MXPA99000642A

MXPA99000642A - Derivatized rodamine tint and its copolime

Info

Publication number: MXPA99000642A
Application number: MXPA/A/1999/000642A
Authority: MX
Inventors: J Ward William; s johnson Brian; R Cramm Jeffrey; e reed Peter
Original assignee: Nalco Chemical Company
Priority date: 1996-07-17
Filing date: 1999-01-15
Publication date: 1999-09-20

Abstract

The present invention relates to: Rhodamine B esters of hydroxy-C2-C6 lower alkyl acrylates are described. Specifically, the hydroxy-lower alkyl radical is a linear lower hydroxy-lower alkyl radical having the hydroxy group attached to the terminal carbon atom. Copolymers of diallyldimethylammonium chloride containing 0.01-2 mole percent of these steamers of Rhodamine B and their use to treat industrial water are also shown

Description

AGENT 8T0L0GTC0 FOR THE TREATMENT AND DIAGNOSIS OEL C NCER AND COMPOSITION THAT CONTAINS IT The present application claims the benefits of US Provisional Patent Application No. 60 / 022,125, filed July 18, 1996, and is a partial continuation of the US Patent Application Serial No. 08 / 8388,682, registered in April 9, 1997, which claims the benefit of the USA Provisional Patent Application with Serial No. 60 / 016,976, registered in May 1996.

FIELD OF THE INVENTION The present invention concerns the treatment and diagnosis of cancer with biological agents.

BACKGROUND OF THE INVENTION Despite improved treatments for certain forms of cancer, it continues to be one of the leading causes of death in the United States. Since the likelihood for complete elimination of cancer is, in most cases, largely increased * by early diagnoses, it is highly desirable that clinicians be able to detect cancers before substantial tumor development. However, the development of methods? 9? 49 that allow the rapid and safe detection of many forms of cancers continues to be a challenge for the average community. An illustrative way of cancer is prostate cancer.

Prostate cancer is the most common cancer in men with an estimated 317,000 cases in 1996 in the United States. It is the second cause of death among men who die of neoplasia with an estimated 40,000 deaths per year. Early detection and treatment is needed to limit the mortality caused by cancer. prostate.

Detection of Cancer of the Prostate.

When metastasis spreads, prostate cancer has a distinctive predilection for bones and lymph nodes. Saitoch et al, "Metastatic Patterns of Prostate Cancer." Correlation Between Sites and Number of Organs Involved, "Cancer, 54: 3078-3084 (1984)." At the same time clinical diagnoses, more than 25% of patients have metastasis in bone demonstrable by radionuclide exploration Murphy, GP, et al. , "The National Survey Of Prostate Cancer In The United States By The American College Of Surgeons," J. Urol., 127: 928-939 (1982) Clinical assessments of nodes involved have proven to be difficult. such as computed tomography ("CT") or magnetic resonance imaging ("MR"), are assumed to be incapable of distinguishing metastatic prostate cancer involving lymph node involvement by other criteria such as size (eg,> 1 cm.). Accordingly, by definition, these imaging modalities are inherently insensitive in detecting diseases of small volume (<1 cm.), As well as non-specific in the detection of e enlarged adenopathies. A recent study assessed the accuracy of MR in patients with clinically localized prostate cancer. R-f in et al., "Comparison Of Magnetic Resonance Iraging And Ultrasonography In Staging Early Prostate Cancer", N. Engel. J. Med., 323: 621-626 (1990). In this study, 194 patients underwent MR and 185 of these patients had a lymph node dissection. 23 (13 o) of the patients were pathologically involved in lymph nodes. MR was suspected in 1 of these 23 cases, resulting in a sensitivity of 4%. Similar results have also been observed with CT scan. Gasser et al., "MRl And Ultrasonography In Ataging Prostate Cancer," N. Engl. J. Med. (Correspondence), 324 (7): 49-495 (1991).

Elevation of serum acid phosphatase activity in patients who had metastasized carcinoma in the prostate was first reported by Gutman et al., J. Clin. Invest. 17: 473 (1938). In prostate cancer, prostatic acid phosphatase is released from the cancerous tissue into the blood stream with the result that the total level of acid phosphatase can in most cases be increased above normal values. Numerous studies of this enzyme and its relation to prostate cancer have been made for some time, for example, Yam, Amer. J. Méd. 56: 504 (1974). However, the measurement of acid phosphatase in serum is elevated in approximately 65-90% of patients who have carcinoma of the prostate with bone metastasis; in approximately 30% of patients without roetgenological evidence of bone metastasis; and in approximately 5-10% of patients lacking clinically demonstrable metastases.

Previous attempts in the matter to develop a specific test for prostatic acid phosphatase have been found only with limited success, because techniques dependent on the activity of the enzyme on a so-called "specific" substrate can not take into account other biochemical and immunochemical differences between the many acid phosphatases that are not related to the activity of the enzyme of prostate origin. In the case of isoenzymes, for example, genetically defined enzymes that have the same characteristic enzymatic activity and a similar molecular structure but differing in the amino acid and / or content sequences and, therefore, immunochemically distinguishable, it might seem inherently impossible to distinguish different forms of isoenzymes merely for the selection of a particular substrate. It is not, therefore, surprising that none of these prior methods in the art is highly specific for the determination of prostatic acid phosphatase activity; for example, see Cancer 5: 236 (1952); J. Lab. Clin. Med. 82: 486 (1973); Clin. Chem. Minutes. 44:21 (1973); and J. Physiol. Chem. 356: 1775 (1975).

In addition to the aforementioned problems of non-specificity that seem to be inherent in many of the previous reagents used in the area, for the detection of acid phosphatase of the prostate, there have been reports of elevated serum acid phosphatase associated with other diseases, which further complicate the problem of obtaining an accurate clinical diagnosis of prostate cancer. For example, Tuchman et al., Am. J. Méd. 27: 959 (1959) observed that serum acid phosphatase levels appear to be elevated in patients with Gaucher's disease.

Due to the difficulties inherent in developing a "specific" substrate for acid phosphatase of the prostate, several investigators have developed immunochemical methods for the detection of acid phosphatase in the prostate. However, previously reported immunochemical methods have their own impediments that have excluded their widespread acceptance. For example, Shulman et al., Immunology 93; 474 (1964) described an immunodiffusion test for the detection of acid phosphatase in the human prostate. Using antiserum prepared from a pristatic fluid antigen obtained by rectal massage of patients with prostatic disease, no cross-reactivity was observed with the precipitin line in the double diffusion technique against normal kidney, testis, liver, and lung extracts. However, this method has the disadvantages of limited sensitivity, equal to the large amount of antigen used, and the use of antiserum that can cross-react with another, antigenically unrelated to the serum of the components of the protein present in the prostatic fluid.

WO 79/00475 of Chu et al. Describe a method for the detection of isoenzyme patterns of prostatic acid phosphatase associated with prostate cancer that highlights many of the above-mentioned drawbacks. However, practical problems arose due to the need for a source of cancerous prostate tissue from which relevant patterns of diagnosis of the prostatic acid phosphatase isoenzyme associated with prostate cancer are extracted for the preparation of their antibodies.

Recently, considerable efforts have been expended to identify enzyme or antigen markers for various types of malignancies with an eye toward the development of specific diagnostic reagents. The ideal marker for tumor could exhibit, among other characteristics, specificity of tissue or of type cells. Previous researchers have shown the frequency of specific antigens-human prostate tissue.

Treatment of Prostate Cancer As described in W. J. Catalona, "Management of Cancer of the Prostate, "New Engl. J. Med., 331 (15): 996-1004 (1994), the management of prostate cancer can be achieved by periods of monitoring, curative treatment and relief.

For men with a life expectancy of less than 10 years, it is appropriate surveillance periods in which low-intensity, early-stage prostate cancer is discovered in time for a prostatectomy for benign hyperplasia. Such cancers rarely progress during the first five years after detection. On the other hand, for younger men, healing treatment is often more appropriate.

Where the prostate cancer was located and the patient's life expectancy is ten years or more, radical prostatectomy offers the best opportunity for the eradication of the disease. Historically, the drawback of this procedure is that more cancers extend beyond the limit of the operation by the time it was detected. However, the use of specific antigen-prostate tests has allowed the early detection of prostate cancer. As a result, the surgery is less extensive with very few complications. Patients with bulky tumors, of high grade and less typically are successively treated by radical prostatectomy.

After surgery, if there are serum concentrations of specific antigen-prostate detectable, persistent cancer is indicated. In many cases concentrations of specific antigen-prostate can be reduced by radiation treatment. However, this concentration often inses again in two years.

Radiation therapy has also been widely used as an alternative for radical prostatectomy. Patients usually treated by radiation therapy are those who are older and less healthy and those with higher-grade tumors, more clinically advanced. Particularly preferred methods are external beam therapy which involves three dimensions, traditional radiation therapy in which the radiation field is derived according to the treated volume; interstitial radiation therapy in which seeds of radioactive compounds are implanted using ultrasound guides; and a combination of external beam therapy and interstitial radiation therapy.

For treatment of patients with locally advanced disease, hormone therapy prior to or after radical prostatectomy or radiation therapy has been used. Hormone therapy is the main form of treatment for men with disseminated prostate cancer. Orchiectomy reduces serum testosterone levels, while treatment with estrogen is similarly beneficial. Estrogen diethyl ester is another hormone therapy used that has the disadvantage that it causes cardiovascular toxicity. When the gondotropin-releasing agonist hormones are administered, the testosterone levels are finally reduced.

Flutamide and other non-steroids, anti-androgen agents block the binding of testosterone to its intracellular receptors. As a result, it blocks the effect of testosterone, insing testosterone concentrations in the serum and allowing patients to remain potent - a significant problem after radical prostatectomy and radiation treatments.

Cytotoxic chemotherapy is largely ineffective in prostate cancer treatments. Its toxicity makes such therapies unfit for elderly patients.

In addition, prostate cancer is relatively resistant to cytotoxic agents.

Use of Monoclonal Antibodies in the Detection and Treatment of Cancer in the Prostate.

Theoretically, radiolabeled monoclonal antibodies ("mAbs") offer the potential to improve both the sensitivity and specificity of prostate cancer detection in lymph nodes and elsewhere. While many mAbs have been previously prepared against antigens related to the prostate, none of these mAbs were specifically generated with an objective image in mind. However, the clinical need leads to the evaluation of some of these mAbs as possible imaging agents. Vihko et al, "Radioimaging of Prostatic Carcinoma With Prostatic Acid Phosphatase-Specific Antibodies", Biotechnology in Diagnostics, 131-134 (1985); Babaian et al., "Radioimmunological Imaging of Meta-tic Prostatic Cancer With 111-Indium-Labeled Moroclonal Antibody PAY 276", J. Urol., 137: 439-443 (1987); Leroy et al., "Radioimmunodetection of Ly ph Node Invasion In Prostatic Cancer: The Use of Iodine 123 (123-1) - Marked Monoclonal Anti-Prostatic Acid Phosphatase (PAP) 227 A F (ab ') 2 fragments of In Vivo Antibody, "Cancer, 64: 1-5 (1939); Meyers and collaborators, "Developmenet Of Monoclonal Antibody Imaging Of Metastatic Prostatic Carcinoma ", The Prostate, 14: 209-220 (1989).

In some cases, monoclonal antibodies developed for the detection and / or treatment of prostate cancer recognize specific antigens for malignant prostatic tissues. Such antibodies are therefore used to distinguish malignant prostatic tissues (for treatment or detection) from benign prostatic tissues, See Patent No. 4,902,615 to Freeman et al.

Other monoclonal antibodies react with surface antigens on all epithelial cells of the prostate either cancerous or benign. See US Patent Nos. 4,446,122 and Re 33,405 of Chu et al., US Patent No. 4,863,851 of McEwan et al., And US Patent No. 5,055,404 of Ueda et al. However, the antigens detected by these monoclonal antibodies are present in singre, and therefore, they compete with localized tumor antigens for the monoclonal antibodies. These causes support the rumor that makes the use of such antibodies unsuitable for in vivo imaging. In therapy, such antibodies, if JQ binds to a cytotoxic agent, could be harmful to other organs.

Horoszewicz et al., "" Monoclonal Antibodies to a New Antigen Marker in Epithelial Prostatic Cells and Serum of Prostate Cancer Patients, "Anticancer Research, 7: 927-936 (1987) (" Horoszewicz ") and US Patent No. 5,162,504 to Horoszewicz describe an antibody, designated 7E11, which recognizes prostate-specific membrane antigens ("PSMA"). Israeli et al., "Molecular Cloning of a Complementary DNA Encoding to Prostate-specific membrane antigen", Cancer Research, 53: 227-230 ( 1993) ("Israeli") describes the cloning and sequencing of PSMA and reports that PSMA is specific to the prostate and shows an increase in expression levels in metastatic sites and hormone-recalcitrant stages. Other studies have indicated that PSMA is more strongly expressed in prostate cancer cells in relation to cells of the normal prostate or of a prostate with benign hyperplasia. In addition, PSMA is not found in serum (Troyer et al., "Detection and Characterization of the Prostate-Specific Membrane Antigen (PSM?) In Tissue Extracts and Body Fluids ", Int. J. Cancer, 62: 52-558 (1995)).

These characteristics make PSMA an attractive target for antibody, a mediator target for imaging and prostate cancer therapy. Imaging studies using indium-labeled 7E11 have indicated that the antibody locates both prostate and sites of metastasis completely well. In addition, 7E11 appears to have clearly better sensitivity to detect lesions compared to other commonly available imaging techniques, such as CT and MR imaging or bone scan. Bander, "Current Status of Monoclonal Antibodies for Imaging and Therapy of Prostate Cancer, "Sem. In Oncology, 21: 607-612 (1994).

However, the use of 7E11 and other known PSMA antibodies to mediate imaging and therapy had several disadvantages. First, PSMA is an integral membrane protein known to have a short intercellular stem and a large extracellular region. The biochemical and mapping characterization (Troyer et al., "Biochemical Characterization and Mapping of the 7E11-C5.3 Epitope of, the Prostate-specific Membrane Antigen", Urol. Oncol., 1: 29-37 (1995)) has demonstrated that the antigenic or epitope site to which the 7E11 antibody binds is present in the intracellular portion of the molecule. Because antibody molecules under normal circumstances do not cross the cell membrane unless they bind the extracellular portion of the molecule and become translocated intracellularly, the 7E11 antibody does not have access to its target antigenic site in some other healthy way, in cells feasible Consequently, imaging using 7E11 is limited to the detection of dead cells in tumor deposits. Additionally, the therapeutic use of antibody 7E11 is limited, because only cells that are already dead can be effectively targets.

Although inadequacy and problems for the diagnosis and treatment of a particular type of cancer are the focus of the preceding discussion, prostate cancer is simply a representative model. The diagnosis and treatment of numerous other cancers have similar problems.

