetic peptides a-antichymotrypsin-bound PSA (ACT-PSA) (c) or an assay based on the antibody epitope (52,53). Methods of antibody that measures only ACT-PSA (0). The response of the latter engineering including Single chain antibodies have been assay is in arbitrary fluorescence units since no ACT-PSA well Summarized by Borrebaeck (54). These improvements standard exists. A. Injection of purified seminal PSA which have improved the tumor target to background ratio and 35 elutes at fraction 39 corresponding to a molecular weight of reduced the incidence of human antimouse antibody 33 kDa. No ACT-PSA is detected. B. Injection of a breast response. extract from the woman receiving the oral contraceptive The present invention provides a method for detecting, Brevicon(R). The PSA assay detects two peaks, one at frac locating and treating non-prostatic endocrine tumors involv tion 39 (free PSA, major peak) and one at fraction 30 (100 ing PSA as the tumor marker and optionally further option 40 kDa, minor peak). The latter peak is ACT-PSA as confirmed ally improves this method by first priming the endocrine by the ACT-PSA assay. The identity of the minor peak at tumors to produce PSA. fraction 21 (650 kDa) is unknown. This data confirms that over 80% of the breast tissue PSA is in the free, 33 kDa SUMMARY OF THE INVENTION form. The HPLC column was calibrated with molecular According to aspects of the invention, a method for 45 weight standards eluting at fraction 21 (660 kDa); 28 (160 detecting and locating non-proStatic endocrine cancers in kDa); 37 (44 kDa); 42 (17 kDa) and 49 (1.4 kDa). Vivo by injecting the human Subject parenterally with an FIG 2 is a Western blot analysis. Samples were electro entity that has been constructed to target PSA, that is either phoresed on 8 to 16% gradient polyacrylamide minigels a polyclonal or monoclonal antibody, or fragments thereof, under reducing conditions, electrotransferred to nitrocellu or constructs thereof including but not limited to, Single 50 lose membranes and probed with a rabbit polyclonal anti chain antibodies, bifunctional antibodies, molecular recog PSA antibody. Detection was achieved by using a horserad nition units, and peptides or entities that mimic peptides, ish peroxidase-conjugated goat anti-rabbit antibody and where the tumor targeting agent is labelled either directly, or chemiluminescence. Lane 1. Molecular weight markers. indirectly with a chelating agent, with one of ''I, ‘I, I, Lane 2. Purified seminal PSA dissolved in bovine serum 11, In, 99mTc, 90Y, 18.Re, 188Re, 32p, 15°Sm, 7Ga, 201T, 77Br 55 albumin. The PSAband appears at 33 koa (just above the 31 or F and is imaged with a photoScanning device, or where kDa marker). Lane 3. Supernatant from a prostatic carci the tumor targeting agent is labelled with either gadolinium, noma cell line (LNCaP) producing PSA. Lane 4. PSA terbium, tin, iron, or isotopes thereof and attached positive normal breast extract from the woman receiving covalently to create a paramagnetic conjugate for the pur Brevicon, containing a band at 33 kDa. Lane 5. Another pose of magnetic resonance imaging. 60 normal breast extract tested negative for PSA by the immu According to a further aspect of the invention, an in vivo nofluorometric procedure. Lane 6. An amniotic fluid tested method for imaging endocrine cancer in non-prostatic tissue for comparison. of a patient comprises: FIG. 3 is production of PSA by the breast carcinoma cell injecting a patient with biological binding units which line MCF-7. Cells were grown to confluency and then bind to PSA produced by non-prostatic tissue of the 65 Stimulated with varying concentrations of either norethin patient, said PSA-binding units being labelled with drone (1) or ethinyl (2) at the final concentrations imaging agents; indicated, in the absence of fetal calf Serum from the culture 6,068,830 S 6 medium. PSA was measured in the culture Supernatant 10 therapeutic effect. For example, a peptide created to recog days post No PSA was detected in cell cultures grown nize PSA delivering Re to the tumour site thereby deliv identically but either non-stimulated or stimulated with the erS localized radiation to ablate the disease. solvent alone (ethyl ). Norethindrone stimulates PSA By way of a further example, a peptide may be developed production at concentrations as low as 10'M. 5 that mimics the epitope for anti-PSA and binds to PSA and PSA receptors. This peptide may be produced on a com FIG. 4 is the gamma camera image of a female SCID mercially available synthesizer, using FMOC solid phase mouse the left leg of which was injected with chemistry. In one application, either tyrosine, lysine, or stimulated T47-D human breast cancer cells. The image was phenylalanine is included in the peptide to which an NS obtained 21 hours after injection of 10 MBq technetium chelate is complexed as per U.S. Pat. No. 4,897.255. The 99m-labelled B80 anti-PSA monoclonal antibody via the tail anti-PSA peptide conjugate is then combined with a vein. The image is an anterior view with the head at the top radiolabel, for example, either sodium '"Te pertechnetate and the left leg extended to the Side. (Na''"TeO) or sodium Reperrhenate(Na'ReO) and FIG. 5 is the gamma camera image of a female SCID may be used to locate a PSA producing tumor. mouse the left leg of which was injected with non-stimulated 15 The invention also provides the use of anti-PSA antibod T47-D human breast cancer cells. The image was obtained ies covalently combined with radioactive, cytotoxic or che 21 hours after injection of 10 MBq technetium-99m-labelled motherapeutic molecules and considers using these antibod B80 anti-PSA monoclonal antibody via the tail vein. The ies in immunoabsorption procedures to Separate benign from image is an anterior View with the head at the top and the left malignant cells. Further, the concept of passive immuno leg extended to the Side. therapy with antidotypic antibodies is now possible. DETAILED DESCRIPTION OF THE This invention includes a method for detecting and locat PREFERRED EMBODIMENTS ing non-prostatic endocrine cancers in Vivo by injecting a human Subject parenterally with an entity that has been This invention originated with the Surprising discovery constructed to target PSA, that is either a polyclonal or that the presence of PSA in human female breast is indica 25 monoclonal antibody, or fragments thereof, or constructs tive of breast tumour. PSA was never thought to exist in thereof including, but not limited to, Single chain antibodies, females because PSA has always been thought to be asso bifunctional antibodies, molecular recognition units, and ciated with the male prostate. This discovery and its use in peptides or entities that mimic peptides, where the tumour the prognosis of female breast cancer is described in appli targeting agent is labelled either directly, or indirectly with cant's international application PCT CA94/00267 filed May a chelating agent, with one I, I, I, In, ''Tc, Y, 13, 1994. Re, Sm, 7Ga, PP, PT1, 77Br or F and is imaged From the discovery of PSA in breast tumors, we have now with a photoScanning device, or where the tumour targeting determined that cancer cells with Steroid receptors can be agent is labelled with either gadolinium, terbium, tin, iron or stimulated to produce PSA. It was discovered that normal isotopes thereof and attached covalently to create a para breast tissue cells and non-PSA producing breast tumor cell 35 magnetic conjugate for the purpose of magnetic resonance lines (our earlier British patent application S.N. 9401491.6 imaging. A further application of the radioimaging technique filed Jan. 26, 1994) could be stimulated to produce PSA. is in the field of radioimmunoguided Surgery, whereby a Furthermore, non-PSA producing ovarian tumor cells could hand-held Scintigraphic probe detector enables a Surgeon to also be stimulated to produce PSA. Hence, the existence of identify and remove localized metastatic disease (60). PSA in a localized region of the body is indicative of a 40 A list of radioisotopes which can be used in the above cancer tumor. Procedures to then image the localized con application is as follows: ’7Ac, At, 'Ba, Ba, Be, centration of PSA are therefore very useful in cancer diag 204Bi, 205 Bi, 206Bi, 76Br, 77Br, 82Br 109Cd, *7Ca, 11C, 14C, nosis and prognosis. 36C, 18Cr, 51Cr, °Cu, “Cu, 7Cu, 165Dy, 153Eu, 18F, 153Gd, Prior to the present invention, agents developed to target Ga, 7Ga, Ga, 7°Ga, 198Au, 3H, 166Ho, 11 In, 113In, PSA were used for diagnostic and therapeutic purposes for 45 115min, 123, 125I, 131I, 189Ir, 191mir, 192Ir, 194Ir, 5°Fe, 55Fe, prostate cancer only. This invention now provides for the use Fe, 177Lu, 15O, 191m-191Os, 109Pd, 32p, 33P, 12K, 226Ra, of radioisotopic or non-isotopic elements containing immu 186Re, 188Re, 82mRb, 153Sm, “Sc, *7Sc, 7°Se, 7Se, Ag, noconjugates directed against PSA in tumours for targeting 2°Na, 2'Na, 89Sr. 35S, 38S, 177Ta, 96Te, 99mTc, 201T, 202T1, non-prostatic endocrine cancer in Vivo. The procedure is 113Sn, 117mSn, 121 Sn, 166Yb, 169Yb, 175Yb. SSY, 90Y, 62Zn, also used for the localization and monitoring of metastases 50 Zn. by either nuclear-based gamma camera or magnetic reso These entities which are constructed to target PSA, as nance imaging. Another advantage of the invention is the aforementioned, can also deliver a toxic agent for therapeu application of reagents directed against PSA labelled with a tic purposes against breast cancer, where the toxic agent is therapeutically effective radionuclide, drug or toxin for the a radioisotope that emits Auger electrons, and/or C. particles, purpose of therapeutic intervention of breast cancer. 