WO2008021322A2

WO2008021322A2 - Selective alpha particle-mediated depletion of tumor vasculature with vascular normalization

Info

Publication number: WO2008021322A2
Application number: PCT/US2007/017919
Authority: WO
Inventors: David A. Scheinberg; Michael R. Mcdevitt; Jaspreet S. Jaggi
Original assignee: Sloan-Kettering Institute For Cancer Research
Priority date: 2006-08-14
Filing date: 2007-08-13
Publication date: 2008-02-21
Also published as: WO2008021322A3

Abstract

Provided herein are methods of inhibiting neovascularization of malignant tissue in a subject by contacting or administering an alpha-emitting radioconjugate, such as a radioimmunoconjugate, for example, an antibody, targeting the vascular endothelium. Also provided are methods of treating a cancer, such as a prostate cancer, in a subject by administering the alpha-emitting radioimmunoconjugate. Alternatively, a prostate cancer may be treated by the sequential administration of an 225Ac-immunoconjugate and a chemotherapeutic agent.

Description

SELECTIVE ALPHA PARTICLE-MEDIATED DEPLETION OF TUMOR VASCULATURE WITH VASCULAR NORMALIZATION

Federal Funding Legend

This invention was produced in part using funds obtained through the National Institutes of Health Grant Nos. ROl-CA 55349 and P01-33049. Consequently, the federal government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Field of the Invention The present invention relates generally to the fields of nuclear medicine and oncology. More specifically, the present invention provides selective methods to deplete tumor vasculature using alpha particle emittors.

Description of the Related Art Inhibition of tumor angiogenesis is an emerging treatment strategy for solid tumors

[I]. Endothelium-targeting peptides, antibodies, antibody fragments and nanoparticles have been used to target the tumor vasculature in various preclinical and clinical studies [2-4]. The ultimate goal of these anti-angiogenic strategies is to inhibit endothelial cell proliferation in tumors via either targeted delivery of toxins, cytotoxic drugs or radiation to endothelial cells, interference with intercellular signaling pathways in endothelial cells, e.g. anti-VEGF therapies, [5-9] or disruption of endothelial cell interaction with the extracellular matrix, e.g. αvβ3 integrin inhibitors [10]. Endothelial cells, unlike cancer cells, are generally genetically and phenotypically stable and do not mutate readily; therefore, development of drug-resistance is not a major concern in therapies directed against endothelial cells [11]. Tumor growth inhibition via anti-angiogenic therapy has certain practical limitations to its implementation [12]. A second wave of angiogenesis initiated by the residual tumor cells can ensue when an anti-angiogenic treatment is discontinued, leading to a late resurgence in tumor growth [13-14]. Therefore, a combination of anti-angiogenic therapy and cytotoxic therapy that targets the tumor cells directly has been suggested to prevent tumor recurrence. However, destruction of tumor vasculature following anti-angiogenic therapy can decrease blood flow to tumors and potentially prevent the delivery of antitumor therapeutics to the tumor cells [12]. Recently, it was shown that antiangiogenic therapies may transiently increase the efficiency of the tumor vasculature, and that administration of cytotoxic therapy in that period may result in enhanced cytotoxic drug delivery to tumor cells [15]. Therefore, optimal scheduling of antiangiogenic and chemotherapy may be required to overcome the pharmacokinetic barriers and could potentially result in long-term tumor remissions.

Vascular endothelial (VE) cadherin is a vascular endothelial cell-specific adhesion molecule that is expressed constitutively throughout the entire vasculature and takes part in the formation of adherens junctions between adjacent endothelial cells [16}. The monoclonal antibody E4G10, is specific for an epitope exposed only on the monomelic, unengaged form of VE cadherin. This allows for selective targeting of endothelial cells in nascent tumor vasculature as well as of VE cadherin positive endothelial progenitor cells (EPCs) in bone marrow and peripheral circulation. Since E4G10 does not bind established vasculature, no vascular leak and hemorrhage is observed in normal organs of mice after E4G10 administration [17]. This allows for selective targeting of endothelial cells in nascent tumor vasculature as well as of VE-cadherin positive endothelial progenitors [16-17].

Alpha particles are a form of extremely potent, short ranged and high energy cytotoxic radiation capable of selectively killing individual cells. Actinium-225 is a molecular-sized generator of an alpha particle-emitting isotope cascade [18-19]. During decay actinium-225 releases three alpha-particle emitting daughters, i.e., francium-221, astatine-217 and bismuth-213 [18]. Thus, there is a significant need in the art for improvements in the area of tumor antiangiogenic radiotimmunotherapies. Specifically, the present invention is deficient in methods of specifically targeting tumor endothelial neovasculature with alpha-emitting radioimmunoconjugates to normalize the same. The present invention fulfills this long-standing need and desire in the art.

