Treatment of prostate cancer by inhibitors of ATP synthase

FIELD OF INVENTION

[0002] This invention concerns methods for treating or preventing prostate cancer by inhibitors of ATP synthase activity.

BACKGROUND OF THE INVENTION

[0003] Prostate cancer is the most common cancer in men with an estimated 244,000 cases in 1995 in the United States. It is the second leading cause among men who die from neoplasia with an estimated 44,000 deaths per year. Prompt detection and treatment is needed to limit mortality caused by 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 watchful waiting, curative treatment, and palliation.

[0004] A number of approaches have been developed to treat prostate cancer. Where prostate cancer is localized and the patient's life expectancy is 10 years or more, radical prostatectomy offers the best chance for eradication of the disease. Historically, the drawback of this procedure is that most cancers had spread beyond the bounds of the operation by the time they were detected. After surgery, if there are detectable serum prostate-specific antigen concentrations (PSA), persistent cancer is indicated. In many cases, prostate-specific antigen concentrations can be reduced by radiation treatment. However, this concentration often increases again within two years.

[0005] Cytotoxic chemotherapy is largely ineffective in treating prostate cancer. Its toxicity makes such therapy unsuitable for elderly patients. In addition, prostate cancer is relatively resistant to cytotoxic agents.

[0006] In view of the deficiency of the existing treatment approaches, it is of great significance to pursue new methods of treatment that particularly target the prostate tumor cells, such as anti-prostate tumor agents. The present invention is intended to use inhibitors of ATP synthase activity as such anti-tumor agents.

[0007] ATP synthase is the enzyme catalyzing the synthesis of ATP. It is seen as spherical projections from the inner membrane surface of mitochondria, consisting of two subcomplexes: F0 and F1. F1 consists of five different polypeptide chains with the stoichiometry α3β3γδε. The F0 subcomplex 11 different subunits and forms a hydrophobic unit that spans the inner mitochondrial membrane. The F0 complex has the ability to translocate protons across the membrane from their high potential on the outside, and when coupled to the F1 subcomplex, ATP synthesis can be achieved. (Harold, F. M., The Vital Force: A Study of Bioenergetics (W. H. Freeman, 1986), pp. 238) (which is hereby incorporated by reference).

[0008] Apart from the traditional role in ATP production, recent studies suggest that ATP synthase may play critical roles in tumor cell metastases. Studies have shown that ATP synthase plays important roles in angiogenesis (the generation of new blood vessels required by tumor to expand beyond a prevascular size). Angiostatin, a potent antagonist of angiogenesis, was found to bind ATP synthase on the surface of human endothelial cells (Moser, T. L., et al., Proc. Natl. Acad. Sci. USA: Vol. 96, pp 2811-2816, 1999). These authors propose that all components of F1 ATP synthase catalytic core are present on the endothelial cell surface since the complex is able to synthesize ATP. In addition, the surface-associated enzyme is active in ATP synthesis as shown by dual-label TLC and bioluminescence assays. ATP synthase activities of the enzyme are inhibited by angiostatin as well as by antibodies directed against the α- and β-subunits of ATP synthase in cell-based and biochemical assays. These experimental results suggest that the inhibitors of ATP synthase activity, such as antibodies against ATP synthase, function as antagonists of angiogenesis. (Moser, T. L., et al., Proc. Natl. Acad. Sci. USA: Vol. 98, pp 6656-6661, 2001). In addition, β-subunit of ATP synthase is also found to differentially express on the surface of some tumor cell lines. It is speculated that ATP synthase is an important ligand in the effector phase of a cytolytic pathway, and plays a role in lymphocyte-induced tumor destruction. (Das, B., et al., J. Exp. Med. Vol. 180, pp 273-281, 1994).

[0009] So far no investigation has been conducted to explore the possible therapeutic application of inhibitors of ATP synthase in prostate cancer treatment. This invention is directed to methods for treating and preventing prostate cancer by using the inhibitors of ATP synthase.

SUMMARY OF THE INVENTION

[0010] The present invention is directed to a method of treating or preventing prostate cancer in a subject in need of such a treatment by administering an inhibitor of ATP synthase in a pharmaceutically effective amount. The present invention also provides for a pharmaceutical composition comprising an inhibitor of ATP synthase, preferably, said inhibitor is an anti-ATP synthase antibody

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]
FIG. 1. Comparison of lipid rafts prepared from normal prostate cells and three prostate carcinoma cell lines, DU 145, LNCaP, and PC-3. Equal amounts of protein from lipid raft preparations were separated by SDS-PAGE and then visualized by silver staining. Differentially expressed proteins are marked.

[0012]
FIG. 2. Comparison of lipid rafts prepared from LNCaP and normal prostate cells by 2-dimensional electrophoresis. Equal amounts of protein from lipid raft preparations were separated by 2-dimensional electrophoresis and then visualized by silver staining. Protein spots that are present in the LNCaP sample, but not the normal prostate sample are denoted with arrows. Protein spots that have been identified by peptide mass profiling are labeled with numbers (see Table 1).

[0013]
FIG. 3. ATP synthase is present in lipid rafts preparations from prostate cancer cell lines, but not from normal prostate cells. Equal amounts of lipid raft proteins prepared from normal prostate cells and three prostate carcinoma cell lines (LNCaP, DU 145, and PC-3) were separated by SDS-PAGE and then electrotransferred onto a PVDF membrane. Western blotting was performed using an antibody specific for ATP synthase followed by HRP-conjugated goat anti-mouse IgG. The membrane was developed using enhanced chemiluminescence.