55 and/or B particles, and/or neutrons, and/or other Sub-atomic Antibodies or chemical entities created to recognize PSA particles, or toxic compounds including but not limited to, are used to carry elements to image PSA Secreting cancer diphtheria toxin, ricin toxin, adriamycin, chlorambucil, or cells and locate the disease. These antibodies or chemical daunorubicin. Further toxins which can be used are ricin and entities are included in the term biological binding unit, its derivatives and fragments, Monensin, Verrucarin A, which term is used to refer to patient compatible entities 60 Abrin and its derivatives, Vinca alkaloids, Tricothecenes, which bind to PSA and comprise antibodies or their and Pseudomonas eXotoxin A. Further drugs for use as toxic derivatives, molecular recognition units and peptides. Anti agents are as follows: Leuco Vorin, Folinic acid, bodies encompass monoclonal and polyclonal antibodies Methotrexate, Mitomycin C, Neocarzinostatin, Vinblastine, and their derivatives and fragments and include Single chain Mitomycin, Melphalan, Mechlorethamine, Fluorouracil, antibodies, bifunctional antibodies and other antibody con 65 Fluoxuriding, Idarubicin, Doxorubicin, Epirubicin, Structs. Further, these biological binding units may deliver Cisplatin, Carmustine, Cyclophosphamide, Bleomycin, Vin particle emitting radionuclides, drugs or toxins to promote a cristine and Cytarabine. 6,068,830 7 8 A list of radioisotopes, which can be used for treating natants was further characterized by HPLC and Western blot endocrine cancers, is as follows: ’7Ac, ''At, ''Ba, 77Br, analysis as shown in FIGS. 1 and 2. Additionally, we were 10°Cd, 5 Cr, 7Cu, 165Dy, 15°Eu, 153Gd, 198Au, 16Ho, able to amplify by reverse transcliption-polymerase chain 113m.In 115m In 123 125 131 189 191m Ir 192Ir 194Ir 52Fe reaction (RT-PCR), prostate specific antigen mRNA from 55Fe 59Fe i.7Lu 109p. 32B 226Ra, 186Re issRe 5°Sm. the stimulated but not the non-stimulated cells and verify its 46Sc. 17Sc.72se, 75se, ioAg. 39Sr, 35s, 17. 117S, 12iSn, identify by Southern hybridization and Sequencing of the 166Yb. 169Yb, 90Y, 212Bi, 11°Sb, '7Hg, 7Ru, 100Pd, 101mRh. PCR product (58). The same procedures confirmed the 212Pb. presence of PSA in breast tumors positive for the protein. Since PSA was found to be associated with more benign We recently demonstrated that among 99 ovarian tumor breast tumors, it was possible that PSA could be expressed extracts tested only three were positive for PSA and the by normal breasts either under physiological circumstances concentration of the PSA was 0.048, 0.034 and 0.0076 or after Steroid Stimulation, Such as described in the ng/mg. Subsequently, a patient with ovarian cancer who also aforesaid British patent application. Cytosolic extracts were underwent liver transplantation and was receiving oral pred prepared as previously described (56) from eighteen normal nisone tablets during the period of ovarian tumor removal breast tissues removed from nine woman (left and right 15 was tested for the presence of PSA in the ovarian tumor. breast) during breast reduction Surgery. PSA immunoreac Remarkably, this ovariant tumor contained 15 ng of PSA/mg tivity was measured in these extracts using a highly specific protein, which is a very high amount. Combined with our and sensitive immunofluorometric technique (57) and by previous results on breast tumor cell line tissue culture two widely used commercial PSA assays. Breast extracts systems (British Patent Application S.N. 9401491.7) in from eight of the nine women were found to contain <0.03 which we found that glucocorticosteroids can stimulate PSA ng of PSA per mg of total protein and were considered production, it is apparent that the ovarian tumor, generally negative for PSA. Surprisingly, two breast extracts from the unable to produce PSA, can be stimulated by steroids like same woman (left and right breast) had relatively high , to produce very high levels of PSA in the tumor. concentrations of PSA (0.11 and 1.53 ng/mg). None of the The tumors, which may be imaged and treated in this eight PSA-negative women was receiving oral contracep 25 invention, have Steroid receptors which are Stimulated in the tives or other . The women with PSA-positive presence of Steroids to express the PSA gene and thus breasts was receiving only one , Brevicon(E), a produce PSA. The PSA-binding units can then bind to highly prescribed oral contraceptive containing 1 mg nore cancer cells producing PSA which allows these cancers to be thindrone (a progestin) and 0.035 mg ethinyl estradiol per treated and localized. tablet. The PSA-positive and negative results in the breast Following this demonstration of stimulation of PSA gene extracts by the immunofluorometric procedure were verified expression in breast carcinoma cell lines and ovarian by using two widely used commercial PSA methods, tumors, it is a further feature of the invention that Stimulat namely, the IMX(R) from Abbott Labs, Abbott Park, Chicago, ing cancers to express PSA would either enable detection of Ill. and the TandemE)-E kit from Hybritech Inc., San Diego, previously undetectable tumors or would improve radioim Calif. Additionally, one highly positive extract was Serially 35 aging of previously detectable tumors. Indeed as shown with diluted in female serum from 2- to 32-fold and analyzed by the normal breast tissue of the patient receiving the oral immunofluorometry and the IMx assay. Identical results contraceptive, Steroid receptor positive tissues could be were obtained. induced to produce PSA which would enable these tissues to The highly positive breast extract was also Subjected to be radioimaged. Further, the production of PSA by the high performance liquid chromatography (FIG. 1) and frac 40 non-PSA producing normal breast and ovarian tumor tissues tions were analyzed by two immunofluorometric procedures after Stimulation will enable radioimaging of non-PSA pro which measure either total PSA (free PSA plus PSA bound ducing endocrine tumors after Stimulation. While normal to a 1-antichymotrypsin) or specifically the PSA-a- endocrine tissue can be stimulated to produce PSA, it will antichymotrypsin (ACT) complex (S). Over 80% of the total not be produced at the high levels produced by Stimulated PSA in normal breast was in the free, 33 kDa form; a small 45 endocrine cancer cells. Thus, priming a patient with a steroid proportion was present as PSA-ACT complex (100 kDa). which induces PSA gene expression to, in turn, produce PSA Another minor Species, containing PSA and ACT was also at the tumor Site, provides increased binding of the entity detected (660 kDa), but its identity is unknown. The pres targeting PSA to endocrine tumors and improves the radio ence of PSA in the highly positive breast tumor extract was imaging or therapeutic delivery of toxic agents. further confirmed by Western blot analysis (FIG. 2). This 33 50 Gamma camera images of SCID mice with norgestrel kDa form of PSA, present in normal breasts stimulated by stimulated (FIG. 4) and non-stimulated (FIG. 5) T47-D oral contraceptives, is similar to the PSA form found in human breast cancer cells injected into the muscle of the left breast tumors (1). In male serum, the majority of PSA is leg were taken. Images were obtained 21 hours after injec present as PSA-ACT complex with a molecular weight of tion of 10 MBq technetium-99m-labelled B80 anti-PSA 100 kDa (57). 55 monclonal antibody via the tail vein. Images are anterior In order to study the oral contraceptive-induced PSA Views, heat at top, with left leg, which contains the tumor production further, T-47D and MCF-7 breast carcinoma cell cells, extended to the Side and immobilized. An upper lines were cultured in the absence of any Steroid threshold of 5% was used to mask the residual radioactivity or in the presence of norethindrone or ethinyl estradiol at in the liver and the abdomen. (That is, the top 5% of counts various concentrations (FIG.3). No PSA was detected in the 60 were Subtracted from all images to allow the leg tumor to be tissue culture Supernatants in the absence of Steroid hor viewed.) The leg containing the tumor is seen in the lower mones after 11 days of confluent cultures. Ethinyl estradiol right portion of each image. Quantification of this pair of stimulated low levels of PSA production at concentrations digital images showed that the Stimulated tumor contained at 210M. Norethindrone was effective in mediating intense least 15% more radioactivity than the control tumor. PSA gene expression at concentrations as low as 10"M. 65 TiSSue counting results were obtained for blood, normal Other progestins were also effective in mediating PSA gene muscle and either the stimulated or control T47-D tumor and expression. The identity of PSA in the tissue culture Super are provided in Table 1. All values are percent injected dose 6,068,830 9 10 per gram tissue, expressed as a mean tStandard deviation for have PSA Secreting breast tumors. A dose escalating regime 4 animals or individual values for 2 animals. Table 1 can be followed, over a period of months, if there is an demonstrates that the T47-D tumor cells had at least double absence of any untoward response. Patient Sera can be the counts of the control. Furthermore, the stimulated tumor examined via in vitro methods for the development of cells have increased counts over that of the non-stimulated 5 antiidiotypic antibodies. The development of anti-idiotypic tumor cells. Thus radioimaging was improved by Stimula antibodies results in enhancement or beneficial modification tion to increase PSA production. of the patient's immune response and thereby elicits an anti-tumor response (51). TABLE 1. 