EP1261371A1 - Membrane estrogen receptor-directed therapy in breast cancer - Google Patents
Membrane estrogen receptor-directed therapy in breast cancerInfo
- Publication number
- EP1261371A1 EP1261371A1 EP01912991A EP01912991A EP1261371A1 EP 1261371 A1 EP1261371 A1 EP 1261371A1 EP 01912991 A EP01912991 A EP 01912991A EP 01912991 A EP01912991 A EP 01912991A EP 1261371 A1 EP1261371 A1 EP 1261371A1
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- Prior art keywords
- membrane
- receptor
- cell
- esfrogen
- cells
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2869—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against hormone receptors
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/31—Chemical structure of the backbone
- C12N2310/315—Phosphorothioates
Definitions
- the present invention is directed to methods of inhibiting the signalling of a membrane- associated estrogen receptor and diagnoses and treatments for mammalian pathologies.
- Cancers of the breast are one of the leading causes of death among women, with the cumulative lifetime risk of a woman developing breast cancer estimated to be 1 in 8. Consequently, the identification of new therapeutic modalities is of significant interest to health care professionals.
- breast cancer arises f om the uncontrolled division of breast cells which can spread into and destroy normal tissues.
- Growth of breast cells is normally regulated by hormones such as estrogen which bind to specific receptors that are present in more than two-thirds of breast cancers.
- these hormone receptors localize in the nucleus and are activated by binding estrogen and subsequently, with DNA in order to communicate growth-promoting signals to specific genes in the DNA of the cell.
- New findings also provide evidence that changes in cancer-related genes, such as those coding for growth factor receptors, may lead to the emergence of estrogen-independent activation of hormone receptors. Specifically, the events leading to the activation of the estrogen receptor signal are associated with disregulated cell division.
- Antiestrogen therapy is one well-known example of these approaches, and it is often used to treat breast cancer and to prevent the recurrence of disease.
- Antiestrogen therapy is one well-known example of these approaches, and it is often used to treat breast cancer and to prevent the recurrence of disease.
- many patients do not respond to this therapy, and most treated patients eventually become resistant to antiestrogens.
- antiestrogens that are now available can result in abnormal uterine growth and thromboembolic events.
- the failure of antihormone therapy in the clinic appears to be due to many factors, including the emergence of estrogen-independent growth that is no longer responsive to treatment with antiestrogen agonists.
- estrogen receptor molecules occur in association with plasma membranes, these molecules may interact with other molecules involved in oncogenesis such as transmembrane erb B growth factor receptors.
- HER-2/erb-B2 receptors occurs in many human breast cancers, and the enzyme activity of HER-2 may play a role in the activation of estrogen receptor even in the absence of estrogen.
- this signaling complex may offer an important new target for therapeutic intervention.
- the present invention is directed to methods of inhibiting the signalling of a membrane- associated estrogen receptor by exposing the membrane-associated estrogen receptor to an inhibitory molecule that inhibits membrane-associated estrogen receptor signalling.
- Illustrative embodiments of the invention provided herein comprise methods of diagnosing and/or treating an individual suspected of suffering from a cancer which expresses membrane-associated estrogen receptor comprising the steps of administering to said individual a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent, and a diagnostically or therapeutically effective amount of a compound consisting of an inhibitory ligand such as an anti-membrane-associated estrogen receptor immunoglobulin polypeptide.
- the antitumor efficacy of two different synthetic inhibitors of MAP kinase-activating enzymes (PD 98059 and U0126) in estrogen-stimulated human breast cancer cells is also disclosed. Both synthetic inhibitors elicit the blockade of estrogen-induced MAP kinase activation in breast cancer cells. This effect of the inhibitors leads to blockade of estrogen-induced cell proliferation.
- the antiestrogen ICI 182780, can block cell proliferation stimulated by the membrane-associated estrogen receptor.
- ⁇ hosphoinositol-3 kinase PI3K
- LY294002 LY294002
- Akt kinase an effector immediately downstream of PI3K that may promote enhanced cell survival.
- membrane-associated estrogen receptor activity can be inhibited by anti-membrane estrogen receptor antibodies, antiestrogens (ICI 182,780) and inhibitors of signaling pathways, such as MAP kinase and PI3/Akt kinase, that originate at the surface membrane.
- the invention consists of a method for inhibiting the growth of a breast cancer cell which expresses the estrogen receptor associated with its cell membrane by contacting the cell with an amount of anti-estrogen receptor immunoglobulin polypeptide sufficient to inhibit cell growth.
- the anti-estrogen receptor immunoglobulin polypeptide recognizes and binds the ligand binding domain of the estrogen receptor.
- the anti-estrogen receptor immunoglobulin polypeptide is selected from the group consisting of monoclonal antibodies Ab 1 and Ab 2.
- a related embodiment of the invention consists of a method for treating a mammalian cancer cell which expresses estrogen receptor associated with its cell membrane by contacting the cancer cell with the anti-estrogen receptor immunoglobulin polypeptide under conditions which allow the antiestrogen receptor immunoglobulin polypeptide to bind to the estrogen receptor associated with the surface membrane of the cancer cell to a degree sufficient to inhibit the growth of the cancer cell.
- the anti-estrogen receptor immunoglobulin polypeptide is selected from the group consisting of monoclonal antibodies Ab 1 and Ab 2.
- Yet another related embodiment of the invention consists of a method of inhibiting the growth of a breast cancer cell having disregulated cell growth comprising the steps of confirming the presence of a estrogen receptor in the membrane of the breast cancer cell, providing a estrogen receptor immunoglobulin polypeptide specific for an epitope within the ligand binding domain of the estrogen receptor, the anti-estrogen receptor immunoglobulin polypeptide being selected to produce inhibition of breast cancer cell growth, and then contacting the cell with the anti-estrogen receptor immunoglobulin polypeptide under conditions which allow the anti-estrogen receptor immunoglobulin polypeptide to bind to the estrogen receptor associated with the surface membrane of the breast cancer cell to a degree sufficient to inhibit the growth of the breast cancer cell.
- the anti-estrogen receptor immunoglobulin polypeptide is selected from the group consisting of monoclonal antibodies Ab 1 and Ab 2.
- Another embodiment of the invention disclosed herein includes an injectable pharmaceutical composition for treatment of a mammalian cancer tumor having cells which express estrogen receptor in association with their cell membranes consisting of an anti-estrogen receptor immunoglobulin polypeptide specific to an epitope on a ligand binding domain of the estrogen receptor; the antiestrogen receptor immunoglobulin polypeptide being selected for its ability to inhibit tumor growth; and a pharmaceutically acceptable injection vehicle.
- the anti-estrogen receptor immunoglobulin polypeptide is selected from the group consisting of monoclonal antibodies Ab 1 and Ab 2.
- kits for use in methods for inhibiting the growth of breast tumor cells which express an estrogen receptor comprising a container, a composition contained within the container, wherein the composition includes an anti-estrogen receptor immunoglobulin polypeptide and instructions for using the anti-estrogen receptor immunoglobulin polypeptide in vivo or in vitro.
- the anti-estrogen receptor immunoglobulin polypeptide is selected from the group consisting of monoclonal antibodies Ab 1 and Ab 2.
- Yet another embodiment of the invention disclosed herein includes a method of radioimaging metastasized breast cancer cells comprising the steps of first administering to an individual suspected of having metastasized breast cancer cells, a pharmaceutical composition that consists of a pharmaceutically acceptable carrier or diluent, and conjugated compound that comprises an antiestrogen receptor immunoglobulin polypeptide and a radioactive active moiety wherein the conjugated compound is present in an amount effective for diagnostic use in humans suffering from breast cancer and then detecting the localization and accumulation of radioactivity in the individual's body.
- a pharmaceutical composition that consists of a pharmaceutically acceptable carrier or diluent, and conjugated compound that comprises an antiestrogen receptor immunoglobulin polypeptide and a radioactive active moiety wherein the conjugated compound is present in an amount effective for diagnostic use in humans suffering from breast cancer and then detecting the localization and accumulation of radioactivity in the individual's body.
- FIG. 1 is a schematic showing the postulated cellular mechanism of action of estrogen (E2) and growth factors in breast cancers with estrogen receptor (ER).
- E2 estrogen receptor
- ER estrogen receptor
- FIG. 1 fails to account for numerous, rapid cellular responses to estrogen treatment (see TABLE 1).
- estrogen stimulates growth of breast cells, stable transfection of cells with the classical ER elicits a paradoxical estrogen-induced inhibition of growth (69, 70).
- estrogen may also bind to a membrane-associated ER, with potential for stimulation of estrogenic responses via an alternate pathway.
- membrane estrogen receptors may be known molecules (kinases, ion channels) with previously unknown binding sites for estrogen, new isoforms of ER- ⁇ or ER- ⁇ in membranes, classical ER complexed with other membrane- associated proteins or truly novel membrane-associated proteins.
- FIG. 2 shows the distribution and relative specific activities of enzymes and specific [ 3 H]estradiol-17 ⁇ binding in plasma membrane and other subtractions of MCF-7 breast cancer cells.
- Cells were grown in estrogen-free media prior to harvesting, then disrupted using controlled homogenization methods as before (48,49).
- A) The yield of marker enzymes and E 2 ⁇ binding in each fraction is expressed as a percentage of that in the cell homogenate, with mean + SE of data from 3 experiments shown.
- FIG. 3 is a photograph showing that estradiol-17B conjugated to fluorescein-labeled albumin (E 2 ⁇ -BSA-FITC) binds at the surface membrane of MCF-7 breast cancer cells.
- E 2 ⁇ -BSA-FITC estradiol-17B conjugated to fluorescein-labeled albumin
- DCC dextran-coated charcoal
- BSA-FITC shows a low level of background cell fluorescence of MCF-7 PAR cells (CONTROL).
- Active ligand, E 2 ⁇ -BSA-FITC labels surface membranes of MCF-7 PAR cells (PARENT:E2).
- E2 ⁇ -BSA-FITC is a steroid conjugate considered to be membrane-impermeant (52,60,61,94).
- FIG. 4 is a graph showing that MCF-7 breast cancer cells proliferate in response to short-term treatment with membrane-impermeable estradiol-17 ⁇ -bovine serum albumin conjugate (E2-BSA) or with free estradiol, but not with control BSA alone (PO.001, t-test).
- E2-BSA membrane-impermeable estradiol-17 ⁇ -bovine serum albumin conjugate
- free estradiol but not with control BSA alone (PO.001, t-test).
- Dextran-coated, charcoal-treated E2-BSA a macromolecular complex used previously for affinity-isolation of estrogen receptor, is considered to be membrane-impermeant on brief exposure to cells (52,60,61, 94).
- the proliferative effect of E2-BSA is blocked by treatment of cells with the pure antiestrogen, ICI 182,780.
- FIG. 1 membrane-impermeable estradiol-17 ⁇ -bovine serum albumin conjugate
- FIG. 5 is a graph showing that estrogen elicits preferential growth of human breast cancer cells selected for expression of membrane-associated estrogen receptor.
- breast cancer cells were fractionated in vitro on the basis of their capacity to bind or not bind with 17 ⁇ -estradiol-17-hemisuccinyl-albumin covalently bound to an inert support (46,47).
- Isolated MCF-7 breast cancer cells were cultivated in estrogen-free media for 72 hrs and then incubated for 30 min at 22 C with immobilized estradiol at a prevailing concentration of approximately 0.5 nM as described before (47). These conditions were shown before to permit selection of cells with high affinity interactions with estradiol at the surface membrane (46,47).
- FIG. 6 is a Western blot showing that the activation of HER-2 growth factor receptor promotes physical association of HER-2 receptor with estrogen receptor (ER).
- MCF-7 breast cancer cells were treated in vitro for 5-60 minutes with 10 nM heregulin, a ligand known to activate HER- 2/HER-3 receptors (3). Lysates were prepared and processed as described before (3). Samples were immunoprecipitated with anti-HER-2 antibody (IP:HER-2 receptor) prior to electrophoresis and Western blotting with anti-ER antibody H222 (IB: estrogen receptor). Estrogen receptor normally occurs as a 65- to 70-kd protein (3). The experiment shown here is representative of results from 4 other experiments.
- FIG. 7 shows that treatment of human breast cancer cells with antisense oligonucleotides targeting nuclear ER mRNA reduce the expression of total cellular ER.
- the groups include: CON (control), AS (antisense phosphorothioate 5' GGGTCATGGTCATGG ; SEQ ID NO: 1) and Ms (missense control).
- FIG. 8 is a graph showing that treatment of human breast cancer cells with antisense oligonucleotides to intracellular estrogen receptor suppress expression of membrane-associated receptors with specific high-affinity binding for estrogen.
- the antisense phosphorothioate oligonucleotide was synthesized as 5'-GGGTCATGGTCATGG-3' (SEQ ID NO: 1), and a missense control was used for comparison. Specific estradiol- 17 ⁇ binding to plasma membrane fractions was done by established methods.
- FIG. 9 is a graph showing that treatment of human breast cancer cells with ER antisense oligonucleotides reduce the expected cellular growth response to 2 nM estradiol- 17 ⁇ .
