US 12,691,179 B2Grant
Somatostatin receptor-based cancer therapy
Issue Date:2026-07-28
•8 Claims
•14 Drawing Sheets
Abstract
Methods and composition for cell-based therapy as well as somatostatin receptor-based therapy are described. For example, in certain aspects methods for administering an anti-tumor therapy using a signaling defective somatostatin receptor mutant are described. Furthermore, the invention provides compositions and methods involve a somatostatin constitutively active somatostatin receptor mutant.
Metadata
Assignee
- Vikas Kundra
Inventor
- Vikas Kundra
Application Information
Application Number:US 18/149,414
Filing Date:2023-01-03
Priority Date:2010-09-08
Art Unit:1638
Classifications
IPC:
A61K47/46A61K35/28A61K38/17A61K38/31A61K47/64A61K47/69A61K51/00A61K51/08C07K14/71C12N15/88
Patent Drawings (14 sheets)
Description
[0001] This application is a divisional of U.S. application Ser. No. 16/659,986, filed Oct. 22, 2019, now U.S. Pat. No. 11,541,119, which is a divisional of U.S. application Ser. No. 15/647,840, filed Jul. 12, 2017, now U.S. Pat. No. 10,493,162, which is a divisional of U.S. application Ser. No. 13/821,541, filed Jun. 26, 2013, now U.S. Pat. No. 9,731,023, which is a national phase application under 35 U.S.C. § 371 of International Application No. PCT/US2011/050803, filed Sep. 8, 2011, which claims priority to U.S. Provisional Application No. 61/380,920 filed on Sep. 8, 2010, the entire content of each of which is hereby incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] This application contains a Sequence Listing XML, which has been submitted electronically and is hereby incorporated by reference in its entirety. Said XML Sequence Listing, created on Jan. 3, 2023, is named KUNDP0002USD3.xml and is 57,075 bytes in size.
1. FIELD OF THE INVENTION
[0003] The present invention relates generally to the field of molecular biology, cell therapy, gene therapy, and cancer therapy. More particularly, it concerns methods for treatment of a subject by administration of cells comprising somatostatin receptor such as a signaling deficient somatostatin receptor mutant. The invention also generally pertains to methods for therapy that involves constitutively active somatostatin receptor mutants.
2. DESCRIPTION OF RELATED ART
[0004] Therapeutic treatment of many human disease states is limited by the systemic toxicity of the therapeutic agents used. Cancer therapeutic agents in particular exhibit a very low therapeutic index, with rapidly growing normal tissues such as skin and bone marrow typically affected at concentrations of agent that are not much higher than the concentrations used to kill tumor cells. For example, gene therapy and cellular therapies have great promise, but they suffer from a lack of methodology for specifically targeting and localization of a gene therapy and/or a cell expressing a recombinant nucleic acid within an organism. Treatment and diagnosis of cancer would be greatly facilitated by the development of compositions and methods for targeted delivery to cancer cells.
[0005] Somatostatin receptors are known to be expressed in a large number of human tumors and represent the basis for in vivo tumor targeting. However, not all tumors express somatostatin receptors. In a pre-clinical tumor model, a recombinant somatostatin receptor type 2 (SSTR2) chimera can serve as a reporter gene of gene expression that can be quantified in vivo and can also serve as a growth inhibitor when targeting cancer. However, this SSTR-mediated signaling, such as growth inhibition, may or may not be desirable in different therapy purposes wherein the SSTR-specific targeting is envisioned.
[0006] Therefore, there exists a need of novel methods for targeted therapy, especially cancer therapy.
SUMMARY OF THE INVENTION
[0007] Aspects of the present invention overcome a major deficiency in the art by providing novel methods and compositions for treating a disease or a condition with cell therapy, gene therapy, radiotherapy, additional anti-tumor therapy, or a combination thereof.
[0008] For targeted cellular therapy, cells having an exogenous expression construct may be delivered to a disease site, such as a tumor, and a therapeutic may target the cells and exert a bystander therapeutic effect to the tumor. For example, a radiotherapeutic may bind the delivered cells, more specifically, the exogenous expressed product in the delivered cells, and kill the neighboring tumor by bystander effect. Alternatively, a thermal therapeutic such as a photothermal therapeutic that bind the delivered cells may kill the neighboring cells upon activation, such as laser-activatable nanoparticles.
[0009] In particular aspects, the treatment methods may be based on somatostatin receptor or its signaling mutants. The signaling mutants may comprise signaling defective mutants or constitutively active mutants. They can be used either on their own or with other agents for therapy. The invention is in part based on the discovery that a radioligand can be therapeutically effective even in cells expressing a signaling deficient SSTR mutant. Radiotherapy has been used to target tumors endogenously expressing SSTR or after gene transfer of wild-type SSTR, but not mutant receptors. The advantages of an SSTR-binding therapeutic such as a radioligand used in combination with an SSTR mutant include, but are limited to, augmentation of the effect of a growth inhibiting mutant, bystander effect, and enablement of cellular therapy using a signaling deficient reporter. In some cell types, SSTR-mediated signaling may not reconstitute, for example, for growth inhibition; tumor growth may be inhibited by other mechanisms such as radiation-induced damage in cells without functional SSTR signaling but expressing SSTR's. It also enables use of agents that may not activate SSTR signaling but bind SSTR's such as antagonists or binding agents that are neither agonists or antagonists.
[0010] Accordingly, in a first embodiment there is provided a method of treating a tumor using a somatostatin receptor such as a signaling defective somatostatin receptor mutant for tumor targeting and therapy. Such a method may be a cell-based therapy, a gene therapy, a radiotherapy, a chemotherapy, an immunotherapy, or a combination thereof.
[0011] In the aspects of a cell-based therapy, the method may comprise providing one or more cells capable of localizing to a tumor. The cells may comprise an expression construct comprising a nucleic acid sequence encoding a somatostatin receptor (SSTR) mutant. In a preferred aspect, the mutant may be a signaling defective mutant to avoid the growth inhibition of delivery cells via a normal somatostatin receptor-mediated signaling. The method may further comprise introducing the cells to a subject having a tumor. Introducing the cells to the subject can be by any method known to those of ordinary skill in the art. For a targeted therapy, the method may further comprise administering to the subject an anti-tumor therapeutic that specifically binds the somatostatin receptor or the cells introduced into the subject and effects a damage on the tumor, for example, through a bystander effect. The therapeutic may be coupled to a ligand that specifically binds the somatostatin receptor, which may be the native form, the signaling defective or constitutively active somatostatin receptor mutant for tumor targeting. The anti-tumor therapeutic may kill the tumor cells which might not express the SSTR by a bystander effect, such as radiotherapy.
[0012] In a further aspect, if the delivered cells or their progeny cells become malignant these cells expressing mutant SSTR may be treated with a therapy targeting the expressed mutant SSTR. This additional benefit serves as an important safeguard against undesired effects of cellular therapy.
[0013] In the aspects of a gene therapy, there may be provided a treatment method comprising providing an expression construct comprising a nucleic acid sequence encoding a signaling altered somatostatin receptor mutant, particularly a constitutively active mutant, and introducing the expression construct into the tumor, for example, directly (e.g., via intratumoral injection) or by a tumor targeting moiety-encoding sequence comprised in the expression construct. The signaling altered mutant may have reduced or enhanced signaling as compared with a native receptor, such as a signaling defective mutant or a constitutively active mutant. The mutant may have transmembrane domains III-VII of a native receptor, therefore retaining the ability to bind a native ligand or an analog.
[0014] The method may further comprise administering to the subject an anti-tumor therapeutic coupled to a ligand that specifically binds the somatostatin receptor or its mutant. A “targeting moiety” is a term that encompasses various types of affinity reagents that may be used to enhance the localization or binding of a substance to a particular location in an animal, including organs, tissues, particular cell types, diseased tissues or tumors. Targeting moieties may include peptides, peptide mimetics, polypeptides, antibodies, antibody-like molecules, nucleic acids, aptamers, and fragments thereof.
[0015] Such a mutant may have at least trans-membrane domains III-VII of a native somatostatin receptor, which may be responsible for binding to its native ligand or any known SSTR binding partners. The mutant may have a mutation that diminishes or eliminates the ability to inhibit cellular growth as compared with a native receptor. The mutant may have a C-terminal deletion. The deletion may include a deletion of an intracellular signaling domain. For example, such a C-terminal deletion include deletion of an amino acid sequence with at least about 80, 85, 90, 95, 99, 99.5, or 100% homology (or any intermediate ranges) to a C-terminal 55-amino acid sequence of human SSTR2 (i.e., amino acid positions 315 to 349 of SEQ ID NO:6) as compared with a corresponding native somatostatin receptor. The mutant may be a truncated mutant, such as a somatostatin receptor that is truncated carboxy terminal to amino acid position 314, particularly human SSTR2 delta 314, which is a human SSTR truncated carboxy terminal to amino acid 314 (having the sequence of amino acids 1-314 of SEQ ID NO:6).
[0016] The mutant may be a mutant of a somatostatin receptor type 1, 2, (2A, 2B), 3, 4, or 5. In further particular embodiments, the mutant is a somatostatin receptor type 2 (SSTR2) mutant. The mutant may be a mutant of a somatostatin receptor of human, mice, rat, primates, or any other mammals. The mutant can be truncated at either the N-terminus or the C-terminus. In some embodiments, there is a truncation at both the N-terminus and the C-terminus. In certain embodiments, the mutant has altered signaling including being signaling defective, altered internalization, or a combination thereof. Altered signaling may include an increase in signaling, a decrease in signaling, or an inciting of signaling pathways different from those incited by the native receptor.
[0017] The cell that is so provided can be any cell known to those of ordinary skill in the art, but in particular embodiments the cell is a stem cell or an immune cell. A “stem cell” generally refers to any cell that has the ability to divide for indefinite periods of time and to give rise to specialized cells. In particular aspects, the invention takes advantage of the tendency for tumors, especially invasive tumors, to attract stem cells or immune cells such as white blood cells. Stem cells thus have the ability to localize to and be incorporated into tumors.
[0018] For example, the stem cell can be an embryonic stem cell, a somatic stem cell, a germ stem cell, an epidermal stem cell, or a tissue-specific stem cell. Examples of tissue-specific stem cells include, but are not limited to, a neural stem cell, a keratinocyte stem cell, a renal stem cell, a embryonic renal epithelial stem cell, an embryonic endodermal stem cell, a hepatocyte stem cell, a mammary epithelial stem cell, a bone marrow-derived stem cell, a skeletal muscle stem cell (satellite cell), a limbal stem cell, a hematopoietic stem cell, a mesenchymal stem cell, peripheral blood mononuclear progenitor cell, a splenic precursor stem cell, and an oesophageal stem cell.
[0019] In some other aspects, the cells may be immune cells. The immune cell may traffic to the tumor by antigen-specific localization. An “immune cell” is any cell associated with generation of an immune response, such as a monocyte, a granulocyte, or a lymphocyte. An “immune cell” is defined herein to refer to a cell that recognizes and responds against microorganisms, viruses, and substances recognized as foreign and potentially harmful to the body. The granulocyte may be a neutrophil, a basophil, or an eosinophil. The lymphocyte may be a T cell, a B cell, or a NK cell. The immune cell may be a stem cell whose progeny includes any of the aforementioned cells associated with generation of an immune response. Particular examples of immune cells include T cells and B cells.
[0020] The immune cells may be active against tumor cells expressing a particular antigen. The immune cells may be engineered to localize to a tumor expressing an antigen that bind the immune cells. Methods of therapy involving immune cells involve techniques well-known to those of ordinary skill in the art.
[0021] The cell can be obtained from any source, both natural and artificial. For example, the cell is a mammalian cell, more specifically, a human cell or a mouse cell. In some embodiments, the cell is an autologous cell. For example, the cell may be a stem cell obtained from a subject, wherein the cell is reintroduced into the subject following the transfer into the cell of the expression construct. In other embodiments, the cell is an allogeneic cell, or a cell obtained from a subject that is distinct from the subject to whom the cell is introduced, but from the same species. In still further embodiments, the cell is a xenogeneic stem cell, or a cell from a different species than the recipient subject.
[0022] For example, introducing the cell to the subject may involve intravenous administration, intracardiac administration, intradermal administration, intralesional administration, intrathecal administration, intracranial administration, intrapericardial administration, intraumbilical administration, intraocular administration, intraarterial administration, intraperitoneal administration, intraosseous administration, intrahemmorhage administration, intratrauma administration, intratumor administration, subcutaneous administration, intramuscular administration, intravitreous administration, direct injection into a normal tissue or organ, direct injection into a diseased tissue or organ like a tumor, topical administration, or any other method of local or systemic administration known to those of ordinary skill in the art.
[0023] In some embodiments, the nucleic acid includes more than one coding region. For example, the nucleic acid may include a second coding region. For example, the second coding region may be a protein tag gene, a reporter gene, a therapeutic gene, a signaling sequence, or a trafficking sequence. Information regarding somatostatin fusion proteins can be found in U.S. Patent App. Pub. No. 20020173626, herein specifically incorporated by reference. A particular example of a fusion protein may be a sequence expressing both an SSTR mutant and a reporter gene, such as an SSTR mutant linked to a luciferase.
[0024] The term “reporter,” “reporter gene” or “reporter sequence” as used herein refers to any genetic sequence or encoded polypeptide sequence that is detectable and distinguishable from other genetic sequences or encoded polypeptides present in cells. A “therapeutic gene” as used herein refers to any genetic sequence or encoding polynucleotide sequence that is known or suspected to be of benefit in the treatment or prevention of disease in a subject. A “signaling sequence” is defined herein to refer to any genetic sequence or encoded polynucleotide sequence that is involved in signal transduction or cell differentiation. A “trafficking sequence” as used herein refers to any genetic sequence or encoded polypeptide sequence that is involved in the transit of cells from one site in a subject to a different site in the subject. Reporter sequences, therapeutic genes, signaling sequences, and trafficking sequences are well-known to those of ordinary skill in the art, and are discussed at length elsewhere in this specification.
[0025] In some embodiments, the first coding region and the second coding region are linked by an IRES or a bidirectional promoter sequence. A “bidirectional promoter sequence” refers to a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription of both the first and the second coding region are controlled. One of ordinary skill in the art would be familiar with bidirectional promoter sequences, such as those set forth in Trinklein et al., 2004 and at world wide web via stanford.eduMdschroed/bidirectional/index.shtml.)
[0026] The nucleic acid encoding a somatostatin receptor or its mutant may be operably linked to a first promoter. In certain aspects, a second promoter sequence is operatively linked to the second coding region. A promoter sequence used herein, such as the first promoter sequence and the second promoter sequence, may be any promoter sequence, such as a constitutive promoter sequence, a tissue-specific promoter sequence, a lineage-specific promoter, or a function-specific promoter sequence. For example, the first promoter sequence and the second promoter sequence may be of the same type (e.g., both constitutive promoter sequences) or may differ in type (e.g., first promoter sequence is a constitutive promoter sequence, and second promoter sequence is a tissue-specific promoter sequence).