1O EXAMPLE 1. Blood O.96 - 0.14 Normal muscle O.129 O.O29 Breast tumors were Snap-frozen in liquid nitrogen. Frozen T47-D control tumor O.228-O.231 Sections (5 um thick) were placed on clean glass slides and (range 2 animals) processed for immunohistochemistry. Slides to be stained T47D- stimulated tumor 0.266-0.405 (range 2 animals) for PSA were fixed immediately in 10% buffered formalin 15 for 10 min. and then placed in phosphate-buffered Saline. Staining was performed with a polyclonal anti-PSA antibody It was proven that the norgestrel-stimulated T-47D cells in from Medix Biotech, Foster City, Calif. and further com the above experiment were producing PSA from the detec pleted by use of the avidin-biotin technique (ABC) using a tion of PSA in the cell culture Supernatant. Furthermore, kit from Vector Laboratories, Burlingame, Calif. Immunop T-47D and MCF-7 tumor cell lines were injected into SCID eroxidase Staining was, according to the manufacturer's mice to develop tumors, which mice were then injected with recommendation (58). Tumors positive or negative for PSA estrogen and/or norgestrel. The results (Table 3) demon by the immunofluorometric procedure were used. The slides strated that human PSA was found in the serum of mice were examined under a light microscope. Tumors, negative injected with norgestrel, however, estrogen blocked the for PSA showed no staining while tumors positive for PSA effect of the norgestrel. Estrogen was previously shown to 25 exhibited Strong brown cytoplasmic Staining with the immu block the effect of progestin on PSA production in the tumor noperoxidase technique. This experiment demonstrated that cell lines tested in our earlier British patent application S.N. PSA in breast tumors can be localized by antibodies against 94O14917. PSA. The breast tumor cell line tissue culture system (described in British patent application S.N. 9401491.7) suggested that EXAMPLE 2 any Steroid having either , , Cell Preparation , progestin or activity can regulate the The following cell line was obtained from the ATCC: PSA gene; however, cannot mediate Such action. T47-D, an estrogen dependent cell line derived from breast These results combined with the results from steroid stimu cancer. The cell line was checked for freedom from micro lation of PSA expression in normal breast cells and non-PSA 35 bial contaminants and all operations were carried out asep producing breast tumor cell lines and ovarian tumor, indicate tically. T47-D cells were cultured in C. Medium with 10% that to Stimulate tumors for radioimaging applications, any fetal calf serum in sterile culture flaskS. Cells were then natural or Synthetic Steroid falling under the above catego incubated at 37 C. and growth was checked visually using ries of activity would be suitable. A to induce a microScope. Two Sets of T47-D cells were prepared, one 40 Set Stimulated with norgestrel, a progestin, and the other Set expression of the PSA gene is found in Table 2. non-stimulated. The stimulated set were stimulated with a A list of non-prostatic endocrine tumors which can be Solution of norgestrel (concentration 1 millimole in ethanol), stimulated to express the PSA gene either initially or to at the concentration of 1 micromolar in fresh media for a increase PSA expression is as follows: breast tumors, ova period of 48 h. PSA was detected in the Supernatant of the rian tumors, lung carcinomas, meningiomas, endometrial 45 norgestrel-stimulated cells Supernatant after 72 h and the carcinomas, colon carcinomas, Salivary gland tumors, cer concentration of PSA increased from 0.013 tug/L to 0.037 Vical carcinomas, uterine carcinomas, adrenal tumors, renal lug/L at 96 h. carcinomas and melanomas. The Steroids, which Stimulate Cells were Scraped into a centrifuge tube and Spun down the production of PSA, may be naturally present in the body in a laboratory centrifuge at approximately 240 G for Six or may be introduced artificially by injection. 50 minutes. Cells were re-Suspended using the Sterile culture An alternative aspect of this invention, as it relates to in medium and re-spun, the procedure was repeated a total of Vivo antibody binding, is a method of passive immuno three times. For a 0.2 ml pellet of cells, 0.1 ml was injected therapy to non-prostatic endocrine cancer where PSA Subcutaneously into the left leg, slightly below the knee, in antibodies, or constructs thereof including, but not limited female SCID mice. The amount of cells injected into the to, chimeric or human antibodies, or their fragments, Single 55 SCID mice were estimated to be between 2.5x10° and chain antibodies, molecular recognition units, and peptides 1x107. The experiment was repeated once using trypsin to or entities that mimic peptides are administered parenterally. avoid clumping of the harvested cells, in which case the Antiidiotypic antibodies may then be produced in reaction to Stimulation with norgestrel was increased to 2 micromolar in this administration. fresh culture medium to overcome any effect due to trypsin. Antidiotypic antibodies are antibodies directed against 60 The results of imaging shown in FIGS. 4 and 5 were the idiotype (the antigen combining region or variable obtained using cells harvested by Scraping, whereas the region) of another antibody, in this case anti-PSA antibody. results of Table 1 were obtained using cells harvested with These antiidiotypic antibodies in turn Stimulate the produc trypsin. tion of antibodies against the idiotype of the antidiotypic Preparation of the Monoclonal Antibody antibodies. Thus this Sub-population of antiidiotypic anti 65 The anti-PSA monoclonal antibody, B80, was obtained bodies bind the initial antigen, PSA. For example, human from Biomira Inc., Edmonton, Alberta. The antibody was anti-PSA antibodies can be administered to patients that prepared for radioimaging using the method described by 6,068,830 11 12 Joiris et al. (55) with 2 iminothiolane. Three solutions are first prepared: 5 milligram/ml Solution of B 80 in physi TABLE 2 ological Saline; 10 milligram/ml Solution of 2 iminothiolane; and a phosphate buffer solution containing 0.25M of phos LIST OF STEROIDS phate adjusted to pH 7.4. 10 micorliters of each of these 33-Acetoxy-9(11), 16-allopregnadien-20-one Solutions was combined and allowed to stand for 30 minutes 21-Acetoxyallopregnan-3,20-dione at about 25 C. This solution was then combined with 100 3f-Acetoxy-5-androsten-17-one 33-Acetoxybismorallocholanic acid microliters of '"TcO (approximately 10 mci/ml or 370.4 3f-Acetoxybisnor-5-cholenic acid MBq/mL) and 100 microliters of a dilute stannous gluco 21-Acetoxy-3C,17-dihydroxy-53-pregnan-11,20-dione heptonate Solution containing 5 micrograms of Stannous 3C-Acetoxy-5-etianic acid chloride. This solution was allowed to incubate for 10 3f-Acetoxy-5?-etianic acid 3f-Acetoxy-5-etienic acid minutes at 25 C. The solution was then purified using a 33-Acetoxyetiocholenic acid Sephadex G-50 column which has been preblocked with a 21-Acetoxy-17-hydroxyallopregnan-3,11,20-trione 1% solution of human serum albumen in 0.9% NaCl. The 33-Acetoxy-5,16-pregnadien-20-one 15 12C-Acetoxypregnan-320-dione column was eluted using 0.9% NaCl. The hottest fraction, as 21-Acetoxypregnanedione determined by measurements using a Capintec Dose Cali Acetoxypregnanolone brator was kept and diluted to 50 Mbd/ml using 0.9% NaCl. 17-Acetoxypregnenolone 0.2 ml, a solution equal to 10 MBq was the injected dose. 21-Acetoxypregnenolone 33-Acetoxy-16,(53)-pregnen-20-one Imaging Studies 11C-Acetoxyprogesterone 0.2 ml of '"Tc radiolabelled B 80 antibody was injected 17-Acetoxyprogesterone intravenously into the tail vein of the SCID mice prepared as 17-Acetoxyprogesterone 3-ethyleneketal -Acetoxyprogesterone described above. Imaging Studies were carried out using a - Siemans Orbiter, model 7400 gamma camera with a low drenosterone energy all purpose collimator using a magnification factor of etiocholane 2.5. Data was collected using a Picker PCS I-II nuclear 25 dosterone dosterone 21- medicine computer. Counts were obtained using a 128x128 dosterone 3-CMO matrix. Imaging Studies were carried out with the mouse dosterone 3-CMO:BSA conscious in a lucite restraining device with the leg con dosterone 18,21-diacetate taining the tumor taped to the base plate. The restraining dosterone diacetate 3-CMO dosterone diacetate 3-CMO:BSA device was placed directly on the collimator. Initial images dosterone 21-hemisuccinate were carried out between 1 and 7 hours post injection using dosterone 21-hemisuccinate:BSA a five minute data acquisition and repeated at 20 hours using ocholesterol a 15 minute acquisition. Quantification was performed by odihydrocorticosterone drawing a region of interest around the tumor. After correc odihydrocortisol 35 Odihydrocortisone tion for background counts, comparisons of the norgestrel Odihydrocortisone acetate Stimulated and control tumors were made using counts per odihydro substance “S” pixel values. O d ihydrotestosterone Following the final imaging Session, the mice were sac rificed by cervical dislocation and Samples of blood, leg egnanolone acetate muscle containing tumor cells, and control muscle from the 40 rahydro compound “A rahydro compound “B” opposite leg were obtained, weighed and assayed in a rahydro compound “E” gamma well counter (Capintec). From the knowledge of the rahydro compound “F” dose administered and counting efficiency, the percent dose rahydrocorticosterone per gram was calculated. rahydrocortisol 45 rahydrocortisone rahydro-11-dehydrocorticosterone EXAMPLE 3 rahydrodesoxycorticosterone rahydrodesoxycorticosterone 21-acetate T-47D tumor cells or MCF-7 tumor cells were implanted rahydro-11-desOxycortisol into SCID mice and left to develop as tumors over several rahydro DOC 50 rahydro DOC 21-acetate weekS. Mice were injected Subcutaneously with estrogen rahydrohydrocortisone and/or norgestrel in ethanol (100 uL of a 10 M solution per rahydro substance “Q mouse) and serum was collected after 48 hours. The results otetrahydro substance “S” O TH “A from measuring PSA within the serum by the fluorescence O TH “B” method (57) are found in Table 3. 55 O TH “E” O TH"E' diacetate TABLE 3 o TH “F” O TH “F” diacetate Serum PSA o Th"S Assay, Androstadienedione Experiment Tumor Type Estrogen Norgestrel Fluorescence Androstadienedione 3-ethylenol ether 60 1. T47D -- 281.8 2 T47D -- -- 2661 Androstatriendione 3 MCF-7 -- -- 1912 4 MCF-7 -- 6893 5 MCF-7 -- 2O67 Androstenolone 6 MCF-7 2642 65 4-Androsten-17B-ol-one enol diacetate 6,068,830 13 14