SUMMARY OF THE INVENTION

The present invention is directed to a method for inhibiting neovascularization of a malignant tissue in a subject. The method comprises contacting vascular endothelium associated with the malignant tissue one or more times with an alpha-emitting radioconjugate targeted thereto thereby inhibiting neovascularization in the subject. In a related method a further step comprises contacting the malignant tissue with one or more of a chemotherapeutic agent or therapeutic radiation one or more times.

The present invention also is directed to a method for treating a cancer in a subject. The method comprises administering a pharmacologically effective amount of an alpha-emitting radioconjugate targeted to a cell adhesion molecule specific for tumor vascular endothelium associated with the cancer to inhibit tumor neovascularization, thereby treating the cancer in the subject. In a related method a further step comprises administering a pharmacologically effective amount of one or more of a therapeutic agent or therapeutic radiation to the subject. In another related method a further step comprises administering the alpha-emitting radioconjugate one or more times. The present invention is directed further to a method for treating prostate cancer in a subject. The method comprises administering a pharmacologically effective amount of an ²²⁵Ac- immunoconjugate specific for VE cadherin to the subject, wherein the ²²⁵Ac-immunoconjugate inhibits neovascularization of a prostate tumor thereby treating the prostate cancer. In a related method a pharmacologically effective amount of one or more of a therapeutic agent or therapeutic radiation may be administered one or more times to the subject. In another related method a further step comprises administering the ²²⁵Ac- immunoconjugate one or more times.

The present invention is directed further still to a related method for treating prostate cancer in a subject. The method comprises sequentially administering pharmacologically effective amounts of an ²²⁵Ac- immunoconjugate specific for VE cadherin and a chemotherapeutic agent to the subject, thereby treating the prostate cancer. In a related method a further step comprises administering one or both of the ²²⁵Ac-immunoconjugate and chemotherapeutic agent one or more times.

Other and further aspects, features and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention. These embodiments are given for the purpose of disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages and objects of the invention, as well as others which will become clear, are attained and can be understood in detail, more particular descriptions of the invention are briefly summarized. The above may be better understood by reference to certain embodiments thereof which are illustrated in the appended drawings. These drawings form a part of the specification. It is to be noted; however, that the appended drawings illustrate preferred embodiments of the invention and therefore are not to be considered limiting in their scope.

Figures 1A-1C depict the characterization of E4G 10. Figure IA is a flow cytometric analysis showing the binding of E4G10 to H5V cells, a mouse endothelioma cell line in comparison to binding of the positive control anti-CD31 or isotype control antibody. Figure IB is X-SPECT gamma camera images of mice (prone with nose at top) at 24, 48 and 72 hours post- injection with "¹In labeled E4G10. Figure 1C shows the biodistribution of ¹¹¹In labeled E4G10 at specified time-points post-injection. Data are mean ± s.e.m. %ID/g = percentage of injected dose per gram of tissue.

Figures 2A-2E demonstrate that ²²⁵Ac-E4G10 therapy inhibits the growth of LnCap celts prostate tumors. Figure 2A shows a flow cytometric analysis depicting the lack of E4G10 binding to LnCap cells; J591, mouse-anti prostate specific membrane antigen is the positive control. Mouse and rat isotype controls also were evaluated. Figure 2B shows in situ (left) and excised tumor (right) in a representative dual control (DC) and ^22SAc-B4G 10 treated animal. Figure 2C shows tumor volume in various treatment groups at described time-points. Figure 2D shows serum prostate specific antigen (PSA) levels in the three treatment groups at 22 days post-implantation with 5 million LnCap cells. Figure 2E is a Kaplan Meier curve showing enhancement of survival with ²²⁵Ac-E4G10 treatment. Data in Figures 2C-2D are mean ± s.e.m. Scale bar, 1 cm.

Figures 3A-3D demonstrate the effect of ²²¹Ac~E4G10 therapy on tumor histology, vascularity and apoptosis. In Figure 3A light microscopy depicts numerous RBC-filled vascular spaces (arrows) in dual control tumor and fewer, but relatively normal -looking vessels (arrowheads) in ^22SAc-E4G10 treated tumor. Figure 3B shows the immunohistochemical staining of tumor- sections for vWF, an endothelial cell marker (top) and TUNEL staining of tumor sections to detect apoptosis (bottom). Quantification of vWF staining (Figure 3C) and apoptosis (Figure 3D) in 4 randomly selected fields are shown. Data are mean ± s.e.m.

Figures 4A-4B demonstrate that ^2zsAc-E4G10 treatment results in a relatively normal remaining tumor vasculature. Figure 4A shows the greater coverage of tumor blood vessels (CD31 positive) by pericytes (β-SMA-positive cells) in ^22SAc-E4G10 treated tumor relative to dual control. Figure 4B are transmission electron micrographs of blood vessels in dual control and ²²⁵Ac- E4G10 treated tumor. The dual control tumor contains extravasated RBC-filled vascular spaces that are not lined with endothelial cells, whereas blood vessels in ²²⁵Ac-E4G10 treated tumor display a continuous endothelial lining (arrow) resting on a basement membrane (BM) that is shared with the surrounding pericyte (P). Scale bar is 50 μm.