- Treatment groups include: control cells (Cn), control cells treated with estradiol (Cn/E), missense oligonucleotide treated cells (Ms), Ms-treated cells with exposure to estradiol (Ms/E), ER antisense-treated cells (As), and As-treated cells exposed to estradiol (As/E).
- FIG. 10 is a schematic showing potential sites for estrogen action in the cell.
- Estrogen may interact with a membrane-associated estrogen receptor (ER), leading to signal transduction to the cell interior by interactions with receptor tyrosine kinases (RTK), MAP kinase cascades or G-protein related pathways. Interference with estrogen-ER interactions or with the activity of downstream signaling pathways may block estrogen-stimulated effects in the cell.
- ER membrane-associated estrogen receptor
- RTK receptor tyrosine kinases
- MAP kinase cascades or G-protein related pathways.
- FIG. 11 is a graph showing that the MAPK (MEK) inhibitor, PD 98059, reduces estrogen- stimulated MAPK activity.
- MAPK activity was assessed in human breast cancer cells after treatment with estradiol (E2) alone or combined with 20 ⁇ M PD 98059 (E2/PD) over 30 min. Results are presented as percent of appropriate controls.
- FIG. 12 is a gel showing that the MAPKK (MEK) inhibitor, PD 98059 (PD), reduces estrogen
- E2 (E2)-induced serine phosphorylation of estrogen receptor. Serine phosphorylation of estrogen receptor in human breast cancer cells was assessed at 15-60 min after treatment with E2 alone or in combination with PD 98059
- FIG. 13 is a graph showing that MAPKK (MEK) inhibitor PD 98059 (PD) reduces estrogen (E2)- induced growth of breast cancer cells.
- MCF-7 human breast cancer cells were treated in vitro with 20 micromolar PD 98059, a synthetic inhibitor of the MAPK-activating enzyme, MAPK/ERK kinase (MEK) (Proc. Natl. Acad. Sci. USA 92 : 7686-7689, 1995) in the presence and absence of estradiol-17B at 2 nM. Control (CN); estradiol (E2); PD 98059 alone (CN/PD); estradiol and PD 98059 (E2/PD).
- FIG. 14A is table which provides information on properties and sources of the various antibodies used in the methods of the invention.
- FIG. 14B is a schematic of the estrogen receptor-alpha protein structure which outlines the various identified functional domains within this molecule.
- FIG. 15 shows binding of [ 3 H] estradiol- 17 ⁇ by plasma membranes from MCF-7 human breast cancer cells.
- This curve shows the difference between the two curves in panel a and represents the specific binding of hormone by plasma membranes.
- FIG. 16 shows the identification of estrogen receptor in subcellular fractions of MCF-7 cells by Western blot and ligand-blot analyses. Proteins from cell subtractions were analyzed by polyacrylamide gel electrophoresis and transferred to nitrocellularose membranes.
- E 2 ⁇ -POD binding is shown in the absence (none) and presence (E 2 ⁇ ) of free estradiol-17B at a 10-fold molar excess in order to assess specific steroid binding (103).
- FIG. 17 is a gel showing that estrogen treatment promotes enhanced association between ER- ⁇ and Cavatellin-2/Flotillin-2/ESA in MCF-7 human breast cancer cells.
- Breast cancer cells were treated with vehicle alone (VH) or 10 nM 17 ⁇ -estradiol (E 2 ⁇ ) for 1, 5, 10 and 20 min. Thereafter, cells were lysed and immunoprecipitated (IP) with anti-estrogen receptor-alpha (ER- ⁇ ) antibody. Immunoprecipitates were subjected to Western blot (IB) with anti-Cavatellin-2/Flotillin-2/ESA antibody
- FIG. 18 shows post-receptor signal transduction induced by estradiol in vitro.
- MAPK mitogen-activated protein kinase
- E 2 ⁇ -BSA covalently linked to BSA
- E 2 ⁇ -BSA 0.5 ⁇ M
- MAPK phosphorylation within 2 min.
- Akt kinase activation was measured by densitometric analysis of phosphorylated GSK-3 ⁇ / ⁇ .
- MCF-7 cells were treated with vehicle (CN) or stimulated with 10 nM estrogen (E 2 ⁇ ) or 0.5 ⁇ M E 2 ⁇ -BSA for 20 min.
- Cells were preincubated with anti LBD Ab2 (Ab2), ER antagonist ICI 182,780 (ICI) or the PI3-kinase inhibitor LY294002 (LY) before addition of E 2 ⁇ -BSA.
- FIG. 19 shows that estradiol- 17B conjugated to fluorescein-labeled albumin (E 2 ⁇ -BSA-FITC) shows binding at the surface membrane of MCF-7 breast cancer cells.
- E 2 ⁇ -BSA-FITC was pre-treated with dextran-coated charcoal to remove any traces of free estradiol. Thereafter, intact cells were labeled with 1 ⁇ M E 2 ⁇ -BSA-FITC, a membrane-impermeant complex (52,60,61,94), to assess membrane binding and then analyzed by fluorescent microscopy and flow cytometry.
- E 2 ⁇ -BSA-FITC Active ligand
- BSA-FITC Control binding with inactive ligand
- BSA-FITC shows a low level of background cell fluorescense.
- S 2 ⁇ - BSA-FITC Surface membrane labeling by E 2 ⁇ - BSA-FITC is competitively reduced by co-incubation with antibody to LBD of ER (Abl).
- MCF-7 cells were permeabilized with 0.1 % Triton X-100 to allow visualization of ER binding in the nucleus.
- FIG. 20 shows the inhibition of MCF-7 breast cancer cell growth by a monoclonal antibody directed against the LBD of nuclear ER.
- FIG. 21 is a graph showing Inhibition of MCF-7 human breast cancer cell growth by the MEK
- estrogen-induced growth of cancer cells is blocked by treatment with antibodies directed to the ligand-binding domain of the nuclear form of estrogen receptor. Moreover, this effect correlates closely with acute estrogen-induced activation of mitogen- activated protein kinase (MAPK) and Akt kinase signaling, and estrogen-induced growth of cancer cells is also blocked by synthetic inhibitors of MAP kinase-activating enzymes.
- MAPK mitogen- activated protein kinase
- Akt Akt kinase signaling
- estrogen-promoted growth of human breast cancer xenografts in nude mice was significantly reduced by treatment in vivo with antibodies directed to the ligand-binding domain of the estrogen receptor.
- antibody directed to membrane-associated estrogen receptor may be one strategy for disrupting cell growth.
- other therapeutic strategies may include the use of agents that can act as partial agonists or antagonists of the membrane receptor (for example, ICI 182,780) or drugs that block estrogen-induced activation of MAPK or PI3K/Akt kinase signaling cascades that emanate from stimulation of the membrane receptor. Specific evidence for the efficacy of these alternate approaches is presented in the Examples below.
- the present invention is directed to methods of inhibiting the signalling of a plasma membrane- associated estrogen receptor by contacting the membrane-associated estrogen receptor with an inhibitory ligand that binds to the ligand-binding domain of the membrane-associated estrogen receptor thereby inhibiting plasma membrane-associated estrogen receptor signalling.
- the invention provides a new approach to the treatment of cancers that express membrane-associated estrogen receptors, for example breast cancers.
- cancers that express membrane-associated estrogen receptors
- the treatment of human breast cancers with antibodies directed to the ligand binding domain of the human estrogen receptor can block the growth of breast cancer cells that bear estrogen receptors.
- treatments targeted to downstream signaling events after stimulation of the membrane estrogen receptor also appear to suppress tumor growth.
- this approach to treatment with blood-borne reagents potentially represents an important addition to available treatment options.
- the invention disclosed herein challenges the current dogma in endocrinology and oncology which holds that estrogen receptors are exclusively intracellular proteins and largely localized to the nucleus of the cell. Specifically, we demonstrate that the use of anti-estrogen receptor immunoglobulin polypeptides targeting the membrane-associated estrogen receptor expressed by these cells elicits a potent antitumor effect. These observations of membrane-associated estrogen receptors on human breast tumor cells may be equally relevant to other estrogen-regulated processes in diverse tissues such as bone, brain, uterus and vascularure.
- the invention disclosed herein has advantages over existing treatment practices: Patients with breast cancer are usually offered multimodality therapy, including surgery, radiation and adjuvant treatment. Depending on tumor stage and estrogen receptor status of the tumor at the time of presentation, adjuvant therapy may include antiestrogen treatment (tamoxifen) or cytotoxic chemotherapy. All current adjuvant treatments are encumbered by numerous side-effects that often reduce the patient's quality of life. Membrane-associated estrogen receptor targeted therapy could provide a new alternative with fewer side-effects and improved quality of life for the affected patients. Optimal embodiments of the methods of the inventions entail those with blockade of estrogen receptor function and tumor cell growth by use of circulating monoclonal antibodies to the estrogen receptor may offer the best outcome.
- HER-2/neu a monoclonal antibody directed to the surface membrane receptor termed HER-2/neu. See e.g. U.S. Patent Nos. 5,772,997, 5,824,311 and 5,773,476 which are incorporated herein by reference.
- blockade of intracellular signaling emanating from activation of the membrane-associated estrogen receptor would offer another method of the invention for inhibition of tumor cell growth.
- polypeptide or protein hi question is at least about 30% identical to an entire naturally occurring protein or a portion thereof, usually at least about 70% identical, and preferably at least about 95% identical.
- substantially purified protein As used herein, the terms “isolated”, “substantially pure” and “substantially homogenous” are used interchangeably and describe a protein that has been separated from components which naturally accompany it. Typically, a monomeric protein is substantially pure when at least about 60 to 75% of a sample exhibits a single polypeptide backbone. Minor variants or chemical modifications typically share the same polypeptide sequence. A substantially purified protein will typically comprise over about 85 to 90% of a protein sample, more usually about 95%, and preferably will be over about 99% pure.
- Protein purity or homogeneity may be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a single polypeptide band on a polyacrylamide gel upon staining. For certain purposes high resolution will be needed and HPLC or a similar means for purification utilized.
- a polypeptide is substantially free of naturally-associated components when it is separated from the native contaminants which accompany it in its natural state.
- a polypeptide which is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally-associated components.
- Proteins may be purified to substantial homogeneity by standard techniques well known in the art, including selective precipitation with such substances as ammonium sulfate, column chromatography, immunopurif ⁇ cation methods, and others. See, for instance, R. Scopes, Protein Purification: Principles and Practice, Springer- Verlag: New York (1982), which is incorporated herein by reference.
- immunoglobulin polypeptide refers to molecules which have specific immunoreactive activity. Antibodies are typical embodiments of immunoglobulin polypeptides.
- antibody refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes.
- Immunoglobulin genes include those coding for the light chains, which may be of the kappa or lambda types, and those coding for the heavy chains. Heavy chain types are alpha, gamma, delta, epsilon and mu.
- the carboxy terminal portions of immunoglobulin heavy and light chains are constant regions, while the amino terminal portions are encoded by the myriad immunoglobulin variable region genes.
- the variable regions of an immunoglobulin are the portions that provide antigen recognition specificity.
- the specificity resides in the complementarity determining regions (CDRs), also known as hypervariable regions, of the immunoglobulins.
- CDRs complementarity determining regions
- the immunoglobulins may exist in a variety of forms including, for example, Fv, Fab, F(ab'), F(ab') 2 , and other fragments, as well as single chains (e.g., Huston, et al., Proc. Nat. Acad. Sci. U.S.A., 85:5879-5883 (1988) and Bird, et al, Science 242:423-426 (1988), which are incorporated herein by reference).
- Huston, et al., Proc. Nat. Acad. Sci. U.S.A., 85:5879-5883 (1988) and Bird, et al, Science 242:423-426 (1988 which are incorporated herein by reference.
- Hood, et al. "Immunology", Benjamin, N.Y
- Monoclonal antibodies are well known in the art and may be obtained by various techniques familiar to those skilled in the art.
- a monoclonal antibody is an antibody produced by a clonal, immortalized cell line separate from cells producing antibodies with a different antigen binding specificity. Briefly, spleen cells from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell (see, Kohler and Milstein, Eur. J. Immunol. 6:511-519 (1976)).
- “Chimeric” antibodies are those antibodies encoded by immunoglobulin genes that have been genetically engineered so that the light and heavy chain genes are composed of immunoglobulin gene segments belonging to different species.
- the variable (V) segments of the genes from a mouse monoclonal antibody may be joined to human constant (C) segments.
- Such a chimeric antibody is likely to be less antigenic to a human than antibodies with mouse constant regions as well as mouse variable regions.
- chimeric antibody also refers to an antibody that includes an immunoglobulin that has a human-like framework and in which any constant region present has at least about 85-90%, and preferably about 95%> polypeptide sequence identity to a human immunoglobulin constant region, a so-called “humanized” immunoglobulin (see, for example, PCT Publication WO 90/07861, which is incorporated herein by reference).
- a so-called "humanized” immunoglobulin see, for example, PCT Publication WO 90/07861, which is incorporated herein by reference.