[0027] In further embodiments, the nucleic acid further includes a third coding region. Preferably, the third coding region is operatively linked to a third promoter sequence. The first coding region, the second coding region, and the third coding region may either be independent or operably linked by one or more IRES or bidirectional promoter sequences. The first promoter sequence, the second promoter sequence, and the third promoter sequence may be individually selected from the group consisting of a constitutive promoter sequence, a tissue-specific promoter sequence, a lineage-specific promoter, and a function-specific promoter sequence. As discussed above, the promoter sequences may be of the same type or be of distinct types.
[0028] The nucleic acid encoding the somatostatin receptor or its mutant may or may not further comprise a nucleic acid sequence encoding a protein tag. The protein tag may be any protein tag known to those of ordinary skill in the art. A protein tag may be fused to the N-terminal end or C-terminal end of the somatostatin receptor or its mutant. The protein tag may or may not have enzymatic activity. In embodiments wherein the protein tag has enzymatic activity, the protein tag may be, for example, hemagglutinin A, beta-galactosidase, thymidine kinase, transferrin, myc-tag, VP 16, (His)6-tag, FLAG, or chloramphenicol acetyl transferase.
[0029] The method may father comprise introducing the expression construct into the cells. The expression construct that is transferred into the cell may or may not be comprised in a delivery vehicle. A “delivery vehicle” is defined herein to refer an entity that associates with a nucleic acid and mediates the transfer of the nucleic acid into a cell. Any delivery vehicle is contemplated by the present invention. In some embodiments, for example, transferring the expression construct into the cell comprises contacting the cell with the delivery vehicle.
[0030] For example, the delivery vehicle may include but is not limited to a polypeptide, a lipid, a liposome, lipofectamine, a plasmid, a viral vector, a phage, a polyamino acid such as polylysine, a prokaryotic cell, or a eukaryotic cell. In particular embodiments, the delivery vehicle is a viral vector. The viral vector can be any viral vector known to those of ordinary skill in the art. For example, the viral vector may be a lentiviral vector, a baculoviral vector, a parvovirus vector, a semiliki forest virus vector, a Sindbis virus vector, a lentivirus vector, a retroviral vector, a vaccinia viral vector, an adeno-associated viral vector, a picornavirus vector, an alphavirus vector, or a poxviral vector. In some embodiments, the viral vector is a lentiviral vector. Transferring the expression construct into the cell can be by any method known to those of ordinary skill in the art. For example, transferring the expression construct may involve performing electroporation or nucleofection of the cell in the presence of the expression construct.
[0031] In some embodiments, the method further comprises sorting of the cell from other cells following the transfer of the expression construct. “Sorting” refers to separation of a cell containing the expression construct from other cells that do not contain the expression construct. Sorting can be performed by any method known to those of ordinary skill in the art, and may rely on the presence of the expression construct. For example, sorting may be performed by fluorescence activated cell sorting (FACS), column chromatography, and/or magnetic resonance beads.
[0032] For tumor treatment, a therapeutic coupled to a ligand that specifically binds to the somatostatin receptor or its mutant may be administered to the subject by targeting the somatostatin receptor or its mutant expressed within or near tumor. An “anti-tumor therapeutic” may be defined herein to refer to a cytotoxic or cytostatic agent. The therapeutic may comprise a therapeutic radionuclide, a chemotherapeutic, a tumor suppressor, an inducer apoptosis, an enzyme, an antibody, an antibody fragment, an siRNA, a hormone, a prodrug, an immunostimulant or any therapeutic that have a bystander effect. In a particular aspect, the therapeutic may be a therapeutic radionuclide, such as a β− particle emitter, for example, 177Lu or 90Y. However, many imaging radionuclides have not be found to be therapeutically effective and may not be used as therapeutic radionuclides, such as 111-In and 99mTc.
[0033] In further embodiments, a therapeutic or a detectable moiety is coupled to a ligand that specifically binds the somatostatin receptor or its mutant to localize a therapy or tracking expression. A “ligand” is defined herein to refer to an ion, a peptide, a oligonucleotide, a molecule, or a molecular group that binds to another chemical entity or polypeptide to form a larger complex. For example, in some embodiments, the ligand is a nucleic acid, such as a DNA molecule or an RNA molecule, a protein, a polypeptide, a peptide, an antibody, an antibody fragment, or a small molecule. For example, the ligand may be somatostatin or an analogue thereof, such as octreotate, octreotide, lanreotide, or sandostatin. The ligand may further comprise a chelator molecule, such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or diethylene triamine pentaacetic acid (DTPA). In particular embodiments, the ligand may be [90Y-DOTA]-octreotate or [177Lu-DOTA]-octreotate.
[0034] The method may further comprise tracking the cells or gene expression in the subject. The method may comprise imaging the cells, particularly imaging a reporter encoded by a sequence comprised in the expression construct or a detectable moiety that will bind the somatostatin receptor expressed in the cells. In further embodiments, the method may comprise further administering a detectable moiety couple to a ligand that specifically binds the somatostatin receptor, such as its mutant.
[0035] A “detectable moiety” is defined herein to refer to any molecule or agent that can emit a signal that is detectable by imaging. For example, the detectable moiety may be a protein, a radioisotope, a fluorophore, a visible light emitting fluorophore, a near infrared light emitting fluorophore, infrared light emitting fluorophore, a metal, a ferromagnetic substance, a paramagnetic substance, a superparamagnetic substance, an electromagnetic emitting substance, a substance with a specific MR spectroscopic signature, an X-ray absorbing or reflecting substance, a small molecule, or a sound altering substance. In certain particular embodiments, the detectable moiety is a radioisotope. In particular embodiments, the detectable moiety is 111-In octreotide.
[0036] In some embodiments, the method for tracking the location of a cell in a subject further comprises detecting expression of the mutant or the reporter by assaying for an association between the mutant or reporter expressed by the cell and a detectable moiety. For example, the association between the cell and the detectable moiety comprises binding of the detectable moiety by the cell, binding of a ligand operably coupled to the detectable moiety by the cell, cellular uptake of the detectable moiety, or cellular uptake of a ligand operably coupled to the detectable moiety.
[0037] Contacting the cell with a detectable moiety may occur either prior to or after the cell is introduced into the subject. In particular embodiments, the cell is contacted with a detectable moiety prior to introduction of the cell into the subject. In some embodiments of the present invention, the cells are contacted with a detectable moiety that binds to the SSTR mutant or the reporter prior to introducing the cells into the subject, and in other embodiments, the cells are contacted with the detectable moiety that binds to the reporter or mutant after the cells are introduced into the subject.
[0038] Any imaging technique known to those of ordinary skill in the art can be applied in imaging the detectable moiety. In some embodiments, for example, the imaging technique is an invasive imaging technique. An “invasive imaging technique” is defined herein to refer to any imaging technique that involves removal of tissue from a subject or insertion of a medical device into a subject. Invasive imaging techniques may involve, for example, performance of a biopsy of tissue in conjunction with an imaging technique such as fluorescence microscopy, or insertion of a catheter or endoscope into a subject for purposes of imaging.
[0039] In particular embodiments, the imaging technique is a non-invasive imaging technique. A “non-invasive imaging technique” is defined herein as an imaging technique that does not involve removal of tissue from a subject or insertion of a medical device into a subject. One of ordinary skill in the art would be familiar with non-invasive imaging techniques. Examples include MRI, MR spectroscopy, radiography, CT, ultrasound, planar gamma camera imaging, SPECT, PET, other nuclear medicine-based imaging, optical imaging using visible light, optical imaging using luciferase, optical imaging using a fluorophore, other optical imaging, imaging using near infrared light, imaging using infrared light, photoacoustic imaging or thermoacoustic imaging.
[0040] In a further aspect, there may be provided a constitutively active human somatostatin receptor mutant, particularly a SSTR type 2 mutant. The constitutively active SSTR could inhibit function such as cell growth or hormone secretion without the need for a ligand. Thus, the cost of the ligand may be saved. Ligands such as clinically used octreotide are contraindicated in some patients and side effects may include inhibition of gastrointestinal and pancreatic function causing digestive problems such as loose stools, nausea, gas, and gallstones and sometimes diabetes and hepatitis. The primary current treatment for acromegaly is somatostatin analogues, octreotide and lanreotide. Signaling constitutively active SSTR gene therapy may both inhibit growth hormone secretion and shrink or eliminate the tumor and an appropriate radioligand may increase efficacy of tumor kill. This paradigm may be used in tumors that express somatostatin receptors such as other neuroendocrine tumors like carcinoid or pancreatic tumors. In addition, the advantage of the gene or cellular therapy approach is that it may be effective in most every tumor, including those that do not normally express somatostatin receptors. The growth suppressing receptor mutant in the constitutively active form could eliminate the need to provide octreotide after gene therapy, making the process less cumbersome, more cost effective, and avoiding toxicity of the octreotide.
[0041] The mutant may be a constitutively active form of a native human somatostatin receptor and have a C-terminal deletion. For example, the mutant is an SSTR mutant. In particular, the C-terminal deletion is a deletion of C-terminal amino acids after position 340 (delta 340), such as an SSTR delta 340 mutant having the sequence of amino acids 1-340 of SEQ ID NO:6. There may also be provided an isolated nucleic acid molecule comprising a sequence encoding the mutant and an expression vector (i.e., expression construct) comprising the isolated nucleic acid molecule.
[0042] There may further be provided a method of treating a disease or a condition wherein SSTR-mediated signaling is desired or needed, for example, inhibition of cell growth or inhibition of hormone secretion is desired. Non-limiting examples of the disease or condition may include cancer (including growth, angiogenesis, invasion, and apoptosis), glucose regulation (such as hyperglycemia of diabetes), neuronal and neuromuscular transmission, gastric acid secretion, growth (such as acromegaly), and ocular neovascularization.
[0043] The method may comprise the steps of: a) providing a therapeutic composition comprising an isolated nucleic acid molecule comprising a sequence encoding a constitutively active SSTR mutant; and b) administering a therapeutically effective amount of the therapeutic composition to the subject. The disease or condition may be a tumor, such as cancer (including growth, angiogenesis, invasion, and apoptosis), glucose regulation (such as hyperglycemia of diabetes), neuronal and neuromuscular transmission, gastric acid secretion, growth (such as acromegaly), and ocular neovascularization
[0044] For example, the isolated nucleic acid molecule further comprises a second coding sequence, such as a protein tag gene, a reporter gene, a therapeutic gene, a signaling sequence, or a trafficking sequence.
[0045] In a further aspect, the method may comprise administering to the subject a therapeutic agent such as an anti-tumor therapeutic coupled to a ligand that specifically binds the somatostatin receptor, particularly a signaling altered somatostatin receptor mutant, such as signaling defective mutant or a constitutively active mutant, depending on whether SSTR-mediated signaling is desired. The isolated nucleic acid molecule may be comprised in a gene delivery vehicle. Examples of a gene delivery vehicle include, but are not limited to, a lipid, a liposome, lipofectamine, a plasmid, a viral vector, a phage, a polyamino acid, a particle, calcium phoshate, or DEAE-dextran. The administration of a gene delivery vehicle may include pulmonary administration, endobronchial administration, topical administration, intraocular administration, parenteral administration, intranasal administration, intratracheal administration, intrabronchial administration, intranasal administration, or subcutaneous administration. For tumor targeting, the isolated nucleic acid molecule may further comprise a tumor-selective promoter or a sequence encoding a tumor targeting moiety.
[0046] The subject to be treated may be any animal, such as a human, a mouse, a rat, or any other mammal. The subject may have a tumor such as a neuroendocrine tumor, breast tumor, lung tumor, prostate tumor, ovarian tumor, brain tumor, liver tumor, cervical tumor, colon tumor, renal tumor, skin tumor, head and neck tumor, bone tumor, esophageal tumor, bladder tumor, uterine tumor, lymphatic tumor, stomach tumor, pancreatic tumor, testicular tumor, or lymphoma.
[0047] The disease to be treated can be any disease known to those of ordinary skill in the art. For example, the disease may be a hyperproliferative disease, an infectious disease, an inflammatory disease, a degenerative disease, a congenital disease, a genetic disease, an immunological disease, trauma, poisoning, or a disease associated with toxicity. In particular embodiments, the disease is a hyperproliferative disease. The hyperproliferative disease may be benign or malignant. In particular embodiments, the hyperproliferative disease is cancer. The cancer may be any type of cancer. For example, the cancer may be breast cancer, lung cancer, prostate cancer, ovarian cancer, brain cancer, liver cancer, cervical cancer, colon cancer, renal cancer, skin cancer, head and neck cancer, bone cancer, esophageal cancer, bladder cancer, uterine cancer, lymphatic cancer, stomach cancer, pancreatic cancer, testicular cancer, lymphoma, or leukemia. In further embodiments, the disease is type I diabetes or type II diabetes.
[0048] In still further embodiments, the disease is a cardiovascular disease. For example, the cardiovascular disease may be cardiomyopathy, ischemic cardiac disease, congestive heart failure, congenital cardiac disease, traumatic cardiac disease, toxic cardiac disease, pericarditis, or genetic cardiac disease.
[0049] Alternatively, the disease may be a neurological disease, such as Parkinson's disease, Alzheimer disease, amyotrophic lateral sclerosis, or multiple sclerosis. The neurological disease may be a neurodegenerative disease, spinal cord disease, traumatic neurological disease, infectious disease, or inflammatory disease. The disease may also be an immunological disease, such as transplant rejection, autoimmune disease, immune complex disease, vasculitis, or HIV infection.
[0050] In particular, the condition to be treated may involve any conditions related to growth hormone (GH) excess, such as acromegaly. Acromegaly is characterized by excessive levels of GH in the blood, often resulting from an adenoma of the anterior pituitary. Acromegaly is associated with significant risk of morbidity (soft-tissue swelling, arthralgia, headache, perspiration, fatigue, CV disorders), insulin resistance and diabetes, vision problems resulting from optic nerve compression by the adenoma, and premature mortality. Most of the biological impacts and symptoms related to GH excess are mediated through IGF-1, which is secreted by the liver as well as many other target organs as a result of GH receptor activation. The growth hormone excess may be related to diseases or conditions involve a gastrointestinal system, neurons, or pituitary gland.
[0051] Embodiments discussed in the context of methods and/or compositions of the invention may be employed with respect to any other method or composition described herein. Thus, an embodiment pertaining to one method or composition may be applied to other methods and compositions of the invention as well.
[0052] As used herein the terms “encode” or “encoding” with reference to a nucleic acid are used to make the invention readily understandable by the skilled artisan; however, these terms may be used interchangeably with “comprise” or “comprising” respectively.