The present invention is directed towards overcoming the deficiencies of previous antibodies of the specialty in diagnosis and treatment or cancer of the prostate and other types.

BRIEF DESCRIPTION OF THE INVENTION One aspect of the present invention concerns a method of removing or destroying cancer cells, the process involves providing a biological agent which, when contacted with an extracellular region of prostate-specific membrane antigens, recognizes the extracellular region of the antigen of specific membrane of prostate. These biological agents are contacted with vascular endothelial cells near cancer cells under effective conditions to allow both binding of the biological agent to the vascular endothelial cells next to cancer cells and destruction or removal of the cancer cells. The biological agent can be used alone or can be associated with a substance effective to destroy or remove the cancer cells on the binding of the biological agent to endothelial cells that are close to the cancer cells.

In a particularly preferred embodiment of the method of removing or killing cancer cells according to the present invention, the biological agent, when contacted with an extracellular region of prostate-specific membrane antigen, binds to and is internalized with the antigen. of prostate-specific membrane of such cells. Preferred biological agents for use in the method of removing or destroying cancer cells according to the present invention are antibodies or binding portions thereof, probes or ligands. The methods of the present invention are particularly used in the destruction or extirpation of renal, urothelial, colon, rectal, lung, and breast cells and liver metastatic adenocarcinoma cells.

Another aspect of the present invention concerns the method of detecting cancerous tissue in a biological sample. This method involves providing a biological agent that, when contacted with an extracellular region of prostate-specific membrane antigen, binds to the extracellular region of the prostate-specific membrane antigen. The biological agent is associated with an effective marker to allow the detection of vascular endothelial cells close to or in the cancerous tissue above the binding of the biological agent to the vascular endothelial cells close to or in the cancerous tissue. The biological sample is contacted with the biological agent having a marker under effective conditions to allow binding of the biological agent to vascular endothelial cells close to or in the cancerous tissue in the biological sample. The presence of cancerous tissue in the biological sample is detected by detection of the marker.

In a particularly preferred embodiment of the method of detecting cancerous tissue in accordance with the present invention, the biological agent is one which, when contacted with an extracellular region of prostate-specific membrane antigen, binds to and is internalized with a prostate-specific membrane antigen. Preferred biological agents for use in the method of detecting cancerous tissue according to the present invention are antibodies or binding portions thereof, probes, or ligands. The method is especially used in the detection of renal, urothelial, colon, rectal, lung, and breast cancerous tissues of metastatic adenocarcinoma of the liver.

Yet another aspect of the present invention concerns a method of excision or destruction of normal, benign hyperplastic, and prostate epithelial cancer cells. The process involves providing a biological agent that recognizes an extracellular region of a prostate antigen with a specific membrane. The biological agent can be used alone or it can be associated with an effective substance to destroy the cells on the link of the biological agent to the cells. These biological agents are then contacted with the cells under conditions effective to allow both bonds of the biological agent to the extracellular region of the prostate-specific membrane antigen and destroy or remove the cells.

In a particularly preferred embodiment of the method of ablation or destruction of normal, benign hyperplastic, and prostate epithelial cells according to the present invention, the biological agent binds to and is internalized with the prostate-specific membrane antigen of such cells. Preferred biological agents for use in the method of removal or destruction of normal, benign hyperplastic, and prostate cancer epithelial cells according to the present invention are antibodies or binding portions thereof, probes, or ligand.

Another aspect of the present invention concerns a method of detecting normal, hyperplastic benign, and cancerous prostate epithelial cells or portions thereof in a biological sample. This method involves providing a biological agent that binds to an extra-terminal region of the prostate-specific membrane antigen. The biological agent is associated with an effective marker to allow the detection of the cells or portions thereof on bonds of a biological agent of the cells or portions thereof. The biological sample is contacted with the biological agent having a marker under conditions effective to allow binding of the biological agent to the extracellular region of the prostate-specific membrane antigen of any of the cells or portions thereof in the biological sample. The presence of any cells or portions thereof in the biological sample is detected by detection of the tag.

In a particularly preferred embodiment of the method of detecting normal, benign hyperplastic, and prostate cancer epithelial cells according to the present invention, the biological agent binds to and is internalized with the prostate-specific membrane antigen of such cells. Preferred biological agents for use in the detection method of normal, benign hyperplastic, and prostate cancer epithelial cells according to the present invention are antibodies or binding portions thereof, probes, or ligands.

Another aspect of the present invention corresponds to a biological people that recognizes an extracellular region of prostate-specific membrane antigen. In a preferred embodiment, the isolated biological agent binds to and is internalized with the prostate-specific membrane antigen. Preferred isolated biological agents that recognize an extracellular region of prostate-specific membrane antigen according to the present invention are isolated anti-bodies, or binding portions thereof, probes or ligands. Hybridoma cell lines that produce monoclonal antibodies of these types are also presented.

The biological agents of the present invention recognize the extracellular region of normal cell, benign hyperplastic, and prostate epithelial cancer antigens. The antibody other than 7E11, which recognizes an epitope of associated prostate antigens that are exposed extracellularly only after cell lysis, the biological agents of the present invention bind to antigenic epitopes that are exposed extracellularly in living prostate cells. Using the biological agents of the present invention, living cells, normal non-fixed, benign hyperplastic, and cancerous prostate epithelial can be objectified, which makes the treatment and diagnosis more effective. In a preferred embodiment for treatment of prostate cancer, the biological agents of the present invention also bind to and are internalized with the prostate-specific membrane antigen, which allows the use of therapeutics of cytotoxic agents that act intracellularly.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is an immunoelectronic micrograph of gold-labeled monoclonal antibody J591 on the surface of LNCaP cells after incubation at 4 ° C.

Figure 2 is an immunoelectronic micrograph of LNCaP cells treated with gold-labeled monoclonal J591 antibody after 10 minutes of incubation at 37 ° C.

Figure 3 is an immunoelectronic micrograph of LNCaP cells treated with gold-labeled monoclonal J591 antibody after 10 minutes of incubation at 37 ° C.

Figure 4 is an immunoelectronic micrograph of LNCaP cells treated with gold-labeled monoclonal J591 antibody after 15 minutes of incubation at 37 ° C.

Figure 5 is an immunoelectronic micrograph of LNCaP cells treated with gold-labeled monoclonal J591 antibody after 15 minutes at 37 ° C showing J591 in endosomes.

Figure 6 summarizes the sequence formation strategy of heavy chain J591 antibody.

Figure 7 shows the heavy chain nucleotide sequence of the monoclonal antibody J591 (designated (SEQ ID NO. 1), the nucleotide sequence of the corresponding inverse, non-coding filaments (designated SEQ ID No. 2), and the corresponding deduced amino acid sequences (designated SEQ ID Nos. 3, 4 and 5).

Figure 8 is a comparison of the heavy chain of monoclonal antibody J591 with the consensual sequence for Heavy Chain of Mouse Subgroup IIA.

Figure 9 summarizes the sequence formation strategy of the kappa light chain of the monoclonal antibody J591.

Figure 10 shows the nucleotide sequences of the kappa light chain of the monoclonal antibody J591 (designated SEQ ID No.), the nucleotide sequence of the corresponding inverse, non-coding filament (designated SEQ ID No. 10), and the corresponding deduced amino acid sequences (designated SEQ ID Nos. 11, 12, and 13).

Figure 11 is a comparison of the kappa light chain of monoclonal antibody J591 with the consensual sequence for Kappa Chains of Mouse Subgroup V.

Figures 12A-12F are micrographs (250 X amplification) that show the immunohistochemical reactivity of mAb J591 in neovasculature of several carcinomas.

DETAILED DESCRIPTION OF THE INVENTION One aspect of the present invention concerns a method of removal or destruction of normal, benign hyperplastic, and prostate cancer epithelial cells.

The process involves providing a biological agent, such as an antibody or binding portion thereof, probes, or ligands, that bind to an extracellular region of prostate-specific membrane antigen (e.g., a portion of prostate-specific membrane antigen that is external a) such cells, the biological agent can be used alone or can be associated with a substance effective to destroy the cells on the binding of the biological agent to the cells. These biological agents are then contacted with the cells under conditions effective to allow both bonds, from a biological agent to the extracellular region of the prostate-specific membrane antigen and destruction or removal of the cells. In its preferred form, such contact is carried out in a living mammal by administration of the biological agent to the mammal under conditions effective to allow both binding of the biological agent to the extracellular region of the prostate-specific membrane antigen and destruction or removal of the cells . Such administration can be carried out orally or parenterally.

In a particularly preferred embodiment of the method of extirpation or destruction of normal, benign hyperplastic, and epithelial cells is cancer prostate according to the present invention, the biological agent that binds to and is internalized with the prostate-specific membrane antigen of such cells. Again, the biological agent can be used alone. Alternatively, the biological agent may be associated with a substance effective to kill cells on the binding of the biological agent to the prostate-specific membrane antigen and on the internalization of the biological agent with the prostate-specific membrane antigen.

The mechanisms by which the biological agent is internalized with the prostate-specific membrane antigen is not critical to the practice of the present invention. For example, the biological agent can induce the internalization of the prostate-specific membrane antigen. Alternatively, the internalization of the biological agent may be the result of routine internalization of the prostate-specific membrane antigen.

The biological agents described above (for example, biological agents, such as an antibody or binding portion thereof, probes or ligands which, when contacted with an extracellular region of prostate-specific membrane antigen, recognize the extacellular region of the prostate. prostate-specific membrane antigen and, preferably, internalized with it) can be used to remove or destroy cancer cells. In this aspect of the present invention, the biological agent can be used alone or can be associated with a substance effective to kill the cancer cells on top of the binding of the biological agent of vascular endothelial cells close to them. These biological agents are brought into contact with vascular endothelial cells close to the cancer cells. The contact is carried out under conditions that are effective to allow the binding of the biological agent to the vascular endothelial cells close to the cancer cells and, in addition, they are effective in destroying or removing the cancer cells. The mechanism by which cancer cells are destroyed or excised is not critical to the practice of the present invention. For example, cancer cells can be destroyed or directly removed by the biological agent, a consequence of their proximity to vascular endothelial cells to which the biological agent binds. Alternatively, the biological agent may destroy, remove, or otherwise change the properties of the vascular endothelial cells to which it binds so that the blood flows to the nearby cancer cells and is stopped or otherwise reduced, thereby cause cancer cells to be destroyed or removed. Accordingly, the method of the present invention is particularly useful for destroying or ablating vascular endothelial cells in cancerous tissues as well as cancer cells contained in cancerous tissue.

In a particularly preferred embodiment of the method of removing or killing cancer cells according to the present invention, the biological agent used is one which, when contacted with an extracellular region of prostate-specific membrane antigen, binds to and is internalized with the extracellular region of the prostate-specific membrane antigen. The methods of the present invention are particularly useful for destroying or removing cancerous prostate epithelial cells as well as other cancer cells other than prostate, examples of cancer cells that are not prostate epithelial cells are renal, urothelial, colon cells, rectal, lung, and breast cancer cells and metastatic adenocarcinoma of the liver. However, the method of the present invention can be used to destroy or remove any cell that expresses an extracellular region of prostate-specific membrane antigen or a portion of it whose subsistence is dependent on cells expressing an extracellular region of specific membrane antigen. Prostate or a portion thereof, the method of the present invention is particularly useful for destroying or removing cancer cells, because cancer endothelial cells that supply blood to cancerous tissues (e.g., tumors, sets of cancer cells, or other cancerous masses) express an extracellular region of prostate-specific membrane antigen, regardless of the type of cancer involved. In contrast, vascular endolteial cells supplying blood to normal tissues do not express an extracellular region of prostate-specific membrane antigen.

Another aspect of the present invention concerns a method of detecting normal, benign hyperplastic, and cancerous epithelial cells or portions thereof in a biological sample. This method involves providing a biological agent, such as an antibody or binding portion thereof, probes, or ligands, that bind to an extracellular region of prostate-specific membrane antigen of such cells. The biological agent is associated with an effective marker to allow the detection of cells or portions (e.g., prostate-specific membrane antigen or fragments thereof released from such normal, hyperplastic benign, and cancerous cells) of these upon binding to the prostate. biological agent to the cells or portions thereof. The biological sample is contacted with the biological agent having a marker under effective conditions to allow binding of the biological agent to the extracellular region of the prostate-specific membrane antigen of any of the cells or portions thereof in the biological sample. The presence of any cells or portions thereof in the biological sample is detected by detection of the tag. It is their preferred form that such contact is carried out in a living mammal and involves the administration of the biological agent to the mammal under effective conditions to allow the binding of the biological agent to the prostate-specific membrane antigen of any of the cells or portions thereof. these in the biological sample. Again such administration can be carried out orally or parenterally.

The method of the present invention can be used to screen patients for diseases associated with the presence of normal, benign hyperplastic, and cancerous epithelial cells or portions thereof.

Alternatively, it can be used to identify the recurrence of such diseases, particularly when the disease is localized in a biological material of the patient. For example, the recurrence of prostatic disease in the prostate fossa can be found following radical prostatectomy. Using the method of the present invention, this recurrence can be detected by administering a short range of radiolabelled antibodies to the mammal and then detecting the marker rectally, such as a transrectal probe detector.

Alternatively, the contacting step can be carried out in a serum or urine sample or other bodily fluids, such as to detect the presence of PSMA in the body fluid. When the contact is carried out in a serum or urine sample, it is preferred that the biological agent recognizes that there is no circulation of other antigens other than PSMA, in the blood. Since intact prostatic cells do not excrete or secrete PSMA in the extracellular domain, the detection of PSMA in serum, urine, or other body fluids generally indicates that prostate cells are being lysed. Accordingly, the biological agents and methods of the present invention can be used to determine the effectiveness of the prostate cancer treatment protocol by monitoring the level of PSMA in serum, urine, or other body fluids.

In a particularly preferred embodiment of the method of detecting normal, benign hyperplastic, and prostate epithelial cancer cells according to the present invention, the biological agent, such as the antibody or binding portions thereof, probes, or ligands, bind to and are internalized with the enterocyte membrane antigen of the prostate of such cells. Again, the biological agent is associated with an effective marker to allow the detection of the cells or portions thereof, such as the binding of the biological agent to, and internalization of the biological agent with the specific membrane antigen of the prostate.

Another aspect of the present invention concerns the method of detecting cancerous tissue in a biological sample. This method involves providing the biological agent described above (e.g., a biological agent, such as an antibody or binding portion thereof, probe or ligand that, when contacted with an extracellular region of prostate-specific membrane antigen, recognizes the extracellular region of the prostate-specific membrane antigen ). The biological agent is associated with a marker that is effective to allow the detection of vascular endothelial cells close to or in the cancerous tissue over the binding of the biological agent to vascular endothelial cells close to or in the cancerous tissue. The biological sample is then contacted with the biological agent having the marker, the contact is carried out under conditions effective to allow the binding of the biological agent to vascular endothelial cells close to or in the cancerous tissue in the biological sample. The presence of cancer cells or portions thereof in the biological sample is detected by detection of the mark.