[0014]
FIG. 4. ATP synthase is present in rafts prepared from many different cancer cell lines. Equal amounts of lipid raft proteins prepared from various cancer cell lines (BeWo, Colo205, HT-29, JEG-3, KG-1, LS 180, MCF-7, LNCaP, NCI-H292, PANC-1, RT-4, and THP-1) were separated by SDS-PAGE and then electrotransferred onto a PVDF membrane. Western blotting was performed as described in FIG. 3.

[0015]
FIG. 5. Flow cytometric analysis of ATP synthase cell surface expression. LNCaP cells were resuspended in 100 μL PBS/5% fetal calf serum with 1 μg anti-ATP synthase (middle) or anti-Trop-1 (EpCAM) (right). Cells were also stained with a negative control antibody (left). Cells were washed and bound antibody was detected with PE-conjugated goat anti-mouse IgG. Cells were then analyzed by flow cytometer.

[0016]
FIG. 6. Flow cytometric analysis of ATP synthase expression in the AML cell line THP-1. THP-1 cells were resuspended in 100 μL PBS/5% fetal calf serum with 1 μg anti-ATP synthase α subunit (middle) or β subunit (right). Cells were also stained with a negative control antibody (left). Cells were washed and bound antibody was detected with PE-conjugated goat anti-mouse IgG. Cells were then analyzed by flow cytometer.

[0017]
FIG. 7. ATP synthase is localized to the cell surface of LNCaP prostate carcinoma as visualized by immunofluorescence. LNCaP cells, grown on glass coverslips, were stained with antibodies specific for ATP synthase (top) or Trop-1 (EpCAM) (bottom). Bound antibody was detected with Alexa 488-conjugated goat anti-mouse IgG. Coverslips were mounted onto slides and examined with a Nikon Optiphot 2 microscope and photographed. No staining is observed on cells that have been stained with the secondary antibody alone (data not shown).

[0018]
FIG. 8. Anti-ATP synthase inhibits LNCaP cell proliferation. LNCaP cells (20,000 cells/well) were plated into a 96 well tissue culture plate. After cells were allowed to grow undisturbed for two days, antibodies (5 μg/ml anti-ATP synthase, anti-Trop-1 (EpCAM) (323/A3), or anti-MHC class II (Mu1D10)) were added and incubated with the cells for 24 hours. AlamarBlue reagent was added to assess cell proliferation. Fluorescence was detected at λex=530 nm, λem=590 nm. Data are expressed as the mean+/−SEM of 4 replicates.

[0019]
FIG. 9. Anti-ATP synthase inhibits LNCaP colony formation in soft agar. LNCaP cells were plated in soft agar and treated with anti-ATP synthase or anti-Trop-1 (EpCAM) (5 μg/ml) for up to 20 days. Colonies were counted under an inverted phase-contrast microscope and a group of 5 or more cells were counted as a colony.

[0020]
FIG. 10. Anti-ATP synthase induces apoptosis in THP-1 cells. THP-1 cells were treated with anti-ATP synthase or anti-Trop-1 (EpCAM) (5 μg/mL) for 24 hours. Cells were then harvested at the indicated times after the induction of apoptosis and were stained with FITC-conjugated annexin V and propidium iodide. Flow cytometry was used to assess percentage of apoptosis (annexin V+ and propidium iodide−/+ cells).

[0021]
FIG. 11. Comparison of lipid rafts from various cancer cell lines. Equal amounts of protein from lipid raft preparations were separated by SDS-PAGE and then visualized by silver staining.

[0022]
FIG. 12. Table 1. shows five identified proteins from LNCaP lipid raft 2-D samples by peptide mass profiling.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Definitions:

[0024] As used herein, the term “antibody” or “immunoglobulin” refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Full-length immunoglobulin “light chains” (about 25 Kd or 214 amino acids) are encoded by a variable region gene at the NH2-terminus (about 110 amino acids) and a kappa or lambda constant region gene at the COOH-terminus. Full-length immunoglobulin “heavy chains” (about 50 Kd or 446 amino acids), are similarly encoded by a variable region gene (about 116 amino acids) and one of the other aforementioned constant region genes, e.g., gamma (encoding about 330 amino acids).

[0025] One form of immunoglobulin constitutes the basic structural unit of an antibody. This form is a tetramer and consists of two identical pairs of immunoglobulin chains, each pair having one light and one heavy chain. In each pair, the light and heavy chain variable regions are together responsible for binding to an antigen, and the constant regions are responsible for the antibody effector functions. In addition to antibodies, immunoglobulins may exist in a variety of other forms including, for example, Fv, Fab, and (Fab′)2, as well as bifunctional hybrid antibodies (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)) and in single chains (e.g., Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85, 5879-5883 (1988) and Bird et al., Science, 242, 423-426 (1988), which are incorporated herein by reference). (See, generally, Hood et al., “Immunology”, Benjamin, N.Y., 2nd ed. (1984), and Hunkapiller and Hood, Nature, 323, 15-16 (1986), which are incorporated herein by reference).

[0026] By “a pharmaceutically effective” amount of a drug or pharmacologically active agent or pharmaceutical formulation is meant a nontoxic but sufficient amount of the drug, agent or formulation to provide the desired effect.

[0027] A “subject,” “individual” or “patient” is used interchangeably herein, which refers to a vertebrate, preferably a mammal, more preferably a human.

[0028] The term “genetically altered antibodies” means antibodies wherein the amino acid sequence has been varied from that of a native antibody. Because of the relevance of recombinant DNA techniques to this invention, one need not be confined to the sequences of amino acids found in natural antibodies; antibodies can be redesigned to obtain desired characteristics. The possible variations are many and range from the changing of just one or a few amino acids to the complete redesign of, for example, the variable or constant region. Changes in the constant region will, in general, be made in order to improve or alter characteristics, such as complement fixation, interaction with membranes and other effector functions. Changes in the variable region will be made in order to improve the antigen binding characteristics.