[0053] As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one.
[0054] The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” As used herein “another” may mean at least a second or more.
[0055] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.
[0056] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0057] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
[0058] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
[0059] FIG. 1 : Human HA-SSTR2D314 does not inhibit cellular growth upon ligand stimulation; whereas HA-SSTR2D340 inhibits growth even without ligand stimulation, similar to inhibition by wild-type receptor upon activation with ligand. 3000 cells HT1080 cells (fibrosarcoma cells that do not endogenously express SSTR2) stably transfected with HA-SSTR2 wild-type (FL) or C-terminal deletions were grown in the presence of 5% FBS with or without 100 nM of the somatostatin analogue, sandostatin. Three days later, MTT assay was used to determine cell number.
[0060] FIG. 2 : Human HA-SSTR2D340 Inhibits growth even without ligand stimulation. Two days after transient transfection with HA-SSTR2D351 or HA-SSTR2D340, 3000 HT1080 cells (fibrosarcoma cells that do not endogenously express SSTR2) were grown in the presence of 5% FBS with or without 100 nM of the somatostatin analogue sandostatin. Three days later, MTT assay was used to determine cell number. Stable transfectants, wild-type receptor (SSTR2FL) was used as a positive control for ligand responsive growth inhibition and HA-SSTR2D314 does not inhibit growth upon ligand stimulation. HA-SSTR2D351 or HA-SSTR2D340 demonstrated equivalent amounts of expression (data not shown).
[0061] FIGS. 3A-B : Exposure to 90Y-DOTA-octreotate (DOTATATE) results in death of HT1080 cells expressing wild-type or signaling deficient SSTR2 receptors, but not vector transfected cells. Stably transfected HT1080 cells were exposed to different amounts of 90Y-DOTATATE for 1 hour and 72 hours. Dose dependent and time dependent killing was observed in HT1080 cells expressing wild-type or signaling deficient HA-SSTR2 receptors, but not vector transfected cells. Cell death was assessed by trypan blue uptake. *p<0.05 vs Vector.
[0062] FIG. 4 : Exposure to 90Y-DOTA-octreotate (DOTATATE) results in bystander cell death of tumor cells. Human mesenchymal stem cells (HS5) stably expressing wild-type or signaling deficient HA-SSTR2 receptors or transfected with vector were admixed with untransfected HT1080 cells and then exposed to 90Y-DOTATATE for 24 hours. Then, live (green)/dead (red) staining was performed. Rosettes (clusters) of dead cells were seen in wells containing mesenchymal stem cells stably expressing wild-type or signaling deficient HA-SSTR2 receptors but not in wells containing mesenchymal stem cells transfected with vector.
[0063] FIG. 5 : The somatostatin analogue octreotide inhibited growth of tumors expressing wild-type receptor, but not the signaling deficient receptor or tumors derived from vector transfected cells. Stably transfected HT1080 cells were implanted subcutaneously in nude mice (three tumors, HA-wt, HA-D314, vector, per animal). When tumors were palpable, daily injections of the somatostatin analogue sandostatin were initiated and tumors were measured by calipers every two days. Receptor activation by octreotide inhibited growth of tumors expressing wild-type receptor, but no decrease in tumor size was seen with the signaling deficient receptor. *p<0.05 SSTR2FL vs Vector. Thus, receptor activation can result in inhibition of tumor growth.
[0064] FIG. 6A : High doses of the imaging agent 111-In-octreotide inhibits tumor growth via the biologic effect of octreotide activating SSTR2 and not the effect of radioactivity (primarily gamma rays) on the tumor. The imaging agent 111-In-octreotide inhibited growth of tumors expressing the wild-type receptor, but not the signaling deficient receptor or tumors derived from vector transfected cells. Stably transfected HT1080 cells were implanted subcutaneously in nude mice (three tumors, HA-wt, HA-D314, vector, per animal). When tumors were palpable, 0.9 mCi of 111-In octreotide was given IV on day 4 and day 6. Tumors were measured by calipers every two days. *p<0.05 SSTR2FL vs Vector. Thus, receptor activation can result in inhibition of tumor growth.
[0065] FIG. 6B : Expression of SSTR2 can be imaged In tumors expressing wild-type or signaling deficient HA-SSTR2. Gamma-camera imaging of mice at 24 and 48 hours after the first and 24 hours after the second 111-In octreotide injection. Uptake (degree of receptor expression) is similar in tumors expressing wild-type or signaling deficient HA-SSTR2 at 24 hours and wanes in both at 48 hours. Increased uptake is again seen 24 hours after the second 111-In octreotide injection. Minimal uptake is seen in tumors derived from cells transfected with vector. Thus, both wild-type and signaling deficient SSTR2 uptake 111-In octreotide. (Arrows denote the indicated structures.)
[0066] FIGS. 7A-7B : 90Y-octreotate (primarily beta emitter) inhibited growth of tumors expressing wild-type or signaling deficient receptors, but not tumors derived from vector transfected cells. Stably transfected HT1080 cells were implanted subcutaneously in nude mice (three tumors, HA-wt, HA-D314, vector, per animal). When tumors were palpable, 90Y-octreotate was given once at a dose of 1 mCi (FIG. 7A ) or twice at doses of 2 mCi and 1 mCi (FIG. 7B ). Tumors were measured by calipers every two days. *p<0.05 HA-SSTR2FL or HA-D314 vs Vector. Tumors expressing the signaling deficient receptor decreased in size in a dose dependent manner due to the effect of radioactivity on the tumor. Reduction in size of tumors expressing wild-type HA-SSTR2 was due to a combination of effects of receptor activation by the somatostatin analogue octreotate and effects of radioactivity. This suggests that tumor growth may be inhibited by other mechanisms such as radiation-induced damage in cells without functional SSTR signaling but expressing SSTR's.
[0067] FIG. 8 : Stem cells expressing the signaling deficient mutant SSTR2D314 incorporate into tumor and can be targeted with a therapeutic for Inhibiting tumor growth. 90Y-octreotate (primarily beta emitter) inhibited growth of human tumors incorporating HS5 human mesenchymal stem cells expressing SSTR2D314, but not control HS5 cells.
[0068] FIG. 9 : Stem cells expressing the signaling deficient mutant SSTR2D314 home to and incorporate into tumor and can be targeted with a therapeutic for inhibiting tumor growth. 90Y-octreotate (primarily beta emitter) inhibited growth of human tumors to which human mesenchymal stem cells expressing HA-SSTR2D314 homed and incorporated, but not control HS5 cells.
[0069] FIG. 10 : After intracardiac injection, human MSC's traffic to HeyA8 tumors; and expression of the signaling deficient SSTR2 mutant in such MSC's can be distinguished. (HS5 cells expressing signaling deficient human SSTR2 (HS5-HA-hSSTR2-SD) injected intracardiac localized to Hey A8 tumors in mice and could be visualized one day after 111-In-octreotide injection (arrow, middle panel, top) whereas background signal was seen with control HS5 cells transfected with vector (arrow, middle panel, bottom). After imaging, tumors were removed and evaluated, confirming the imaging findings. Right panel: Biodistribution demonstrated increased uptake of 111-In-octreotide in tumors from animals injected intracardiac with HS5 cells expressing signaling deficient human SSTR2 (HA-hSSTR2-SD) compared to injected with HS5 cells transfected with vector. Inset: Western blot using an antibody to the HA domain of signaling deficient HA-SSTR2 demonstrated expression of the reporter by HS5 cells in tumors from animals injected intracardiac with HS5 cells expressing signaling deficient human SSTR2 (top arrow, left, Anti-HA-11) but not with HS5 cells transfected with vector (top arrow, right, Anti-HA-li). Beta-actin, a control house-keeping protein expected to be found in all tumors, was seen in both tumor lysates (bottom arrow, Anti-beta-actin) as expected.)
[0070] FIG. 11 : 90Y-octreotate inhibited growth of embryonic stem cell-derived tumors expressing wild-type or signaling deficient SSTR2-based receptors, but not tumors derived from wild type ES cells. Thus, even if delivered cells go awry, those made to express SSTR2-based genes can be targeted therapeutically. Mouse ES cells (these are pluripotent stem cells that can be used to create transgenic mice and like most ES cells result in teratomas when injected in nude mice) were transfected with a plasmid containing HA-SSTR2 or HA-SSTR2delta314. Clones were then selected for expression. The cells were then injected into nude mice subcutaneously and tumor size was measured by calipers. After tumors were present in three locations per mouse in all 8 mice, the animals were injected i.v. with 90-Y labeled octreotate two days and 5 days later.
[0071] FIG. 12 . Constitutively active SSTR2delta340 inhibits the growth of tumor cells. Nude mice were Injected subcutaneously with HeyA8 tumors cells and then each tumor was injected with adenovirus expressing HA-SSTR2delta340 or control inserts (8 tumors per group). Tumor size was measured by calipers. *p<0.05, tumors injected with adv-HA-SSTR2delta340 and given saline vs tumors injected with adv-GFP and given saline; for clarity only the comparison of these two groups is illustrated with *.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
I. Introduction
[0072] The invention relates to somatostatin receptors or their mutants that can be used either on their own or with other agents for therapy. Somatostatin receptors are involved in a variety of processes such as cancer (including growth, angiogenesis, invasion, and apoptosis), glucose regulation (such as hyperglycemia of diabetes), neuronal and neuromuscular transmission, gastric acid secretion, growth (such as acromegaly), and ocular neovascularization. In most of these processes, SSTR's have an inhibitory effect that can be therapeutically beneficial.
[0073] Signaling altered somatostatin receptors may be used for therapeutic uses that affect or are deficient in affecting functions such as growth inhibition and secretion inhibition. These may act directly, for example, after gene transfer and the expressed constitutively active mutant may have a therapeutic effect such as growth or secretion inhibition. The gene transfer of a signaling altered mutant may serve to localize the expression to the area of interest or systemically.
[0074] On the other hand, the signaling defective mutants may act indirectly, for example, through cellular therapy. In this aspect, the signaling defective or deficient mutants are desirable to not to affect the function of the delivered cells, which may home to the area of interest. In particular, maintenance of delivered cell migration and reproduction is important.
[0075] With either gene or cellular therapy, the receptor mutants may be used to localize additional therapies, such as therapies that affect cells expressing the SSTR mutant (e.g., a cytotoxic or cytostatic agent to the cell expressing an SSTR mutant), or a therapy that has bystander effect on nearby cells (such as a radioactive substance).
[0076] For example, with cellular therapy of tumors, the therapeutic cell could migrate to and then incorporate within the tumor, and via SSTR mutant could localize a substance (for example, a radioligand) that by bystander effect would aid in killing nearby tumor cells.
[0077] With gene therapy, adding a radioligand could increase kill of the cells expressing the gene and nearby tumor cells that are either not expressing the mutant or are expressing it at a low level. Gene expression may be heterogeneous even within the same tumor after gene transfer.
[0078] The SSTR mutant alone or SSTR mutant binding therapeutic agents may also be used to augment the effect of other therapeutic genes that may, for example, be dual expressed or linked to the SSTR mutants (such as via an internal ribosome entry site (IRES)) to enhance therapeutic efficacy compared to either gene alone.
II. Somatostatin Receptor Mutants
[0079] Somatostatin receptor (SSTR), belongs to the family of G protein-coupled receptors with seven transmembrane domains. SSTR2 can serve as a reporter of gene expression that can be quantified in vivo (Yang et al, 2005). Somatostatin receptors are over-expressed on a variety of tumors (John et al, 1996), and somatostatin receptor imaging can identify a variety of neuroendocrine malignancies, including carcinoid, islet cell tumor, pheochromocytoma, paraganglioma, small-cell lung cancer, and medullary thyroid cancer (Termanini et al, 1997; Lamberts et al., 2001; Kwekkeboom et al, 2000). For imaging, radiopharmaceutical analogs of the naturally occurring ligand, somatostatin, may be used. Upon activation, somatostatin initiates a variety of signaling events that affect cellular functions such as secretion, chemotaxis, and growth suppression. These effects have been exploited using therapeutic analogs of somatostatin, for example, to ameliorate or prevent carcinoid syndrome (Nikou et al., 2005; Ducreux et al., 2000; Wymenga et al., 1999).
[0080] For somatostatin receptor, in vitro studies suggest that the sixth and seventh transmembrane domains are essential for binding octreotide. Transmembrane domains three through five may also be important because a cysteine-cysteine disulfide bond is predicted between transmembrane domains three and extracellular domain two. Transmembrane domains three through seven have been predicted to cooperate in forming the pocket for binding octreotide.
[0081] For signaling, the C-terminus and intracytoplasmic domains of SSTR2 appear to be involved. As stated above, for both rat SSTR2 and human SSTR5, deletion analysis has demonstrated that the cytoplasmic C-terminus regulates inhibition of cAMP production. In particular embodiments of the present invention, the SSTR mutant amino acid sequence is a truncated recombinant somatostatin receptor amino acid sequence. Truncation can be at either the N-terminus or the C-terminus or both termini. For example, deletion of the SSTR2 after amino acid 314 is signaling defective and can be used for targeted therapy and imaging. In other aspects, deletion of the SSTR2 after amino acid 340 is constitutively active and can be used for targeted SSTR-mediated therapy.
[0082] There are six somatostatin receptor types, 1, 2A and 2B, 3, 4, and 5. Types 2A and 2B are alternate splice variants that are identical, except that type 2A has a longer intracytoplasmic carboxy-terminus (Petersenn et al., 1999). In certain embodiments, the somatostatin receptor mutant is a mutant of somatostatin receptor type 1, 2, 2A, 2B, 3, 4, or 5. Information pertaining to somatostatin receptors can be found in U.S. Patent Application Pub. No. 2002/0173626, which is herein specifically incorporated by reference in its entirety. Somatostatin receptors may be of any animal species, such as mouse, human, rat, pig, etc. Exemplary information regarding sequences of human or mouse somatostatin receptors is set forth in Table 1:
[0083]
| TABLE 1 | |||
|---|---|---|---|
| Exemplary SSTR Sequence Summary | |||
| Nucleic Acid | Accession | ||
| Sequence | Amino Acid | No.* for | |
| SSTR Type | (mRNA or cDNA) | Sequence | mRNA sequence |
| 1 (Human) | SEQ ID NO: 1 | SEQ ID NO: 2 | BC035618 |
| 1 (Mouse) | SEQ ID NO: 3 | SEQ ID NO: 4 | NM_009216 |
| 2 (Human) | SEQ ID NO: 5 | SEQ ID NO: 6 | BC019610 |
| 2A (Mouse) | SEQ ID NO: 7 | SEQ ID NO: 8 | NM_001042606 |
| 2B (Mouse) | SEQ ID NO: 9 | SEQ ID NO: 10 | AF008914 |
| 3 (Human) | SEQ ID NO: 11 | SEQ ID NO: 12 | BC096829 |
| 3 (Mouse) | SEQ ID NO: 13 | SEQ ID NO: 14 | BC120843 |
| 4 (Human) | SEQ ID NO: 15 | SEQ ID NO: 16 | BC117270 |
| 4 (Mouse) | SEQ ID NO: 17 | SEQ ID NO: 18 | U26176 |
| 5 (Human) | SEQ ID NO: 19 | SEQ ID NO: 20 | AY081193 |
| 5 (Mouse) | SEQ ID NO: 21 | SEQ ID NO: 22 | AF004740 |
| *GenBank Accession Number pertains to nucleic acid sequence and encoded protein. | |||
[0084] Detailed information regarding the splice variants of human SSTR2 and its genomic structure can be found in Petersenn et al., 1999, herein specifically incorporated by reference. Additional information regarding the sequence of SSTR2 can be found in Yamada et al. (1992) and Vanetti et al. (1992).
