WO2012050892A2

WO2012050892A2 - Methods for stimulating, increasing or enhancing killing of a cell that expresses luteinizing hormone releasing hormone (lhrh) receptors

Info

Publication number: WO2012050892A2
Application number: PCT/US2011/053684
Authority: WO
Inventors: Hector Alila; Rajasree Solipuram; Sita Aggarwal; William Hansel
Original assignee: Esperance Pharmaceuticals, Inc.; Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College
Priority date: 2010-09-29
Filing date: 2011-09-28
Publication date: 2012-04-19
Also published as: WO2012050892A3

Abstract

The invention relates to compositions and methods for stimulating, increasing, or enhancing luteinizing hormone-releasing hormone (LHRH) receptor (LHRH-R) expression on cells with a first agent, to target cells for arrest, inhibition, reduction or decrease of cell growth, survival, or proliferation, or increased, stimulated, induced or enhanced cell killing, lysis or apoptosis with a second agent that binds to LHRH-R

Description

METHODS FOR STIMULATING, INCREASING OR ENHANCING KILLING OF A CELL THAT EXPRESSES LUTEINIZING HORMONE RELEASING HORMONE (LHRH)

RECEPTORS

Related Application

[0001] This application claims the benefit of priority of application serial no. 61/387,904 filed

September 29, 2010, which is expressly incorporated herein by reference in its entirety.

Technical Field

[0002] The invention relates to stimulating, increasing, or enhancing luteinizing hormone-releasing hormone (LHRH) receptor (LHRH-R) expression on cells with a first agent, to target cells for lolling with a second agent that binds to such receptors (e.g., LHRH-R). Such second agents include LHRH-R ligands and binding moieties, for example, LHRH, LHRH, fragments, LHRH analogues, and antibodies and subsequences that bind to LHRH-R. LHRH-R expressing cells, such as undesirable or aberrant cells, including proliferative or hyperproliferative cells and cellular disorders, including proliferative disorders such as tumors, cancers, neoplasia and metastases and malignancies can be treated.

Introduction

[0003] The need to develop new therapeutics for treatment of primary tumors and metastases is clearly evident when the five year survival rate of cancer patients is considered: Only 10-40 % for patients with lung, colorectal, breast, pancreatic, ovarian, endometrial and prostate cancer survive if diagnosed with distant metastatic disease.

[0004] Conventional cancer chemotherapy lacks specificity resulting in general toxicity of both normal and cancer cells. Targeted cancer cell therapies based on unique molecules expressed by cancer cells provide safer approaches that do not harm normal cells.

Summary

[0005] The invention is based, at least in part on agents that stimulate, induce, increase or enhance expression of luteinizing hormone releasing hormone (LHRH) receptors (LHRH-R), or a receptor that binds to LHRH, and agents that bind to (i.e., target) such as LHRH-R expressing cells. Such agents, referred to herein for convenience as "first" agents can be used to stimulate, induce, increase or enhance cell expression of a receptor that binds to LHRH (e.g., LHRH-R) thereby increasing the tolling of such receptor expressing cells by second agents that bind to the receptors. By increasing expression of receptors, such as LHRH-R, on such cells, the cells are at increased susceptibility to cytotoxicity (e.g., killing) by the second agent. For example, contact of a cell with stimulated, induced, increased or enhanced expression of a receptor that binds to LHRH (e.g., LHRH-R) with a second agent (e.g., an LHRH-R binding moiety or ligand) that binds to the receptor, causes inhibition or reduction of cell growth, proliferation or survival, and can additionally lead to apoptosis, killing or death of the receptor (e.g., LHRH-R) expressing cell.

[0006] Thus, in accordance with the invention there are provided methods of stimulating, inducing, increasing and enhancing cell cytotoxicity (e.g., cell killing, lysis or apoptosis). In one embodiment, a method includes contacting a cell in vitro, ex vivo or in vivo with a first agent that stimulates, induces, increases or enhances expression of luteinizing hormone releasing hormone (LHRH) receptors (LHRH- R), or a receptor that binds to LHRH, and contacting such cells with a second agent that binds to the receptor in an amount sufficient to decrease, reduce or inhibit growth, proliferation or survival of the cell.

[0007] Such methods can be used for treatment of undesirable or aberrant cell proliferation or hyperproliferative disorders of such LHRH-R expressing cells, including proliferative disorders, such as tumors, cancers, neoplasia and metastases and malignancies. In accordance with the invention, there are also provided methods of decreasing, reducing or inhibiting proliferation of a hyperproliferative cell in vitro, ex vivo and in vivo that expresses a receptor that binds to luteinizing hormone-releasing hormone (LHRH) or an LHRH fragment or analogue. In one embodiment, a hyperproliferative cell is contacted with a first agent to stimulate, induce, increase or enhance cell expression of LHRH-R and contacted with a second agent binds to LHRH-R in an amount sufficient to decrease, reduce or inhibit proliferation of the hyperproliferative cell.

[0008] There are further provided methods of reducing or inhibiting proliferation of a neoplastic, tumor, cancer or malignant cell that expresses a receptor that binds to LHRH, LHRH fragment or LHRH analogue. In one embodiment, a cell is contacted with a first agent to stimulate, induce, increase or enhance cell expression of LHRH-R and contacted with a second agent that binds to LHRH-R in an amount sufficient to decrease, reduce or inhibit proliferation of the neoplastic, tumor, cancer or malignant cell.

[0009] There are moreover provided methods of treating a subject having or are at risk of a

hyperproliferative disorder. Methods include arresting, decreasing, reducing or inhibiting undesirable or aberrant cell proliferation or a cellular proliferative disorder in a subject in need of treatment, wherein at least a portion of the undesirable or aberrant proliferating cells or cells of said cellular proliferative disorder express a receptor that binds to LHRH, LHRH fragment or LHRH analogue. In one embodiment, a method includes administering to a subject a first agent to stimulate, induce, increase or enhance cell expression of LHRH-R, and a second agent that binds to the LHRH-R in an amount sufficient to arrest, decrease, reduce or inhibit the undesirable or aberrant cell proliferation or cellular proliferative disorder.

[0010] A number of non-metastatic and metastatic neoplastic, cancer, tumor and malignant cells express or over express a receptor that binds to LHRH, LHRH fragments or LHRH analogues. For example, many non-metastatic and metastatic neoplasias, cancers, tumors and malignancies express receptors that bind to LHRH, LHRH fragments or LHRH analogues, including, for example, luteinizing hormone- releasing hormone I (aka, gonadotropin-releasing hormone I), luteinizing hormone-releasing hormone Π (aka, gonadotropin-releasing hormone Π) or lamprey eel luteinizing hormone-releasing hormone (luteinizing hormone-releasing hormone HI, aka, gonadotropin-releasing hormone III) and fragments and analogs thereof. Such non-metastatic and metastatic neoplastic, cancer, tumor and malignant cells can be targeted for treatment.

[0011 j In accordance with the invention, methods of treating a subject having or at risk of having a neoplasia, tumor, cancer or malignancy (metastatic, non-metastatic or benign) are provided. Methods include treating a neoplasia, tumor, cancer or malignancy in a subject in need of treatment, wherein at least a portion of the cells of said neoplasia, tumor, cancer or malignancy express a receptor that binds to LHRH, an LHRH fragment or an LHRH analogue. In one embodiment, a method includes adniinistering to a subject a first agent to stimulate, induce, increase or enhance cell expression of LHRH-R, and a second agent that binds to the LHRH receptor in an amount sufficient to treat the neoplasia, tumor, cancer or malignancy.

[0012] Still further provided are methods of decreasing, reducing or inhibiting metastasis of a neoplasia, tumor, cancer or malignancy to other sites, or formation or estabhshment of metastatic neoplasia, tumor, cancer or malignancy at other sites distal from a primary neoplasia, tumor, cancer or malignancy in a subject, wherein at least portion of the cells of said neoplasia, tumor, cancer or malignancy express a receptor that binds to LHRH, an LHRH fragment or an LHRH analogue. In one embodiment, a method includes admirustering to a subject a first agent to stimulate, induce, increase or enhance cell expression of LHRH-R, and a second agent that binds to the LHRH receptor in an amount sufficient to decrease, reduce or inhibit metastasis of the neoplasia, tumor, cancer or maHgnancy to other sites, or decrease, reduce or inhibit formation or estabhshment of metastatic neoplasia, tumor, cancer or maHgnancy at other sites distal from the primary neoplasia, tumor, cancer or malignancy.

[00131 First and second agents according to the invention include ligands and binding moieties such as hormones, fragments and analogues of hormones. First and second agents according to the invention also include peptides, polypeptides, proteins, steroids, nucleic acids and carbohydrates. Such peptides, polypeptides and proteins (amino acid sequences), include or consist of amino acid sequences of about 1 to 10, 10 to 20, 15 to 20, 20 to 30, 30 to 40, 40 to 50, 60 to 70, 70 to 80, 80 to 90, 90 to 100 or more amino acids. Such peptides, polypeptides and proteins (amino acid sequences) also include or consist of one or more L- or D-amino acids. D-amino acids can be 1 to 10, 10 to 20, 15 to 20, 20 to 30, 30 to 40, 40 to 50, 60 to 70, 70 to 80, 80 to 90, 90 to 100 or more residues, and can be at any particular residue.

[0014] Non-limiting exemplary first agents according to the invention include follicle-stimulating hormone (FSH) or an FSH analogue, epidermal growth factor (EGF) or an EGF analogue, estradiol or an estradiol analogue or mimetic such as diethylestradiol, estrone, estriol, genistein or phytoestrogen; or testosterone or a testosterone analogue such as dihydrotestosterone, methyltestosterone or fluoxymesterone, among others.

[0015] Second agents according to the invention bind to LHRH-R, and are also referred to herein as binding ligands or moieties, e.g., LHRH-R ligands or LHRH-R binding moieties. Second agents that bind to LHRH-R are cytotoxic, that is they can arrest, reduce, inhibit or decrease cell survival, growth or proliferation, or possess an ability to lyse cells, kill cells or stimulate or induce cell apoptosis. Binding of a second agent to cell surface expressed receptor that binds to LHRH, LHRH fragment or LHRH analogue (e.g., LHRH-R) causes arrest, reduction, inhibition or decreased cell growth or proliferation, and can additionally lead to apoptosis, lysis, killing or death of such receptor expressing cell.

[0016] Second agents according to the invention can include, but are not required to include, a toxic moiety or entity, such as a chemotherapeutic drug, radionuclide, toxin (e.g., bacterial toxin), pore forming peptide, or cell cytotoxic or lytic moiety. Examples of second agents that can include, but are not required to include a toxic moiety or entity, as such second agents inherently can be toxic towards cells, are antibodies and subsequences thereof that bind to LHRH-R. Examples of second agents that typically include a toxic moiety or entitiy include hormones, fragments and analogues thereof that bind to LHRH- R, which such agents can include one or more of a toxic moiety or entitiy, such as a chemotherapeutic drug, radionuclide, toxin (e.g., bacterial toxin), pore forming peptide, or cell cytotoxic or lytic moiety.

[0017] Typically, LHRH-R expressed by cells has an extracellular region to which the second agents can bind. Non-limiting LHRH-R regions include an ammo-terminal 1 to 29 amino acid sequence, or ammo-terminal 1 to 50 amino acid sequence of LHRH receptor.

[0018] Second agents that bind to LHRH-R can but need not, compete for and therefore inhibit reduce, decrease or block binding of LHRH, an LHRH (binding) fragment, or an LHRH analogue to LHRH-R. Second agents that bind to LHRH-R can also but need not, compete for and therefore inhibit, reduce, decrease or block binding of an antibody, or subsequence thereof, to LHRH-R. Such second agents can also include an LHRH receptor agonist or antagonist, i.e. a ligand or binding moiety that inhibits or stimulates an activity of LHRH-R.

[0019] Second agents according to the invention therefore include LHRH receptor ligands and binding moieties, or in the case of an LHRH receptor ligand that comprises a protein or polypeptide, subsequences thereof. Non-limiting examples of ligands and binding moieties include a hormone, a hormone analogue, a fragment of a hormone or a hormone analogue that binds to an LHRH receptor. Non-limiting examples of hormone analogues that bind to LHRH-R include mifepristone, flutaminde, lupron, zoladex, supprelin, synatel triptorelin, buserelin, centrorelix, ganirelix, abarelix, antide, teverelix and degarelix (Fe200486) (see, e.g., Table 1).

[0020J Second agents further include antibodies and subsequences thereof that bind to a receptor that binds to LHRH, or LHRH fragments or analogues. The antibody or binding subsequence thereof targets such cells. Antibodies and subsequences thereof include polyclonal and monoclonal antibodies. Antibodies and subsequences thereof include human, humanized and primatized, as well as chimeric forms. Antibodies and subsequences thereof include IgG, IgA, IgM, or IgD. Exemplary non-limiting examples of subsequences include Fab, Fab', F(ab')₂, Fv, Fd, single-chain Fv (scFv), disulfide-linked Fvs (sdFv), V_L, V_H, Camel Ig, V-NAR, VHH, trispecific (Fab₃), bispecific (Fal¾), diabody ((V_L-V_H)2 or (V_H- VL)₂), triabody (trivalent), tetrabody (tetravalent), minibody ((SCF_V-CH3)₂), bispecific single-chain Fv (Bis-scFv), IgGdeltaCH2, scFv-Fc, (scFv^-Fc, affibody, aptamer, avimer and nanobody.

[0021] As set forth herein, invention second agents can include, or be fused, linked or conjugated to, but are not required to include or be fused, linked or conjugated to, a toxic moiety or entity, such as a chemotherapeutic drug, radionuclide, toxin (e.g., bacterial toxins), pore forming peptide, or cell cytotoxic or lytic moiety. Thus, LHRH receptor ligands, binding moieties, including hormones, subsequences and analogues thereof, and antibodies and subsequences thereof, can be fused, linked or conjugated to a toxic moiety or entity, but need not be fused, linked or conjugated to a toxic moiety or entity.

[0022] Exemplary cell cytotoxic and lytic moieties include cecropins, defensins, melittins, sarcotoxins, magainin peptides, and subsequences, analogues and hybrids (chimera) thereof. Exemplary cell cytotoxic and lytic moieties also include polypeptide, peptide and protein sequences that form an alpha helix, an amphipathic alpha helical structure, or is cationic Exemplary cell cytotoxic and lytic moieties further include polypeptide, peptide and protein sequences that form an amphipathic alpha helical structure of at least 30% of the length of the polypeptide, peptide or protein. Such cell cytotoxic and lytic domains that include or consist of a polypeptide, peptide or protein have about 1 to 10, 10 to 20, 15 to 20 (i.e., 15, 16, 17, 18, 19 or 20 amino acids), 20 to 30, 30 to 40, 40 to 50, 60 to 70, 70 to 80, 80 to 90, 90 to 100 or more amino acids.

[0023] Non-limiting additional examples of a cell cytotoxic or lytic domain also include a peptide, such as a sequence (e.g., a sequence that includes or consists of a 10 to 100 amino acid sequence) that also includes or consists of an amino acid sequence selected from KFAKFAKKFAKFAK (Phorl4), KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKFA,

KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKFAKKFAKF, KFAKFAKKFAKFAKKFAKFA and KFA1 PAKXFAKFAKKFAKFAK (Phor21), or a subsequence thereof; or an amino acid sequence selected from KFAKFAKKFAKFAK (Phorl4), KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKFA, KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKFAKKFAKF, KFAKFAKKFAKFAKKFAKFA and KFAKFAKKFAKFAKKFAKFAK (Phor21) having one or more of the K residues substituted with any of an F or L residue, one or more of the F residues substituted with any of a K, A or L residue, or one or more of the A residues substituted with any of a K, F or L residue, or a subsequence thereof.

[0024] LHRH receptor ligands and binding moieties, and subsequences and analogues thereof according to the invention, optionally fused, linked or conjugated to a toxic moiety or entity, such as a cell cytotoxic or lytic moiety, can have little or no detectable hemolytic activity against human red blood cells, in vitro or in vivo. In particular embodiments, an LHRH receptor ligand or binding moiety, or a subsequence or analogue thereof, including a ligand or binding moiety fused, linked or conjugated to a toxic moiety or entity, such as a cell cytotoxic or lytic moiety, has a hemolytic activity of more than about 500 μΜ, or more than about 250 uM, or more than about 100 μΜ, or more than about 75 μΜ, or more than about 50 μΜ, or more than about 25 μΜ, or more than about 10 μΜ, or more than about 5 μΜ, against human red blood cells, in vitro or in vivo.

[0025] Second agents that include, or are fused, linked or conjugated to a toxic moiety or entity (e.g., a chemotherapeutic drug, radionuclide, toxin (e.g., bacterial toxin), pore forming peptide, or cell cytotoxic or lytic moiety) can be joined by a covalent bond, or a peptide (e.g., one or more A, S or G amino acid residues, or from 1 to 50 L- or D-amino acid residues in length) or non-peptide linker (e.g., a linear carbon chain). When joined to a polypeptide, peptide or protein sequence, such as a hormone or antibody, or subsequence or analogue thereof fused, linked or conjugated to a toxic moiety or entity, they can be positioned at either the NH₂-terminus or the C-terminus relative to each other. Thus, conjugation or linkage can occur at the NHo-terminus or at the C-tenninus.

[0026] As set forth herein, cells that express receptors that bind to LHRH, LHRH fragments and LHRH analogues (e.g., LHRH-R) targeted in accordance with the invention include undesirably or aberrantly proliferating cells, such as hyperprohferating and hyperproliferative cells (e.g., cells of hype rohferative disorders). Non-limiting examples of such cells include, for example, benign hyperplastic cells, and non-metastatic and metastatic neoplastic, tumor, and cancer cells. Non-limiting examples of benign hyperplastic cells include benign prostate hyperplasia, endometrial hyperplasia, breast lobular or ductal hyperplasia, endometriosis or uterine fibroid. Non-limiting examples of metastatic and metastatic cancers, tumors and malignancies include a solid or liquid tumor, myeloma, lymphoma, leukemia, carcinoma, sarcoma, melanoma, adenoma, adenocarcinoma, neural, glioma, glioblastoma, meningioma, neuroblastoma, retinoblastoma, astrocytoma, oligodendrocytoma, mesothelioma, reticuloendothelial, lymphatic or haematopoietic neoplasia, tumor, cancer or malignancy.