- all parts of such a “humanized” immunoglobulin, except possibly the complementarity determining regions (CDR's) are substantially identical to corresponding parts of one or more native human immunoglobulin sequences.
- framework residues may also be replaced with those within or across species especially if certain framework residues are found to affect the structure of the CDRs.
- a chimeric antibody may also contain truncated variable or constant regions.
- the term "framework region”, as used herein, refers to those portions of immunoglobulin light and heavy chain variable regions that are relatively conserved (i.e., other than the CDR's) among different immunoglobulins in a single species, as defined by Kabat, et al., (1987): Sequences of Proteins of Immunologic Interest, 4th Ed., US Dept. Health and Human Services, which is incorporated herein by reference).
- a "human-like framework region” is a framework region that in each existing chain comprises at least about 70 or more amino acid residues, typically 75 to 85 or more residues, identical to those in a human immunoglobulin.
- HER-1 (EGF) family most frequently implicated in human cancer (1-3, 4-8).
- the HER (erb B) receptor family includes the HER-2 (erb B2) protein, a 185-kD transmembrane tyrosine kinase encoded by HER-2 oncogene, (9-11), the HER-3 protein (12) and HER-4 receptor (13,14).
- Overexpression of HER-2 or related growth factor receptors is estimated to occur in two-thirds of sporadic breast cancers (1), while amplification or overexpression of HER-2 is found in 25-30%> of breast cancers in women and 4 /o of breast cancers in men (15-18).
- HER-2 overexpression is a marker of poor prognosis (15- 19) and is associated with the failure of antiestrogen therapy (3,20-31).
- Receptors for estrogen also occur in a family of potentially oncogenic receptors. Sequence similarities between the erb A gene product of avian erythroblastosis virus and ER provide evidence that these two proteins likely evolved from a common gene (32). Erb A genes cannot induce cell transformation alone, but cooperate with the viral erb B oncogenes in cell transformation (33). With this lineage of cooperativity between erb A and erb B genes, it is not surprising to find reports of significant cross-communication and interaction between erb B (HER) pathways and estrogen receptor signaling (3,24,27,34-36). It is generally held that the biologic activity of estrogen in the breast is mediated through the specific high-affinity estrogen receptor located in breast cell nuclei (1,37). In the absence of estrogen, ER is considered to associate with proteins that prevent its interaction with the cellular transcription apparatus. Upon estrogen binding, the receptor undergoes an activating conformational change that promotes association with target genes, thus permitting regulation of gene transcription.
- Figure 1 shows a schematic of the postulated cellular mechanism of action of estrogen (E2) and growth factors in breast cancers with estrogen receptor (ER).
- E2 estrogen receptor
- ER estrogen receptor
- Figure 1 shows a schematic of the postulated cellular mechanism of action of estrogen (E2) and growth factors in breast cancers with estrogen receptor (ER).
- E2 estrogen binding to ER in the nucleus promotes receptor dimer formation and receptor phosphorylation that may enhance binding to nuclear estrogen-responsive elements (ERE) and coactivator proteins, leading, in turn, to initiation of specific gene transcription.
- EEE nuclear estrogen-responsive elements
- coactivator proteins leading, in turn, to initiation of specific gene transcription.
- the latter model fails to account for numerous, rapid cellular responses to estrogen treatment (see TABLE 1).
- estrogen stimulates growth of breast cells, stable transfection of cells with the classical ER elicits a paradoxical estrogen-induced inhibition of growth (69,70).
- Membrane-associated estrogen receptors may be known molecules (kinases, ion channels) with previously unknown binding sites for estrogen, new isoforms of ER ⁇ or ERB in membranes, classical forms of ER complexed with other membrane- associated proteins, truly novel membrane proteins, or a combination of these options.
- membrane ER may activate one or more of several pathways, including interaction with growth factor membrane receptors such as HER-2 tyrosine kinase receptor or activation of G-proteins and adenylate cyclase, inositol phosphate, calcium homeostasis and or MAP kinase (see TABLE 1 for details).
- growth factor membrane receptors such as HER-2 tyrosine kinase receptor or activation of G-proteins and adenylate cyclase, inositol phosphate, calcium homeostasis and or MAP kinase (see TABLE 1 for details).
- Antiestrogen therapy is commonly used for treatment of patients with breast cancers that express estrogen receptor (ER).
- ER estrogen receptor
- the efficacy of endocrine treatment depends on the close regulation of breast cell growth by estrogens and peptide growth factors.
- the disease usually becomes resistant to estrogens, and most patients no longer respond to antiestrogen therapy.
- New options for antiestrogen treatment are clearly needed, and alternative therapies may now derive from findings showing that a portion of ER molecules occur in plasma membranes of human breast cancer cells and may interact with transmembrane erb B/HER growth factor receptors.
- HER-2/erb B-2 receptors It is known that expression of HER-2/erb B-2 receptors occurs in many human breast cancers, and the protein kinase activity of HER-2 may play a role in the ligand-independent activation of ER. If active cross-communication between ER and HER-2 growth factor receptor occurs and leads to promotion of cancer growth, this signaling complex may offer an important new target for therapeutic intervention. Since overexpression of HER-2 receptor in breast cancer is associated with the failure of antiestrogen therapy in the clinic, understanding the biologic basis of the association between membrane esfrogen receptors and HER-2 receptors may also help to improve decisions on patient management and to increase patient survival. As illustrated below, the disclosure herein both provides insight into the biology of breast cancer as well as providing novel methods for inhibiting the growth of breast cancer cells.
- endocrine therapy is an important intervention in women with breast cancers that express the estrogen receptor (ER).
- ER estrogen receptor
- Treatment with tamoxifen and other antiestrogens has increased the survival of breast cancer patients, and these agents are now used in breast cancer prevention.
- the success of endocrine therapy in breast cancer is dependent on the close regulation of breast cell growth by steroid hormone and growth factor receptors (1,2).
- steroid hormone and growth factor receptors (1,2).
- breast cancer it usually becomes resistant to estrogens, and, consequently, most patients no longer respond to tlierapy with tamoxifen or other antiestrogens.
- new information on the existence of an alternate estrogen signaling pathway in breast cancer cells allows the design of novel and more effective antihormone treatments for human breast cancers (3).
- the disclosure herein provides evidence for the existence of receptors for estrogen in association with plasma membranes of human breast cancer cells.
- plasma membranes were isolated from human breast cancer cells with and without overexpression of HER-2 receptor.
- Activity of estrogen receptors was found by ligand binding and immunoassay with antibody to ER.
- antisense oligonucleotides directed to nuclear ER block expression of both nuclear and membrane forms of ER.
- membrane ER may originate, in part, from the same transcripts that produce the intracellular ER. Purification of membrane-associated estrogen receptors may be achieved by affinity chromatography, with recovered receptor to be used for preparation of new monoclonal antibodies and for further molecular characterization.
- membrane esfrogen receptors play a role in promoting the growth of human breast cancers.
- These studies provide new evidence of signal transduction by membrane ER, including interactions with HER-2 trans -membrane receptor and other signaling pathways such as mitogen-activated protein kinase.
- HER-2 trans -membrane receptor and other signaling pathways such as mitogen-activated protein kinase.
- antisense oligonucleotides we find a blockade of the growth of breast cancer cells.
- the disclosure herein further provides evidence that treatments directed to membrane-associated estrogen receptors lead to the blockade of breast cancer cell growth.
- cells with defined levels of ER- ⁇ , membrane ER and HER-2 receptors show significant growth inhibition in vitro after treatment with antibodies directed to the ligand binding domain of ER.
- Treatment of breast cancer cells with membrane-impermeant estradiol leads to the rapid stimulation of MAP kinase activity in the cell interior, an event that may be associated with the promotion of tumor cell growth.
- estradiol estradiol linked to BSA
- membrane-associated binding sites for estrogen may mediate rapid effects of estradiol- 17 ⁇ that contribute to proliferation of human breast cancers.
- estradiol- 17 ⁇ binding-sites After controlled homogenization and fractionation of MCF-7 breast cancer cells, the bulk of specific estradiol binding is found in nuclear fractions.
- a significant portion of specific, high-affinity estradiol- 17 ⁇ binding-sites are also enriched in plasma membranes.
- These estradiol binding-sites co-purify with 5'- nucleotidase, a plasma membrane-marker enzyme, and are free from major contamination by cytosol or nuclei.
- Electrophoresis of membrane fractions allowed detection of a primary 67-kDa protein and a secondary 46-kDa protein recognized by estradiol-17 ⁇ and by a monoclonal antibody directed to the ligand-binding domain of the nuclear form of esfrogen receptor.
- Estrogen-induced growth of MCF-7 breast cancer cells in vitro was blocked by treatment with the antibody to estrogen receptor and correlated closely with acute hormonal activation of mitogen-activated protein kinase and Akt kinase signaling.
- Estrogen-promoted growth of human breast cancer xenografts in nude mice was also significantly reduced by treatment 777 V7Vo with the estrogen receptor antibody.
- membrane- associated forms of estrogen receptor plays a role in promoting intracellular signaling for hormone- mediated proliferation and survival of breast cancers and offer a new target for antitumor therapy.
- human breast cancer cells contain a membrane- associated binding site for estrogen that closely resembles nuclear ER. Activation of this membrane- associated receptor appears to promote rapid stimulation of MAPK and PI3K/Akt kinase signaling and later cell proliferation. Biologic activity of the membrane-associated receptor can be diminished 777 vitro and in vivo by antibody directed against the ligand-binding domain of nuclear ER. The results provide evidence that estrogens may initiate membrane-associated signaling events leading to modulation of the growth and survival of breast cancers.
- the plasma membrane E 2 ⁇ binding-sites constitute about 20% of total cell binding-sites for the steroid, a level of membrane concentration comparable to that found for other known transmembrane hormone receptors (92).
- monoclonal antibodies against the LBD of nuclear ER can identify membrane- associated ER in MCF-7 cells, a finding consistent with studies with other cell types (56,61,94).
- the primary membrane-associated protein reactive with antibodies to LBD of nuclear ER and with E 2 ⁇ ligand is 67-kDa, a molecular size comparable to that of nuclear ER, but additional protein species, notably at 46-kDa, were also detected (32,93).
- ER- ⁇ Two forms of ER- ⁇ with molecular masses of 67- and about 46-kDa occur in target cells, including vascular endothelial cells (94) and MCF-7 cells (95), and in HeLa cells transfected with ER cDNA (32).
- target cells including vascular endothelial cells (94) and MCF-7 cells (95), and in HeLa cells transfected with ER cDNA (32).
- the nature of the truncated receptor form may be due, in part, to limited protein degradation or to alternative translation (95).
- the disclosure herein confirms membrane-initiated signal transduction by ER in breast cancer cells, including interactions with signaling pathways such as MAPK and PI3K/Akt kinase.
- These stimulatory effects of free E 2 ⁇ appear to be equaled by E 2 ⁇ -BSA 777 vitro.
- the serine/threonine kinase Akt a downstream effector of PI3-kinase, has been implicated in cell survival and prevention of apoptosis in MCF-7 cells (96).
- the finding that such regions of membrane associated ER are exposed and that antibodies targeting that particular domain of the esfrogen receptor have the greatest inhibitory effects is also unexpected.
- the estrogen receptor is generally involved in the activation of cell growth, one could not predict the finding that molecules that interact with this receptor (particularly those that interact with the ligand binding domain) would inhibit receptor-mediated activity.
- An illustrative embodiment of the invention provided herein comprises a method of inhibiting the signalling of a plasma membrane-associated estrogen receptor comprising contacting the membrane-associated estrogen receptor with an inhibitory ligand that binds to the ligand binding domain of the membrane-associated estrogen receptor thereby inhibiting membrane-associated estrogen receptor signalling.
- the inhibitory ligand may not cross the cell membrane and in this way limits the non or less specific effects of ligands which cross this barrier.
- the inhibitory ligand is selected from the group consisting of monoclonal antibodies Ab 1 and Ab 2 (see FIG. 14A).
- Yet another embodiment of the invention consists of inhibiting the signalling of a membrane- associated estrogen receptor with an inhibitory ligand and further contacting the cell with an anti-HER- 2 immunoglobulin polypeptide under conditions which allow the anti-HER-2 immunoglobulin polypeptide to bind to HER-2 on the surfaces of the cancer cell to a degree sufficient to inhibit the growth of the cancer cell.
- Yet another embodiment of the invention is an assay for identifying and/or characterizing a ligand that can inhibit membrane-associated estrogen receptor signalling.
- the disclosure provided herein identifies a set of standard ligands which provide parameter means for identifying and assessing novel molecules.
- this aspect of the invention provides a process for identifying and/or assessing the ability of a novel molecule to inhibit membrane-associated estrogen receptor signalling in a comparative assay that compares the novel molecule's measured activity with that of one of the inhibitory ligand identified herein (e.g. monoclonal antibodies Ab 1 and Ab 2).
- a specific embodiment of the invention consists of a method for identifying a compound which inhibits membrane-associated estrogen receptor signalling comprising the steps of contacting a membrane- associated estrogen receptor with a test compound, and determining whether said compound inhibits the activation and/or signalling of the membrane-associated estrogen receptor.