[0085] Upon activation, SSTR2 regulates signaling such as cAMP (Schwartkop et al., 1999) and cGMP production. The latter appears to regulate cell proliferation (Lopez et al., 2001). Gambhir et al. (1999) found that a D2 receptor mutant deficient in regulating cAMP can still be imaged. No functional (phenotypic changes) cellular changes were assessed such as effects on proliferation. In COS-7 cells, activation of human SSTR2 results in decreased cAMP production and activation of phospholipase C and calcium mobilization fully or partially, respectively, via a pertussis toxin sensitive G-protein. Through cAMP, somatostatin can regulate secretion. In 32D hematopoietic cells, cAMP appears to be required for SSTR2 mediated chemotaxis. The cytoplasmic C-terminus of the somatostatin receptor is involved in regulating cAMP. Deletion of amino acids beyond 349 of rat SSTR2 increases basal cAMP inhibition in human embryonic kidney (HEK 293) cells. Deletion of amino acids beyond 318 of human SSTR5 eliminates inhibition of cAMP in Chinese hamster ovary (CHO KI) cells.
[0086] Inhibition of proliferation by SSTR2 involves multiple downstream mediators including phosphatases. The tyrosine phosphatase SHP-I is regulated by SSTR2, but SHP-I does not appear to regulate cAMP in the breast carcinoma line MCF-7. Upstream of SHP-I are reported to be inhibitory G proteins, the tyrosine phosphatase SHP-2 and the tyrosine kinase Src. SHP-2 interacts with SSTR2 tyrosine 228 in the context LCYLFI in the third intracellular domain and tyrosine 312 in the context of ILYAFL in transmembrane domain 7 next to the C-terminus. The phosphatases may have direct effect on phosphorylation of the somatostatin receptor itself, stimulatory growth factors or other downstream effectors. Phosphatidyl inositol, Ras, Rapl, B-raf, MEK1 and 2, Map kinase/Erk 1 and 2 have been implicated in SSTR2 mediated signaling in CHO DG44 cells; but in neuroblastoma cells, Ras did not appear to be involved and Map kinase/Erk 1 and 2 activity decreased, instead of increased as in CHO DG44 cells.
[0087] Also downstream of SHP-I is the neuronal nitric oxide synthase (nNOS) and guanylate cyclase, both of which appear necessary for SSTR2 mediated inhibition of proliferation in CHO cells and mouse pancreatic acinar cells. The inhibition may also involve other phosphotyrosine phosphatases and more downstream effectors such as cyclin dependent kinase inhibitor p27kip1. Somatostatin also regulates transcription factors such as c-jun, c-fos and AP-I.
[0088] Among the receptor subtypes, human type 2 (SSTR2; Kluxen et al., 1992; Bell et al. 1993; Panetta et al., 1994; O'Carroll et al., 1993; Yamada et al., 1992) has the highest affinity for the most common clinically used somatostatin imaging analog, 111Indium-labeled octreotide. This radiopharmaceutical, approved for whole body imaging, and 99mTc labeled analogs, approved for lung imaging, are used in clinical practice to detect tumors over-expressing somatostatin receptors, such as neuroendocrine tumors. This radiopharmaceutical and 99mTc-labeled analogs are used in clinical practice to detect tumors that endogenously over-express somatostatin receptors (John et al., 1996) and have been used in animals models to image tumors that express exogenously introduced SSTR2, for example, as a reporter gene (Kundra et al., 2002).
[0089] The normal biodistribution and dosimetry of radiolabeled somatostatin analogs used for imaging clinically has been well studied. The radiopharmaceutical is normally found in the kidneys, bladder, liver, spleen and bowel after intravenous injection. At the tracer doses used for imaging, no side-effects greater than placebo are found and patients are routinely imaged serially. Clinically, increased SSTR2 expression renders even small tumors detectable. PET based agents are also being developed.
[0090] The nucleic acid encoding the SSTR amino acid sequence may encode a mutated SSTR sequence, a functional SSTR protein domain, a stably expressed non-functional SSTR, an SSTR polypeptide, or an SSTR polypeptide equivalent, each of which may include one or more transmembrane, extracellular, intracellular, extracellular loop(s) and/or intracellular loop(s). The mutation may be a deletion, point mutation, insertion, truncation, or frame shift mutation. The source of the nucleic acids may be derived from genomic DNA, i.e., cloned directly from the genome of a particular organism, mRNA from a particular organism, and/or synthesized by use of various methods including but not limited to PCR™.
[0091] In some embodiments, the nucleic acid may be complementary DNA (cDNA). cDNA is DNA prepared using messenger RNA (mRNA) as a template. Thus, a cDNA does not contain any interrupted coding sequences and usually contains almost exclusively the coding region(s) for the corresponding protein. In other embodiments, the nucleic acid may be produced synthetically.
[0092] It may be advantageous to combine portions of the genomic DNA with cDNA or synthetic sequences to generate specific constructs. For example, where an intron is desired in the ultimate construct, a genomic clone may need to be used. Introns may be derived from other genes in addition to SSTR. The cDNA or a synthesized polynucleotide may provide more convenient restriction sites for the remaining portion of the construct and, therefore, would be used for the rest of the sequence.
[0093] The present invention also includes nucleic acids encoding mutated SSTR polypeptide equivalents. These nucleic acids encoding SSTR mutant polypeptide equivalents may be naturally-occurring homologous nucleic acid sequences from other organisms. A person of ordinary skill in the art would understand that commonly available experimental techniques can be used to identify or synthesize nucleic acids encoding SSTR mutant polypeptide equivalents. The present invention also encompasses chemically synthesized mutants of these sequences. Another kind of sequence variant results from codon variation. Because there are several codons for most of the 20 normal amino acids, many different DNAs can encode SSTR mutants.
III. Cells for Therapeutic Uses
[0094] Cells having the somatostatin receptors or their mutants can be used in various therapeutic applications. In particular embodiments, the cells may be delivered to a tumor or be incorporated into a tumor. An anti-tumor therapeutic or a detectable moiety that target the cells may be administered to the subject. The targeting may be achieved by specific association between a receptor ligand coupled to the therapeutic or the detectable moiety and the introduced receptor mutant in the cells.
[0095] The cells that are employed in the methods of the present invention can be any type of cell, such as an eukaryotic cell. In some embodiments, the cells are stem cells, progeny cells thereof, fibroblast cells or immune cells, such as immune progenitor cells or white blood cells.
[0096] The cells such as stem cells or immune cells can have transferred an expression construct encoding an SSTR or its mutant prior to introduction of the cells to the subject at any stage in the preparation. In particular embodiments, the mutant may be a truncated SSTR2, such as SSTR2delta314.
[0097] The term “stem cell” generally refers to any cells that have the ability to divide for indefinite periods of time and to give rise to specialized cells. The definition of “stem cell” includes, but is not limited to: a) totipotent cells such as an embryonic stem cell, an extraembryonic stem cell, a cloned stem cell, a parthenogenesis derived cell, a cell reprogrammed to possess totipotent properties, or a primordial germ cell; b) a pluripotent cell such as a hematopoietic stem cell, an adipose derived stem cell, a mesenchymal stem cell, a cord blood stem cell, a placentally derived stem cell, an exfoliated tooth derived stem cells, a hair follicle stem cell or a neural stem cell; and c) a tissue specific progenitor cell such as a precursor cell for the neuronal, hepatic, nephrogenic, adipogenic, osteoblastic, osteoclastic, alveolar, cardiac, intestinal, or endothelial lineage.
[0098] The cells that are employed in the methods of the present invention can be obtained from any source known to those of ordinary skill in the art. In some embodiments, for example, the cells are stem cells obtained from a donor. In other embodiments, the cells are obtained from the subject who is to receive the cells as part of a therapeutic procedure. The cells can be derived, for example, from tissues such as pancreatic tissue, liver tissue, smooth muscle tissue, striated muscle tissue, cardiac muscle tissue, bone tissue, bone marrow tissue, bone spongy tissue, cartilage tissue, liver tissue, pancreas tissue, pancreatic ductal tissue, spleen tissue, thymus tissue, Peyer's patch tissue, lymph nodes tissue, thyroid tissue, epidermis tissue, dermis tissue, subcutaneous tissue, heart tissue, lung tissue, vascular tissue, endothelial tissue, blood cells, bladder tissue, kidney tissue, digestive tract tissue, esophagus tissue, stomach tissue, small intestine tissue, large intestine tissue, adipose tissue, uterus tissue, eye tissue, lung tissue, testicular tissue, ovarian tissue, prostate tissue, connective tissue, endocrine tissue, and mesentery tissue. In particular embodiments, the cells that are employed in the methods of the present invention are hematopoietic stem cells. The hematopoietic stem cells can be obtained, for example, from the blood or bone marrow of a subject. Further, stem cells of different tissue types (other than hematopoetic stem cells) can be obtained from the blood.
[0099] The stem cells to be expanded can be isolated from any organ of any mammalian organism, by any means known to one of skill in the art. The stem cells can be derived from embryonic or adult tissue. One of skill of the art can determine how to isolate the stem cells from the particular organ or tissue of interest, using methods known in the art. In a particular embodiment, the stem cells are isolated from same as prior paragraph. For example, the stem cells can be obtained from blood or bone marrow.
[0100] One of skill in the art will be able to determine a suitable growth medium for initial preparation of stem cells. Commonly used growth media for stem cells includes, but is not limited to, Iscove's modified Dulbecco's Media (IMDM) media, DMEM, KO-DMEM, DMEM/F12, RPMI 1640 medium, McCoy's 5 A medium, minimum essential medium alpha medium (.alpha.-MEM), F-12K nutrient mixture medium (Kaighn's modification, F-12K), X-vivo 20, Stemline, CClOO, H2000, Stemspan, MCDB 131 Medium, Basal Media Eagle (BME), Glasgow Minimum Essential Media, Modified Eagle Medium (MEM), Opti-MEM I Reduced Serum Media, Waymouth's MB 752/1 Media, Williams Media E, Medium NCTC-109, neuroplasma medium, BGJb Medium, Brinster's BMOC-3 Medium, CMRL Medium, CO2—Independent Medium, Leibovitz's L-15 Media, and the like.
[0101] If desired, other components, such as growth factors, can be added. Exemplary growth factors and other components include, but are not limited to, thrombopoietin (TPO), stem cell factor (SCF), IL-I, IL-3, IL-7, flt-3 ligand (fit-3L), G-CSF, GM-CSF, Epo, FGF-I, FGF-2, FGF-4, FGF-20, IGF, EGF, NGF, LIF, PDGF, bone morphogenic proteins (BMP), activin-A, VEGF, forskolin, glucocorticords, and the like. Furthermore, the media can contain either serum such as fetal calf, horse, or human serum, or more preferably, serum substitution components. Numerous agents have been introduced into media to alleviate the need for serum. For example, serum substitutes have included bovine serum albumin (BSA), insulin, 2-mercaptoethanol and transferrin (TF). One of ordinary skill in the art would be familiar with these supplementary components that can be added to the media.
[0102] The stem cells can then be stored for a desired period of time, if needed. Stem cell storage methods are well-known to those of skill in the art.
[0103] The stem cells can be sorted prior to administration by methods known in the art, using, for example, antibody technology such as fluorescence activated cell sorting (FACS), magnet activated cell sorting methods (e.g., magnetic resonance beads), column chromatography, or to isolate cells having the desired stem cell markers, or to remove unwanted, contaminating cell types having unwanted cell markers. For example, stem cells expressing an SSTR mutant amino acid sequence can be isolated from cells that do not expression the SSTR mutant amino acid sequence using any of these techniques.
[0104] Other cells that could be introduced into a subject for therapy include immune cells. For example, immune cells may be applied in the treatment of cancer. Examples of immune cells include T cells and B cells. For example, using positron emission tomography (PET), Koehne et al (2003) demonstrated in vivo that Epstein-Barr virus (EBV)-specific T cells expressing herpes simplex virus-1 thymidine kinase (HSV-TK) selectively traffic to EBV+ tumors expressing the T cells' restricting HLA allele. Furthermore, these T cells retain their capacity to eliminate targeted tumors. Capitalizing on sequential imaging, Dubey et al. (2003) demonstrated antigen specific localization of T cells expressing HSV-TK to tumors induced by murine sarcoma virus/Moloney murine leukemia virus (M-MS V/M-MuL V).
IV. Therapeutic Agents and Methods
[0105] “Treatment” and “treating” refer to administration or application of a drug or therapy (such as protein, nucleic acid, gene therapy, or cell-based therapy) to a subject or performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit of a disease or health-related condition.
[0106] The term “therapeutic benefit” used throughout this application refers to anything that promotes or enhances the well-being of the subject with respect to the medical treatment of his condition. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease.
[0107] A “disease” or “health-related condition” can be any pathological condition of a body part, an organ, or a system resulting from any cause, such as infection, genetic defect, trauma, and/or environmental stress. The cause may or may not be known. Examples of such conditions include cancer and diabetes.
[0108] “Prevention” and “preventing” are used according to their ordinary and plain meaning to mean “acting before” or such an act. In the context of a particular disease or health-related condition, those terms refer to administration or application of an agent, drug, or remedy to a subject or performance of a procedure or modality on a subject for the purpose of blocking the onset of a disease or health-related condition.
A. Cell-Based Therapy
[0109] The cells of the invention may be applied to treat subjects requiring the repair or replacement of body tissues resulting from disease or trauma. Treatment may entail the use of the cells of the invention to produce or induce new tissue, and the use of the tissue thus produced, according to any method presently known in the art or to be developed in the future. For example, the cells of the invention may be given systemically, implanted, injected or otherwise administered directly to the site of tissue damage so that they will produce or induce new tissue in vivo.