[0027] Cells that express receptors that bind to LHRH, LHRH fragments and LHRH analogues (e.g., LHRH-R) that can be targeted in accordance with the invention also include non-proliferating or dormant cancer cells. Methods of the invention are therefore directed to proliferating (dividing) cells, such as normal, undesirably or aberrantly proliferating cells, or non-proliferating (non-dividing) or dormant cancer cells. The cells targeted according to the invention can be drug resistant cells, such as multi-drug resistant cells.

[0028] Cells that express receptors that bind to LHRH, LHRH fragments and LHRH analogues (e.g., LHRH-R) that can be targeted in accordance with the invention can be present in or affect a lung, head or neck, nasopharynx, throat, nose or sinuses, brain, spine, breast, adrenal gland, pituitary gland, thyroid, lymph, gastrointestinal (mouth, esophagus, stomach, duodenum, ileum, jejunum (small intestine), colon, rectum), genito-urinary tract (uterus, ovary, cervix, endometrial, bladder, testicle, penis, prostate), kidney, pancreas, liver, bone, bone marrow, lymph, blood, muscle, skin or stem cell. Particular non-limiting examples of cells that express receptors that bind to LHRH, LHRH fragments and LHRH analogues (e.g., LHRH-R) that can be targeted in accordance with the invention include breast, ovarian, uterine, cervical, prostate, testicular, pancreatic, skin, blood cells, adrenal gland, pituitary, thyroid and endometrial cells.

[0029] Target cells may be within a cell mass or plurality of other cells that do not detectably express receptors that bind to LHRH, LHRH fragments and LHRH analogues (e.g., LHRH-R). Such target cells may also be within a cell mass or plurality of other cells that express less LHRH-R than the target LHRH-R expressing cells, and are therefore less susceptible to an activity of a second agent, such as anti- cell survival, growth, proliferative (e.g., killing, lysis, apoptosis) or anti-metastatic activity. Such cells may include cells that express a low amount of LHRH-R but do not exhibit significant or detectable increased expression of LHRH-R when treated with a first agent. Nevertheless, invention methods are applicable to mixed populations of cells, in vitro, ex vivo and in vivo, that contain LHRH-R expressing cells, non-LHRH-R expressing cells and cells that express less LHRH-R than target LHRH-R expressing cells which may or may not be less susceptible to an arrest, decrease, inhibition or reduction of cell survival, growth, proliferation, or increased, stimulated, enhanced or induced lolling, lysis, apoptosis, etc.).

[0030] Methods performed include, among others, contacting, administering or treating a subject in need of arresting, decreasing, inhibiting, reducing or preventing growth, proliferation, survival, differentiation, or activity of a cell, such as a proliferating cell, for example, an undesirably or aberrantly proliferating cell (e.g., a hyperproliferative cell). Exemplary subjects include a subject having or at risk of having undesirable or aberrant cell proliferation; a subject having or at risk of having a benign hyperplasia; or a non-metastatic or metastatic neoplasia, cancer, tumor or malignancy (e.g., a solid or liquid tumor, myeloma, lymphoma, leukemia, carcinoma, sarcoma, melanoma, neural, reticuloendothelial and haematopoietic neoplasia).

[0031] As set forth herein, methods include treating a subject having or at risk of having a metastasis. A method of the invention includes reducing or inhibiting growth, survival, proliferation, mobility or invasiveness of tumor or cancer cells that potentially or do develop metastases (e.g., disseminated tumor cells); reducing or inhibiting formation or establishment of metastases arising from a primary tumor or cancer to one or more other sites, locations or regions distinct from the primary tumor or cancer; reducing or inhibiting growth or proliferation of a metastasis at one or more other sites, locations or regions distinct from the primary tumor or cancer after the metastasis has formed or has been established; or reducing or inhibiting formation or establishment of additional metastasis after the metastasis has been formed or established. [0032] Neoplasia, tumor, cancer and malignancy treatable in accordance with the invention include solid cellular mass, hematopoietic cells, or a carcinoma, sarcoma (e.g. lymphosarcoma, liposarcoma, osteosarcoma, chondrosarcoma, leiomyosarcoma, rhabdomyosarcoma or fibrosarcoma), lymphoma, leukemia, adenoma, adenocarcinoma, melanoma, glioma, glioblastoma, meningioma, neuroblastoma, retinoblastoma, astrocytoma, oUgodendrocytoma, mesothehoma, reticuloendothelial, lymphatic or haematopoietic (e.g., myeloma, lymphoma or leukemia) neoplasia, tumor, cancer or malignancy.

[0033] Neoplasia, tumor, cancer and malignancy treatable in accordance with the invention can be present in or affect a lung (small cell lung or non-small cell lung cancer), head or neck, nasopharynx, throat, nose or sinuses, brain, spine, breast, adrenal gland, pituitary gland, thyroid, lymph, gastrointestinal (mouth, esophagus, stomach, duodenum, ileum, jejunum (small intestine), colon, rectum), gemto-urinary tract (uterus, ovary, cervix, endometrial, bladder, testicle, penis, prostate), kidney, pancreas, liver, bone, bone marrow, lymph, blood, muscle, skin or stem cell neoplasia, tumor, cancer, or malignancy.

[0034] Methods may be practiced with other treatments or therapies (e.g., surgical resection, radiotherapy, ionizing or chemical radiation therapy, chemotherapy, immunotherapy, local or regional thermal (hyperthermia) therapy, or vaccination). Such treatments or therapies can be administered prior to, substantially contemporaneously with (separately or in a mixture), or following administration of a first or second agent, or mixture thereof.

[0035] First and second agents can be delivered or administered separately, e.g., the first agent prior to, during or after the second agent. Typically, a first agent will be delivered or administered prior to a second agent. A period of time, for example, 1-6, 6-12, 12-24, 24-48, 48-72 minutes, hours, days, weeks or months can intervene between one administration and a subsequent adrninistration. First and second agents can also be delivered or administered as a mixture. Such first and second agents, and mixtures thereof, can include compositions, such as a pharmaceutically acceptable carrier or excipient appropriate for delivery or aciministration to or in vivo contact with a subject, or a mixture of a first and/or second agent with an anti-cell proliferative or immune stimulating agent, for example.

[0036] First or second agents, and mixtures thereof, can be administered in a unit dosage form. In one embodiment, a first and/or second agent are/is a unit dosage in an amount effective to treat a subject having or at risk of undesirable cell proliferation or a hyperproliferative disorder.

[0037] Methods of the invention include providing a subject with a benefit. In particular

embodiments, a method of treatment results in partial or complete destruction of the neoplastic, tumor, cancer or malignant cell mass, volume, size or numbers of cells, stimulating, inducing or increasing neoplastic, tumor, cancer or malignant cell necrosis, lysis or apoptosis, reducing neoplasia, tumor, cancer or malignancy volume size, cell mass, inhibiting or preventing progression or an increase in neoplasia, tumor, cancer or malignancy volume, mass, size or cell numbers, or prolonging lifespan; results in reducing or decreasing severity, duration or frequency of an adverse symptom or complication associated with or caused by the neoplasia, tumor, cancer or malignancy, results in reducing or decreasing pain, discomfort, nausea, weakness or lethargy; or results in increased energy, appetite, improved mobility or psychological well being.

Description of Drawings

[0038] Figure 1 shows reduced tumor weight changes from baseline. MIAPaCa2 pancreatic cancer cell xenografts were removed from the nude mice and weighed at necropsy. Tumor weights of baseline (untreated mice) compared with those treated with vehicle, LHRH + Phorl8, EP-100 (0.2 mg/kg and 2 mg/kg, n=7). EP-100 significantly (*P<0.05) reduced tumor weights compared to vehicle treated controls to levels not different from the baseline values (P>0.05).

[0039] Figure 2 shows LHRH receptor gene expression in pancreatic cancer cell lines. mRNA (2 μg) isolated from cancer cells was subjected to real time RT-PCR. The graph represents relative quantitation of LHRH receptor gene expression in pancreatic cancer cell lines, MIAPaCa2, BxPC-3 and PANC-1, compared to prostate cancer cell line, PC-3 (positive control). MIAPaCa2 and BxPC-3 cells show >6- fold higher expression of LHRH receptor gene (*P<0.05) than PC-3 cells. ¾A C-1 cells show 2-fold higher expression than PC-3 cells, which is least among the pancreatic cancer cell types. The values are means±SEM.

[0040] Figure 3 shows that FSH increases LHRH receptor gene expression in PANC- 1 pancreatic cancer cells. PANC-1 cells were treated with FSH (0, 10 and 30 ng ml ) in steroid free medium with 5% charcoal treated FBS for 24 h. 2ug of mRNA isolated from cancer cells was subjected to real time RT- PCR. The graph represents relative quantitation of LHRH receptor gene expression in PANC-1 cells compared to prostate cancer cell line, PC-3. PANC-1 cells treated with 10 and 30 ng/ml FSH show 3-fold higher expression of LHRH receptor gene compared to PANC-1 cells without FSH. The values are meartfcSEM.

[0041] Figure 4 shows IC₅₀ of EP-100 performed with IAPaCa2, PANC-1 and BxPC-3 cells treated with EP-100. Cell viability assays were performed with MIAPaCa2, PANC-1 and BxPC-3 cells (5,000 cells/well) treated with EP-100 (0-200 μΜ) for 48 h. Cell viability (% Control) was assessed by the MTT assay and was plotted against the EP-100 concentration. The values are means ± SD of triplicate samples. The IC₅₀ values of EP-100 for MIAPaCa2, PANC-1 and BxPC-3 are 6.47±0.34 μΜ, 5.42±1.34 μΜ and 4.08±0.76 μΜ (**P<0.01 MIAPaCa2 vs BxPC-3).

[0042] Figures 5A-5B show that pre-treatment of PANC-1 and BxPC-3 cells with FSH increases cytotoxicity of EP-100. Cells (5,000/well) were incubated in DMEM supplemented with 0.2% dextran charcoal treated FBS for 24 h and pre-treated with FSH (10 or 30 ng/mL) alone for 24 h followed by EP 100 alone (4 or 6 μΜ) or EP-100 (4 or 6 μΜ) plus FSH (10 or 30 ng/mL) for another 24 h. Vehicle treated cells served as control. Cell viability was assessed; percent (%) cytotoxicity was calculated and plotted. The values are means ± SD of triplicate samples. Pre-treatment of PANC-1 and BxPC-3 cells with FSH at both dose levels increased the cytotoxicity of EP-100 (*P<0.05, **P<0.01, ***P<0.001 and ^"^FO.OOl)

[0043] Figure 6 shows that pre-treatment of MIAPaCa2, PANC-l and BxPC-3 cells with LHRH decreases cytotoxicity of EP-100. Cells (5,000 cells/well) were treated with LHRH (10 μΜ) and EP-100 (10 μΜ) for 48 h. Viability was assessed by the MTT assay. The values are means ± SEM of triplicate samples. Pre-incubation of all cell lines with LHRH decreased cytotoxicity of EP-100 (***,

Λ^ΛΛΡ<0.001 vs EP-100 alone).

[0044] Figures 7A-7B show tumor volumes during A) treatment; and B) at necropsy. EP- 100 at the 0.02 and 0.2 mg/kg levels, with or without pre-treatment with FSH, significantly regressed the tumor volumes. ***P<0.001 vs vehicle, ⁺⁺⁺P<0.001 vs baseline,^†††P<0.001 0.02 mg kg vs 0.2 mg/kg and ^ΛΛΡ<0.01 0.02 mg/kg vs 0.2 mg/kg pre-treated with FSH.

[0045] Figures 8A-8B show that EP-100 decreases tumor weight of PANC-l xenografts in nude mice.

A) EP-100 at 0.02 and 0.2 mg/kg, with or without FSH pre-treatment, significantly decreased tumor weights. (**P<0.01, ***P<0.001 vs vehicle, ^AP<0.05 0.2 mg/kg vs 0.2 mg/kg pre-treated with FSH); and

B) Pre-treatment with FSH significantly decreased the tumor weights from baseline in EP-100 treated nude mice. (⁺P<0.05, ⁺⁺⁺P<0.001 vs baseline). FSH pretreatment of mice receiving EP-100 at 0.2 mg kg further decreased the tumor weights compared to EP-100 alone at 0.2 mg/kg (^ΛΡ <0.05)

Detailed Description

[0046] The invention relates to agents that stimulate, induce, increase or enhance expression of luteinizing hormone releasing hormone (LHRH) receptors (LHRH-R), or a receptor that binds to LHRH, LHRH fragments or LHRH analogues, and agents that bind to (i.e., target) such receptor (e.g., LHRH-R) expressing cells for arresting, decreasing, reducing or inWbiting growth, survival or proliferation of such receptor (e.g., LHRH-R) expressing cells. Increasing expression of such receptor (e.g., LHRH-R) on cells increases, enhances, induces or promotes susceptibiUty to cytotoxicity (e.g., killing) by a second agent (e.g., ligand or binding moiety). Contact of a cell with stimulated, induced, increased or enhanced expression of a receptor that binds to LHRH with a second agent (e.g., binding moiety or ligand) that binds to the receptor that binds to LHRH, causes arrest, decrease, inhibition or reduction of cell growth, survival or proliferation, and can additionally lead to killing, lysis, apoptosis, or death of such receptor (e.g., LHRH-R) expressing cells. Thus, in accordance with the invention there are provided methods of stimulating, inducing, increasing and enhancing cell cytotoxicity (e.g., cell Idlling) towards cells that are capable of expressing a receptor that binds to LHRH, LHRH fragments or LHRH analogues (e.g., LHRH-R), i.e., that can be induced to express LHRH-R, cells that express LHRH-R and cells that express greater amounts of a receptor that binds to LHRH, LHRH fragment or LHRH analogue (e.g., LHRH-R) in response to a first agent. [0047] First agents according to the invention stimulate, induce, increase or enhance cellular LHRH-R expression, half-life or stability of LHRH-R RNA or protein, or the amount of cell surface LHRH-R. Non-limiting exemplary first agents include folhcle-stimulating hormone (FSH) or an FSH analogue, epidermal growth factor (EGF) or an EGF analogue, estradiol or an estradiol analogue, or testosterone or a testosterone analogue.

[0048] Second agents according to the invention, also referred to herein as binding moieties or ligands, bind to LHRH-R. Second agents that bind to LHRH-R are cytotoxic, that is they can arrest, reduce, inhibit or decrease cell survival, growth or proliferation, or possess an ability to lyse cells, kill cells or increase, stimulate or induce cell apoptosis. Thus, a second agent can arrest, reduce, inhibit or decrease cell survival, growth or proliferation, or can lyse cells, kill cells or increase, enhance, stimulate or induce cell apoptosis.

[0049] In order to be cytotoxic towards cells, second agents according to the invention can include, but are not required to include, a toxic moiety or entity, such as a chemotherapeutic drug, radionuclide, toxin (e.g., bacterial toxin), pore forming or amphpathic peptide, or cell cytotoxic or lytic moiety. Examples of second agents that can include, but are not required to include a toxic moiety or entity, as the second agent can inherently be toxic towards cells, are antibodies and subsequences thereof that bind to LHRH- R. Examples of second agents that typically include a toxic moiety or entity include hormones, fragments and analogues thereof that bind to LHRH-R.

[0050] Second agents according to the invention therefore include those linked, fused or conjugated to a toxic moiety or entity, such as hormones, fragments and analogues of hormones linked, fused or conjugated to a toxic moiety or entity. Second agents that bind to LHRH-R can be agonists or antagonists. Second agents according to the invention can also but need not, compete for and therefore inhibit, reduce, decrease or block binding of a hormone, antibody, or fragment or analogue thereof, to LHRH-R.

[0051] Receptors that can be targeted include receptors that bind to luteinizing hormone-releasing hormone (LHRH), LHRH (binding) fragments, or LHRH analogues. Non-limiting specific examples of such receptors bind to luteinizing hormone-releasing hormone I (aka, gonadotropin-releasing hormone I), luteinizing hormone-releasing hormone Π (aka, gonadotropin-releasing hormone II) or lamprey eel luteinizing hormone-releasing hormone (luteinizing hormone-releasing hormone ΙΠ, aka, gonadotropin- releasing hormone III).

[0052] A "receptor" that binds to LHRH, LHRH (binding) fragments, or LHRH analogues, is typically present on a surface of a cell (e.g., a membrane receptor) or within a cell. A receptor may associate with the cell membrane surface or traverse the cell membrane. For example, a receptor protein can have a transmembrane domain that traverses the cell membrane, optionally with a portion that is cytoplasmic or extracellular, or both. Receptors therefore include full length intact native receptors, such as LHRH-R, containing an extracellular, transmembrane or cytoplasmic portion, as well as truncated forms or fragments thereof (e.g., an extracellular, transmembrane or cytoplasmic portion or subsequence of the receptor alone, or in combination). For example, a soluble LHRH-R typically lacks a

transmembrane region and may optionally also lack all or a part of the native extracellular or cytoplasmic region (if present in native receptor). Such truncated forms and fragments can retain at least partial binding to a ligand or binding moiety such as a hormone, a hormone fragment, an analogue or an antibody or subsequence thereof. Cells to target therefore include cells that express any receptor to which LHRH, LHRH (binding) fragments, or LHRH analogues bind.

[0053] Second agents that bind such receptors to which LHRH, LHRH (binding) fragments, and

LHRH analogues bind can therefore recognize any "epitope" on the receptors. Particular epitopes are located on the extracellular domain or portion of receptors, such as LHRH-R, that bind to luteinizing hormone-releasing hormone (LHRH), LHRH (binding) fragments, or LHRH analogues. In particular, embodiments second agents bind to the first 1 to 29 or 1 to 50 amino acid residues of LHRH receptor (aka, Gonadotropin-Releasing Hormone Receptor, GnRH Receptor). Such second agents include those that can inhibit, reduce, decrease or block binding of LHRH, an LHRH (binding) fragment, or an LHRH analogue to the receptor. Second agents therefore useful in accordance with the invention include agents that bind to an extracellular portion of LHRH-R (e.g., that bind to the first 1 to 29 or first 1 to 50 amino acid residues of LHRH-R), and agents that can inhibit, reduce, decrease or block binding of LHRH, an LHRH (binding) fragment, or an LHRH analogue to LHRH-R.