Figures 5A-5C demonstrate that a combination of ^22SAc-E4G10 treatment results in a relatively normal remaining tumor vasculature. Figure SA shows tumor volume in the four treatment groups over time. Data are mean ± s.e.m. Figure 5B shows a Kaplan Meier survival curve of treated animals showing significant enhnacement of animal survival when ²²⁵Ac-E4G10 therapy is followed by a course of paclitaxel. Figure 5C shows the absence of histopathologic damage in normal organs, assessed 10 days after cessation of ²²⁵Ac-E4G10 treatment. DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term "a" or "an", when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one", but it is also consistent with the meaning of "one or more", "at least one", and "one or more than one". Some embodiments of the invention may consist of or consist essentially of one or more elements, method steps, and/or methods of the invention. It is contemplated that any method, compound, drug, or composition described herein can be implemented with respect to any other method, compound, drug, or composition described herein. As used herein, the term "or" " in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or".

As used herein, the term "contacting" refers to any suitable method of bringing the alpha-emitting radioconjugate or radioimmunoconjugate described herein or other chemotherapeutic agent or radiation into contact with a malignant tissue, a tumor, or a cell comprising the same. In vitro or ex vivo this is achieved by exposing the malignant tissue or tumor or tumor cells in a suitable medium to the radioimmunoconjugate or other chemotherapeutic agent or radiation. For in vivo applications, any known method of administration is suitable as described herein.

As used herein, the term "treating" or the phrase "treating a tumor" or "treating tumor cells" includes, but is not limited to, halting the growth of the tumor or tumor cell(s), killing the tumor or tumor cell(s), or reducing the number of tumor cells or the size of the tumor. Halting the growth refers to halting any increase in the size or the number of tumor cells or tumor or to halting the division of the tumor cells. Reducing the size refers to reducing the size of the tumor or the number of or size of the tumor cells. As would be apparent to one of ordinary skill in the art, the term "tumor" refers to a mass of malignant neoplastic cells or a malignant tissue comprising the same. Also, as would be apparent to a skilled artisan, the term "cancer" refers to the type(s) or location(s) of the malignant disease associated with formation of a particular tumor. As particularly used herein halting the growth of or reducing the size of a tumor and the tumor cells comprising the same refers to inhibiting, preventing, stopping, or reducing tumor neovascularization or killing tumor endothelial cells and/or tumor cells.

As used herein, the term "subject" refers to any target of the treatment, preferably a mammal, more preferably a human.

In one embodiment of the present invention there is provided a method of inhibiting neovascularization of a malignant tissue in a subject, comprising contacting vascular endothelium associated with the malignant tissue one or more times with an alpha-emitting radioconjugate targeted thereto thereby inhibiting neovascularization in the subject. Further to this embodiment the method comprises contacting the malignant tissue with one or both of a therapeutic agent or therapeutic radiation one or more times. In this further embodiment the therapeutic agent may be a chemotherapeutic agent, a monoclonal antibody or fragment thereof, or a hormonal agent. Also, the alpha-emitting radioconjugate may contact the vascular endothelium prior to, concurrently with or after one or both of the chemotherapeutic agent or the therapeutic radiation.

In these embodiments the the cell-specific adhesion molecule may be VE-cadherin. In one example the VE-cadherin is in monomeric form. Also, in these embodiments the alpha- emitting radioconjugate may be a radioimmunoconjugate. An example of an alpha-emitting radioimmunoconjugate is ^22SAc-E4G10. Furthermore, in these embodiments the malignant tissue may be a prostate tumor, a colon tumor, a kidney tumor, a lung tumor, a breast tumor, an ovarian tumor, a pancreatic tumor, or a brain tumor .

In another embodiment of the present invention there is provided a method of treating a cancer in a subject, comprising administering a pharmacologically effective amount of an alpha-emitting radioconjugate targeted to a cell adhesion molecule specific for tumor vascular endothelium associated with the cancer to inhibit tumor neovascularization, thereby treating the cancer in the subject.

Further to this embodiment the method comprises administering the alpha-emitting radioconjugate one or more times. Further still to this embodiment the method comprises administering one or both of a pharmacologically effective amount of a therapeutic agent or therapeutic radiation one or more times to the subject. The therapeutic agent may be a chemotherapeutic agent, a monoclonal antibody or fragment thereof, or a hormonal agent. A non- limiting example of a chemotherapeutic agent is paclitaxel, doxorubicin, mitomycin-C, bleomycin, cisplatin, tamoxifen, vincristine, vinblastine, 6-mercaptopurine, or 5-fluorouracil. A non-limiting example of a hormonal agent is an androgen antagonist. In this further embodiment the alpha- emitting radioconjugate may be administered prior to, concurrently with or after administration of one or both of the chemotherapeutic agent or the therapeutic radiation.