- a molecule that inhibits the activation of membrane-associated estrogen receptor is useful for inhibiting membrane- associated estrogen receptor associated cellular activities.
- a product identified by such methods There are a variety of art accepted methods for measuring the inhibition of a membrane- associated estrogen receptor, a number of which are described herein.
- Typical preferred embodiments described herein include the inhibition of the estrogen-induced growth of MCF-7 breast cancer cells 777 vitro as described in Example 8 and the observing an inhibition in the growth of MCF-7 breast cancer xenografts in nude mice as described in Example 9.
- inhibition of membrane- associated esfrogen receptor mediated signaling is measured by observing an inhibition in the induction of ERK MAP kinase activation.
- ligand binding to the membrane-associated esfrogen receptor is measured generally by a radioligand binding assay.
- the invention disclosed herein also provides an assay for identifying and/or assessing other membrane-associated estrogen receptor inhibitory ligands, by, for example, providing a set of standard inhibitory ligands which provide parameter means for identifying and assessing novel molecules.
- this aspect of the invention provides a process for identifying and/or assessing the ability of a novel molecule to bind to membrane-associated estrogen receptor in a comparative assay that compares the novel molecule's measured activity with that of one of the inhibitory ligands identified herein.
- a number of analytical assays for analyzing ligand-receptor interactions are well known in the art and include for example the growth inhibition and activation assays discussed in the Examples below.
- a related method for identifying a compound which inhibits membrane-associated esfrogen receptor signalling comprising the steps of contacting a membrane-associated estrogen receptor with a test compound, determining whether said compound binds to said membrane-associated estrogen receptor; and if the compound binds to membrane-associated estrogen receptor, determining whether said compound inhibits membrane- associated estrogen receptor activation and/or signaling. Also included in this embodiment is a product identified by such methods.
- the methods for inhibiting the growth of a breast cancer cell which expresses the esfrogen receptor on its cell membrane by contacting the cell with an amount of anti-estrogen receptor immunoglobulin polypeptide sufficient to inhibit cell growth have a number of a advantages over existing methods.
- the anti-estrogen receptor polypeptides of the present invention will not recognize cells which do not express this molecule at their surface membrane, the methods disclosed herein have a greater specificity that methods which utilize the less specific art accepted compositions traditionally used in the treatment of breast cancer.
- the toxicity of these lineage specific, extracellular molecules is less than the toxicity observed with other, less specific molecules used in the treatment of cancers.
- the present invention includes methods of diagnosing and treating an individual suspected of suffering from breast cancer comprising the steps of administering to said individual a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent, and a diagnostically or therapeutically effective amount of a compound consisting of an anti-esfrogen receptor immunoglobulin polypeptide.
- the methods disclosed herein include a number of different embodiments.
- the invention consists of a method for inhibiting the growth of a breast cancer cell which expresses the esfrogen receptor in association with its surface membrane by contacting the cell with an amount of anti-estrogen receptor immunoglobulin polypeptide sufficient to inhibit cell growth.
- the anti-esfrogen receptor immunoglobulin polypeptide recognizes and binds the ligand binding domain of the estrogen receptor.
- the anti-estrogen receptor immunoglobulin polypeptide is selected from the group consisting of monoclonal antibodies Ab 1 and Ab 2.
- a related embodiment of the invention consists of a method for treating a mammalian cancer cell which expresses esfrogen receptor in association with its plasma membrane by contacting the cancer cell with the anti-estrogen receptor immunoglobulin polypeptide under conditions which allow the anti-esfrogen receptor immunoglobulin polypeptide to bind to the estrogen receptor associated with the plasma membranes of the cancer cell to a degree sufficient to inhibit the growth of the cancer cell.
- the anti-estrogen receptor immunoglobulin polypeptide is selected from the group consisting of monoclonal antibodies Ab 1 and Ab 2.
- Yet another related embodiment of the invention consists of a method of inhibiting the growth of a breast cancer cell having disregulated cell growth comprising the steps of confirming the presence of a estrogen receptor associated with the surface membrane of the breast cancer cell, providing a esfrogen receptor immunoglobulin polypeptide specific for an epitope within the ligand binding domain of the esfrogen receptor, the anti-esfrogen receptor immunoglobulin polypeptide being selected to produce inhibition of breast cancer cell growth and then contacting the cell with the anti-estrogen receptor immunoglobulin polypeptide under conditions which allow the anti-estrogen receptor immunoglobulin polypeptide to bind to the estrogen receptor associated with the surface membrane of the breast cancer cell to a degree sufficient to inhibit the growth of the breast cancer cell.
- the confirmatory step is useful in contexts where the expression of membrane esfrogen receptor is in question.
- an anti-estrogen receptor immunoglobulin polypeptide is used to confirm the presence of this molecule, for example one that has the same specificity as the molecule used in the therapeutic method.
- the anti-estrogen receptor immunoglobulin polypeptide is selected from the group consisting of monoclonal antibodies Ab 1 and Ab 2.
- a second therapeutic molecule used in the treatment of cancers for example an anti-HER-2 immunoglobulin polypeptide or an anti-HER-1/EGF receptor immunoglobulin polypeptide, under conditions which inhibit the growth of the cancer cell.
- Another embodiment of the invention consists of a method of inhibiting the activation of a cellular process that is induced via a membrane-associated estrogen receptor by contacting a cell with an anti-esfrogen receptor immunoglobulin polypeptide under conditions which allow the anti-estrogen receptor immunoglobulin polypeptide to bind to esfrogen receptor associated with the surface membrane of the cell to a degree sufficient to inhibit the activation of a cellular process that is induced via the membrane-associated estrogen receptor.
- This embodiment is consistent with observations that the pharmacology of esfrogen receptors is complex and that subtle differences in their structure, as well as their cellular milieu in which they are acting (e.g. the cellular membrane), can have marked effects on specific responses within the cell (see e.g.
- Another embodiment of the invention disclosed herein includes an injectable pharmaceutical composition for treatment of a mammalian cancer tumor having cells which express estrogen receptor associated with their cell membranes consisting of an anti-estrogen receptor immunoglobulin polypeptide specific to an epitope on a ligand binding domain of the estrogen receptor; the anti- esfrogen receptor immunoglobulin polypeptide being selected for its ability to inhibit tumor growth; and a pharmaceutically acceptable injection vehicle.
- the anti-esfrogen receptor immunoglobulin polypeptide is selected from the group consisting of monoclonal antibodies Ab 1 and Ab 2.
- kits for use in methods for inhibiting the growth of breast tumor cells winch express an estrogen receptor comprising a container, a composition contained within the container, wherein the composition includes an anti-estrogen receptor immunoglobulin polypeptide and instructions for using the anti-estrogen receptor immunoglobulin polypeptide 777 vivo or 777 vitro.
- the anti-estrogen receptor immunoglobulin polypeptide is selected from the group consisting of monoclonal antibodies Ab 1 and Ab 2.
- Yet another embodiment of the invention disclosed herein includes a method of radioimaging metastasized breast cancer cells comprising the steps of first administering to an individual suspected of having metastasized breast cancer cells, a pharmaceutical composition that consists of a pharmaceutically acceptable carrier or diluent, and coupled compound that comprises an anti-estrogen receptor immunoglobulin polypeptide and a radioactive active moiety wherein the coupled compound is present in an amount effective for diagnostic use in humans suffering from breast cancer and then detecting the localization and accumulation of radioactivity in the individual's body.
- the anti-estrogen receptor immunoglobulin polypeptides of the present invention may be obtained through a variety of sources including commercial suppliers.
- the anti-estrogen receptor immunoglobulin polypeptides of the present invention may be prepared by immunizing an animal with a purified or partially purified human esfrogen receptor.
- the animals immunized can be any one of a variety of species which are capable of immunologically recognizing epitopes characteristic of the human estrogen receptor, such as murine, porcine, equine, etc.
- Monoclonal antibodies of the invention may be prepared by immortalizing nucleic acid sequences which encode immunoglobulin polypeptides or portions thereof that bind specifically to antigenic determinants characteristic of the human estrogen receptor.
- the immortalization process can be carried out by hybridoma fusion techniques, by viral transformation of antibody-producing lymphocytes, recombinant DNA techniques, or by techniques that combine cell fusion, viral transformation and/or recombinant DNA methodologies.
- cells producing human anti-esfrogen receptor monoclonal antibodies are immortalized using, e.g., Epstein-Barr virus (EBV) transformation techniques.
- EBV Epstein-Barr virus
- B lymphocytes derived from peripheral blood, bone marrow, lymph nodes, tonsils, etc. of patients, preferably those immunized with the estrogen receptor or portions thereof, are immortalized using EBV according to methods such as those described in U.S. Pat. No. 4,464,465, and Chan et al., J. Immunol. 136:106 (1986), which are incorporated herein by reference.
- Human anti-estrogen receptor monoclonal antibodies can also be prepared by a variety of other ways, e.g., using a combination of EBV or other viral transformation and hybridoma fusion techniques.
- the hybridomas can be created by fusing stimulated B cells, obtained from a individual immunized with the esfrogen receptor or a portion thereof, with a mouse/human heterohybrid fusion partner, a variety of which have been described. See, e.g., U.S. Pat. No. 4,624,921 and James and Bell, J. Immunol. Meths. 100:5-40 (1987), which are incorporated herein by reference.
- a mouse/human fusion partner can be constructed by fusing human lymphocytes stimulated or transformed by EBV with readily available mouse myeloma lines such as NS-1 or P3NS-1, in the presence of, e.g., polyethylene glycol.
- the hybridomas or lymphoblastoid cells which secrete antibody of interest can be identified by screening culture supernatants for antibody that binds to the esfrogen receptor. More preferably, a screening assay may be employed to detect those antibodies which inhibit, for example, estrogen- mediated mitogenesis.
- Cells which possess the desired activity are cloned and subcloned in accordance with conventional techniques and monitored until stable, immortalized lines producing the anti- esfrogen receptor monoclonal antibody of interest are identified.
- monoclonal antibody is meant an antibody produced by a clonal, immortalized cell line separate from cells producing antibodies with a different antigen binding specificity.
- monoclonal antibodies are produced isolated from other monoclonal antibodies and, accordingly, in substantially pure form (relative to other antibodies) and at a concentration generally greater than normally occurring in sera from the animal species which serves as a B cell source.
- DNA sequences which encode a human anti-estrogen receptor immunoglobulin polypeptide or portions thereof that specifically bind to the estrogen receptor, or a specific domain of the estrogen receptor (such as the ligand binding domain) by screening a DNA library from human B cells according to a general protocol as disclosed by Huse et al., Science 246:1275-1281 (1989), incorporated herein by reference, and then cloning and amplifying the sequences which encode the anti-esfrogen receptor antibodies (or binding fragments) of the desired specificity.
- the immunoglobulins may then be produced by introducing an expression vector containing the appropriate immunoglobulin gene, or portion thereof, into an appropriate host cell.
- the host cell line is then maintained under conditions suitable for high level expression of the immunoglobulin nucleotide sequences, and, as desired, the collection and purification of the light chains, heavy chains, light/heavy chain dimers or intact antibodies, binding fragments or other immunoglobulin forms may follow.
- Suitable host cells include microorganisms, but mammalian or insect tissue cell culture may be preferable for producing the monoclonal antibody of the present invention (see, E. Winnacker, "From Genes to Clones," VCH Publishers, N.Y., N.Y. (1987), which is incorporated herein by reference).
- suitable mammalian host cell lines capable of secreting intact immunoglobulins have been developed in the art, and include the Chinese hamster ovary (CHO) cell line, but preferably hybridomas or transformed B-cells will be used. Bacterial phage or yeast systems may also be employed. Once expressed, the whole antibodies, their dimers, individual light and heavy chains, or other immunoglobulin forms of the present invention can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like (see, generally, R. Scopes, Protein Purification, Springer-Verlag, N.Y. (1982), incorporated herein by reference).
- polypeptides may then be used therapeutically (including exfracorporeally) or in developing and performing assay procedures, immunofluorescent stainings, and the like.
- therapeutically including exfracorporeally
- immunofluorescent stainings and the like.
- the immunoglobulin polypeptides produced according to the present invention may be of the IgG, IgM, IgA or IgD isotype, and may further be any of the appropriate subclasses thereof, such as, e.g., IgGi, IgG 2 , IgG 3 , or IgG .
- "class-switching" of the isolated immunoglobulin polypeptides may be readily accomplished. In this method genes encoding the constant regions which determine the isotype of the immunoglobulin molecule of interest are replaced by genes encoding a desired isotype or subclass, as generally described in European patent publication EP 314,161, incorporated herein by reference. Class-switched immunoglobulins may also be isolated by selecting cells which have undergone spontaneous switching using selection methods known in the art.
- substantially human an antibody or binding fragment thereof comprised of amino acid sequences which are at least about 50% human in origin, at least about 70 to 80% more preferred, and about 95-99% or more human most preferred, particularly for repeated administrations over a prolonged period as may be necessary to treat established estrogen-mediated cell proliferation disorders.