[0054] In particular aspects, a second agent specifically binds to a mammalian (e.g., human) LHRH receptor. A representative human LHRH receptor sequence to which a second agent binds includes, but is not limited to, a full length or a subsequence or an amino acid variant of:

1 A SASPEQN QNHCSAINNS IPLMQGNLPT LTLSGKIRVT VTFFLFLLSA TF ASFLL L

61 QKWTQKKEKG KKLSR KLLL KHLTLANLLE TLIVMPLDGM WNITVQ YAG ELLCKVLSYL

121 KLFS YAPAF MMWISLDRS LAITRPLALK SNSKVGQSMV GLAWILSSVF AGPQLYIFRM

181 IHLADSSGQT KVFSQCVTHC SFSQWWHQAF YNFFTFSCLF IIPLFIMLIC NAKIIFTLTR

241 VLHQDPHELQ LNQSK NIPR ARLKTLKMTV AFATSFTVCW TPYYVLGIWY FDPEMLNRL

301 SDPVNHFFFL FAFLNPCFDP LIYGYFSL.

[0055] A specific non-limiting example of an LHRH fragment that binds to LHRH receptor is set forth as: QHWSYGLRPG. Thus, an LHRH fragment, such as QHWSYGLRPG, can comprise a second agent. Additional exemplary LHRH fragments and analogues that bind to LHRH-R, and can therefore comprise second agents, are as set forth in Table 1 below:

Table 1 : Exemplary Amino acid sequence of LHRH isoforms and analogues

LHRH forms Naturally occurring LHRH

1 2 3 4 5 6 7 8 9 10 mLHRH pGlu His Trp Ser Tyr Gly Leu Arg Pro Gly NH₂ (LHRH-I)

cLHRH pGlu His Trp Ser His Gly Trp Tyr Pro Gly NH₂ (LHRH-II)

ILHRH pGlu His Trp Ser His Asp Trp Lys Pro Gly NH₂ (LHRH-III)

rLHRH pGlu His Trp Ser Tyr Gly Leu Trp Pro Gly NH₂ cfLHRH pGlu His Trp Ser His Gly Leu Pro Pro Gly NH₂

LHRH agonists:

Lupron^[ar(TAP) pGlu His Trp Ser Tyr D Leu Leu Arg Pro NEt

Zoladex^[a] pGlu His Trp Ser Tyr D Ser Leu Arg Pro Gly NH₂ (Zeneca) (tBu)

Supprelin^[a) pGlu His Trp Ser Tyr D His Leu Arg Pro Gly NH₂ (Roberts) (ImBzJ)

Synarel^[a] (Searle) pGlu His Trp Ser Tyr D Nal Leu Arg Pro Gly NH₂

Triptorelin^[al pGlu His Trp Ser Tyr D Trp Leu Arg Pro Gly NH₂ (Ferring)

Buserelin^[aI pGlu His Trp Ser Tyr D Ser Leu Arg Pro NEt (Hoechst) (tBu)

LHRH antagonists:

Centroelix^[ar D Nal D Cpa D Pal Ser Tyr D Cit Leu Arg Pro D Ala NH₂ (Asta Medica)

Ganirelix^!al D Nal D Cpa D Pal Ser Tyr D hArg Leu D hArg Pro D Ala NH2 (Organon) (B)₂ (Et)2

Abarelix^M D Nal D Cpa D Pal Ser N Me D Asn Leu Lys Pro D Ala NH2 (Praecis) Tyr (iPr)

Antide^[cl D al D Cpa D Pal Ser Lys D Cit Leu Lys Pro D Ala NH2 (Ares Seronon) (Nic) (iPr)

Teverelix^[d] D Nal D Cpa D Pal Ser Tyr D hCit Leu Lys Pro D Ala NH2 (Ardana) (iPr)

Fe 200486^[cl D Nal D Cpa D Pal Ser Aph D Aph Leu Lys Pro D Ala NH2 (Feeing) (Hor) (Gin) (iPr)

Nal-Glu^[cl (NIH) D al D Cpa D Pal Ser Arg DGIu(AA) Leu Arg Pro D Ala NH2

From Leuschner C. LHRH Conjugated Magnetic Nanoparticles for Diagnosis and Treatment of Cancers, Chapter 6, pp 174-231. Kumar, Challa S. S. R. (ed.) Nanomaterials for Cancer Diagnosis - Nanotechnologies for the Life Sciences (Volume 7), 2007, Wiley- VCH, Weinheim, Germany

[0056] Second agents according to the invention also include antibodies and subsequences thereof that bind to receptors that bind to LHRH, LHRH (binding) fragments, or LHRH analogues, such as LHRH-R. The term "antibody" refers to a protein that binds to other molecules (antigens) via heavy and/or light chain variable domains, V_H and/or V_L, respectively. An "antibody" refers to any monoclonal or

polyclonal immunoglobulin molecule, such as IgG, IgA, IgD, IgM, and any subclass thereof. Exemplary subclasses for IgG are IgGi, IgG₂, IgG₃ and IgG₄. Antibodies include full-length antibodies that include two heavy and two light chain sequences. Antibodies can have kappa or lambda light chain sequences, either full length as in naturally occurring antibodies, mixtures thereof (i.e., fusions of kappa and lambda chain sequences), and subsequences/fragments thereof. Naturally occurring antibody molecules contain two kappa or two lambda light chains.

[0057] Antibodies and subsequences thereof include mammalian, primatized, humanized and fully human antibodies and subsequences thereof. Antibodies and subsequences thereof include those produced or expressed by or on transformed cells or hybridomas, or B cells, or those produced synthetically or by other organisms (plant, insect, bacteria, etc.).

[0058] A "monoclonal" antibody refers to an antibody that is based upon, obtained from or derived from a single clone, including any eukaryotic, prokaryotic, or phage clone. A "monoclonal" antibody is therefore defined structurally, and not the method by which it is produced.

[0059] Antibodies and subsequences thereof can be produced or are available commercially or from other sources. For example, antibodies that bind to receptors that bind to LHRH, LHRH (binding) fragments, or LHRH analogues, such as LHRH-R are commercially available. For example, Ab-3 is a mouse monoclonal IgGl antibody available from Thermo Scientific (Fremont California, Clone

Designation GNRH03), with an epitope aa 1-29 (MAN-SASPEQNQNHCSAINNSIPLMQGNLPY) and binds to human GnRH/LHRH receptor. Additional examples of antibodies are available from Santa

Cruz Biotechnology Inc. (Santa Cruz, California), and include GnRHR (AT2.G7), an IgGi mouse monoclonal antibody raised against a mixture of synthetic peptides from different regions of GnRH receptor of human origin; GnRH receptor (GRX-6) an IgGi mouse monoclonal antibody raised against a synthetic peptide corresponding to amino acids 1-31 of the GnRH/LHRH receptor protein of human origin, and which binds to GnRH/LHRH receptor of human origin; GnRH/LHRH receptor (GRX-5) an

IgGi mouse monoclonal antibody raised against a synthetic peptide corresponding to amino acids 1-31 of

GnRH/LHRH receptor of human origin; GnRH LHRH receptor (GRX-8) an IgGi mouse monoclonal antibody which binds to GnRH/LHRH receptor of human origin. Isotype GnRHR2 (67-R) an IgG_2a mouse monoclonal antibody raised against recombinant protein of GnRHR2 of human origin. Further examples of antibodies are available from antibodies-online GmbH (SchloB-Rahe-Str. 15, 52072 Aachen,

Germany), and include a mouse monoclonal antibody (IgGi) that binds to human gonadotropin-releasing hormone receptor (GnRHR) (Order number ΑΒΓΝ201809) produced with an immunogen peptide corresponding to amino acids 1 to 29 of the human GnRH/LHRH receptor; a mouse monoclonal antibody (IgGi) that binds to human GnRH/LHRH receptor (Order number ΑΒΓΝ210677) produced with a synthetic peptide immunogen corresponding to amino acids 1 to 29 of the human GnRH/LHRH receptor protein conjugated to bovine serum albumin, and which reacts with GnRH receptors in the anterior pituitary; a mouse monoclonal antibody that binds to human GnRH/LHRH receptor (Order number ΑΒΓΝ243903); IgG mouse monoclonal antibodies produced by immunization with a mixture of peptides from different regions of GnRH/LHRH receptor protein and that binds to human GnRH receptor

(Order number ΑΒΓΝ243903, Order number ΑΒΓΝ330374, Clone 8A162, which recognizes specifically

GnRH receptor from human, and Order number ABIN330377, Clone 8L72, which exhibits species cross-reactivity); IgGi mouse monoclonal antibody produced by immunization with a synthetic peptide of amino acids 1-29 (MANSASPEQNQNHCSAINNSIPLMQ- GNLPY) from the N-terminal region of the human GnRH/LHRH receptor protein and that specifically binds to human GnRH/LHRH Receptor (Order number ΑΒΓΝ337150, Clone 3F193); IgG mouse monoclonal antibodies produced by immunization with highly pure human GnRH/LHRH receptor protein and that specifically bind to human GnRH/LHRH receptor (Order numbers ABIN345094 and ABIN345095, Clones M0201127 and M0201128); and IgGi mouse monoclonal antibody produced by immunization with a synthetic peptide amino acids 1 to 29 of human GnRH/LHRH receptor extracellular region and that specifically binds to human GnRH/LHRH receptor extracellular domain (Order number ABIN345539, Clone A9E4).

Antibodies and subsequences therefore include the foregoing specific examples, as well as antibodies and subsequences that compete for binding of any of the antibodies disclosed herein to LHRH/GnRH receptor.

[0060] Additional specific non-limiting antibodies and subsequences thereof have a binding affinity for LHRH-R within about ¾ 10^"2 M to about ¾ 10^~15 M, or within about ¾ 10^"5 M to about ¾ 10^'12 M. In more particular embodiments, an antibody has binding affinity for LHRH-R with a dissociation constant (KD) less than 5xl0^"2 M, 10^"2 M, 5xl0^"3 M, 10^"3 M 5x10"* M, 10 M 5xl0^"5 M, 10^"5 M 5X10^"6 M, 10^"6 M 5xl0^"7 M, 1⁽T⁷ M 5xl0^'8 M, lO^"8 M 5xl0^"9 M, 10^'9 M 5xl0^"10 M, 10^~10 M 5X10^"11 M, 10^"11 M 5xl0^"12 M, 10^"12 M 5xl0^'13 M, 10^"13 M 5xl0^"14 M, 10 ¹⁴ M 5xl0^"15 M, and 10^"15 M.

[0061] Binding affinity can be detennined by association (K_a) and dissociation (¾) rate. Equihbrium affinity constant, KD, is the ratio of Κ Κ^ Association (K_a) and dissociation (K_<j) rates can be measured using surface plasmon resonance (SPR) (Rich and Myszka, Curr. Opin. Biotechnol. 11 :54 (2000);

Englebienne, Analyst. 123: 1599 (1998)). Instrumentation and methods for real time detection and monitoring of binding rates are known and are commercially available (BiaCore 2000, Biacore AB, Upsala, Sweden; and Malmqvist, Biochem. Soc. Trans. 27:335 (1999)). KD values can be defined as the antibody concentration required to saturate one half (50%) of the binding sites on LHRH-R.

[0062] Antibodies also include subsequences that bind to receptors that bind to LHRH, LHRH (binding) fragments, or LHRH analogues, such as LHRH-R. An antibody subsequence (or fragment) refers to a portion of a full length antibody that retains at least partial antigen binding capability of a comparison full length antibody. Antibodies, and antibody subsequences, including single-chain antibodies, can include all or a portion of heavy or light chain variable region sequences (e.g., CDR1, CDR2 or CDR3 in a heavy chain variable region sequence or in a light chain variable region sequence, optionally including flanking framework regions, FRs) alone or in combination with all or a portion of one or more of the following: hinge region, C_HI , C_H2, and C_H3 domains.

[0063[ Non-limiting representative antibody subsequences include but are not limited to Fab, Fab',

F(ab')₂, Fv, Fd, single-chain Fv (scFv), disulfide-linked Fvs (sdFv), V_L, V_H, Camel Ig, V-NAR, VHH, trispecific (Fab₃), bispecific (Fab2), diabody ((V_L-V_H)₂ or (V_H-V_L)₂), triabody (trivalent), tetrabody (tetravalent), minibody ((SCF_V-CH3)2), bispecific single-chain Fv (Bis-scFv), IgGdeltaCH2, scFv-Fc, (scFv)2-Fc, affibody, aptamer, avimer or nanobody, or other antigen binding subsequences of an intact immunoglobulin.

[0064] A mammalian antibody is an antibody which is produced by a mammal, transgenic or non- transgenic, or a non-mammalian organism engineered to produce a mammalian antibody, such as a non- mammalian cell (bacteria, yeast, insect cell), animal or plant.

[0065] A "human" antibody means that the amino acid sequence of the antibody is fully human, i.e., human heavy and human light chain variable and human constant regions. Thus, all of the amino acids are human or exist in a human antibody. An antibody that is non-human may be made fully human by substituting the non-human amino acid residues with amino acid residues that exist in a human antibody. Amino acid residues present in human antibodies, CDR region maps and human antibody consensus residues are known in the art (see, e.g., Kabat, Sequences of Proteins of Immunological Interest, 4^th Ed.US Department of Health and Human Services. Public Health Service (1987); Chothia and Lesk (1987). A consensus sequence of human V_H subgroup III, based on a survey of 22 known human V_H HI sequences, and a consensus sequence of human V_L kappa-chain subgroup I, based on a survey of 30 known human kappa I sequences is described in Padlan Mol. Immunol. 31 : 169 ( 1994); and Padlan Mol. Immunol. 28:489 (1991). Human antibodies therefore include antibodies in which one or more amino acid residue has been substituted with one or more amino acids present in any other human antibody.

[0066] A "humanized" antibody, means that the amino acid sequence of the antibody has non-human amino acid residues (e.g., mouse, rat, goat, rabbit, etc.) of one or more complementarity determining regions (CDRs) that specifically bind to the desired antigen in an acceptor human immunoglobulin molecule, and one or more human amino acid residues in the Fv framework region (FR), which are amino acid residues that flank the CDRs. Such antibodies typically have reduced immunogenicity and therefore a longer half-life in humans as compared to the non-human parent antibody from which one or more CDRs were obtained or are based upon.

[0067] Antibodies include those referred to as "primatized," which are "humanized" except that the acceptor human immunoglobulin molecule and framework region amino acid residues may be any primate amino acid residue (e.g., ape, gibbon, gorilla, chimpanzees orangutan, macaque), in addition to any human residue. Human FR residues of the immunoglobulin can be replaced with corresponding non-human residues. Residues in the CDR or human framework regions can therefore be substituted with a corresponding residue from the non-human CDR or framework region donor antibody to alter, generally to improve, antigen affinity or specificity, for example. A humanized antibody may include residues, which are found neither in the human antibody nor in the donor CDR or framework sequences.

For example, a FR substitution at a particular position that is not found in a human antibody or the donor non-human antibody may be predicted to improve binding affinity or specificity human antibody at that position. Antibody framework and CDR substitutions based upon molecular modeling are well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions (see, e.g., U.S. Patent No. 5,585,089; and Riechmann et al, Nature 332:323 (1988)).

[0068] First and second agents can include or consist of an amino acid sequence. Exemplary lengths of such sequences are from about 5 to 10, 10 to 20, 20 to 25, 25 to 50, 50 to 100, 100 to 150, 150 to 200, or 200 to 300 or more amino acid residues in length. In more particular embodiments, an amino acid sequence includes or consists of about 1 to 10, 10 to 20, 15 to 20 (i.e., 15, 16, 17, 18, 19 or 20 amino acids), 20 to 30, 30 to 40, 40 to 50, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 110, 110 to 120, or more amino acids.

[0069] The term "bind," or "binding," when used in reference to a ligand or binding moiety (e.g., hormone or antibody), means that the ligand or binding moiety interacts at the molecular level with all or a part of a sequence or an antigenic epitope present on a receptor that binds to LHRH, an LHRH

(binding) fragment, or an LHRH analogue. Specific binding is selective for an epitope present in an LHRH receptor. Ligands and binding moieties thereof include specific or selective binding to LHRH receptor or an LHRH receptor subunit. Specific and selective binding can be distinguished from nonspecific binding using assays known in the art (e.g., immunoprecipitation, ELISA, flow cytometry. Western blotting).

[0070J Second agents can optionally include, but are not required to be, fused, linked or conjugated to a second, third, fourth, fifth, sixth, seventh, etc. domain (e.g., a toxic moiety or entity). Such domains can impart a distinct or complementary function or activity. Such domains also can be composed of non- natural structural components, for example: a) residue linkage groups other than a natural amide bond; b) non-natural residues in place of naturally occurring amino acid residues; and c) residues which induce secondary structural mimicry, i.e., induce or stabilize a secondary structure. Such domains include cyclic structures such as an end-to-end amide bond between the amino and carboxy-terminus of the molecule or intra- or inter-molecular disulfide bond(s). Such domains also include, for example, sugar or carbohydrate residues, phosphate groups, fatty acids, lipids, etc.

[0071} Non-limiting examples of toxic moieties or entities that can be included in a second agent, such as by fusion, linkage or conjugation, include a chemotherapeutic drug, radionuclide, toxin (e.g., bacterial toxin), pore forming or amphipathic peptide, or cell cytotoxic or lytic moiety. Such second agents fused, linked or conjugated to a toxic moiety or entity, such as a cytotoxic or a lytic moiety, or not fused, linked or conjugated to a toxic moiety or entity, such as a cytotoxic or a lytic domain can be used to arrest, reduce, inhibit or decrease growth, survival or proliferation, or lyse, kill or stimulate or induce apoptosis of one or more target LHRH-R producing or expressing cells. [0072] Non-limiting examples of radionclides and radioisotopes, include, for example, ⁴⁷Sc⁶⁷Cu, ⁷²Se, ⁸⁸Y, ^Sr, ⁹⁷Ru, "Tc, ¹⁰⁵Rh, ^{l u}In, ¹²⁵1, ¹³¹I, ^,49Tb, ^,53Sm, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁹⁴Os, ²⁰³Pb, ^{2I 1}At, ²¹²Bi,²¹³Bi, ²¹²Pb, ²²³Ra,²²⁵Ac, ^{2 7}Ac, and ^{2 8}Th.

