AU2006235421A1 - Conjugated anti-PSMA antibodies - Google Patents

Description

WO 2006/110745 PCT/US2006/013473 CONJUGATED ANTI-PSMA ANTIBODIES RELATED APPLICATIONS This application is related to U.S. Provisional Application 60/669,347, filed April 8, 2005, which is herein,incorporated by reference in its entirety. 5 FIELD OF THE INVENTION The present invention relates to the development of methods and tools effective for treating, preventing, and diagnosing cancer. Specifically, the present invention is directed to methods of treating, preventing, and diagnosing cancer comprising using antibodies which immunospecifically bind to prostate specific membrane antigen and are conjugated to a 10 radioisotope via a MeO-DOTA linkage. BACKGROUND OF THE INVENTION Prostate cancer is among the most significant medical problems in the United States, as the disease is now the most common malignancy diagnosed in American males. The American Cancer Society estimates that for the year 2000, 180,400 new cases of prostate cancer were 15 diagnosed with 31,900 deaths from the disease. Five year survival rates for patients with prostate cancer range from 88% for those with localized disease to 29% for those with metastatic disease. The rapid increase in the number of cases appears to result in part from an increase in disease awareness as well as the widespread use of clinical markers such as the secreted proteins prostate specific antigen (PSA) and prostatic acid phosphatase (PAP) (Chiaroda (1991) Cancer Res. 51, 20 2498-2505). The prostate gland is a site of significant pathology affected by conditions such as benign growth (BPH), neoplasia (prostatic cancer) and infection (prostatitis). Prostate cancer represents the second leading cause of death from cancer in man (Chiaroda (1991) Cancer Res. 51, 2498 2505). However the prostate is the leading site for cancer development in men. The difference 25 between these two facts relates to prostatic cancer occurring with increasing frequency as men age, especially in the ages beyond sixty at a time when death from other factors often intervenes. Also, the spectrum of biologic aggressiveness of prostatic cancer is great, so that in some men following detection the tumor remains a latent histologic tumor and does not become clinically significant, whereas in the other it progresses rapidly, metastasizes and kills the patient in a 30 relatively short two to five year period (Chiaroda (1991) Cancer Res. 51, 2498-2505; Warner et al. (1991) Urologic Clinics of North America 18, 25-33).