- chimeric antibodies or chimeric immunoglobulin polypeptides that specifically bind to the human estrogen receptor and thus inhibit binding of esfrogen to the receptor are also within the scope of the present invention.
- a typical therapeutic chimeric antibody would be a hybrid protein consisting of the variable (V) or antigen-binding domain from a mouse immunoglobulin specific for a human esfrogen receptor antigenic determinant, and the constant (C) or effector domain from a human immunoglobulin, although domains from other mammalian species may be used for both variable and constant domains.
- chimeric antibody also refers to antibodies coded for by immunoglobulin genes in which only the complementarity determining regions (CDR's) are transferred from the immunoglobulin that specifically recognizes the antigenic determinants, the remainder of the immunoglobulin gene being derived from a human (or other mammalian, as desired) immunoglobulin gene.
- CDR's complementarity determining regions
- This type of chimeric antibody is referred to as a "humanized” (in the case of a human immunoglobulin gene being used) antibody.
- the hypervariable regions of the variable domains of the anti-estrogen receptor immunoglobulin polypeptides comprise a related aspect of the invention.
- the hypervariable regions in conjunction with the framework regions (those portions of immunoglobulin light and heavy chain variable regions that are relatively conserved among different immunoglobulins in a single species), enable the anti-esfrogen receptor immunoglobulin polypeptides to recognize and thus bind to the human estrogen receptor.
- the hypervariable regions can be cloned and sequenced.
- these regions that confer specific recognition of the esfrogen receptor can then be cloned into a vector for expression in a host as part of another immunoglobulin molecule or as a fusion protein, e.g., a carrier molecule which functions to enhance immunogenicity of the cloned idiotope
- a carrier molecule which functions to enhance immunogenicity of the cloned idiotope
- human antibody is meant to include antibodies of entirely human origin as well as those which are substantially human, unless the context indicates otherwise.
- non-human antibodies As the generation of human anti-estrogen receptor monoclonal antibodies may be difficult with conventional immortalization techniques, it may be desirable to first make non-human antibodies and then transfer via recombinant DNA techniques the antigen binding regions of the non-human antibodies, e.g., the Fab, complementarity determining regions (CDRs) or hypervariable regions, to human constant regions (Fc) or framework regions as appropriate to produce substantially human molecules.
- CDRs complementarity determining regions
- Fc constant regions
- variable regions or CDRs for producing the chimeric immunoglobulins of the present invention may be similarly derived from monoclonal antibodies capable of binding to the human estrogen receptor, and will be produced in any convenient mammalian system, including, mice, rats, rabbits, human cell lines, or other vertebrates capable of producing antibodies by well known methods.
- Variable regions or CDRs may be produced synthetically, by standard recombinant methods including polymerase chain reaction (PCR) or through phage-display libraries. For phage display methods, see for example, McCafferty et al.
- Completely human antibodies can also be produced in fransgenic animals.
- the desired human immunoglobulin genes or gene segments can be isolated, for example by PCR from human B cells, the DNA cloned into appropriate vectors for expression in eukaryotic cells and the cloned DNA introduced into animals to produce transgenics. Animals suitable for the production of transgenics expressing human immunoglobulins include mice, rats, rabbits and pigs with rodents being preferred.
- mice and other animals for the preparation of transgenics that express human immunoglobulins should preferably have one or more of their endogenous immunoglobulin loci inactivated or "knocked-out" to facilitate identification and isolation of the human antibodies (See e.g., Lonberg, et al. Nature 368:856-859 (1994)).
- modified immunoglobulins can be readily designed and manufactured utilizing various recombinant DNA techniques well known to those skilled in the art.
- modifications of the genes may be readily accomplished by a variety of well-known techniques, such as PCR and site-directed mutagenesis (see, Gillman and Smith, Gene 8:81-97 (1979) and S. Roberts et al., Nature 328:731-734 (1987), both of which are incorporated herein by reference).
- polypeptide fragments comprising only a portion of the primary immunoglobulin structure may be produced.
- Immunoglobulin polypeptide also encompasses a truncated immunoglobulin chain, for example, a chain containing less constant region domains than in the native polypeptide.
- truncated polypeptides can be produced by standard methods such as introducing a stop codon into the gene sequence 5' of the domain sequences to be deleted. The truncated polypeptides can then be assembled into truncated antibodies.
- anti-idiotypic antibodies can be produced by using a specific immunoglobulin as an immunogen in accordance with standard techniques. For example, infection or immunization with a estrogen receptor polypeptide, or fragment thereof, induces a neutralizing immunoglobulin, which has on its Fab variable region combining site an image of the estrogen receptor polypeptide that is unique to that particular immunoglobulin, i.e., an idiotype. Immunization with such an anti-esfrogen immunoglobulin polypeptide induces an anti-idiotype antibody, which has a conformation at its combining site that mimics the structure of the original estrogen receptor antigen. These anti-idiotype antibodies may therefore be used to treat estrogen- mediated diseases.
- anti-esfrogen receptor immunoglobulin polypeptides of the invention find utility in therapeutic and diagnostic methods and compositions.
- anti-esfrogen receptor immunoglobulin polypeptides are used as a soluble ligand for human estrogen receptor, masking the receptor or otherwise inhibiting esfrogen molecules from binding to the receptor, and thereby inhibiting the undesired cell migration and proliferation.
- Antibodies as used herein also include bispecific antibodies which can be produced such as by the methods described in the following references: Glennie et al. J. Immunol. 139:2367-2375 (1987); Segal et al. Biologic Therapy of Cancer Therapy of Cancer Updates 2(4): 1-12 (1992); and Shalaby et al. J. Exp. Med. 175:217-225 (1992).
- the anti-esfrogen receptor immunoglobulin polypeptides of the invention will generally be used intact, or as immunogenic fragments, such as F v , Fab, F(ab'), or F(ab') 2 fragments.
- the fragments may be obtained from antibodies by conventional techniques, such as by proteolytic digestion of the antibody using, e.g., pepsin or papain, or by recombinant DNA techniques in which a gene or portion thereof encoding the desired fragment is cloned or synthesized, and expressed in a variety of hosts.
- esfrogen receptors permit some specificity for localizing therapeutic and diagnostic agents that are coupled to anti-esfrogen receptor immunoglobulin polypeptides that target breast cancer cells.
- the present invention provides pharmaceutical compositions and methods for imaging and thereby will more definitively diagnose metastasis. Further, the present invention provides pharmaceutical compositions, comprising therapeutic agents and methods for specifically targeting and eliminating breast cancer cells.
- Immunoglobulin polypeptides in whole or in part, may be utilized alone or coupled with active molecules such as the functional regions from other genes (e.g., enzymes), or other molecules such as toxins, labels and targeting moieties to produce fusion proteins (e.g., "immunotoxins") having novel properties. In these cases of gene fusion, the two components are present within the same polypeptide chain.
- the immunoglobulin or fragment thereof may be chemically bonded to the toxin or label by any of a variety of well-known chemical procedures.
- the linkage may be by way of heterobifunctional cross-linkers, e.g., SPDP, carbodiimide, glutaraldehyde, or the like.
- Anti-esfrogen receptor immunoglobulin polypeptides are conjugated to active agents by a variety of well-known techniques readily performed without undue experimentation by those having ordinary skill in the art. The technique used to conjugate the anti-esfrogen receptor immunoglobulin polypeptide to the active agent is dependent upon the molecular nature of the anti-esfrogen receptor immunoglobulin polypeptide and the active agent.
- assays may be performed to ensure that the conjugated molecule retains the activities of the moieties.
- the esfrogen receptor binding assay described above may be performed using the conjugated compound to test whether it is capable of binding to the esfrogen receptor.
- the activity of the active moiety may be tested using various assays for each respective type of active agent. Radionuclides retain their activity, i.e. their radioactivity, irrespective of conjugation.
- active agents which are toxins, drugs and targeting agents
- standard assays to demonstrate the activity of unconjugated forms of these compounds may be used to confirm that the activity has been retained.
- Conjugation may be accomplished directly between the anti-esfrogen receptor immunoglobulin polypeptide and the active agent or linking, intermediate molecular groups may be provided between the anti-estrogen receptor immunoglobulin polypeptide and the active agent.
- Crosslinkers are particularly useful to facilitate conjugation by providing attachment sites for each moiety.
- Crosslinkers may include additional molecular groups which serve as spacers to separate the moieties from each other to prevent either from interfering with the activity of the other.
- the active moieties may be an imaging agent.
- Imaging agents are useful diagnostic procedures as well as the procedures used to identify the location of metastasized cells. Imaging can be performed by many procedures well-known to those having ordinary skill in the art and the appropriate imaging agent useful in such procedures may be conjugated to an anti-esfrogen receptor immunoglobulin polypeptide by well-known means. Imaging can be performed, for example, by radioscintigraphy, nuclear magnetic resonance imaging (MRI) or computed tomography (CT scan). The most commonly employed radionuclide imaging agents include radioactive iodine and indium. Imaging by CT scan may employ a heavy metal such as iron chelates. MRI scanning may employ chelates of gadolinium or manganese.
- PET positron emission tomography
- radionuclides useful in imaging procedures include: 3 K, 52 Fe, 57 Co, 67 Cu, 67 Ga, 68 Ga, 77 Br, SI Rb, m In, U3 In, 123 I, I25 1, 127 Cs, 129 Cs, 131 1, 132 1, 197 Hg, 203 Pb and 206 Bi.
- the active moieties may be a therapeutic agent such as a chemotherapeutic drug.
- a therapeutic agent such as a chemotherapeutic drug.
- One having ordinary skill in the art may conjugate an anti-estrogen receptor immunoglobulin polypeptide peptide to a chemotherapeutic drug using well-known techniques. For example, Magerstadt, M. Antibody Conjugates and Malignant Disease. (1991) CRC Press, Boca Raton, USA, pp. 110-152) which is incorporated herein by reference, teaches the conjugation of various cytostatic drugs to amino acids of antibodies. Such reactions may be applied to conjugate chemotherapeutic drugs to anti-estrogen receptor immunoglobulin polypeptides, including esfrogen receptor binding peptides, with an appropriate linker.
- Anti-esfrogen receptor immunoglobulin polypeptides which have a free amino group such as esfrogen receptor binding peptides may be conjugated to active agents at that group.
- these functional groups that is free amino and carboxylic acids, are targets for a variety of homobifunctional and heterobifunctional chemical crosslinking agents which can crosslink these drugs directly to the single free amino group of esfrogen.
- one procedure for crosslinking anti-esfrogen receptor immunoglobulin polypeptides which have a free amino group such as esfrogen receptor binding peptides, for example anti-estrogen receptor immunoglobulin polypeptides such as monoclonal antibodies Ab 311 and Ab 10 to active agents which have a free amino group such as methofrexate, doxorubicin, daunorubicin, cytosinarabinoside, cisplatin, vindesine, mitomycin and bleomycin, or alkaline phosphatase, or protein- • or peptide-based toxin employs homobifunctional succinimidyl esters, preferably with carbon chain spacers such as disuccinimidyl suberate (Pierce Co, Rockford
- the anti-esfrogen receptor immunoglobulin polypeptide and the toxin may be produced as a single, fusion protein either by standard peptide synthesis or recombinant DNA technology, both of which can be routinely performed by those having ordinary skill in the art.
- two peptides, the anti-estrogen receptor immunoglobulin polypeptide and the peptide- based toxin may be produced and/or isolated as separate peptides and conjugated using crosslinkers.
- conjugation of estrogen receptor binding peptides and toxins can exploit the ability to modify the single free amino group of an esfrogen receptor binding peptide while preserving the receptor-binding function of this molecule.
- One having ordinary skill in the art may conjugate an anti-esfrogen receptor immunoglobulin polypeptide to a radionuclide using well-known techniques. For example, Magerstadt, M. (1991) Antibody Conjugates And Malignant Disease, CRC Press, Boca Raton, Fla., and Barchel, S. W. and Rhodes, B. H., (1983) Radioimaging and Radiotherapy, Elsevier, NY, N.Y., each of which is incorporated herein by reference, teach the conjugation of various therapeutic and diagnostic radionuclides to amino acids of antibodies.
- Such reactions may be applied to conjugate radionuclides to anti-esfrogen receptor immunoglobulin polypeptides or to anti-esfrogen receptor immunoglobulin polypeptides including anti-estrogen receptor immunoglobulin polypeptides with an appropriate linker.
- Suitable labels include, for example, radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescers, chemilumdnescers, magnetic particles. See, for examples of patents teaching the use of such labels, U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241, all of which are incorporated by reference.
- Immunotoxins including single chain molecules for use in the disclosed methods, may be produced by recombinant means. Production of various immunotoxins is well-known with the art, and methods can be found, for example in "Monoclonal Antibody-Toxin Conjugates: Aiming the Magic Bullet," Thorpe et al, Monoclonal Antibodies in Clinical Medicine, Academic Press, pp. 168-190 (1982); E. Vitetta, Science (1987) 238:1098-1104; and G. Winter and C. Milstein, Nature (1991) 349:293-299; all incorporated herein by reference. A variety of cytotoxic agents are suitable for use in immunotoxins.