[0073] Cell cytotoxic or lytic moieties include peptides and mimetics that form an amphipathic alpha- helix, synthetic membrane disrupting or pore forming peptides of various structures, such alpha-helical, cationic, linear or lytic peptides containing amino acid derivatives. Particular examples, include melhtin, cecropins, magainins, dermaseptins, pardaxin, amoebapore, catelicidins, drosocins, abaecinds, androctonin, alpha defensins, beta defensins, defensin 4, lactoferrin (and derivatives

sarcotoxins, and bacterial toxins such as pseudomonas exotoxin or derivatives, diphtheria toxins or derivatives, antibiotics such as calicheamicin, anthracyclines such as doxorubicin or its derivatives, auristatins or analogs, maytansinoids or analogs, etc.

[0074] Typically, an amphipathic alpha-helix contains mostly hydrophilic amino acids on one side of the alpha-helix and the other side contains mostly hydrophobic amino acids. The amino acid sequence of an amphipathic alpha helix alternates between hydrophilic and hydrophobic residues every 3 to 4 residues.

A PNNPNNP repeat pattern or motif is predicted to form an amphipathic alpha-helix where P represents a positively charged amino acid residue and N a neutral amino acid residue. A PNNPNNP repeat pattern provides a cationic binding site for binding to a negatively charged cell membrane.

[0075] Non-limiting examples of a cell cytotoxic or lytic moieties also include a sequence that includes or consists of an amino acid sequence selected from KFAKFAKKJFAKFAK (Phorl4),

KFAKFAKKFAKFAKK, KFAKFAK FAKFAKKF, KFAKFAKKFAKFAKKFA,

KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKFAKKFAKF, KFAKFAKKFAKFAKKFAKFA and

KFAKFAKKFAKFAKKFAKFAK (Phor21); or an amino acid sequence selected from

KFAKFAKKFAKFAK (Phorl4), KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF,

KFAKFAKKFAKFAKKFA, KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKFAKKFAKF,

KFAKFAKKFAKFAKKFAKFA and KFAKFAKKFAKFAKKFAKFAK (Phor21).

[0076] Additional non-limiting examples of a cell cytotoxic or lytic domain also includes a 10 to 100 amino acid sequence that includes an amino acid sequence selected from KFAKFAKKFAKFAK

(Phor 14), KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKFA,

KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKFAKKFAKF, KFAKFAKKFAKFAKKFAKFA and

KFAKFAKKFAKFAKKFAKFAK (Phor21); or an amino acid sequence selected from

KFAKFAKKFAKFAK (Phor 14), KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF,

KFAKFAKKFAKFAKKFA, KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKFAKKFAKF,

KFAKFAKKFAKFAKKFAKFA and KFAKFAKKFAKFAKKFAKFAK (Phor21).

[0077] Such cell cytotoxic and lytic domains can have one or more residues substituted. For example, a sequence that includes or consists of KFAKFAKKFAKFAK (Phorl4), KFAKFAKKFAKFAKK,

KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKFA, KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKFAKKFAKF, KFAKFAKKFAKFAKKFAKFA and

KFAKPAKKFAJ FAKKFAKFAK (Phor21) can have one or more of the K residues substituted with any of an F or L residue, one or more of the F residues substituted with any of a K, A or L residue, or one or more of the A residues substituted with any of a K, F or L residue, for example.

[0078] Further non-limiting examples of cytotoxic (lytic) domains that can be joined to a ligand or binding moiety include or consist of lytic peptides as described in Boman et al., Curr. Top. Microbiol. Immunol, 94/95:75-91 (1981); Boman et al, Aram. Rev. Microbiol., 41:103 (1987); Zasloff, Pro Natl. Acad. Sci. USA, 84:3628 (1987); Ganz et al., J. Chin. Invest., 76: 1427 (1985); and Lee et al., Proc. Natl. Acad. Sci. USA, 86:9159 (1989). Lytic peptides and their sequences are also described in Yamada et al., Biochem. J., 272:633 (1990); Taniai et al., Biochimica Et Biophysica Acta, 1132: 203 (1992); Boman et al, Febs Lett, 259:103 (1989); Tessier et al., Gene, 98:177 (1991); Blondelle et al., Biochemistry, 30: 4671 (1991); Andreu et al., Febs Lett., 296: 190 (1992); Macias et al., Can. J. Microbiol, 36:582 (1990); Rana et al., Biochemistry, 30:5858 (1991); Diamond et al., Proc. Natl. Acad. Sci. USA, 90:4596 ff (1993); Selsted et al, J. Biol. Chem., 268:6641 ff (1993); Tang et al, J. Biol. Chem., 268:6649 ff (1993); Lehrer et al., Blood, 76:2169 (1990); Ganz et al., Sent. Resp. Infect. I, pp. 107-117 (1986); Kagan et al., ?roc. Natl Acad. Sci. USA, 877:210 (1990); Wade et al., Proc. Natl. Acad. Sci. USA, 87:4761 (1990); Romeo et al., J. Biol. Chem., 263:9573 (1988); Jaynes et al, WO 89/00199 (1989); Jaynes, WO 90/12866 (1990); and Berkowitz, WO 93/01723 (1993).

[0079] Cecropins, such as cecropin A, cecropin B, and cecropin D, are typically small, highly homologous, basic peptides. The ammo-terminal half of various cecropins contains a sequence that will form an amphipathic alpha-helix, and the caiix>xy-terminal half of the peptide comprises a hydrophobic tail. A cecropin-like peptide has been isolated from porcine intestine (Lee et al., Proc. Natl. Acad. Sci. USA, 86:9159 (1989)). Cecropin peptides have been reported to kill a number of animal pathogens other than bacteria (Jaynes et al, FASEBJ., 2878-2883 (1988); Arrowood et al., J. Protozool., 38, No. 6, 161S- 163S (1991); and Arrowood et al., Antimicrob. Agents Chemother., 35: 224 (1991)). Normal mammalian cells do not appear to be adversely affected by cecropins, even at high concentrations (Jaynes et al., Peptide Research, 2, No. 2, pp. 1-5 (1989); and Reed et al, Mol. Reprod. Devel, 31, No. 2, pp. 106-113 (1992)). The synthetic lytic peptide known as S-l (or Shiva 1) has been reported to destroy intracellular Brucella abortus-, Trypanosoma cruzi-, Cryptosporidium parvum-, and infectious bovine herpes virus I (IBR)-infected host cells, with little or no toxic effects on noninfected mammalian cells (Jaynes et al., Peptide Research, 2, No. 2, pp. 1-5 (1989); Wood et al., Proc. Ann. Amer. Soc. Anim. Set, Utah State University, Logan, Utah J. Anim. Sci. (Suppl. 1), vol. 65, p. 380 (1987); Arrowood et al, J. Protozool, 38, No. 6, pp. 161S-163S (1991); Arrowood et al., Antimicrob. Agents Chemother., 35:224 (1991); and Reed et al., Mol. Reprod. Devel, 31, No. 2, pp. 106-113 (1992)).

[0080] Defensins, originally found in mammals, are typically small peptides containing six to eight cysteine residues (Ganz et al., J. Clin. Invest., 76: 1427 (1985)). Extracts of normal human neutrophils contain three defensin peptides: human neutrophil peptides HNP-1, HNP-2, and HNP-3. Defensin peptides have also been reported in insects and higher plants (Dimarcq et al., EMBOJ., 9:2507 (1990); and Fisher et al., Proc. Natl. Acad. Sci. USA, 84:3628 (1987).

[0081] Sarcotoxins are typically slightly larger peptides, and have been purified from the fleshfly Sarcophaga peregrine (Okada et al., J. Biol. Chem., 260:7174 (1985). Although highly divergent from the cecropins and defensins, sarcotoxins appear to have a similar antibiotic function.

[0082] Other lytic peptides have been reported in amphibians. Two peptides from the African clawed frog, Xenopiis laevis, named PGS and Gly^Lys²² PGS (Gibson et al, J. Biol. Chem., 261:5341 (1986); and Givannini et al., Biochem. J, 243: 113 (1987)). Xenopus-derived peptides have been reported to have antimicrobial activity, which had been renamed magainins (Zasloff, Proc. Natl. Acad. Sci. USA, 84:3628 (1987)). In other reports, the in vitro use of a magainin to selectively reduced the viability of the parasite Bonamia ostreae at doses that did not affect cells of the flat oyster Ostrea edulis (Morvan et al., Mol. Mar. Biol, 3:327 (1994)).

[0083] Still further non-hmiting examples of a cell cytotoxic or lytic domain also include or consist of a FALALKALKKALKKLKKALKKAL (hecate), Phe Ala Phe Ala Phe Lys Ala Phe Lys Lys Ala Phe Lys Lys Phe Lys Lys Ala Phe Lys Lys Ala Phe (Dl A21); Phe Ala Lys Lys Phe Ala Lys Lys Phe Lys Lys Phe Ala Lys Lys Phe Ala Lys Phe Ala Phe Ala Phe (D2A21): Lys Arg Lys Arg Ala Val Lys Arg Val Gly Arg Arg Leu Lys Lys Leu Ala Arg Lys lie Ala Arg Leu Gly Val Ala Phe (D5C); and Lys Arg Lys Arg Ala Val Lys Arg Val Gly Arg Arg Leu Lys Lys Lee Ala Arg Lys lie Ala Arg Leu Gly Val Ala Lys Leu Ala Gly Leu Arg Ala Val Leu Lys Phe (D5C1) sequence, or a subsequence thereof.

[0084] Still additional cytotoxic moieties or domains linked, fused or conjugated to a second agent include or consist of synthetic peptides disclosed in U.S. Patent Nos. 6,656,334; 6, 635,740 and

5,789,542.

[0085] Yet additional exemplary domains include domains facilitating isolation, which include, for example, metal chelating peptides such as polyhistidine tracts and histidine-tryptophan modules that allow purification on immobilized metals; protein A domains that allow purification on immobilized immunoglobulin; and domain utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle WA). Optional inclusion of a cleavable sequence such as Factor Xa or enterokinase between a purification domain and the antibody or fragment can be used to facilitate purification. For example, an expression vector can include a nucleic acid encoding sequence such that an antibody or fragment is linked to six histidine residues followed by a thioredoxin and an enterokinase cleavage site. The histidine residues facilitate detection and purification of the antibody or fragment while the enterokinase cleavage site provides a means for purifying the antibody or fragment from the remainder of the protein (see e.g., Kroll, DNA Cell. Biol. 12:441 (1993)).

[0086] The terms "fusion" or "chimeric" or "conjugate" and grammatical variations thereof, when used in reference to a molecule, such as a second agent, means that the molecule contains portions or sections that are derived from, obtained or isolated from, or are based upon or modeled after two different entities that are distinct from each other as they do not typically exist together in nature. That is, for example, one portion of the fusion or conjugate, such as a ligand or binding moiety (e.g., LHRH-R binding hormone or antibody), includes or consists of a portion that binds to LHRH-R, and a second portion that includes or consists of a toxic moiety or entity, such as a cell cytotoxic or a lytic moiety, each of first and second portions structurally distinct.

[0087] Ligands and binding moieties, and toxic moieties and entities, such as chemotherapeutic drugs, radionuclides, toxins (e.g., bacterial toxins), pore forming peptides, cell cytotoxic and lytic moieties can be positioned at either the ]SH₂-terminus or the C-terminus relative to each other. Thus, a toxic moiety or entity, such as chemotherapeutic drug, radionuclide, toxin (e.g., bacterial toxins), pore forming peptide, or cell cytotoxic or lytic moiety, can be linked, fused or conjugated (typically covalently) at the NH₂- terminus or at the C-teirninus of the binding moiety or ligand.

[0088] A second agent with one or more additional toxic moieties or entities can be fused, linked or conjugated indirectly or directly, by a covalent or by a non-covalent bond. Non-limiting examples of covalent bonds are amide bonds, non-natural and non-amide chemical bonds, which include, for example, glutaraldehyde, N-hyc oxysuccinirnide esters, Afunctional maleimides, N, N'- dicyclohexylcarbc)diimide (DCC) or Ν,Ν'-diisopropylcarbodiimide (DIC). Linking groups alternative to amide bonds include, for example, ketomethylene (e.g., -C(=0)-CH₂- for -C(=0)-NH-), aminomethylene (CH₂-NH₂), ethylene, olefin (CH=CH), ether (CH₂-0), thioether (CH₂-S), tetrazole (CN₄-), thiazole, retroamide, thioamide, or ester (see, e.g., Spatola (1983) in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins. Vol. 7, pp 267-357, "Peptide and Backbone Modifications," Marcel Decker, NY).

[0100] Linkers further include chemical moieties connecting the molecules. Examples include moieties that react with free or semi-free amines, oxygen, sulfur, hydroxy or carboxy groups. These linkers may be cleavable by chemical or enzymatic means, such as disulfide bridges, hydrazone bonds, and di- or terra- amino acid sequences. Heterobifunctional reagents containing an arnine-reactive group at one end and a disulfide bond with a good leaving group at the other end can serve as a linker. Such reagents include SPDP, N-succinirnidyl 3-(2-pyridyldithio)proprionate; LC-SPDP; SMPT,

succinimidyloxycairxmyl-a-mem^ PDTP, 3-(2-pyridyldithiol)propionate; cystamine; Ellman's Reagent, and S-sulfonate formation using sodium sulfite in the presence of sodium tetrathionate. Another group of heterobifunctional reagents produce a noncleavable thioether bond. This group includes SLAB, N-succinimidyl(4-iodoacetyl) aminobenzoate; SMCC, succinimidyl-4-(N- maleimideomethyl)cyclohexane- 1 -carboxylate; MBS, m-maleirnideobenzoyl-N-hydroxysuccinimide ester; and SMPB, succinimidyl-4-(r maleimidophenyl)butyrate, Linkers such as DTP A,

diemylenerriaminepentaacetic acid, can be used to bind radioisotopes, for example, metallic ions (e.g., cations) that bind to groups on the protein. [0089] One or more domains, such as those in a fusion, conjugate or chimera, can be separated by an intervening region, such as a hinge, spacer or linker positioned between an antibody or fragment and second molecule (e.g., a cell cytotoxic or lytic domain). A particular non-limiting example is a single or multi-carbon chain. Multi-carbon chains include carboxylic acids (e.g., dicarboxylic acids) such as glutaric acid, succinic acid and adipic acid. Another particular example of an intervening region is an amino acid, peptide or a non-peptide hinge, spacer or linker. Peptide hinge, spacer or linker sequences can be any length, but typically range from about 1 to 10, 10 to 20, 15 to 20, 20 to 30, 30 to 40, 40 to 50 amino acid residues. In particular embodiments, a peptide hinge, spacer or linker is from 1 to 25 L- or D- amino acid residues, or 1 to 6 L- or D-arnino acid residues. Particular amino acid residues included in intervening region can include one or more of or C, A, S or G amino acid residues. Specific non-limiting examples of intervening regions include a peptide sequence within or set forth as, GSGGS, ASAAS, or an aliphatic carbon chain of any length (e.g., 2-50 carbons), such as CX (where X is the number of carbons), e.g., C6, or CCCCCC. Other peptide linkers include but are not limited to GS, AF, FK, VK, FFK, FA, GSGRSA, RVRRSV, SS, Cit-V, F-Cit, at various lengths. Derivatives of amino acids and peptides can be positioned between the first and second domain. A specific non-limiting example of an amino acid derivative is a lysine derivative, or a 6 carbon linker such as a-amino-caproic acid.

[0090] As used herein the terms "amino acid sequence," "protein," "polypeptide" and "peptide" are used interchangeably to refer to two or more amino acids, or "residues," covalently linked by an amide bond or equivalent. Amino acid sequences can be linked by non-natural and non-amide chemical bonds including, for example, those formed with glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides, or N, N'-dicyclohexylcarrx)diirnide (DCC). Non-amide bonds include, for example, ketomethylene, aminomethylene, olefin, ether, thioether and the like (see, e.g., Spatola in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp 267-357 (1983), "Peptide and Backbone Modifications," Marcel Decker, NY).

]0091] As set forth herein, amino acid sequences include one or more L-amino acid sequences, D- amino acid sequences and amino acid sequences composed entirely of L- or D-amino acid residues, and mixtures of L-amino acids and D-amino acids. L- or D-amino acid content can therefore be about 1 to 10, 10 to 20, 15 to 20 (i.e., 15, 16, 17, 18, 19 or 20 amino acids), 20 to 30, 30 to 40, 40 to 50, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 110, 110 to 120, or more or all amino acids, and can be at any particular residue of the sequence. For example, L- or D-amino acids can be at any residue, such as a K, F or A residue. Amino acid sequences can be a linear or a cyclic structure, conjugated to a distinct moiety, form intra or intermolecular disulfide bonds, and also form higher order multimers or oligomers with the same or a different amino acid sequence, or other molecules.

[0092] Amino acid sequences, such as ligands, binding domains, toxic moieties and entities include modifications or variations, such as amino acid substitutions, additions or deletions. Thus, an amino acid sequence can incorporate any number of conservative or non-conservative amino acid substitutions, as long as such substitutions do not destroy activity (e.g., LHRH-R binding). Thus, for example, a modified hormone or antibody, or a fragment or analogue thereof, can retain at least partial LHRH-R binding activity, or cell growth, survival or proliferation decrease, inhibition or reduction, killing, lysis or apoptosis, of an unmodified hormone, antibody or fragment or analogue thereof.

[0093] A "conservative substitution" is a replacement of one amino acid by a biologically, chemically or structurally similar residue. Biologically similar means that the substitution is compatible with a biological activity. Structurally similar means that the amino acids have side chains with similar length, such as alanine, glycine and serine, or having similar size, or the structure of a cytotoxic or lytic domain, such as an amphipathic alph helix. Chemical similarity means that the residues have the same charge or are both hydrophilic or hydrophobic. Particular examples include the substitution of one hydrophobic residue, such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, serine for threonine, etc. Routine assays can be used to determine whether a variant has activity, e.g., binding activity or cell growth or prohferation inhibition or reduction, killing or apoptosis, activity.