WO 2006/110745 PCT/US2006/013473 In prostate cancer cells, two specific proteins that are made in very high concentrations are prostatic acid phosphatase (PAP) and prostate specific antigen (PSA) (Henttu et al. (1989) Bioch. Biophys. Res. Comm. 160, 903-908; Nguyen et al. (1990) Clin. Chem. 35, 1450-1455; Yong et al. (1991) Cancer Res. 51, 3748-3752). These proteins have been characterized and have 5 been used to follow response to therapy. With the development of cancer, the normal architecture of the gland becomes altered, including loss of the normal duct structure for the removal of secretions and thus the secretions reach the serum. Measurement of serum PSA is suggested as a potential screening method for prostatic cancer. Indeed, the relative amount of PSA and/or PAP in the cancer changes as compared to normal or benign tissue. 10 PAP was one of the earliest serum markers for detecting metastatic spread (Nguyen et al. (1990) Clin. Chem. 35, 1450-1455). PAP hydrolyses tyrosine phosphate and has a broad substrate specificity. Tyrosine phosphorylation is often increased with oncogenic transformation. It has been hypothesized that during neoplastic transformation there is less phosphatase activity available to inactivate proteins that are activated by phosphorylation on tyrosine residues. In 15 some instances, insertion of phosphatases that have tyrosine phosphatase activity has reversed the malignant phenotype. PSA is a protease and it is not readily appreciated how loss of its activity correlates with cancer development (Henttu et al. (1989) Bioch. Biophys. Res. Comm. 160, 903-908; Yong et al. (1991) Cancer Res. 51, 3748-3752). The proteolytic activity of PSA is inhibited by zinc. Zinc 20 concentrations are high in the normal prostate and reduced in prostatic cancer. Possibly the loss of zinc allows for increased proteolytic activity by PSA. As proteases are involved in metastasis and some proteases stimulate mitotic activity, the potentially increased activity of PSA could be hypothesized to play a role in the tumors metastases and spread (Liotta (1986) Cancer Res. 46, 1 7). Both PSA and PAP are found in prostatic secretions. Both appear to be dependent on the 25 presence of androgens for their production and are substantially reduced following androgen deprivation. Prostate-specific membrane antigen (PSMA), which appears to be localized to the prostatic membrane, has also been identified as a marker for prostate cancer. PSMA is expressed in virtually all prostate cancers (Bostwick et al. (1998) Cancer, 82, 2256-2261). Although recent 30 studies have shown that it is also expressed by the small intestine epithelial (brush-border) cells, proximal renal tubule cells and salivary glands, the level of expression in these normal tissue is 100 to 1,000 fold less that in prostate tissue, and these PSMA expressing normal cells are not typically exposed to circulating antibodies due to their brush-border/luminal location (Nanus et al. (2003) Journal of Urology, 170, S84-S89). In addition, in contrast to other prostate related 35 antigens such as PSA, PMSA, which is a type II integral membrane cell surface protein, it is not 2 WO 2006/110745 PCT/US2006/013473 secreted and, therefore, is an excellent target for monoclonal antibody therapy (Nanus et al. (2003) Journal of Urology, 170, S84-S89). This antigen was identified as the result of generating monoclonal antibodies to a prostatic cancer cell, LNCaP (Horoszewicz et al. (1993) Cancer Res., 53, 227-230). LNCaP is a 5 cell line established from the lymph node of a hormone refractory, heavily pretreated patient (Horoszewicz et al. (1983) Cancer Res. 43, 1809-1818). This cell line was found to have an aneuploid human male karyotype. It maintained prostatic differentiation functionality in that it produced both PSA and PAP. It possessed an androgen receptor of high affinity and specificity. Mice were immunized with LNCaP cells and hybridomas were derived from sensitized animals. 10 A monoclonal antibody was derived and was designated 7E1 1-C5 (Horoszewicz et al. (1993) Cancer Res. 53, 227-230). The antibody staining was consistent with a membrane location and isolated fractions of LNCaP cell membranes exhibited a strongly positive reaction with immunoblotting and ELISA techniques. This monoclonal antibody was also used for detection of immunoreactive material in 15 serum of prostatic cancer patients (Horoszewicz et al. (1993) Cancer Res. 53, 227-230). The immunoreactivity was detectable in nearly 60% of patients with stage D-2 disease and in a slightly lower percentage of patients with earlier stage disease, but the numbers of patients in the latter group were small. Patients with benign prostatic hyperplasia (BPH) were negative. Patients with no apparent disease were negative, but 50 to 60% of patients in remission, yet with active 20 stable disease or with progression, demonstrated positive serum reactivity. Patients with non prostatic tumors did not show immunoreactivity with 7E 11-C5. The 7E1 1-C5 monoclonal antibody is now used as a molecular imaging agent and is the first and currently the only commercial product targeting PSMA. Prostascint@ consists of 7E1 1 C5 linked to the radioisotope Indium-111. Due to the selective expression of PSMA by prostate 25 cancer cells, Prostascint@ can image the extent and spread of prostate cancer using a common gamma camera. U.S. Patent 5,162,504 discloses and claims the monoclonal antibody 7E11-C5 and the hyrbirdoma cell line that produces it. U.S. Patents 4,671,958; 4,741,900 and 4,867,973 disclose and claim antibody conjugates, methods for preparing such conjugates, methods for using such conjugates for in vivo imaging, testing and therapeutic treatment, and methods for delivering 30 radioisotopes by linking them to such antibodies. There are at least three other anti-PSMA antibodies in the art that have been conjugated to radioisotopes and at least one tested for the treatment of prostrate cancer. J591, J415, and J591 are monoclonal antibody binds with high affinity to an extracellular epitope of PSMA and localizes specifically in PSMA (Smith-Jones et al. (2000) Cancer Res. 60, 5237-5243). These antibodies 3 WO 2006/110745 PCT/US2006/013473 have been labeled with 13I and " In via a DOTA linkage. The average DOTA to antibody ratio for these antibodies was 5:1, with little apparent loss of immunoreactivity. Conjugation average of eight DOTA molecules to J591 resulted in a 20% reduction in immunoreactivity (Smith-Jones et al. (2000) Cancer Res. 60, 5237-5243). Thus, the art indicates that there is an upper limit of 5 DOTA to antibody ratio without the antibody losing immunoreactivity. SUMMARY OF THE INVENTION The invention encompasses novel compositions which comprise an antibody which immunospecifically binds to prostate specific membrane antigen (PSMA), wherein said antibody is conjugated to a radioisotope via a MeO-DOTA linkage (henceforth known as "conjugated 10 antibody" or "conjugated antibodies"). In a further embodiment, the ratio of MeO-DOTA to antibody is about 9:1 or greater. The invention also encompasses methods for preventing, treating, or managing cancer in a subject which comprises administering an antibody which immunospecifically binds to prostate specific membrane antigen (PSMA), wherein said antibody is conjugated to a radioisotope via a 15 MeO-DOTA. In a further embodiment, the MeO-DOTA to antibody ratio is about 9:1 or greater. In an even further embodiment, the MeO-DOTA to antibody ratio is about 9:1, or greater, with little, if any, loss of immunoreactivity. The conjugated antibodies of the invention can be administered in combination with one or more other cancer therapies. In particular, the present invention provides methods of 20 preventing, treating, or managing cancer in a subject comprising administering to said subject a therapeutically or prophylactically effective amount of one or more conjugated antibodies of the invention in combination with the administration of a therapeutically or prophylactically effective amount of one or more chemotherapies, hormonal therapies, biological therapies/immunotherapies and/or radiation therapies, other than the administration of the 25 conjugated antibody of the invention, and/or in combination with surgery. The conjugated antibody of the invention can be administered concurrently to a subject in separate pharmaceutical compositions or in the same composition. In addition, it is also contemplated that the conjugated antibody of the invention can be administered prior to the administration of other therapies or after the administration of other therapies. The therapeutic agents may be administered to a 30 subject by the same or different routes of administration. The invention also includes methods and compositions for the treatment of cancer in a mammal, including a human, comprising administering to said mammal an amount of a cytotoxic agent, or a pharmaceutical composition comprising an amount of the cytotoxic agent, that is 4 WO 2006/110745 PCT/US2006/013473 effective in enhancing the binding of a monoclonal antibody to an epitope on the cytoplasmic domain of PSMA. In one embodiment, the cytotoxic agent will induce apoptosis of the malignant cell, and/or increase permeability, and/or otherwise disrupt the cell membrane. In another embodiment, the cytotoxic agent is administered to a mammal prior to or simultaneously with the 5 conjugated antibodies of the invention. In this aspect of the invention, the cytotoxic agent may disrupt the cancer cell(s), thereby expressing the cytoplasmic domain on the PSMA antigen. The invention further provides diagnostic methods to evaluate or diagnose an individual with a malignant cell expressing PSMA using the conjugated antibodies of the invention. In particular embodiments, the diagnostic methods of the invention provide methods of imaging and 10 localizing malignant cells expressing PSMA, methods of diagnosis and prognosis using tissues and fluids distal to the primary tumor site (as well as methods using tissues and fluids of the primary tumor and tissues and/or using tissues and fluids surrounding the primary tumor), for example, whole blood, sputum, urine, serum, fine needle aspirates (i.e., biopsies). In other embodiments, the diagnostic methods of the invention provide methods of imaging and localizing 15 metastases and methods of diagnosis and prognosis in vivo. In such embodiments, primary tumors are detected using the conjugated antibody of the invention. The antibodies of the invention may also be used for immunohistochemical analyses of frozen or fixed cells or tissue assays. The invention further provides a novel, efficient method to conjugate antibodies which 20 immunospecifically bind to prostate specific membrane antigen (PSMA) with MeO-DOTA. In addition, the invention further provides an efficient method to complex the conjugated antibody with a radioisotope. The invention also provides pharmaceutical compositions comprising one or more monoclonal antibodies of the invention either alone or in combination with one or more other 25 agents useful for cancer therapy. In another embodiment, kits comprising-the pharmaceutical compositions or diagnostic reagents of the invention are provided. BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a Coomassie-stained one-dimensional 4 to 20% SDS-PAGE gel of eight lots of 7E1 1 30 C5 conjugates (conjugated to a radioisotope via a MeO-DOTA linkage) prepared under conditions as set forth in Table 1. Arrows indicate heavy chain (50 kDA) and light chain (25 kDA). 5 WO 2006/110745 PCT/US2006/013473 Figure 2 (A) is size exclusion chromatogram of 7E11-C5 antibody and (B) a representative chromatogram of its MeO-DOTA conjugate (lot 200402495-5/8). Relative peak areas of unmodified protein and conjugates are summarized in Table 3. Figure 3A-D are size exclusion chromatograms of different complexations of MeO-DOTA 5 Cyt351, 200402495-29, under the indicated condition. Relative peak areas of unmodified protein and conjugates are summarized in Table 5. Figure 4 is a size exclusion chromatogram of a high specific activity conjugate preparation at 30 minutes. Relative peak areas of unmodified protein and conjugates are summarized in Table 6. DETAILED DESCRIPTION 10 Radiation is an effective cancer treatment, but it is difficult to direct and can be devastating to nontargeted parts of the body. Indeed, treatments are often limited by its non selectivity, resulting in toxicity on the normal tissues. Monoclonal antibodies, or fragments thereof, on the other hand, are adept at selectively targeting diseased cells. The ability of antibodies to exploit antigenic differences between normal and malignant tissues and to exact a 15 variety of antitumor responses offers significant advantages to conventional forms of therapy. However, antibodies alone often have inadequate therapeutic effectiveness. Conjugating a radioisotope to a monoclonal antibody, or fragments thereof, can solve both problems. Monoclonal antibodies are highly specific and can be used as vehicles to deliver substances to specific target sites. Thus, the conjugation of a monoclonal antibody to a 20 radioisotope is an effective way to target specific cells, thus reducing the side effects of radiotherapy. The invention described relates to compositions comprising an antibody, or fragments thereof, which immunospecifically binds to prostate specific membrane antigen (PSMA), wherein said antibody, or fragment(s) thereof, is conjugated to a radioisotope via a MeO-DOTA 25 linkage. In one embodiment of the invention, the anti-PSMA monoclonal antibody is 7E1 1-C5. In another embodiment the conjugated 7E1 1-C5 antibody is complexed with 177 Lu. In a preferred embodiment, the conjugated 7E 11-C5 is complexed with 1 77 Lu at a ratio of isotope to antibody of about 9:1 or greater. The invention also discloses methods of using, formulating and making the antibodies of the invention. 