[0110] In addition, the stem cells, the mature cells produced from these stem cells, and the cell lines derived from these stem cells can be used: (1) to screen for the efficacy and/or cytotoxicity of compounds, allergens, growth/regulatory factors, pharmaceutical compounds, etc.; (2) to elucidate the mechanism of certain diseases; (3) to study the mechanism by which drugs operate; (4) to diagnose, monitor and treat cancer in a patient; (5) for gene therapy; and (6) to produce biologically active products, to name but a few uses.
[0111] In addition, immune cells can be applied in methods of therapy. These cells may be active against cells expressing a particular antigen. Methods of therapy involving immune cells involve techniques well-known to those of ordinary skill in the art.
[0112] Certain embodiments of the present invention involve introducing a pharmaceutically acceptable dose of cells encoding an SSTR mutant. Pharmaceutical compositions of the present invention comprise a therapeutically or diagnostically effective amount of the cells of the present invention. The phrases “pharmaceutical or pharmacologically acceptable” or “therapeutically effective” or “diagnostically effective” refers to compositions of cells of the present invention that do not produce an unacceptable adverse, allergic or other untoward reaction when administered to a subject, such as, for example, a human or a laboratory animal (e.g., mouse, rat, dog), as appropriate. One of ordinary skill in the art would be familiar with protocols known in the art for the administration of cells (such as stem cells) to a subject for the treatment of a disease. For example, see U.S. Pat. Nos. 5,139,941, 5,670,148, 7,078,032, and 6,927,060, each of which is hereby specifically incorporated by reference.
B. Therapeutic Nuclides
[0113] In certain embodiments, therapeutics such as therapeutic nuclides may be used in targeted therapy. The therapeutic nuclides may include radiopharmaceuticals such as alpha-emitting particles, beta-emitting particles, or Auger electrons as described in detail below.
[0114] The therapeutic nuclide may be coupled to a carrier that specifically binds to a somatostatin receptor or a mutant thereof. The method may further involve administration of a therapeutic radionuclide into the body by intravenous injection in liquid or aggregate form, ingestion while combined with food, inhalation as a gas or aerosol, or injection of a radionuclide that has undergone micro-encapsulation. Most diagnostic radionuclides emit gamma rays and therefore may be excluded from therapeutic nuclides in certain aspects, while the cell-damaging properties of beta particles are used in therapeutic applications.
[0115] Refined radionuclides for use in nuclear medicine may be derived from fission or fusion processes in nuclear reactors, which produce radionuclides with longer half-lives, or cyclotrons, which produce radionuclides with shorter half-lives, or take advantage of natural decay processes in dedicated generators, i.e., molybdenum/technetium or strontium/rubidium, or 68Ga.
[0116] Alpha-particle emitters—Over the past 40 years, the therapeutic potential of several alpha particle-emitting radionuclides has been assessed. These particles (i) are positively charged with a mass and charge equal to that of the helium nucleus, their emission leading to a daughter nucleus that has two fewer protons and two fewer neutrons; (ii) have energies ranging from 5 to 9 MeV and corresponding tissue ranges of approximately five mammalian-cell diameters; and (iii) travel in straight lines. The linear energy transfer (LET, in keV/μm, which reflects energy deposition and, therefore, ionization density along the track of a charged particle) of these energetic and doubly charged (+2) particles is very high (~80-100 keV/μm) along most of their up-to-100-μm path before increasing to ~300 keV/μm toward the end of the track (Bragg peak). Consequently, in the case of cell irradiation, the therapeutic efficacy of alpha-particle emitters depends on (i) distance of the decaying atom from the targeted mammalian cell nucleus—vis-à-vis the probability of a nuclear traversal; and (ii) role of heavy ion recoil of the daughter atom, in particular when the alpha particle emitter is covalently bound to nuclear DNA. Of equal importance are the contribution(s) from bystander effects and the magnitude of cross-dose (from radioactive sources associated with one cell to an adjacent/nearby cell—see below) as this will vary considerably depending on the size of the labeled cell cluster and the fraction of cells labeled.
[0117] The application of alpha-particle-emitting radionuclides as targeted therapeutic agents continues to be of interest. When such radionuclides are selectively accumulated in the targeted tissues (e.g., tumors), their decay should result in highly localized energy deposition in the tumor cells and minimal irradiation of surrounding normal host tissues. The investigation of the therapeutic potential of alpha-particle emitters has focused mainly on five radionuclides: astatine-211 (211At), bismuth-212 (212Bi), bismuth-213 (213Bi), radium-223 (223Ra), and actinium-225 (225Ac).
[0118] Beta-particle emitters—Beta particles are negatively charged electrons emitted from the nucleus of decaying radioactive atoms (one electron/decay), that have various energies (zero up to a maximum) and, thus, a distribution of ranges. After their emission, the daughter nucleus has one more proton and one less neutron. As these beta particles traverse matter, they lose their kinetic energies and eventually follow a contorted path and come to a stop. Because of their small mass, the recoil energy of the daughter nucleus is negligible. Additionally, the LET of these energetic and negatively (−1) charged particles is very low (~0.2 keV/μm) along their up-to-a-centimeter path (i.e., they are sparsely ionizing), except for the few nanometers at the end of the range. Consequently, their therapeutic efficacy predicates the presence of very high radionuclide concentrations within the targeted tissue. The long range of these emitted electrons leads to the production of cross-fire, a circumstance that negates the need to target every cell within the tumor, so long as all the cells are within the range of the decaying atoms. As with alpha particles, the probability of the emitted beta particle's traversing the targeted cell nucleus depends to a large degree on (i) the position of the decaying atom vis-d-vis the nucleus—specifically nuclear DNA—of the targeted tumor cell; (ii) its distance from the tumor cell nucleus; and (iii) the radius of the latter. Obviously, intranuclear localization of therapeutic radiopharmaceuticals is highly advantageous and, if possible, should always be sought.
[0119] Historically, studies of radionuclide-based tumor therapy have been carried out mainly with energetic beta-particle emitters. The exposure of cells in vitro to beta particles leads, in general, to survival curves that have a distinct shoulder and a DO of several thousand decays. Despite the rather low in vitro cytotoxicity, these radionuclides continue to be pursued for targeted therapy, mainly due to their availability and favorable physical characteristics (e.g., energy and range of the emitted electrons leading to cross-fire irradiation; physical half-lives compatible with the biologic half-lives of the carrier molecules) (Table 2).
[0120]
| TABLE 2 | |||||
|---|---|---|---|---|---|
| Beta-particle emitters | |||||
| Radionuclide | Half-life | Eβ−(max) (keV)I | Rβ−(max) (mm)H | ||
| 33P | 25.4 | d | 249 | 0.63 | |
| 177Lu | 6.7 | d | 497 | 1.8 | |
| 67Cu | 61.9 | h | 575 | 2.1 | |
| 131I | 8.0 | d | 606 | 2.3 | |
| 186Re | 3.8 | d | 1,077 | 4.8 | |
| 165Dy | 2.3 | h | 1,285 | 5.9 | |
| 89Sr | 50.5 | d | 1491 | 7.0 | |
| 32P | 14.3 | d | 1,710 | 8.2 | |
| 166Ho | 28.8 | h | 1854 | 9.0 | |
| 188Re | 17.0 | h | 2,120 | 10.4 | |
| 90Y | 64.1 | h | 2,284 | 11.3 | |
[0121] Nonenergetic Particles—During the decay of many radioactive atoms, a vacancy is formed (most commonly in the K shell) as a consequence of electron capture (EC) and/or internal conversion (IC). Each of these vacancies is rapidly filled by an electron dropping in from a higher shell. The process leads to a cascade of atomic electron transitions that move the vacancy toward the outermost shell. These inner-shell electron transitions result in the emission of characteristic X-ray photons or an Auger, Coster-Kronig, or super Coster-Kronig monoenergetic electron (collectively called Auger electrons). Typically, an average of 5 to 30 Auger electrons—with energies ranging from a few eV to approximately 1 keV—are emitted per decaying atom. In addition to producing low-energy electrons, this form of decay leaves the daughter atom with a high positive charge resulting in subsequent charge-transfer processes. The very low energies of Auger electrons have two major consequences: (i) these light, negatively (−1) charged particles travel in contorted paths and their range in water is from a fraction of a nanometer up to ~0.5 μm; and (ii) multiple ionizations (LET: 4-26 keV/μm) occur in the immediate vicinity (few nanometers) of the decay site, reminiscent of those observed along the path of an alpha particle. Finally, the short range of Auger electrons necessitates their close proximity to the radiosensitive target (DNA) for radiotherapeutic effectiveness. This is essentially a consequence of the precipitous drop in energy density as a function of distance in nanometers. Examples of therapeutic Auger-electron emitters include 125I, 123I, 77Br, 111In, and 195mPt.
C. Therapeutic Genes
[0122] In certain embodiments of the present invention, the mutant somatostatin receptor-coding sequence or may be operably linked to a therapeutic gene. In further embodiments, a somatostatin receptor ligand may be coupled to a therapeutic gene expression construct to be targeted to somatostatin mutant-expressing cells.
[0123] A “therapeutic gene” is a gene which can be administered to a subject for the purpose of treating or preventing a disease. For example, a therapeutic gene can be a gene administered to a subject for treatment or prevention of diabetes or cancer. Examples of therapeutic genes include, but are not limited to, Rb, CFTR, pi 6, p21, p27, p57, p73, C-CAM, APC, CTS-I, zacl, scFV ras, DCC, NF-I, NF-2, WT-I, MEN-I, MEN-II, BRCAl, VHL, MMACl, FCC, MCC, BRCA2, IL-I, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-I 1 IL-12, GM-CSF, G-CSF, thymidine kinase, mda7, fus1, interferon α, interferon β, interferon γ, ADP, p53, ABLI, BLCl, BLC6, CBFAl, CBL, CSFIR, ERBA, ERBB, EBRB2, ETS1, ETS2, ETV6, FGR, FOX, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCL1, MYCN5 NRAS, PIM1, PML, RET, SRC, TALI, TCL3, YES, MADH4, RBl, TP53, WTI, TNF, BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, ApoAI, ApoAIV, ApoE, Rapl A, cytosine deaminase, Fab, ScFv, BRCA2, zacl, ATM, HIC-I, DPC-4, FHIT, PTEN, ING1, NOEY1, NOEY2, OVCA1, MADR2, 53BP2, IRF-I, Rb, zacl, DBCCR-I, rks-3, COX-I, TFPI, PGS, Dp, E2F, ras, myc, neu, raf, erb. fms, trk, ret, gsp, hst, abl, ElA, p300, VEGF, FGF, thrombospondin, BAI-I, GDAIF, or MCC.
[0124] In certain embodiments of the present invention, the therapeutic gene is a tumor suppressor gene. A tumor suppressor gene is a gene that, when present in a cell, reduces the tumorigenicity, malignancy, or hyperproliferative phenotype of the cell. This definition includes both the full length nucleic acid sequence of the tumor suppressor gene, as well as non-full length sequences of any length derived from the full length sequences. It being further understood that the sequence includes the degenerate codons of the native sequence or sequences which may be introduced to provide codon preference in a specific host cell. Examples of tumor suppressor nucleic acids within this definition include, but are not limited to APC, CYLD, HIN-I, KRAS2b, pló, pl9, p21, p27, p27mt, p53, p57, p73, PTEN, Rb, Uteroglobin, Skp2, BRCA-I, BRCA-2, CHK2, CDKN2A, DCC, DPC4, MADR2/JV18, MEN1, MEN2, MTS1, NF1, NF2, VHL, WRN, WT, CFTR, C-CAM, CTS-I, zacl, scFV, MMACl, FCC, MCC, Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), 101F6, Gene 21 (NPRL2), or a gene encoding a SEM A3 polypeptide and FUS1. Other exemplary tumor suppressor genes are described in a database of tumor suppressor genes at world wide web at cise.ufl.edu/~yyl/HTML-TSGDB/Homepage.litml. This database is herein specifically incorporated by reference into this and all other sections of the present application. Nucleic acids encoding tumor suppressor genes, as discussed above, include tumor suppressor genes, or nucleic acids derived therefrom (e.g., cDNAs, cRNAs, mRNAs, and subsequences thereof encoding active fragments of the respective tumor suppressor amino acid sequences), as well as vectors comprising these sequences. One of ordinary skill in the art would be familiar with tumor suppressor genes that can be applied in the present invention.
[0125] In certain embodiments of the present invention, the therapeutic gene is a gene that induces apoptosis (i.e., a pro-apoptotic gene). A “pro-apoptotic gene amino acid sequence” refers to a polypeptide that, when present in a cell, induces or promotes apoptosis. The present invention contemplates inclusion of any pro-apoptotic gene known to those of ordinary skill in the art. Exemplary pro-apoptotic genes include CD95, caspase-3, Bax, Bag-1, CRADD, TSSC3, bax, hid, Bak, MKP-7, PERP, bad, bcl-2, MSTI, bbc3, Sax, BIK, BID, and mda7. One of ordinary skill in the art would be familiar with pro-apoptotic genes, and other such genes not specifically set forth herein that can be applied in the methods and compositions of the present invention. The therapeutic gene can also be a gene encoding a cytokine. The term ‘cytokine’ is a generic term for proteins released by one cell population which act on another cell as intercellular mediators. A “cytokine” refers to a polypeptide that, when present in a cell, maintains some or all of the function of a cytokine. This definition includes full-length as well as non-full length sequences of any length derived from the full length sequences. It being further understood, as discussed above, that the sequence includes the degenerate codons of the native sequence or sequences which may be introduced to provide codon preference in a specific host cell.
[0126] Examples of such cytokines are lymphokines, monokines, growth factors and traditional polypeptide hormones. Included among the cytokines are growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor, prostaglandin, fibroblast growth factor; prolactin; placental lactogen, OB protein; tumor necrosis factor-α and -β; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor, integrin; thrombopoietin (TPO); nerve growth factors such as NGF-β; platelet-growth factor, transforming growth factors (TGFs) such as TGF-α and TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-α, -β, and -γ; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-I, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-IO IL-I 1, IL-12; IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-24, LIF, G-CSF5, GM-CSF, M-CSF, EPO, kit-ligand or FLT-3. Other examples of therapeutic genes include genes encoding enzymes. Examples include, but are not limited to, ACP desaturase, an ACP hydroxylase, an ADP-glucose pyrophorylase, an ATPase, an alcohol dehydrogenase, an amylase, an amyloglucosidase, a catalase, a cellulase, a cyclooxygenase, a decarboxylase, a dextrinase, an esterase, a DNA polymerase, an RNA polymerase, a hyaluron synthase, a galactosidase, a glucanase, a glucose oxidase, a GTP ase, a helicase, a hemicellulase, a hyaluronidase, an integrase, an invertase, an isomerase, a kinase, a lactase, a lipase, a lipoxygenase, a lyase, a lysozyme, a pectinesterase, a peroxidase, a phosphatase, a phospholipase, a phosphorylase, a polygalacturonase, a proteinase, a peptidase, a pullanase, a recombinase, a reverse transcriptase, a topoisomerase, a xylanase, a reporter gene, an interleukin, or a cytokine. Further examples of therapeutic genes include the gene encoding carbamoyl synthetase I, ornithine transcarbamylase, arginosuccinate synthetase, arginosuccinate lyase, arginase, fumarylacetoacetate hydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin, glucose-6-phosphatase, low-density-lipoprotein receptor, porphobilinogen deaminase, factor VIII, factor IX, cystathione beta.-synthase, branched chain ketoacid decarboxylase, albumin, isovaleryl-CoA dehydrogenase, propionyl CoA carboxylase, methyl malonyl CoA mutase, glutaryl CoA dehydrogenase, insulin, beta.-glucosidase, pyruvate carboxylase, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, H-protein, T-protein, Menkes disease copper-transporting ATPase, Wilson's disease copper-transporting ATPase, cytosine deaminase, hypoxanthine-guanine phosphoribosyltransferase, galactose-1-phosphate uridyltransferase, phenylalanine hydroxylase, glucocerbrosidase, sphingomyelinase, α-L-iduronidase, glucose-6-phosphate dehydrogenase, HSV thymidine kinase, or human thymidine kinase.