In all these embodiments the cell-specific adhesion molecule may be VE-cadherin. In one example the VE-cadherin is in monomeric form. Also, in these embodiments, the alpha- emitting radioconjugate may be a radioimmunoconjugate. An example of an alpha-emitting radioimmunoconjugate is a humanized or chimeric ²²⁵Ac-E4G10. Furthermore the cancer may be prostate cancer, colon cancer, kidney cancer.a lung cancer, breast cancer, ovarian cancer, pancreatic cancer, or brain cancer.

In yet another embodiment of the present invention there is provided a method of treating prostate cancer in a subject, comprising administering a pharmacologically effective amount of an 225-Ac-immunoconjugate specific for VE cadherin to the subject, wherein the ³²⁵Ac- immunoconjugate inhibits neovascularization of a prostate tumor thereby treating the prostate cancer. Further to this embodiment the method comprises administering the ²²⁵Ac- immunoconjugate one or more times. Further still to this embodiment the method comprises administering one or both of a pharmacologically effective amount of a therapeutic agent or therapeutic radiation one or more times to the subject. In these embodiments the VE cadherin may be in monomeric form. The ²²⁵Ac-immunoconjugate, the therapeutic agent, the number of times of administration thereof and the timing of administration with a therapeutic agent or therapeutic radiation is as described supra.

In yet another embodiment of the present invention there is provided a method of treating prostate cancer in a subject, comprising sequentially administering pharmacologically effective amounts of an ^22SAc-immunoconjugate specific for VE cadherin and a chemotherapeutic agent to the subject, thereby treating the prostate cancer. Further to this embodiment the method comprises administering one or both of the ^22SAc-immunoconjugate and chemotherapeutic agent one or more times. The VE cadherin, the ²²⁵Ac- immunoconjugate, the chemotherapeutic agent and the timing of administation of the ²²⁵Ac- immunoconjugate and chemotherapeutic agent is as described supra.

Provided herein are methods for selectively targeting tumor endothelial neovasculature using radiolabeled conjugates to inhibit formation of new tumor vasculature thereby normalizing the existing tumor vasculature. Neither the tumor per se or its supporting microenvironment require targeting. Furthermore, these methods are effective to suppress tumor growth, enhance tumor cell apoptosis and prolong survival, without gross or histopathological toxicity in normal tissues or their vasculatures.

Particularly, the radioconjugate may be a radioimmunoconjugate, such as, but not limited to, an antibody or antibody fragment, including monoclonal, chimeric or humanized antibodies, targeting tumor endothelial cells. Methods of generating antibodies and antibody fragments are well known in the art. For example, but not limited to, the antibody may be a humanized or chimeric version or fragment thereof of the monoclonal antibody E4G10 which is specific for the unengaged form of VE cadherin. The radiolabel is an alpha-emittor, preferably, actinium-225, which as a chelate is coupled to the monoclonal antibody or other targeting moiety. Chelators for actinium-225, for example a bifunctional chelator, and other alpha-emittors and methods of forming the chelate, the radioconjugate and the radioimmunoconjugate are known in the art. The alpha-emittor labeled radiooconjugate may inhibit tumor neovascularization in a tumor tissue in vitro or in a tumor in vivo.

Thus, the present invention provides methods of treating a tumor or a cancer by administering to a subject a pharmacologically effective amount of the radioconjugate or radioimmunoconjugate, for example, but not limited to_> 225-actinium-E4G10 humanized or chimeric monoclonal antibody or a pharmaceutical composition thereof. The cancer may be any vascularized cancer, for example, but not limited to, prostate cancer, colon cancer, kidney cancer, lung cancer, ovarian cancer, breast cancer, pancreatic cancer, or a brain cancer.

In addition, the radioimmunoconjugate may be administered in conjunction with one or more other therapeutics and/or in conjunction with radiation therapies. For example, other therapeutics may include chemotherapeutic agents and/or other antibodies or fragments thereof and may be monoclonal antibodies, humanized antibodies or chimeric antibodies and/or a therapeutic hormonal agent, e.g., androgen antagonists. Without being limiting, chemotherapeutic agents may comprise antitumor antibiotics, e.g., doxorubicin, mitomycin-C, or bleomycin, alkylating agents, e.g., cyclophosphamide, nitrosoureas, e.g., lomusitne or carmustine, antimetabolites, e.g., 6- mercaptopurine or 5-fluorouracil, plant alkaloids, e.g., paclitaxel, vincristine or vinblastine, or steroid hormones, e.g., tamoxifen. A non-limiting example of a chemotherapeutic agent is paclitaxel.

The radioimmunoconjugate may be administered in a single dose or multiple doses. Furthermore, the radioimmunoconjugate may be administered prior to, concurrently or after administration of one or more chemotherapeutics and/or therapeutic radiation. The number of doses and timing of administration of the radioimmunoconjugate effects normalization of tumor vasculature which, when timed with chemotherapy, maximizes the delivery of the chemotherapeutic and/or timed with radiation therapies improves radiation sensitivity of tumors. It is contemplated that delivering the two treatment modalities in a carefully planned temporal fashion would result in a synergistic effect on tumor cell killing.