30 Conjugated antibodies The invention comprises antibodies, or fragments thereof, which immunospecifically bind to prostate specific membrane antigen (PSMA), wherein said antibody is conjugated to a 6 WO 2006/110745 PCT/US2006/013473 radioisotope via a MeO-DOTA linkage. MeO-DOTA, oc-(5-isothiocyanato-2-methoxyphenyl) 1,4,7,10-teraazacyclododeczane- 1,4,7,1 0-tretraacetic (Dow Chemical Company) is a bifunctional chelant. A bifunctional chelant is a molecule that has, in addition to chelating functionality, the ability to be conjugated (linked) to a biotargeting molecule (e.g. monoclonal antibody). MeO 5 DOTA provides metal complexes with high stability, thereby reducing the incidence of background during imaging procedures or damage to non-targeted tissues in radioimmunotherapy. In one embodiment of the invention, the antibody is 7E 11-C5. In another embodiment the MeO DOTA linked antibody is complexed to a radioisotope which is selected from the group consisting of 3 H, 1 4 C, 1 8 F, 1 9 F, 31 P, 32 P, 35 S, 1311, 1251, 1231 6 4 Cu, 1 87 Re, 1"In, 90 Y, 99 Tc, 1"Lu. In another 10 embodiment the antibody comprises 7E1 1-C5 conjugated to 1"Lu via MeO-DOTA. In a preferred embodiment the MeO-DOTA to antibody ratio is about 9:1 or greater. An antibody is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains (an IgM antibody consists of five of the basic heterotetramer unit along with an additional polypeptide called J chain, and therefore contain ten antigen binding 15 sites, while secreted IgA antibodies can polymerize to form polyvalent assemblages comprising two to five of the basic four chain units along with J chain). The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains and the methods of the current invention include the use of antibodies with either a kappa or lambda L chain. Depending on the amino acid 20 sequence of the constant domain of their heavy chains (CH), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated alpha, delta, epsilon, gamma and mu, respectively. The gamma and alpha classes are further divided into subclasses on the basis of relatively minor differences in CH sequence and function, e.g., humans express the following subclasses: IgGi, 25 IgG2, IgG3, IgG4, IgAl and IgA2. The methods of the present invention include the use of antibodies, including monoclonal antibodies, from any of the above classes and/or subclasses. As used herein, the term "variable" refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies. The variable domain mediates antigen binding and defines specificity of a particular antibody for its particular antigen. However, the 30 variability is not evenly distributed across the 11 0-amino acid span of the variable domains. Instead, the variable regions consist of relatively invariant stretches called framework regions (FR) of about fifteen to thirty amino acids separated by shorter regions of extreme variability called "hypervariable regions" that are each about nine to twelve amino acids long. The variable domains of native heavy and light chains each comprise four framework regions, largely adopting 35 a beta-sheet configuration, connected by three hypervariable regions, which form loops 7 WO 2006/110745 PCT/US2006/013473 connecting, and in some cases forming part of, the beta-sheet structure. The hypervariable regions in each chain are held together in close proximity by the framework region and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Public 5 Health Service, National Institutes of Health). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC). The term "hypervariable region" when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region generally 10 comprises amino acid residues from a "complementarity determining region" or "CDR" which contributes to the specificity of the antibody. The term "antibodies or fragments thereof' as used herein refers to antibodies or fragments thereof that specifically bind to a PSMA polypeptide or a fragment of a PSMA polypeptide and do not specifically bind to other non-PSMA polypeptides. Preferably, antibodies 15 or fragments that immunospecifically bind to a PSMA polypeptide or fragment thereof do not non-specifically cross-react with other antigens (e.g., binding cannot be competed away with a non-PSMA protein, e.g., BSA in an appropriate immunoassay). Antibodies or fragments that immunospecifically bind to an PSMA polypeptide can be identified, for example, by immunoassays or other techniques known to those of skill in the art. Antibodies of the invention 20 include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, intrabodies, diabodies, multispecific antibodies (including bi-specific: antibodies), human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scFv) (including bi-specific scfvs), single chain antibodies, Fab' fragments, F(ab') 2 fragments, disulfide-linked Fvs (sdFv), and anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments 25 of any of the above. In particular, antibodies of the present invention include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds to an PSMA antigen (e.g., one or more complementarity determining regions (CDRs) of an anti-PSMA antibody). The term "monoclonal antibody" as used herein refers to an antibody obtained from a 30 population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts and includes antibody fragments as defined herein. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different 35 determinants (epitopes), each monoclonal antibody is directed against a single determinant on the 8 WO 2006/110745 PCT/US2006/013473 antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies useful in the present invention may be prepared by the hybridoma 5 methodology first described by Kohler et al. (1975) Nature 256, 495 or may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see U.S. Patent 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al. (1991) Nature 352:624-628 and Marks et al. (1991) J. Mol. Biol. 222, 581-597, for example. 10 As used herein, an "intact" antibody is one which comprises an antigen-binding site as well as a CL and at least heavy chain constant domains, CHI and Cm and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variant thereof. Preferably, the intact antibody has one or more effector functions. 15 An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen binding CDR or variable region of the intact antibody. Examples of antibody fragments include Fab, Fv, Fab' and F(ab') 2 fragments; diabodies; linear antibodies (see U.S. Patent 5,641,870 and Zapata et al. (1995) Protein Eng. 8, 1057-1062); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. 20 Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, a designation reflecting the ability to crystallize readily. The Fab fragment consists of an entire L chain along with the variable region domain of the H chain (VH), and the first constant domain of one heavy chain (CHI). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin 25 treatment of an antibody yields a single large F(ab') 2 fragment which roughly corresponds to two disulfide linked Fab fragments having divalent antigen-binding activity and is still capable of cross-linking antigen. Fab' fragments differ from Fab fragments by having additional few residues at the carboxy terminus of the CHI domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) 30 of the constant domains bear a free thiol group. F(ab') 2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known. 9 WO 2006/110745 PCT/US2006/013473 The Fe fragment comprises the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, which region is also the part recognized by Fc receptors (FcR) found on certain types of cells. As used herein, "Fv" is the minimum antibody fragment which contains a complete 5 antigen-recognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (three loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three 10 CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site. As used herein, "Single-chain Fv" also abbreviated as "sFv" or "scFv" are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and 15 VL domains which enables the sFv to form the desired structure for antigen binding (see Rosenburg et al. (1994) The Pharmacology of Monoclonal Antibodies, Springer-Verlag, pp. 269 315). As used herein, the term "diabodies" refers to small antibody fragments prepared by constructing sFv fragm-ents (see preceding paragraph) with short linkers (about 5 to about 10 20 residues) between the VH and VL domains such that inter-chain but not intra-chain pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., fragment having two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossover" sFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Diabodies are described more fully in, for example, WO 93/11161 and Hollinger et al. (1993) Proc. Natl. Acad. 25 Sci. USA 90, 6444-6448. An "isolated antibody" is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous 30 components. In preferred embodiments, the antibody will be purified to greater than 95% by weight of antibody, and most preferably more than 99% by weight. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step. 10 WO 2006/110745 PCT/US2006/013473 In one embodiment of the invention, the conjugated antibody binds to an epitope on the cytoplasmic domain of a protein specific to cancer cells (i.e., a cancer cell marker). In another embodiment, the conjugated antibody includes, but is not limited to, an antibody which binds to an epitope on the cytoplasmic domain of PSMA, including but not limited to, the 7E1 1-C5 5 monoclonal antibody as described in U.S. Patent 5,162,504 which is herein incorporated by reference in its entirety. The hybridoma cell line which produces the 7E11-C5 monoclonal antibody has been deposited with the American Type Culture Collection under Deposit No. HB10494. The conjugated antibodies used in the methods of the invention include "chimeric" 10 antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they 15 exhibit the desired biological activity (see U.S. Patent 4,816,567 and Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81, 6851-6855). Chimeric antibodies of interest herein include, but are not limited to "humanized" antibodies comprising variable domain antigen-binding sequences derived from a non-human mammal (e.g., murine) and human constant region sequences. Antibodies of the invention may also comprise a fully human antibody sequence. In another embodiment, 20 antibodies of the invention may be a fully human antibody. Diagnostic methods Antibodies, or fragments thereof, of the invention can be used as diagnostic or detectable agents. In a preferred embodiment, the antibody, or fragments thereof, immunospecifically bind to prostate specific membrane antigen (PSMA), wherein said antibody is conjugated to a 25 radioisotope via a MeO-DOTA linkage. Antibodies of the invention can be useful for monitoring or prognosing the development or progression of a cancer as part of a clinical testing procedure, such as determining the efficacy of a particular therapy. Additionally, such antibodies can be useful for monitoring or prognosing the development or progression of cancerous conditions. In another embodiment, following initial administration of the conjugated antibody of the 30 invention, the cancer cells can be imaged and the relative amount of cancerous cells determined by any available means. The invention includes diagnostic methods to detect cancer and/or assess the effect therapeutic agents on cancer cells in an organ or body area of a patient. The present methods include administration of a composition comprising a detectable amount of an anti PSMA antibody conjugated to a radioisotope via MeO-DOTA to a patient before and after 11 WO 2006/110745 PCT/US2006/013473 therapy. In a further embodiment, the MeO-DOTA to antibody ratio is about 9:1 or greater. In an even further embodiment, the MeO-DOTA to antibody ratio is about 9:1, or greater, with little, if any, loss of immunoreactivity. Subsequent to administration of the therapeutic agent, an additional amount of detectable monoclonal antibody can be administered to determine the 5 relative amount of cancer cells remaining following treatment. Comparison of the before and after treatment images can be used as a means to assess the efficacy of the treatment wherein a decrease in the number of cancer cells imaged following treatment is indicative of an efficacious treatment regimen. As used herein, the term "detectable amount" refers to the amount of labeled conjugated 10 antibody which binds to PSMA administered to a patient that is sufficient to enable detection of binding of the labeled monoclonal antibody to one or more malignant cancer cells in a tumor. As used herein, the term "imaging effective amount" refers to the amount of the labeled antibody administered to a patient that is sufficient to enable imaging of binding of the antibody to one or more malignant cancer cells in a tumor. 15 The methods of the invention comprise conjugated antibodies of the invention which, in conjunction with non-invasive neuroimaging techniques such as magnetic resonance spectroscopy (MRS) or imaging (MRI), or gamma imaging such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT), are used to identify and quantify abnormal cells in vivo including malignant cells in tumors. The term "in vivo imaging" refers to 20 any method which permits the detection of labeled monoclonal antibody as described above. For gamma imaging, the radiation emitted from the tumor or area being examined is measured and expressed either as total binding, or as a ratio in which total binding in one tissue is normalized to (for example, divided by) the total binding in another tissue or the entire body of the same subject during the same in vivo imaging procedure. Total binding in vivo is defined as the entire signal 25 detected in a tumor or tissue by an in vivo imaging technique without the need for correction by a second injection of an identical quantity of labeled compound along with a large excess of unlabeled, but otherwise chemically identical compound. As used herein, the terms "subject" or "patient" refers to a mammal, preferably a human, and most preferably a human suspected of having abnormal cells, including malignant cells in a tumor. 