[0029] The term “humanized antibody” or “humanized immunoglobulin” refers to an immunoglobulin comprising a human framework, at least one and preferably all complimentarily determining regions (CDRs) from a non-human antibody, and in which any constant region present is substantially identical to a human immunoglobulin constant region, i.e., at least about 85-90%, preferably at least 95% identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of one or more native human immunoglobulin sequences. See, e.g. Queen et al., U.S. Pat. Nos. 5,5301,101; 5,585,089; 5,693,762; and 6,180,370 (each of which is incorporated by reference in its entirety).

[0030] The term “chimeric antibody” refers to an antibody in which the constant region comes from an antibody of one species (typically human) and the variable region comes from an antibody of another species (typically rodent).

[0031] The term “inhibit growth of cancer (tumor) cells” refers to any action that may decrease the growth of a cancer cell. The inhibition may reduce the growth rate or the size of cancer cells, or inhibit or prevent proliferation, growth, or migration of cancer cells. The inhibition may inhibit the colony formation of cancer cells due to the anchorage-independent growth. Preferably, such an inhibition at the cellular level may reduce the size, deter the growth, reduce the aggressiveness, or prevent or inhibit metastasis of a tumor (cancer) in a patient.

[0032] The term “colony formation” refers to the number of tumor (cancer) cell colonies formed due to the anchorage-independent tumor (cancer) cell growth. A variety of methods can be used to measure the “colony formation”, such as by counting the number of the formed colonies formed (see Examples).

[0033] The present invention provides for a method of treating or preventing prostate cancer.

[0034] In a preferred embodiment of the present invention, an inhibitor of ATP synthase activity is administered to a subject in need of such a treatment in a pharmaceutically effective amount.

[0035] The present invention also provides for a method of inhibiting growth of a cancer cell comprising contacting an inhibitor of ATP synthase with said cancer cell. Preferably, said inhibiting reduces the colony formation of a cancer cell by more than 50%, 60%, 70%, 80% or 90%, or as high as 95%. More preferably, said cancer cell is a prostate cancer cell.

[0036] Preferably, the inhibitor is a protein.

[0037] More preferably, the protein directly interacts with ATP synthase.

[0038] More preferably, the protein is an anti-ATP synthase antibody, including but not limited to an antibody recognizing the α-subunit of ATP synthase, or an antibody recognizing the β-subunit of ATP synthase.

[0039] Preferably, the anti-ATP synthase antibody can inhibit cancer cell proliferation by more than 10%, more preferably by more than 20%. More preferably, said cancer cell is a prostate cancer cell.

[0040] Preferably, the anti-ATP synthase antibody can inhibit cancer cell colony formation by more than 50%, 60%, 70%, 80% more preferably by more than 90%, or by as high as about 95%. Preferably, said cancer cell is a prostate cancer cell.

[0041] The present invention provides a method of inducing apoptosis of leukemia cells comprising contacting an antibody with said leukemia cells, wherein said antibody binds to or neutralizes ATP synthase. Preferably, said antibody induces apoptosis of the leukemia cells by more than 20%. Preferably, said leukemia cells are acute myelogenous leukemia cells. More preferably, said leukemia cells are cells derived from THP-1 cell line.

[0042] The present invention provides a method of treating eukemia in a subject in need of such a treatment comprising administering into said subject an antibody against ATP synthase in a pharmaceutically effective amount.

[0043] The anti-ATP synthase antibodies of the present invention may be in a polyclonal or monoclonal form and may bind to any epitope or subunit of ATP synthase. Anti-ATP synthase antibodies of all species of origins are included. Non-limiting exemplary anti-ATP synthase antibodies include antibodies derived from human, chicken, goats, and rodents (e.g., rats, mice, hamsters and rabbits), including transgenic rodents genetically engineered to produce fully human antibodies (see, e.g., Lonberg et al., WO93/12227 (1993) and Kucherlapati, et al., WO91/10741 (1991)), which are herein incorporated by reference in their entirety).

[0044] The polyclonal antibodies can be produced by immunization of host animals by ATP synthase. The polyclonal antibodies are secreted into the bloodstream and can be recovered using known techniques. Purified forms of these antibodies can, of course, be readily prepared by standard purification techniques, preferably including affinity chromatography with Protein A, anti-immunoglobulin, or the antigen itself. In any case, in order to monitor the success of immunization, the antibody levels with respect to the antigen in serum will be monitored using standard techniques such as ELISA, RIA and the like.

[0045] The monoclonal antibodies can be produced by conventional hybridoma methodology known in the art. In particular, after the immunization with ATP synthase, the host animal may be sacrificed and the lymphocytes of said animal are isolated. The lymphocytes can produce or be capable of producing antibodies that specifically bind to the protein used for immunization. Lymphocytes then are fused with myeloma cells using suitable fusing agents to form hybridomas cells that produce the desired monoclonal antibody.

[0046] The antibodies may also be produced by using the method of lipid raft proteomics or lipid raft immunization, which is disclosed in U.S. Ser. No. 60/331,965, hereby incorporated by reference in its entirety. In particular, the method for identifying anti-tumor agents comprises immunizing an animal with lipid rafts from the interested cancer cells, such as prostate cancer cell, creating hybridomas from the immunized animal; screening the hybridomas, and purifying and identifying the hybridoma antibodies (see more details in U.S. Ser. No. 60/331,965).