- Cytotoxic agents can include radionuclides, such as Iodine-131, Yttrium-90, Rhenium- 188, and Bismuth-212; a number of chemotherapeutic drugs, such as vindesine, methofrexate, adriamycin, and cisplatinum; and cytotoxic proteins such as ribosomal inhibiting proteins like pokeweed antiviral protein, Pseudomonas exotoxin A, ricin, diphtheria toxin, ricin A chain, etc., or an agent active at the cell surface, such as the phospholipase enzymes (e.g., phospholipase C).
- radionuclides such as Iodine-131, Yttrium-90, Rhenium- 188, and Bismuth-212
- chemotherapeutic drugs such as vindesine, methofrexate, adriamycin, and cisplatinum
- the present invention provides pharmaceutical compositions that comprise the compounds of the invention and pharmaceutically acceptable carriers or diluents.
- the pharmaceutical composition of the present invention may be formulated by one having ordinary skill in the art. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, A. Osol, a standard reference text in this field, which is incorporated herein by reference.
- conjugated compounds of the present invention can be used alone or in combination with other diagnostic, therapeutic or additional agents.
- additional agents include excipients such as coloring, stabilizing agents, osmotic agents and antibacterial agents.
- inhibitory molecules of the present invention such as anti-esfrogen receptor antagonists
- ICI 182,780 or signaling inhibitors of membrane-associated esfrogen receptor-induced signal fransduction pathways (including MAP kinase, PI3K/Akt kinase, adenylate cyclase and G-protein coupled, and nitric oxide signaling pathways), may be obtained through a variety of sources including commercial suppliers.
- anti-membrane estrogen receptor reagents of the present invention may be prepared by the design and synthesis of selective membrane-associated estrogen receptor modulators of steroidal or non-steroidal nature (see Piefras et al., Proc. Am. Assoc. Cancer Res. 40 : 637, 1999).
- Such uses include for example, the determination of membrane-associated esfrogen receptor ligand concentrations, the development of membrane-associated esfrogen receptor ligand agonists and antagonists and the characterization of the physiological roles of these ligands and their receptors. Consequently, a wide variety of protocols based on methods pertaining to such ligand-receptor interactions are described for these purposes (see, e.g., U.S. Patent No. 5,871,909, U.S. Patent No. 5,030,576, U.S. Patent No. 6,110,737 and U.S. Patent No. 6,040,290).
- one aspect of the present invention relates to a method of detecting metastasized breast cancer cells in an individual suspected of suffering from metastasized breast cancer by radioimaging.
- Such individuals may be diagnosed as suffering from metastasized breast cancer and the metastasized breast cancer cells may be detected by administering to the individual, preferably by intravenous administration, a pharmaceutical composition that comprises a pharmaceutically acceptable carrier or diluent and a conjugated compound that comprises an anti-esfrogen receptor immunoglobulin polypeptide and an active moiety wherein the active moiety is a radioactive and detecting the presence of a localized accumulation or aggregation of radioactivity, indicating the presence of cells with membrane esfrogen receptors.
- a pharmaceutical composition that comprises a pharmaceutically acceptable carrier or diluent and a conjugated compound that comprises an anti-esfrogen receptor immunoglobulin polypeptide and an active moiety wherein the active moiety is a radioactive and detecting the presence of a localized accumulation or aggregation of radioactivity, indicating the presence of cells with membrane esfrogen receptors.
- the pharmaceutical composition comprises a pharmaceutically acceptable carrier or diluent and a conjugated compound that comprises an anti- esfrogen receptor immunoglobulin polypeptide and an active moiety wherein the active moiety is a radioactive.
- the pharmaceutical composition comprises a pharmaceutically acceptable carrier or diluent and a conjugated compound that comprises an anti-esfrogen receptor immunoglobulin polypeptide and an active moiety wherein the active moiety is a radioactive agent selected from the group consisting of: 43 K, 52 Fe, 57 Co, 67 Cu, 67 Ga, 68 Ga, 77 Br, 81 Rb, I U In, U3 In, 123 I, I25 I, 127 Cs, 129 Cs, 131 I, 132 I, 197 Hg, 203 Pb and 206 Bi.
- a radioactive agent selected from the group consisting of: 43 K, 52 Fe, 57 Co, 67 Cu, 67 Ga, 68 Ga, 77 Br, 81 Rb, I U In, U3 In, 123 I, I25 I, 127 Cs, 129 Cs, 131 I, 132 I, 197 Hg, 203 Pb and 206 Bi.
- the individual being treated may be diagnosed as having metastasized breast cancer or may be diagnosed as having localized breast cancer and may undergo the treatment proactively in the event that there is some metastasis as yet undetected.
- the pharmaceutical composition contains a diagnostically effective amount of the conjugated composition.
- a diagnostically effective amount is an amount which can be detected at a site in the body where cells with esfrogen receptors are located without causing lethal side effects on the individual.
- one aspect of the present invention relates to a method of treating individuals suspected of suffering from breast cancer. Such individuals may be treated by administering to the individual a pharmaceutical composition that comprises a pharmaceutically acceptable carrier or diluent and a anti-esfrogen receptor immunoglobulin polypeptide.
- the pharmaceutical composition includes a conjugated compound that comprises an anti-esfrogen receptor immunoglobulin polypeptide and an active moiety wherein the active moiety is a therapeutic agent.
- the pharmaceutical composition comprises a pharmaceutically acceptable carrier or diluent and a conjugated compound that comprises an anti- esfrogen receptor immunoglobulin polypeptide and an active moiety wherein the active moiety is a radiostable active agent.
- the active moiety is selected from the group consisting of: methofrexate, doxorubicin, daunorubicin, cytosinarabinoside, etoposide, 5-4 fluorouracil, melphalan, chlorambucil, cis-platinum, vindesine, mitomycin, bleomycin, purothionin, macromomycin, 1,4- benzoquinone derivatives, frenimon, ricin, ricin A chain, Pseudomonas exotoxin, diphtheria toxin, Clostridium perfringens phospholipase C, bovine pancreatic ribonuclease, pokeweed antiviral protein, abrin, abrin A chain, cobra venom factor, gelonin, saporin, modeccin, viscumin, volkensin, alkaline phosphatase, nifroimidazole, mefronidazole and misoni
- the pharmaceutical composition comprises a pharmaceutically acceptable carrier or diluent and a conjugated compound that comprises an anti- esfrogen receptor immunoglobulin polypeptide and an active moiety wherein the active moiety is a radioactive agent.
- the pharmaceutical composition comprises a pharmaceutically acceptable carrier or diluent and a conjugated compound that comprises an anti-esfrogen receptor immunoglobulin polypeptide and an active moiety wherein the active moiety is a radioactive agent selected from the group consisting of: K, Fe, Co, Cu, Ga, Ga, Br, 81 Rb, m In, 113 In, 123 1, 125 1, 127 Cs, 129 Cs, I31 1, 132 1, 197 Hg, 203 Pb and 206 Bi.
- a radioactive agent selected from the group consisting of: K, Fe, Co, Cu, Ga, Ga, Br, 81 Rb, m In, 113 In, 123 1, 125 1, 127 Cs, 129 Cs, I31 1, 132 1, 197 Hg, 203 Pb and 206 Bi.
- the individual being treated may be diagnosed as having metastasized breast cancer or may be diagnosed as having localized breast cancer and may undergo the freatment proactively in the event that there is some metastasis as yet undetected.
- the pharmaceutical composition contains a therapeutically effective amount of the conjugated composition.
- a therapeutically effective amount is an amount which is effective to cause a cytotoxic or cytostatic effect on metastasized breast cancer cells without causing lethal side effects on the individual.
- Another embodiment of the present invention relates to a method of treating individuals suspected of suffering from metastasized breast cancer.
- the individual being treated may be diagnosed as having metastasized breast cancer or may be diagnosed as having localized breast cancer and may undergo the freatment proactively in the event that there is some metastasis as yet undetected.
- the pharmaceutical composition contains a therapeutically effective amount of the coupled composition.
- a therapeutically effective amount is an amount which is effective to cause a cytotoxic or cytostatic effect on metastasized breast cancer cells without causing lethal side effects on the individual.
- compositions for pharmaceutical compositions, the anti-esfrogen receptor immunoglobulin polypeptides of the invention as described herein can be administered to an individual having a estrogen-mediated cellular proliferation disorder.
- compositions are administered to a patient in an amount sufficient to effectively block cell receptors, and thereby cure or at least partially arrest the cellular proliferation and its symptoms and/or complications.
- Amounts effective for this use will depend on a variety of factor, for example, the nature of the anti-esfrogen receptor immunoglobulin polypeptide composition, the manner of adminisfration, the stage and severity of the disease being treated, the weight and general state of health of the patient, and the judgment of the prescribing physician.
- anti-esfrogen receptor immunoglobulin polypeptide and peptide compositions derived therefrom may be employed in serious disease states, that is, life-threatening or potentially life threatening situations. In such cases, it is possible and may be felt desirable by the treating physician to administer substantial excesses of these compositions.
- human anti-esfrogen receptor monoclonal antibodies or substantially human anti- esfrogen receptor monoclonal antibodies of the invention are most preferred under these circumstances.
- concentration of anti-estrogen receptor immunoglobulin polypeptides of the invention in the pharmaceutical formulations can vary widely, i.e., from less than about 1%, usually at or at least about 10-15%) to as much as 50%) or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of adminisfration selected.
- a typical pharmaceutical composition for intravenous infusion could be made up to contain 250 ml of sterile Ringer's solution, and 100 mg of anti-esfrogen receptor immunoglobulin polypeptide.
- the antiestrogen receptor immunoglobulin polypeptides and fragments thereof can also be administered via liposomes.
- the anti-esfrogen receptor immunoglobulin polypeptides can serve to target the liposomes to particular tissues or cells displaying the human esfrogen receptor.
- Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like.
- the immunoglobulin polypeptide or fragment to be delivered is incorporated as part of the liposome, alone or in conjunction with a molecule which is, for example, toxic to the target cells.
- a liposome suspension containing an immunoglobulin polypeptide can be administered intravenously, locally, topically, etc. in a dose which varies according to, inter alia, the manner of adminisfration, the peptide being delivered, and the stage of disease being treated.
- conventional nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
- routes of adminisfration include those selected from the group consisting of intravenous, infraarterial, infraperitoneal, local administration into the blood supply of the organ in which the tumor resides or directly into the tumor itself.
- Intravenous adminisfration is the preferred mode of administration. It may be accomplished with the aid of an infusion pump.
- the dosage administered varies depending upon factors such as: the nature of the active moiety; the nature of the conjugated composition; pharmacodynamic characteristics; its mode and route of administration; age, health, and weight of the recipient; nature and extent of symptoms; kind of concurrent freatment and frequency of freatment.
- compositions can be carried out with dose levels and pattern being selected by the treating physician.
- the pharmaceutical formulations should provide a quantity of anti-esfrogen receptor immunoglobulin polypeptide of the invention sufficient to effectively treat the patient.
- Adminisfration should begin at the first indication of undesirable cellular proliferation or shortly after diagnosis, and continue until symptoms are substantially abated and for a period thereafter. In well established cases of disease, loading doses followed by maintenance doses will be required.
- the pharmaceutical compositions for therapeutic freatment are intended for parenteral, topical, oral or local administration.
- the pharmaceutical compositions are administered parenterally, e.g., intravenously, subcutaneously, inttadermally, or intramuscularly.
- compositions for parenteral adminisfration which comprise a solution of the anti-esfrogen receptor immunoglobulin polypeptide dissolved or suspended in an acceptable carrier, preferably an aqueous carrier.
- an acceptable carrier preferably an aqueous carrier.
- aqueous carriers may be used, e.g., water, buffered water, 0.4%) saline, 0.3%> glycine, hyaluronic acid and the like.
- These compositions may be sterilized by conventional, well known sterilization techniques, or may be sterile filtered.
- the resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to adminisfration.
- compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, ttiethanolamine oleate, etc.
- auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, ttiethanolamine oleate, etc.
- kits are also provided by the invention.
- Such kits may comprise a carrier means being compartmentalized to receive in close confinement one or more container means such as vials, tubes, and the like, each of the container means comprising one of the separate elements to be used in the method.
- one of the container means may comprise an anti-esfrogen receptor immunoglobulin polypeptide that is or can be coupled to an active moiety.
- kits of the invention will typically comprise the container described above and one or more other containers comprising materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
- a label may be present on the container to indicate that the composition is used for a specific therapy or non-therapeutic application, and may also indicate directions for either 777 v7 ' vo or 7?7 vitro use, such as those described above.
- ER- ⁇ ligand-binding domain
- LBD ligand-binding domain
- Ab2 against LBD from amino acids 302-595
- Ab3 against hinge- region, HSP 90- and DNA-binding domains from amino acids 280-335 (NeoMarkers).