Before contacting the complete biological sample with the biological agent, the use of a portion of the biological sample is contemplated. For example, a tissue biopsy sample may be contacted with the biological agent to determine the presence of cancerous tissues in the sample of tissue biopsies as well as in the larger biological sample since it is taken.

Alternatively, the biological agent can be contacted with a serum or urine sample to hit any of the vascular endothelial cells that express an extracellular region of prostate-specific membrane antigen, which are presented here.

Since vascular endothelial cells that express an extracellular region of prostate-specific membrane antigen are found in the vasculature of cancerous tissues but not in the vasculature of normal tissues, the detection of the mark in a serum or urine sample, indicates the presence of cancerous tissue in the broad biological sample since it is taken (eg, a patient).

In a particularly preferred embodiment of the method of detecting cancerous tissues according to the present invention, the biological agent employed is one which, when contacted with an extracellular region of prostate-specific membrane antigen, binds and is internalized with the specific membrane antigen of the prostate. The methods of the present invention can be used to detect cancerous epithelial cells of the prostate as well as cancerous tissues containing cancer cells other than prostate cancer epithelial cells. Examples of cancerous tissues containing cancer cells other than prostate cancer epithelial cells that can be detected with the methods of the present invention include renal, urothelial, colon, rectal, lung, and breast cancerous tissues and metastatic adenocarcinoma cancerous tissues. of the liver As indicated above, appropriate biological agents to either destroy, remove, or detect normal and cancerous, benign hyperplastic cells, and cancerous prostate epithelial cells include antibodies, such as monoclonal or polyclonal antibodies. In addition, fragments of antibodies, media antibodies, hybrid derivatives, probes, and other molecular constructions can be used. These biological agents, such as antibodies, binding portions thereof, probes, or ligands, bind to extracellular regions of prostate-specific membrane antigen or portions thereof in normal, hyperplastic benign, and prostate cancer epithelial cells, biological agents link to all these cells, not only for cells that are fixed or cells whose intracellular antigenic regions are otherwise exposed to the extracellular domain. Consequently, the binding of biological agents is concentrated in areas where there are prostate epithelial cells, regardless of whether these cells are either fixed or unfixed, feasible or necrotic. Additionally or alternatively, these biological agents, such as antibodies, binding portions thereof, probes, or ligands, bind to and are internalized with prostate-specific membrane antigens or portions thereof in normal, benign hyperplastic, and cancerous prostate epithelial cells.

The production of monoclonal antibodies can be effected by techniques that are well known in the art.

Basically, the process involves first obtaining immune cells (lymphocytes) from the spleen of a mammal (e.g., mouse) that has previously been immunized with the antigen of interest either in vivo or in vi tro. The secreting lymphocytes of the antibody are then fused with myeloma cells (mouse or transformed cells, which are capable of replicating indefinitely in cell culture, thereby producing an immortal, immunoglobulin secreting cell line.) The resulting fused cells, or hybridomas, are cultured, and the resulting colonies examined for the production of the desired monoclonal antibodies.

The colonies that produce such antibodies are cloned, and developed either in vivo or in vi tro to produce large amounts of antibodies. A description of the theoretical bases and practical methodology of fusing cells is sequentially located in Kohler and Milstein, Nature 256: 495 (1975), which is hereby incorporated by reference.

Mammalian lymphocytes are immunized by in vivo immunization of the animal (e.g., a mouse) with the protein or polypeptide of the present invention. Such immunizations are repeated as necessary at intervals of up to several weeks to obtain a sufficient antibody titer. After the last increase of antigens, the animals are sacrificed and the spleen cells are removed.

Fusion with mammalian myeloma cells or other fusion partners capable of replicating indefinitely in cell culture is effected by standard and well-known techniques, for example, by the use of polyethylene glycol ("PEG") or other fusion agents (See Milstein and Kohler , Eur. J. Immunol., 6: 511 (1976), which is incorporated herein by reference). This immortal cell line, which is preferably murine, but can also be derived from cells of other mammalian species, including but not limited to rats and humans, is selected to be deficient in enzymes necessary for the utilization of certain nutrients, be capable of rapid development, and by having good fusion capacity. Many such cell lines are known to those skilled in the art, and others are regularly described.

Procedures for producing polyclonal antibodies are also well known. Typically, such antibodies can be produced by administering the protein or polypeptide of the present invention subcutaneously to New Zealand white rabbits that have been first bled to obtain pre-immune serum. The antigens can be injected up to a total volume of 100 μl "per site" in six different sites. Each injected material will contain the synthetic surfactants pluronic polyols as adjuvants, or powdered acrylamide gel containing the protein or the polypeptide before electrophoresis of the SDS-polyacrylamide gel. The rabbits are then bled two weeks before the first injection and periodically boosted with the same antigen three times every six weeks. A serum sample is then collected ten days before each reinforcement. Polyclonal antibodies are recovered from the serum by affinity chromatography using the corresponding antigen to capture the antibody. Finally, the rabbits are euthanized with pentobarbital 150 mg./Kg IV. These and other methods for culturing polyclonal antibodies are set forth in E. Harlow, et al., Editors, Antibodies: A Laboratory Manual (1988), which is incorporated herein by reference.

In addition to using the complete antibodies, the processes of the present invention encompass the use of binding portions of such antibodies. Such linking portions include Fab fragments, F (ab ') 2 fragments, and Fv fragments.

These antibody fragments can be made by conventional methods, such as proteolytic fragmentation procedures, as described in J. Goding, Monoclonal Antibodies: Principies and Practice, pp. 98-118 (N. Y. Academic Press 1983), which is incorporated herein by reference Alternatively, the processes of the present invention can utilize probes or ligands found either in nature or synthetically prepared by recombinant DNA procedures or other biological or molecular methods. Appropriate probes or ligands are molecules that bind to the extracellular regions of prostate-specific membrane antigens identified by the monoclonal antibodies of the present invention. Other appropriate probes or ligands are molecules that bind to and are internalized with prostate-specific membrane antigens. Such probes or ligands can be, for example, proteins, peptides, lectins or nucleic acid probes.

It is particularly preferred to use the monoclonal antibodies identified below in Table 1.

TABLE 1 Name of the Anti ATCC Designation for Monoclonal Bodies the Hybridoma Cell Line E99 HB-12101 J415 HB-12109 J533 HB-12127 J591 HB-12126 These antibodies can be used alone or as a component in a mixture with other antibodies or other biological agents to treat cancers or image formation of cancerous tissues (particularly the endothelial cells there) or prostate epithelial cells with characteristic variants on the antigenic surface .

Although, either the biological agents are used for treatment or diagnosis, they may be administered orally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by nasal instillation, intracavity or intravesicular instillation, intraocularly, intraarterially, intralesionally, or by Application on mucous membranes, such as, of the nose, throat, and bronchial tubes. They may be administered alone or with pharmaceutically and physiologically acceptable carriers, excipients, or stabilizers, and may be in solid or liquid form such as tablets, capsules, powders, solutions, suspensions, or emulsions.

The dosage unit of the solid forms can be of the conventional type. The solid form may be a capsule, such as an ordinary gelatin type containing the biological agent, such as an antibody or binding portion thereof, of the present invention and inert fillers such as lactose, sucrose, or corn starch. In another embodiment, these compounds are tableted with bases for comventional tablets such as lactose, lactose, sucrose, or corn starch in combination acacia-like binders, corn starch, or gelatin, disintegrating agents such as, corn starch, potato starch, or alginic acid, and a lubricant similar to stearic acid or magnesium stearate.

The biological agent of the present invention can also be administered in injectable dosages in solution or in suspension of these materials in a physiologically acceptable diluent with a pharmaceutical vehicle. Such vehicles include sterile liquids such as water and oils, with or without the addition of a surfactant and other physiologically and pharmaceutically acceptable carriers, which include adjuvants, excipients or stabilizers. Illustrative oils are those originating in petroleum, of animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil, in general, water, saline, aqueous dextrose and sugar-related solutions, and glycols such as, propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions.

For use in aerosols, the biological agent of the present invention in solution or in suspension can be packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants such as propane, butane, or isobutane with conventional adjuvants. The materials of the present invention may also be administered in a non-pressurized form such as a nebulizer or atomizer.

Biological agents can be used to detect cancerous tissues (particularly the vascular endothelial cells of the present) and normal, hyperplastic benign, and prostate cancer epithelial cells in vivo. This is achieved by labeling the biological agent, administering the labeled biological agent to a mammal, and then by imaging the mammal.

Examples of useful markers for diagnostic imaging in accordance with the present invention are radiolabels such as mi, mi, 99mTc, 32P, 12 &I, 3H, 34C, and i? RR, fluorescent labels such as fluorescein and rhodamine, markers active to nuclear magnetic resonance, isotopes that emit positrons detectable by the positron emission tomography scanner ("PET"), chemiluminescent such as luciferin, and enzymatic labels such as peroxidase or phosphates.

Short-range radiation emitters such as isotopes detectable by short-range detector probes, such as a transrectal probe, can also be employed, these isotopes and transrectal detection probes, when used in combination, are especially useful in detecting recurrences in the prostatic fossa and pelvic nodal disease. The biological agent can be labeled with such reagents using techniques known in the art.

For example, see Wensel and Meares, Radioimmunoimaging and Radioimmunotherapy, Elsevier, New York (1983), which is incorporated herein by reference, by techniques relating to the radiolabelling of antibodies. See also, D. Colcher et al., "Use of Monoclonal Antibodies as Radiopharmaceuticals for the localization of Human Carcinoma Xenografts in Athymic Mice", Meth. Enzymol. 121: 802-816 (1986), which is incorporated herein by reference.

A radiolabelled biological agent of this invention can be used for in vi tro diagnostic tests. The specific activity of a tainted biological agent, such as a taguised antibody, binding portion thereof, probe or ligand, depends on the half-life, the isotopic purity of the radioactive label and on how the label is incorporated into the biological agent, in the Table 2 lists several commonly used isotopes, their specific activities and half-life times, in immunoassay tests, the greater the specific activity, in general the better the sensitivity.

TABLE 2 Isotopes Specific Activity of Average Life Pure Isotope (Curies / Mol) i4C 6.25 X 101 5720 years 3H 2.01 X 10. 12.5 years i25l 2.18 X lOe 60 days Methods for labeling the biological agents with radioactive isotopes listed in Table 2 are generally known in the art. Tritium labeling procedures are described in US Patent No. 4,302,438, which is incorporated herein by reference. Iodatives, tritium labeling, and 35S labeling procedures specially adapted for murine monoclonal antibodies are described by Goding, J. W. (supra, pp 124-126) and the references cited herein, which are incorporated herein by reference. Other methods for iodinating biological agents, such as antibodies, binding portions thereof, probes, or ligands, are described by Hunter and Greenwood, Nature 144: 945 (1962), David et al., Biocheraistry 13: 1014-1021 (1974), and US Patent Nos. 3,867,517 and 4,376,110, which are incorporated herein by reference. Radiolabel elements that are useful in imaging include 123I, mi, mln, and 99mTc, for example. Procedures for iodination of biological agents are described by Greenwood, F. et al., Biochem. J. 89: 114-123 (1963); Marchalonis, J., Biochem. J. 113: 299-305 (1969); and Morrison, M. et al., Immunochemistry, 289-297 (1971), which are incorporated herein by reference. Methods for labeling with 9mTc are described by Rhodes, B. et al. in Burchiel, S. et al. (eds.) Tumor Imaging: The Radioimmunochemical Detection of Cancer, New York: Masson 111-123 (1982) and references cited herein, which are incorporated herein by reference. Appropriate procedures for labeling per ml of biological agents are described by Hnatowich, D. J. et al., J. Immul. Methods, 65: 147-157 (1983), Hnatowich, D. et al., J. Applied Radiation, 35: 554-557 (1984), and Buckley, RG et al., FEBS 166: 202-204 (1984), which they are incorporated herein by reference. ' In the case of radiolabelled biological agents, the biological agent that is administered to the patient is located in the tumor in the presence of the antigen with which the biological agent reacts, and "image formed" is detected in vivo using known techniques such as radionuclear scan using for example, a gamma or emission tomography camera. See, for example, A. R. Bradwell et al., "Developments in Antibody Imaging," Monoclonal Antibodies for Cancer Detection and Therapy, R. W. Baldwin et al., (Eds.), Pp. 65-85 (Academic Press 1985), which is incorporated herein by reference. Alternatively, a scanner of transaxial positron emission tomography, such as the so-called Pet IV located in "brookhaven National Laboratory, can be used when the radiolabel emits positrons (for example, uC,? ßF, isO, and 13N).

Chlorophoric and chromophoric labeled biological agents can be prepared from standard portions known in the art. Since antibodies and other proteins absorb light rays having wavelengths greater than about 310 nm, the fluorescent portions could be selected to have a substantial absorption of wavelengths of about 310 nm and preferably above 400 nm. A variety of suitable fluorescers and chromophores are described by Stryer, Science, 162: 526 (1968) and Brand, L. et al., Annual Review of Biochemistry, 41: 843-868 (1972), which are incorporated herein by reference . Biological agents can be labeled with fluorescent chromophore groups by conventional methods such as those set forth in US Patent Nos. 3,940,475, 4,289,747, and 4,376,110, which are incorporated herein by reference.

A group of fluorescers having a number of the desired properties described above are Xanthene tinctures, which include fluorescein derived from 3,6-dihydrohydroxy-9-henilxantenhydrole and resminines and rhodamines derived from 3,6-diamino-9-phenylxantenhydro and lysanima rhodamine B. Rhodamine and fluorescein derived from 9-0-carboxyphenylxantenhydrole have a 9-0-carboxyphenyl group. Fluorescein compounds having reactive coupling groups such as amino and isothiocyanate groups such as fluorescein isothiocyanate and fluorescamine are readily available. Another group of fluorescent compounds are the naphthylamines, which have an amino group in position a or β.

Biological agents can be labeled with fluorochromes or chromophores by the procedures described by Goding, J. (supra, pp 208-249). Biological agents can be labeled with an indicator group containing the 19 F atom, active NMR, or a plurality of such atoms since (i) substantially all naturally abundant fluoride atoms are 19 F and isotopes. consequently substantially all compounds containing fluoride are NMR-active; (ii) many chemically active polyfluorinated compounds such as trifluoroacetic anhydride are commercially available at relatively low cost, and (iii) many fluorinated compounds have been found medically acceptable for use in humans such as perfluorinated polyethers used to transport oxygen as a replacement for hemoglobin. Before allowing such incubation times, an NMR determination of the whole body is carried out using an apparatus such as one of those described by Pykett, Scientific American, 246: 78-88 (1982), which is incorporated herein by reference , to locate and form images of cancerous tissues (particularly the vascular endothelial cells of the present) and prostate epithelial cells.