[0047] The present invention also includes genetically altered antibodies that are functionally equivalent to the anti-ATP synthase antibodies. Modified antibodies providing improved stability and/or therapeutic efficacy are preferred. Examples of modified antibodies include those with conservative substitutions of amino acid residues, and one or more deletions or additions of amino acids which do not significantly deleteriously alter the antigen binding utility. Substitutions can range from changing or modifying one or more amino acid residues to complete redesign of a region as long as the therapeutic utility is maintained. Antibodies of this invention can be modified post-translationally (e.g., acetylation, and phosphorylation) or can be modified synthetically (e.g., the attachment of a labeling group). The genetic modification can be achieved by the standard molecular cloning techniques known in the art.

[0048] The genetically altered antibodies also include chimeric antibodies that derived from the anti-ATP synthase antibodies. Preferably, the chimeric antibodies comprise a variable region derived from a mouse or rat and a constant region derived from a human so that the chimeric antibody has a longer half-life and is less immunogenic when administered to a human subject. The method of making chimeric antibodies is known in the art.

[0049] Preferably, the genetically altered anti-ATP synthase antibodies used in the present invention include humanized version of the antibodies described herein. More preferably, said humanized antibody comprising CDRs of a mouse donor immunoglobulin and heavy chain and light chain frameworks of a human acceptor immunoglobulin. The method of making humanized antibody is disclosed in U.S. Pat. Nos.: 5,530,101; 5,585,089; 5,693,761; 5,693,762; 6,180,370, each of which is incorporated by reference in its entirety.

[0050] The fragments of the antibodies disclosed herein, which retain the binding specificity to ATP Synthase, are also included in the present invention. Examples include, but are not limited to, the heavy chains, the light chains, and the variable regions as well as the truncated chains (truncated at the carboxyl end), which is particularly useful for immunoscintigraphic procedures. Examples of truncated chains include, but are not limited to Fab fragment (consisting of the VL, VH, CL and CH1 domains); the Fd fragment (consisting of the VH and CH1 domains); the Fv (consisting of VL and VH domains of a single arm of an antibody); dab fragment (consisting of a VH domain); isolated CDR regions; F(ab′)2 fragment, a bivalent fragment (comprising two Fab fragments linked by a disulphide bridge at the hinge region). The truncated chains can be produced by conventional biochemistry techniques, such as enzyme cleavage, or recombinant DNA techniques, each of which is known in the art.

[0051] The variable region of the antibodies and its humanized version of the present invention may be fused to functional regions from other genes (e.g., enzymes, U.S. Pat. No. 5,004,692, which is incorporated by reference in its entirety) to produce fusion proteins (e.g., immunotoxins) or conjugates having novel properties. When used therapeutically, the antibodies disclosed herein may be used in unmodified form or may be modified with an effector moiety that delivers a toxic effect, such as a drug, cytotoxin (preferably, a protein cytotoxin or a Fe domain of the monoclonal antibodies), radionuclide, etc (see, e.g., U.S. Pat. No. 6,086,900, which is incorporated by reference in its entirety).

[0052] Conjugates that are immunotoxins including conventional antibodies have been widely described in the art. The toxins may be coupled to the antibodies by conventional coupling techniques or immunotoxins containing protein toxin portions can be produced as fusion proteins. The conjugates of the present invention can be used in a corresponding way to obtain such immunotoxins. Illustrative of such immunotoxins are those described by Byers, B. S. et al. Seminars Cell Biol 2:59-70 (1991) and by Fanger, M. W. et al. Immunol Today 12:51-54 (1991). (See, generally, “Chimeric Toxins,” Olsnes and Phil, Pharmac. Ther., 25, 355-381 (1982), and “Monoclonal Antibodies for Cancer Detection and Therapy,” eds. Baldwin and Byers, pp. 159-179, 224-266, Academic Press (1985),

[0053] In addition, the inhibitor can also be a compound that inhibits the enzyme activity of ATP synthase, including but not limited, to an antagonist for ATP synthase. More examples include compounds that may interact with ATP synthase signaling pathway and down-regulates the activity of ATP synthase. The understanding of the mechanism of ATP synthase in tumor activity provides the useful information for the search of this kind of compounds. Several existing theories explain the possible role of ATP synthase on tumor cell surface. First of all, cell-surface-generated ATP may be transported into the cell to provide a source of energy in the tumor microenvironment where Po2 levels are very low. Alternatively, cell surface-generated ATP may act through the P2Y receptor to activate Ca2+-dependent signaling cascades which increase DNA synthesis. The third theory is that ATP synthase may pump out protons which in turn acidifies the tumor microenvironment and as a consequence, pH-dependent enzymes produced by tumor cells are able to cut through the extracellular matrix and metathesize. Any of these theories provides a starting point to search for the ideal compounds or molecules as ATP synthase inhibitor. The suitable compounds can be sought by using the conventional techniques known to a skilled artisan in the field of organic chemistry and biochemistry.

[0054] In addition, the inhibitor can inhibit the protein expression of ATP synthase. The inhibitor is a nucleic acid including but not limited to an antis-sense nucleic acid of the nucleic acid sequence encoding part or full or having substantial sequence similarity of ATP synthase. The DNA sequence of ATP synthase is known in the art. Subsequently, anti-sense nucleic acid probe of ATP synthase DNA, and the optimal condition of the anti-sense blocking can be developed by using the related techniques known to a skilled artisan in the field of molecular biology.

[0055] The present invention also provides for a pharmaceutical composition comprising an inhibitor of ATP synthase activity. The pharmaceutical composition can further comprise a pharmaceutically acceptable carrier.

[0056] The effective treatment of prostate cancer by ATP synthase inhibitors can include various stages, such as androgen-dependent prostate cancer and androgen-independent prostate cancer.