Dosage formulations of the radioconjugate, the radioimmunoconjugate or the labeled E4G 10 antibody and other therapeutic agents may comprise conventional non-toxic, physiologically or pharmaceutically acceptable carriers or vehicles suitable for the method of administration. Methods of administration are well-known and standard in the art. Determination of which specific activity of the actinium-225 and which dosage of the radioconjugate or radioimmunoconjugate are well within the purview of one of ordinary skill in the art, as are routes of administration for the same. Also, an artisan of ordinary skill in this art can determine which chemotherapeutics known and standard in the art and/or what radiation regimen is appropriate for the cancer. Such determination is based on, at least in part, on the subject's health, the progression or remission of the cancer, the route of administration and the formulation used.

The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. EXAMPLE 1

Methods

Animals

Male BALB/c and athymic nude mice (NCr nu/nu), 4-12 weeks of age, were obtained from Taconic, Germantown, NY. All animal studies were conducted according to the NIH Guide for the Care and Use of Laboratory .

Flow cytometry

Row cytometric analysis of H5V and LnCap cells was performed with anti-CD31 (Pharmingen, San Diego, CA)₁ E4G10, J591 (an ti -prostate specific membrane antigen or anti- PSMA) or isotype control antibodies (R&D systems, Minneapolis, MN) and fluorochrome-labeled secondary antibodies. Samples were acquired on an FC500 cytometer (Beckman Coulter, Fullerton, CA) and analyzed with FlowJo software (Tree Star Inc., Ashland, OR).

Preparation, quality control and administration of radioimmunoconjugates

²²⁵Ac (Oak Ridge National Laboratory, Oak Ridge, TN) and "¹In (Perkin Elmer, Boston, MA) were conjugated to E4G10 or non-specific rat IgG2a isotype antibody using a two-step labeling method, as described [20-21]. Routine quality control of the labeled antibody was performed using instant thin layer chromatography to estimate the radio-purity and cell binding assay to determine the immunoreactivity. Mice were anesthetized and then injected intravenously via the retro-orbital venous plexus with the radioimmunoconjugate. The injected volume was 100 μl and the antibody dose was 0.6-0.7 μg per 50 nCi injection. Typical radiochemical purity was 95- 99%.

Gamma Camera imaging and biodistribution

For gamma-imaging, anesthetized animals were imaged, in prone position, on X- SPECT ™ scanner (Gamma Medica, Northridge, CA), a dedicated rodent imaging device, at specified time-points post-injection with 230μCi of ^mIn-E4G10. Images were acquired in a 56x56x16 image matrix using photopeak energy windows of 172 keV ± 10% and 273 keV + 10% and no zoom. For organ distribution studies, mice were sacrificed at indicated time-points post- injection with ^ιnIn-E4G10 (3μCi) and their blood and the specified organs were harvested. The organs were washed in distilled water, blotted dry on gauze, weighed and the activity of 11 Hn (15- 550 keV window) was measured using a gamma-counter (COBRA II, Packard Instrument Company, Meriden, CT). Samples of the injectate (100 μl) were used as decay correction standards. Percentage of injected dose of ,,,In per gram of tissue weight (%ID/g) was calculated for each animal and the mean %ID/g was determined at each time-point, as described previously [21-22]. Tumor implantation in mice

LNCaP prostate tumor cell line was obtained from the American Type Culture Collection (Rockville, MD). The LNCaP cells were grown in RPMI 1640 medium supplemented with L-glutamine, 10% fetal bovine serum and penicillin-streptomycin in an atmosphere of 5% CO2 and air at 37 degrees C. The cells were harvested and 1 million or 5 million cells were injected in 200 μL matrigel (BD Biosciences, Palo Alto, CA) into the right flank of the animal. Animals were checked twice weekly for the development of palpable tumors at the site of injection.

Tumor therapy studies

In the first ^22SAc-E4G10 monotherapy study, mice were engrafted with 1 million LNCaP cells. The test group received 50 nCi of ^22SAc labeled E4G10. Controls included vehicle (received 1% human serum albumin), unlabeled E4G10 (received 7 μg E4G10), ²²⁵Ac labeled isotype control (received 50 nCi (0.6 μg) of ^22SAc labeled irrelevant rat IgG2a). Treatments were administered at 3, 5, 7 and 10 days post-implantation of xenografts. In the second ²²⁵Ac-E4G10 monotherapy study, mice were injected with 5 million LNCaP cells and treated on days 3, 5, 7 and 10 days post xenograft implantation with either vehicle (received 1% human serum albumin), 50 nCi of 225Ac labeled irrelevant isotype control IgG mixed with 7 μg of unlabeled specific E4G10 (dual control) or 50 nCi of 225Ac labeled E4G10. For the combination therapy study, ²²⁵Ac labeled E4G10 or isotype control antibody (50 nCi) was administered at 16, 18, 21, and 23 days post- implantation with 5 million LNCaP cells. Paclitaxel (20 mg/kg i.p.) was administered to the specified groups on days 27, 30, 34 and 37.