In cases where it is important to distinguish between regions containing living or dead prostate epithelial cells or to distinguish between living and dead prostate epithelial cells, the antibodies of the present invention (or other biological agents of the present invention), labeled as described above, it can be co-administered in conjunction with an antibody or other biological agent that recognizes only living or only dead prostate epithelial cells labeled with a marker that can be distinguished from the label used to label the subject antibody. of the two marks at various locations or times, temporal and spatial concentration variations of normal living or dead cells, benign hyperplastic, and prostate cancer epithelium and can be investigated.In particular, this method can be carried out using antibodies marked of the present and invention, which recognize both dead or living prostate epithelial cells, and labeled 7E11 antibodies, which recognize only dead prostate epithelial cells.

Biological agents can also be used to destroy or remove normal and cancerous cells, benign hyperplastics, and prostate cancer epithelial cells in vivo. This involves using the biological agents themselves or with a ctotoxic drug to which the biological agents of the present invention (eg, biological agents that recognize normal cells, benign hyperplastics, and cancerous prostate epithelial cells) are associated. This involves administering the biological agents associated with a cytotoxic drug to a mammal that requires such treatment. In the case of cancerous prostate epithelial cells, since biological agents recognize prostate epithelial cells, any such cells to which the biological agents bind are destroyed. Although such administration can destroy normal prostate epithelial cells, this is not problematic, because the prostate is not required to live or survive. Although the prostate can indirectly contribute to fertility, this does not appear to be one in patients receiving the treatment of the present invention, in the case of cancerous tissues, since the biological agents recognize vascular endothelial cells that are close to cancer cells, binding agents of the biological agent / cytotoxic drug complex so that these vascular endothelial cells destroy them, thereby cutting off the blood flow to the next cancer cells and thereby destroying or removing these cancer cells.

Alternatively, the biological agents, by virtue of their binding to vascular endothelial cells that are close to the cancer cells, are located close to the cancer cells. Thus by the use of appropriate biological agents (including those containing substances effective to destroy cells indiscriminately but only over a narrow range), cells in cancerous tissues (which include cancer cells) can be selectively destroyed or excised.

The biological agents of the present invention can be used to deliver a variety of cytotoxic drugs including therapeutic drugs, a compound that emits radiation, plant molecules, fungi, or bacterial origin, biological proteins, and mixtures thereof. Cytotoxic drugs can be cytotoxic drugs that act intracellularly, such as low-range radiation emitters, which include, for example, reduced range, high energy to emitter.

Enzymatically active toxins and fragments thereof are exemplified by diphtheria toxin A fragment, non-binding active fragments of diphtheria toxin, exotoxin A (from pseudomonas aeruginosa), ricin A chain, abrin A chain, modecin chain A, a -sacrine, certain proteins aleurites fordii, certain proteins Diantin, proteins Phytolacca americana (PAP, PAPII and PAP-S), inhibitor Morodica charantia, curcin, crotina, inhibitor Saponaria officinalis, gelotin, mitogilin, restrictocin, fenomycin, and enomycin, for example . Methods for preparing enzymatically active polypeptides of immunotoxins are described in WO84 / 03508 and WO85 / 03508, which are incorporated herein by reference. Certain portions of cytotoxins are derived from adriamycin, chlorambucil, daunomycin, methotrexate, neocarcinostatin, and platinum, for example.

Procedures for conjugating biological agents with cytotoxic agents have been previously described. Procedures for conjugating the biological agents with the cytotoxic agents that have been previously described. Methods for conjugating chlorambucil with antibodies are described by Flechner, I., European Journal of Cancer, 9: 741-745 (1973); Ghose, T. et al., British Medical Journal, 3: 495-499 (1972); and Szekerke, M. et al., Neoplasma, 19: 211-215 (1972), which are incorporated herein by reference. Procedures to conjugate daunomycin and adriamycin to antibodies are described by Hurwitz, E. et al., Cancer Research, 35: 1175-1181 (1975), which are incorporated herein by reference. Methods for preparing conjugated antibody-ricin are described in US Patent No. 4,414,148 and by Osawa, T. et al., Cancer Surveys, 1: 373-388 (1982) and references cited herein, which are incorporated herein by reference. reference. Coupling of processes thus also described in EP 86309516.2, which is incorporated herein by reference.

In a particularly preferred embodiment of the present invention, especially well suited to destroy or excise normal, benign hyperplastic, and cancerous prostate epithelial cells, a first biological agent is conjugated to a pro-drug activator. The pro-drug activator is conjugated to a second biological agent according to the present invention, preferably one that binds to a non-suitable site on the prostate-specific membrane antigen molecule. If two biological agents bind in suitable or unfit binder sites it can be determined by conventional selective binding tests. For example, monoclonal antibodies J591, J533, and E99 bind to suitable binding sites on the prostate-specific membrane antigen molecule, monoclonal antibodies J415, on the other side, bind to a binding site that is unfit for the site in the prostate. that J591, J533, and E99 linked. Thus, for example, the first biological agent may be one of J591, J533, and E99, and the second biological agent may be J415. Alternatively, the first biological agent may be J415, and the second biological agent may be one of J591, J533, and E99. Drug-appropriate drug pairs suitable for use in the practice of the present invention are described in Blackely et al., "ZD2767, An Improved System for Antibody-directed Enzyme Prodrug Therapy That Results in Tumor The biological agent of the present invention can be used and marketed together with equipment, as a team, to detect the particular marker.

Biological agents of the present invention can be used in conjunction with other treatments including surgery, radiation, cryosurgery, thermotherapy, hormonal treatment, chemotherapy, vaccines, and other immunotherapies.

Also encompassed in the present invention is a method of destruction or extirpation that involves the use of biological agents by prophylaxis. For example, these materials can be used to prevent or delay the development or progress of prostate cancer or other cancers.

The use of therapeutic methods of the present invention to treat cancer of the prostate and other cancers has a number of benefits. Since the biological agents according to the present invention only target cancerous cells (such as cells from cancerous tissues containing vascular endothelial cells) and prostate epithelial cells, other tissues are excepted. As a result treatment with such biological agents is ensured, particularly for Regressions in Colorectal Tumor Xenografts ", Cancer Research, 56: 3287-3292 (1996), which is incorporated herein by reference.

Alternatively, the biological agent can be coupled to emitters of high energy radiation, for example, a radioisotope, such as me, an emitter?, Which, when it has located the site of a tumor, results in destruction of several cell diameters. See, for example, SE Method, "Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy, RW Baldwin et al., (Eds.) Pp 303-316 (Academic Press 1985), which is incorporated to the present as a reference Other available radioisotopes include a-emitters, such as 212Bi, 233Bi, and 2i: At, and β emitters, such as Isre and 90. Radiation therapy is expected to be particularly effective, because prostate epithelial cells and cells Vascular endothelial cells in cancers are relatively radiosensitive, in which the biological agents are used alone to destroy or remove cancer cells or prostate epithelial cells, such destrucc ions or ablations can be effected to initiate immune functions in endogenous hosts such as complement mediation or antibody-dependent cellular cytotoxicity. patients of advanced age. Treatment according to the present invention is expected to be particularly effective, because it directs high levels of biological agents, such as antibodies or binding portions of these, probes, or ligands, of the spinal cord and lymph nodes in which the metastasis of cancer in the prostate and the metastasis of any other cancer predominate. In addition, the methods of the present invention are particularly well suited for the treatment of cancer in the prostate, because local tumors of prostate cancer tend to be small in size and, therefore, are easily destroyed by cytotoxic agents. the treatment according to the present invention can be effectively monitored with clinical parameters, such as, in the case of prostate cancer, prostate-specific serum antigen and / or pathological features of a patient's cancer, including, stage, incision Gleason, extracapsular, seminal, vesicular or perineural invasion, positive margins, involved lymph nodes, etc. alternatively, these parameters can be used to indicate when such treatment could be employed.

Because the biological agents of the present invention bind to living prostate cells, therapeutic methods for the treatment of prostate cancer use these biological agents which are much more effective than those which are targeted by lysed prostate cells. For the same reasons, diagnostic and imaging methods that determine the location of normal, benign hyperplastic, or cancerous prostate epithelial cells (as well as vascular endothelial cells in cancers) are improved by the use of the biological agents of the present invention. In addition, the ability to differentiate between living and dead prostate cells may be advantageous, especially for monitoring the effectiveness of a particular treatment regimen.

Hybridomas E99, J415, J533, and J591 have been deposited according to, and in satisfaction of, the requirements of the Budapest Treaty in the "International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure with the American Type Culture Collection ( "ATCO" at 12301 Parklawn Drive, Rockville, Maryland 20852. Hybridoma E99 was deposited on May 2, 1996, and received ATCC Designation No. HB-12101. Hybridoma J415 was deposited on May 30, 1996, and received the ATCC Designation. Number: HB-12109. Hybridomas J533 and J591 were deposited on June 6, 1996, and received the ATCC Designation Number HB-12127 and HB-12126, respectively.

The present invention is further illustrated by the following examples.

EXAMPLES Example 1 - Human tissues Recent specimens of benign and malignant tissues were obtained from the Department of Pathology of the New York Hospital Cornell University Medical Center ("NYH-CUMC"), Example 2 - Cultivation of Fabrics Cell lines cultured from human cancers were obtained from the Laboratory of Urological Oncology of NYH-CUMC. PC-3 cell lines of prostate cancer (Mickey, DD, et al., "Characterization of a Human Prostate Adenicarcinoma Cell Line (DU145) As A Monolayer Culture And As A Solid Tumor In Athypic Mice", Prog. Clin. Biol. Res., 37: 67-84 (1980), incorporated herein by reference), DU-145 (Mickey, DD, et al., "Characterization Of A Human Prostate Adenocarcinoma Cell Line (DU145) As A Monolayer Culture And As A Solid Tumor In Athypic Mice, "Prog. Clin. Biol. Res., 37: 67-84 (1980), which is incorporated herein by reference), and LNCaP (Horoszewicz, JS, et al.," LNCaP Model Of Human Prostatic Carcinoma ", Cancer Res., 43: 1809-1818 (1983), which is incorporated herein by reference) was obtained from the American Type Culture Collection (Rockville, MD). Hybridomas were initially cloned in RPMI-1640 medium supplemented with 10% FCS, 0.1 mM non-essential amino acids, 2 mM L-glutamine, 100 units / ml. of penicillin, 100 μg / ml. of streptomycin and HAT medium (GIBCO, Grand Island, NY). Subclones were cultured in the same medium without aminopterin.

Example 3 - Preparation of Mouse Monoclonal Antibodies.

BALB females of mice were immunized intraperitoneally with LNCaP (6X106 cells) three times at 2 week intervals. An intraperitoneal booster immunization was administered with fresh prostate epithelial cells that have been developed in vitro.

Three days later, spleen cells were fused with Mouse SP-2 myeloma cells using standard techniques (Ueda, R. et al., "Cell Surface Antigens Of Human Renal Cancer Defined by Mouse Monoclonal Antibodies: Identification of Tissue-Specific Kidney Glycoproteins ", Proc. Nati, Acad. Sci. USA, 78: 5122-5126 (1981), which is incorporated herein by reference.) Supernatants of clones Results were examined by viable rosette cytotoxicity and complement tests against viable LNCaP clones that were positive by these assays were examined by immunochemistry in contrast to normal kidney, colon, and prostate Clones that were LNCap * / NmlKid- / colon- / Prostate + were selected and subcloned 3 times by limiting dilution.The immunoglobulin class of culture supernatant of each clone was determined by immunodiffusion using rabbit specific antiserum (Calbiochem, San Diego, CA) .Mambs were purified using the MAPS-II package ( Bio-Rad, Richmond, CA).

Example 4 -Biotinylation of mAbs Purified mAbs were dialyzed in 0.1 M NaHC 3 for 2 hours. One ml. of mAb in 1 mg / ml. It was mixed with 0.1 ml. of biotinamidacaproate N-hydroxysuccinamide ester (Sigma) in dimethylsulfoxide (1 mg./ml.) and stirred for 4 hours at room temperature. Unbound biotin was removed by dialysis in contrast regulated phosphate buffered saline ("PBS").

Example 5 - Immunohistochemical Staining of Prostate Tissues.

Cryostatic sections of prostate tissues were placed in Falcon rings dish 3034 covered plates (Becton-Dickenson, Lincoln Park, NJ) previously coated with 0.45% gelatin solution as described in Marusich, M. F., "A Rapid Method for Processing Very Large Numbers of Tissue Sections for Immunohistochemical Hybridoma Screening ", J. Immunol Methods, 11: 143-145 (1988), which is incorporated herein by reference.The dishes were stored at -80 ° C. % paraformaldehyde in PBS for 10 minutes at room temperature, and, after rinsing with PBS, the endogenous peroxidase activity was blocked by treatment with 0.3% hydrogen peroxide in PBS for 10 minutes at room temperature. were incubated with 2% BSA in PBS for 20 minutes, mAbs were added for 60 minutes at room temperature.The slides were extensively washed with PBS and incubated with rabbit anti-mouse Ig conjugated peroxidase (DAKO Corp., Santa Barbara, CA) diluted 1: 100 in 10% normal human serum in PBS for 60 minutes at room temperature.After a diaminobenzidine reaction, the spots on the sections were counted with hematoxylin.

Example 6 - Serological Analysis The hemadsorption test mixed with anti-mouse immunoglobulin was performed as described by Ueda, R., et al., "Cell Surface antigens of Human Renal Cancer Defined By Mouse Monoclonal Antibodies: Identification Of Tissue-Specific Kidney Glycoproteins ", Proc. Nati, Acad. Sci.

USA, 78: 5122-5126 (1981), which is incorporated herein by reference. To prepare the indicator cells, the anti-mouse Ig (DAKO Corp.) was conjugated to human type RBC using 0.01% chromium chloride. Antibodies were incubated with target cells at room temperature for one hour. The target cells were then washed, and the indicator cells added for 1 hour.

Example 7 - Immunoprecipitation LNCaP cells (2X107) were biotinylated with biotin-NHSS (at a final concentration of 5 mM) for 30 minutes on ice. After washing, the biotinylated cells were resuspended in 1 ml. of lysis buffer (20 mM Tris / HCL pH 8.0, 1 mM EDTA, 1 mM PMSF, 1% Triton X-100) for 30 minutes on ice. The suspension was centrifuged at 1500g X 100 min. at 4 ° C, and the supernatant was centrifuged at 12,000 rpm X 15 minutes at 4 ° C. The resulting lysate was preabsorbed with rabbit anti-mouse IgG-coated goat for 1 hour at 4 ° C. The preabsorbed lysate was incubated with mAb the night before at 4 ° C. Ig anti-mouse rabbit or goat-coated agarose beads were added for 2 hours at 4 ° C and then washed. The beads were resuspended in Tris-Base / NaCl, added to the control sample with 2-mercaptoethanol, and boiled for 5 minutes. After centrifugation, the supernatant was run on 12% SDS-PAGE gel. The gel was transferred to a nitrocellulose membrane that was blocked and stained with straptavidin-peroxidase. The membrane was developed with diaminobenzidine ("DAB").