- Cells were maintained in esfrogen-free conditions 48 h before the experiment. In selected studies, cells were pre-incubated 90 min with U0126 (25 ⁇ M) , a selective inhibitor of MEK1 and MEK2 (101) or for 2 h with anti-LBD Ab-2 (10 ⁇ g/ml) before freatment with esfrogens. Protein samples were separated by SDS-PAGE and then transferred to a nitrocellulose membrane for immunodetection with anti-phospho-p44/p42 MAP kinase (Thr202/ Tyr204) polyclonal antibody (New England Biolabs, Beverly, MA, USA), using the Pierce Western blotting system.
- Akt activity was measured by Western blot using the Akt kinase assay kit (Cell Signaling Technology, Beverly, MA, USA). After growth in estrogen-free conditions, cells were pre-incubated with anti-LBD Ab-2 (10 ⁇ g/ml), anti-PI(3) kinase inhibitor LY294002 (lO ⁇ M) (102) or ICI 182,780 (1 ⁇ M) (Astra Zeneca, Newark, DL, USA), followed by freatment with 10 nM E 2 ⁇ or 0.5 ⁇ M E 2 ⁇ -BSA for 20 min. Lysates were incubated overnight with anti-Akt kinase antibody.
- FIG. 21 is a graph showing Inhibition of MCF-7 human breast cancer cell growth by the MEK 1/2 inhibitor U0126.
- MCF-7 cells were inoculated subcutaneously at 5 x 10 7 cells / animal in the mid-back region of 3-mo-old female athymic mice (Charles River, Wilmington, MA, USA) primed with E 2 ⁇ in a biodegradable binder as before (91). Treatment was initiated when tumors grew to >30 mm 3 . Animals were randomized by weight and tumor size at the start of the experiment, with 6-8 animals included in each freatment group. Antibody and control solutions were administered by infraperitoneal injection. Anti-ER LBD Ab2 was given at a dose of 3.5 mg/kg in 6 doses at 4-day intervals (over 26 days). Control injections with mouse IgGi (Pharmingen, San Diego, CA, USA) were given on an identical freatment protocol.
- EXAMPLE 2 Plasma membrane ER in human breast cancer cells
- estrogen-binding activity In addition to nuclear binding of esfradiol, estrogen-binding activity also shows significant co-localization with the plasma membrane marker enzyme, 5 '-nucleotidase.
- enzyme-linked immunoassay of esfrogen receptor (74,75) in particle-free extracts of whole homogenate and plasma membrane revealed that 15% of homogenate ER localized to plasma membranes.
- the PM fraction After purification of plasma membranes from the crude nuclear fraction by use of discontinuous-sucrose density gradient centtifugation, the PM fraction showed enhanced activity of 5 '-nucleotidase, a plasma membrane marker enzyme, to about 23 -times that of homogenate (Fig. 2a, b).
- Specific [ 3 H]E 2 ⁇ binding in plasma membranes was enriched to 28-times homogenate activity and represented 22%> of homogenate binding.
- This data shows that specific E 2 ⁇ binding co- purifies with a plasma membrane marker protein in membrane fractions from breast cancer cells. LDH activity, highly enriched in cytosol, is not significantly detected in PM (Fig. 2 a, b).
- a secondary band at 46-kDa and minor bands at 62-kDa and 97-kDa were detected in PM and other cell fractions by use of Western blot (Fig. 16a) and ligand-blotting (Fig. 16b). Identification of estrogen receptor forms in association with caveola-related membrane domains
- Caveolae and caveola-related domains are liquid-ordered regions of plasma membrane that are enriched in molecules that play important roles in intracellular signal ttansduction (Smart et al., Mol. Cell. Biol. 19: 7289, 1999). These molecules include receptor tyrosine kinases (HER-1, HER-2), components of the ras-MAPK pathways, PKC's, G-protern coupled receptors, etc. Consequently, caveolae and caveola-related domains function as critical preassembled signaling complexes for cross- communication between distinct signaling components.
- membrane-associated esfrogen receptor forms in MCF-7 cells interact directly with cavatellin-2/flotillin-2, an integral membrane protein found in caveola-related domains (Volonte et al., J. Biol. Chem. 274: 12702, 1999) (see FIG. 17).
- the membrane-associated esfrogen receptor appears to occur in association with a plasma membrane subcompartment that plays a crucial role in intracellular signal fransduction (FIG. 17).
- EXAMPLE 3 Interaction of membrane-associated estrogen receptor with immobilized estradiol and alteration of membrane-associated ER in human breast cancer cells with HER-2 overexpression.
- E 2 ⁇ -BSA-FITC fluorescein-labeled E 2 ⁇ -BSA
- this macromolecular complex shows evidence of ligand specificity, with significant reduction ( ⁇ 0.01) of E 2 ⁇ -BSA-FITC binding by competition with equimolar amounts of free E 2 ⁇ , E 2 ⁇ -BSA, tamoxifen or ICI 182, 780, while the related steroid congener, progesterone, is not effective.
- Surface binding of E 2 B-BSA-FITC is also significantly diminished by competition with antibody to the LBD of nuclear ER, providing evidence of some immunologic identity of the membrane site with nuclear ER (Fig. 19 c, e).
- Fig. 19 c, e As expected, after permeabilization of cells by disruption of plasma membrane with detergent, intense labeling of ER in cell nuclei is found and occurs in 96% of breast cancer cells (Fig.
- MCF-7 human breast cancer cells with (MCF-7 HER-2) or without (MCF-7 PAR) HER-2 receptor overexpression (3) were freated with l ⁇ M E 2 ⁇ -BSA-FITC, a membrane-impermeant compound with relatively high binding affinity for ER (52,61). Labeling of membrane ER was found in both breast cancer cell lines (see FIG. 3), with 78% of MCF-7 PAR cells and 44% of MCF-7 HER-2 cells showing cell surface ER labeling (see TABLE 2 below).
- MCF-7 PAR cells membrane- associated binding of E 2 ⁇ -BSA-FITC was competitively reduced by free esfradiol, tamoxifen, ICI 182,780, excess E 2 ⁇ -BSA, and by 1 ⁇ g/ml monoclonal antibody Ab 1 to the ligand binding domain of ER- ⁇ , but not by progesterone (see also, FIG. 19).
- breast cells were permeabilized with 0.05% Triton X-100, and 98% of MCF-7 PAR cells and 79% of MCF-7 HER-2 cells showed uptake of the esfradiol conjugate.
- membrane ER was estimated to be 3% of that found in MCF-7 PAR cells but was increased to 12% of that detected in MCF-7 HER-2 cells (see TABLE 2).
- E 2 -BSA-FITC Fluorescent esfradiol-BSA conjugate
- EXAMPLE 4 Membrane ER stimulates proliferation of breast cancer cells
- E 2 -BSA E2-BSA
- BSA BSA
- 10 nM ICI 182,780 with 200 nM E 2 -BSA or 20 nM esfradiol for only 10 minutes.
- cells were rinsed 3 times, cultivated in esfrogen-free media for an additional 48 hrs and counted. The results are expressed as percent control (vehicle alone) and show that E 2 -BSA and free estradiol, but not BSA, stimulate cell growth (PO.001, t-test).
- the proliferative effect of E 2 ⁇ -BSA is blocked by freatment of cells with the pure antiestrogen, ICI 182,780.
- Isolated MCF-7 breast cancer cells were cultivated in esfrogen-free media for 72 hrs and then incubated for 30 min at 22°C with immobilized esfradiol at a prevailing concentration of approximately 0.5 nM as described before (47). These conditions were shown before to permit selection of cells with high affinity interactions with esfradiol at the cell surface (46,47). No significant binding of cells was observed when inert supports coupled only to albumin were used, indicating specificity of cell binding to immobilized esfradiol.
- Breast cancer cells bound to immobilized esfrogen (E2-binding) were dislodged from the fibers in the presence of excess E2 ⁇ and recovered mtact by cenfrifugation (47).
- Corresponding cells which had not become bound to immobilized esfradiol (non-binding), as well as cells not selected for binding to immobilized esfrogen (unfractionated) were processed and recovered under parallel conditions. All cell groups were than cultivated for 3 days in esfrogen-free media, followed by treatment with or without 2 nM esfradiol- 17 ⁇ for 72 hrs. Cell numbers in all groups were quantitated and expressed relative to the initial cell number at the start of the treatments. The increment in esfrogen-induced cell growth in E2-binding cells was significantly greater than that found in unfractionated and non-binding cell populations (P .001, t-test, 3 experiments).
- EXAMPLE S ER associates directly with HER-2 receptor To further evaluate prior reports of cross-communication between ER and HER-2 receptors
- MCF-7 PAR cells were treated 777 vitro with heregulin, a ligand for activation of HER- 2/HER-3 receptor heterodimers (3).
- Figure 6 shows that the activation of HER-2 growth factor receptor promotes physical association of HER-2 receptor with esfrogen receptor (ER).
- MCF-7 breast cancer cells were freated in vitro for 5-60 minutes with 10 nM heregulin, a ligand known to activate HER-2/HER-3 receptors (3). Lysates were prepared and processed as described before (3). Samples were immunoprecipitated with anti-HER-2 antibody (IP: HER-2 receptor) prior to electrophoresis and Western blotting with anti-ER antibody H222 (IB: estrogen receptor).
- IP anti-HER-2 antibody
- IB estrogen receptor
- Esfrogen receptor normally occurs as a 65- to 70-kd protein (3).
- the experiment shown here is representative of results from 4 other experiments. In independent experiments in which samples were immunoprecipitated with anti- ER antibody prior to electrophoresis and Western blotting with anti-HER-2 antibody, a similar association between ER and HER-2 receptors was found.
- EXAMPLE 6 Antisense oligonucleotides to intracellular ER suppresses membrane ER and tumor growth
- the antisense phosphorothioate oligonucleotide was synthesized as 5'-GGGTCATGGTCATGG-3' (SEQ ID NO: 1), and a missense control was used for comparison. Specific esfradiol-17 ⁇ binding to plasma membrane fractions was done by established methods. In addition, MCF-7 breast cancer cells display marked growth inhibition following ER antisense treatment (see FIG. 9). These results provide evidence consistent with the finding that membrane-associated esfrogen receptors in breast cancer cells may derive, in part, from the same transcripts that yield the intracellular forms of ER.
- EXAMPLE 7 Inhibition of estrogen -stimulated MAP Idnase blocks growth of breast cancer cells
- MAP kinase mitogen- activated protein kinase
- Estrogen freatment leads, in turn, to increased serine phosphorylation of the estrogen receptor at 15 minutes, and this estrogen effect is also suppressed by prior exposure of cells to PD 98059 (see FIG. 12).
- This blockade of esfrogen-induced MAP kinase activation by PD 980959 results in the suppression of estrogen-induced growth of MCF-7 breast cancer cells (see FIG. 13).
- EXAMPLE 8 Monoclonal antibodies to intracellular ER block growth-stimulatory action of membrane ER
- E 2 ⁇ -BSA proliferative effect of E 2 ⁇ -BSA is blocked by freatment of cells with lCI 182,780, a pure antiestrogen (P ⁇ 0.001), or by prior exposure to anti-ER Abl (P ⁇ 0.05) or Ab2 (P ⁇ 0.001) (Fig. 20 ).
- Rapid effects ofE and E ⁇ -BSA on activation of MAPK and Akt kinase in breast cancer cells Post-receptor signal ttansduction events, such as stimulation of MAPK, extracellular signal- regulated Idnase ERK-1 (p44) and ERK-2 (p42) (43, 104), may contribute to proliferative effects of E 2 ⁇ in breast cells.
- E 2 ⁇ but not 17 ⁇ -esfradiol (E 2 ⁇ ) promotes phosphorylation of MAPK isoforms, with effects evident within 2 min (Fig. 18a).
- E 2 ⁇ 17 ⁇ -esfradiol
- MCF-7 cells were freated with E 2 ⁇ linked to BSA, a macromolecular complex considered to be membrane-impermeant (52, 60, 61, 94).
- EXAMPLE 9 Inhibition of breast tumorigenesis in vivo by antibody to ligand-binding domain of ER- ⁇ The antitumor activity of antibodies to ER- ⁇ was evaluated further using MCF-7 tumors 77 VO. MCF-7 cells were grown as subcutaneous xenografts in female afhymic mice primed with E 2 ⁇ to promote growth of these estrogen-dependent cells (3). Antibody or control treatments were initiated when tumors grew to >30 mm 3 . Anti-ER Ab2 was administered in 6 doses over a 26-day period.
- EXAMPLE 10 Assessing the existence and identity of receptors for estrogen in membranes of breast cancer cells Many studies document the existence of membrane estrogen receptors, but the identity of these receptors remains elusive. These receptors may be known molecules (kinases, G proteins, ion channels) with unknown binding sites for estrogen, new isoforms of ER ⁇ or ER ⁇ in membranes (38,61), classical ER complexed with other membrane-associated proteins or truly novel membrane proteins (65). This work can determine which interpretation is correct. By use of 'controlled subcellular fractionation' methods (3,48,71), plasma membranes can be isolated from MCF-7 human breast cancer cells with and without overexpression of HER-2 receptor (FIG. 2).