Tumor size was measured with calipers, and tumor volume was calculated by the formula 0.52 x dl2 x d2, where dl is the smaller diameter and d2 is the larger diameter. Animals were followed over long term for survival advantage. Mice were bled retro-orbitally on described days and serum prostate specific antigen (PSA) was determined using an immunoassay kit (Alpco diagnostics, Windham, NH).

Histopathologic toxicity studies BALB/c mice (n=5) were injected four times with lOOnCi ²²⁵Ac-E4G10 (twice the dose at same schedule as the tumor therapy experiments). Animals were sacrificed 10 days after last injection and their lungs, kidneys, heart, liver and spleen were excised, fixed and examined by light microscopy. Anatomic Pathology and Immunohistology

Tumors and normal organs from mice were harvested, formalin-fixed and paraffin- embedded. Three micron sections were stained with hematoxylin and eosin (H&E), Periodic-acid Schiff (PAS) and Masson's trichrome, and evaluated with an Olympus BX45 light microscope, as described [23]. Eight micron tumor-sections were immunostained with goat anti-CD31 (SantaCruz Biotechnology, Santa Cruz, CA) and mouse anti-smooth muscle actin (α-SMA; Sigma, St. Louis, MO) as primary antibodies, biotinylated secondary antibodies and streptavidin-fluorophores as tertiary reagents. Images were acquired on a Leica TCS SP2 AOBS confocal laser-scanning microscope. Apoptosis was detected in 8 μm tumor-sections using the TUNEL assay (In situ cell death detection kit; Roche). Immunoperoxidase staining was performed for von Willebrand factor and caspase-3, using rabbit anti-vWF (Dako, Carpinteria, CA) and rabbit anti-cleaved caspase-3 (Cell signaling technology, Beverly, MA), and imaged on a Zeiss Axiovert 200 microscope. Acquired images were evaluated using ImageJ software available at the NIH website. For each of the four random fields (571x428um) of tumour sections stained with vWF, the number of pixels of positive staining was divided by the total number of pixels, and expressed as a percentage. The degree of apoptosis was estimated in each randomly selected field (1142x857 microns) by calculating the percentage of TUNEL positive cells out of the total number of cells as measured by nuclear counterstaining.

Electron Microscopy

Pieces of tumor tissue were fixed in 4% paraformaldehyde, post-fixed in 1% Osmium tetroxide and later embedded in epon. Ultra-thin sections (200-400 microns) were cut on nickel grids, stained with uranyl acetate and lead citrate and examined using an electron microscope (Hitachi H-7500, Pleasanton, CA).

EXAMPLE 2 E4G10 binds to cultured endothelial cells but not to established vasculature

The binding specificity of the monoclonal antibody E4G10 for endothelial cells of the neovasculature was determined in vitro and in vivo by its binding to H5V mouse endothelioma cells, and by the lack of specific uptake in normal tissues by imaging and biodistribution studies. In flow cytometric studies, E4G10 bound with high affinity to H5V cells (Fig IA). X-SPECT gamma camera images at various time-points post-injection with ¹¹¹In trace-labeled E4G10 showed no organ-specific uptake of the radioactivity in BALB/c mice (Fig. I B). The radioactivity gradually cleared from the blood pool and other vascularized organs such as heart and lungs. At later time- points, the radioactivity remained only at the sites of IgG catabolism such as the liver and spleen. Detailed quantitation of the biodistribution was performed by sacrificing animals at defined time- points post-injection with ^{l π}In-E4G10 and measuring the radioactivity in harvested organs (Fig. 1C). Therefore, the post-mortem data confirmed the lack of specific uptake of E4G10 in normal tissues seen in whole body imaging study.

EXAMPLE 3 225AC-B4G10 inhibits the growth of prostate cancer xenografts in mice

E4G10 did not bind the human LNCaP prostate tumor cells in flow cytometric studies (Fig. 2A). The therapeutic efficacy of the 225Ac generator labeled E4G10 was tested in two separate experimental trials in athymic male mice that were xenografted with human LNCaP prostate tumors. 225Ac-E4G10 was therapeutically effective and significantly inhibited the growth of tumors. None of the control treatments had any significant effects on tumor growth (Figs. 2B- 2C). Serum PSA, a surrogate marker for total body prostate tumor cell burden [25], was used to confirm the anti -tumor effects and was significantly lower ( p < 0.001 vs. dual control; One way ANOVA and Bonferroni's post-hoc analysis) in ²²³Ac-E4G10treated animals as compared to the controls (Fig. 2D). As a consequence of the anti-tumor effect, the median survival of ²²⁵Ac-E4G10 treated animals was longer relative to the control groups (Fig. 2E). Therefore, even though E4G10 did not bind to the LNCap tumors directly, treatment with ^22SAc-labeled E4G10 resulted in an inhibition of tumor growth, lower serum PSA and enhanced survival in prostate cancer xenograft- bearing mice.