Sequential immunoprecipitation was similar except that the lysate was initially pre-clarified with a mAb the night before at 4 ° C. A second mAb was then used to immunoprecipitate the pre-clarified lysate. approximately 2000 clones were examined, of which four clones were selected as described in example 3 above. After subcloning, the supernatants of the 4 hybridomas, E99, J415, J533, and J591, were assayed by immunofluorescence against viable LNCaP (for example non-fixed), immunoprecipitation, and immunoprecipitation to confirm reactivity to PSMA.

The immunofluorescence study utilizes the objetibo cell LNCaP (originally described in Horoszewics, which is incorporated herein by reference, to make the 7E11 antibody and the prototype cell line for PSMA expression) shows that the E99 antibody binds to and converts to viable cells Immunofluorescent LNCaP. This is in contrast to antibody 7E11, which as originally observed in Horoszewicz, which is incorporated herein by reference, gives only poor or no binding for viable LNCaP cells but exhibits strong binding once the cells are fixed. (destroyed).

The reactivities of the four mAbs with normal human tissues were examined immunohistochemically; These results are presented in Table 3.

TABLE 3 Reactivity of mAbs with normal human tissues by indirect staining of the immunoperoxidase Fabrics E99 J415 J533 J591 Prostate * Kidney Glomerulus Prox. Tubule Ureter? ? ? Bladder? ? ? Test? ? ? Uterus Esophagus? ? ? Small intestine ? ? ? ? Stomach? ? ? Colon? ? ? Spleen? ? ? ? Thyroid ? ? ? ? Lung? ? ? ? Pancreas ? ? ? ? Liver? ? ? ? * BPH 0-3 + 0-3 + 0-4 + 0-4 + * Prostate Cancer 0-3 + 0-3 + 0-4 + 0-4 + * LNCaP (scid) 3+ 3+ 4+ 4+ * LuCaP (scid) 0-2 + 0-2 + 0-3 + 0-3 + * positive; Z weak, heterogeneous; ? negative The sequential immunoprecipitation studies above showed that 7E11, E99, J415, J533, and J591 bind to the same molecule, for example PSMA.

E eg 8 - Analysis by Western Spotting To confirm that the E99, J415, J533, and J591 antibodies precipitate an identical band to the 7E11 antibody (by e emc-or PSMA), Western Blot Analysis was performed. Seed plasma (400 μg / band) or LNCaP lysates were loaded on 12% SDS-Page gel bands. After electrophoresis, the gels are transferred to nitrocellulose membranes. The membranes were blocked with 5% dry milk / Tris-regulated saline-tween 20 ("TBST") for 60 minutes at room temperature. After washing, the membranes were incubated with primary mAb for 60 minutes at room temperature. After washing, the membranes were incubated with sheep antiserum-Ig-peroxidase 1/5000 in 5% dry milk / TBST for 60 minutes at room temperature. After repeated washing, the membrane was developed using a chemiluminescent tag designated "ECL" (Amersham Life Sciences, International, Arlington Heights, Illinois) in accordance with the manufacturer's instructions. The results of the Western Spotting experiment are presented in Table 4.

TABLE 4 Sample 7E11 E99 J415 J533 J591 Prostate 100KD 100KD 100KD 100KD 100KD (seminal) band band band band fluid band LNCaP 100KD & 100KD & 100KD & 100KD & 100KD cell lysate 200KD 200KD 200KD 200KD 200KD bands bands bands bands "bands Example 9 - Reactivity of mAb in the External Region of PSMA To confirm the surface (external) cell expression of the PSMA detected, recent, viable LNCaP cells were tested, without fixation, in vitro, by immunofluorescence. LNCaP cells were washed and incubated with mAb for 1 hour at room temperature and then with an anti-mouse-fluorescein Ig (DAKO Corp., Santa Barbara, CA). The slides were read with a fluorescent microscope. The negative control consisted of an irrelevant matched isotype of mAb, while an anti-class MHC mAb served as a positive control.

The results of immunofluorescence and rosette assays are presented in Table 5.

TABLE 5 Comparison of 7E11 with new mAbs LNCaP 7E11 E99 J415 J533 J591 viable cells Immunofluorescence neg. 3+ 3+ 4+ 4+ Rosette test neg + + + + LNCaP-fixed +++ ++++ +++ ++++ Example 10 - Capacity Studies A capacity study was carried out to determine whether J591, J533, E99, and J415 detected the same or different antigenic sites (epitopes) of the prostate-specific membrane antigen molecule using the following procedure.

Plates were coated with LNCaP cell line lysing as a source of prostate-specific membrane antigen and washed to remove non-binding material. The "Cold" Monoclonal antibody (unlabeled) was incubated on the plate for 1 hour at room temperature to allow binding of its antigenic site. Subsequently, a second monoclonal antibody, labeled with either biotin or 125I, was added for an additional hour. The plates were washed to remove non-binding material. The amount of the second monoclonal antibody associated with the prostate-specific membrane antigen of the coated plate was determined by avidin-alkaline phosphatase in an enzyme-bound immunoassay (in the case of the second biotin-labeled monoclonal antibody) or by physically counting the plate in a gamma counter (in the case of the second monoclonal antibody 1251-labeled).

The controls consisted of using the same monoclonal antibody both cold and labeled to define "100 % capacity "or using the monoclonal antibody in a totally different molecule (eg, monoclonal antibody Y-56, which detects inhibin, a prostate-related protein different from prostate-specific membrane antigen) to define" 0% of capacity" .

The results indicated that J591, J533, and E99 each interfere, compete, or block the link of someone else but does not block the J415 link and vice versa. 7E11 / CYT356, known to bind PSMA at a different (intracellular) site, did not block any of J591, J533, E99, or J415.

Pairs of monoclonal antibodies that have to bind in non-capable sites allow. the development of antibody sandwich assays for detecting soluble antigens, such as solubilized prostate-specific membrane antigen or fragments thereof, in, for example, body fluids. For example, the antigen (e.g., prostate-specific membrane antigen or a fragment thereof) could be "captured" from body fluid with J591 and, in another step, detected by labeled J415.

In another context, for example treatment, one could increase the binding antibody by the use of a combination of non-cpacious monoclonal antibodies. For example, assuming that the non-capable sites are each represented once on the specific membrane antigen molecule of the prostate, adding a combination of J591 plus J415 could bind twice as many monoclonal antibody molecules as both monoclonal antibodies alone. Linking two non-capable antigenic binding sites can also result in increased antigen cross-linking and, possibly, increased internalization. Also, since the two detected sites are physically located in. the same membrane-specific antigen molecule of the prostate, the binding of two monoclonal antibody molecules as well as one molecule of prostate-specific membrane antigen puts the two monoclonal antibody molecules in close proximity to each other, a presentation which provides optimal drug-prodrug interaction. For example, the monoclonal antibody J591 can be conjugated to a prodrug activator. Since the prodrug and activator bind in close proximity only to the site of prostate-specific membrane antigen-expressing cells (eg, prostate cancer cells), the activation of prodrugs in the active form could take place only in those sites .

Example 11 - Microscopy Co-focal microscopy and immunoelectron microscopy demonstrated that E99, J591, J533, and J415 are associated with the clathrin-coated membrane cell and then rapidly internalize into endosomes (cytoplasmic vesicles). Figures 1-4 are immunoelectron micrographs that track the interaction of the gold-labeled monoclonal antibody J591 with the cell surface as a function of time. In these figures, the location of the monoclonal antibody is indicated by the black dots.

Viable LNCaP cells were incubated with J591 for one hour at 4 ° C. The cells were washed and then incubated at 37 ° C for 0, 5, 10, or 15 minutes, after which time they were fixed and processed by immunoelectron microscopy. Figure 1 shows the previous cell at 37 ° C incubation. J591 can be seen associated with the cell by the external appearance of the cell membrane. In this figure "M" indicates the mitochondria of the cell, and "N" indicates its nucleus. Figure 2 shows the cell after incubation at 37 ° C for 5 minutes. The arrow indicates the formation of clathrin-coated spots. In Figure 3, which shows the cell after 10 minutes of incubation at 37 ° C, widening or endocytocytosis of the clathrin-coated spots could be seen as indicated by the arrow. Figure 4 shows that, after incubation at 37 ° C for 15 minutes, the monoclonal antibody J591 is contained in endocytic vesicles in the cell, as indicated by the arrows. As can be seen in Figure 5, after incubation at 37 ° C for 15 minutes, the monoclonal antibody J591 is also contained in endosomes, as indicated by the arrows.

Example 12 - Sequence Formation of the Variable Region of the Monoclonal Antibody J591 The total RNA was prepared from 10? J591 murine hybridoma cells. A sample of the conditioned medium of these cells was tested by binding of the J591-specific antigen in prostate cells. The conditioned medium was positive for both ELISA and Western Spotting for antigen binding. cDNA VH and VK were prepared using reverse transcriptase and mouse constant region k and mouse IgG constant region primers. The first strands of cDNAs were amplified by PCR using a variety of primers from mouse reporter sequences (6 for VH and 7 for VK). The amplified cDNAs were purified on the gel and cloned into the bluepT7 vector.

The obtained VH and VK clones were examined for correct insertion by PCR, and the DNA sequence of selected clones was determined by the dideoxy chain termination method.

Excluding the primer region (as the sequence thereof depends on the sequence of the primer that was used.) This sequence was obtained from clones produced with three different 5 'primers.A: clone had a base pair change in the reporter sequence, and one clone contained an aberrant PCR.Using the strategy of sequence formation in figure 6, the nucleotide sequence of the heavy chain was obtained.It is designated SEQ ID No. 1 and is presented in figure 7, in parallel with the sequence nucleotide of the corresponding inverse, non-coding filaments (designated SEQ ID No. 2) These sequences include part of the reporter sequence and part of the antibody constant region The corresponding amino acid sequences deduced from J591 VH, designated SEQ ID NO. No. 3, SEQ ID No. 4, and SEQ ID No. 5, are also shown in Figure 7. The coding strand of the J591 heavy chain variable region (exclusive of the components of the secu indicator and constant region) has the following nucleotide sequence (designated SEQ ID No. 6): GAGC-T CAGCTGCAACAGTCTGGACCTGAACTGGTGAAGCCTGGGACTTCAGTGAGG ATATCCTGCAAGACTTCTGGATACACATTCACTGAATATACCATACACTGGGtsAAG CAGAGCCATGGAAAGAGCCTTGAGTGGATTGsAAACATCAATCCTAACAATGGTsXGT ACCACCTACAATCAGAAGTtcsAGGACAAGsCCACATTGACTGTAGACAAGTCCTCC AGTACAGCCTACATGGAGCTCCGCAGCCTAACATCTGAGGATTCTGCAGTCTATTAT TCA TGTGCAGCTGGTTGGAACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCC The opposite, non-coding strand of the variable region of the heavy chain J591 (exclusive of the components of the reporter sequence and the constant region) has the following nucleotide sequence (designated SEQ ID No. 7): TGAGGAGACTGTGAGAGT3GTGCCTTGGCCCCAGTAGTCAAAGTTCCAACCAGCTGC ACAATAATAGACTGCAGAATCCTCAGATGTTAGGCTGCGGAGCTCCATGTAGGCTGT ACTGGA0GAC7TGTCTACAGTCAATGTGGCCTTGTCCTCGAACTTCTGATTG7AGGT GGTACCAC ATTGTTAGGATTGATGTTTCCAATCCACTCAAGGCTCTTTCCATGGCT CTGCTTCACCCAGTGTATGGTATATTCAGTGAATGTGTATCCAGAAGTCTTGCAGCA TATC TCACTGAAGTCCCAGGCTTCACCAGTTCAGGTCCAGACTGTTGCAGCTGGAC The protein sequence corresponding to the variable region of the heavy chain J591 (exclusive of the components of the reporter sequence and the constant region) has the following nucleotide sequence (designated SEQ ID No. 8):? V? iQQSGPELVKPGTSVRISCKTSGYTFTEYTIHWVKQSHGKSLEWIGNINPNNGG TTY Q FE KAT TVDKSSSTAYMEL SLTSEDSAVYYCAAGWNFDYWGQGTT TVS The J591 VH is in the Subgroup IIA of Heavy Mouse Chains (Kabat et al., Sequences of Proteins of Immunological Interest, US Department of Healt and Human Services (1991) ("Kabat"), which is incorporated herein by reference). The sequence of J591 VH is compared to the consensus sequence for this subgroup in Figure 8.

In contrast to VH, more than a VK sequence was obtained. Of the 15 VK clones examined, four gave the sequence of an aberrant mouse Igk from the fusion partner (Carol et al., Molecular Immunology, 25: 991-995 (1988), which is incorporated herein by reference). These clones were originated from two specific 5 'primers. No additional work was done with these clones. Of the remaining clones, ten gave identical nucleotide sequences, and one clone, VK17, gave an alternative VK sequence. The ten identical clones were originated from three 5 'primers (different from the two that gave the aberrant sequence), one of which also produced VK17. The strategy of sequence formation used is shown in Figure 9.