TABLE 2-continued TABLE 2-continued

LIST OF STEROIDS LIST OF STEROIDS Androsterone acetate holesteryl ethylether Androsterone benzoate holesteryl formate Androsterone chloroformate holesteryl glucuronide, sodium salt Androsterone-CMO holesteryl hemisuccinate Androsterone glucuronide holesteryl heptanoate Androsterone hemisuccinate holesteryl hexadecanoate Androsterone propionate holesteryl hydrocinnamate Androsterone sodium sulfate holesteryl hydrogen Androsterone tosylate holesteryl iso-butyrate Anhydroxyprogesterone holesteryl laurate Apocholic acid holesteryl methylcarbonate Becomethasone holesteryl methyl ether Becomethasone 21-acetate holesteryl myristate Beclomethasone 17,21-dipropionate 15 holesteryl nonanoate Becomethasone 21-hemisuccinate holesteryl octanoate Beclomethasone 21-propionate holesteryl pelargonate holesteryl B-phenylpropionate Betamethasone 21-acetate holesteryl n-propionate Betamethasone-17,21-dipropionate holesteryl pyridinium sulfate Betamethasone 21-disodium phosphate holesteryl sodium sulfate Betamethasone 21-hemisuccinate holesteryl stearate Betamethasone 17-valerate holesteryl tosylate 3,4-Bis(4-hydroxyphenyl)-hexane holesteryl valerate 3,4-Bis-(4-hydroxyphenyl)-3-hexene holic acid Bolderone holic acid methyl ester Bolderone sulfate, sodium salt holic acid sodium salt 2C-Bromo-5C-cholestan-3-one 25 Cincho 4-Bromoequilenin Cistestosterone 4-Bromoequilin Coprostane 2-Bromoestradiol Coprostanol 4-Bromoestradiol Coprostanol acetate 16C-Bromoestradiol Coprostanol benzoate 16C-Bromo-17C-estradiol Coprostan-3-one 16C-Bromoestrone Coprostenol 16B-Bromoestrone Coprosterol 2-Bromoethynylestradiol Cortexolone 4-Bromoethynylestradiol Cortexone 17-Bromopregnenolone 17-Caproxyprogesterone 35 Corticosterone acetate Chenodeoxycholic acid Corticosterone 21-acetate, 3-CMO Chenodeoxycholic acid 3-Hemisuccinate Corticosterone 21-acetate, 3-CMO:BSA Chenodeoxycholic acid methyl ester Corticosterone 3-CMO Chenodeoxycholic acid sodium salt Corticosterone 3-CMO:BSA 3B-Chloro-5C-cholestane Corticosterone diethyleneketal 3B-Chloro-5-cholestene Corticosterone hemisuccinate 21-Chloro-17-hydroxyprogesterone 40 Corticosterone hemisuccinate:BSA Cholaic acid holestanol Cortisol acetate -Cholestanol Cortisol 21-acetate, 3-CMO holestanol Cortisol 21-acetate, 3-CMO:BSA holestanol acetate Cortisol 3-CMO holestanol benzoate 45 Cortisol 3-CMO:BSA holestanol hemisuccinate Cortisol glucuronide estanol propionate Cortisol glucuronide, sodium salt holestanol tosylate Cortisol hemisuccinate Cholestan-3?-ol chloride Cortisol 17-valerate OleStaCe A1- C Cholestan-3-one enol acetate 50 Cortisone 5C-Cholestan-3?-yl chloride holestanyl chloride Cortisone 21-acetate, 3-CMO OleStee Cortisone 21-acetate, 3-ethyleneketal O estenol Cortisone diethyleneketal Cholesten-3-ol chloride Cortisone 21-hemisuccinate Ole:SeOile 55 Cortisone 21-sodium sulfate 4 Cholesten-3-one enol acetate Cortol holesterilene B-Cortol holesterin Cortolone holesterol B-Cortolone holesteryl acetate DES holesteryl acetoacetate DHEA holesteryl benzoate 60 DHEA acetate holesteryl n-butyrate DOCA holesteryl caprylate DOCA 3-ethyleneketal holesteryl chloride DOC 21-aldehyde hemiacetal holesteryl chloroacetate DOC 3-CMO holesteryl chloroformate DOC 3-CMO:BSA holesteryl cinnamate 65 DOC glucuronide holesteryl n-decylate DOC hemisuccinate 6,068,830 15 16

TABLE 2-continued TABLE 2-continued

LIST OF STEROIDS LIST OF STEROIDS DOC propionate 21-Desoxycortisol DPA 21-Desoxycortisone 16-Dehydroallopregnanolone 17-Desoxycortol 16-Dehydroallopregnanolone acetate 17-Desoxy-f-cortol 24-Dehydrocholesterol 17-Desoxy-f-cortolone Dehydrocholic acid 11-Desoxy-17-hydroxycorticosterone 1-Dehydrocortisone Desoxymethasone 11-Dehydrocorticosterone 11-Dehydrocorticosterone acetate 11-Dehydrocorticosterone hemisuccinate Dexamethasone hemisuccinate Dehydrocortisol Dexamethasone hemisuccinate:BSA Dexamethasone 21-mesylate Dehydroepiandrosterone acetate Dexamethasone phosphate disodium salt Dehydroepiandrosterone glucuronide 15 Dianabol Dehydroepiandrosterone potassium sulfate 2,4-Dibromoestradiol Dehydroepiandrosterone propionate Dehydroepiandrosterone sodium sulfate 3C,12C-Diformyloxydesoxycholic acid Dehydroepiandrosterone tosylate Dihydroandrosterone 6-Dehydro-17C-estradiol Dihydrocholesterol 6-Dehydroestradiol Dihydrocholesterol acetate 6-Dehydroestradiol diacetate Dihydrocholesterol benzoate 7-Dehydro-17C-estradiol Dihydrocholesterol hemisuccinate 7-Dehydro-17B-estradiol Dihydrocholesterol methyl ether 7-Dehydro-17B- Dihydrocholesterol propionate 16-Dehydroestradiol diacetate Dihydrocholesterol tosylate 6-Dehydroestrone 5C-Dihydrocortexone 6-Dehydroestrone acetate 25 5C-Dihydrocorticosterone 6-Dehydroestrone benzoate 5C-Dihydrocortisol 6-Dehydroestrone benzyl ether 5C- acetate 6-Dehydroestrone methyl ether 20B-Dihydrocorticosterone 8-Dehydroestrone 20B-Dihydrocortisol Dehydroisoandrosterone Dihydrocortisone Dehydroisoandrosterone acetate 5C-Dihydrocortisone -Dehydromethyltestosterone 5 B-Dihydrocortisone 6-Dehydro-5C-pregnan-3B-ol 11,20-dione Dihydrocortisone acetate 6-Dehydro-5C-pregnan-3B-ol 11,20-dione acetate 5C-Dihydro-11-dehydrocorticosterone 6-Dehydropregnanolone Dihydro-11-desoxycortisol 6-Dehydropregnanolone acetate 17B-Dihydroequilenin 6-Dehydro-5C-pregnan-3B-ol 20-one 35 17B-Dihydroequilenin diacetate 6-Dehydro-5C-pregnan-3B-ol 20-one acetate 17C-Dihydroequilenin 6-Dehydro-5f-pregnan-3?-ol 20-one 17B-Dihydroequilenin 6-Dehydro-5f-pregnan-3?-ol 20-one acetate 17B-Dihydroequilenin diacetate 6-Dehydropregnenolone 20B-Dihydro Kendall's compound “B” 6-Dehydropregnenolone acetate 5C-Dihydro substance “Q 6-Dehydropregnenolone acetate, Oxime 5C-Dihydro substance “S” 6-Dehydropregnenolone oxime 40 5C- 6-Dehydroprogesterone Dihydrotestosterone -Dehydrotestosterone Dihydrotestosterone acetate -Dehydrotesrosterone acetate Dihydrotestosterone benzoate -Dehydrotestosterone benzoate Dihydrotestosterone 3-CMO -Dehydrotestosterone 3-CMO Dihydrotestosterone 3-CMO:BSA -Dehydrotestosterone hemisuccinate 45 Dihydrotestosterone chloroformate -Dehydrotestosterone propionate Dihydrotestosterone cyclopentylpropionate -Dehydrotestosterone sodium sulfate Dihydrotestosterone enanthate 6-Dehydrotestosterone Dihydrotestosterone glucuronide 6-Dehydrotestosterone acetate Dihydrotestosterone hemisuccinate 6-Dehydrotestosterone benzoate Dihydrotestosterone hemisuccinate:BSA 6-Dehydrotestosterone 3-CMO 50 Dihydrotestosterone hexahydrobenzoate 6-Dehydrotestosterone hemisuccinate Dihydrotestosterone propionate 6-Dehydrotestosterone propionate Dihydrotestosterone tosylate Deoxycholic acid 5 B-Dihydrotestosterone Deoxycholic acid diacetate 5 B-Dihydrotestosterone acetate Deoxycholic acid sodium salt 5 B-Dihydrotestosterone 3-CMO Desmosterol 55 5 B-Dihydrotestosterone hemisuccinate Desmosterol acetate 5 B-Dihydrotestosterone propionate 3C,12C-Dihydroxycholanic acid DesoxycorticOsterone 5 B-Dihydroxycorticosterone DesoxycorticOsterone acetate 3C,7c-Dihydroxy-12-ketocholanic acid Desoxycorticosterone acetate 3-CMO 3C,12C-Dihydroxynorcholanate Desoxycorticosterone acetate 3-CMO:BSA 3?,17-Dihydroxy-5-pregnen-3-one DesoxycorticOsterone acetate, 3-ethyleneketal 60 Dihydro Reichstein's substance “S” DesoxycorticOsterone 21-aldehyde hemiacetal 3,6-Diketocholanic acid Desoxycorticosterone 3-CMO 3,7-Diketocholanic acid Desoxycorticosterone 3-CMO: BSA 3,12-Diketocholanic acid DesoxycorticOsterone glucuronide 7,12-Diketolithocholic acid DesoxycorticOsterone hemisuccinate 7,12-Diketolithocholic acid sodium salt DesoxycorticOsterone propionate 65 6,16-Dimethyl-16-dehydroprogesterone 11-Desoxycortisol 2,4-Dinitroestradiol 6,068,830 17 18