30 For purposes of in vivo imaging, the type of detection instrument available is a major factor in selecting a given label. For instance, radioactive isotopes are particularly suitable for in vivo imaging in the methods of the present invention. The type of instrument used will guide the selection of the radioisotope. For instance, the radioisotope chosen must have a type of decay detectable by a given type of instrument. Another consideration relates to the half-life of the 35 radioisotope. The half-life should be long enough so that it is still detectable at the time of 12 WO 2006/110745 PCT/US2006/013473 maximum uptake by the target, but short enough so that the host does not sustain deleterious radiation. The isotopically-labeled monoclonal antibody can be detected using gamma imaging where emitted gamma irradiation of the appropriate wavelength is detected. Methods of gamma imaging include, but are not limited to, positron emission tomography (PET) imaging or for single 5 photon emission computerized tomography (SPECT). Preferably, for SPECT detection, the chosen radiolabel will lack a particulate emission, but will produce a large number of photons. For PET detection, the radiolabel will be a positron-emitting radioisotope which will be detected by the PET camera. In the present invention, conjugated antibodies are useful for in vivo detection and 10 imaging of tumors. These compounds are to be used in conjunction with non-invasive neuroimaging techniques such as magnetic resonance spectroscopy (MRS) or imaging (MRI), positron emission tomography (PET), and single-photon emission computed tomography (SPECT). In accordance with this invention, the conjugated antibody may be labeled (complexed) with any acceptable radioisotope. For example, including, but are not limited to, 3 H, 15 14C, 18F, 1F, 3P, 32P, "s, 1I, 15I, 131, 64Cu, 1 87 Re, "'In, 90 Y, 99 Tc, 77 Lu using techniques described below or known in the art. The diagnostic methods of the present invention may use isotopes detectable by nuclear magnetic resonance spectroscopy for purposes of in vivo imaging and spectroscopy. Elements particularly useful in magnetic resonance spectroscopy include, but are not limited to, ' 9 F and 1 3 C. 20 Suitable radioisotopes for purposes of this invention include beta-emitters, gamma-emitters, positron-emitters and x-ray emitters. These radioisotopes include, but are not limited to, 1 "In, 1311, 123, 18 F, 11 C, 75 Br and 7 6 Br. Suitable stable isotopes for use in Magnetic Resonance Imaging (MRI) or Spectroscopy (MRS), according to this invention include, but are not limited to, ' 9 F and 1 3 C. Suitable 25 radioisotopes for in vitro identification and quantification of abnormal cells including tumor cells, in a tissue biopsy or post-mortem tissue include 121, "C and 3 H. Examples of these radiolabels include, but not limited to, 64 Cu or ' 8 F for use in PET in vivo imaging, 123I or 1311 for use in SPECT imaging in vivo, 1F for MRS and MRI and 3 H or 1 "C for in vitro methods. However, any conventional method for visualizing diagnostic probes can be utilized in accordance with this 30 invention. Generally, the dosage of the isotopically-labeled monoclonal antibody will vary depending on considerations such as age, condition, sex, and extent of disease in the patient, contraindications, if any, concomitant therapies and other variables, to be adjusted by the skilled artisan. Dosage can vary from 0.00 1 mg/kg to 1000 mg/kg, preferably 0.1 mg/kg to 100 mg/kg. 13 WO 2006/110745 PCT/US2006/013473 Administration to the patient may be local or systemic and accomplished intravenous, intra arterial, intra-thecal (via the spinal fluid), intra-cranial or the like. Administration may also be intra-dermal or intra-cavitary, depending upon the body site under examination. After a sufficient time has elapsed for the labeled monoclonal antibody to bind with the 5 abnormal cells, for example thirty minutes to forty-eight hours, the area of the subject under investigation is examined by routine imaging techniques such as MRS/MRI, SPECT, planar scintillation imaging, PET, and emerging imaging techniques, as well. The exact protocol will necessarily vary depending upon factors specific to the patient, as noted above, and depending upon the body site under examination, method of administration and type of label used; the 10 determination of specific procedures would be routine to the skilled artisan. For tumor imaging, preferably, the amount (total or specific binding) of the bound isotopically-labeled monoclonal antibody is measured and compared (as a ratio) with the amount of isotopically-labeled monoclonal antibody bound to the tumor following chemotherapeutic treatment. In another embodiment, the conjugated antibodies of the invention can be used for 15 diagnosis and prognosis by using tissues and fluids distal to the primary tumor site (as well as methods using tissues and fluids of the primary tumor and/or tissue and fluids surrounding the tumor). Antibodies of the invention can be used to assay PMSA levels in a biological sample using classical immunohistological methods as known to those of skill in the art (e.g., see Jalkanen et al. (1985) J. Cell. Biol. 101, 976-985; and Jalkanen et al. (1987) J. Cell. Biol. 105, 20 3087-3096). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (1251, 121), carbon ( 4 C), sulfur ( 3 sS), tritium (3H), indium (1 21 In), and technetium ("Tc). 25 In still further embodiments, the present invention provides diagnostic kits, including both immunodetection and imaging kits, for use with the immunodetection and imaging methods described above. Methods of Treatment The invention encompasses a method for treating cancer which comprises a malignant 30 cell expressing PSMA in a patient in need thereof comprising administering a conjugated antibody which specifically binds to PSMA expressed by a malignant cell. In one embodiment, the conjugated antibody binds to a cytoplasmic epitope on the PSMA. In another embodiment, the conjugated antibody includes, but is not limited to, the 7E11-C5 monoclonal antibody as 14 WO 2006/110745 PCT/US2006/013473 described in U.S. Patent 5,162,504 herein incorporated by reference in its entirety. The hybridoma cell line which produces the 7E 11 -C5 monoclonal antibody has been deposited with the American Type Culture Collection under Deposit No. HB 10494. In a preferred embodiment, the antibody is conjugated with a radioisotope via a MeO-DOTA linkage. In an even further 5 embodiment, the MeO-DOTA to antibody ratio is about 9:1 or greater. In a preferred embodiment, the MeO-DOTA to antibody ratio is about 9:1, or greater, with little, if any, loss of immunoreactivity. The amount of the conjugated antibody composition of the invention which will be effective in the treatment, prevention or management of cancer can be determined by standard 10 research techniques. For example, the dosage of the composition which will be effective in the treatment, prevention or management of cancer can be determined by administering the composition to an animal model such as, e.g., the animal models disclosed herein or known to those skilled in the art. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. 15 Selection of the preferred effective dose can be determined (e.g., via clinical trials) by a skilled artisan based upon the consideration of several factors which will be known to one of ordinary skill in the art. Such factors include the disease to be treated or prevented, the symptoms involved, the patient's body mass, the patient's immune status and other factors known by the skilled artisan to reflect the accuracy of administered pharmaceutical compositions. 20 The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the cancer, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. For antibodies, the dosage administered to a patient is typically 0.1 mg/kg to 100 mg/kg 25 of the patient's body weight. Preferably, the dosage administered to a patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more preferably 1 mg/kg to 10 mg/kg of the patient's body weight. Generally, human and humanized antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human or humanized antibodies and less frequent 30 administration is often possible. In specific embodiments, patients with prostate cancer are administered an effective amount of one or more conjugated antibodies of the invention. In another embodiment, the antibodies of the invention can be administered in combination with an effective amount of one or 15 WO 2006/110745 PCT/US2006/013473 more other agents useful for prostate cancer therapy including but not limited to: external-beam radiation therapy, interstitial implantation of radioisotopes (i.e., 1251, palladium, iridium), leuprolide or other LHRH agonists, non-steroidal antiandrogens (flutamide, nilutamide, bicalutamide), steroidal antiandrogens (cyproterone acetate), the combination of leuprolide and 5 flutamide, estrogens such as DES, chlorotrianisene, ethinyl estradiol, conjugated estrogens U.S.P., DES-diphosphate, radioisotopes, such as strontium-89, the combination of external-beam radiation therapy and strontium-89, second-line hormonal therapies such as aminoglutethimide, hydrocortisone, flutamide withdrawal, progesterone, and ketoconazole, low-dose prednisone, or other chemotherapy regimens reported to produce subjective improvement in symptoms and 10 reduction in PSA, PAP and/or PMSA levels including docetaxel, paclitaxel, estramustine/docetaxel, estramustine/etoposide, estramustine/vinblastine, and estramustine/paclitaxel. Given the invention, certain preferred embodiments will encompass the administration of lower dosages in combination treatment regimens than dosages recommended for the 15 administration of single agents. The invention provides for any method of administrating lower doses of known prophylactic or therapeutic agents than previously thought to be effective for the prevention, treatment, management or amelioration of cancer. Preferably, lower doses of known anti-cancer therapies are administered in combination with lower doses of conjugated monoclonal antibodies 20 of the invention. The invention also includes methods and compositions for the treatment of cancer in a mammal, including a human, comprising administering to said mammal an amount of a cytotoxic agent, or a pharmaceutical composition comprising an amount of the cytotoxic agent, that is effective in enhancing the binding of an antibody to an epitope of PSMA, for instance a 25 cytoplasmic epitope. Said cytotoxic agent can be administered prior to or simultaneously with said antibody. In a preferred embodiment, said antibody binds to an epitope on the cytoplasmic domain of PSMA. In a further embodiment, said antibody is conjugated with MeO-DOTA. In a specific embodiment said antibody is 7E1 1-C5 and is conjugated to MeO-DOTA. In another embodiment, the cytotoxic agent will induce apoptosis of the malignant cell, and/or increase 30 permeability, and/or otherwise disrupt the cell membrane. In a further aspect of the invention, the cytotoxic agent is administered to a mammal prior to or simultaneously with the conjugated antibodies of the invention. In this aspect of the invention, the cytotoxic agent disrupts the cancer cell(s), thereby expressing the cytoplasmic domain on the PSMA antigen. Exposure of the cytoplasmic domain allows for targeting of the remaining cancer cells with the conjugated 16 WO 2006/110745 PCT/US2006/013473 antibody of the invention. (See, co-pending U.S. application 60/643,589 filed on January 14, 2005, which is herein incorporated by reference). Examples of chemotherapeutic agents, include but are not limited to, BCNU, cisplatin, gemcitabine, hydroxyurea, paclitaxel, temozomide, topotecan, fluorouracil, vincristine, 5 vinblastine, procarbazine, dacarbazine, altretamine, cisplatin, methotrexate, mercaptopurine, thioguanine, fludarabine phosphate, cladribine, pentostatin, fluorouracil, cytarabine, azacitidine, vinblastine, vincristine, etoposide, teniposide, irinotecan, docetaxel, doxorubicin, daunorubicin, dactinomycin, idarubicin, plicamycin, adriamycin, mitomycin, bleomycin, tamoxifen, flutamide, leuprolide, goserelin, aminoglutethimide, anastrozole, amsacrine, asparaginase, mitoxantrone, 10 mitotane and amifostine. In another embodiment, the therapeutic method comprises administration of the conjugated antibody of the invention in combination with radiation for the treatment of cancer. In particular, the radiation is designed to disrupt the cell membrane of the cancer cell to expose PSMA, for instance the cytoplasmic domain of PSMA. Once the cytoplasmic domain is exposed, 15 an antibody can bind to PSMA. In a preferred embodiment, said antibody binds to an epitope on the cytoplasmic domain of PSMA. In a further embodiment, said antibody is conjugated with MeO-DOTA. In a specific embodiment said antibody is 7E11-C5 and is conjugated to MeO DOTA. The methods of the invention are also designed to induce apoptosis (cell death) in cancer cells, reduce the incidence or number of metastases, and reduce tumor size. Tumor cell resistance 20 to radiotherapy agents represents a major problem in clinical oncology. Thus, in the context of the present invention, it also is contemplated that combination therapy with such an antibody could be used on radiation resistant tumors to improve the efficacy of the radiation therapy. (See, co-pending U.S. application 60/643,5 89 filed on January 14, 2005, which is herein incorporated by reference). 25 Types of cancer that can be treated the methods of the invention include solid tumors. Examples of solid tumors include, but are not limited to, endothelial cell carcinoma. Examples of endothelial cell carcinoma include, but are not limited to, renal cell carcinoma, colon carcinoma, transitional cell carcinoma, lung carcinoma, breast carcinoma and prostatic adenocarcinoma. Examples of renal cell carcinoma include, but are not limited to, clear cell carcinoma, 30 papillary carcinoma, chromophobe carcinoma, collecting duct carcinoma and unclassified carcinoma. Examples of lung carcinoma include, but are not limited to, adenocarcinoma, alveolar cell carcinoma, squamous cell carcinoma, large cell and small cell carcinoma. Examples of breast carcinoma include, but are not limited to, adenocarcinoma, ductal carcinoma in situ, lobular carcinoma in situ, invasive ductal carcinoma, medullary carcinoma and mucinous carcinoma. 17 WO 2006/110745 PCT/US2006/013473 Another example of solid tumor treatable by the methods of the invention includes endothelial cell sarcoma. In one embodiment, the sarcoma is a soft tissue sarcoma. Metatstatic tumors are also treatable. Method of Making the Antibodies of the Invention 5 U.S. patents 5,435,990 and 5,652,361 disclose methods of making and using MeO-DOTA and other bifunctional chelators. U.S. patents 5,435,990 and 5,652,361 are herein incorporated by reference in their entireties for all purposes. The modification of antibodies for the addition of MeO-DOTA, in one embodiment, may be accomplished by formation of a covalent linkage with an amino acid residue of the protein and 10 a functional group of the bifunctional chelator which is capable of binding proteins. MeO-DOTA could also be attached to the antibody via a linker molecule. Examples of linker molecules useful for conjugating MeO-DOTA to a polypeptide are disclosed in, for example, DeNardo et al. (1998) Clin Cancer Res. 4, 2483-2490; Peterson et al. (1999) Bioconjug. Chem. 10, 553-557; and Zimmerman et al. (1999) Nucl. Med. Biol. 26, 943-950, which are hereby incorporated by 15 reference in their entirety. U.S. Patents 5,652,361 and 5,756,065, disclose chelating agents that may be conjugated to antibodies. The method by which the disclosed chelating agents are conjugated are also disclosed in U.S. Patents 5,652,361 and 5,756,065. Any suitable process that results in the formation of the conjugates of this invention is within the scope of this invention. However, one 20 important aspect for an efficient reaction is the antibody to conjugate ratio. Once the conjugate and the antibody are purified and ready for conjugating both the antibody and are added to a reaction mixture. The conjugate to antibody ratio should be approximately 5:1, 10:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 150:1, 200:1, 250:1, respectively. The pH of the reaction mixture should be about 10 to about 5, preferably about 9 to about 8. More preferably the pH 25 should be about 8.5. The conjugation reaction should be carried out at a temperature range from about 20'C to about 37*C. Up to 5, 6, 7, 8, 9, 10, 11, 12 or more MeO-DOTA chelates could be bound to the antibody following the described protocol. In one embodiment, the MeO-DOTA to antibody ratio is about 9:1 or greater. In another embodiment, the MeO-DOTA to antibody ratio is about 9:1, or greater, with little loss, if any, of immunoreactivity. 