The nucleic acid sequence of J591 VK corresponding to the ten identical clones (designated SEQ ID No. 9) is shown in Figure 10, in parallel with the nucleic acid sequence of the corresponding counterpart, non-coding strand (deleting SEC). ID No. 10) and the deduced amino acid sequences, which are designated SEC. Id. No. 11, SEC. ID. No. 12, and SEC. ID. No. 13. These sequences include part of the reporter sequence and part of the antibody constant region. The coding filament of J591 of the light chain variable region (kappa) (exclusive of the components of the indicator sequence and the constant region) corresponding to the ten identical clones has the following nucleotide sequence (designated SEQ ID No. 14): AACATTsTAATGACCCAATCTCCCAAATCCATsTCCATGTCAGT -. GGAGAGAGGGTC ACCTTGACCTGCAAGGCCAGTGAGAATGTGGTTACTTATGTTTCCTGGTATCAACAG AAACCAGAGCAGTCTCCTAAACTGCTGATATACGsGGCATCCAACCGGTACACTGGs GTCGCCGATCGCTTCACAGGCAGTGGATCTGCAACAGATTTCACTCTGACCATCAGC AGTGTGCAGGCTGAAGACCTTGCAGATTATCACTGTGGACAGsGTTACAGCTATCCG TACACGTTCGsAsGGGGGACCAAGCTGGAAATAAAA The opposite, non-coding filament of the light chain variable region J591 (kappa) (exclusive of the components of the reporter sequence and the constant region) corresponding to the ten identical clones has the following nucleotide sequence (designated SEC. ID No. 15): TTTTATTTCCAGCTTGGTCCCCCCTCCGAACGTGTACGGATAGCTGTAACCCTGTCC ACAGTGATAATCTGCAAGGTCTTCAGCCTsCACACTGCTGATGGTCAGAGTGAAATC TGTTGCAGATCCACTGCCTGTGAAGCGATCGGGGACCCCAGTGTACCGGTTGGATGC CCCGTATATCAGCAGTTTAGGAGACTGCTCTGGTTTCTGTTGATACCAGGAAACATA AGTAACCACATTCTCACTGGCCTTGCAGGTCAAGGTGACCCTCTCTCCTACTGACAT GGACATGGATTTGGGAGATTGGGTCATTACAATGTT The protein sequence corresponding to the J591 of light chain variable region (kappa) (exclusive of the components of the reporter sequence and the constant region) corresponding to the ten identical clones has the following nucleotide sequence (designated SEQ ID NO: n. 16): NIVMTQSPKSMSMSVGERVTLTCKASENWTYVSWYQQKPEQSPKLLIYGASNRYTG VPDRFTGSGSATDFTLTISSVQA? DLA YHCGQGYSYPYTFGGGTKL? IK The coding filament of the light chain variable region J591 (kappa) (exclusive of the components of the sequence of the reporter sequence and the constant region) corresponding to the clone VK17 has the following nucleotide sequence (designated SEQ ID NO. 17): GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTC AGCATCATCTGTAAGGCCAGtCAAGATGTssGTACTGCTGTAGACTsstATCAACAG AAACCAGGACAATCTCCTAAACTACTGATTTATTGGGCATCCACTCGGCACACTGGA GTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGACTTCACTCTCACCATTACT AATGTTCAGTCTGAAGACTTGGCAGATTATTTCTGTCAGCAATATAACAGCTATCCT CTCACGTTCGGTGCTGGGACCATGCTGGACCTGAAA The opposite, non-coding filament of J591 of light chain variable region (kappa) exclusive of the components of the indicator sequence and of the constant region) corresponding to clone VK17 has the following nucleotide sequence (designated SEQ ID NO: No. 18): TTTCAGGTCCAGCATGGTCCCAGCACCGAACGTGAGAGGATAGCTGT7ATATTGCTG ACAGAAATAATCTGCCAAGTCTTCAGACTGAACATTAGTAATGGTGAGAGTGAAGTC TGTCCCAGATCCACTGCCtGTGAAGCGATCAGGGACTCCAGTGTGCCGAGTGGATGC CCAATAAATCAGTAGTTTAGGAGATTGTCCTGGTTTCTGTTGATACCAGTCtACAGC AGTACCCACATCTTGACTGGCCTTACAGATGATGCTGACCCTGTCTCCTACTGATGTGGACATGAATTTGTGAGACTGGGTCATCACAATGTC The protein sequence corresponding to J591 of light chain variable region (kappa) (exclusive of Iso components of the reporter sequence and of the constant region) corresponding to clone VK17 has the following nucleotide sequence (designated SEQ ID No. 19). : DIVMTQSHKFMSTSVGDRVSIICKASQDVGTAVDWYQQKPGQSPKLLIYWASTRHTG VPDRFtGSGSGTDFTLTITNVQSEDLADYFCQQYNSYPLTFGAGTMLDLK The J591 VK is in the V subgroup of the Kappa Mouse Chains (Kabat, which is incorporated herein by reference). The sequence of the J591VK corresponding to the ten identical clones is compared to the consensual sequence for the subgroup of figure 11.

J591's are preferred ones having heavy chain variable region DNA coding for filament sequences corresponding to SEQ. ID. No. 6 and sequences of non-coding filaments (opposite) corresponding to SEC. ID. No. 7. The heavy chain variable region of J591 preferably has an amino acid sequence corresponding to SEC. ID. No. 8. The light chain variable region of J591 preferably has a DNA encoding the filament sequence corresponding to SEQ. ID. No. 17, a non-coding DNA of the (opposite) filament sequence corresponding to SEC. ID. No. 18, and an amino acid sequence corrected to the SEC. ID. No. 19 Example 13 - Immunohistochemical Staining of Normal and Cancer Tissues Tissues with cancer from 23 carcinomas were pre-cooled in liquid nitrogen, rapidly frozen in OCT compound (Miles, Elkhart, Indiana) on dry ice, and stored at -80 ° C. Cryostatic tissue sections (5μm) were fixed in cold acetone (4 ° C) for 10 minutes. mAbs (5μg / ml or supernatants hybridomas) were incubated for 1 hour at room temperature. The binding antibody was detected using rabbit anti-mouse Ig-peroxidase (Dako, Carpinteria, California) as a secondary antibody and DAB (Sigma, San Luis, Missouri) as a chromogen. Isotype-matched irrelevant antibody was used as a negative control. mAbs J591, J533, J415, and E99 reacted strongly with vascular endothelium in all 23 carcinomas studied, including 9/9 renal carcinomas, 5/5 urothelial carcinomas, 6/6 colon carcinomas, 1/1 lung carcinomas, and 1 / 1 breast carcinomas, and 1/1 metastatic adenoma of the liver. Figures 2A- "F, respectively, show the immunohistochemical reactivity of mAb J591 of the neovasculature of renal, urothelial, colon, lung, and breast carcinomas, and metastatic adenocarcinoma of the liver.

Although the invention has been described in detail for the purpose of illustration, it is to be understood that such details are for that purpose only and that variations may be made by those skilled in the art without departing from the spirit and scope of the invention which is defined by claims of the present.