[0057] For the purpose of treatment of disease, the appropriate dosage of the above inhibitors will depend on the severity and course of disease, the patient's clinical history and response, the toxicity of the inhibitors, and the discretion of the attending physician. The inhibitors are suitably administered to the patient at one time or over a series of treatments. The initial candidate dosage may be administered to a patient. The proper dosage and treatment regime can be established by monitoring the progress of therapy using conventional techniques known to the people skilled of the art.

[0058] Dosage levels of the order of from about 10−7 M to about 10−1 M, preferably in the range 10−5 to 10−1M, are useful in the treatment. The pharmaceutically effective amount ranges between about 0.01 to about 1000 mg, preferably between about 0.05 to about 100 mg, and most preferably between about 0.5 to about 50 mg for single doses. The amount of active ingredients that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors, including the activity of the specific inhibitor employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy, and can be determined by those skilled in the art.

[0059] There are various methods of administering the inhibitors. There are various methods of administering the inhibitors. The inhibitor may be administered to a patient intravenously as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intra-cerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, inhalation routes, or other delivery means known to the people skilled in the art.

[0060] Preferably, pharmaceutical compositions of the present invention are useful for parenteral administration, i.e., subcutaneously, intramuscularly and particularly, intravenously. The compositions for parenteral administration commonly comprise a solution of the inhibitor of ATP synthase, preferably the anti-ATP synthase antibody, or a cocktail thereof dissolved in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, e.g., water, buffered water, 0.4% saline, 0.3% glycine and the like. These solutions are sterile and generally free of particulate matter. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, histidine and arginine. The concentration of the inhibitors (preferably antibodies) in these formulations can vary widely, i.e., from less than about 0.01%, usually at least about 0.1% to as much as 5% by weight and are selected primarily based on fluid volumes, and solubilities in accordance with the particular mode of administration selected.

[0061] The present invention also provides for a method of detecting prostate cancer, comprising detecting the presence of ATP synthase in the prostate cells of a subject in need of such detection. The present invention also provides a method of detecting breast cancer, colon cancer, or leukemia (preferably, acute myelogenous leukemia) comprising detecting presence of ATP synthase in the breast cells, colon cells, and T-cells respectively. Since our data show that ATP synthase is differentially expressed on the surface of the prostate cancer cells-, and leukemia cells but not in normal cells, an antibody against ATP synthase can be used as a bio-marker for detecting these types of cancers. The antibody includes, but is not limited to, the antibody against α-subunit of ATP synthase.

[0062] The present invention also provides for a diagnostic kit comprising anti-ATP synthase antibodies. Such a diagnostic kit further comprises a packaged combination of reagents in predetermined amounts with instructions for performing the diagnostic assay. Where the antibody is labeled with an enzyme, the kit will include substrates and co-factors required by the enzyme. In addition, other additives may be included such as stablizers, buffers and the like. The relative amounts of the various reagents may be varied widely to provide for concentrations in solution of the reagents that substantially optimize the sensitivity of the assay. Particularly, the reagents may be provided as dry powders, usually lyophilized, including excipients that, on dissolution, will provide a reagent solution having the appropriate concentration.

[0063] The inhibitors of the present invention may also be employed for the inhibition of cancer cell growth, or for the treatment of other types of cancer or neoplasm or malignant tumors found in mammals, including carcinomas and sarcomas. Examples of cancers are cancer of the brain, breast, cervix, bladder, colon, head & neck, kidney, lung, non-small cell lung, melanoma, mesothelioma, ovary, sarcoma, stomach, uterus, and medulloblastoma. Preferably, the inhibitors may be employed to detect or treat disorders including, but not limited to, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small-cell lung tumors, primary brain tumors, stomach cancer, colon cancer, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, adrenal cortical cancer, and leukemia.

[0064] Though the inhibitors of the present invention are primarily concerned with the treatment of human subjects, they may also be employed for the treatment of other mammalian subjects such as dogs and cats for veterinary purposes.

[0065] Additionally, the inhibitors can be utilized alone in substantially pure form, or together with chemotherapeutic agents, as are known to those of skill in the art (see, e.g., Cancer: Principles and Practice of Oncology, 5th ed., Devita et al., Lippincott-Ravel Publishers, 1997). Other therapies that may be used in conjunction with treatment with the antibodies include administration of anti-sense nucleic acid molecules or biologicals, such as additional therapeutic antibodies, as well as radiation and/or surgery (see, e.g., WO0034337). Thus, the treatment of the present invention is formulated in a manner allowing it to be administered serially or in combination with another agent for the treatment of cancer.

[0066] The following examples are offered by way of illustration and not by way of limitation. The disclosure of all citations in the specification is expressly incorporated herein by reference.

EXAMPLES

Example 1

[0067] This example describes the identification of anti-tumor targets for the treatment of prostate cancer by using lipid raft proteomics.

[0068] In the present invention, molecules critical to the treatment of prostate cancer were sought by initially detecting differential expression of proteins in normal cells and the prostate tumor cell lines. Lipid rafts of each cell line were isolated and studied subsequently.

[0069] Materials and Methods

[0070] a. Lipid Raft Preparation

[0071] Lipid rafts were prepared as described in Green et al, J. Cell Biol. 146, 673-682 (1999). Briefly, cells (8.0×106 cells/sample) were lysed in 0.1% vol/vol Brij-58, 20 mM Tris HCl, pH 8.2, 140 mM NaCl, 2 mM EDTA, 25 μg/ml aprotinin, 25 μg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride for 10 minutes on ice. Cells were homogenized using 10 strokes of a Dounce homogenizer, then lysed 20 minutes more on ice. The resulting lysate was adjusted to 40% wt/wt sucrose and applied onto a 60% wt/wt sucrose cushion. A sucrose step-gradient consisting of 25% wt/wt sucrose and 5% wt/wt sucrose were layered on top of the lysate. Gradients were centrifuged 18 hours at 170,000×g at 4° C. in a SW55 rotor. Fractions (0.2 ml) were taken from the top of the gradient. Lipid rafts float to the interface of the 25% and 5% sucrose layers (Fractions 7 and 8). The amount of protein in each fraction was determined using the BCA Protein Assay Kit. Protein was concentrated by centrifugation at 2000×g in Vivaspin 6 PES membrane columns (molecular weight cut off=10,000 kDa).