[0094] Specific examples include a substitution, deletion or addition of one or more amino acid (e.g., 1-3, 3-5, 5-10, 10-20, or more) residues of the sequences. A modified sequence can have 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or more identity to a reference sequence.

[0095] The term "identity" and "homology" and grammatical variations thereof mean that two or more referenced entities are the same. Thus, where two amino acid sequences are identical, they have the same amino acid sequence. "Areas, regions or domains of identity" mean that a portion of two or more referenced entities are the same. Thus, where two amino acid sequences are identical or homologous over one or more sequence regions, they share identity in these regions.

[0096] The extent of identity between two sequences can be ascertained using a computer program and mathematical algorithm known in the art. Such algorithms that calculate percent sequence identity (homology) generally account for sequence gaps and mismatches over the comparison region. For example, a BLAST (e.g., BLAST 2.0) search algorithm (see, e.g., Altschul et al., J. Mol. Biol. 215:403 (1990), publicly available through NCBI) has exemplary search parameters as follows: Mismatch -2; gap open 5; gap extension 2. For polypeptide sequence comparisons, a BLASTP algorithm is typically used in combination with a scoring matrix, such as PAM100, PAM 250, BLOSUM 62 or BLOSUM 50. FASTA (e.g., FASTA2 and FAST A3) and SSEARCH sequence comparison programs are also used to quantitate the extent of identity (Pearson et al., Proc. Natl. Acad. Sci. USA 85:2444 (1988); Pearson, Methods Mol Biol. 132:185 (2000); and Smith et al., J. Mol. Biol. 147:195 (1981)). Programs for quantitating protein structural similarity using Delaunay-based topological mapping have also been developed (Bostick et al., Biochem Biophys Res Commun. 304:320 (2003)). [0097] Peptides and

can be produced and isolated using methods known in the art. Peptides can be synthesized, whole or in part, using chemical methods known in the art (see, e.g., Caruthers (1980). Nucleic Acids Res. Symp. Ser. 215; Horn (1980); and Banga, A.K., Therapeutic Peptides and Proteins, Formulation, Processing and Delivery Systems (1995) Technomic Publishing Co., Lancaster, PA). Peptide synthesis can be performed using various solid-phase techniques (see, e.g., Roberge Science 269:202 (1995); Merrifield, Methods Enzymol. 289:3(1997)) and automated synthesis may be achieved, e.g., using the ABI 431 A Peptide Synthesizer (Perkin Elmer) in accordance with the manufacturer's instructions. Peptides and peptide mimetics can also be synthesized using combinatorial methodologies. Synthetic residues and polypeptides incorporating mimetics can be synthesized using a variety of procedures and methodologies known in the art (see, e.g., Organic Syntheses Collective Volumes, Gilman, et al. (Eds) John Wiley & Sons, Inc., NY). Modified peptides can be produced by chemical modification methods (see, for example, Belousov, Nucleic Acids Res. 25:3440 (1997);

Frenkel, Free Radic. Biol. Med. 19:373 (1995); and Blommers, Biochemistry 33:7886 (1994).

[0098] First and second agents, with or without additional domains or molecules, such as a toxic moiety or entity, include isolated and purified forms. The term "isolated," when used as a modifier of a composition, means that the composition is made by the hand of man or is separated, substantially completely or at least in part, from the naturally occurring in vivo environment. Generally, an isolated composition is substantially free of one or more materials with which it normally associates with in nature, for example, one or more protein, nucleic acid, lipid, carbohydrate, cell membrane. The term "isolated" does not exclude alternative physical forms of the composition, such as multimers/oligomers, variants, modifications or derivatized forms, or forms expressed in host cells produced by the hand of man. The term "isolated" also does not exclude forms in which there are combinations therein (e.g., pharmaceutical formulations and combination compositions), any one of which is produced by the hand of man.

[0099] An "isolated" composition can also be "purified" when free of some, a substantial number of, most or all of the materials with which it typically associates with in nature. Thus, an isolated first or second agent thereof that also is substantially pure does not include other components such as polypeptides or polynucleotides present among millions of other sequences, such as proteins of a protein library or nucleic acids in a genomic or cDNA library, for example. A "purified" composition can be combined with one or more other molecules.

[00100] Second agents, including a toxic moiety or entity fused, linked or conjugated thereto, such as a toxic moiety or entity, bind to a receptor that binds to LHRH, an LHRH (binding) fragment, or an LHRH analogue, in solution or in solid phase, on cells in vitro or in vivo or in situ. A receptor that binds to LHRH, an LHRH (binding) fragment, or an LHRH analogue can also be present in vivo, such as expressed or produced by one or more cells in vivo, in vitro, in primary cell isolates, passaged cells, cultured cells, immortalized cells and cells ex vivo. [00101] Second agents, including a toxic moiety or entity fused, linked or conjugated thereto, such as a cell cytotoxic or lytic domain, can be used to target cells for decreasing or inhibiting cell survival, growth or proliferation, or stimulating, inducing or increasing lysis, cell death or apoptosis, in vitro, ex vivo and in vivo. In methods of the invention, target cells can or do express or produce receptor(s) that bind to a LHRH, LHRH (binding) fragment, or LHRH analogue, such as LHRH-R. For example a cell that expresses a receptor that binds to LHRH, an LHRH (binding) fragment or an LHRH analogue can be targeted by the second agent and thereby be preferentially (selectively) lysed or killed compared to cells that express none or less of the LHRH receptor.

[00102] Methods of the invention are therefore considered selective since they do not substantially bind to, and therefore do not substantially target (e.g., kill or lyse) cells that do not express a receptor that binds to LHRH or an LHRH fragment or analogue, e.g., an LHRH-R. Methods of the invention therefore include more particular methods that do not result in more than 10%, 20%, or 30% of non- target cells within a given area or volume being killed or lysed, typically less than 10%, 20%, or 30% of non-target cells within a given area or volume being killed or lysed (e.g., 0-30%).

[00103] In accordance with the invention, there are provided in vitro, ex vivo and in vivo methods of reducing or irihibiting proliferation of a cell(s) that is capable of expressing a receptor that binds to LHRH, an LHRH fragment or LHRH analogue, e.g., an LHRH-R, or expresses a receptor that binds to LHRH, an LHRH fragment or LHRH analogue, e.g., an LHRH-R. In one embodiment, a method includes contacting a cell with first and second agents in an amount sufficient to arrest, reduce, decrease, or inhibit growth, proliferation or survival of the cell.

[00104] Non-limiting examples of cells capable of or that do express a receptor that binds to LHRH, an LHRH fragment or LHRH analogue (e.g., an LHRH-R) include hyperproliferative cells, and cells that exhibit aberrant or undesirable hyperproliferation. In particular non-limiting examples, such cells include benign, non-metastatic and metastatic neoplastic, cancer, tumor and malignant cells, as well as disseminated neoplastic, cancer, tumor and malignant cells and dormant neoplastic, cancer, tumor and malignant cells. Cells that express LHRH-R, or express LHRH-R at elevated levels relative to non-target normal or

cells provide selectivity for such cells. Thus, an LHRH-R binding second agent can bind to LHRH-R expressed in or produced by a target cell, such as a hyperprohferative cell (e.g., benign, and non-metastatic and metastatic neoplasias, cancers, tumors and malignancies, and disseminated and dormant neoplastic, cancer, tumor and malignant cells), but not detectably expressed or is produced or expressed at relatively lower levels by a normal or non-target cell, thereby preferentially targeting the cells (e.g., hyperproliferative cells).

[00105] Receptor expressing or producing target cells may be present within a population of nonreceptor expressing cells, or cells that express little receptor such that they are not substantially killed by a second agent that binds to the receptor (e.g., LHRH-R). Accordingly, the methods of the invention can selectively target cells that express or produce receptor(s) that bind to LHRH, LHRH (binding) fragment, or LHRH analogue.

[00106] The term "contacting" means direct or indirect binding or interaction between two or more entities (e.g., between an antibody or fragment thereof and a cell). Contacting as used herein includes in solution, in solid phase, in vitro, ex vivo, in a cell and in vivo. Contacting in vivo can be referred to as administering, administration, delivering or delivery.

[00107] Accordingly, methods of treating undesirable or aberrant cell proliferation and hyperproliferative disorders are provided. In one embodiment, a method includes adrninistering to a subject (in need of treatment) a first agent to stimulate, induce, increase or enhance cell expression of a receptor that binds to LHRH, LHRH fragment or LHRH analogue (e.g., LHRH-R), and a second agent that binds to the receptor in an amount sufficient to treat the undesirable or aberrant cell proliferation or hyperproliferative disorder.

[00108] A "hyperproliferative disorder" refers to any undesirable or aberrant cell survival (e.g., failure to undergo programmed cell death or apoptosis), growth or proliferation. Such disorders include benign hyperplasias, non-metastatic and metastatic neoplasias, cancers, tumors and malignancies. Undesirable or aberrant cell proliferation and hyperproliferative disorders can affect any cell, tissue, organ in a subject. Undesirable or aberrant cell proliferation and hyperproliferative disorders can be present in a subject, locally, regionally or systemically. A hyperproliferative disorder can arise from a multitude of tissues and organs, including but not limited to breast, lung (e.g., small cell or non-small cell), thyroid, head and neck, brain, nasopharynx, throat, nose or sinuses, lymphoid, adrenal gland, pituitary gland, thyroid, lymph, gastrointestinal (mouth, esophagus, stomach, duodenum, ileum, jejunum (small intestine), colon, rectum), genito-urinary tract (uterus, ovary, vagina cervix, endometrium, fallopian tube, bladder, testicle, penis, prostate), kidney, pancreas, liver, bone, bone marrow, lymph, blood, muscle, skin, and stem cells, which may or may not metastasize to other secondary sites, regions or locations.

[00109] In accordance with the invention, methods of treating a metastatic or non-metastatic tumor, cancer, malignancy or neoplasia are provided. Such methods of treating a metastatic or non-metastatic tumor, cancer, malignancy or neoplasia, include treating a subject in need of treatment due to having or at risk of having a metastatic or non-metastatic tumor, cancer, malignancy or neoplasia, and methods of increasing effectiveness or improving an anti-proliferative, anti-tumor, anti-cancer, anti-neoplasia or anti- malignancy, therapy are provided. In respective embodiments, a method includes administering to a subject with or at risk of a metastatic or non-metastatic tumor, cancer, malignancy or neoplasia, a first agent that can stimulate, induce, increase or enhance expression of LHRH receptors by cells, or a receptor that binds to LHRH, and administering a second agent that binds to LHRH receptors, sufficient to treat the metastatic or non-metastatic tumor, cancer, malignancy or neoplasia; administering to the subject a first agent that can stimulate, induce, increase or enhance expression of LHRH receptors by cells, or a receptor that binds to LHRH, and administering a second agent that binds to LHRH receptors, sufficient to treat the subject; and administering to a subject that is undergoing or has undergone metastatic or non-metastatic tumor, cancer, malignancy or neoplasia therapy, a first agent that can stimulate, induce, increase or enhance expression of LHRH receptors by cells, or a receptor that binds to LHRH, and administering a second agent that binds to LHRH receptors, sufficient to increase effectiveness of the anti-proliferative, anti-tumor, anti-cancer, anti-neoplasia or anti-mahgnancy therapy.

[00110] Such disorders can affect virtually any cell, organ or tissue type, e.g., carcinoma, sarcoma, melanoma, neural, and reticuloendothelial or haematopoietic neoplastic disorders (e.g., myeloma, lymphoma or leukemia). Accordingly, methods are applicable to metastatic or non-metastatic tumor, cancer, malignancy or neoplasia of any cell, organ or tissue origin.

[00111] As used herein, the terms "neoplasia" and "tumor" refer to a cell or population of cells whose growth, proliferation or survival is greater than growth, proliferation or survival of a normal counterpart cell, e.g. a cell proliferative or differentiative disorder. A tumor is a neoplasia that has formed a distinct mass or growth. A "cancer" or "malignancy" refers to a neoplasia or tumor that can invade adjacent spaces, tissues or organs. A "metastasis" refers to a neoplasia, tumor, cancer or malignancy that has disseminated or spread from its primary site to one or more secondary sites, locations or regions within the subject, in which the sites, locations or regions are distinct from the primary tumor or cancer.

Typically, target cells of a neoplasia, tumor, cancer, or malignancy, or a metastasis thereof, express or produce receptors) that bind to LHRH, LHRH (binding) fragment, or LHRH analogue.

[00112] Specific non-limiting examples of cells, tissue and organ which express or produce receptors) that bind to LHRH, LHRH (binding) fragment, or LHRH analogue and can develop neoplasia, tumor, cancer, malignancy or metastasis are listed in Table 2:

27

601557964vl Kidney yes Sion Vardi 1992 28/28 biopsies

RA, ICC, T-PCR

Brain yes Van Groeningen 1998

[00113] Neoplastic, tumor, cancer and malignant cells (metastatic or non-metastatic) include dormant or residual neoplastic, tumor, cancer and malignant cells. Such cells typically consist of remnant tumor cells that are not dividing (G0-G1 arrest). These cells can persist in a primary site or as msserninated neoplastic, tumor, cancer or malignant cells as a minimal residual disease. These dormant neoplastic, tumor, cancer or malignant cells remain asymptomatic, but can develop severe symptoms and death once these dormant cells proliferate. Invention methods can be used to reduce or inhibit proliferation of dormant neoplastic, tumor, cancer or malignant cells, which can in turn inhibit or reduce tumor or cancer relapse, or tumor or cancer metastasis or progression.

[00114] A metastatic or non-metastatic tumor, cancer, malignancy or neoplasia may be in any stage, e.g., early or advanced, such as a stage I, II, ΙΠ, IV or V tumor. A metastatic or non-metastatic tumor, cancer, malignancy or neoplasia may have been subject to a prior treatment or be stabilized (non-progressing) or in remission.

[00115] In terms of metastasis, invention methods can be used to reduce, decrease or inhibit metastasis of a primary tumor or cancer to other sites, or the formation or establishment of metastatic tumors or cancers at other sites distal from the primary tumor or cancer thereby inhibiting or reducing tumor or cancer relapse or tumor or cancer progression. Thus, methods of the invention include, among other things, 1 ) reducing or inhibiting growth, survival, proliferation, mobility or invasiveness of tumor or cancer cells that potentially or do develop metastases; 2) reducing or inhibiting formation or establishment of metastases arising from a primary tumor or cancer to one or more other sites, locations or regions distinct from the primary tumor or cancer; 3) reducing or inhibiting growth, survival, or proliferation of a metastasis at one or more other sites, locations or regions distinct from the primary tumor or cancer after a metastasis has formed or has been established; and 4) reducing or inhibiting formation or establishment of additional metastasis after the metastasis has been formed or established.

[00116] Cells of a metastatic or non-metastatic tumor, cancer, malignancy or neoplasia may be aggregated in a "solid" cell mass or be dispersed or diffused. A "solid" tumor refers to cancer, neoplasia or metastasis that typically aggregates together and forms a mass. Specific non-limiting examples include visceral tumors such as melanomas, breast, pancreatic, uterine and ovarian cancers, testicular cancer, including seminomas, gastric or colon cancer, hepatomas, adrenal, renal and bladder carcinomas, lung, head and neck cancers and brain tumors/cancers.

[00117] Carcinomas, which refer to malignancies of epithelial or endocrine tissue, include respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. Exemplary carcinomas include those forming from the uterus, cervix, lung, prostate, breast, head and neck, colon, pancreas, testes, adrenal, kidney, esophagus, stomach, liver and ovary. The term also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues. Adenocarcinoma includes a carcinoma of a glandular tissue, or in which the tumor forms a gland like structure.

[00118] Sarcomas refer to malignant tumors of mesenchymal cell origin. Exemplary sarcomas include for example, lymphosarcoma, liposarcoma, osteosarcoma, chondrosarcoma, leiomyosarcoma, rhabdomyosarcoma and fibrosarcoma.

[00119] Neural neoplasias include glioma, glioblastoma, meningioma, neuroblastoma, retinoblastoma, astrocytoma and oligodendrocytoma.

[00120] A "liquid tumor," which refers to neoplasia that is dispersed or is diffuse in nature, as they do not typically form a solid mass. Particular examples include neoplasia of the reticuloendothelial or hematopoietic system, such as lymphomas, myelomas and leukemias. Non-limiting examples of leukemias include acute and chronic lymphoblastic, myeloblastic and multiple myeloma. Typically, such diseases arise from poorly differentiated acute leukemias, e.g., erythroblastic leukemia and acute megakaryoblastic leukemia. Specific myeloid disorders include, but are not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML). Lymphoid malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM). Specific malignant lymphomas include, non-Hodgkin's lymphoma and variants such as diffuse large B cell lymphoma, Mantle cell lymphoma, Follicular lymphoma, peripheral T cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Sternberg disease.

[00121] As disclosed herein, undesirable or aberrant cell proliferation or hyperproliferative disorders can occur in uterus, breast, vagina, cervix, fallopian tube and prostate gland. Endometriosis occurs when cells of the uterus grow outside of the uterus and into other areas, such as ovaries, bladder or bowel. Fibroids and polyps can affect uterus, breast, vagina, cervix and fallopian tube. Benign prostatic hyperplasia (BPH) occurs when stromal or epitheUal cells of the prostate gland hyperproliferate resulting in increased size of the gland in men.

[00122] Target cells include cells that participate in or are required for reproduction or fertility. Thus, in accordance with the invention, there are provided methods of reducing fertility of an animal. In one embodiment, a method includes administering to an animal, a first agent that can stimulate, induce, increase or enhance expression of LHRH receptors by cells, or a receptor that binds to LF1RH, and administering to the animal a second agent that binds to LHRH receptors, sufficient to reduce fertility. [00123] Undesirable or aberrant cell proliferation or hyperproliferative disorders can also occur in endometrium. Thus, in accordance with the invention, there are provided methods of treating endometriosis. In one embodiment, a method includes administering to an animal a first agent that can stimulate, induce, increase or enhance expression of LHRH receptors by cells, or a receptor that binds to LHRH, and administering to the animal a second agent that binds to LHRH receptors, sufficient to treat endometriosis.