LIST OF SEQUENCES (1. GENERAL INFORMATION: (i) APPLICANT: Cornell Research Foundation, Inc. (ii) TITLE OF THE INVENTION: TREATMENT AND DIAGNOSIS CER (iii) NUMBER OF SEQUENCES: 19 (iv) ADDRESS FOR CORRESPONDENCE: (A) RECIPIENT: Nixon, Hargrave, Devans & Doyke LLP (B) STREET: Clinton Square, P. O. Box 1051 (C) CITY: Rochester (D) STATE: New York (E) COUNTRY: U.S.A. (F) POSTAL AREA: 14603-1051 (v) TYPE OF READING COMPUTER (A) MIDDLE TYPE: diskette (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentin Relay No. 1.0, Version No. 1.3 (vi) CURRENT DATES OF THE APPLICATION: (A) APPLICATION NUMBER (B) REGISTRATION DATE: (C) CLASSIFICATION: (vii) APPLICATION PRIORITY DATE (A) APPLICATION NUMBER: US 06 / 022,125 (B) REGISTRATION DATE: July 18, 1996 (vii) APPLICATION PRIORITY DATE (A) APPLICATION NUMBER: US 08 / 838,632 (B) REGISTRATION DATE: AUGUST 9, 1997 (viii) OFFICER / INFORMATION AGENT: (A) NAME: Goldman, Michael L. (B) REGISTRATION NUMBER: 30,727 (C) REFERENCE NUMBER / CÉDULA: 19603/1174 iix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (716) 263-13-04 (B) TELEFAX: (716) 263-1600 INFORMATION OF SEQ ID NO: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 391 base pairs (B) TYPE: Nucleic acid (C) TYPE OF FILAMENT: one only (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 1: tCTCCTStCA GGAACTsCAs GTstCCTCTC TsAsGTCCAG CTGCAACAGT CTGGACCTGA 60 ACTGGTGAAG CCTGGGACTt CAGTGAGGAT ATCCTGCAAG ACTTCTGGAT ACACATTCAC 120 T3AATATACC ATACACTGGG TGAAGCAGAs CCATGGAAAG AGCCrtGAGT OGATtsGAAA 180 CATCAATCC AACAATGGTG GTACCACCTA CAATCAGAAG TTCGAGGACA AGGCCACATT 240 GACT3TAGAC AAGTCCTCCA GTACAGCCTA CATGGAGCTC CGCAGCCTAA CATCTGAGGA 300 7 CTGCAGTC TATTATTGTG CAGCTGGTTG GAACTTGGAC TACTGGGGCC AAGGCACCAC 360 TCTCACAGTC TCCTCAGCCA AAACGACACC C 391 (2) SEQUENCE INFORMATION SEQ ID NO: 2 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 391 base pairs (B) TYPE: nucleic acid (C) TYPE OF FILAMENT: one only (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: GGGTGTCGTT TTGGCTGAGG AGACTGTGAG AGTGGTGCCT TGGCCCCAGT AGTCAAAGTT 60 CCAACCAGCT GCACAATAAT AGACTGCAGA ATCCTCAGAT GTTAGGCtsC GGAGCTCCAT 120 3TAGGCTGTA CTGGAGGACT TGTCTACAGT CAATGTGGCC TTGTCCTCGA ACTTCTGATT 180 3TAGGTGGTA CCACCATTGT TAGGATTGAT GTTTCCAATC CAC CAAGGC TCTTTCCATG 240 GCTC GCTTC ACCCAGTGTA TGGTATATTC AGTGAATGTG TATCCAGAAG TCTtGCAGGA 300 ATCCTCACT GAAGTCCCAG GCTTCACCAG TTCAGGTCCA GACTGTTGCA GCTGGACCTC 360 AGAGAGGACA CCT3CAGTTC CTAGCAGGAG A 391 (2) SEQUENCE INFORMATION SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 123 amino acids (B) TYPE: amino acids (C) TYPE OF FILAMENT: (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3 Ser Pro Val Arg Asn Cys Arg Cys Pro Leu Gly Pro Ala Ala Thr Val 1 5 10 15 Trp Thr Thr Gly Glu Wing Trp Asp Phß Ser Glu Asp He Leu Gln Asp 20 25 30 Phe Trp lie His llß His lie Tyr His Thr Leu Gly Glu Wing Glu Pro 35 40 45 Trp Lys Glu Pro Val Asp Trp Lys His Gln Ser Gln Trp Trp Tyr His 50 55 60 Leu Gln Ser alu Val Arg Gly Gln sly His He Asp Cys Arg Gln Val 65 70 75 80 Leu Gln Tyr Ser Leu His Gly Wing Pro Gln Pro Asn lie Gly Phe Cys 85 90 95 Be Leu Leu Leu Cys Be Trp Leu slu Leu Leu Gly Pro Pro Arg His 100 ios 110 H: s Ser His Ser Leu Leu Ser GIn Asn Asp Thr 115 120 [2] INFORMATION OF SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 130 amino acids (B) TYPE: amino acid (C) TYPE OF FILAMENT: (D) TOPOLOGY: linear (li) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRITION: SEQ ID NO: 4 Leu Leu Ser Gly Thr Wing Gly Val Leu Ser Glu Val Gln Leu Gln Gln 1 5 10 15 Ser Gly Pro Glu Leu Val Lys Pro Gly Thr Ser Val Arg He Ser Cys 25 30 Lys Thr Ser sly Tyr Thr Phe Thr slu Tyr Thr He His Trp Val Lys 35 40 45 Gln Ser His Gly Lys Ser Leu Glu Trp He Gly Asn He Asn Pro Asn 50 55 60 Asn Gly a and Thr Thr Tyr Asn sln Lys Phe slu Asp Lys Ala Thr Leu 65 70 75 80 Thr Val Asp Lys Being Ser Thr Wing Tyr Mßt Glu Leu Arg Being Leu 85 90 95 Thr Ser Glu Asp Ser Wing Val Tyr Tyr Cys Wing Wing Gly Trp Asn Phe 100 105 110 Asp Tyr Trp Gly Gln sly Thr Thr Leu Thr Val Ser Ser Wing Lys Thr 115 120 125 Thr Pro 130 (2) INFORMATION OF SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 125 amino acids (B) TYPE: amino acid (C) TYPE OF FILAMENT: (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: Leu Ser Cys sln Olu Leu Gln Val Ser Ser Leu Arg Ser Ser Cys Asn 1 5 10 15 Ser Leu Asp Leu Asn Trp Ser Leu Gly Leu Gln Gly Tyr Pro Ala Arg 25 30 Leu Leu Asp Thr His Ser Leu Asn He Pro Tyr Thr Oly Ser Arg Ala 35 40 45 ee Glu Arg Ala Leu Ser Oly Leu Glu Thr s. n - 50 c = nr S r r le Leu Thr Met Val 5S 60 val P or Pro Thr He Arg Ser Ser Arg Thr Arg Pro H 1-ij.sa L.eeuu Tihnrr s 70 ser 7S 30: o Pro Val without Pro Thr T as rp be Sßr Ala Ala His Leu Arg He Leu 90 Wave be He Ue Val Gln Leu Val Gly Thr Leu Thr Thr Gly u. Lys lü5 110 Pro Leu Ser Gln Pro Sßr Gln Pro Lys Arg His Pro 120 125 (2) INFORMATION OF SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 345 base pairs (B) TYPE: nucleic acid (C) TYPE OF FILAMENT: one only (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: CDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: GAGGTCCAGC TGCAACAGTC TGsACCTGAA CTGGTGAAOC CTGGsACTTC AGTOAOGATA 60 TCCT3CAAGA CTTCTsOATA CACATTCACT GAATATACCA TACACTCOOT OAAGCAGAGC 120 CATGGAAAGA GCCTTGAGTG GATTGGAAAC ATCAATCCTA ACAATGGTGG TACCACCTAC 130 AATCAGAAGT TCGAGGACAA GGCCACATTG ACTGTAGACA AGTCCTCCAG TACAGCCTAC 240 ATGGAGCTCC GCAGCCTAAC ATCTGAGGAT TCtsCAGTCT ATTATTGTGC AGCTGGTTGG 300 AACTTTGACT "-ACTOGOGCCA AGGCACCACT CTCACAGTCT CCTCA 345 (2) INFORMATION OF SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 345 base pairs (B) TYPE: nucleic acid (C) TYPE OF FILAMENT: one only (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7 TGAGGAGACT GTGAGAGTGs TGCCTTGsCC CCAGTAGTCA AAGTTCCAAC CAGCTGCACA 60 ATAATAGACT GCAGAATCCT CAGATGTTAG GCTGCGGAGC TCCATGTAGG CTGTACTGGA 120 GGACTTGTCT ACAGTCAATG TGsCCTTGTC CTCGAACTTC TGATTGTAGG TGGTACCACC 180 ATTGTTAOGA TTGATGTTTC CAATCCAC7C AAGGCTCTTT CCATGGCTCT GCTTCACCCA 240 stOTATGGTA TATTCAGTGA ATGTGTATCC AGAAGTCTTG CAGGATATCC TCACTGAAGT 300 CCCAGOCTTC ACCAGTTCAG GtCCAGACTG TTGCAGCTGG ACCTC 345 (2) INFORMATION OF SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 115 amino acids (B) TYPE: amino acid (C) TYPE OF FILAMENT: only one; D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRITION: SEQ ID NO: 8: Olu Val Oln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Thr 1 5 10 15 Ser Val Arg He Ser Cys Lys Thr Sßr Gly Tyr Thr Phß Thr Glu Tyr 20 25 30 Thr He His Trp Val Lys Gln Sßr His Gly Lys Sßr Leu Glu Trp He 35 40 45 Gly Asn He Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe = "55 60 Gu Asp Lys Ala thr Leu Thr Val Asp Lya Sßr Ser Ser Thr Ala Tyr 7S 80 • read Olu Leu Arg Ser Leu Thr Ser ßlu Asp Ser Ala Val Tyr Tyr Cvs 85 90 95 * Wing Wing Gly Trp Asn Phe Asp Tyr Trp Gly sln Gly Thr Thr Leu Thr 100 1 10055 -1-10- Val Ser Ser 115 (2) INFORMATION OF SEQ ID NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 363 base pairs (B) TYPE: nucleic acid (C) TYPE OF FILAMENT: one only (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9: TTATATGGAO CTGATGGGAA CATTGTAATG ACCCAATCTC CCAAATCCAT GTCCATGTCA 60 GTAGGAGAsA GGGTCACCTT GACCTCCAAG GCCAGTGAGA ATGTGGTTAC TTATGTTTCC 120 TGGTATCAAC AGAAACCAOA GCAGTCTCCT AAACTGCTGA TATACGGGGC ATCCAACCGG TC 180 TACACT3GGG CCGATCG CTTCACAGGC AGTGGATCTG CAACAGATTT CACTCTGACC 240 ATCAGCAOTC TGCAGGCTGA AGACCTTGCA GATTATCACT GTGGACAGGG TTACAGCTAT 300 CCGTACACGT TCGGAGGGGG GACCAAGCTG GAAATAAAAC sGGCTGATGC TGCACCAACT 360 GTA 363 (2) INFORMATION OF SEQ ID NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 363 base pairs (B) TYPE: nucleic acid (C) TYPE OF FILAMENT: one only (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 10 TACAGTTGGT GCAOCATCAG CCCGTTTtAT TTCCAGCTTG GTCCCCCCTC CGAACGTGTA 60 C3GATAGCTG TAACCCTGTC CACAGTGATA ATCTGCAAGs TCTTCAGCCT GCACACTGCT 120 GATGOTCAGA sTGAAATCTG TTGCAGATCC ACTGCCTGTG AAGCGATCOG GGACCCCAGT 180 GTACCGGTTG GATGCCCCGT ATATCAGCAG TTTAGGAGAC TGCTCTGGTT TCTGTTGATA 2 0 CCAGGAAACA TAAGTAACCA CATTCTCACT sGCCTTGCAG GTCAAGGTGA CCCTCTCTCC 300 TACTGACATG GACATGsATT TGGGAGATTG OsTCATTACA ATOTTCCCAT CAGCTCCATA 360 TAA 363 (2) INFORMATION OF SEQ ID NO: 11: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 121 amino acids (B) TYPE: amino acid (C) TYPE OF FILAMENT: (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein 0 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 11 Leu Tyr sly Wing Asp sly Asn He Val Mßt Thr Oln Ser Pro Lys Ser 1 10 15 Met Ser Mee Ser Val aly Glu Arg Val Thr Leu Thr Cys Lys Wing Ser 20 25 30] S Glu Asn Val Val Thr Tyr Val Ser Trp Tyr Gln Gln Lvs Pro slu Gln 35 40 45 Ser Pro Lys Leu Leu He Tyr Gly Wing Ser Asn Arg Tyr Thr Gly Val 50 55 60 Pro Asp Arg Phß Thr Gly Sßr Gly Ser Wing Thr Asp Phe Thr Leu Thr 65 70 75 80 He Ser Ser Val s Ala Slu Asp Leu Wing Asp Tyr His Cys Gly Gln 85 90 95 Gly Tyr Ssr Tyr Pro Tyr Thr Phs Oly Gly Gly Thr Lys Leu slu He 100 105 no Lys Arg Ala Asp Ala Ala Pro Thr Val 115 120 (2) INFORMATION OF SEQ ID NO: 12 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 114 amino acids (B) TYPE: amino acid (C) TYPE OF FILAMENT: (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 12: Tyr Met slu Leu Met Gly Thr Leu Pro Asn Leu Pro Asn Pro Cys Pro 1 Year - 15 Cys Gin Glu Arg Oly Ser Pro Pro Ala Arg Pro Val Arg Mßt Trp Leu 20 25 3rd Leu Mee Phe Pro Gly He Asn Arg Asn Gln Ser Ser Leu Leu Asn Cvs 40 5 I Tyr Thr sly His Pro Thr Gly Thr Leu Gly Sßr Pro He Wing Gla Ser 55 55 Wing Val Asp Leu Oln Gln He Ser Leu Pro Ser Wing Val Cys Ars Leu 65 70 75 3 80 Lys Thr Leu Gln He He Thr Val Asp Arg Val Thr Wing He Ara Thr 85 90 953 Arg Ser Olu Gly Gly Pro Ser Trp Lys Asn Gly Leu Mee Leu His Gln 100 105 no Leu Tvr (2) INFORMATION OF SEQ ID NO: 13: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 116 amino acids (B) TYPE: amino acid (C) TYPE OF FILAMENT: (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 13: He He Trp Ser Trp Glu His Cys Asn Asp Pro He Ser Gln He His 1 5 10 15 Val His Val Ser Arg Arg Glu Gly His Leu Asp Leu Gln Gly Gln slu 20 25 30 Cys Oly Tyr Leu Cys Phe Leu Val Ser Thr Olu Thr Arg Ala Val Ser 35 40 45 Thr Wing Asp He Arg Oly He Gln Pro Val His Trp Gly Pro Arg Sßr 50 55 60 Leu His Arg Gln Trp He Cys Asn Arg Phe His Ser Asp His Wave Gln oS 70 75 30 Cys Ala Gly Arg Pro Cys Arg Leu Ser Leu Trp Thr sly Leu sln Leu 85 90 95 Ser Val His Val Arg Arg Gly Asp sln Wing Gly Asn Lys Thr Gly Cys 100 IOS no Cys Thr Asn Cys 115 (2) INFORMATION OF SEQ ID NO: 14: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 321 base pairs (B) TYPE: nucleic acid (C) TYPE OF FILAMENT: one only (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 14 AACATTGTAA TGACCCAATC TCCCAAATCC ATGTCCATGT CAGtAGGAGA GAGGGTCACC 60 T GACCTGCA AGGCCAGTGA GAATGTGGTT ACTTATGTTT CCTGGTATCA ACAGAAACCA 120 GAGCAGTCTC CTAAACTGCT GATATACGGG GCATCCAACC GGTACACTGG OGTCCCCGAT 180 CGCTTCACAG sCAGTGGATC TGCAACAGAT TTCACTCTOA CCATCAGCAO TaTGCAOGCT 240 OAAGACCn G CAGATTATCA CTGTsGACAa GOTGACAGCT ATCCGTACAC sTTCGGAGGG 300 GGGACCAAGC TGGAAATAAA A 32? (2) INFORMATION OF SEQ ID NO: 15: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 321 base pairs (B) TYPE: nucleic acid (C) TYPE OF FILAMENT: one only (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 15: T-GTTAT-GTCC AscrTssTCc CCCC CCGAA CGTGTACGGA TAOC OTAAC CCTGTCCACA 60 GTGATAATCT sCAAGGTCTT CAGCCTGCAC ACrGCTGATG GTCAGAGTGA AATCTßTTGC 120 AGATCCACTG cctstsAAsc OATCGOGOAC CCCAGTGTAC CGGTTGGATG CCCCGTATAT 180 CAGCAGTTGA GGAGACTGCT CTGGTTGCTG TTGATACCAG GAAACATAAs TAACCACATT 240 CTCACTGGCC TTGCAGGTCA AGGTGACCCT CTCTCCTACT GACATCOACA TsGATTTGGß 300 AGATTGGGTC ATTACAATGT T 321 (2) INFORMATION OF SEQ ID NO: 16: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 107 amino acids (B) TYPE: amino acid (C) TYPE OF FILAMENT: (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 16: Asn He Val Mee Thr Gln Sßr Pro Lya sßr Mee Sßr Mee Ser Val Gly 1 5 10? S Glu Arg Val Thr Leu Thr Cys Lya Wing Ser Glu Aan Val Val Thr Tyr 20 25 30 Val Ser Trp Tyr sln Gln Lya Pro slu Gln Being Pro Lya Leu Leu? Le 3S 40 45 Tyr G and Wing Being Aan Arg Tyr Thr sly Val Pro Aap Arg Ph Th Thr Oly 50 55 60 Ser Gly Being Wing Thr Asp Phe Thr Leu Thr Be Ser Ser Val Gln Wing 65 70 75 80 slu Asp Leu Ala Aap Tyr His Cyß sly Gln Gly Tyr Ser Tyr Pro Tyr 85 90 95 Thr Phß Gly Gly Gly Thr Lya Leu Glu He Lys 100 105 (2) INFORMATION OF SEQ ID NO: 17: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 321 base pairs (B) TYPE: nucleic acid (C) TYPE OF FILAMENT: one only (D) TOPOLOGY: linear (ii) TYPE OF MOLECUL: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 17 GACATTGTGA TGACCCAGTC TCACAAATTC ATGTCCACAT CAGTAGGAOA CAsGGTCAGC SO ATCATCTGTA AGGCCAGTCA AsATGTGssT ACTGCTsTAs ACTGGTATCA ACAGAAACCA 120 GGACAATCTC CTAAACTACT GATTTATTsG GCATCCACTC GsCACACTGG AGTCCCTGAT 180 CGCTTCACAG GCAGTGGATC TGGGACAGAC TTCACTCTCA CCATTACTAA TGTTCAsTCT 240 OAAGACTTss CAGATTATTT CTGTCAGCAA TATAACAGCT ATCCTCTCAC GTTCGsTGCT 300 GGGACCATGC TGGACCTGAA A 321 (2) INFORMATION OF SEQ ID NO: 18: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 321 base pairs (B) TYPE: nucleic acid (C) TYPE OF FILAMENT: one only (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA Xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 18 TTTCAGGTCC AGCATGGTCC CAGCACCGAA CGTGAGAGGA TAGCTGTTAT ATTGCTaACA 60 GAAATAATCT GCCAAGTCTT CAGACTGAAC ATTAGTAATG GTGAGAGTGA AGTCTGTCCC 120 AGATCCACTG CCTGTGAAGC GATCAOGOAC TCCAGTGTGC CGAGTGGATG CCCAATAAAT - 180 CAGTAGTTT? - GßAGATTOTC CTGGTTTCTG TTGATACCAG TCTACAGCAG TACCCACATC 240 TTGACTGGCC TTACAGATGA TGCTGACCCT GTCTCCTACT sATGTGGACA TGAATTTGTG 300 AGACTGGGTC ATCACAATGT C 321 (2) INFORMATION OF SEQ ID NO: 19: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 107 amino acids (B) TYPE: amino acid (C) TYPE OF FILAMENT: (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 19: Aap He Val Met Thr Gln Ser Hia Lya Phe Mßt Sßr Thr Sßr Val Gly 1 5 10? S Aap Arg Val Sßr He He Cys Lya Wing Sßr sln Asp Val Gly Thr Wing 20 25 30 Val Asp Trp Tyr Gln Gln Lys Pro sly sln Being Pro Lys Leu Leu He 35 40 45 Tyr Trp Wing Being Thr Arg His Thr sly Val Pro Asp Arg Phe Thr Oly 50 55 60 Being Gly Being Gly Thr Aap Phß Thr Leu Thr He Thr Asn Val sln Sßr 65 70 75 80 Olu Asp Leu Ala Asp Tyr Phe Cya sln Oln Tyr Asn Ser Tyr Pro Leu 85 90 95 Thr Phe sly Ala sly Thr Met Leu Asp Leu Lys 100 105 It is noted that in relation to this date the best method known by the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention.

Having described the invention as above, it is claimed as property in the following,