[0072] b. Electrophoresis and Western Blotting

[0073] Lipid raft proteins were separated by SDS-PAGE on a 4-20% gradient gel and then electrotransferred onto a polyvinylidene difluoride membrane (PVDF). The membrane was blocked for 1.5 hours at room temperature in PBS with 5% milk. The membrane was then incubated with 0.4 μg/ml mouse anti-ATP synthase (Molecular Probes, catalog #A-11144) in PBS with 1% BSA and 0.5% Tween-20 for 2 hours at room temperature. After extensive washing, the membrane was incubated with HRP-conjugated goat antibodies specific for mouse IgG for 1 hour at room temperature in PBS with 1% BSA and 0.5% Tween-20. After extensive washing, blot was developed using enhanced chemiluminescence followed by fluorography.

[0074] c. N-terminal Sequencing

[0075] Proteins to be sequenced were separated by SDS-PAGE on a 4-20% gradient gel and then electrotransferred onto a PVDF membrane. The membrane was stained for 2 minutes using colloidal Coomassie and then destained in water. The resulting bands were excised and subjected to N-terminal Edman sequencing as described by Miller, Methods: A Companion to Methods in Enzymology 6, 315 (1994). Results were confirmed using matrix assisted laser desorption ionization-time of flight (MALDI-TOF) peptide-mass profiling.

[0076] d. MALDI-TOF Peptide-mass Profiling

[0077] Proteins to be analyzed were separated by SDS-PAGE on a 4-20% gradient gel. Alternatively, 2-dimensional electrophoresis was used to further separate lipid raft proteins using an IPGphor Isoelectric Focusing System according to the manufacturers protocol (Amersham Pharmacia Biotech, Piscataway, N.J.). Proteins were visualized by staining the resulting gel with 0.05% Coomassie Blue R250, 50% methanol, 10% acetic acid in water followed by destaining in 15% methanol, 10% acetic acid in water.

[0078] Protein band or spot of interest was excised with a razor blade and equilibrated in 100 mM Tris HCl, pH 8.5 at room temperature for 45 minutes. The solution was replaced with 150 μL of 2 mM DTT in 100 mM Tris-HCl, pH 8.5. The samples were incubated with agitation for 30 minutes at 60° C. The solution was replaced with 150 82 L of 20 mM iodoacetic acid in 100 mM Tris-HCl, pH 8.5. The samples were incubated in the dark at 37° C. for 30 minutes. The solution was replaced with 150 μL of equal parts 100 mM Tris-HCl, pH 8.5 and acetonitrile. The tubes were shaken vigorously at 37° C. for 45 minutes. This step was repeated until the gel bands were clear. The solution was removed and the gel slices were dried in a SpeedVac on low vacuum strength for 15 minutes. The gel bands were re-swelled with 0.25-0.5 μg of a concentrated endo-protease Lysine-C or trypsin solution, then covered with 50-80 μL of 100 mM Tris-HCl, pH 8.5, 10% acetonitrile and incubated with agitation for 18 hours at 37° C. After digestion the samples were stored at 4-8° C.

[0079] The digest solution was removed from the micro-centrifuge tube, acidified with 10% trifluoroacetic acid (TFA) in water v/v to 1% TFA v/v, desalted and concentrated using a C18 Zip-Tip. The micro-column eluate was combined 1:1 with 10 mg/ml alpla-Cyano-4-hydroxycinamic acid in 60% acetonitrile and spotted on a MALDI-TOF sample plate. A close external calibrant with the approximate concentration of the sample was spotted adjacent to the sample position on the MALDI plate. The calibrant was prepared by diluting a pre-made calibration mix consisting of angiotensin II fragment 1-7 and adrenocorticotropic hormone fragment 18-39 with Zip-Tip eluant. The diluted calibration mixture was combined 1:1 with matrix solution. The sample and calibrant spots were dried simultaneously at room temperature.

[0080] MALDI mass fingerprints were acquired on a Perceptive Biosystems Voyager DE Pro MALDI-TOF in reflector mode with delayed extraction and positive polarity. Approximately 100-300 shots from a 20 KV laser were accumulated. During spectrum acquisition a resolution calculator was employed to ensure accurate calibration. After the spectra were acquired and calibrated, the monoisotopic masses were automatically selected using the de-isotoping function on Voyager Software. The calibrated monoisotopic peak lists were exported into ProteinProspector MS-Fit version 3.2.1 and searched against the largest non-redundant database available from the National Center for Biotechnology Information (NCBInr).

[0081] e. Flow Cytometry Staining

[0082] Flow cytometry was used to screen hybridoma supernatants for the presence of cell surface binding antibodies. The cells (2×105) were resuspended in 100 μL ice cold PBS with 10 μL tissue culture supernatant on ice for 1 hour. After extensive washing, cells were incubated with phycoerythrin-conjugated goat antibodies specific for mouse IgG for 30 minutes on ice. Cells were washed again and cell surface bound antibody was detected using a Becton Dickenson FACScan. Additionally, hybridoma supernatants were similarly screened on many cancer cell lines or whole blood to test for specificity.