EXAMPLE 4

Effects of ²²⁵Ac-E4G10 treatment on tumor histology

To dissect the mechanism of growth inhibition by ²²⁵Ac-E4G10, dual control and ²²⁵Ac-E4G10 treated animals were sacrificed at 14 and 22 days after tumor implantation (four animals per group at each time-point), and their tumors were excised and analyzed. The tumors in control animals were grossly hemorrhagic and on light microscopy, displayed infiltration of tumor cell masses by a network of markedly dilated, poorly defined, anastomosing vascular spaces filled with extravasated RBCs (Fig. 3A). In contrast, ²²⁵Ac-F^GlO treated tumors showed groups of cohesive tumor cells separated by bands of acellular hyalinized stroma containing small, discrete and wellformed capillary vessels, which were lined by endothelial cells resting on a basement membrane as visualized with trichrome stain.

Immunostaining for vWF, an endothelial cell marker, was significantly greater in the control tumors (p = 0.0002; Student's t-test) relative to the ^22SAc-E4G10 treated ones (Fig. 3B- 3C). Additionally, TUNEL assay showed a significantly greater percentage of apoptotic cells in the ²²⁵Ac-E4G10 treated tumors (p = 0.0125; Student's t-test) relative to the control tumors (Figs. 3B & 3D). The TUNEL assay data was confirmed by cleaved caspase-3 immunohistochemistry (data not shown).

EXAMPLE 5 2²⁵Ac-E4G10 treatment leads to a relatively normalized tumor vasculature

To investigate whether treatment with ²²⁵Ac-E4G10, in addition to inhibiting tumor angiogenesis, also resulted in normalization of the residual tumor vasculature, tumor cross-sections were dual immunostained with CD31 (endothelial cell marker) and α-SMA (mural cell marker). The majority of the vascular endothelial cells in ²²⁵Ac-E4G10 treated tumor had pericyte coverage, whereas little coverage was observed in the tumor treated with the control agents (Fig. 4A). Transmission electron microscopy revealed sinusoid like blood vessels in dual control tumor, which were lined by tumor cells and filled with extravasated erythrocytes (RBCs, Fig. 4B). In contrast, most vessels in ^22SAc-E4G10 treated tumors appeared mature and were lined by a continuous layer of endothelial cells resting on a basement membrane and surrounded by a pericyte.

EXAMPLE 6 Sequential administration of ²²⁵Ac-E4G10 and paclifoxel enhances the anti-tumor response

The structural normalization of residual tumor vasculature following ^22SAc-E4G10 treatment prompted us to ask whether administration of a cytotoxic drug in that timeperiod would enhance the overall anti-tumor response via greater accessibility of the drug to tumor cells. Monotherapy with ²²⁵Ac-E4G10 significantly inhibited tumor growth and enhanced animal survival compared to controls as was observed in previous experiments (Figs. 5A-5B). However, subsequent bi-weekly administration of paclitaxel for two weeks, starting four days after the last ^2MAc-E4G10 injection resulted in a significant enhancement of the anti-tumor response compared to ²²⁵Ac-E4G10 monotherapy. Median survival for the specific combination treatment group was 182 days versus 113 days for the animals that received ²²⁵Ac-E4G10 alone or 84 days for animals that received ^22S Ac- labeled isotype antibody and paclitaxel.

Three animals each from the 225Ac-E4G10 and ²²⁵Ac-isotype control (IgG2a) group were sacrificed before commencement of paclitaxel therapy for histopathologic analyses of the tumor vasculature. As observed earlier (Fig. 3A), the tumor vasculature in ^22SAc-E4G10 treated animals, though less extensive than that seen in ^22SAc-isotype treated animals, displayed a relatively greater structural maturity (data not shown). Figure 5C shows the absence of histopathologic damage in normal organs, assessed 10 days after cessation of ^22SAc-E4G10 treatment.

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Any publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. Further, these publications are incorporated by reference herein to the same extent as if each individual publication was specifically and individually incorporated by reference.

One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The present examples along with the methods, procedures, treatments, molecules, and specific compounds described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention as defined by the scope of the claims.

Claims

WHAT IS CLAIMED IS:

1. A method for inhibiting neovascularization of a malignant tissue in a subject, comprising: contacting vascular endothelium associated with the malignant tissue one or more times with an alpha-emitting radioconjugate targeted thereto thereby inhibiting neovascularization in the subject.

2. The method of claim 1 , further comprising: contacting the malignant tissue with one or both of a therapeutic agent or therapeutic radiation one or more times.

3. The method of claim 2, wherein the therapeutic agent is a chemotherapeutic agent, a monoclonal antibody or fragment thereof, or a hormonal therapeutic agent.

4. The method of claim 2, wherein the alpha-emitting radioconjugate contacts the vascular endothelium prior to, concurrently with or after one or both of the chemotherapeutic agent or radiation.