[00124] Undesirable or aberrant cell proliferation or hyperproliferative disorders can also cause benign prostate hyperplasia, breast ductal or lobular hyperplasia, or uterine endometriosis, fibroid or polyps. Thus, in accordance with the invention, there are provided methods of treating fibroids and polyps of an animal. In one embodiment, a method includes administering to the animal a first agent that stimulates, induces, increases or enhances expression of LHRH receptors by cells, or a receptor that binds to LHRH, and admiinstering to the animal a second agent that binds to LHRH receptors, sufficient to treat the fibroid or polyp.

[00125] As also disclosed herein, undesirable or aberrant cell proliferation or hyperproliferative disorders can occur in prostate. Thus, in accordance with the invention, there are provided methods of treating benign prostate hyperplasia or metastatic prostate neoplasia. In one embodiment, a method includes administering to an animal an amount of a first agent that stimulates, induces, increases or enhances expression of LHRH receptors by cells, or a receptor that binds to LHRH, and administering to the animal a second agent that binds to LHRH receptors, sufficient to treat benign prostate hyperplasia or metastatic prostate neoplasia.

[00126] Any composition, treatment, protocol, therapy or regimen having an anti-cell proliferative activity or effect can be combined with or used in combination in the invention methods. Methods of the invention therefore include anti-proliferative, anti-tumor, anti-cancer, anti-neoplastic and anti-metastatic treatments, protocols and therapies, which include any other composition, treatment, protocol or therapeutic regimen that inhibits, decreases, arrests, slows, reduces or prevents a hyperproliferative disorder, such as tumor, cancer, malignant or neoplastic growth, progression, metastasis, proliferation or survival, or worsening in vitro or in vivo. Particular non-limiting examples of an anti-proliferative (e.g., tumor) therapy include chemotherapy, immunotherapy, radiotherapy (ionizing or chemical), local thermal (hyperthermia) therapy, surgical resection and vaccination.

[00127] First or second agents can be administered prior to, substantially contemporaneously with or following administration of the anti-cell proliferative, anti-neoplastic, anti-tumor, anti-cancer, anti- metastatic or immune-enhancing treatment or therapy. A period of time, for example, 1-6, 6-12, 12-24, 24-48, 48-72 minutes, hours, days, weeks or months can intervene between one administration or treatment and a subsequent administration or treatment. First or second agents can be administered as a combination composition with the anti-cell proliferative, anti-neoplastic, anti-tumor, anti-cancer, anti- metastatic or immune-enhancing treatment or therapy, metastatic or non-metastatic tumor, cancer, malignancy or neoplasia.

[00128] Anti-proliferative, anti-neoplastic, anti-tumor, anti-cancer and anti-metastatic compositions, therapies, protocols or treatments include those that prevent, disrupt, interrupt, inhibit or delay cell cycle progression or cell proliferation; stimulate or enhance apoptosis or cell death, inhibit nucleic acid or protein synthesis or metabolism, inhibit cell division, or decrease, reduce or inhibit cell survival, or production or utilization of a necessary cell survival factor, growth factor or signaling pathway

(extracellular or intracellular). Non-hmiting examples of chemical agent classes having anti-cell proliferative, anti-neoplastic, anti-tumor, anti-cancer and anti-metastatic activities include alkylating agents, anti-metabolites, plant extracts, plant alkaloids, nitrosoureas, hormones, nucleoside and nucleotide analogues. Specific examples of drugs having anti-cell proliferative, anti-neoplastic, antitumor, anti-cancer and anti-metastatic activities include cyclophosphamide, azathioprine, cyclosporin A, prednisolone, melphalan, chlorambucil, mechlorethamine, busulphan, methotrexate, 6-mercaptopurine, thioguanine, 5-fluorouracil, cytosine arabinoside, 5-azacytidine (5-AZC) and 5-azacytidine related compounds, bleomycin, actinomycin D, mithramycin, mitomycin C, carmustine, lomustine, semustine, streptozotocin, hydroxyurea, cisplatin, carboplatin, oxiplatin, mitotane, procarbazine, dacarbazine, taxol, taxotere, vinblastine, vincristine, doxorubicin and dibromomannitol, topoisomerase inhibitors (irinotecan, topotecan, etoposide, teniposide), gemcitabine, pemetrexed etc.

[00129] Additional agents that are applicable in the invention methods and uses are known in the art and can be employed. For example, biologicals such as antibodies, cell growth factors, cell survival factors, cell differentiative factors, cytokines and chemokines can be administered. Non-limiting examples of monoclonal antibodies include rituximab (Rituxan®), trastuzumab (Herceptin®), bevacizumab

(Avastin®), cetuximab (Erbitux®), alemtuzumab (Campath®), panitumumab (Vectibix®), ibritumomab tiuxetan (Zevalin®), tositumomab (Bexxar®) etc., which can be used in combination with, inter alia, a fusion construct in accordance with the invention. Other targeted drugs that are applicable for use with the fusion constructs are imatinib (Gleevec®), gefitinib (Iressa®), bortzomib (Velcade®), lapatinib (Tykerb®), sunitinib (Sutent®), sorafenib (Nexavar®), nilotinib (Tasigna®), Erlotinib hydrochloride (Tarceva®) etc.

[00130] Additional non-limiting examples include immune-enhancing treatments and therapies, which include cell based therapies. Such therapies include lymphocytes, plasma cells, macrophages, dendritic cells, T-cells, NK cells or B-cells; an antibody, a cell growth factor, a cell survival factor, a cell differentiative factor, a cytokine or a chemokine, etc.

[00131] Methods of the invention may be practiced prior to (i.e. prophylaxis), concurrently with or after evidence of the presence of undesirable or aberrant cell proliferation or a hyperproliferative disorder, disease or condition begins (e.g., one or more symptoms). Administering a first agent that can stimulate, induce, increase or enhance expression of LHRH receptors by cells, or a receptor that binds to LHRH, and a second agent that binds to LHRH receptors, prior to, concurrently with or immediately following development of a symptom of undesirable or aberrant cell proliferation or a hyperproliferative disorder may decrease the occurrence, frequency, severity, progression, or duration of one or more symptoms of the undesirable or aberrant cell proliferation or a hyperproliferative disorder, disease or condition in the subject. In addition, administering a first agent that can stimulate, induce, increase or enhance expression of LHRH receptors by cells, or a receptor that binds to LHRH, and a second agent that binds to LHRH receptors, prior to, concurrently with or immediately following development of one or more symptoms of the undesirable or aberrant cell proliferation or a hyperproliferative disorder, disease or condition may inhibit, decrease or prevent the spread or dissemination of hypeφroliferating cells (e.g., metastasis) to other sites, regions, tissues or organs in a subject, or establishment of hypeφroliferating cells (e.g., metastasis) at other sites, regions, tissues or organs in a subject.

[00132] Methods of the invention, such as treatment methods, can provide a detectable or measurable therapeutic benefit or improvement to a subject. A therapeutic benefit or improvement is any measurable or detectable, objective or subjective, transient, temporary, or longer-term benefit to the subject or improvement in the condition, disorder or disease, an adverse symptom, consequence or underlying cause, of any degree, in a tissue, organ, cell or cell population of the subject. Therapeutic benefits and improvements include, but are not limited to, reducing or decreasing occurrence, frequency, severity, progression, or duration of one or more symptoms or complications associated with a disorder, disease or condition, or an underlying cause or consequential effect of the disorder, disease or condition. Methods of the invention therefore include providing a therapeutic benefit or improvement to a subject.

[00133] In a method of the invention in which a therapeutic benefit or improvement is a desired outcome, first and second agents can be administered in a sufficient or effective amount to a subject in need thereof. An "amount sufficient" or "amount effective" refers to an amount that provides, in single or multiple doses, alone or in combination, with one or more other compositions (therapeutic agents such as a chemotherapeutic or immune stimulating drug), treatments, protocols, or therapeutic regimens agents, a detectable response of any duration of time (long or short term), a desired outcome in or a benefit to a subject of any measurable or detectable degree, for any duration of time (e.g., for hours, days, months, years, or cured) over a period of time. The doses or "sufficient amount" or "effective amount" for treatment (e.g., to provide a therapeutic benefit or improvement) typically are effective to ameliorate a disorder, disease or condition, or one, multiple or all adverse symptoms, consequences or complications of the disorder, disease or condition, to a measurable extent, although reducing or inhibiting a progression or worsening of, or stabilizing, the disorder, disease or condition or a symptom, is considered a satisfactory outcome. For multiple doses, a period of time, for example, 1-6, 6-12, 12-24, 24-48, 48-72 minutes, hours, days, weeks or months can intervene between one administration and a subsequent administration. [00134] The term "ameliorate" means a detectable objective or subjective improvement in a subject's condition. A detectable improvement includes a subjective or objective reduction in the occurrence, frequency, severity, progression, or duration of a symptom caused by or associated with a disorder, disease or condition, an improvement in an underlying cause or a consequence of the disorder, disease or condition, or a reversal of the disorder, disease or condition.

[00135] Treatment can therefore result in inhibiting, reducing or preventing a disorder, disease or condition, or an associated symptom or consequence, or underlying cause; inhibiting, reducing or preventing a progression or worsening of a disorder, disease, condition, symptom or consequence, or underlying cause; or further deterioration or occurrence of one or more additional symptoms of the disorder, disease condition, or symptom. Thus, a successful treatment outcome leads to a "therapeutic effect," or "benefit" or inhibiting, reducing or preventing the occurrence, frequency, severity, progression, or duration of one or more symptoms or underlying causes or consequences of a condition, disorder, disease or symptom in the subject. Treatment methods affecting one or more underlying causes of the condition, disorder, disease or symptom are therefore considered to be beneficial. Stabilizing or inhibiting progression or worsening of a disorder or condition is also a successful treatment outcome.

[00136] A therapeutic benefit or improvement therefore need not be complete ablation of any one, most or all symptoms, complications, consequences or underlying causes associated with the condition, disorder or disease. Thus, a satisfactory endpoint is achieved when there is an incremental improvement in a subject's condition, or a partial reduction in the occurrence, frequency, severity, progression, or duration, or inhibition or reversal, of one or more associated adverse symptoms or complications or consequences or underlying causes, worsening or progression (e.g., stabilizing one or more symptoms or complications of the condition, disorder or disease), of one or more of the physiological, biochemical or cellular manifestations or characteristics of the disorder or disease, over a short or long duration of time (hours, days, weeks, months, etc.).

[00137] In particular emrx>diments, a method of treatment results in partial or complete destruction of a metastatic or non-metastatic tumor, cancer, malignant or neoplastic cell mass, volume, size or numbers of cells; results in stimulating, inducing or increasing metastatic or non-metastatic tumor, cancer, malignant or neoplastic cell necrosis, lysis or apoptosis; results in reducing metastatic or non-metastatic tumor, cancer, malignant or neoplastic volume, size, cell mass; results in inhibiting or preventing progression or an increase in metastatic or non-metastatic tumor, cancer, malignant or neoplastic volume, mass, size or cell numbers; results in inhibiting or decreasing the spread or dissemination of hyperproUferating cells (e.g., metastasis) to other (secondary) sites, regions, tissues or organs in a subject, or establishment of hyperproliferating cells (e.g., metastasis) at other (secondary) sites, regions, tissues or organs in a subject; or results in prolonging lifespan of the subject. In additional particular embodiments, a method of treatment results in reducing or decreasing severity, duration or frequency of an adverse symptom or complication associated with or caused by the metastatic or non-metastatic tumor, cancer, malignancy or neoplasia.

[00138] An amount sufficient or an amount effective can but need not be provided in a single administration and, can but need not be, administered alone or in combination with another composition (e.g., chemotherapeutic or immune enhancing or stimulating agent), treatment, protocol or therapeutic regimen. For example, the amount may be proportionally increased as indicated by the need of the subject, status of the disorder, disease or condition treated or the side effects of treatment. In addition, an amount sufficient or an amount effective need not be sufficient or effective if given in single or multiple doses without a second composition (e.g., chemotherapeutic or immune stimulating agent), treatment, protocol or therapeutic regimen, since additional doses, amounts or duration above and beyond such doses, or additional compositions (e.g., chemotherapeutic or immune stimulating agents), treatments, protocols or therapeutic regimens may be included in order to be considered effective or sufficient in a given subject. Amounts considered sufficient also include amounts that result in a reduction of the use of another treatment, therapeutic regimen or protocol.

[00139] An amount sufficient or an amount effective need not be effective in each and every animal or subject treated, prophylactically or therapeutically, nor a majority of treated animals or subjects in a given group or population. As is typical for treatment or therapeutic methods, some animals and subjects will exhibit greater or less response to a given treatment, therapeutic regimen or protocol. An amount sufficient or an amount effective refers to sufficiency or effectiveness in a particular animal or subject, not a group or the general population. Such amounts will depend in part upon the condition treated, such as the type or stage of undesirable or aberrant cell proliferation or hyperproliferative disorder (e.g., a metastatic or non-metastatic tumor, cancer, malignancy or neoplasia), the therapeutic effect desired, as well as the individual subject (e.g., the bioavailability within the subject, gender, age, etc.).

[00140] Particular non-limiting examples of therapeutic benefit or improvement for undesirable or aberrant cell proliferation, such as a hyperproliferative disorder (e.g., a metastatic or non-metastatic tumor, cancer, malignancy or neoplasia) include a reduction in cell size, mass or volume, inhibiting an increase in cell size, mass or volume, a slowing or inhibition of worsening or progression, stimulating cell necrosis, lysis or apoptosis, reducing or inhibiting neoplastic or tumor malignancy or metastasis, reducing mortality, and prolonging lifespan of a subject. Thus, inhibiting or delaying an increase in cell size, mass, volume or metastasis (stabilization) can increase lifespan (reduce mortality) even if only for a few days, weeks or months, even though complete ablation of the metastatic or non-metastatic tumor, cancer, malignancy or neoplasia has not occurred. Adverse symptoms and complications associated with a hyperproliferative disorder (e.g., a metastatic or non-metastatic tumor, cancer, malignancy or neoplasia) that can be reduced or decreased include, for example, pain, nausea, discomfort, lack of appetite, lethargy and weakness. A reduction in the occurrence, frequency, severity, progression, or duration of a symptom of undesirable or aberrant cell proliferation, such as a hyr^rproliferative disorder (e.g., a metastatic or non-metastatic tumor, cancer, malignancy or neoplasia), such as an improvement in subjective feeling (e.g., increased energy, appetite, reduced nausea, improved mobility or psychological well being, etc.), are therefore all examples of therapeutic benefit or improvement.

[00141] For example, a sufficient or effective amount of first and second agent is considered as having a therapeutic effect if administration results in less chemotherapeutic drug, radiation or immunotherapy being required for treatment of undesirable or aberrant cell proliferation, such as a hyperproliferative disorder (e.g., a metastatic or non-metastatic tumor, cancer, malignancy or neoplasia).

[00142] The term "subject" refers to animals, typically mammalian animals (mammals), such as humans, non human primates (apes, gibbons, chimpanzees, orangutans, macaques), domestic animals (dogs and cats), farm animals (horses, cows, goats, sheep, pigs) and experimental animal (mouse, rat, rabbit, guinea pig). Subjects include animal disease models, for example, animal models of undesirable or aberrant cell proliferation, such as a hyperproliferative disorder (e.g., a metastatic or non-metastatic tumor, cancer, malignancy or neoplasia) for analysis in vivo.

[00143] Subjects appropriate for treatment include those having or at risk of having undesirable or aberrant cell growth or proliferation, or a metastatic or non-metastatic tumor, cancer, malignant or neoplastic cell, those undergoing as well as those who are undergoing or have undergone antiproliferative (e.g., undesirable or aberrant cell proliferation, metastatic or non-metastatic tumor, cancer, malignancy or neoplasia) therapy, including subjects where the tumor is in remission. "At risk" subjects typically have risk factors associated with undesirable or aberrant cell proliferation, development of hyperplasia (e.g., a tumor).

[00144] Particular examples of at risk or candidate subjects include those with cells that express a receptor that binds to LHRH, LHRH (binding) fragment, or an LHRH analogue, particularly where cells targeted for inhibition or reduction of growth or proliferation, necrosis, lysis, killing or destruction express or produce greater numbers or amounts of LHRH-R than non-target cells. Such cells can be selectively or preferentially targeted for for inhibition or reduction of growth or proliferation, necrosis, lysis or killing.

[00145] At risk subjects also include those that are candidates for and those that have undergone surgical resection, chemotherapy, immunotherapy, ionizing or chemical radiotherapy, local or regional thermal (hyperthermia) therapy, or vaccination. The invention is therefore applicable to treating a subject who is at risk of a metastatic or non-metastatic tumor, cancer, malignancy or neoplasia or a complication associated with a metastatic or non-metastatic tumor, cancer, malignancy or neoplasia, for example, due to metastatic or non-metastatic tumor, cancer, malignancy or neoplasia reappearance or regrowth following a period of stability or remission.

[00146] Risk factors include gender, lifestyle (diet, smoking), occupation (medical and clinical personnel, agricultural and livestock workers), environmental factors (carcinogen exposure), family history (autoimmune disorders, diabetes, etc.), genetic predisposition, etc. For example, subjects at risk for developing melanoma include excess sun exposure (ultraviolet radiation), fair skin, high numbers of naevi (dysplastic nevus), patient phenotype, family history, or a history of a previous melanoma.

Subjects at risk for developing cancer can therefore be identified by lifestyle, occupation, environmental factors, family history, and genetic screens for tumor associated genes, gene deletions or gene mutations. Subjects at risk for developing breast cancer lack Brcal, for example. Subjects at risk for developing colon cancer have early age or high frequency polyp formation, or deleted or mutated tumor suppressor genes, such as adenomatous polyposis coli (APC), for example.

[00147] Subjects also include those precluded from other treatments. For example, certain subjects may not be good candidates for surgical resection, chemotherapy, immunotherapy, ionizing or chemical radiotherapy, local or regional thermal (hyperthermia) therapy, or vaccination. Thus, candidate subjects for treatment in accordance with the invention include those that are not a candidate for surgical resection, chemotherapy, immunotherapy, ionizing or chemical radiotherapy, local or regional thermal (hyperthermia) therapy, or vaccination.