[0083] f. Immunofluorescence

[0084] LNCaP cells were grown on glass coverslips undisturbed for two days. Cells were fixed with 3% paraformaldehyde in PBS for 15 minutes. After being washed with PBS, cells were incubated in 50 mM NH4Cl in PBS for 10 minutes. After washing with PBS, cells were subsequently incubated with 5% goat serum in PBS for 30 minutes followed by staining with 5 μg/ml anti-ATP synthase or anti-Trop-1 (EpCAM) in 2.5% goat serum in PBS for 1 hour at room temperature. Cells were stained with anti-MHC class II (Kostelny et al Int. J. Cancer 93, 556 (2001)) as a negative control. After washing with PBS, bound antibody was detected by incubating cells with Alexa 488-conjugated goat antibodies specific for mouse IgG in 2.5% goat serum in PBS for 30 minutes at room temperature. After extensive washing, glass coverslips were mounted in a solution containing Mowiol 4-88, glycerol, and 150 mM Tris HCl, pH 8.5. Slides were placed at 4° C. overnight before viewing. Cells were analyzed by fluorescence microscopy on a Nikon Optiphot 2 microscope.

[0085] g. LNCaP Proliferation

[0086] LNCaP cells were plated at 20,000 cells/well into a 96 well tissue culture plate. After cells were allowed to grow undisturbed for two days, antibodies (5 μg/ml anti-ATP synthase, anti-Trop-1 (EpCAM), or anti-MHC class II) were added and incubated with the cells for 24 khours. Cell proliferation was measured using the AlamarBlue vital dye indicator assay. AlamarBlue reagent was added to each well and the plates were incubated for 3 to 4 hours at 37° C. to allow for fluorescence development. Fluorescence was detected at λex=530 nm, λem=590 nm. Data are expressed as the mean+/−SEM of 4 replicates.

[0087] h. Soft Agar Colony Formation Assay

[0088] For anchorage-independent cell growth, a soft agar colony formation assay was performed in a six-well plate. Each well contained 2 mL of 1% agar in complete medium as the bottom layer. The top layer contained 2 mL 0.5% agar in complete medium, 1000-10000 LNCaP cells, and 5 μg/mL mAb (anti-ATP synthase, or anti-Trop-1). One mL complete medium was added and the cultures were maintained at 37° C. in a humidified 5% CO2 atmosphere for up to 20 days. One mL complete medium was added once a week. Media was removed and the colonies were stained with 0.005% crystal violet in PBS for 2 hours. The number of colonies was determined by counting them under an inverted phase-contrast microscope at 100×, and a group of 10 or more cells were counted as a colony.

[0089] Results and Discussion

[0090] a. Differential Expression of ATP Synthase in Prostate Tumor Cells

[0091] Lipid rafts were extracted from normal prostate cells and three widely used prostate cancer cells—LNCaP, DU145, and PC-3. The protein expression of each sample was compared using one-dimensional electrophoresis together with silver staining. Several protein bands in the one-dimensional electrophoresis gel appeared only in the prostate cancer cell lines, indicating that they are candidate proteins related to the prostate cancer. One of the candidate proteins with molecular weight of approximately 50 kD (denoted by an arrow with a “*” in FIG. 1) was selected for N-terminal sequencing. The sequencing result indicated that it was the β-subunit of ATP synthase.

[0092] The differential protein expression was also confirmed by two-dimensional electrophoresis and then visualized by silver staining. Protein spots that are present in the LNCaP sample, but not the normal prostate sample are denoted with arrows (16 spots identified), as shown in FIG. 2. Many of these lipid raft proteins were excised and subjected to MALDI-TOF peptide mass profiling analysis. The identities of 5 of them are shown in Table 1. The locations of these proteins on the 2-D gel are labeled with numbers as shown in FIG. 2. We found 2 subunits of ATP synthase (α and β subunits), 2 voltage-dependent anion channel/porin proteins, adenine nucleotide translocator, and prohibitin. All of these 5 identified proteins are mitochondria proteins but somehow are associated with lipid rafts of LNCaP cells. It is not known how many of these lipid raft-associated proteins are exposed to the outer surface of the cell membrane and therefore are accessible to antibodies. As several anti-ATP synthase antibodies are commercially available, we used one of them to confirm that some ATP synthase molecules are located in lipid rafts and they are accessible to antibodies.

[0093] Western blot analysis of lipid rafts using anti-α-subunit ATP synthase antibody further confirmed the correlation between the ATP synthase and prostate cancer. As shown in FIG. 3, positive staining appeared in all three prostate cancer cell lines, but not the normal cells, indicating that ATP synthase was present in lipid rafts of prostate cancer cells but not normal cells. In addition, similar Western blot analysis of lipid rafts from cancer cells of different origins indicates that some cancer cell lines, such as the AML cell lines KG-1 and THP-1, the breast cancer cell line MCF-7, the colon cancer cell line LS180, and the bladder cancer cell line RT4 also express ATP synthase in lipid rafts (FIG. 4). ATP synthase was also showed to be expressed on the surface of the androgen-independent cell line DU 145 by a similar analysis.

[0094] b. Cell Surface Localization of ATP Synthase

[0095] To investigate the surface localization of the ATP synthase, prostate tumor cell line LNCaP was analyzed by FACS staining and immunofluorescence microscopy. As shown in FIG. 5, FACS staining by anti-ATP synthase antibody in LNCaP tumor cells revealed about 25.9% of cells had ATP synthase cell surface expression, Staining with an antibody against Trop-1 (EpCAM), a cell surface protein expressed on cancer cells, showed surface localization in 92.8% of the cells. Similar analysis of the AML cell line THP-1 by flow cytometry also showed that these cells have ATP synthase on their cell surface (FIG. 6).

[0096] The surface localization of ATP synthase was further evidenced by the immunofluorescence microscopy. As shown in FIG. 7, immunostaining of LNCaP cells with anti-ATP synthase gave rise to positive cell surface staining. The colocalization of Trop-1 (EpCAM) and ATP synthase indicated that ATP synthase was indeed expressed on the surface of prostate tumor cells.