[0127] Therapeutic genes also include genes encoding hormones. Examples include, but are not limited to, genes encoding growth hormone, prolactin, placental lactogen, luteinizing hormone, follicle-stimulating hormone, chorionic gonadotropin, thyroid-stimulating hormone, leptin, adrenocorticotropin, angiotensin I, angiotensin II, β-endorphin, β-melanocyte stimulating hormone, cholecystokinin, endothelin I, galanin, gastric inhibitory peptide, glucagon, insulin, lipotropins, neurophysins, somatostatin, calcitonin, calcitonin gene related peptide, β-calcitonin gene related peptide, hypercalcemia of malignancy factor, parathyroid hormone-related protein, parathyroid hormone-related protein, glucagon-like peptide, pancreastatin, pancreatic peptide, peptide YY, PHM, secretin, vasoactive intestinal peptide, oxytocin, vasopressin, vasotocin, enkephalinamide, metorphinamide, alpha melanocyte stimulating hormone, atrial natriuretic factor, amylin, amyloid P component, corticotropin releasing hormone, growth hormone releasing factor, luteinizing hormone-releasing hormone, neuropeptide Y, substance K, substance P, or thyrotropin releasing hormone.
[0128] As will be understood by those in the art, the term “therapeutic gene” includes genomic sequences, cDNA sequences, and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins, and mutants. The nucleic acid molecule encoding a therapeutic gene may comprise a contiguous nucleic acid sequence of about 5 to about 12000 or more nucleotides, nucleosides, or base pairs. “Isolated substantially away from other coding sequences” means that the gene of interest forms part of the coding region of the nucleic acid segment, and that the segment does not contain large portions of naturally-occurring coding nucleic acid, such as large chromosomal fragments or other functional genes or cDNA coding regions. Of course, this refers to the nucleic acid segment as originally isolated, and does not exclude genes or coding regions later added to the segment by human manipulation.
[0129] Encompassed within the definition of “therapeutic gene” is a “biologically functional equivalent” therapeutic gene. Accordingly, sequences that have about 70% to about 99% homology of amino acids that are identical or functionally equivalent to the amino acids of the therapeutic gene will be sequences that are biologically functional equivalents provided the biological activity of the protein is maintained.
V. Detection of Introduced Cells and Genes
[0130] In addition to cell-based therapy, methods and compositions for tracking of delivered cells may be also provided. The cells may be delivered to a site of interest by direct injection, targeting moieties, or intrinsic properties of cells themselves, such as the propensity of stem cells to migrate toward a tumor. Tracking of therapeutic cell compositions may help decide the dosage, efficacy, and location of the cells and improve targeting efficiency. The cells may be tracked by detecting the expression of reporters expressed in the delivered cells directly or by further administering a detectable moiety that specifically binds to the reporters.
A. Reporters
[0131] In certain embodiments, the introduced SSTR mutant gene may be expressed in the cells and serve as reporters for tracking the cells. In other embodiments of the present invention, the expression construct comprises a coding region that encodes a reporter other than an SSTR mutant. The term “reporter,” “reporter gene” or “reporter sequence” as used herein refers to any genetic sequence or encoded polypeptide sequence that is detectable and distinguishable from other genetic sequences or encoded polypeptides present in cells. Preferably, the reporter sequence encodes a protein that is readily detectable either by its presence, its association with a detectable moiety or by its activity that results in the generation of a detectable signal. In particular, reporters that can be imaged non-invasively or with non-invasive techniques are envisioned.
[0132] In some embodiments, a reporter nucleic acid may encode a polypeptide having a tag. In association with this embodiment, the method may further comprise the step of contacting the host cell with a fluorescently labeled antibody specific for the tag, thereby labeling the host cell, which may be detected and/or isolated by FACS or other detection, sorting or isolation methods.
[0133] In various embodiments, a nucleic acid sequence of certain aspects of the invention comprises a reporter nucleic acid sequence or encodes a product that gives rise to a detectable polypeptide. A reporter is or encodes a reporter molecule which is capable of directly or indirectly generating a detectable signal. Generally, although not necessarily, the reporter gene includes a nucleic acid sequence and/or encodes a detectable polypeptide that is not otherwise produced by the cells. However, non-host species specific (e.g., non-human) reporters expressed in a human subject can incite an immune response that can kill the introduced cells.
[0134] Many reporter genes have been described, and some are commercially available for the study of gene regulation (e.g., Alam and Cook, 1990, the disclosure of which is incorporated herein by reference). Signals that may be detected include, but are not limited to color, fluorescence, luminescence, isotopic or radioisotopic signals, cell surface tags, cell viability, relief of a cell nutritional requirement, cell growth and drug resistance. Reporter sequences include, but are not limited to, DNA sequences encoding β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, membrane bound proteins including, for example, G-protein coupled receptors (GPCRs), CD2, CD4, CD8, the influenza hemagglutinin protein, symporters (such as NIS) and others well known in the art, to which high affinity antibodies or ligands directed thereto exist or can be produced by conventional means, and fusion proteins comprising a membrane bound protein appropriately fused to an antigen tag domain from, among others, hemagglutinin or Myc.
[0135] In various embodiments, the desired level of expression of at least one of the reporter sequence is an increase, a decrease, or no change in the level of expression of the reporter sequence as compared to the basal transcription level of the reporter sequence. In a particular embodiment, the desired level of expression of one of the reporter sequences is an increase in the level of expression of the reporter sequence as compared to the basal transcription level of the reporter sequence.
[0136] In various embodiments, the reporter sequence encodes unique detectable proteins which can be analyzed independently, simultaneously, or independently and simultaneously. In certain embodiments, the reporter sequence encodes a protein that can be visualized non-invasively such as the SSTR2 Δ314.
[0137] In other embodiments, the host cell may be a eukaryotic cell or a prokaryotic cell. Exemplary eukaryotic cells include yeast and mammalian cells. Mammalian cells include human cells and various cells displaying a pathologic phenotype, such as cancer cells.
B. Detectable Moieties
[0138] In certain embodiments of the invention, a reporter, such as an SSTR mutant amino acid sequence, may be imaged by detecting its association with a detectable moiety. A “detectable moiety” is defined herein to refer to any molecule that can attach, either directly or indirectly, to a reporter. Examples of detectable moieties are set forth above. For example, in some embodiments, the detectable moiety comprises a ligand.
[0139] A ligand is defined herein to refer to an ion, a peptide, a oligonucleotide, aptamer, a molecule, a small molecule, or a molecular group that binds to another chemical entity or polypeptide to form a larger complex. In the context of the present invention, the ligand may bind to a reporter or to an amino acid sequence attached to the reporter sequence (e.g., such as a protein tag fused to the N-terminal end or C-terminal end of the reporter amino acid sequence) to form a larger complex. Any ligand known to those of ordinary skill in the art is contemplated for use as a ligand in the context of the present invention. In some embodiments of the present invention, a ligand may be contacted with the cell for imaging. The ligand may or may not be internalized by the cell. Where a reporter has become localized to the cell surface, the ligand, in these embodiments, may bind to or associate with the reporter. Any method of binding of the ligand to the reporter is contemplated by the present invention. In certain other embodiments, a ligand may become internalized by a cell. Once internalized the ligand may, but need not, bind to or associate with the reporter or a second reporter within the cell.
[0140] The detectable moiety may be a molecule or part of a molecule that has properties or is conjugated to a moiety such that it is capable of generating a signal that can be detected. Any imaging modality known to those of ordinary skill in the art can be applied to image a ligand. In some embodiments, the ligand is capable of binding to or being coupled to a molecule or part of a molecule that can be imaged. For example, the ligand may be capable of binding to or be coupled to a radionuclide, and the radionuclide can be imaged using nuclear medicine techniques known to those of ordinary skill in the art. For example, the ligand may be 111In-octreotide. Information regarding imaging using 111In-octreotide can be found in U.S. Patent App. Pub. No. 20020173626, herein specifically incorporated by reference. In other embodiments, for example, the ligand is capable of binding to or being coupled to a contrast agent that can be detected using imaging techniques well-known to those of ordinary skill in the art. For example, the ligand may be capable of binding to or being coupled to a CT contrast agent, an ultrasound agent, an optical agent, or an MRI contrast agent. In certain embodiments of the present invention, a detectable moiety can bind to the reporter, and the ligand in turn generates a signal that can be measured using an imaging modality known to those of ordinary skill in the art. In other embodiments, the ligand can bind to a protein tag that is fused to the reporter. Thus, for example, imaging would involve measuring a signal from the ligand, and this in turn would provide for localization of the reporter sequence within the cell or within a subject.
[0141] A variety of valent metal ions, or radionuclides, are known to be useful for radioimaging and can be employed as detectable moieties. Examples include, but are not limited to 67Ga, 68Ga, ““1Tc, 111In, 123I, 125I, 131I, 169Yb, 60Cu, 61Cu, 64Cu, 62Cu, 201Tl, 72A, and 157Gd. In certain embodiments of the present invention, the nucleic acid for use in the imaging methods of the present invention encodes an amino acid sequence that can be radiolabeled in vivo. Radiolabeling of the encoded reporter sequence can be direct, or it can be indirect, such as by radiolabeling of a ligand that can bind the protein tag or reporter sequence. Radiolabeled agents, compounds, and compositions provided by the present invention are provided having a suitable amount of radioactivity.
[0142] Once the encoded sequence is radiolabeled, it can be imaged for visualizing a site, such as a tumor in a mammalian body. In accordance with this invention, the radiolabel is administered by any method known to those of ordinary skill in the art. For example, administration may be in a single unit injectable dose, administered as a radiolabeled ligand. Any of the common carriers known to those with skill in the art, such as sterile saline solution or plasma, may be utilized. Generally, a unit dose to be administered has a radioactivity of about 0.01 mCi to about 300 mCi, preferably 5 mCi to about 30 mCi. The solution to be injected at unit dosage is usually from about 0.01 mL to about 10 mL.
[0143] After intravenous administration of the radiolabeled reagent, imaging of the organ or tumor in vivo can take place, if desired, in minutes, hours or even longer, after the radiolabeled reagent is introduced into a patient. In some instances, a sufficient amount of the administered dose may accumulate in the area to be imaged within about 0.01, 0.05, 0.1, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 minutes, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours or any intermediate ranges.
C. Imaging Methods
[0144] Imaging of cells or a detectable moiety may be performed using any method known to those of ordinary skill in the art. Examples include PET, SPECT, and gamma scintigraphy. In gamma scintigraphy, the radiolabel is a gamma-radiation emitting radionuclide and the radiotracer is located using a gamma-radiation detecting camera (this process is often referred to as gamma scintigraphy). The imaged site is detectable because the radiotracer is chosen either to localize at a pathological site (termed positive contrast) or, alternatively, the radiotracer is chosen specifically not to localize at such pathological sites (termed negative contrast). Some aspects of the present invention pertain to methods for tracking the location of a cell in a subject that involve detecting the location of the cell in the subject by contacting the cell with a detectably moiety that binds to the SSTR mutant that is expressed in the cell.
[0145] Detection of the expressed SSTR mutant amino acid sequence can be performed by any method known to those of ordinary skill in the art. For example, the reporter may be imaged by administration of a detectable moiety to a subject, wherein the detectable moiety is directed to the reporter amino acid sequence. In other embodiments, the detectable moiety is a radiolabeled probe, such as 111In-octreotide. In further embodiments, the detectable moiety is a probe that can be imaged optically, such as by fluorescence, near infrared, infrared, MR, or ultrasound. Any method known to those of ordinary skill in the art for measuring a signal derived from a reporter or an associated detectable moiety that attaches to the reporter is contemplated for inclusion in the present invention. Exemplary methods of detecting are as follows.
[0146] A variety of nuclear medicine techniques for imaging are known to those of ordinary skill in the art. Any of these techniques can be applied in the context of the imaging methods of the present invention to measure a signal from the reporter. For example, gamma camera imaging is contemplated as a method of imaging that can be utilized for measuring a signal derived from the reporter. One of ordinary skill in the art would be familiar with techniques for application of gamma camera imaging. In one embodiment, measuring a signal can involve use of gamma-camera imaging of an 111In or 99mTc conjugate, in particular 111In-octreotide or 99mTc-somatostatin analogue. Single photon emission tomography (SPECT) may also be performed for three dimensional localization.
[0147] Computerized tomography (CT) is contemplated as an imaging modality in the context of the present invention. By taking a series of X-rays, sometimes more than a thousand, from various angles and then combining them with a computer, CT made it possible to build up a three-dimensional image of any part of the body. A computer is programmed to display two-dimensional slices from any angle and at any depth. The slices may be combined to build three-dimensional representations.
[0148] In CT, intravenous injection of a radiopaque contrast agent can assist in the identification and delineation of soft tissue masses when initial CT scans are not diagnostic. Similarly, contrast agents aid in assessing the vascularity of a soft tissue or bone lesion. For example, the use of contrast agents may aid the delineation of the relationship of a tumor and adjacent vascular structures.
[0149] CT contrast agents include, for example, iodinated contrast media. Examples of these agents include iothalamate, iohexol, diatrizoate, iopamidol, ethiodol, and iopanoate. Gadolinium agents have also been reported to be of use as a CT contrast agent (see, e.g., Henson et al, 2004). For example, gadopentate agents have been used as a CT contrast agent (discussed in Strunk and Schild, 2004).