TABLE 2-continued TABLE 2-continued

LIST OF STEROIDS LIST OF STEROIDS 7B-Estradiol 3-benzoate, 17-valerate Diosgenin acetate 7B-Estradiol 3-benzyl ether Diosgenin benzoate 7B-Estradiol 17-cyclopentylpropionate E 1 7B-Estradiol diacetate E 2 7B-Escradiol dibenzoate E3 7B-Estradiol dicyclopentylpropionate E 4 7B-Estradiol diglucuronide C-Ecdysone 7B-Estradiol dihemisuccinate ectrocortin 7B-Estradiol dipalmitate pi-allocholesterol 7B-Estradiol diphosphate, disodium salt pi-allotetrahydro “B” 7B- piandrosterone 7B-Estradiol disodium sulfate piandrosterone acetate 7B-Estradiol 17-enanthate piandrosterone benzoate 15 7B-Estradiol 3-glucuronide piandrosterone chloroformate 7B-Estradiol 17-glucuronide piandrosterone 17-CMO 7B-Estradiol 3-hemisuccinate piandrosterone glucuronide 7B-Estradiol 17-hemisuccinate piandrosterone hemisuccinate 7B-Estradiol 17-hemisuccinate:BSA piandrosterone potassium sulfate 7B-Estradiol 17-hexahydrobenzoate piandrosterone propionate 7B-Estradiol 3-methyl ether piandrosterone sodium sulfate 7B-Estradiol 17-phenylpropionate piandrosterone tosylate 7B-Estradiol 3-phosphate, disodium salt picholestanol 7B-Estradiol 17-phosphate, disodium salt picholesterol 7B-Estradiol 3-sodium sulfate pi compound “F” 7B-Estradiol 17-sodium sulfate picoprostanol 7B-Estradiol 17-stearate picoprostanol acetate 25 7B-Estradiol 17-valerate picoprostanol benzoate picoprostanol hemisuccinate Estriol 3-acetate picoprostanol propionate Estriol 16-acetate picoprosterol Estriol 16,17-diacetate pidihydrocholesterol Estriol 3,17-disodium sulfate 16- Estriol 16,17-disodium sulfate 16-Epiestriol 3-methylether Estriol 3-glucuronide, sodium salt 16-Epiestriol triacetate Estriol 3-hemisuccinate 17-Epiestriol Estriol 16-hemisuccinate 17-Epiestriol triacetate Estriol 3-methyl ether 16,17-Epiestriol Estriol 3-phosphate, disodium salt 16,17-Epiestriol triacetate 35 Estriol 3-sodium sulfate Estriol 17-sodium sulfate Epitestosterone acetate Epitestosterone benzoate Estriol tripropionate Epitestosterone hemisuccinate 1-Epi Tetrahydro compound “B” 1-Epi-Tetrahydro compound “F” 1-Epi-Terrahydrocorticosterone 40 Estrone benzyl ether 1-Epi- Estrone 17-enol acetate, 3-methyl ether 1-Epi-Tetrahydrohydrocortisone Estrone enol diacetate 1-Epi-TH “B” Estrone ethyl ether 1-Epi-TH “F” Estrone glucuronide, sodium salt Epoxypregnanolone Estrone hemisuccinate Epoxypregnenolone 45 Estrone methoxime Epoxypregnenolone acetate 6C, 17-epoxyprogesterone Estrone phosphate, disodium salt d- Estrone propionate d-Equilenin acetate Estrone trimethylacetate d-Equilenin benzoate histerone d-Equilenin benzyl ether 50 24 b-Ethylcholesterol d-Equilenin etheyl ether 17C-Ethyl-19-nor- d-Equilenin methyl ether E hynodiol E hynylandrostanolone Equilin acetate E hynylandrostendiol Equilin benzoate E hynylandrostenolone Equilin methylether 55 17C-Ethynyldihydrotestosterone 4.22-Ergostadien-3-one E hynylestradiol Ergosterol 17C-Ethynylestadiol 3-acetate Esmillagenin hynylestradiol 3-methyl ether 17C-Ethynyl-19-nor-testosterone 17C-Estradiol hynyl testosterone 17C-Estradiol 3-acetate iadienic acid 17C-Estradiol 17-acetate 60 iadienic acid 3-acetate 17C-Estradiol diacetate iadienic acid methyl ester 17B-Estradiol ianic acid 17B-Estradiol 3-acetate ienic acid 17B-Estradiol 17-acetate ienic acid acetate 17B-Estradiol 17-acetate, 3-benzoate ienic acid methyl ester 17 B-Estradiol 17-acetate, 3-methyl ether 65 iocholane 17B-Estradiol 3-benzoate iocholan-3C,17o-diol 6,068,830 19 20

TABLE 2-continued TABLE 2-continued

LIST OF STEROIDS LIST OF STEROIDS iocholan-3C,17B-diol 6?-Hydroxycorticosterone iocholan-3C,17B-diol diacetate 7-Hydroxycorticosterone iocholan-3?,17C-diol 6?-Hydroxycortisol iocholan-3?,17-diol 8-Hydroxy-11-deoxycorticosterone iocholan-3?,17-diol diacetate 3C-Hydroxy-7,12-dikerocholanic acid iocholan-3C,6C-diol-17-one 8-Hydroxy DOC iocholan-3?,11.f3-diol-17-one 2-Hydroxyestradiol iocholan-3,17-dione 2-Hydroxyestradiol 17-acetate iocholan-3C-ol 2-Hydroxyestradiol 3-methyl ether iocholan-3C-ol-11,17-dione 4 -Hydroxyestradiol iocholanolone 6C-Hydroxyestradiol iocholanolone acetate 1C-Hydroxyestradiol iocholanolone benzoate 6C-Hydroxy-17f-estradiol iocholanolone 17-CMO 15 6?-Hydroxy-17B-estradiol iocholanolone glucuronide 2-Hydroxyestriol iocholanolone hemisuccinate 5C-Hydroxyestriol iocholanolone potassium sulfate 2-Hydroxyestrone iocholanolone propionate -Hydroxyestrone iocholanolone sodium sulfate 6C-Hydroxyestrone iocholan-3?-ol-17-one 2-Hydroxyestrone-3-methyl ether iocholan-17B-ol-3-one 3?-Hydroxy-5C-etianic acid iocholan-3?-ol-17-one acetate 3?-Hydroxy-5C-etianic acid methyl ester iocholan-3?-ol-17-one benzoate 3C-Hydroxy-5f-etianic acid iocholan-3?-ol-17-one hemisuccinate 3C-Hydroxy-5f-etianic acid methyl ester iocholan-3?-ol-17-one propionate 3?-Hydroxy-5?-etianic acid iocholenic acid acetate 3?-Hydroxy-5?-etianic acid methyl ester ucinonide 25 11B-Hydroxyetiocholanolone umethasone 16C-Hydroxyetiocholanolone uocinolone acetonide 3?-Hydroxy-5-etiocholenic acid 2-Fluoroestradiol 3C-Hydroxy-6-ketocholanic acid 11C-Hydroxymethyltestosterone 6?-Hydroxyprednisolone Flurand renolide 17-Hydroxypregnanolone Furocortisone 16C-Hydroxypregnenolone Fucosterol 17-Hydroxypregnenolone Glycochenodeoxycholic acid 17-Hydroxypregnenolone 3-acetate Glycochenodeoxycholic acid sodium salt 21-Hydroxypregnenolone Glycocholanic acid 2C-Hydroxyprogesterone Glycocholic acid 35 6C-Hydroxyprogesterone Glycocholic acid potassium salt 68-Hydroxyprogesterone Glycocholic acid sodium salt 68-Hydroxyprogesterone acetate Glycodehydrocholic acid 68-Hydroxyprogesterone hemisuccinate Glycodehydrocholic acid sodium salt 68-Hydroxyprogesterone hemisuccinate:BSA Glycodeoxycholic acid 11C-Hydroxyprogesterone Glycodeoxycholic acid sodium salt 11C-Hydroxyprogesterone acetate Glycohyodeoxycholic acid 40 11C-Hydroxyprogesterone hemisuccinate Glycohyodeoxycholic acid sodium salt 11C-Hydroxyprogesterone hemisuccinate:BSA Glycolithocholic acid 11C-Hydroxyprogesterone tosylate Glycolithocholic acid sodium salt 11f8-Hydroxyprogesterone Hecogenin 12C-Hydroxyprogesterone Hecogenin acetate 16C-Hydroxyprogesterone Hetero-1- diacetate 45 17-Hydroxyprogesterone 6?-Hydrocortisol 17-Hydroxyprogesterone 3-CMO 17-Hydroxyprogesterone 3-CMO:BSA 11C-Hydrocortione 18-Hydroxyprogesterone 19-Hydroxyprogesterone Hydrocortisone acetate, 3-CMO 20C-Hydroxyprogesterone Hydrocortisone acetate, 3-CMO:BSA 50 20?-Hydroxyprogesterone Hydrocortisone 3-CMO 21-Hydroxyprogesterone Hydrocortisone 3-CMO:BSA 6?-Hydroxytestosterone Hydrocortisone glucuronide 7C-Hydroxytestosterone Hydrocortisone hemisuccinate 11C-Hydroxytestosterone Hydrocortisone hemisuccinate:BSA 11C-Hydroxytestosterone hemisuccinate 3B-Hydroxy-5C-androstan-17-one 55 11C-Hydroxytestosterone hemisuccinate:BSA 4-Hydroxyandrostenedione 11B-Hydroxytestosterone 7C-Hydroxyandrostenedione 16C-Hydroxytestosterone 19-Hydroxy-4-androsten-3,17-dione 16?-Hydroxytestosterone 17 B-Hydroxy-4-androsten-3-one 19-Hydroxytestosterone 16C-Hydroxyandrosterone 11C-Hydroxytigogenin 3B-Hydroxy-bisnor-5-cholenic acid Hyocholic acid 3B-Hydroxy-5-cholenic acid 60 Hyocholic acid methyl ester 4-Hydroxycholesterol Hyodeoxycholic acid 7C-Hydroxycholesterol Hyodeoxycholic acid methyl ester 7-Hydroxycholesterol Iodocholesterol 19-Hydroxycholesterol Isoallospirostan-3?,12-diol 20C-Hydroxycholesterol Isoandrosterone 25-Hydroxycholesterol 65 14-iso-Equilenin acetate Hydroxycholic acid 14-iso-Equilenin methyl ether 6,068,830 21 22