30 Binding of a therapeutic radioactive isotope to the bifunctional chelator (complexing) may be accomplished either with the bifuntional chelator, MeO-DOTA, that is not complex to an antibody or, alternatively, a composition consisting of MeO-DOTA conjugated to an antibody. The complexing reaction should be accomplished at a pH of about 7 to about 4, preferably about 5 18 WO 2006/110745 PCT/US2006/013473 to about 6. More preferably the pH should be about 5.5. The complexing reaction may be accomplished in an acetate buffer (e.g. sodium acetate, potassium acetate) at a molar concentration of at least about 50 mM, 100 mM, 150 mM, 200 mM, or 250 mM or more. Preferably the molar concentration should be about 100 mM. The complexing reaction should 5 carried out at a temperature range from about 20'C to about 37 0 C. The percent of radionucleotide complexed with DOTA should be between about 80% and about 100%, preferably, between about 87% to about 95%. The radioisotope that can be complexed with MeO-DOTA include, but are not limited to, 3 H, 1 4 C, ' 8 F, 1 9 F, 31 P, 32 P, 35s, 1311, 1251, 1231, 6 4 Cu, 1 8 7 Re, 1 "In, 9Y, 99 Tc, 177Lu or any radioisotope 10 suitable for imaging. Pharmaceutical Compositions The compositions of the invention include bulk drug compositions useful in the manufacture of pharmaceutical compositions (e.g., impure or non-sterile compositions) and pharmaceutical compositions (i.e., compositions that are suitable for administration to a subject or 15 patient) which can be used in the preparation of unit dosage forms. Such compositions comprise a prophylactically or therapeutically effective amount of a prophylactic and/or therapeutic agent disclosed herein or a combination of those agents and a pharmaceutically acceptable carrier. Preferably, compositions of the invention comprise a prophylactically or therapeutically effective amount of one or more antibodies of the invention and a pharmaceutically acceptable carrier or an 20 agent that reduces expression (e.g., antisense oligonucleotides) and a pharmaceutically acceptable carrier. In a further embodiment, the composition of the invention further comprises an additional therapeutic, e.g., anti-cancer, agent. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other 25 generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered 30 intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or 19 WO 2006/110745 PCT/US2006/013473 emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Generally, the ingredients of compositions of the invention are supplied either separately 5 or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline 10 can be provided so that the ingredients may be mixed prior to administration. The compositions of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, 15 triethylamine, 2-ethylamino ethanol, histidine, procaine, etc. Various delivery systems are known and can be used to administer the antibody of the invention or the combination of a conjugated antibody of the invention and a prophylactic agent or therapeutic agent useful for preventing or treating cancer, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the antibody or antibody 20 fragment, receptor-mediated endocytosis (see for example, Wu and Wu (1987) J. Biol. Chem. 262, 4429-4432). Methods of administering a prophylactic or therapeutic agent of the invention include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural, and mucosal (e.g., intranasal, inhaled, and oral routes). In a specific embodiment, prophylactic or therapeutic agents of the invention are 25 administered intramuscularly, intravenously, or subcutaneously. The prophylactic or therapeutic agents may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. 30 In a specific embodiment, it may be desirable to administer the prophylactic or therapeutic agents of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion (such as limb perfusion), by injection, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. 20 WO 2006/110745 PCT/US2006/013473 Kits The invention provides a pharmaceutical pack or kit comprising one or more containers filled with an monoclonal antibody of the invention. In one embodiment the kit comprises an anti-PSMA antibody conjugated to a radioisotope via MeO-DOTA to a patient before and after 5 therapy. In a further embodiment, the MeO-DOTA to antibody ratio is about 9:1 or greater. In an even further embodiment, the MeO-DOTA to antibody ratio is about 9:1, or greater, with little, if any, loss of immunoreactivity. Additionally, one or more other prophylactic or therapeutic agents useful for the treatment of a cancer can also be included in the pharmaceutical pack or kit. The invention also provides a pharmaceutical pack or kit comprising one or more containers filled 10 with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. 15 The present invention provides kits that can be used in the above methods. In one embodiment, a kit comprises one or more a monoclonal antibodies of the invention. In another embodiment, a kit further comprises one or more other prophylactic or therapeutic agents useful for the treatment of cancer, in one or more containers. In one embodiment, the antibody of the invention is an antibody which immunospecifically binds to prostate specific membrane antigen 20 (PSMA), wherein said antibody is conjugated to a radioisotope via a MeO-DOTA linkage. In a specific embodiment said antibody is 7E1 1-C5 conjugated to 1 77 Lu via MeO-DOTA. In another embodiment, the conjugated 7E 11 -C5 is complexed with 177 Lu at a ratio of isotope to antibody of about 9:1 or greater. In certain embodiments, the other prophylactic or therapeutic agent is a chemotherapeutic. In other embodiments, the prophylactic or therapeutic agent is a biological or 25 hormonal therapeutic. Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples describe embodiments of the present invention, and are not to be construed as limiting 30 in any way the remainder of the disclosure. 21 WO 2006/110745 PCT/US2006/013473 EXAMPLES Example 1. Isolation and Characterization of 7E11-C5 1.1 Cell Lines and Tissues The LNCaP cell line was established from a metastatic lesion of human prostatic 5 carcinoma. The LNCaP cells grow readily in vitro (up to 8 x105 cells/cm 2 ; doubling time, 60 hours), form clones in semisolid media, and show an aneuploid (modal number, 76 to 91) human male karyotype with several marker chromosomes. The malignant properties of LNCaP cells are maintained. Athymic nude mice develop tumors at the injection site (volume-doubling time, 86 hours). Functional differentiation is preserved; both cultures and tumor produce a prostate acid 10 phosphatase (PAP) and prostate specific antigen (PSA). High-affinity specific androgen receptors are present in the cytosol and nuclear fractions of cells in culture and in tumors. Estrogen receptors are demonstrable in the cytosol. The model is hormonally responsive. In vitro, a dihydro-testosterone modulates cell growth and stimulates acid phosphatase production. In vivo, the frequency of tumor development and the mean time of tumor appearance are significantly 15 different for either sex. LNCaP cells, therefore, meet criteria of a versatile model for immunological studies of human prostatic cancer in the laboratory. Seven malignant cell lines of human origin were obtained from the Memorial Sloan Kettering Institute and included: DU-145 and PC-3 derived from prostatic cancer; MCF-7, derived from pleural effusion of scirrhous carcinoma of the breast (Soule et al. (1973) J. Natl. 20 Cancer Inst. 51, 1409-1416); MeWo, malignant melanoma; RT-4, transitional cell carcinoma; HT-29, adenoma of the colon and A209, rhabdomyosarcoma. Four other cell lines (two malignant and two normal), isolated and established at Roswell Park Memorial Institute were also used: TT, thyroid medullary carcinoma, pancreatic cancer, BG-9 and MLD - both normal diploid neonatal foreskin fibroblast (see Horoszewicz et al. (1978) Infect. Immun. 19, 720-726; Chen et 25 al. (1982) Human Pancreatic Adenocarcinoma 18, 24-32; Leong et al. (1982) Advances in Thyroid Neoplasia 1984, 95-108). All of the above cell lines were routinely maintained in RPMI medium 1640 (Roswell Park Memorial Institute) supplemented with 10% heat inactivated fetal bovine serum, 1 mM L-glutamine, and 50 gg/ml of penicillin and streptomycin (Gibco). Fresh normal, benign and malignant prostate cancer tissues were obtained either from the 30 Department of Surgery or the Department of Pathology at Roswell Park Memorial Institute. The tissues were quick frozen in M-1 embedding matrix (Lipshaw Corp) and stored at -80'C. 1.2. Immunization and Cell Fusion 22 WO 2006/110745 PCT/US2006/013473 Ten week old male Balb/c mice (West Seneca Laboratoryreceived intraperitoneal injections (2 x 107 cells/0.2ml) of washed (three times in RPMI medium 1640) live LNCaP cells suspended in RPMI medium 1640, at monthly intervals for 3 months. Three days before fusion, the mice received an intraperitoneal challenge of 2 x 107 cells in RPMI medium 1640 and an 5 intravenous injection of the plasma membrane isolated from 1 x 108 LNCaP cells. Cell fusion was carried out using a modification of the procedure developed by Kohler and Milstein (1975) Nature 256, 495-497. Mouse splenocytes (1 x 108 cells) were fused in HyBRL-Prep 50% polyethylene glycol 1450 (Bethesda Research Laboratories) with 5 x 107 mouse myeloma cells (P3 x 63Ag8.653). Fused cells were distributed to ten 96-well culture plates (Falcon, Oxnard, 10 CA) and grown in hypoxanthine/aminopterin/thymidine (HAT) medium at 37'C with 7.5% CO 2 in a humid atmosphere. Fourteen days later, supernatants were assayed for binding activity to plasma membrane isolate from LNCaP cells and MLD (normal human fibroblasts) using the Enzyme Linked Immunosorbent Assay (ELISA) with anti-mouse IgG p-galactosidase linked F(ab') 2 fragment from sheep (Amersham Corp.) or goat anti-mouse IgG horseradish peroxidase 15 conjugate (Bio-Rad Laboratories) in a primary screen. Dried membrane isolate (400 ng/well) instead of whole LNCaP cells was used in the primary screening process because of poor attachment of the LNCaP cells to the plastic wells. To circumvent this problem, immunofiltration on a disposable microfold system (V&P Scientific) using whole LNCaP cells was used as a confirmatory assay as described in Section 1.5. In addition, the dot-immunobinding assay on 20 nitrocellulose membrane (Section 1.4) was used to screen the supernatants of hybridomas for reactivity against LNCaP cell cytosol (100,000 x g supernatants) and crude plasma membrane preparation. To determine the specificity spectrum of the cultures showing reactivity with the plasma membranes and/or whole LNCaP cells, the culture fluids were further tested by ELISA on a panel of an additional nine viable, normal and neoplastic human cells lines as described in 25 Section 1.5. 1.3. Isolation of Plasma Membrane-Enriched Fraction Plasma membrane-enriched fractions were obtained from LNCaP cells and normal human diploid fibroblast strain MLD by modification of published methods (Kartner et al. (1977) J. Membrane Biol. 36, 191-211). Briefly, MLD cells in roller bottles or LNCaP cells in plastic 30 culture flasks were gently rinsed 4 times with phosphate buffered saline (PBS). The cells were then rinsed once with hypotonic lysing buffer (3 mM Hepes [hydroxyethylpiperazine ethanesulfonic acid] (pH 7.0), 0.3 mM MgCl 2 , 0.5 mM CaCl 2 ) and the buffer discarded. Fresh lysing buffer (5-25 ml) was added to each bottle or flask and the cells allowed to swell for 30 minutes at room temperature. The swollen cells were removed from the surface and disrupted by 35 manual shaking. The progress of disruption was monitored by phase microscopy of a sample 23 WO 2006/110745 PCT/US2006/013473 droplet. Gentle trituration (8-10 times) with a 10 ml pipette was used to complete disruption of the LNCaP cells. Vigorous shaking and pipetting were necessary to completely break-up the MLD cells. Phenyl-methylsulfonyl fluoride (PMSF, 0.5 mM) (Calbiochem) was added to minimize proteolysis. The disrupted cell suspensions were centrifuged at 100 x g to remove nuclei and 5 incompletely disrupted cell clumps. The nuclei pellet was washed once with the lysing buffer and after centrifugation the supernatant was combined with the first supernatant and centrifuged at 3,000 x g for 10 minutes, at 4'C. The pellet consisting of mitochondria and debris was discarded and the supernatant designated as membrane lysate was layered over a discontinuous density gradient composed of 15 ml each of 10, 30 and 38% sucrose (w/v) and centrifuged at 60,000 x g 10 for 2-1/2 hours in an SW 25.2 rotor (Beckman). Material banding at the interface between 10% and 30% sucrose layers was removed by aspiration, washed free of sucrose using lysing buffer and pelleted by centrifugation at 36,000 x g for 60 minutes. Pellets were resuspended in PBS and aliquots taken for assay of protein and the enzyme phosphodiesterase-I (EC3.1.3.35) as a marker for plasma membranes. The 10/30 plasma membrane isolate was used in the screening assays for 15 the hybridoma supernatants. All fractions were dispensed and stored as single-use aliquots at 90 0 C. 1.4. Dot-Immunobinding Assay The dot-immunobinding assay was used to screen large numbers of supernatants of hybridomas producing monoclonal antibodies (Hawkes et al. (1982) Anal. Biochem. 119, 142 20 147). The crude plasma membrane isolate, the 10/30 plasma membrane isolate and/or the cytosol fractions containing the cellular antigen were dotted (1-3 g1) on a washed nitrocellulose filter paper grid (Bio-Rad). The protein concentration of the "antigen" ranged from between 0.1 to 0.1 mg/ml. After thorough drying, the filter was washed in Tris Buffered Saline (TBS, 50 mM Tris HCl, 200 mM NaCl, pH 7.4). Treatment of the filter paper with 3% (w/v) bovine serum albumin 25 (Sigma, St. Louis, MO) in TBS for 15 minutes at room temperature resulted in the blockage of nonspecific antibody binding sites on the filter and on the walls of the plastic vessel used to carry out the reaction. The filter paper was then incubated with hybridoma supernatant or purified monoclonal antibody (2-20 pl) for 60 minutes in several changes of TBS, the blocking step was repeated. A second antibody (F(ab') 2 goat anti-mouse IgG) conjugated to horseradish peroxidase 30 (Bio-Rad) (diluted 1:1000 in blocking solution) was added and incubation was carried out at 37 0 C for 120 minutes. After washing with TBS the peroxidase activity was developed with 4-chloro-1 napthol (0.6 mg/ml in TBS, Merck Inc.) and hydrogen peroxide (0.01% v/v). A positive reaction appeared as a blue colored dot against the white filter background. 