- MCF-7 cells have been stably fransfected with a vector containing the full-length cDNA of human HER-2 gene isolated from a primary breast cancer (3,15-17). These cells are termed MCF-7 HER-2.
- the vector for insertion of HER-2 into human cells contains full-length HER-2 gene ligated into a replication-defective refroviral expression vector, pLXSN (3,15-17).
- pLXSN replication-defective refroviral expression vector
- a vector devoid of HER-2 but with neomycin phosphofransferase gene for selection with G418 was packaged in an identical fashion and served as a control to infect MCF-7 cells (MCF-7 PAR).
- MCF-7 HER-2 cells with 2-5 copies of HER-2 gene per cell can be used in these studies.
- Cells can be routinely plated in RPMI medium 1640 (GIBCO) with 2mM glutamine and 1% penicillin G-sfreptomycin-fungizone. For standard plating, medium with 10% heat-inactivated fetal bovine serum can be used.
- ER ⁇ activity of ER ⁇ can be determined by two independent approaches, ligand binding with [ 3 H]-esfradiol-17 ⁇ (3) and enzyme-linked immunoassay (ELISA) (74,75). Differences in the two approaches may be found if membrane ER originate from a transcript other than classical ER ⁇ . If this occurs, one skilled in the art can consider use of ligand affinity blotting techniques to characterize alternative estrogen-binding membrane molecules (FIG. 16)(60,61). Prior studies show that MCF-7 cells do not contain significant levels of ER ⁇ (76). Verification of plasma membrane purity can be confirmed as before by use of enzyme markers (48,71) and ELISA assay of HER-2 (77).
- Synthetic oligonucleotide probes can be designed from peptide sequences of membrane ER and can then be used to isolate clones corresponding to ER from randomly primed cDNA libraries as described before (82). The availability of these molecular tools for cell fransfection studies (82, 83) could rapidly advance our understanding of the role of membrane-associated ER in breast cancer.
- One skilled in the art can also prepare plasma membranes for comparison from MCF-7 cells freated with ER antisense oligonucleotide (0.35 ⁇ M for 24 h) in order to suppress expression of ER ⁇
- EXAMPLE 11 Assessing the role of membrane estrogen receptors in promoting growth of breast cancers
- tyrosine phosphorylation of HER-2 can be assessed using immunoprecipitation of cell lysate protein with agarose-conjugated anti-phosphotyrosine monoclonal antibody (Upstate), followed by immunoblotting with anti-HER-2 antibody (3).
- Physical interaction of HER-2 receptor with membrane ER, ER ⁇ and ER ⁇ , following heregulin or estrogen stimulation, can be assessed further using methods described elsewhere (3) (FIG. 6).
- MAP kinase activation Activity of MAP kinase in response to estradiol in MCF-7 and confrol cells can be evaluated further 7 ' ⁇ z vitro using a specific antibody recognizing the active, phosphorylated forms of p44/p42 MAPK (Erkl/ Erk 2) for Western immunoblot analyses as before (40).
- Western immunoblots of the same cell extracts from MCF-7 and confrol cells can also be done using an antibody that recognizes both nonphosphorylated forms of p44/ ⁇ 42 MAP kinase in order to assess whether freatment alters MAP kinase activity and/or expression (FIG. 11).
- Intracellular calcium concentrations can be measured by using the ratiometeic fluorescent indicator dye Fura 2-AM, a membrane-permeant acetoxymethyl ester form of Fura 2 (Molecular Probes). Confluent MCF-7 cell monolayers grown on coverglasses can be incubated with 10 ⁇ M Fura 2-AM using established protocols, with calculation of calcium concentration as before (40). As required, alternate measures of calcium flux can also be considered (39).
- G protein activation To assess activation of G proteins by membrane ER, binding of guanosine 5'-3-0-(thio)friphos ⁇ hate (GTP ⁇ S) to Gas or Gaq can be tested in membrane preparations as described before (40,61).
- GTP ⁇ S guanosine 5'-3-0-(thio)friphos ⁇ hate
- Adenylate cyclase activity To assess activation of adenylate cyclase activity by membrane ER, cAMP generation can be measured by established procedures (40,41,61)
- Inositol phosphate production Generation of inositol phosphate (E ⁇ canbe assayed as before (40,61).
- Ligands for testing in these experiments can include free estradiol- 17B, membrane- impermeant esfradiol- 17B-BS A (61), BSA (control), esfradiol-17 ⁇ (stereospecif ⁇ c confrol), and progesterone (steroid confrol).
- ligands in some experiments can be used in combination with antibodies to membrane ER or to classical ER functional domains (H- 222, Abbott; domain-specific Neomarkers antibodies) to determine the accessibility of membrane ER to modulation by cell surface-reactive agents; antibodies to HER-2 (Herceptin); or antiestrogens, including tamoxifen, the pure anti-esfrogen, ICI 182,780, or raloxifene.
- H- 222 Abbott
- domain-specific Neomarkers antibodies antibodies to determine the accessibility of membrane ER to modulation by cell surface-reactive agents
- antibodies to HER-2 Herceptin
- antiestrogens including tamoxifen, the pure anti-esfrogen, ICI 182,780, or raloxifene.
- the association of membrane ER signaling with franscriptional events and with cell growth (61) can be assessed further by established methods as below: Transient transfection of breast cancer cells with ERE-CAT reporter gene constructs:
- a reporter plasmid containing palindromic ERE and the chloramphenicol acetylfransferase (CAT) gene can be used in these studies (ERE-CAT; 3).
- Substitution of the basic reporter plasmid pBLCAT2 for pERE-BLCAT in selected experiments can provide a confrol for specificity of the DNA-binding site in the regulatory sequence of the reporter gene.
- the plasmid pCMV which contains the ⁇ -galactosidase gene, can be used as an internal control for transfection efficiency (3).
- Cells can be teansfected as before (3). Activity of test reagents can be assessed using transfected cells with or without ERE-CAT.
- CAT protein can be quantitated in cell extracts using a non-radioactive enzyme-linked immuno-sorbant assay (5'-3', Boulder,CO) as before (3).
- CAT reporter activity can be normalized for protein in each sample.
- results of the latter studies and ongoing research in this area 38,76
- one skilled in the art may also consider assessing the fransactivation properties of membrane ER in the context of an API site, using fransfection of an API reporter plasmid in cells expressing membrane ER. Activation of growth in human breast cancer cells :
- MCF-7 cells can be cultivated in esfrogen-free media for 72h prior to the start of proliferation experiments.
- ligands for testing can include free esfradiol- 17 ⁇ , estradiol- 17B-BSA (membrane-impermeant [61]), BSA (confrol), estradiol- 17 ⁇ (confrol), and progesterone (confrol), and growth can be quantitated by cell counts at the end of the experiment as before (3) (FIG. 20).
- EXAMPLE 12 Investigating alternate treatments to prevent breast cancer progression in models of breast cancer
- Erb B/HER-reactive peptides are known to modulate ER- dependent transcription in the absence of esfrogen, in part by downstream pathways involving MAP kinase (86-88).
- activation of HER-2 signaling by heregulin supports growth of estrogen- dependent MCF-7 cells in the absence of esfrogen (3).
- Blockade of esfrogen-induced growth of MCF-7 cells by tyrosine kinase inhibitors further attests to the importance of tyrosine kinases in esfrogen action (89).
- Phosphorylation of ER itself on tyrosine and serine residues elicits functional changes in the interaction of ER with ERE (3,87,88).
- ERE ERE
- the franscriptional activity of ER may be modulated independent of esfrogen by phosphorylation of ER through growth factor signaling pathways (3,86-89; see FIG.1).
- FIG. 1 With the emergence of these novel modes of esfrogen action (FIG. 1), one skilled in the art can evaluate new approaches to antiestrogen therapy that may offer patients fewer side-effects and greater antitumor efficacy. A number of important breast cell models can be available for use in these experiments.
- MCF-7 breast cancer cells with expression of ER- ⁇ , with and without overexpression of HER-2 or HER-l/EGFR (3) b) ER-null breast cells and daughter cells stably teansfected with cDNA for ER- ⁇ (3,61) c) MCF-7 cells freated with ER antisense oligonucleotide for suppression of ER- ⁇ expression (84,85) d) ER-null COS-7 and CHO cells; COS-7 and CHO daughter cells stably fransfected with ER- ⁇ cDNA (3, 61) e) COS-7 and CHO daughter cells stably fransfected with cDNA for ER- ⁇ as well as cDNA for either HER-1, HER-2, HER-3 or HER-2/HER-3 together (90).
- ER-null COS-7 and CHO cells stably fransfected with cDNA for either HER-1, HER-2, HER-3 or HER-2/HER-3 together can be available for these studies and can also be used to examine the report of direct interactions between esfradiol and HER receptors (see 54) f) ER-positive breast cancer cells selected for binding to immobilized esfradiol (46,47; see FIG. 4)
- Cell models with qualitative and quantitative differences in membrane ER- ⁇ and HER receptor expression can be tested for their differential growth sensitivities to antiestrogens, including tamoxifen, ICI 182,780 and raloxifene.
- Patient management decisions in the clinic may be greatly aided by a simple understanding of how esfrogen and erb B/HER growth factor pathways function to promote growth of the cancer cell and how different levels of receptor expression affect the response to standard antiestrogen therapies.
- Esfrogen-induced activation of mitogen-activated protein kinase requires mobilization of intracellular calcium. Proc. Natl. Acad. Sci. USA, 96: 4686-4691. 41.) Szego, C. and Davis, J. (1969). Adenosine 3',5'-monophos ⁇ hate in rat uterus : acute elevation by esfrogen. Proc. Natl. Acad. Sci. USA, 58 : 1711-1715.
- ERs Cell membrane and nuclear estrogen receptors (ERs) originate from a single transcript: studies of ER ⁇ and Er ⁇ expressed in Chinese Hamster Ovary cells. Mol. Endocrinol., 13 : 307-319. 62.) Szego, CM. and R.J. Piefras (1981). Membrane recognition and effector sites in steriod hormone action. In: Biochemical Actions of Hormones, Vol. VIII (G. Litwack, editor), Academic Press, New York, pp.307-464.
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MXPA02011093A (en) | 2000-05-10 | 2004-08-19 | David A Sirbasku | Compositions and methods for the diagnosis, treatment and prevention of steroid hormone responsive cancers. |
EP1490502B1 (en) * | 2001-11-14 | 2013-01-09 | Signe BioPharma Inc. | Screening method for predicting susceptibility to breast cancer |
WO2003082322A1 (en) * | 2002-03-28 | 2003-10-09 | Medvet Science Pty. Ltd. | A method of modulating cellular activity |
SE0401302D0 (en) * | 2004-05-21 | 2004-05-21 | Forskarpatent I Syd Ab | Inhibition of recognized phosphorylation |
FR2873699B1 (en) * | 2004-07-29 | 2009-08-21 | Pierre Fabre Medicament Sa | NOVEL ANTI-IGF ANTIBODIES IR RT USES THEREOF |
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US8900588B2 (en) | 2010-01-08 | 2014-12-02 | Les Laboratories Servier | Methods for treating breast cancer |
US20220008636A1 (en) * | 2020-07-12 | 2022-01-13 | Henry J. Smith | Apheresis to remove interfering substances |
CN115369092A (en) * | 2022-09-30 | 2022-11-22 | 北京大学 | Quantitative screening method of estrogen receptor antagonist based on cell impedance sensing |
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Title |
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ABBONDANZA CIRO ET AL: "Characterization and epitope mapping of a new panel of monoclonal antibodies to estradiol receptor" STEROIDS, vol. 58, no. 1, 1993, pages 4-12, XP001183746 ISSN: 0039-128X * |
KUNISUE H ET AL: "ANTI-HER2 ANTIBODY ENHANCES THE GROWTH INHIBITORY EFFECT OF ANTI-OESTROGEN ON BREAST CANCER CELLS EXPRESSING BOTH OESTROGEN RECEPTORS AND HER2" BRITISH JOURNAL OF CANCER, LONDON, GB, vol. 82, no. 1, January 2000 (2000-01), pages 46-51, XP001008510 ISSN: 0007-0920 * |
MOREY ANJALI K ET AL: "Estrogen and progesterone inhibit vascular smooth muscle proliferation" ENDOCRINOLOGY, vol. 138, no. 8, 1997, pages 3330-3339, XP002304840 ISSN: 0013-7227 * |
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SOMJEN DALIA ET AL: "Nongenomic effects of an anti-idiotypic antibody as an estrogen mimetic in female human and rat osteoblasts" JOURNAL OF CELLULAR BIOCHEMISTRY, vol. 65, no. 1, 1997, pages 53-66, XP002304841 ISSN: 0730-2312 * |
WITTERS LOIS M ET AL: "Enhanced anti-proliferative activity of the combination of tamoxifen plus HER-2-neu antibody" BREAST CANCER RESEARCH AND TREATMENT, vol. 42, no. 1, 1997, pages 1-5, XP002304672 ISSN: 0167-6806 * |
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