[0097] Moreover, FACS staining of LNCaP tumor cells growing in Matrigel® demonstrated a much higher percentage of cells exhibiting a positive surface staining of ATP synthase, confirming that ATP synthase cell surface expression can be modulated by cellular environment, and this may play important roles in the real biological environment (data not shown).

[0098] c. Inhibition of Prostate Cancer Cell Proliferation in the Presence of Anti-ATP Synthase

[0099] As shown in FIG. 8, LNCaP tumor cell proliferation can be reduced by antibodies specific for ATP synthase. This reduction in cellular proliferation ranges from 12.5% to 27.8%. This substantial inhibition of cell proliferation suggests a potential of anti-ATP synthase antibody as an anti-tumor agent for treating prostate cancer.

[0100] The above experiments demonstrate that ATP synthase is closely related to the prostate cancer cells. It is localized on the surface of prostate tumor cells, and may play a key role in the biological activities of prostate cancer cells. Blocking the activity of ATP synthase by antibodies inhibits the prostate tumor cell growth, suggesting the possibility of clinical application of ATP synthase inhibitors as anti-tumor agents for treating prostate cancer.

[0101] d. Inhibition of Prostate Cancer Cell Colony Formation in Soft Agar in the Presence of Anti-ATP Synthase

[0102] Transformed cancer cells are resistant to anchorage-independent growth inhibition and are able to growth in soft agar without attaching to cell matrix. Formation of colonies (three-dimensional growth under tissue culture growth conditions) of cancer cells in soft agar is often correlated to the aggressiveness of the tumor in vivo. To assess whether anti-ATP synthase has any anti-cancer activity, we used it inhibit LNCaP colony formation in vitro. As shown in FIG. 9, LNCaP colony formation can be reduced by an antibody specific for ATP synthase (anti-α subunit) at 5 μg/ml. This reduction in colony formation by anti-ATP synthase could be as high as 95%, whereas the action of an anti-Trop-1 antibody (Ep-CAM) antibody was less impressive, at about 68%. This substantial inhibition of colony formation suggests a potential of anti-ATP synthase antibody as an anti-tumor agent for treating prostate cancer.

[0103] e. AML Cell Death Induction in the Presence of Anti-ATP Synthase

[0104] The anti-cancer activity of anti-ATP synthase can also be demonstrated in the AML cell line THP-1, which expresses ATP synthase on the cell surface. Incubation of THP-1 cells with anti-ATP synthase at 5 μg/ml for 24 hours led to substantial cell death as assayed by flow cytometry (FIG. 10). Compared to PBS or an irrelevant antibody control, the specific killing induced by anti-ATP synthase was 25%. This anti-AML activity suggests a potential of antiATP synthase antibody as anti-leukemia or lymphoma agent to treat hematological malignancies.

[0105] The above experiments demonstrate that ATP synthase expression is closely related to certain cancer cells. It is localized on the surface of prostate and AML cancer cells, and may play a key role in the biological activities of prostate and AML cancer cells. Blocking the activity of ATP synthase by antibodies inhibits the prostate cancer cell growth and colony formation, and induces cell death in AML cancer cells, suggesting the possibility of clinical application of ATP synthase inhibitors as anti-tumor agents for treating prostate cancer and AML.

Example 2

[0106] This example describes the treatment of prostate cancer by antibodies specific for ATP synthase in well-established androgen-dependent and androgen-independent prostate cancer xenografts.

[0107] Six to ten week old male nude NCR nu/nu mice are inoculated subcutaneously in the mid-scapular region with 5×106 androgen-dependent LNCaP cells. Cells that are injected are reconstituted with basement membrane in the form of Matrigel as described (Sato et al Cancer Res. 5, 1584-1589 (1997)). To maintain serum testosterone levels, male mice are implanted with 12.5 mg sustained release testosterone pellets subcutaneously prior to receiving the tumor cell inoculation. Antibodies specific for ATP synthase (Molecular Probes, cat #A-11144 and A-21299) are given intraperitoneally on day 2 and 4. Tumors are measured every three to four days with vernier calipers. Tumor volumes are calculated by the formula π/6×(larger diameter)×(smaller diameter)2. For the androgen-independent prostate cancer xenograft studies, DU 145 or PC-3 cells are used.

Example 3

[0108] This example describes the human prostate cancer therapeutic regime by using the inhibitors of ATP synthase.

[0109] The patient's cancer biopsy sample is stained positively for the expression of ATP synthase on the cancer cell surface by immunohistochemistry for the patient to be eligible for treatment. Anti-ATP synthase antibodies are administered either intravenously or subcutaneously in a dose range from 0.05 to about 25 mg/kg. Patients receive at least 4 weekly doses. Tumor size is monitored by CAT scan or MRI prior to therapy and post therapy. Reduction of the tumor size is the primary indication of the drug's efficacy. Tumor shrinkage by 50% or more is considered as a partial response. Complete disappearance of the tumor is considered as a complete response. For prostate cancer patients, PSA level prior to therapy and post therapy is also monitored as a secondary indication of treatment efficacy.

[0110] Although the invention has been described with reference to the presently preferred embodiments, it should be understood that various modifications can be made without departing from the spirit of the invention.

[0111] All publications, patents, patent applications, and web sites are herein incorporated by reference in their entirety to the same extent as if each individual patent, patent application, or web site was specifically and individually indicated to be incorporated by reference in its entirety.

	Number	Date	Country
	60329178	Oct 2001	US
	60331965	Nov 2001	US

Treatment of prostate cancer by inhibitors of ATP synthase

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