TABLE 2-continued TABLE 2-continued

LIST OF STEROIDS LIST OF STEROIDS Isoergosterone 2-Methoxyethynylestradiol Iso-Sarsasapogenin 16C.-Methyl-17-acetoprogesterone Kendall's compound “A Methyl-3C-Acetoxycholanate Kendall's compound “B” Methyl-7c-acetoxy-3,12-diketocholanate Kendall's compound “C Methyl-3C-acetoxy-12C-hydroxycholanate Kendall's compound “E” Methyl-3C-acetoxy-12-ketocholanate Kendall's compound “E” acetate Methylacetoxylithocholate Kendall's compound “F” Methylandrostanediol Kendall's compound “G” Methylandrostanolone Kendall's compound “H” Methylandrostendiol Kendall's desoxy compound “B” Methylchenodeoxycholic diacetate 11-Ketoandrosterone Methylchenodeoxycholate Ketocholanic acid Methyl cholate 3-Ketocholanic acid 15 Methyl cholate 3,7-diacetate 6-Ketocholestanol 6.C.-Methylhydrocortisone 6-Ketocholestanol acetate Methyldehydrocholate 7-Ketocholestanol 9(11)-Methyl dehydrotestosterone 6-Ketocholestenone Methyldeoxycholate 7-Ketocholesterol 6.C.-Methyl-11-desoxycortisol 7-Ketocholesterol acetate 17C-Methyldihydrotestosterone 3-CMO 18-Ketocorticosterone Methyldihydrotestosterone 7-Ketodeoxycholic acid Methyl-3C,12C-dihydroxynorcholanate 6-Keto-17ot-estiadiol Methyl-3C,12C-diol diacetoxynorcholanate 6-Keto-17ot-estradiol 6-CMO 6-Methyldiosgenin 6-Keto-17ot-estradiol 6-CMO:BSA 6-Methyldiosgenin acetate 6-Keto-17B-estradiol 6-Methylepoxypregnenolone 6-Keto-17B-estradiol 6-CMO 25 7C-Methylestradiol 6-Keto-17B-estradiol 6-CMO:BSA Methylestradiol 3-methyl ether 16-Keto-17B-estradiol 1-Methylestrone 6-Ketoestriol 7C-Methylestrone 6-Ketoestriol 6-CMO 6.C.-Methyl-17-hydroprogesterone 6-KetOestriol 6-CMO:BSA 16C.-Methyl-17-hydroxyprogesterone 6-Ketoestriol triacetate Methylhydroxytigogenin 6-Ketoestrone Methylhydroxytigogenin 3-acetate 6-Ketoestrone acetate Methylhyodeoxycholate 6-Ketoestrone 6-CMO Methyl lithocholate 6-KetOestrone 6-CMO:BSA 7C-Methyl-19-nor-testosterone 16-Ketoestrone 17C-Methyl-19-nor-testosterone 6-Ketoethynylestradiol 35 Methyloxyprogesterone 6-Ketoethynylestradiol 6-CMO 6.C.- 6-Ketoethynylestradiol 6-CMO:BSA 6.C.-Methylprednisolone acetate 3-Keto-5C-etianic acid 6.C.-Methylprednisolone hemisuccinate 3-Keto-5f-etianic acid 6.C.-Methylprednisolone sodium succinate 3-Ketoetiocholanic acid 16?-Methylprednisone 3-Ketoetiocholanic acid methyl ester 6-Methylpregnenolone 1-Ketoetiocholanolone 40 6-Methylpregnenolone acetate 3-Keto-4-etiocholenic acid 16C.-Methylpregnenolone 3-Keto-4-etiocholenic acid ethyl ester 16?-Methylpregnenolone 3-Keto-4-etiocholenic acid methyl ester 16C.-Methylprogesterone 1-Ketoisoandrosterone 16C.-Methyl substance “S” 6-Ketolithocholic acid 16C.-Methyl substance “S” acetate 7-Ketolithocholic acid 45 2-Ketolithocholic acid A1-Methyltestosterone 2-Ketolithocholic acid acetate, methyl ester 17C-Methyl-A1-testosterone 2-Ketolithocholic acid benzoate, methyl ester 2-Methyoxyestradiol 1-Ketopregnanolone 2-Methyoxyestradiol 3-methyl ether 1-Ketopregnanolone acetate Methyl-3?,12C-diacetoxycholanate 7-Ketopregnenolone 50 Methyl-3?,12C-diacetoxydeoxycholanate 6-Ketoprogesterone Murocholic acid 1-Ketoprogesterone C-Muricholic acid 1-Ketotestosterone B-Muricholic acid 6-Ketotestosirerone 6-Ketotestosterone acetate Neocholestene 1-Kerotigogenin 55 Nilevar Lanosterol 6-Nitrocholesteryl acetate Lithocholic acid 6-Nitrocholesteryl benzoate Lithocholic acid acetate 2-Nitroestradiol Lithocholic acid acetate methyl ester 4-Nitroestrone 19-Nor-4-androsten-3,17-dione acetate 19-Nor-4-androsten-17C-ethyl-17?-ol-3-one 60 19-Nor-4-androsten-17C-ethynyl-17?-ol-3-one 19-Nor-4-androsten-17B-ol-3-one 4-Methoxyestradiol 19-Nor-Androsterone 2-Methoxyestrapentol Nordeoxycholic add 4-Methoxyestriol Nordeoxycholic acid diacetate 2-Methoxyestrone Nordeoxycholic acid diacetate, methyl ester 4-Methoxyestrone 65 Nordeoxycholic acid methyl ester 2-Methoxyestrone 3-methyl ether 6,068,830 23 24