[00148] First and second agents thereof may be formulated in a unit dose or unit dosage form. In a particular embodiment, a first or second agent is in a unit dose or unit dosage predicted or calculated to be effective to treat a subject having undesirable or aberrant cell prohferation or a hyperproliferative disorder. In an additional particular embodiment, a first or second agent is in a unit dose or unit dosage predicted or calculated to be effective to treat a subject having a metastatic or non-metastatic tumor, cancer, malignancy or neoplasia. In a further particular embodiment, a first or second agent in a unit dose or unit dosage predicted or calculated to be effective to reduce fertility of a subject. Exemplary unit doses range from about 25-250, 250-500, 500-1000, 1000-2500 or 2500-5000, 5000-25,000, 5000- 50,000 ng; and from about 25-250, 250-500, 500-1000, 1000-2500 or 2500-5000, 5000-25,000, 5000- 50,000

[00149] Compositions and methods of the invention may be contacted or provided in vitro, ex vivo or in vivo. Compositions can be administered to provide the intended effect as a single or multiple dosages, for example, in an effective or sufficient amount. Exemplary doses range from about 25-250, 250-500, 500-1000, 1000-2500 or 2500-5000, 5000-25,000, 5000-50,000 pg/kg; from about 50-500, 500-5000, 5000-25,000 or 25,000-50,000 ng/kg; and from about 25-250, 250-500, 500-1000, 1000-2500 or 2500- 5000, 5000-25,000, 5000-50,000 μg/kg, on consecutive days, or alternating days or intermittently.

Single or multiple doses can be administered on consecutive days, alternating days or intermittently.

[00150] Compositions can be administered and methods may be practiced via systemic, regional or local aclministration, by any route. For example, a first or second agent can be administered systemically, regionally or locally, intravenously, orally (e.g., ingestion or inhalation), intramuscularly,

intraperitoneally, intradermally, subcutaneously, intracavity, intracranially, transdermally (topical), parenterally, e.g. transmucosally or rectally. Compositions and methods of the invention including pharmaceutical formulations can be administered via a (micro)-encapsulated delivery system or packaged into an implant for administration.

[00151] First and/or second agents can be included in pharmaceutical compositions. A pharmaceutical composition refers to "pharmaceutically acceptable" and "physiologically acceptable" carriers, diluents or excipients. As used herein, the term "pharmaceutically acceptable" and "physiologically acceptable," when referring to carriers, diluents or excipients includes solvents (aqueous or non-aqueous), detergents, solutions, emulsions, dispersion media, coatings, isotonic and absorption promoting or delaying agents, compatible with pharmaceutical administration and with the other components of the formulation.

[00152] Pharmaceutical compositions can be formulated to be compatible with a particular route of administration. Compositions for parenteral, intradermal, or subcutaneous administration can include a sterile diluent, such as water, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents. The preparation may contain one or more preservatives to prevent microorganism growth (e.g., antibacterial agents such as ben2yl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as emylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose).

[00153] Pharmaceutical compositions for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and polyetheylene glycol), and suitable mixtures thereof. Fluidity can be maintained, for example, by the use of a coating such as lecithin, or by the use of surfactants.

Antibacterial and antifungal agents include, for example, parabens, chlorobutanol, phenol, ascorbic acid and thimerosal. Including an agent that delays absorption, for example, aluminum monostearate and gelatin can prolonged absorption of injectable compositions.

[00154] Additional pharmaceutical formulations and delivery systems are known in the art and are applicable in the methods of the invention (see, e.g., Remington's Pharmaceutical Sciences (1990) 18th ed., Mack Publishing Co., Easton, PA; The Merck Index (1996) 12th ed., Merck Publishing Group, Whitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms, Technonic Publishing Co., Inc., Lancaster, Pa., (1993); and Poznansky, et al, Drug Delivery Systems, R. L. Juliano, ed., Oxford, N.Y. (1980), pp. 253-315).

[00155] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein. [00156] All applications, publications, patents and other references, GenBank citations and ATCC citations cited herein are incorporated by reference in their entirety. In case of conflict, the specification, including definitions, will control.

100157] As used herein, the singular forms "a", "and," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a first agent" or a "second agent" includes a plurality of such first or second agents. Reference to "an LHRH receptor (R)" includes a plurality of such receptors, and so forth.

[00158] As used herein, all numerical values or ranges include fractions of the values and integers within such ranges and fractions of the integers within such ranges unless the context clearly indicates otherwise. Thus, to illustrate, reference to a numerical range, such as a percentage range, 90-100%, includes 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so forth. Reference to a range of 1 to 10 amino acids therefore includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. Reference to a range includes subranges. For example, includes a range of 1 to 10 amino acids therefore includes 1 to 8, 3 to 10, 2-8, 3-7, 5-10, etc. Reference to a series of ranges includes combinations of the upper and lower end of the ranges. For example, reference to a series of ranges from 1 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 60 to 70, 70 to 80, 80 to 90, 90 to 100, includes ranges from 5-20, 5-50, 5-100, 20-50, 20-100, 30-50, 30-100, etc. and, so forth.

[00159] The invention is generally disclosed herein using affirmative language to describe the numerous embodiments. The invention also specifically includes embodiments in which particular subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, procedures, assays or analysis. Thus, even though the invention is generally not expressed herein in terms of what the invention does not include aspects that are not expressly included in the invention are nevertheless disclosed herein.

[00160] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the following examples are intended to illustrate but not limit the scope of invention described in the claims.

Examples

Example 1

[00161] This example includes a description of materials and methods.

[00162] In vitro: Cytotoxic effects of EP-100 (KFAKFAKKFAKFAKKFAKQHWSYGLRPG) were determined using the MTT cell viability assay. Briefly, 5,000 cells/well (MIAPaCa2, PANC-1 and BxPC-3) were plated in 96-well plates in triplicate, allowed to attach overnight and then treated with EP- 100 (0 - 200 μΜ) for 48 h. At the end of exposure, 20

was added to each well. After 4 h of incubation with the MTT reagent, the cells were lysed using a lysis buffer (50% DMF, N,N- Dimethylformamide and 20% Sodium dodecyl sulphate, SDS) at 37 °C overnight and the absorbance at 590 ran, which is directly proportional to the number of live cells, was determined using a microplate reader. Cells treated with vehicle served as a control. The cell viability (% control) was determined and the IC₅₀ was calculated according to the Hill equation using GraphPad Prism (v.4) software.

[00163] To determine whether EP-100 cytotoxicity to pancreatic cancer cells is mediated by LHRH receptors, all three cell lines were pre-incubated with 10 μΜ LHRH for 1 hour and then with 10 μΜ EP- 100 for 48 h and the cell viability (% control) was determined by the MTT assay as described above and percent cytotoxicity was plotted.

[00164] To determine if FSH increases the cytotoxicity of EP-100 to PANC-1 and BxPC-3 cells, the IC₅₀ values of EP-100 were determined using the MTT assay by incubating the PANC-1 and BxPC-3 cells for 48 hours in the presence or absence of FSH (0, 10 and 30 r|g/ml) and increasing concentrations (0-20 μΜ) of EP-100.

[00165] In vivo: Eighty four female nude mice (athymic Balb/C, Charles River Laboratories,

Wilmington, MA) were injected subcutaneously in the interscapular area with lxlO⁶ PANC-1 human pancreatic cancer cells and Matrigel (0.3ml). When the resulting tumors reached 100-200 mm³, the mice were randomly allotted to seven treatment groups (11 /group) as follows: 1) Vehicle treated controls; 2) "Baseline" controls sacrificed at the beginning of the treatment; 3) EP-100 treated (0.02 mg/kg); 4) EP- 100 treated (0.02 mg/kg); pre-treated with FSH (3 μg/day) for 3 days; 5) EP-100 treated (0.2 mg/kg); 6) EP-100 treated (0.2 mg/kg); pre-treated with FSH (3 μg/day) for 3 days; and 7) FSH treated (3 μg/day) for 3 days.

[00166] All mice were caged individually, under controlled light conditions (12/12) and fed a standard irradiated laboratory ration. Treatments (except FSH) were administered by tail vein injections given once a week for three weeks. The mice in groups 4, 6 and 7 were pre-treated with FSH (3 μg/day) subcutaneously, on each of the three days, preceding the EP-100 injections. Mice in the baseline group were sacrificed just prior to the first treatment. Mice in all the remaining groups were necropsied one week following the last injection. Body weights, tumor weights and volumes were recorded and images of all the tumors taken. The following tissues were removed, weighed and fixed for later

histopathological studies: tumors, ovaries, pituitaries, lungs, kidneys, livers, uteri, pancreases and spleen.

Example 2

[00167] This example includes a description of studies demonstrating that LHRH receptor targeting conjugates kill pancreatic cancers in vivo.

[00168] Adrninistration of lytic peptide conjugates LHRH-Phor21, a conjugate of LHRH and Curcumin, and a conjugate of LHRH and a lytic peptide (EP-100) each cause growth inhibition, and in some cases complete regression of pancreatic cancer cell (MIAPaCa2) tumors in nude mice (Figure 1). These compounds were designed to target LHRH receptors over-expressed by the cancer cells and destroy them. LHRH hormone receptors are over-expressed by pancreatic cancer cells, but not expressed by normal pancreatic cells. However, all pancreatic cancer cell lines do not over-express LHRH receptors to the same degree. This could limit the ability of these drugs to bind to and destroy tumors arising from the cells that express fewer receptors.

Example 3

[00169] This example includes a description of studies demonstrating that follicle stimulating hormone (FSH) increases expression of LHRH-receptor, and that EP-100 rapidly kills LHRH-receptor expressing cells.

[00170] Quantitative studies of receptor gene expression showed the following order of LHRH receptor expression in pancreatic cancer cell lines; BxPC-3>MIAPaCa-2>PANC-l (Figure 2). In in vitro studies, treatment of PANC-1 cells in culture with small amounts (10 or 30 ng/ml) of the anterior pituitary hormone, follicle stimulating hormone (FSH), produced a 3-fold increase in LHRH receptor gene expression (Figure 3). Therefore, an analysis of pre-treatment of PANC-1 human pancreatic cancer cells with FSH to enhance the ability of LHRH-receptor targeting EP-100 to destroy these pancreatic cancer cells in vitro was conducted.

[00171] EP-100 (0-200 μΜ) cytotoxicity against MIAPaCa2, PANC-1 and BxPC-3 cells in vitro was determined using TT cytotoxicity assays. The IC50 values of EP-100 for MIAPaCa2, PANC-1 and BxPC-3 were 6.47±0.34 μΜ, 5.42±1.34 μΜ and 4.08±0.76 μΜ (mean±SD) respectively (Figure 4). Maximum cytotoxicity was observed at 20 μΜ and a plateau was observed with EP-100 concentrations > 20 μΜ. A time course study of cytotoxicity for all three cell lines was also performed at 2, 6, 12 and 24 h with EP-100 (0 - 20 μΜ). It was observed that EP-100 kills up to 75% of cells in less than 6h; 24 h exposure with EP-100 resulted in 85 - 90% cell death in all the cell lines. These observations demonstrate that EP-100 is a fast-acting compound. Pretreatment of PANC-1 and BxPC3 pancreatic cancer cells with FSH increased the cytotoxicity of EP-100 (Figures 5 A and 5B).

[00172] To determine if EP-100 targets the LHRH receptors on the pancreatic cancer cell lines for cytotoxicity, an MTT assay was performed in which the pancreatic cancer cells were pre-incubated with 10 μΜ LHRH and then treated with 10 μΜ EP-100 for 48 h (Figure 6). Pre-incubation with LHRH decreased cytotoxicity of EP-100 by 27.08%, 10.94% and 10.73% (***P<0.0001) in MIAPaCa2, PANC- 1 and BxPC-3 cells respectively, indicating that EP-100 targets the LHRH receptors.

Example 4

[00173] This example includes a description of in vivo studies indicating that FSH treatment enhances killing of LHRH-receptor expressing tumors with EP-100. [00174] Tumor volumes during the treatment period and at necropsy are shown in Figures 7A and 7B. Tumor volumes during treatment at both dose levels, with or without FSH pre-treatment, were clearly reduced compared to vehicle treated controls (Figure 7A). Tumor volumes at necropsy were also significantly lower (***P<0.0001) than that of vehicle treated mice (Figure 7B), and significantly lower than that of "Baseline" control mice (' ¹ 'PO.0001). There was a significant difference in tumor volumes at necropsy between mice treated with 0.02 and 0.2 mg/kg (^†††P<0.0001).

[00175] Pre-treatment with FSH significantly lowered the tumor volumes at the higher dose of EP-100, 0.2 mg/kg (^ΛΛΡ<0.001) but not at the lower dose. Images of the tumors taken at the time of necropsy clearly indicate differences in the tumor volumes in the EP-100 treated groups.

[00176] Similarly, tumor weights at necropsy (Figure 8 A) were also significantly reduced in all EP-100 treated groups compared to the vehicle treated controls (**P<0.001 and ***P<0.0001) and to the baseline control group fP<0.05, ^PO.0001). EP-100, at 0.02 mg/kg pre-treated with FSH, 0.2 mg/kg with and without FSH pre-treatment significantly regressed the tumor weights compared to baseline (Figure 8B). FSH pre-treatment improved the efficacy at both dose levels when compared to baseline tumor weights. However, at the lower dose of EP-100 (0.02 mg/kg), pre-treatment with FSH did not significantly reduce tumor weights when compared to 0.02 mg/kg EP-100 alone (P>0.05). At the higher dose of EP-100 (0.2 mg/kg), pre-treatment with FSH significantly reduced tumor weights compared to 0.2 mg/kg EP-100 alone, (PO.05). (Figure 8B)

[00177] There was no significant difference in body weights of mice in any of the treatment groups compared to the control group during the treatment period or at the end of treatment. None of the mice showed any signs of cachexia during the studies.

[00178] Tumor, ovary and pituitary tissues were fixed and embedded in paraffin blocks. Lung, kidney, liver, uteri, pancreatic and splenic tissues were fixed in 10% formalin. Histological analysis of the tumors treated with EP-100 and EP-100 plus FSH revealed marked increases in necrotic cells with pyknotic nuclei and a decrease in the number of dividing cells compared to the tumors obtained from control animals. The ovaries of control, EP-100 and EP-100 plus FSH treated mice all contained primordial, primary, secondary and tertiary follicles and corpora lutea, but ovaries from the FSH pretreated animals contained many mature follicles and were highly vascularized.

[00179] In sum, FSH and other compounds that increase LHRH-receptor can be used with an LHRH- receptor targeting ligand, such as a hormone or antibody, to kill cells that express LHRH-receptor. In particular, EP-100, a conjugate of LHRH and a lytic peptide

(KFAKFAKKFAKFAKKFAKQHWSYGLRPG) is cytotoxic to human pancreatic cancer cells

(MIAPaCa2, PANC-1 and BxPC-3), that over-express LHRH receptor, with IC₅₀ values ranging from 4- 6 μΜ. In vitro, pre-incubation of these cells with LHRH decreases the cytotoxicity of EP-100 indicating that EP-100 targets the LHRH-receptors on these pancreatic cancer cells. EP-100 alone, at 0.02 mg/kg completely prevented the growth and at 0.2 mg kg, caused regression of PANC-1 tumor xenografts in nude mice. FSH pre-treatment, at both dose levels caused significant (P<0.05) regression of tumor weights and volumes compared to the baseline mdicating that FSH pre-treatment improved the ability of EP-100 to target and destroy the PANC-1 tumor xenografts by increasing the number of LHRH receptors.

Claims

What is Claimed:

1. A method of stimulating, increasing or enhancing killing of a cell that expresses luteinizing hormone releasing hormone (LHRH) receptors, comprising contacting the cells with a first agent that stimulates, induces, increases or enhances expression of LHRH receptors, or a receptor that binds to LHRH, and contacting the cells with a second agent that binds to LHRH receptors.

2. The method of claim 1 , wherein said first or second agent comprises a hormone.

3. The method of claim 1 , wherein the first agent comprises folhcle-stimulating hormone (FSH) or an FSH analogue, epidermal growth factor (EGF) or an EGF analogue, a steroid hormone, estradiol or an estradiol analogue, or testosterone or a testosterone analogue.

4. The method of claim 1 , wherein said first or second agent comprises a peptide, polypeptide, protein, steroid, nucleic acid or carbohydrate.

5. The method of claim 1 , wherein said first or second agent consists of or comprises an amino acid sequence of about 1 to 10, 10 to 20, 15 to 20, 20 to 30, 30 to 40, 40 to 50, 60 to 70, 70 to 80, 80 to 90, 90 to 100 or more amino acids.

6. The method of claim 1 , wherein said first or second agent includes one or more L- or D- amino acids.

7. The method of claim 1 , wherein said second agent binds to LHRH receptor extracellular domain or portion of the receptor.

8. The method of claim 1 , wherein said second agent binds to an ammo-terminal 1 to 29 amino acid sequence or ammo-terminal 1 to 50 amino acid sequence of LHRH receptor.

9. The method of claim 1, wherein said second agent inhibits, reduces, decreases or blocks binding of luteinizing hormone-releasing hormone (LHRH), an LHRH (binding) fragment, or an LHRH analogue to the LHRH receptor.

10. The method of claim 1 , wherein the second agent comprises an LHRH receptor ligand or a subsequence thereof.

11. The method of claim 10, wherein said ligand is a hormone, a hormone analogue, a fragment of a hormone or a hormone analogue that binds to an LHRH receptor, or wherein said ligand comprises an LHRH receptor agonist or antagonist.

12. The method of claim 11 , wherein the analogue is selected from mifepristone, flutaminde, lupron, zoladex, supprelin, synatel triptorelin, buserelin, centrorelix, ganirelix, abarelix, antide, teverelix and degarelix (Fe200486).

13. The method of claim 1, wherein the second agent comprises a chemotherapeutic drug, a radionuclide, bacterial toxin or bacterial toxin fragment, or a rx>re-forming peptide.

14. The method of claim 1 , wherein the second agent comprises an antibody or antibody subsequence thereof that binds to an LHRH receptor.