24 WO 2006/110745 PCT/US2006/013473 Immunoblotting of cytosol and membrane fractions indicated that the soluble cytosol fraction of LNCaP cells was not reactive, while sedimentable (approximately 105,000 x g) membrane associated fractions gave strongly positive spots with monoclonal antibody 7E1 1. 1.5. Enzyme Linked Immunosorbent Assay (ELISA) 5 The enzyme linked immunosorbent assay (ELISA) was used for general enzyme immunoassay of antigen and screening for monoclonal antibody production. Target cells were seeded (2-30 x 104 cells/ml) on microtiter plates (Falcon) 4-7 days before assay. Nonspecific binding sites on the plates were blocked with 1% (w/v) swine gelatin in a special media (FL), formulated to keep the cells viable. The FL media consisted of Dulbecco's Modified Eagle 10 Medium (Gibco) supplemented with 15 mM Hepes, 0.3% NaCl, 10 mM NaN 3 and swine gelatin (1% for blocking or 0.3% for washing) pH 7.2-7.4. Hybridoma culture fluids (50 pl per well) were added and incubation was carried out at 371C. for 60 minutes. The plates were washed 4 times with FL media and F(ab') 2 goat anti-mouse IgG conjugated to horseradish peroxidase (1:1250 in 0.3% swine gelatin, 0.01M PBS) was used in place of the p-galactoxidase conjugate. 15 After washing, 100 p1 of substrate (25 ml of 0.1 M citrate buffer pH 5.0, 10 p1 of 30% H202 and 10 mg of a-phenylenediamine (Sigma) was added to each well. The plate was incubated for 30 minutes in the dark, and the reaction stopped with 50 pl of 2 N H2S0 4 /well. The absorbance was determined at 490 nm using the Bio-Tek EIA reader. Dried crude plasma membrane isolates from LNCaP cells or dried cells were used 20 initially in the primary screening procedure of hybridoma culture fluids. Approximately 400 ng of membrane protein in 50 g1 of buffer (S3 mM Hepes, 0.3 mM MgCl 2 , 0.5 mM CaCl 2 ) was dried (35'C overnight) in 96 well flat bottom microtiter plates (Falcon). Nonspecific binding sites on the plates were blocked with 1% swine gelatin in PBS containing 0.1% NaN 3 . With the exception of the wash buffer consisting of 0.01M Hepes and 0.2 pM of PMSF in saline (pH 7.6), all other 25 reagents used were as described above for cell surface enzyme immunoassay. LNCaP cells attach poorly to plastic wells and detach from the plastic surface during the ELISA procedure. To circumvent this problem immunofiltration on a disposable microfold system (V&P Scientific) using whole LNCaP cells was employed as a confirmatory assay for the dried membrane assay. After nonspecific binding sites on the disposable microfold system was 30 blocked with 5% human serum albumin (HSA) in PBS, 2.5 x 104 LNCaP cells in 100 R1 of 5% HSA buffer was deposited on the filter discs with vacuum. After washing the filters with 0.3% gelatin in 0.01M phosphate buffer, the plates were processed as described above. However, after incubation with substrate, the reaction mixture from each well (100 pl) was transferred to 2 area Costar plates (Costar) before spectrophotometric determination on the EIA reader. Hybridomas 25 WO 2006/110745 PCT/US2006/013473 were detected in approximately 500 culture wells. 206 hybridomas were successfully expanded and on primary ELISA screen, 126 reacted with partially purified LNCaP membranes, 92 reacted with intact LNCaP cells 76 reacted with normal human fibroblast-cells and membrane preparations. Further screening by ELISA and by immunoperoxidase staining on a panel of 5 additional 11 viable, normal and neoplastic cell lines and by immunoblotting of cytosol and membranes fractions from LNCaP cells narrowed the field of two cloned hybridoma cell lines of particular interest, including MAb 7E 11 and 9H10. Hybridoma cultures showing specificity restricted to the LNCaP cells and membranes were cloned by limiting dilution and subcloned in agarose (see e.g., Schreier et al.(1980) 10 Hybridoma Techniques pp. 11-15, Cold Spring Harbor Laboratory Press). Stable cultures of antibody-producing hybridomas were expanded in complete media (RPMI 1640 media supplemented with 10% (w/v) heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 pg/ml streptomycin, and 10 gg/ml insulin) and cryopreserved. After cloning, two stable monoclonal hybridoma cell lines were obtained and designated as 7E 11 -C5 and 9H 1 0-A4 respectively. 15 Exhausted culture fluids and mouse ascites fluids were the source of antibodies used for further studies. Ascites fluid from mice carrying the hybridoma cell line was used to obtain large quantities of monoclonal antibodies. Hybridoma cells for ascites fluid production were washed 2 times with RPMI 1640 medium and resuspended at a density of 1-5 x 10 7 cells/ml. Using a 20 gauge needle, 0.2 ml of the cell suspension was injected into the peritoneal cavity of female nude 20 mice. Pristane was not routinely used to precondition the animals. Ascites fluid containing high titers of antibodies was regularly harvested four to five weeks after injection with the hybridoma cells. 1.6. Isotyping of Monoclonal Antibodies Monoclonals 7E1 1-C5 and 9H10-A4 are of the IgGl subclass, as determined by double 25 diffusion gel precipitation with isotype specific antisera (Miles). Consistent with this finding were observations that Protein A conjugated with either fluoroscein or horseradish peroxidase (BIO-RAD) failed to react with smears of LNCaP cells following incubation with either monoclonal. 30 Example 2. Conjugation of 7EC11-C5 Conjugates were prepared at pH 8 and 9 in 0.1 M HEPES buffer, at chelant to protein ratios of 50 and 200, at 20 and 37'C for four hours. Antibody 7E1 1-C5 concentration was 0.022 mM. Conjugates were purified by gel filtration chromatography. Table 1 summarizes the lot 26 WO 2006/110745 PCT/US2006/013473 numbers and reaction conditions. As Table 2 indicates average loading values from 0.2 to 5.3 were observed. Increasing molar ratios, temperature and pH facilitated the conjugation reaction. Table 1. Lot numbers of 7E11-C5 conjugates and conditions of preparations. Lot number Conditions 200402495-5/1 pH=8, 200C, r=50, HEPES 200402495-5/2 pH= 8 , 370C, r=50, HEPES 200402495-5/3 pH=8, 200C, r=200, HEPES 200402495-5/4 pH=8, 370C, r=200, HEPES 200402495-5/5 pH=9, 200C, r=50, HEPES 200402495-5/6 pH=9, 370C, r=50, HEPES 200402495-5/7 pH=9, 200C, r=200, HEPES 200402495-5/8 pH=9, 370C, r=200, HEPES 5 Table 2. Masses & average loading value of MeO-DOTA conjugated to antibodies. Lot #c Condition MassCh Conj. Pure Mass shift: MALDI arge mAb mAb conj-pure ALVb mass mass mass 200402495_5/1 pH=8, 2 0 4C, r=50 M'i 150254 150143 111 0.2 200402495_5/2 pH=8, 37'C, r=50 M+1 150658 150143 515 0.9 200402495_5/3 pH=8, 2 0 -C, r=200 M+' 150652 150143 509 0.9 200402495_5/4 pH=8, 37 C, r=200 M+1 151616 150143 1473 2.6 200402495_5/5 pH=9, 20'C, r=50 MP 150587 150143 444 0.8 200402495_5/6 pH=9, 37C, r=50 MV 151731 150143 1588 2.8 200402495_5/7 pH=9, 2 0 *C, r=200 M+' 151721 150143 1578 2.8 200402495_5/8 pH=9, 37 C, r=200 M 153146 150143 3003 5.3 27 WO 2006/110745 PCT/US2006/013473 aMALDI-MS values were computed from the average value of a z*M+, z= 1, ... 4, where z is the charge number and Mz+ is the mass-to-charge ratio of the (multiple charged) molecular ion, observed in the analysis of the pure or conjugated monoclonal antibodies. bALV is the average loading value. 5 "ysine conjugate. dSingly charged mass. "Doubly charged mass. To determine the apparent molecular weight, the protein samples were analyzed by high resolution SDS-PAGE (Figure 1). Two distinct protein bands were observed for each monoclonal antibody and respective conjugate at approximately 25 and 50 kDa. 10 To determine protein integrity after conjugation, size exclusion chromatography was conducted. Unmodified protein was also analyzed to compare if some aggregation occur after conjugation. Relative peak areas of unmodified protein and conjugates are summarized in Table 3. The chromatogram of the unmodified protein and a representative chromatogram of a conjugate are shown in Figure 2. Unmodified 7E1 1-C5 antibody shows two peaks, one eluting at 15 15.53 minutes (area= 1.79%) and the main peak at 18.13 minutes (area= 98.21%). The area of the minor peak of the conjugates varies from 2.2 to 4.4 %. Table 3. Relative peak areas 7E11-C5 antibody and MeO-DOTA-conjugates, determined from size exclusion chromatograms. Sample Peak area 1 (%) Peak area 2 (%) 7E11-C5 1.79 98.21 200402495-5/1 3.52 96.48 200402495-5/2 2.70 97.30 200402495-5/3 4.38 95.62 200402495-5/4 2.02 97.00 200402495-5/5 3.64 96.36 200402495-5/6 2.85 97.15 200402495-5/7 3.88 96.12 200402495-5/8 2.19 |97.81 28 WO 2006/110745 PCT/US2006/013473 Example 3. Complexation of Conjugated Antibody MeO-DOTA-7E1 1-C5 was complexed with 77 Lu at 2.25 mg/mL protein concentration at pH 5.2 at 20'C in sodium acetate buffer. Buffer concentrations of 50, 100 and 250 mM were studied. Complexation was monitored using size exclusion chromatography with radiometric and 5 UV detection. Samples were analyzed after 30 min reaction time. Results are summarized in Table 4. Table 4. Effect of NaOAc buffer concentration on complexation [Buffer] (mM) % Lu complexed 50 97.24 100 98.50 250 90.32 These data indicated that the 100 mM buffer was optimal for complexation. Therefore 10 the 100 mM buffer was chosen for the further complexations. Example 4. Effect of pH on complexation Complexation was carried out at pH 5.5 and 6 using a 100 mM NaOAc buffer. Representative chromatograms at pH 5.5 are shown in Figure 3. Samples were analyzed at 30 minutes and 2 hours. Aggregation was between 3-4 %, determined by UV. Summary of 15 complexation at pH 5.5 and 6 is in Table 5. Table 5. Summary of complexation using 100 mM sodium acetate buffer Conjugate Lot pH Temp (*C) Time (h) % complexed % Aggregate (UV) 200402495-14 5.2 20 0.5 98.5 3.6 200402495-14 6 20 0.5 76.7 3.1 2.0 77.2 3.0 6 37 0.5 80.1 3.0 2.0 82.5 3.0 2004-02495-29 5.5 20 0.5 97.8 3.4 29 WO 2006/110745 PCT/US2006/013473 Conjugate Lot pH Temp (*C) Time (h) % complexed % Aggregate (UV) 2.0 100 3.4 37 0.5 98.6 3.3 2.0 100 3.3 These data indicate that optimal pH for the complexing reaction is about 5.5. Example 5. Preparation of high specific activity conjugate On the basis of complexations carried out using trace amount of 1 77 Lu (enough 17 Lu to 5 have a good radiometric signal on the HPLC), the following conditions were selected to prepare high specific activity material: 100 mM sodium acetate buffer, 37'C, pH 5.5, protein concentration 2.5 mg/mL. 0.5 mg protein and 656 mCi of 1 77 Lu was used. The reaction was monitor by size exclusion chromatography. Target specific activity was 1 mCi/mg. Table 6 summarizes the results. Calculating with 90 % radioactivity incorporation, the specific activity of 10 the complex-conjugate is 1.18 mCi/mg. Maximum incorporation is reached within 30 min. Aggregation is 3%. Figure 4 shows the chromatogram of the 30 min. time point. Table 6. Time study of the complexation of Lu-177, high specific activity preparation Time (h) % complex % Aggregate (UV) 0.5 89.9 3.0 1 91.8 3.0 2.5 89.8 3.5 Upon review of these data presented above the optimal conditions for conjugating an 15 antibody of the invention, preferably 7E1 1-C5, is incubating said antibody in 0.1 M HEPES buffer at a pH of 8.5 with molar ratio of linker to antibody of approximately 200:1 at 37'C for approximately 4 hours with slight agitation. The resulting antibody will be conjugated with molar ratio of MeO-DOTA to antibody of approximately 9:1 or greater. Optimal complexing reactions occur in 100mM Na-Actetate, pH 5.5 at 20'C for 0.5 hours. The resulting antibody is 1.2mCi/mg 20 of 7E1 1-C5 at 90% complexation. A person skilled in the art will recognize that modifications and variations of these conditions will slightly vary the actual conjugated and complexed antibody composition. 30 WO 2006/110745 PCT/US2006/013473 Example 6: Anti-PSMA-meO-DOTA Immunoconjugate (CYT-500) Anti-PSMA-meO-DOTA Immunoconjugate (CYT-500) is comprised of 7E1 1C5-3 monoclonal antibody (CYT-35 1) that is currently used in the manufacture of its commercial product ProstaScint@. ProstaScint@ is comprised of CYT-351 conjugated via periodate oxidation 5 of the carbohydrate groups located on the heavy chains to the linker-chelator GYK-DTPA HCl [glycyl-tyrosyl-(N-C-diethylenetriaminepentaacetic acid)-lysine hydrochloride] which is complexed with the gamma emiting radioistope "'In. Anti-PSMA-meO-DOTA Immunoconjugate is comprised of CYT-351 covalently conjugated to the linker-chelator meO DOTA [a-(5-isothiocyanato-2-methoxyphenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10 10 tetraacetic acid]. The CYT-351-meO-DOTA immunoconjugate has been shown to be stable in human serum thereby decreasing the chance for secondary toxicities as a result of shed linker and/or radioisotopes. Example 7: 7E11C5-3 Monoclonal Antibody (CYT-351) 15 CYT-351 is a murine IgG1 monoclonal antibody secreted by a murine/murine hybridoma cell line, which was produced by immunizing BALB/c mice with live LNCaP human prostatic adenocarcinoma cells and partially purified LNCaP plasma membranes. The LNCaP cell line used to immunize the mice is a well characterized continuous cell line which was established from a needle biopsy taken from a lymph node metastasis of human prostatic adenocarcinoma. LNCaP 20 cells grow readily in vitro, form clones in semisolid media, show an aneuploid (modal number 76 91) human male karyotype with several marker chromosomes and maintain the malignant properties of an adenocarcinoma. The CYT-3 51 hybridoma was established and originally described by Horoszewicez et al. (1987) Anticancer Res. 7, 927-936 and U.S. Patents 5,162,504 and 5,578,484). Spleen cells from 25 mice immunized with live LNCaP cells were fused with P3X63Ag8.653 murine myeloma cells. The cells were cloned twice by limited dilution cloning and a stable hybridoma, designated hybridoma 7E1 1-C5, was expanded and cryopreserved. This clone secreted a prostate-specific monoclonal antibody of the IgG1 subclass which was originally designated monoclonal antibody 7E1 1-C5. 