TABLE 2-continued TABLE 2-continued

LIST OF STEROIDS LIST OF STEROIDS Norethidrone Smilagenin acetate 19-Nor-4- Sodium cholate Norethynodrel Sodium dehydrocholate Norgestrel Sodium glycochenodeoxycholate Norlutin Sodium glycocholate 19-Norprogesterone Sodium glycodehydrocholate 19-Nortestosterone Sodium glycodeoxycholate 19-Nortestosterone acetate Sodium glycohyodeoxycholate 19-Nortestosterone benzoate Sodium glycolithocholate 19-Nortestosterone 3-CMO Sodium lithocholate 19-Nortestosterone dichloroacetate Sodium taurochenodeoxycholate 19-Nortestosterone hemisuccinate Sodium taurocholanic acid 19-Nortestosterone propionate Sodium taurocholate 19-Nortestosterone sodium sulfate 15 Sodium taurodehydrocholate Sodium taurodeoxycholate Sodium taurohyodeoxycholate Sodium taurolithocholate Stanolone Prednisolone 21-carboxylic acid Stigmastadienone Prednisolone hemisuccinate Srigmasterol Prednisolone 21-phosphate, disodium salt acetate Prednisone Prednisone acetate Taurochenodeoxycholic acid Prednisone hemisuccinate Taurochenodeoxycholic acid sodium salt Prednisone 21-hemisuccinate Nasalt Taurocholanic acid Pregnanediol Taurocholanic acid sodium salt Pregnanediol diacetate 25 Taurocholic acid Pregnanedione Taurocholic acid sodium salt Taurodehydrocholic acid Pregnanetriol 3-glucuronide sodium salt Taurodehydrocholic acid sodium salt Taurodeoxycholic acid Pregnanolone Taurodeoxycholic acid sodium salt Pregnanolone acetate Taurohyodeoxycholic acid Pregnanolone hemisuccinate Taurohyodeoxycholic acid sodium salt 4-Pregnen-113,21-diol-3,20-dione 18-al Taurolithocholic acid Pregnenindiol Taurolithocholic acid sodium salt Testane A1-Testosterone Pregnenolone acetate Oxime 35 A1-Testosterone acetate Pregnenolone 20-CMO A1-Testosterone benzoate Pregnenolone glucuronide A1-Testosterone 3-CMO Pregnenolone hemisuccinate A1-Testosterone hemisuccinate Pregnenolone methyl ether A1-Testosterone hexahydrobenzoate Pregnenolone sodium sulfate A1- A1-Testosterone sodium sulfate Pregnenolone tosylate 40 A1- Testosterone Progesterone Testosterone acetate Progesterone 3-CMO Testosterone benzoate Progesterone 3-CMO:BSA Testosterone 3-CMO Testosterone 3-CMO:BSA Reichstein's epi"U” Testosterone cyclopentylpropionate Reichstein's substance “A 45 Testosterone dichloroacetate Reichstein's substance “C” Testosterone enol diacetate Reichstein's substance “Dehydro C Testosterone 3-ethyleneketal Reichstein's substance “D Reichstein's substance Epi “E” Testosterone glucuronide sodium salt Reichstein's substance “E” Testosterone hemisuccinate Reichstein's substance “Fa 50 Testosterone hemisuccinate:BSA Reichstein's substance “G” Testosterone hexahydrobenzoate Reichstein's substance “H” Testosterone phosphoric acid Reichstein's substance “J” Testosterone potassium sulfate Reichstein's substance “K” Testosterone propionate Reichstein's substance “L Testosterone sodium sulfate Reichstein's substance “M” 55 Testosterone tosylate Reichstein's Suhstance "N Tetrahydro compound “A Reichstein's Suhstance “O'” Tetrahydro compound “B” Reichstein's substance “P” Tetrahydro compound “E” Reichstein's substance “O'” Tetrahydro compound “E” acetate Reichstein's substance “S” Tetrahydro compound “F” Reichstein's substance “T Tetrahydrocortexolone Reichstein's substance “U” 60 Reichstein's substance “V” Tetrahydrocortisol Rockogenin Sirsasapogenin Tetrahydrocortisone acetate Sarsasapogenin acetate Tetrahydro-11-dehydrocorticosterone B-Sitosterol Tetrahydrodesoxycorticosterone B-Sitosterol acetate 65 Tetrahydro-11-desoxycortisol Smilagenin Tetrahydro DOC 6,068,830 25 26 15. Hoffer, P., 1980. Status of gallium 67 in tumor detection, TABLE 2-continued J. Nucl. Med., V 21 p. 394. 16. Johnston, G. S., 1981. Clinical applications of gallium in LIST OF STEROIDS Tetrahydro hydrocortisone oncology, Int.J. Nucl. Med. Biol. V8, p.249. Tetrahydro substance “Q 17. Silberstein, E., B., 1978. Gallium scanning in inflamma Tetrahydro substance “S” tory and neoplastic conditions, Clin. Nucl. Med., V 6, Tetrahydro substance “S” 21-acetate p.63. 18. Hoffer, P., 1978. The utility of gallium-67 tumor imag ing: a comment on the final reports of the cooperative study group. J. Nucl. Med., V 19, p. 1082. 19. Strauss, L. G. and Conti, P. S. 1991. The applications of TH “S 21-acetate PET in clinical oncology. J. Nucl. Med. V 32, N 4, p. Theelol 623-648. Tigogenin 20. Wahl, R. L. 1990. Sequential quantitative FDG/PET Tigogenin acetate assessment of the metastatic response of breast carcino Transdehydroandrosterone 15 Transestriol mas to chemotherapy. J. Nucl. Med. V 31, p.746. 21. Sigurdson, E. R. and Cohen, A. M. 1991. Commentary on applications of PET in clinical oncology. J. Nucl. Med. V 32. p. 649. Triendiol Triketocholanic acid 22. Spicer, J., Duncan, W. P. and Rotert, G. A. 1984. U.S. Urocortisol Pat. No. 4,659.517. Urocortisone 23. Hochberg, J. 1984. U.S. Pat. No. 4,465,676. Ursocholanic acid 24. Pomper, M. G., Van Brocklin, H., and Tieme, A. M. 1990. Ursodeoxycholic acid 11B-methoxy and 16C.-fluoroestradiols: receptor based Wintersteiner's compound “A Wintersteiner's compound “B” imaging agents. J. Med. Chem. 33:3143–3155. Wintersteiner's compound “D 25 25. Katzenellenbogan, J. A., Mcelvany K. D. et al. 1982. Wintersteiner's compound 'F 16C-77Br-bromo-11 (8-methoxyestradiol: a gamma emit Wintersteiner's compound 'G' ting estrogen imaging agent with high uptake and reten tion in target organs. J Nucl. Med. 23:411-419. 26. Hanson, R.N., Franke, L. A. et al. 1984. Preparation and References evaluation of 17o-'I-iodovinyl-11f8-methoxyestradiol 1. Cuschieri, A., 1986. Tumors of the breast: an overview, as a Selective radioligand for tissues containing estrogen In. Comprehensive Textbook of Oncology, Williams & receptors: concise communication, J. Nucl. Med. Wilkins p.1002–1009. 25:998-10O2. 2. Anonymous, 1993. Breast Cancer In: Handbook of U.S. DeSease Incidence and Prevalence Bio. Bus. Int. p.1-15. 27. Lipman, M. E., Do, H. M. T., and Hochberg, R. B. 1991. 3. Harris, J. R., Lippman, M. E., Veronesi, U., Willett, W. 35 Specific estrogen receptor binding and biological effects 1992. Breast cancer. New Engl. J. Med., V 327, p. of 16 C-iodoestradiol on human breast cancer cells. 319-328. Cancer Res. V 41, p.3150–3154. 4. Harris, J. R., Lippman, M. E., Veronesi, U., Willett, W. 28. Zielinski, J. E., Larner, J. M., Hoffer, P. B., and 1992. Breast cancer. New Engl. J. Med., V 327, Hochberg, R. B. 1989. The synthesis of 11B-methoxy p.390–398. 40 (16C.-'I) iodoestradiol and its interaction with estrogen 5. Harris, J. R., Lippman, M. E., Veronesi, U., Willett, W. receptor in vivo and in vitro. J. Nucl. Med. V 30, 1992. Breast cancer. New Engl. J. Med., V 327, p.209-215. p.473–480. 6. Kaplan, W. D. 1988. Introduction: Current status of 29. Ryan, J. W., Rotmensch, J., Pan M-L, et al. 1989. Human tumour imaging In. Antibodies in Radiodiagnosis and biodistribution Studies of a novel non-Steroidal estrogen Therapy, CRC Press p.2-12. 45 (I-123-IBHPE) J. Nucl. Med. V 30, p.883. 7. Lentle, B. C., Scott, J. R., Schmidt, R. P., Hopper, H. R., 30. DeSombre, E. R., Huges A., Shafii, B. et al. 1992. and Catz, Z., 1985, Clinical value of direct tumor Scin Estrogen receptor directed radiotoxicity with Auger tigraphy: a new hypothesis, J. Nucl. Med., V. 26, p. 1215. electron-emitting nuclides: 17 O.( I-11 B 8. Wagner, H. N. 1992. Annual meeting highlights: mol methoxtestradiol and CHO-ER cells. In: Biophysical ecules with messages, J. Nucl. Med., 33:8: p.12 N. 50 aspects of Auger Process. AIP Press p.352–371. 9. Davis, M. A. and Jones, A. G. 1976. comparison of 31. McLaughlin, W. H., Milius, R. A., Pillai, K. M., and 99mTc-phosphonate and phosphonate agents for Skeletal Blumenthal R. D. 1989. Cytotoxicity of receptor mediated imaging, Semin. Nucl. Med.,6: p19. 16C-('I) iodoestradiol in cultured MCF-7 human breast 10. Brady, L. W. and Croll, M. N., 1979. The role of bone cancer cells. J. Natl. Cancer Inst. V 81, p. 437-440. Scanning in the cancer patient, Skel. Radiol., V 3.p217. 55 32. DiZio, J. P., Anderson, C.J. et al. 1992. Technetium- and 11. Waxman, A. D. 1982. Scintigraphic evaluation of diffuse rhenium-labelled progestins: Synthesis, receptor binding hepatic disease, Semin. Nucl. Med., V 12, p 75. 