5 The method of claim 1, wherein the alpha-emitting radioconjugate targets a cell-specific adhesion molecule specific for the vascular endothelium.

6. The method of claim 5, wherein the cell-specific adhesion molecule is VE- cadherin.

7. The method of claim 6, wherein the VE-cadherin is in a monomelic form.

8. The method of claim 1 , wherein the alpha-emitting radioconjugate is a radioimmunoconjugate.

9. The method of claim 1, wherein the alpha-emitting radioimmunoconjugate is a humanized or chimeric ^22SAc-E4G10.

10. The method of claim 1, wherein the malignant tissue is a prostate tumor, a colon tumor, a kidney tumor, a lung tumor, a breast tumor, an ovarian tumor, a pancreatic tumor, or a brain tumor.

11. A method for treating a cancer in a subject, comprising: administering a pharmacologically effective amount of an alpha-emitting radioconjugate targeted to a cell adhesion molecule specific for tumor vascular endothelium associated with the cancer to inhibit tumor neovascularization, thereby treating the cancer in the subject.

12. The method of claim 11 , further comprising: administering the alpha-emitting radiooconjugate one or more times.

13. The method of claim 11 , further comprising: administering one or both of a pharmacologically effective amount of a therapeutic agent or therapeutic radiation one or more times to the subject.

14. The method of claim 13, wherein the therapeutic agent is a chemotherapeutic agent, a monoclonal antibody or fragment thereof, or a hormonal agent.

15. The method of claim 13, wherein the chemotherapeutic agent is paclitaxel, doxorubicin, mitomycin-C, bleomycin, cisplatin, tamoxifen, vincristine, vinblastine, 6- mercaptopurine, or 5-fluorouracil.

16. The method of claim 13, wherein the hormanal agent is an androgen antagonist.

17. The method of claim 13, wherein the alpha-emitting radioconjugate is administered prior to, concurrently with or after administration of one or both of the chemotherapeutic agent or the therapeutic adiation.

18. The method of claim 11, wherein the cell-specific adhesion molecule is VE- cadherin.

19. The method of claim 18, wherein the VE-cadherin is in a monomeric form.

20. The method of claim 1 1, wherein the alpha-emitting radioconjugate is a radioimmunoconjugate.

21. The method of claim 20, wherein the alpha-emitting radioimmunoconjugate is a humanized or chimeric ^22SAc-E4G10.

22. The method of claim 11, wherein the cancer is prostate cancer, colon cancer, kidney cancer,a lung cancer, breast cancer, ovarian cancer, pancreatic cancer, or brain cancer.

23. A method for treating prostate cancer in a subject, comprising: administering a pharmacologically effective amount of an 225-Ac- immunoconjugate specific for VE cadherin to the subject, wherein the ^22SAc-immunoconjugate inhibits neovascularization of a prostate tumor thereby treating the prostate cancer.

24. The method of claim 23, further comprising: administering the ^22SAc-immunoconjugate one or more times.

25. The method of claim 23, further comprising: administering one or both of a pharmacologically effective amount of a therapeutic agent or therapeutic radiation one or more times to the subject.

26. The method of claim 25, wherein the therapeutic agent is a chemotherapeutic agent, a monoclonal antibody or fragment thereof, or a hormonal agent.

27. The method of claim 26, wherein the chemotherapeutic agent is paclitaxel, doxorubicin, mitomycin-C, bleomycin, cisplatin, tamoxifen, vincristine, vinblastine, 6- mercaptopurine, or 5-fluorouracil.

28. The method of claim 26, wherein the hormanal agent is an androgen antagonist.

29. The method of claim 23, wherein the VE-cadherin is in a monomelic form.

30. The method of claim 23, wherein the ^22SAc- immunoconjugate is a humanized or chimeric ^22SAc-E4G10.

31. The method of claim 23, wherein the ^22SAc-imunoconjugate is administered prior to, concurrently with or after one or both of the chemotherapeutic agent or therapeutic radiation.

32. A method for treating prostate cancer in a subject, comprising: sequentially administering pharmacologically effective amounts of an ²²⁵Ac- immunoconjugate specific for VE cadherin and a chemotherapeutic agent to the subject, thereby treating the prostate cancer.

33. The method of claim 32, further comprising: administering one or both of the ^2Z5Ac-immunoconjugate and chemotherapeutic agent one or more times.

34. The method of claim 32, wherein the chemotherapeutic agent is paclitaxel, doxorubicin, mitomycin-C, bleomycin, cisplatin, tamoxifen, vincristine, vinblastine, 6- mercaptopurine, or 5-fluorouracil.

35. The method of claim 32, wherein the VE-cadherin is in a monomelic form.

36. The method of claim 32, wherein the ²²⁵Ac- immunoconjugate is a humanized or chimeric ^22SAc-E4G10.

37. The method of claim 32, wherein the ²²⁵Ac- immunoconjugate is administered prior to, concurrently with or after the chemotherapeutic agent.