30 A culture of the CYT-351 seed stock was used to establish a 100 vial Master Cell Bank (MCB). A single vial of cells was thawed and the cells recovered into a 25 cm 2 flask containing basal cell culture medium supplemented with 2.5% FBS (fetal bovine serum). The cells were subsequently expanded into 75 cm 2 flasks, 150 cm 2 flasks, a 500 ml spinner flask, and finally on 31 WO 2006/110745 PCT/US2006/013473 into a three liter spinner. The cells were harvested and placed into freezing medium (basal medium supplemented with 20% FBS and 10% DMSO). The cells were then aliquoted into 100 vials, each containing approximately 9 x 106 cells and labeled with the designation 2MMO 180 MOO 1 -9M, and subsequently stored in the vapor phase of liquid nitrogen. Ten vials from the 5 serum-grown MCB were used for tests to determine if the preparation was sterile and free of infectious adventitious agents. The results of these tests demonstrated that the CYT-35 1 MCB was sterile and free of infectious adventitious agents Example 8: Methoxy-DOTA linker Methoxy-DOTA (a-(5-isothiocyanato-2-methoxyphenyl)- 1,4,7,10 10 tetraazacyclododecane- 1,4,7,1 0-tetraacetic acid) is prepared from a purely synthetic process. meDOTA and its methods of use and manufacture is disclosed in U.S. Patents 5,435,990 and 5,652,361 both of which are herein incorporated by reference in their entirety. Example 9: CYT-351 Manufacturing Process The cell banks, components, raw materials and manufacturing process used to produce 15 CYT-351 intermediate antibody for use in producing Anti-PSMA-meO-DOTA Immunoconjugate (CYT-500) are done so in accordance with the GMP manufacturing process. The growth/production medium for the CYT-351 hybridoma is a defined, serum-free media available from HyClone Laboratory (HyQ-CCM T M ) and is comprised of 925 basal medium. Cell culture is performed in an AcuSyst-Xcell hollow fiber bioreactor and pH, temperature and 20 oxygen levels monitored throughout the run. Samples are removed to monitor glucose, lactate and CYT-3 51 levels. Media feed is achieved via peristaltic pump. Medium is perfused through the bioreactor and the conditioned medium containing CYT-351 is harvested, clarified by filtration and stored at 2 to 8'C. The production run typically lasts for 60 to 70 days. Each CYT-3 51 harvest is sampled and tested for CYT-3 51 titer, immunoreactivity, 25 endotoxin and bioburden. Prior to purification, pooled harvest samples are tested minimally for: CYT-351 concentration, Mycoplasma, sterility and virus by reverse transcriptase, XC Plaque, S+L- Focus and in vitro viral testing. Harvest and purification of CYT-3 51 are performed in classified rooms with appropriate environmental monitoring to allow for aseptic processing. The CYT-351 harvest is filtered 30 through a 0.45 ptm filter, concentrated to approximately 6 to 12 mg/ml CYT-351 using a Pellicon tangential-flow ultrafiltration device fitted with a 30 kDa cutoff membrane. Following concentration, the concentrated crude CYT-351 product is passed over a Sephadex G-25 column 32 WO 2006/110745 PCT/US2006/013473 to remove low molecular weight moieties. The G-25 column is equilibrated and eluted with 0.7 M ammonium sulfate (pH 8.0 to 8.4). The eluted protein (CYT-3 51) peak is loaded onto a Protein A affinity column equilibrated with 0.7 M ammonium sulfate. The loaded Protein A column is washed with thirty 5 (30) column volumes of 0.7 M ammonium sulfate followed by a short wash with 55 mM sodium acetate (pH 7.0 to 8.5). Bound CYT-351 is eluted from the Protein A column with 55 mM sodium acetate (pH 4.0 to 4.5) and the pH of the eluted product adjusted to 5.1 to 5.3 with 55 mM sodium acetate (pH 7.0 to 8.5). The Protein A purified material is passed over a DEAE Sepharose column equilibrated in 10 55 mM sodium acetate (pH 5.1 to 5.3). This is a passive purification step in that the CYT-351 passes over the column whereas DNA, albumin and other acidic components bind to the support. The CYT-351 peak is then loaded onto a S-Sepharose column equilibrated with 55 mM sodium acetate (pH 5.1 to 5.3). The column is washed with 10 mM sodium phosphate buffer (pH 5.9 to 6.1). The bound CYT-351 is eluted with 10 mM phosphate buffered saline (pH 5.9 to 6.1). 15 Purified CYT-3 51 is filtered through a sterile 0.22 pim, sampled for Quality Control testing and stored at 2 to 8 0 C until needed for conjugation. Sterile filtered bulk CYT-351 has an approved shelf life of three years at 2 to 8*C. Example 10: Manufacturing Process for the Immunoconjugate CYT-500 Prior to conjugation, the purified CYT-351 is passed through a DV-20 (PALL) virus 20 removal filter. The commercial manufacturing process for CYT-351, described above, results in 8.9 log viral removal. An additional 5 to 6 log viral removal is obtained using the DV-20 filter, resulting in approximately 14 log removal. Purified monoclonal antibody CYT-351 is combined with 0.22 pm filtered (cellulose acetate) meO-DOTA in 0.5 M HEPES (pH 8.85). The linker to CYT-351 ratio is 70:1 with a total 25 of 6 grams CYT-351 used for the toxicology lot (clinical lots also are 6 gram CYT-351 scale). The reaction mixture is incubated for three hours at 35 to 37'C with gentle stirring. Following three hours, the reaction mixture is adjusted to 7.0 with 1 M acetic acid to slow the reaction. The resultant product was concentrated from approximately 1900 to 300 ml using a Millipore Labscale TFF system with one Pelicon XL Biomax 50 filter. The concentrate was stored 30 overnight at 2-8'C. The concentrate was chromatographed with 0. 1M sodium acetate (pH 5.5) on a 9 x 90 cm Superose 12 column. The main (product) fraction was collected and concentrated to approximately 21 mg/ml using a Millipore Labscale TFF system with one Pelicon XL Biomax 50 filter. This material (CYT-500) was filtered through a 0.22 Rm filter and stored at 2 to 8 0 C. 33 WO 2006/110745 PCT/US2006/013473 The bulk CYT-500 is stored at 2 to 8C and tested for contaminants before being released for use. Released CYT-35 1 is filtered through a sterile 0.22 pim filter and filled into 10 ml Type 1 borosilicate glass vials and stoppered with presterilized 20 mm stoppers. The filled, unlabeled vials are sealed with 20 mm flip-off crimp, visually inspected and sampled for Quality Control 5 testing. Vials are placed in trays marked "quarantine" pending release. Example 11: 7E11-meO-DOTA Serum Stability An important requirement of antibody-chelating agent immunoconjugates is that they form kinetically inert complexes with metals of interest, in this case, 177 Lu. These complexes must be stable following conjugating to a protein and should stay intact in vivo to avoid secondary 10 toxicities. Similarly, loss of lanthanide metals can result in toxic effects, such as radioactive doses to the liver and bone. Accordingly, we tested serum stability of 77 Lu labeled CYT-500 and compared it to 'In-labeled ProstaScint. Size exclusion chromatography was used to analyze the radioactivity (1 7 7 Lu) loss from the complex-conjugate in serum. Uncomplexed 1 77 Lu associates with serum proteins and tends to 15 elute with the high molecular weight species, similarly to 77 Lu-meO-DOTA- immunoconjugate (1 77 Lu-CYT-500). The fact that serum proteins bind Lu weakly in a non-specific manner allows us to differentiate between the serum protein 77 Lu complex and 1 77 Lu-CYT-500. The weak association between ' 77 Lu and serum proteins can be broken up by DTPA, while DTPA can not transchelate the metal from DOTA type chelates. 20 To determine if one percent metal loss from the complex conjugate can be measured, mixtures of 77 Lu-CYT-500and 1 7 Lu-MeO-DOTA were prepared. The radioactivity in both 1 77 Lu-CYT-500 and ' 7 7 Lu-MeO-DOTA was determined by radioactive counting before mixing them. Two samples were prepared. In the first sample 7% of the total radioactivity came from 1 77 Lu-MeO-DOTA and in the second one 1%. The size exclusion chromatography analysis 25 showed 9.8 and 3.0% of the radioactivity eluting as the low molecular weight component. The chromatographic method and counting gave the same results within the experimental error. 77 Lu-CYT-500 antibody conjugate was incubated in human serum and before HPLC analysis DTPA was added to the sample to complex nonspecifically bound Lu. The results are tabulated in Table 7. During the two week course of the study insignificant metal loss was 30 observed for 1 77 Lu-CYT-500 (98% at day 0 and 96% at day 15) and minimal metal loss was observed for "'In-DTPA-Cyt-351 (98% at day 1 and 91 % at day 15). Radioactivity associated with the high molecular weight components of the mixture, determined by size exclusion chromatography after addition of DTPA. 34 WO 2006/110745 PCT/US2006/013473 Table 7. Stability in Human Serum % Radioactivity associated with high mw Time (day) 77 Lu 177 Lu-CYT-500 SD '..iIn-ProstaScint SD 0 0 97.8 0.8 98.4 0.1 1 0 98.8 0.1 97.1 0.1 2 0 98.5 0.4 97.2 0.1 3 0 96.5 0.2 97.1 0.1 4 0 97.7 0.2 96.2 0.6 6 0 97.1 0.7 96.1 0.7 7 0 97.9 0.5 95.1 1.1 8 0 98.2 0.1 94.7 0.6 9 0 97.0 0.5 94.6 1.1 10 0 98.1 94.2 1.1 15 0 95.6 0.4 91.0 0.4 Example 12: 7E11-meO-DOTA Acute Toxicity Study in Rats The purpose of this study was to determine the potential toxicity (including neurotoxicity) 5 of Anti-PSMA-meO-DOTA Immunoconjugate (CYT-500) when administered once by intravenous injection to male Sprague Dawley rats. Eighty male rats were randomly assigned to one of four groups and administered 100 mM sodium acetate buffer (control article) or Anti PSMA-meO-DOTA Immunoconjugate (CYT-500) at 3, 15 or 30 mg/kg once on Study Day (SD). Forty rats (10/group) were subjected to a full gross necropsy on SD 4; the remaining rats were 10 necropsied on SD 15. An additional 27 rats were assigned to one of the three treated groups (9/group) and blood was collected at selected timepoints for future toxicokinetic profiling. Parameters evaluated included mortality, clinical observations, body weight, food consumption, neurotoxicity, ophthalmology, clinical pathology, gross pathology, absolute and relative organ weights and histopathology. Treatment with Anti-PSMA-meO-DOTA 15 Immunoconjugate (CYT-500) had no effect on mortality, clinical observations, body weight, food consumption, neurotoxicity, ophthalmology, clinical pathology, gross pathology, absolute and 35 WO 2006/110745 PCT/US2006/013473 relative organ weights and histopathology. Therefore, under the conditions of this study the observed no-effect level (NOEL) is at least 30 mg/kg (100x the anticipated human dose). Example 13: 7E11-meO-DOTA Acute Toxicity Study in Dogs The purpose of this study was to determine the potential toxicity of Anti-PSMA-meO 5 DOTA Immunoconjugate (CYT-500) when administered once by intravenous injection to male beagle dogs. Twenty four male dogs were randomly assigned to one of four groups and administered 100 mM sodium acetate buffer (control article) or Anti-PSMA-meO-DOTA Immunoconjugate (CYT-500) at 0.6, 3 or 6 mg/kg once on SD 1. Twelve dogs (three per group) were subjected to a full gross necropsy on SD 4; the remaining 12 dogs were necropsied on SD 10 15. Parameters evaluated included mortality, clinical observations, body weights, food consumption, ophthalmology, cardiology, clinical pathology, gross pathology, absolute and relative organ weights, and histopathology. Treatment with Anti-PSMA-meO-DOTA Immunoconjugate (CYT-500) had no effect on mortality, clinical observations, body weights, food consumption, ophthalmology, cardiology, 15 clinical pathology, gross pathology or absolute and relative organ weights. Test article related findings consisted of vasculitis of the central veins of the liver in treated animals. Lesions were more pronounced in SD 4 animals and, although present, appeared to be resolving in SD 15 animals. The most severe lesions in SD 4 animals were seen in animals treated at 3 or 6 mg/kg (10 and 20x the anticipated human dose, respectively). By SD 15, the lesions were milder 20 overall, suggesting that with additional time resolution may be possible. In conclusion, intravenous injections of Anti-PSMA-meO-DOTA Immunoconjugate (CYT-500) were generally well tolerated. Example 14: 7E11-meO-DOTA Cardiovascular Safety Pharmacology Study The purpose of this study was to evaluate cardiovascular safety following intravenous 25 administration of Anti-PSMA-meO-DOTA Immunoconjugate (CYT-500) in male Beagle dogs. Seven male dogs were given an intravenous injection of 100 mM sodium acetate buffer on SD 1, and Anti-PSMA-meO-DOTA Immunoconjugate (CYT-500) at 0.6 mg/kg on SD 8, 3 mg/kg on SD 15, and 6 mg/kg on SD 22 and 29. Each dose administration was followed by at least a one week wash-out period. Cardiovascular profiling and body temperature data were collected via 30 telemetry following doses on SD 1, 8, 15 and 22. Other parameters evaluated included mortality, clinical observations, and body weights. Treatment with Anti-PSMA-meO-DOTA Immunoconjugate (CYT-500) at doses up to 6 mg/kg had no effects on blood pressure, heart rate, electrocardiographic parameters, body 36 WO 2006/110745 PCT/US2006/013473 temperature, body weights or mortality. One animal experienced anaphylaxis shortly after administration of a 6 mg/kg dose on SD 22. This animal was removed from the study and returned to the stock colony. Symptoms of anaphylaxis were not observed in any other animals following both a single and repeat dose at 6 mg/kg. In conclusion, intravenous injection of Anti 5 PSMA-meO-DOTA Immunoconjugate (CYT-500) at doses up to 6mg/kg were generally well tolerated. Example 15: 7E11-meO-DOTA Respiratory Function Study The purpose of this study was to evaluate respiratory function following intravenous administration of Anti-PSMA-meO-DOTA Immunoconjugate (CYT-500) in male Beagle dogs. 10 Six male dogs were given an intravenous injection of 100 mM sodium acetate buffer on SD 1, and Anti-PSMA-meO-DOTA Immunoconjugate (CYT-500) at 6 mg/kg on SD 4. Parameters evaluated included mortality, clinical observations, body weights and respiratory function assessment. Respiratory function assessment included respiratory rate, saturated blood oxygen levels (SpO2) and end-tidal pressures (ETCO 2 ). 15 Treatment with Anti-PSMA-meO-DOTA Immunoconjugate (CYT-500) had no effect on mortality, clinical observations, body weight, or respiratory function. Therefore under the conditions of this study the no-observed effect-level (NOEL) is at least 6 mg/kg. Although the present invention has been described in detail, it is understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the 20 invention is limited only by the following claims. All cited patents, patent applications and publications referred to in this application are herein incorporated by reference in their entirety. All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all 25 purposes. Many modifications and variations of the present invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only, and the invention is to be limited only by the terms of the appended claims along with the full scope of equivalents to which 30 such claims are entitled. 37