12. Drum, D. E. 1982. Current status of radiocolloid hepatic and in vivo distribution of an 11 B-substituted progestrin Scintiphotography for Space-occupying disease. Semin. labelled with technetium-99m and rhenium-186. J. Nucl. Nucl. Med., V 13, p 64. Med. 33:558-569. 13. Ege, G. N., 1976 Internal mammary lymphoscintigraphy. 60 33. Hansen, J. 1975. U.S. Pat. No. 3,927,193. The rationale, technique, interpretation and clinical appli 34. Goldenberg, M. D., Deland, F., Kim, F. 1978. Use of cation: a review based on 848 cases, Radiology, V 118, radiolabelled antibodies to carcinoembryonic antigen for p.101. the detection and localization of diverse cancers by exter 14. Grove, R. B., Reba, R. C., Eckelman, W. C., and nal photoscanning. N Engl J Med, 298: 1384-1388. Goodyear, M., 1974. Clinical evaluation of radiolabelled 65 35. Kohler, G. and Milstein, C. 1975. Continuous culture of bleomycin for tumor detection, J. Nucl. Med., V15, p. fused cells Secreting antibodies of predefined specificity. 386. Nature, 256:495-497. 6,068,830 27 28 36. Neal, C. E., Baker, M.R., and Texter, J. H. 1992. Prostate 56. Diamandis, EP, YU, H and Sutherland DJ A. Detection imaging with antibodies. Appl. Radiology, 21:39–46. of prostate Specific antigen immunoreactivity in breast 37. Wynant, G. E., Murphy, G. P., and Horoszewicz, J. S. tumors. Breast Cancer Res Treat (in press) (1994). 1991. Immunoscintigraphy of prostatic cancer: prelimi 57. Yu, H and Diamandis EP, Ultrasensitive Time-resolved nary results with 'In-labelled monoclonal antibody 5 immunofluometric assay of prostate Specific antigen in 7E11-C5.3 (CYT-356). Prostate, 18:229–241. Serum and preliminary clinical Strudies. Clin. Chem. 38. Horoszewicz, J. S. U.S. Pat. No. 5,162,504. 1993; 99:2108-14. 58. Monne M, Croce C, Yu H, Diamandis E. P. Molecular 39. Wright, G. L. and Starling, J.J. U.S. Pat. No. 5,153,118. characterization of prostate-specific antigen in breast tis 40. Goldenberg, D. M., DeLand F. H., and Benett, S.J. 1983. Sue. Unpublished data. Radioimmunodetection of prostatic cancer. In Vivo use of 10 59. Hsu S M, Raine L, Fanger H. Use of avidin-biotin radioactive antibodies against prostatic acid phosphatase peroxidase (ABC) in immunoperoxidase techniques. J. for diagnosis and detection of prostatic cancer by nuclear Histochem Cytochem 1981; 29:577–80. imaging. J Amer Med Assoc 2:350-353 60. Martin, E. W., Mojzisik, C. M., Hinkle, G. Heyt al., 41. Wang, E. H., Freidman, P. N., and Prices, C. 1989. Cell (1988) Radioimmunoguided Surgery using monoclonal 57:379-392. 15 antibody, Am. J. Surgery 156:386-92. 42. Lamki, L. M., Buzdar, A. M. et al. 1991. Indium-111 Although preferred embodiments of the invention are labelled B72.3 monoclonal antibody in the detection and described herein in detail, it will be understood by those Staging of breast cancer: a phase 1 Study. J. Nucl. Med. skilled in the art that variations may be made thereto without 32:1326-1332. departing from the Spirit of the invention or the Scope of the 43. Khaw, B. A., Bailes, J. S., Schieder, S. L., Lancaster, J., 20 appended claims. Powers, J., Strauss, H. W., Lasher, J. C., and McGuire, W. What is claimed is: L. 1988. Human breast tumor imaging using 'In labelled 1. An in Vivo method for imaging breast and ovarian monoclonal antibody: athymic mouse model. Eur. J. Nucl. cancers in non-prostatic tissue of a patient comprising: Med. V.14, p.362-366. injecting a patient with a steroid Selected from the group 44. Colcher, D., P. Horan, Nuti, M. et al. 1981. A spectrum 25 consisting of , mineralcorticoids, of monoclonal antibodies reactive with human mammary , and progestin, which induces tumor cells. Proc. Natl. Acad. Sci. V78, p. 3199-3203. the cancer cells to produce prostate Specific antigen, 45. Rainsbury, R. M., Westwood, J. H., Coombes, R. C., Said cancer cells having receptorS for the injected Neville, A. M., McCready, V. R., and Gaset, J. C. 1983. Steroid, Location of metastastic breast carcinoma by a monoclonal 30 injecting a patient with antibodies which bind to prostate antibody chelate labelled with indium-111. Lancet V 1, p. Specific antigen produced by non-prostatic tissue of the 934-938. patient, Said antibodies being labeled with imaging 46. Larson, S.M. 1981. Monoclonal antibodies for diagnosis agents; and therapy. Univ. Wash. Med. V 8, p. 22-28. allowing Said antibodies to incubate in Vivo and bind 47. Zalutsky, M. R. 1988. Antibody-mediated radiotherapy: 35 prostate Specific antigen associated with a cancer, and future prospects. In. Antibodies in Radiodiagnosis and detecting presence of Said imaging agents of bound anti Therapy, CRC Press p.213–235. bodies localized to the cancer. 48. Goldenberg, D. M. 1988. Targeting of cancer with 2. A method of claim 1, wherein the antibodies are radiolabelled antibodies. Arch. Pathol. Lab. Med. V 112 p. Selected from the group consisting of polyclonal antibodies, 580-587. 40 monoclonal antibodies, antibody fragments, antibody 49. Elias, D.J., L. E. Kline, R. O. Dillman, R. M. Trim 1990. constructs, Single chain antibodies and bifunctional antibod Treatment of human B-Lymphoma Xenografts in nude CS. mice with adriamycin-immunoconjugates prepared using 3. The method of claim 1, wherein the antibodies are an acid Sensitive linker. Antibody Immunoconj. and labeled with a radioisotope and the patient is imaged with a Radiophrm. V 3, p.60. 45 photoScanning device. 50. Sivolapenko, C. Moreno, J. Corvalan, W. Smith, A. 4. A method of claim 3, wherein the radioisotope is Ritter and A. Epenetos 1990. Redustion of the anti-mouse selected from the group consisting of ’7Ac, 'AT, 'BA, immunoglobulin response using a bispecific monoclonal 13 Ba. 7Be, 204Bi, 205 Bi, 206Bi, 7°Br, 77Br, 82 BroCd, *7CA, antibody complexed to vinblastine. Antibody Immuno 11C, 14C, 36C1, 48Cr, 51Cr, 62Cu, “Cu, 67Cu, 165Dy, 155Eu, conj. and Radiophrm. V 3, p. 61. 50 18F, 153Gd, Ga, 7Ga, Ga, 7°Ga, 198Au, *H, 16Ho, 11 In, 51. Hertel, A., Donnerstag, B. L. Schulte, A. Noujaim, et al. 113In, 115min, 123, 125I, 131I, 189Ir, 191m Ir, 192Ir, 194Ir, 52Fe, 1992. Therapeutic effects of anti-idiotypic HAMA after 55 Fe, Fe, 177Lu, 15O, 191m-191OS, 109Pd, 32P 33P. 12K, 226Ra, 186Re, 188Re, 82mRb, 153Sm, “Sc, *7Sc, 7°Se, 7Se, immunoscintigraphy in ovarian cancer patients. Eur. J. 'Ag, 2°Na, 2'Na, 89Sr. 35S, 38S, 177Ta, 96Te, 99mTc, 201T, Nucl. Med., V. 19, p. 608. 202T, 115Sn, 117mSn, 12iSn, 166Yb, 169Yb, 175Yb, 88Y. 90Y, 52. King, D. J., Mountain, J. R., Adair, R. J., Owens, R. J. 55 Zn, and Zn. et al. 1992. Tumor localization of engineered antibody 5. A method of claim 4, wherein the radioisotope is fragments Antibody Immunoconj. and Radiophrm. V 5, selected from the group consisting of: "I, I, In, ''"Tc, p. 159–170. 90Y, 186Re, 153Sm, 7Ga, 201T, 77Br, and 18E. 53. Colcher, D., Bird, R., Roselli, M., et al. 1990. In vivo 6. The method of claim 1, wherein the antibodies are tumor targeting of a recombinant Single-chain antigen- 60 labeled with a metal attached covalently to create a para binding protein. J. Nat. Cancer Inst. V. 82, p. 1191-1197. magnetic conjugate and the patient is imaged by magnetic 54. Borrebaeck, C. A. 1992. Antibody Engineering: A prac resonance imaging. tical guide. W. H. Freeman and Company. 7. A method of claim 6, wherein the metal is selected from 55. Joiris, E., Bastin B., and Thornback, J. R. 1991. A new the group consisting of gadolinium, terbium, tin, iron and method for labelling of monoclonal antibodies and their 65 isotopes thereof. fragments with '"Technetium. Nucl. Med. Biol. V. 18, p. 353-356.

UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION

PATENT NO.: 6,068,830

DATED: May 30, 2000

INVENTORS: Eleftherios P. DIAMANDIS, et al.

It is certified that errors appear in the above-identified patent and that said Letters Patent is hereby corrected as shown below: In Claim 4, column 28, line 47, 'AT' has been deleted and in its place --'At has been inserted;

“'BA” has been deleted and in its place --'Ba-- has been inserted;

line 48, after “Br”, --- has been inserted;

“CA" has been deleted and in its place --"Ca-- has been inserted;

line 52, “'"'OS” has been deleted and in its place --'"'Os-- has been inserted.

Signed and Sealed this Twenty-second Day of May, 2001 Zaaé, f-á4.

NICHOLAS P. GODICI

Attesting Officer Acting Director of the United States Patent and Trademark Office