Claims

CLAIMS 1. A method of removing or destroying cancer cells that is characterized in that it comprises: providing a biological agent that is characterized in that when it is brought into contact with an extracellular region of prostate-specific membrane antigen, it binds to the extracellular region of the antigen of the prostate. specific membrane of the prostate and, contact vascular endothelial cells next to the cancer cells with the biological agent under effective conditions to allow both, to bind the biological agent to the vascular endothelial cells next to the cancer cells and the extirpation or destruction of the cancer cells .
2. A method according to claim 1, characterized in that the biological agent destroys or removes the vascular endothelial cells close to the cancer cells, thereby destroying or extirpating the cancer cells by reducing their blood flow.
3. A method according to claim 1, characterized in that the cancer cells are renal cancer cells, urothelial cancer cells, colon cancer cells, rectal cancer cells, lung cancer cells, breast cancer cells, or metastatic adenocarcinoma cancer cells. of the liver 4. A method according to claim 1, characterized in that the biological agent is an antibody or binding portion thereof, probe, oligonucleotide. 5. A method according to claim 1, characterized in that the biological agent, when placed in contact with an extracellular region of prostate-specific membrane antigen, is internalized with the prostate-specific empennagen antigen. 6. A method according to claim 1, characterized in that said contact is carried out in a living mammal and comprises: administering the biological agent to the mammal under effective conditions to allow both to bind the biological agent to vascular endothelial cells near the cells cancerous and destroy cancer cells. 7. A method according to claim 6, characterized in that said administration is carried out orally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, by intracavity or intravesicular instillation, intraocularly, intraarterially, intralesionally, or by application to the mucous membranes. 8. A method according to claim 4, characterized in that an antibody is used to carry out said method, the antibody is selected from the group consisting of a monoclonal antibody and a polyclonal antibody. 9. A method according to claim 8, characterized in that the antibody is selected from the group consisting of a monoclonal antibody E99, a J415, a J533, and a J591. 10. A method according to claim 8, which is caracered because the antibody is a monoclonal antibody produced by a hybridoma cell line having an ATCC access number selected from the group consisting of HB-12101, HB-12109, HB-12127, and HB-12126. 11. A method according to claim 4, characterized in that a binding portion of an antibody is used to carry out said method, the binding portion being selected from the group consisting of a Fab fragment, an F (ab ') fragment. 2, and a Fv fragment. 12. A method of compliance to claim 4, characterized in that the probe or ligand is used in carrying out said method. 13. A method according to claim 1, characterized in that the biological agent is associated with a substance effective to destroy or remove the cancer cells on the binding of the biological agent of vascular endothelial cells close to the cancer cells. 14. A method according to claim 13, which is characterized in that the substance effective to destroy or remove the cancer cells is a cytotoxic drug. 15. A method according to claim 14, characterized in that the cytotoxic drug is selected from the group consisting of therapeutic drugs, a radiation emitting compuersto, plant molecules, fungi, or bacterial origin, biological proteins, and mixtures of these . 16. A method according to claim 4, characterized in that the antibody is effective to initiate an immune function in an endogenous host. 17. A method according to claim 16, which is characterized in that the immune function in the endogenous host is cellular mediator complement cytotoxicity. 18. A method according to claim 16, which is characterized in that the immune function of the endogenous host is antibody-dependent cellular cytotoxicity. 19. A method according to claim 1, characterized in that the biological agent is in a further composition comprising a physiologically acceptable carrier, excipient, or stabilizer. 20. A method according to claim 1, characterized in that the biological agent is in a further composition comprising a pharmaceutically acceptable carrier, excipient, or stabilizer. 21. A method of detecting cancerous tissue in a biological sample that is characterized in that it comprises: providing a biological agent that, when placed in contact with an extracellular region of prostate-specific membrane antigen, binds to the extracellular region of the specific membrane antigen of prostate, which is characterized in that the biological agent is associated with an effective marker to allow the detection of vascular endothelial cells close to or in the cancerous tissue over the binding of the biological agent in the vascular endothelial cells close to or in the cancerous tissue; contacting the biological sample with the biological agent having a marker under effective conditions to allow binding to the biological agent to vascular endothelial cells near or in the cancerous tissue in the biological sample; and detecting the presence of any cancerous tissue in the biological sample by detecting the mark. 22. A method according to claim 21, which is caracerated because the cancerous tissue is renal cancer tissue, cancerous urothelial tissue, cancerous colon tissue, rectal cancerous tissue, cancerous lung tissue, cancerous breast tissue, or metastatic adenocarcinoma cancerous tissue of the liver 23. A method according to claim 21, characterized in that the biological agent is an antibody or binding portion thereof, probe, or ligand. 24. A method according to claim 21, characterized in that the biological agent, when placed in contact with the extracellular region of the prostate-specific membrane antigen, is internalized with the specific membrane antigen of the prostate. 25. A method according to claim 21, characterized in that said contact is carried out in a living mammal and because it comprises: administering the biological agent to the mammal under conditions effective to allow the binding of the biological agent to vascular endothelial cells close to or in the cancerous tissue in the biological sample. 26. A method according to claim 25, characterized in that the marker is a short-range radiation emitter. 27. A method according to claim 25, characterized in that said administration is carried out orally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intraversal instillation, by intracavity instillation or by intravesicular instillation, intraocularly, intraarterially, intralesionally. or by application on mucous membranes. 28. A method according to claim 23, which is caracered because an antibody is used to carry out said method, said antibody being selected from the group consisting of a monoclonal antibody and a polyclonal antibody. 29. A method according to claim 28, characterized in that the antibody is selected from the group consisting of a polyclonal antibody E99, a J415, a J533, and a J591. 30. A method according to claim 28, characterized in that the antibody is a monoclonal antibody produced by a hybridoma cell line having an ATCC Accession number selected from the group consisting of HB-12101, HB-12109, HB-12127, and HB-12126. 31. A method according to claim 23, characterized in that a binding portion of an antibody is used to carry out said method, the binding portion being selected from the group consisting of a Fab fragment, an F (ab ') fragment. 2, and a Fv fragment. 32. A method according to claim 23, characterized in that a probe or lidand is used to carry out said method. 33. A method according to claim 21, characterized in that the marker is selected from the group consisting of a fluorescent label, a radioactive label, an active marker to nuclear magnetic resonance, a luminescent label, and a chromophoric label. 34. A method according to claim 21, characterized in that the biological agent is in a further composition comprising a physiologically acceptable carrier, excipient or stabilizer. 35. A method according to claim 21, characterized in that the biological agent is in a further composition comprising a pharmaceutically acceptable carrier, excipient or stabilizer. 36. A method according to claim 21, characterized in that said contact is carried out in a serum or urine sample. 37. A method according to claim 21, which is chaper- ized because said contact is carried out on a tissue biopsy sample. 38. A method of removing or destroying normal, hyperplastic benign cells, and cancerous prostate epithelial cells, comprising: providing a biological agent that binds an extracellular region of prostate-specific membrane antigen and contacting said cells with the biological agent under conditions effective to allow both, binding of the biological agent to the extracellular region of the prostate-specific membrane antigen and the extirpation or destruction of said cells. 39. A method according to claim 38, characterized in that the biological agent is an antibody or binding portion thereof, probe, or ligand. 40. A method according to claim 38, which is characterized in that the biological agent is internalized with the specific membrane antigen of the prostate. 41. A method according to claim 38, characterized in that said contacting is carried out in a living mammal and comprises: Administering the biological agent to the mammal under effective conditions to allow both, binding of the biological agent to the extracellular region of the mammal. prostate-specific membrane antigen and destruction of said cells. 42. A method according to claim 41, which is characterized in that the biological agent is internalized with the specific membrane antigen of the prostate. 43. A method according to claim 21, characterized in that said administration is carried out orally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, intracavity instillation, or intravesicular instillation, intraocularly, intraarterially, intralesionally, or by application to mucous membranes. 44. A method according to claim 39, characterized in that an antibody is used to carry out said method, the antibody is selected from the group consisting of a monoclonal antibody and a polyclonal antibody. 45. A method according to claim 44, characterized in that the antibody is selected from the group consisting of a polyclonal antibody E99, a J415, a J533, and a J591. 46. A method according to claim 44, characterized in that the antibody is a monoclonal antibody produced by a hybridoma cell line having an ATCC Accession number selected from the group consisting of HB-12101, HB-12109, HB-12127, and HB-12126. 47. A method according to claim 39, which is characterized in that a binding portion of an antibody is used to carry out said method, the binding portion is selected from the group consisting of a Fab fragment, an F (ab ') 2 fragment. , and a Fv fragment. 48. A method according to claim 39, characterized in that the probe or ligand is used to carry out said method. 49. A method according to claim 38, characterized in that the biological agent is associated with a substance effective to destroy or extirpate said cells on the binding of the biological agent to the extracellular region of the prostate-specific membrane antigen of said cells. 50. A method according to claim 49, which is characterized in that the substance effective to destroy said cells is a cytotoxic drug. 51. A method according to claim 50, characterized in that the cytotoxic drug is selected from the group consisting of therapeutic drug, a radiation emitting compound, plant molecules, fungi, or bacterial origin, biological proteins, and mixtures of these . 52. A method according to claim 39, which is characterized in that the antibody is effective to initiate an immune function in an endogenous host. 53. A method according to claim 52, which is characterized in that the immune function of the endogenous host is the cellular cytotoxicity of mediated compelment. 54. A method according to claim 52, which is caracered because the immune function of the endogenous host is the antibody-dependent cellular cytotoxicity. 55. A method according to claim 38, which is caracered because the biological agent is in a further composition comprising a physiologically acceptable carrier, excipient or stabilizer. 56. A method according to claim 38, which is caracerated because the biological agent is in an additional composition comprising a pharmaceutically acceptable carrier, excipient or stabilizer. 57. A method according to claim 38 which is characterized in that it additionally comprises: providing a second biological agent that binds the extracellular region of the prostate-specific membrane antigen and contacting said cells with the second biological agent under effective conditions to allow the binding of the second biological agent to the extracellular region of the prostate-specific membrane antigen. 58. A method according to claim 57, which is characterized in that the biological agent and the second biological agent link to non-capable binding sites on the extracellular region of the prostate-specific membrane antigen. 59. A method according to claim 57, which is caracered because the binary agent is a monoclonal antibody J415 and the second biological agent is a monoclonal antibody E99, a J533, or a J591. 60. A method according to claim 57, which is caracered because the biological agent is associated with a substance effective to destroy or extirpate said cells on the binding of the biological agent to the extracellular region of the prostate-specific membrane antigen of said cells and by activation by an activator and characterized in that the second biological agent is linked to the activator. 61. A method of detecting normal, benign hyperplastic, and cancerous prostate epithelial cells or a portion thereof in a biological sample comprising: providing a biological agent that binds an extracellular region of prostate-specific membrane antigen, which is characterized in that biological agent is associated with a specific marker to allow the detection of said cells or a portion thereof on the binding of the biological agent to said cells or a portion thereof; contacting the biological sample with the biological agent having a marker under conditions effective to allow binding of the biological agents to the extracellular region of the prostate-specific membrane antigen of said cells or a portion thereof in the biological sample; and detecting a presence of any of said cells or a portion thereof in the biological sample by detection of the tag. 62. A method according to claim 61, which is characterized in that the biological agent is an antibody or binding portion thereof, probe, or ligand. 63. A method according to claim 61, which is characterized in that the biological agent is internalized with the prostate-specific membrane antigen. 64. A method according to claim 61, characterized in that said contacting is carried out in a living mammal and comprises: administering the biological agent to the mammal under conditions effective to allow binding of the biological agent to the extracellular region of the antigen of prostate-specific membrane of any of said cells or a portion thereof in the biological sample. 65. A method according to claim 64, characterized in that the marker is a short range radiation emitter. 66. A method according to claim 64, characterized in that said detection is carried out correctly. 67. A method according to claim 64, characterized in that the biological sample is from the mammalian prostatic fossa. 68. A method according to claim 64, characterized in that said detection is carried out after a prostatectomy. 69. A method according to claim 64, which is characterized in that the biological agent is internalized with the prostate-specific membrane antigen. 70. A method according to claim 64, characterized in that said administration is carried out orally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intraversal instillation, intracavity instillation, or intravesicular instillation, intraocularly, intraarticularly, intralesionally, application to mucous membranes. 71. A method according to claim 62, characterized in that an antibody is used to carry out said method, said antibody is selected from the group consisting of a monoclonal antibody and a polyclonal antibody. 72. A method according to claim 71, which is characterized in that the antibody is selected from the group consisting of a monoclonal antibody E99, a J415, a J533 and a J591. 73. A method according to claim 71, characterized in that the antibody is a monoclonal antibody produced by a hybridoma cell line having an Access ATCC Number selected from the group consisting of HB-12101, HB-12109, HB-12127, and HB-12126. 74. A method according to claim 62, characterized in that a binding portion of an antibody is used to carry out said method, the binding portion is selected from the group consisting of the fragment Fab, fragment F (ab ') 2, and a fragment Fv. 75. A method according to claim 62, which is characterized in that a probe or ligand is used to carry out said method. 76. A method according to claim 61, which is characterized in that the marker is selected from the group consisting of a fluorescent label, a radioactive label, an active marker to nuclear magnetic resonance, a luminescent label, and a chromophoric label. 77. A method according to claim 61, characterized in that the biological agent is in a further composition comprising a physiologically acceptable carrier, excipient, or stabilizer. 78. A method according to claim 61, characterized in that the biological agent is in a further composition comprising a pharmaceutically acceptable carrier, excipient or stabilizer. 79. A method according to claim 61, characterized in that said contacting is carried out in a serum or urine sample. 80. An isolated biological agent that binds an extracellular region of prostate-specific membrane antigen. 81. An isolated biological agent according to claim 80, characterized in that said isolated biological agent is an isolated antibody or a binding portion thereof, probe, or ligand. Isolated biological is a binding portion of an antibody selected from the group consisting of a Fab fragment, an F (ab ') 2 fragment, and an Fv fragment. 37. An isolated biological agent according to claim 81, which is characterized in that the isolated biological agent is a probe, or a ligand. 88. An isolated biological agent according to claim 80, characterized in that the biological agent is associated with a cytotoxic drug. 39. An isolated biological agent according to claim 88, characterized in that the cytotoxic drug is selected from the group consisting of a therapeutic drug, a radiation emitter, plant molecules, fungi, or bacterial origin, biological proteins, and mixtures of these. 90. A composition characterized in that it comprises: a biological agent according to claim 88 and a carrier, excipient, or stabilizer mixed with the physiologically acceptable biological agent. 82. An isolated biological agent according to claim 80, characterized in that the biological agent is internalized with the prostate-specific membrane antigen. 83. An isolated biological agent according to claim 81, characterized in that the biological agent is an antibody selected from the group consisting of a monoclonal antibody and a polyclonal antibody. 84. An isolated biological agent according to claim 83, characterized in that the antibody is selected from the group consisting of a monoclonal antibody E99, a J415, a J533, and a J591. 85. An isolated biological agent according to claim 83, characterized in that the antibody is a monoclonal antibody produced by a hybridoma having an ATCC Accession number selected from the group consisting of HB-12101, HB-12109, HB-12127, and HB-12126. 86. An isolated biological agent according to claim 81, which is characterized in that the agent 91. A composition characterized in that it comprises: a biological agent according to claim 88 and a vehicle, excipient or stabilizer mixed with the pharmaceutically acceptable biological agent. 92. An isolated biological agent according to claim 80, characterized in that said biological agent is associated with a marker. 93. A biological agent according to claim 92, characterized in that the marker is selected from the group consisting of a fluorescent label, a biologically active enzyme label, a radioactive label, an active marker to nuclear magnetic resonance, a luminescent label, and a chromophoric marker. 94. A composition characterized Dorpiip rninnran ^. a biological agent according to claim 92 and a carrier, excipient, or stabilizer mixed with the physiologically acceptable biological agent. 95. A composition characterized Dornup mmnronHe; a biological agent according to claim 92 and a carrier, excipient, or stabilizer with the pharmaceutically acceptable biological agent. 96. A cancer screening device for the norpnp rnmnrpnrip: a biological agent according to claim 92 and means for detecting the marker. 97. An equipment according to claim 96, which is characterized in that the label is selected from the rupe consisting of a fluorescent label, a biologically active enzyme label, a radioactive label, a luminescent label, and a chromophoric label. 98. A kit according to claim 96, characterized in that said biological agent is a monoclonal antibody E99, a J415, a J533, or a J591. 99. A kit according to claim 96, characterized in that the biological agent is in a further composition comprising a physiologically acceptable carrier, excipient, or stabilizer. 100. A kit according to claim 96, characterized in that the biological agent is in a further composition comprising a pharmaceutically acceptable carrier, excipient or stabilizer. 101. A kit according to claim 96, characterized in that the cancer is selected from the group consisting of prostate cancer, renal cancer, urothelial cancer, colon cancer, lung cancer, breast cancer, and metastatic adenocarcinoma of the liver. 102. A hybridoma cell line that produces a monoclonal antibody that binds an extracellular region of prostate-specific membrane antigen. 103. A hybridoma cell according to claim 102, which is characterized in that the antibody is internalized with the prostate-specific membrane antigen. 104. A hybridoma cell line according to claim 102, which is characterized in that the monoclonal antibody is a monoclonal antibody E99, a J415, a J533, or a J591. 105. A hybridoma cell line according to claim 102 which is characterized in that the hybridoma cell line has an ATCC Accession number selected from the group consisting of HB-12101, HB-12109, HB-12127, and HB-