[0150] Magnetic resonance imaging (MRI) is an imaging modality that uses a high-strength magnet and radio-frequency signals to produce images. The most abundant molecular species in biological tissues is water. It is the quantum mechanical “spin” of the water proton nuclei that ultimately gives rise to the signal in imaging experiments and other nuclei can also be imaged. In MRI, the sample to be imaged is placed in a strong static magnetic field (1-12 Tesla) and the spins are excited with a pulse of radio frequency (RF) radiation to produce a net magnetization in the sample. Various magnetic field gradients and other RF pulses then act on the spins to code spatial information into the recorded signals. By collecting and analyzing these signals, it is possible to compute a three-dimensional image which, like a CT, SPECT, and PET image, is normally displayed in two-dimensional slices. The slices may be combined to build three-dimensional representations. Contrast agents used in MR or MR spectroscopy imaging differ from those used in other imaging techniques. Their purpose is to aid in distinguishing between tissue components with similar signal characteristics and to shorten the relaxation times (which will produce a stronger signal on T1-weighted spin-echo MR images and a less intense signal on T2-weighted images). Examples of MRI contrast agents include gadolinium chelates, manganese chelates, chromium chelates, and iron particles. Atoms other than H, may be used, such as F and Na. Contrast may also be introduced using hyperpolarization.
[0151] PET and SPECT Imaging modalities that provide information pertaining to information at the cellular level, such as cellular viability, include positron emission tomography (PET) and single-photon emission computed tomography (SPECT). In PET, a patient ingests or is injected with a radioactive substance that emits positrons, which can be monitored as the substance moves through the body. Closely related to PET is single-photon emission computed tomography, or SPECT.
[0152] The major difference between the two is that instead of a positron-emitting substance, SPECT uses a radioactive tracer that emits high-energy photons. SPECT is valuable for diagnosing multiple illnesses including coronary artery disease, and already some 2.5 million SPECT heart studies are done in the United States each year. PET radiopharmaceuticals for imaging are commonly labeled with positron-emitters such as 11C, 13N, 15O, 18F, 82Rb, 62Cu, and or 68Ga. SPECT radiopharmaceuticals are commonly labeled with positron emitters such as 99mTc, 201Tl, and 67Ga, or 111In. Important receptor-binding SPECT radiopharmaceuticals include [123I]QNE, [123I]IBZM, and [123I]iomazenil. These tracers bind to specific receptors, and are of importance in the evaluation of receptor-related diseases
[0153] Optical imaging is another imaging modality that has gained widespread acceptance in particular areas of medicine. Examples include optical labeling of cellular components, and angiography such as fluorescein angiography and indocyanine green angiography of the eyes. Examples of optical imaging agents include, for example, fluorescein, a fluorescein derivative, indocyanine green, Oregon green, a derivative of Oregon green derivative, rhodamine green, a derivative of rhodamine green, an eosin, an erythrosin, Texas red, a derivative of Texas red, malachite green, nanogold sulfosuccinimidyl ester, cascade blue, a coumarin derivative, a naphthalene, a pyridyloxazole derivative, cascade yellow dye, dapoxyl dye. Optical imaging includes near infrared imaging and infrared imaging. Near infrared imaging has more tissue penetration and less background.
[0154] Another biomedical imaging modality that has gained widespread acceptance is ultrasound. Ultrasound imaging has been used to provide real-time cross-sectional and even three-dimensional images of soft tissue structures and blood flow information in the body. High-frequency sound waves and a computer create images of blood vessels, tissues, and organs.
[0155] Ultrasound imaging of blood flow can be limited by a number of factors such as size and depth of the blood vessel. Ultrasonic contrast agents, a relatively recent development, include perfluorine and perfluorine analogs, which are designed to overcome these limitations by helping to enhance grey-scale images and Doppler signals.
[0156] Photoacoustic and thermoacoustic imaging may also be used in certain aspects. Photoacoustic imaging delivers non-ionizing laser pulses. Some of these are absorbed and converted to heat leading to thermoelastic expansion and ultrasonic emission that is detected by an ultrasound transducer. Photoacoustic imaging may be used on native tissue, or with contrast agents. When radio frequency, instead of light, is used to heat tissue, it is referred to as thermoacoustic imaging.
[0157] In certain embodiments, imaging using more than one modality is performed. For example, as set forth above, the imaging modality may include, but are not limited to, CT, MRI, PET, SPECT, ultrasound, or optical imaging. Other examples of imaging modalities known to those of ordinary skill in the art are contemplated by the present invention.
[0158] The imaging modalities may be performed at any time during or after administration of the composition comprising the diagnostically effective amount of the compound that comprises two imaging moieties. For example, the imaging studies may be performed during administration of the dual imaging compound of the present invention, or at any time thereafter. In some embodiments, the first imaging modality is performed beginning concurrently with the administration of the dual imaging agent, or about 1 sec, 1 hour, 1 day, or any longer period of time following administration of the dual imaging agent, or at any time in between any of these stated times. In some embodiments of the present invention a second imaging modality may be performed concurrently with the first imaging modality, or at any time following the first imaging modality. For example, the second imaging modality may be performed about 1 sec, about 1 hour, about 1 day, or any longer period of time following completion of the first imaging modality, or at any time in between any of these stated times. One of ordinary skill in the art would be familiar with performance of the various imaging modalities contemplated by the present invention.
D. Imaging of a Subject Following Stem Cell Administration
[0159] Imaging of a cell and/or its progeny can be performed following introduction of a cell into a subject. For example, imaging can be performed after about 1 second, 1 minute, 1 hour, 1 day, 1 week, 1 month, 1 year, or any longer period of time following administration of the cell. In some embodiments, imaging and biodistribution analysis can be performed as described by Yang et al, 2005. In other embodiments, imaging may be preformed after approximately one and one-half weeks. One of ordinary skill in the art would be familiar with generating a protocol to imaging cells, such as stem cells, following introduction of cells into a subject.
[0160] Imaging of a cell and/or its progeny that include an expressed SSTR mutant can be performed for several purposes. For example, imaging can be performed to follow the transit of cells, such as stem cells, in the body following introduction of the cells into a subject. Imaging can also be used to assess cell viability following introduction of the cells into a subject, and over the course of time. Further, imaging can also be performed to assess stem cell or immune cell localization in a subject. For example, placing the reporter under the control of a constitutive promoter would provide for constant expression that may be used to assess localization and viability of the cell. Imaging can be used to assess trans/differentiation or fusion. For example, placing the reporter under the control of a tissue-selective promoter sequence would provide for expression of a particular reporter only upon trans/differentiation or fusion of a cell or its progeny to a particular tissue/cell type.
[0161] Alternatively, imaging can be performed to assess an immune cell, stem cell or its progeny's expression from a promoter of a gene whose product performs a function of interest following introduction of the cell into a subject. For example, placing a reporter in the expression construct under the control of a function-specific promoter would provide for expression of the reporter in stem cells until trans/differentiation or fusion. Alternatively expression may occur upon differentiation of the cell into a cell capable of performing a specific function. As an example in lymphocytes, T-cell activation may be assessed using promoter elements that initiate transcription upon T-cell activation. Thus, imaging can be applied in a wide variety of contexts that are significant in the context of cell therapy such as stem cell and immune cell therapy.
[0162] The reporter may be linked to a gene of interest, for example by an IRES or a bidirectional promoter, so that expression of the reporter may be used to track not only its own expression, but also that of the gene of interest. Examples of genes of interest include those whose products may function in homing, implantation or differentiation.
[0163] With multiple promoter-reporter constructs transferred into a cell, combinations of the above may be evaluated including in vivo. For example, with multiple promoter-reporter constructs transferred individually or together, viability, localization, differentiation, functional expression, and indirect evaluation of expression of a linked gene of interest may be evaluated. When used in combination, different promoters and different reporters that can be identified either simultaneously or serially may need to be employed. Simultaneous evaluation may be performed for example if the reporter or its detectable moiety have separable characteristics, for example, different energies of emission of gamma rays that can be separated by a gamma camera. In certain embodiments, a combination of more than one imaging technique can be used to determine trafficking, viability, and/or differentiation of the cells. For example, MR and γ-camera imaging can be used to determine the biodistribution of radiopharmaceutical in tumors. Imaging can be performed following administration of a subject with a detectable moiety. Because the detectable moiety will have a limited physical and biological life, imaging of the reporter can be performed repeatedly. In addition, cells that have been exposed to a detectable moiety can be introduced into the subject, and then the subject subjected to one or more imaging techniques following introduction of the cells. Imaging can be performed a single time or more than one time point following introduction of the cells into the subject allowing serial evaluation. Image acquisition can be performed by any method known to those of ordinary skill in the art.
[0164] The reporter within the cells can be detected both in vivo and ex vivo. In certain embodiments, ex vivo evaluation occurs on a biopsy sample of tissue obtained from the subject following introduction of the cell into the subject. Ex vivo evaluation for the reporter can be performed using a variety of techniques including but not limited to autoradiography, immunologic techniques such as immunohistochemistry, ELISA or Western blotting, PCR™, optical, CT, MR, nuclear imaging, or ultrasound. As discussed above, in vivo-imaging can also be performed using any of a variety of modalities known to those of ordinary skill in the art.
VI. Pharmaceutical Preparations and Compostions
[0165] Pharmaceutical compositions of the present invention comprise an effective amount of one or more cell or gene delivery compositions or additional agent dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases “pharmaceutical or pharmacologically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference).
[0166] The cells of the present invention can be introduced to a subject by any method known to those of ordinary skill in the art. Examples include intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticular Iy, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, topically, locally, injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, directly into a heart chamber, directly injected into the organ or portion of organ or diseased site of interest, or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art.
[0167] The actual required amount of a composition of the present invention administered to a subject, such as a patient with a disease, can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
[0168] The present invention contemplates methods of preventing, inhibiting, or treating such diseases or conditions in a subject by administration of a cell that has been transfected with an expression construct comprising a sequence encoding a somatostatin receptor constitutively active mutant. Aspects of the invention also include the use of the methods and compositions of the invention in combination with other therapies, as discussed in greater detail below.
[0169] Diseases to be prevented, treated or diagnosed can be any disease that affects a subject that would be amenable to therapy or prevention through administration of a cell as described herein. For example, the disease may be a disease amenable to stem cell therapy. Examples include cancer, diabetes, cardiovascular disease, neurological disease, neurodegenerative disease, genetic disease, liver disease, infection, trauma, toxicity, or immunological disease. Additional diseases are discussed elsewhere in this specification.
[0170] For example, the disease may be a hyperproliferative disease. A hyperproliferative disease is a disease associated with the abnormal growth or multiplication of cells. The hyperproliferative disease may be a disease that manifests as lesions in a subject. Exemplary hyperproliferative lesions include pre-malignant lesions, cancer, and tumors. The cancer can be any type of cancer including those derived from mesoderm, endoderm, or ectoderm such as blood, heart, lung, esophagus, muscle, intestine, breast, prostate, stomach, bladder, liver, spleen, pancreas, kidney, neurons, myocytes, leukocytes, immortalized cells, neoplastic cells, tumor cells, cancer cells, duodenum, jejunum, ileum, cecum, colon, rectum, salivary glands, gall bladder, urinary bladder, trachea, larynx, pharynx, aorta, arteries, capillaries, veins, thymus, lymph nodes, bone marrow, pituitary gland, thyroid gland, parathyroid glands, adrenal glands, brain, cerebrum, cerebellum, medulla, pons, spinal cord, nerves, skeletal muscle, smooth muscle, bone, testes, epidiymides, prostate, seminal vesicles, penis, ovaries, uterus, mammary glands, vagina, skin, eyes, or optic nerve.
[0171] Other examples of diseases to be treated include return of lost or lack of function such as diabetes where insulin production is inadequate, infectious diseases, genetic diseases, and inflammatory diseases, such as autoimmune diseases. The methods and compositions of the present invention can be applied to deliver an antigen that can be applied in immune therapy or immune prophylaxis of a disease. One of ordinary skill in the art would be familiar with the many disease entities that would be amenable to prevention or treatment using the pharmaceutical compositions and methods set forth herein.
VII. Combination Therapy
[0172] It is an aspect of this invention that the claimed methods for treating cells in a subject can be used in combination with another agent or therapy method.
[0173] In certain embodiments, the disease is cancer, the other agent or therapy is another anti-cancer agent or anti-cancer therapy. Treatment with the claimed somatostatin receptor-based therapeutic agent may precede or follow the other therapy method by intervals ranging from minutes to weeks. In embodiments where another agent is administered, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agents would still be able to exert an advantageously combined effect on the cell. For example, it is contemplated that one may administer two, three, four or more doses of one agent substantially simultaneously (i.e., within less than about a minute) with the therapeutic agents of the present invention. In other aspects, a therapeutic agent or method may be administered within about 1 minute to about 48 hours or more prior to and/or after administering an SSTR-based therapeutic agent or agents of the present invention, or prior to and/or after any amount of time not set forth herein. In certain other embodiments, an SSTR-based therapeutic agent of the present invention may be administered within of from about 1 day to about 21 days prior to and/or after administering another therapeutic modality, such as surgery or gene therapy. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several weeks (e.g., about 1 to 8 weeks or more) lapse between the respective administrations. Various combinations may be employed, the claimed SSTR-based agent is derivative is “A” and the secondary agent, which can be any other therapeutic agent or method, is “B”:
[0174]
| A/B/A B/A/B B/B/A AJAJB A/B/B BIAJA A/B/B/B B/A/B/B | |
| B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A | |
| B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A | |
[0175] Administration of the SSTR-based therapeutic agents of the present invention to a patient will follow general protocols for the administration of chemotherapeutics, taking into account the toxicity, if any, of these agents. It is expected that the treatment cycles would be repeated as necessary. It also is contemplated that various standard therapies, as well as surgical intervention, may be applied in combination with the therapeutic. These therapies include but are not limited to additional drug therapy, chemotherapy, additional radiotherapy, immunotherapy, gene therapy and surgery.
A. Chemotherapy
[0176] Cancer therapies also include a variety of combination therapies with both chemical and radiation based treatments. Chemotherapies include, but are not limited to, for example, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP 16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabien, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate, or any analog or derivative variant of the foregoing.
B. Radiotherapy
[0177] Other factors that cause DNA damage and have been used extensively include what are commonly known as γ-rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated such as microwaves and UV-irradiation. It is most likely that all of these factors effect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells. The terms “contacted” and “exposed,” when applied to a cell, are used herein to describe the process by which a therapeutic construct and a chemo therapeutic or radiotherapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell. To achieve cell killing or stasis, both agents are delivered to a cell in a combined amount effective to kill the cell or prevent it from dividing.
C. Immunotherapy
[0178] Immunotherapeutics, generally, rely on the use of immune modulators, immune effector cells and molecules to cure or palliate disease. In certain embodiments, immune modulators, immune effector cells and molecules target and destroy cancer cells. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may serve as an effector of therapy or it may recruit other cells to actually effect cell killing. The antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionucleotide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells.
[0179] Immunotherapy, thus, could be used as part of a combined therapy, in conjunction with gene therapy. The general approach for combined therapy is discussed below. Generally, the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells. Many tumor markers exist and any of these may be suitable for targeting in the context of the present invention. Common tumor markers include carcino embryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and pi 55.