Claims (47)

1. An antibody, or fragment thereof, which immunospecifically binds to prostate specific membrane antigen (PSMA), wherein said antibody is conjugated to a radioisotope via a MeO DOTA linkage. 5

2. The antibody of claim 1, wherein said monoclonal antibody is 7E1 1-C5.

3. The antibody of claim 2, wherein the MeO-DOTA to antibody ratio is about 9:1 or greater.

4. The antibody of claim 1, wherein said radioisotope is selected from the group consisting of 3 H, 14 C, 18 F, 1 9 F, 31 P, 32 p, 35 S 1311, 1251, 1231 6 4 Cu, 1 87 Re, "IIIn, 9 0 Y, 9 9 Tc, 177 Lu. 10

5. The antibody of claim 1, wherein said antibody is 7E11-C5 and said radioisotope is ' 77 Lu.

6. The antibody of claim 5, wherein said radioisotope and said antibody is at a ratio of about 9:1 or greater.

7. A method for treating cancer which comprises a malignant cell expressing PSMA 15 comprising administering an antibody, or fragment thereof, which immunospecifically binds to prostate specific membrane antigen (PSMA) to a patient in need thereof, wherein said antibody is conjugated to a radioisotope via a MeO-DOTA linkage.

8. The method of claim 7, wherein said antibody is 7E1 1-C5.

9. The method of claim 8, wherein the MeO-DOTA to antibody ratio is about 9:1 greater. 20

10. The method of claim 7, wherein said radioisotope is selected from the group consisting of 'H, 14C, ' 8 F, ' 9 F, 31 P, 32 P, 35 S, 1311, 1251, 1231, 64 Cu, 1 8 7 Re, "In, Y, 9 9 Tc, 1 77 Lu.

11. The method of claim 7, wherein said antibody is 7E11-C5 and said radioisotope is 177 Lu

12. The method of claim 11, wherein said radioisotope and said antibody is at ratio of 25 about 9:1 or greater.

13. A method of imaging a tumor in a patient comprising administering an antibody, or fragment thereof, which immunospecifically binds to prostate specific membrane antigen (PSMA), wherein said antibody is conjugated to a radioisotope via a MeO-DOTA linkage. 38 WO 2006/110745 PCT/US2006/013473

14. A method for diagnosing cancer which comprises a malignant cell expressing PSMA comprising exposing a tissue sample to an antibody, or fragment thereof, which immunospecifically binds to prostate specific membrane antigen (PSMA), wherein said antibody is conjugated to a radioisotope via a MeO-DOTA linkage. 5

15. The method of claim 14, wherein said antibody is 7E1 1-C5.

16. The method of claim 15, wherein the MeO-DOTA to antibody ratio is about 9:1 or greater.

17. The method of claim 14, wherein said radioisotope is selected from the group consisting of 3 H, 1 4 C, ' 8 F, ' 9 F, 3 1 P, 32 p, 35 S, 1311, 1251, 1231, 6 4 Cu, 187 Re, "'In, 9 0 Y, 9 9 Tc, 1 77 Lu. 10

18. The method of claim 14, wherein said antibody is 7E 11-C5 and said radioisotope is 1 77 Lu.

19. The method of claim 14, wherein said radioisotope and said monoclonal antibody is at a ratio of about 9:1 or greater.

20. A pharmaceutical composition comprising an antibody, or fragment thereof, which 15 immunospecifically binds to prostate specific membrane antigen (PSMA), wherein said antibody is conjugated to a radioisotope via a MeO-DOTA linkage.

21. The composition of claim 20, wherein said antibody is 7E1 1-C5 and said radioisotope is 77 Lu.

22. The composition of claim 21, wherein said radioisotope and said antibody is at a ratio 20 of about 9:1 or greater.

23. A kit comprising the antibody of claim 1, 2, 3, 4, 5, or 6 in one or more containers.

24. A method for conjugating an antibody, or fragment thereof, which immunospecifically binds to prostate specific membrane antigen (PSMA) with MeO-DOTA, wherein said method comprises incubating MeO-DOTA and said antibody in a molar ratio about 25 5:1 or greater, respectively.

25. The method of claim 24 wherein said molar ratio is about 10:1, respectively.

26. The method of claim 24 wherein said molar ratio is about 50:1, respectively.

27. The method of claim 24 wherein said molar ratio is about 100:1, respectively. 39 WO 2006/110745 PCT/US2006/013473

28. The method of claim 24 wherein said molar ratio is about 150:1, respectively.

29. The method of claim 24, wherein said molar ratio is about 200:1, respectively.

30. The method of claim 24, wherein said antibody is 7E1 1-C5.

31. A method of complexing the antibody of claim 1, 2, 3, 4, 5, or 6 with a radioisotope, 5 wherein the complexing reaction is conducted at a pH between 4.5 to 6.5.

32. The method of claim 31 wherein the pH is 5.5.

33. A method of complexing the antibody of claim 1, 2, 3, 4, 5, or 6 with a radioisotope, wherein the complexing reaction is carried out in a sodium acetate buffer concentration between 50 mM to 250 mM. 10

34. The method of claim 33 wherein the sodium acetate buffer is 100 mM.

35. The antibody of claim 1, 2, 3, 4, 5, or 6 wherein said antibody is a monoclonal antibody.

36. The antibody of claim 1, 2, 3, 4, 5, or 6 wherein said antibody is a humanized antibody. 15

37. The antibody of claim 1, 2, 3, 4, 5, or 6 wherein said antibody is a human antibody.

38. The antibody of claim 1, 2, 3, 4, 5, or 6 wherein said antibody binds to an intracellular or cytoplasmic epitope or domain.

39. The antibody of claim 1, 2, 3, 4, 5, or 6 wherein said antibody binds to an extracellular epitope or domain. 20

40. The method of claim 7 further comprising administering at least one additional agent.

41. The method of claim 40, wherein said additional agent comprises radiation.

42. The method of claim 40, wherein said additional agent comprises a chemotherapeutic agent.

43. The method of claim 40, wherein said additional agent comprises a cytotoxic agent. 25

44. The method of claim 43, wherein said cytotoxic agent enhances binding to an epitope of PSMA. 40 WO 2006/110745 PCT/US2006/013473

45. The method of claim 44, wherein said epitope is located on the cytoplasmic domain of PSMA.

46. The method of claim 7, wherein said cancer is prostrate cancer.

47. The method of claim 7, wherein said cancer is selected from the group consisting of 5 renal cell carcinoma, colon carcinoma, transitional cell carcinoma, lung carcinoma, breast carcinoma adenocarcinoma, ductal carcinoma, lobular carcinoma, invasive ductal carcinoma, medullary carcinoma and mucinous carcinoma. 41