15. The method of claim 14, wherein the antibody or subsequence thereof is a monoclonal antibody.

16. The method of claim 14, wherein the antibody or subsequence thereof is human, humanized or primatized.

17. The method of claim 14, wherein the antibody or subsequence thereof comprises an IgG, IgA, IgM, IgE, or IgD.

18. The method of claim 14, wherein the antibody or subsequence thereof comprises an Fab, Fab', F(ab')₂, Fv, Fd, single-chain Fv (scFv), disulfide-linked Fvs (sdFv), V_L, V_H, Camel Ig, V-NAR, VHH, trispecific (Fab₃), bispecific (Fab₂), diabody ((V_L-V_H^ or (V_H-V_L).), triabody (trivalent), tetrabody (tetravalent), minibody ((SCF_V-C_H3)₂), bispecific single-chain Fv (Bis-scFv), IgGdeltaCH2, scFv-Fc, (scFv)₂-Fc, affibody, aptamer, avimer or nanobody.

19. The method of claim 11 or 14, wherein the LHRH receptor ligand, subsequence thereof, or the antibody or subsequence thereof is fused or conjugated to a cell cytotoxic or lytic moiety, or is not fused or not conjugated to a cell cytotoxic or lytic moiety.

20. The method of claim 19, wherein the cell cytotoxic or lytic moiety comprises a chemotherapeutic drug, a radionuclide, bacterial toxin or bacterial toxin fragment, or a rx re-forming peptide.

21. The method of claim 19, wherein the cell cytotoxic or lytic moiety comprises a cecropin, defensin, mehttin, sarcotoxin, magainin peptide, or a subsequence, an analog or hybrid (chimera) thereof.

22. The method of claim 19, wherein said cell cytotoxic or lytic moiety comprises a peptide sequence.

23. The method of claim 22, wherein the peptide sequence forms an alpha helix, an amphipathic alpha helical structure, or is cationic.

24. The method of claim 22, wherein the peptide sequence forms an amphipathic alpha helical structure of at least 30% of the length of the peptide.

25. The method of claim 19, wherein said cell cytotoxic or lytic moiety consists of a sequence from about 10 to about 50 L- or D-amino acids, or from about 15 to about 100 L- or D-amino acids.

26. The method of claim 19, wherein said cell cytotoxic or lytic moiety comprises or consists of an amino acid sequence selected from KFAKFAKKFA FAK (Phorl4),

KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF, JAKFAKKFAKFAKKFA,

KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKFAKKFAKF, KFAKFAKKFAKFAKKFAKFA and

KFAKFAKKFAKFAKKFAKFAK (Phor21); or comprises or consists of an amino acid sequence selected from KFAKFAKKFAKFAK (Phorl4), KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF,

KFAKFAKKFAKFAKKFA, KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKFAKKFAKF,

KFAKFAKKFAKFAKKFAKFA and KFAKFAKKFAKFAKKF AKFAK (Phor21) having one or more of the K residues substituted with any of an F or L residue, one or more of the F residues substituted with any of a K, A or L residue, or one or more of the A residues substituted with any of a K, F or L residue.

27. The method of claim 19, wherein said cell cytotoxic or lytic moiety comprises or consists of a 10 to 100 amino acid sequence that includes an amino acid sequence selected from

KFAKFAKKFAKFAK (Phorl4), KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF,

KFAKFAKKFAKFAKKFA, KFAKFAKKFAKFAK FAK, KFAKFAKKFAKFAKKFAKF,

KFAKFAKKFAKFAKKFAKFA and FAKFAKKFAKFAKKFAKFAK (Phor21); or an amino acid sequence selected from KFAKFAKKFAKFAK (Phorl4), KFAKFAKKFAKFAKK,

KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKFA, KFAKFAKKFAKFAKKFAK,

KFAKFAKKFAKFAKKFAKF, KFAKFAKKFAKFAKKFAKFA and

KFAKFAKKFAKFAKKFAKFAK (Phor21); or comprises or consists of a 10 to 100 amino acid that includes an amino acid sequence selected from KFAKFAKKFAKFAK (Phorl4),

KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKFA,

KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKFAKKFAKF, KFAKFAKKFAKFAKKFAKFA and KFAKFAKKFAKFAKKFAKFAK (Phor21); or an amino acid sequence selected from

KFAKFAKKFAKFAK (Phorl4), KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF,

KFAKFAKKFAKFAKKFA, KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKFAKKFAKF,

KFAKFAKKFAKFAKKFAKFA and KFAKFAKKFAKFAKKFAKFAK (Phor21) having one or more of the K residues substituted with any of an F or L residue, one or more of the F residues substituted with any of a K, A or L residue, or one or more of the A residues substituted with any of a K, F or L residue.

28. The method of claim 19, wherein said cell cytotoxic or lytic moiety comprises

FALALKALKKALKKXKKALKKAL (hecate), Phe Ala Phe Ala Phe Lys Ala Phe Lys Lys Ala Phe Lys Lys Phe Lys Lys Ala Phe Lys Lys Ala Phe (D1A21); Phe Ala Lys Lys Phe Ala Lys Lys Phe Lys Lys Phe Ala Lys Lys Phe Ala Lys Phe Ala Phe Ala Phe (D2A21): Lys Arg Lys Arg Ala Val Lys Arg Val Gly Arg Arg Leu Lys Lys Leu Ala Arg Lys lie Ala Arg Leu Gly Val Ala Phe (D5C); and Lys Arg Lys Arg Ala Val Lys Arg Val Gly Arg Arg Leu Lys Lys Lee Ala Arg Lys lie Ala Arg Leu Gly Val Ala Lys Leu Ala Gly Leu Arg Ala Val Leu Lys Phe (D5C1), or a subsequence thereof.

29. The method of claim 19, wherein the LURH receptor ligand, subsequence thereof, or the antibody or subsequence thereof fused or conjugated to a cell cytotoxic or lytic moiety has no detectable hemolytic activity against human red blood cells, in vitro or in vivo.

30. The method of claim 19, wherein the LURH receptor ligand, subsequence thereof, or the antibody or subsequence thereof fused or conjugated to a cell cytotoxic or lytic moiety has a hemolytic activity of more than about 500 uM, or more than about 250 μΜ, or more than about 100 uM, or more than about 75 μΜ, or more than about 50 μΜ, or more than about 25 μΜ, or more than about 10 μΜ, or more than about 5 μΜ, against human red blood cells.

31. The method of claim 19, wherein said LHRH receptor ligand or subsequence thereof, or antibody or subsequence thereof, and said cytotoxic or lytic moiety are joined by a covalent bond, or a peptide or non-peptide linker.

32. The method of claim 19, wherein said LHRH receptor ligand or subsequence thereof, or antibody or subsequence thereof, and said cytotoxic or lytic moiety are joined by a peptide sequence having from 1 to 50 L- or D-amino acid residues, or a linear carbon chain.

33. The method of claim 19, wherein said LHRH receptor ligand or subsequence thereof, or antibody or subsequence thereof, and said cytotoxic or lytic moiety are joined by a peptide sequence comprising one or more A, S or G amino acid residues.

34. The method of claim 19, wherein said LHRH receptor ligand or subsequence thereof, or antibody or subsequence thereof, is positioned at the NH₂-tenninus relative to said cytotoxic or lytic moiety, or wherein said LHRH receptor ligand or subsequence thereof, or antibody or subsequence thereof, is positioned at the COOH-terrninus relative to said cytotoxic or lytic moiety.

35. The method of claim 1, wherein said second agent consists of or comprises an amino acid sequence set forth as: QHWSYGLRPG.

36. The method of claim 14, wherein said antibody or said subsequence thereof competes with one or more of Ab-3 mouse monoclonal antibody, AT2.G7 mouse monoclonal antibody, GRX-6 mouse monoclonal antibody, GRX-5 mouse monoclonal antibody, GRX-8 antibody, 67-R IgG_2a mouse monoclonal antibody, or any of mouse monoclonal antibodies that binds to Human Gonadotropin- Releasing Hormone Receptor (Order number ΑΒΓ 201809, Order number AB 210677, Order number ABIN243903, Order number ΑΒΓΝ243903, Order number ABIN330374, Order number ΑΒΓΝ330377, Order number ABM337150, Order number ABIN345094, Order number ABIN345095, and Order number ABIN345539) for binding to LHRH/GnRH receptor.

37. The method of claim 14, wherein said antibody or said subsequence thereof binds to the epitope to which one or more of Ab-3 mouse monoclonal antibody, AT2.G7 mouse monoclonal antibody, GRX-6 mouse monoclonal antibody, GRX-5 mouse monoclonal antibody, GRX-8 antibody, 67-R IgG_2a mouse monoclonal antibody, or any of mouse monoclonal antibodies that bind to Human Gonadotropin-Releasing Hormone Receptor (Order number ΑΒΓΝ201809, Order number ABIN210677, Order number ΑΒΓΝ243903, Order number ΑΒΓΝ243903, Order number ABIN330374, Order number ΑΒΓΝ330377, Order number AB 337150, Order number ABIN345094, Order number ΑΒΓΝ345095, and Order number ABIN345539) binds.

38. The method of claim 1, wherein the method kills the cell or cells, or induces cell lysis or apoptosis, of a portion of the cells.

39. The method of claim 1, wherein said cell is a hyperproliferative cell.

40. The method of claim 1 , wherein said cell is a metastatic or non-metastatic neoplastic, tumor or cancer cell.

41. The method of claim 1 , wherein said cell is a breast, ovarian, uterine, cervical, prostate, testicular, adrenal, skin, pancreatic, blood, pituitary or endometrial cell.

42. The method of claim 1 , wherein the contact is in vitro, ex vivo or in vivo.

43. The method of claim 1, wherein said first and second agents are in an amount effective to decrease, reduce or inhibit proliferation of the cell, or survival of the cell.

44. The method of claim 1 , wherein said first and second agents are in an amount effective to treat a subject having undesirable cell proliferation or a hyperproUferative disorder.

45. The method of claim 1 , wherein said first and second agents are in an amount effective to treat a subject having a metastatic or non-metastatic tumor, cancer or neoplasia.

46. The method of claim 1 , wherein said first and second agents are in an amount effective to treat a subject having undesirable fibrosis or a fibrotic disorder.

47. A method of treating a subject having a hyperorohferative disorder, comprising administering to the subject an amount of a first agent sufficient to stimulate, induce, increase or enhance expression of LHRH receptors by cells of the hyperproliferative disorder, or a receptor that binds to LHRH, and administering to the subject a second agent that binds to an LHRH receptor, to treat the hyperproliferative disorder.

48. The method of claim 47, wherein the hyperproliferative disorder comprises a neoplasia, tumor, cancer or mahgnancy.

49. A method of reducing or inhibiting metastasis of a neoplasia, tumor, cancer or malignancy to other sites, or formation or establishment of metastatic neoplasia, tumor, cancer or malignancy at other sites distal from a primary neoplasia, tumor, cancer or mahgnancy, comprising administering to the subject an amount of a first agent sufficient to stimulate, induce, increase or enhance expression of LHRH receptors by cells of the hyperproliferative disorder, or a receptor that binds to LHRH, and aclministering to the subject a second agent that binds to an LHRH receptor, to reduce or inhibit metastasis of the neoplasia, tumor, cancer or mahgnancy to other sites, or formation or establishment of metastatic neoplasia, tumor, cancer or mahgnancy at other sites distal from the primary neoplasia, tumor, cancer or mahgnancy.

50. The method of claims 49, wherein the neoplasia, tumor, cancer or mahgnancy is metastatic, non-metastatic or benign.

51. The method of claim 49, wherein the neoplasia, tumor, cancer or malignancy comprises a solid cellular mass.

52. The method of claim 49, wherein the neoplasia, tumor, cancer or mahgnancy comprises a carcinoma, sarcoma, lymphoma, leukemia, adenoma, adenocarcinoma, melanoma, glioma, glioblastoma, meningioma, neuroblastoma, retinoblastoma, astrocytoma, ol igodendrocytoma, mesothelioma, reticuloendothelial, lymphatic or haematopoietic neoplasia, tumor, cancer or mahgnancy.

53. The method of claim 49, wherein the sarcoma comprises a lymphosarcoma, liposarcoma, osteosarcoma, chondrosarcoma, leiomyosarcoma, rhabdomyosarcoma or fibrosarcoma.

54. The method of claim 49, wherein the haematopoietic neoplasia, tumor, cancer or malignancy comprises a myeloma, lymphoma or leukemia.

55. The method of claim 49, wherein the sarcoma comprises a lymphosarcoma, liposarcoma, osteosarcoma, chondrosarcoma, leiomyosarcoma, rhabdomyosarcoma or fibrosarcoma.

56. The method of claim 49, wherein the neoplasia, tumor, cancer or malignancy comprises a lung, thyroid, head or neck, nasopharynx, throat, nose or sinuses, brain, spine, breast, adrenal gland, pituitary gland, thyroid, lymph, gastrointestinal (mouth, esophagus, stomach, duodenum, ileum, jejunum (small intestine), colon, rectum), genito-urinary tract (uterus, ovary, cervix, endometrial, bladder, testicle, penis, prostate), kidney, pancreas, liver, bone, bone marrow, lymph, blood, muscle, or skin neoplasia, tumor, or cancer.

57. The method of claim 56, wherein the lung neoplasia, tumor, cancer or malignancy comprises small cell lung or non-small cell lung cancer.

58. The method of claim 49, wherein the neoplasia, tumor, cancer or malignancy comprises a stem cell neoplasia, tumor, cancer or malignancy.

59. The method of claim 49, wherein the neoplasia, tumor, cancer, or metastasis comprises a stage I, II, ΠΙ, IV or V neoplasia, tumor, cancer, or metastasis.

60. The method of claim 49, wherein the neoplasia, tumor, cancer, or metastasis is progressively worsening, or is in remission.

61. The method of claim 49, wherein the method inhibits or reduces relapse or progression of the neoplasia, tumor, cancer or malignancy.

62. The method of claim 49, further comprising administering an anti-cell proliferative, antineoplastic, anti-tumor, anti-cancer or immune-enhancing treatment or therapy.

63. The method of claim 62, wherein the treatment or therapy comprises surgical resection, radiotherapy, ionizing or chemical radiation therapy, chemotherapy, immunotherapy, local or regional thermal (hyperthermia) therapy, or vaccination.

64. The method of claim 62, wherein the treatment or therapy comprises administering an alkylating agent, anti-metabolite, plant extract, plant alkaloid, nitrosourea, hormone, nucleoside or nucleotide analogue.

65. The method of claim 62, wherein the treatment or therapy comprises administering cyclophosphamide, azathioprine, cyclosporin A, prednisolone, melphalan, chlorambucil,

mecWoremamine, busulphan, methotrexate, 6-mercaptopurine, thioguanine, 5-fluorouracil, cytosine arabinoside, AZT, 5-azacytidine (5-AZC) and 5-azacytidine related compounds, bleomycin, actinomycin D, mithramycin, mitomycin C, carmustine, lomustine, semustine, streptozotocin, hydroxyurea, cisplatin, mitotane, procarbazine, dacarbazine, taxol, vinblastine, vincristine, doxorubicin or dibromomannitol.

66. The method of claims 1 or 49, wherein the first agent is adrninistered prior to, substantially contemporaneously with or following aclministration of the second agent.

67. The method of claims 1 or 49, wherein the subject is a candidate for or has undergone surgical resection, chemotherapy, immunotherapy, ionizing or chemical radiotherapy, local or regional thermal (hyperthermia) therapy, or vaccination.

68. The method of claims 1 or 49, wherein the treatment results in partial or complete destruction of the neoplastic, tumor, cancer or malignant cell mass, volume, size or numbers of cells, stimulating, inducing or increasing neoplastic, tumor, cancer or malignant cell necrosis, lysis or apoptosis, reducing neoplasia, tumor, cancer or malignancy volume size, cell mass, inhibiting or preventing progression or an increase in neoplasia, tumor, cancer or malignancy volume, mass, size or cell numbers, or prolonging lifespan.

69. The method of claims 1 or 49, wherein the treatment results in reducing or decreasing severity, duration or frequency of an adverse symptom or complication associated with or caused by the neoplasia, tumor, cancer or malignancy, or pain, discomfort, nausea, weakness or lethargy.

70. The method of claims 1 or 49, wherein the treatment results in increased energy, appetite, improved mobility or psychological well being.

71. A method of reducing fertility of an animal, comprising aclm stering to the animal an amount of a first agent that stimulates, induces, increases or enhances expression of LHRH receptors by cells, or a receptor that binds to LHRH, and administering to the animal a second agent that binds to LHRH receptors, sufficient to reduce fertility.

72. A method of reducing or treating endometriosis of an animal, comprising administering to the animal an amount of a first agent that stimulates, induces, increases or enhances expression of LHRH receptors by cells, or a receptor that binds to LHRH, and administering to the animal a second agent that binds to LHRH receptors, sufficient to treat endometriosis.

73. A method of reducing or treating benign prostate hyperplasia of an animal, comprising administering to the animal an amount of a first agent that stimulates, induces, increases or enhances expression of LHRH receptors by cells, or a receptor that binds to LHRH, and administering to the animal a second agent that binds to LHRH receptors, sufficient to treat benign prostate hyperplasia.

74. A method of reducing or treating a fibroid or polyp of an animal, comprising administering to the animal an amount of a first agent that stimulates, induces, increases or enhances expression of LHRH receptors by cells, or a receptor that binds to LHRH, and administering to the animal a second agent that binds to LHRH receptors, sufficient to treat the fibroid or polyp.

75. The method of claim 74, wherein the fibroid or polyp is present in breast, uterus, vagina, cervix or fallopian tube.

76. The method of claim 1 , wherein the cell is in a subject.

77. The method of claim 1 , wherein the cell expresses a receptor that binds to luteinizing hormone-releasing hormone I, luteinizing hormone-releasing hormone Π, or lamprey III luteinizing hormone-releasing hormone.

78. The method of claims 1, 49 or 71 to 74, wherein the subject is a mammal.

79. The method of claims 1 , 49 or 71 to 74, wherein the subject is a human.

80. The method of claims 49 or 71 to 74, wherein the first or second agent is administered to the subject locally, regionally, or systemicaUy, or into the neoplasia, tumor, cancer, or metastasis.