D. Nucleic Acid-Based Therapy
[0180] In yet another embodiment, the secondary treatment is an additional gene therapy in which a therapeutic polynucleotide is administered before, after, or at the same time as the nucleic acid composition of the present invention. Delivery of the SSTR-based therapeutic agent in conjunction with a vector encoding a gene product will have a combined therapeutic effect such as an anti-hyperproliferative effect on target tissues.
[0181] RNA interference (RNAi) is a powerful gene-silencing process that holds great promise in the field of cancer therapy. The evolving understanding of the molecular pathways important for carcinogenesis has created opportunities for cancer therapy employing RNAi technology to target the key molecules within these pathways. Major targets for siRNA therapy include oncogenes and genes that are involved in angiogenesis, metastasis, survival, antiapoptosis and resistance to chemotherapy.
[0182] Many gene products involved in carcinogenesis have already been explored as targets for RNAi intervention, and RNAi targeting of molecules crucial for tumor-host interactions and tumor resistance to chemo- or radiotherapy has also been investigated. In most of these studies, the silencing of critical gene products by RNAi technology has generated significant antiproliferative and/or proapoptotic effects in cell-culture systems or in preclinical animal models.
[0183] siRNA can be introduced into the cells by using either chemically synthesized siRNA oligonucleotides (oligos), or vector-based siRNA (shRNA), which allows long lasting and more stable gene silencing. Nanoparticles and liposomes are commonly used carriers, delivering the siRNA with better transfection efficiency and protecting it from degradation. In combination with standard chemotherapy, siRNA therapy can also reduce the chemoresistance of certain cancers, demonstrating the potential of siRNA therapy for treating many malignant diseases.
E. Surgery
[0184] Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative and palliative surgery. Curative surgery is a cancer treatment that may be used in conjunction with other therapies, such as the treatment of the present invention, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy and/or alternative therapies. Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and miscopically controlled surgery (Mohs' surgery). It is further contemplated that the present invention may be used in conjunction with removal of superficial cancers, precancers, or incidental amounts of normal tissue.
VIII. Nucleic Acids
[0185] Aspects of the invention include introducing into a cell with an expression construct comprising at least a first region that is a nucleic acid sequence encoding a somatostatin receptor or its mutant operatively linked to a first promoter sequence. The somatostatin mutant may be constitutively active or signaling defective. In other aspects, expression construct may include one or more additional nucleic acid sequences, such as additional reporters, additional coding regions, or additional promoters
[0186] The term “nucleic acid” is well known in the art. A “nucleic acid” as used herein will generally refer to a molecule (i.e., a strand) of DNA, RNA or a derivative or analog thereof, comprising a nucleobase. A nucleobase includes, for example, a naturally occurring or derivatized purine or pyrimidine base found in DNA (e.g., an adenine “A,” a guanine “G,” a thymine “T” or a cytosine “C”) or RNA (e.g., an A, a G, an uracil “U” or a C). The term “nucleic acid” encompasses the terms “oligonucleotide” and “polynucleotide,” each as a subgenus of the term “nucleic acid.” The term “oligonucleotide” refers to a molecule of between about 3 and about 100 nucleobases in length. The term “polynucleotide” refers to at least one molecule of greater than about 100 nucleobases in length.
[0187] These definitions generally refer to a single-stranded molecule, but in specific embodiments will also encompass an additional strand that is partially, substantially or fully complementary to the single-stranded molecule. Thus, a nucleic acid may encompass a double-stranded molecule or a triple-stranded molecule that comprises one or more complementary strand(s) or “complement(s)” of a particular sequence comprising a molecule
[0188] The term “vector” is used to refer to a carrier into which a nucleic acid sequence can be inserted for introduction into a cell where it can be expressed and/or replicated. The term “expression vector,” “expression construct” or “nucleic acid vector” refers to a nucleic acid containing a nucleic acid sequence or “cassette” coding for at least part of a nucleic acid sequence, also referred to herein as a gene, product capable of being transcribed and “regulatory” or “control” sequences, which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host cell. In addition to control sequences that govern transcription and translation, the expression vectors may contain nucleic acid sequences that serve other functions as well.
[0189] The term “promoter” is used interchangeably with “promoter element” and “promoter sequence.” Likewise, the term “enhancer” is used interchangeably with “enhancer element” and “enhancer sequence.” A promoter, enhancer, or repressor, is said to be “operably linked” to a nucleic acid or transgene, such as a nucleic acid encoding a recombinant seven transmembrane G-protein associated receptor, when such element(s) control(s) or affect(s) nucleic acid or transgene transcription rate or efficiency. For example, a promoter sequence located proximally to the 5′ end of a transgene coding sequence is usually operably linked with the transgene. As used herein, term “regulatory elements” is used interchangeably with “regulatory sequences” and refers to promoters, enhancers, polyadenylation sites and other expression control elements, or any combination of such elements.
A. Promoters
[0190] Promoters are positioned 5′ (upstream) to the genes that they control. Many eukaryotic promoters contain two types of recognition sequences: TATA box and the upstream promoter elements. The TATA box, located 25-30 bp upstream of the transcription initiation site, is thought to be involved in directing RNA polymerase II to begin RNA synthesis at the correct site. In contrast, the upstream promoter elements determine the rate at which transcription is initiated. These elements can act regardless of their orientation, but they must be located within 100 to 200 bp upstream of the TATA box.
[0191] Enhancer elements can stimulate transcription up to 1000-fold from linked homologous or heterologous promoters. Enhancer elements often remain active even if their orientation is reversed (Li et al, 1990). Furthermore, unlike promoter elements, enhancers can be active when placed downstream from the transcription initiation site, e.g., within an intron, or even at a considerable distance from the promoter (Yutzey et al, 1989).
[0192] As is known in the art, some variation in this distance can be accommodated without loss of promoter function. Similarly, the positioning of regulatory elements with respect to the transgene may vary significantly without loss of function. Multiple copies of regulatory elements can act in concert. Typically, an expression vector comprises one or more enhancer sequences followed by, in the 5′ to 3′ direction, a promoter sequence, all operably linked to a transgene followed by a polyadenylation sequence. A “promoter” sequence is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors. The phrases “operatively positioned,” “operatively linked,” “under control,” and “under transcriptional control” mean that a promoter is in a correct functional location and orientation in relation to a nucleic acid sequence to control transcriptional initiation and expression of that sequence. A promoter may or may not be used in conjunction with an “enhancer,” which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence. Together, an appropriate promoter or promoter/enhancer combination, and a gene of interest, comprise an expression cassette. One or more expression cassettes may be present in a given nucleic acid vector or expression vector. In certain aspects, one expression cassette may encode a transactivator that interacts with a promoter of a second expression cassette. The one or more expression cassettes may be present on the same and/or different expression vector.
[0193] A promoter may be one naturally associated with a gene or sequence, as may be obtained by isolating a portion the 5′ non-coding sequences located upstream of the coding segment or exon. Such a promoter can be referred to as “endogenous.” Similarly, an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment.
[0194] In certain aspect of the invention a heterologous promoter may be a chimeric promoter, where elements of two or more endogenous, heterologous or synthetic promoter sequences are operatively coupled to produce a recombinant promoter. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not “naturally occurring,” i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCR™, in connection with the compositions disclosed herein (see U.S. Pat. Nos. 4,683,202 and 5,928,906, each incorporated herein by reference). Such promoters may be used to drive reporter expression, which include, but are not limited to GPCRs, O-galactosidase or luciferase to name a few. Furthermore, it is contemplated the control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.
[0195] A promoter and/or enhancer could be used that effectively directs the expression of the DNA segment in a cell type, organelle, and organism chosen for expression. Those of skill in the art of molecular biology generally know the use of promoters, enhancers, and cell type combinations for protein expression, for example, see Sambrook et al. (2001), incorporated herein by reference. The promoters employed may be constitutive, tissue-selective, inducible, lineage-specific, or function-specific and/or useful under the appropriate conditions to direct expression of the introduced DNA segment, such as is advantageous in the production of proteins, recombinant proteins and/or peptides. The promoter may be heterologous or endogenous or a combination thereof. The position of the promoter/may be varied. It is contemplated that 1, 2, 3, 4, or more expression cassettes may be present in a particular vector or a particular cell with no general preference as to the order of the cassettes in an expression vector. A first, second, third or fourth promoter of an expression cassette may be a constitutive, tissue selective, lineage specific, or function-specific promoter sequence that drives expression of a gene of interest, such as a protein tag gene, a reporter, a signaling sequence, a trafficking sequence, or a therapeutic gene.
[0196] Certain aspects of the invention include promoter sequences that interact with endogenous or exogenous transactivators. In certain aspects a transactivator is a recombinant transactivator. A recombinant transactivator may be expressed in cells into which a nucleic acid of the invention is introduced. Alternatively, a recombinant transactivator or a nucleic acid encoding a recombinant transactivator may be introduced before, with or after a nucleic acid of the invention. In certain aspects, the recombinant transactivator may be encoded in a nucleic acid encoding an imaging or therapeutic agent.
[0197] A promoter may be functional in a variety of tissue types and in several different species of organisms, or its function may be restricted to a particular species and/or a particular normal or diseased tissue or cell type. Further, a promoter may be constitutively active, or it may be selectively activated by certain substances (e.g., a tissue-selective factor), under certain conditions (e.g., hypoxia, or the presence of an enhancer element in the expression cassette containing the promoter), or during certain developmental stages of the organism (e.g., active in fetus, silent in adult). A “function-specific promoter sequence” is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled, wherein the sequence is active in cells and whose products perform a particular function of interest. Examples include insulin, T-cell receptor, immunoglobulin, hormone or paracrine promoters such as vascular endothelial growth factor, structural protein promoters such as dystrophin, intracellular components such as fat or melanin, or extracellular components such as cartilage.
[0198] Promoters useful in the practice of the present invention may be tissue-specific—that is, they are capable of driving transcription of a gene in one or a few normal or diseased tissue(s) while remaining largely “silent” or expressed at relatively low levels in other tissue types. It will be understood, however, that tissue-specific or tissue-selective promoters may have a detectable amount of “background” or “base” activity in those tissues where they are silent. The degree to which a promoter is selectively activated in a target tissue can be expressed as a selectivity ratio (activity in a target tissue/activity in a control tissue). In this regard, a tissue specific promoter useful in the practice of the present invention typically has a selectivity ratio of greater than about 1:1.01, 1:1.1, 1:1.5, 1:2, 1:3, 1:4, 1:5 or more. Preferably, the selectivity ratio is greater than about 1:1.5. The promoter may also function in a reverse manner with decreased activity in the normal or diseased tissue(s) of interest. It will be further understood that certain promoters, while not restricted in activity to a single tissue type, may nevertheless show selectivity in that they may be active in one group of tissues, and less active or silent in another group. Such promoters are also termed “tissue specific” or “tissue selective,” and are contemplated for use with the present invention. For example, promoters that are active in a particular type of tissue may be therapeutically useful in diseases affecting the tissue that may be amenable to stem cell therapy.
[0199] The level of expression of a coding region under the control of a particular promoter can be modulated by manipulating the promoter region. For example, different domains within a promoter region may possess different gene-regulatory activities. The roles of these different regions are typically assessed using vector constructs having different variants of the promoter with specific regions deleted (i.e., deletion analysis) or base pair(s) mutated. Vectors used for such experiments typically contain a reporter sequence, which is used to determine the activity of each promoter variant under different conditions. Application of such a deletion analysis enables the identification of promoter sequences containing desirable activities and thus identifying a particular promoter domain, including core promoter elements, those elements when deleted detrimentally effect characteristics of the promoter, such as but not limited to selectivity or transcription factor binding. This approach may be used to identify, for example, the smallest region capable of conferring tissue specificity, or the smallest region conferring a robust transcriptional response when combined with other promoter elements, such as but not limited to the core CMV promoter or a mini-CMV.
[0200] A number of promoters, described herein, may be particularly advantageous in practicing the present invention. In most instances, these promoters may be isolated as convenient restriction digest fragments suitable for cloning into a selected vector. Alternatively, promoter fragments may be isolated using the polymerase chain reaction or by oligonucleotide synthesis. Cloning of these promoter fragments may be facilitated by incorporating restriction sites at the 5′ ends of the primers.
[0201] The promoter sequence can be any promoter sequence known to those of ordinary skill in the art. Promoter sequences are discussed in detail elsewhere in this specification. For example, the promoter sequence may be a function-specific promoter sequence, a constitutive promoter sequence, or a tissue-selective promoter sequence.
[0202] In particular embodiments, the promoter sequence is a function-specific promoter sequence. A “function specific promoter sequence” is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled, wherein the sequence is active in cells and whose products perform a particular function of interest. Examples of tissue selective promoter sequences include an insulin promoter sequence, T cell receptor promoter sequence, immunoglobulin promoter sequence, hormone or paracrine promoters such as vascular endothelial growth factor promoter sequences, structural protein promoters such as a dystrophin promoter sequence, intracellular component such as fat or melanin promoter sequences, or extracellular component such as cartilage promoter sequences. Other examples include a pBROAD promoter sequence, a c-fos promoter sequence, a c-HA-ras promoter sequence, an intercellular adhesion molecule 2 promoter sequence, and a platelet-derived growth factor (PDGF) promoter sequence.
[0203] The promoter sequence may also be a constitutive promoter sequence. A “constitutive promoter sequence” is defined herein to refer to a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled, wherein the sequence is active in cells of most any lineage. For example, the constitutive promoter sequence may be a beta-actin promoter sequence, an elastase I promoter sequence, a metallothionein (MTII) promoter sequence, a 5 S ribosomal promoter sequence, an Elastase promoter sequence, an Elastase I promoter sequence, a polyoma promoter sequence, a Cytomegalovirus promoter sequence, a retrovirus promoter sequence, a papilloma virus promoter sequence, a fibronectin promoter sequence, a ubiquitin promoter, an actin promoter, an elongation factor 1 alpha, an early growth factor response 1, an eukaryotic initiation factor 4Al, a ferritin heavy chain, a ferritin light chain, a glyceraldehyde 3-phosphate dehydrogenase, a glucose-regulated protein 78, a glucose-regulated protein 94, a heat shock protein 70, a heat shock protein 90, a beta-kinesin, a phosphoglycerate kinase, an ubiquitin B, a beta-actin, RNA virus promoter, DNA virus promoter, adenoviral promoter sequence, a baculoviral promoter sequence, a CMV promoter sequence, a parvovirus promoter sequence, a herpesvirus promoter sequence, a poxvirus promoter sequence, an adeno-associated virus promoter sequence, a semiliki forest virus promoter sequence, an SV40 promoter sequence, a vaccinia virus promoter sequence, a lentivirus promoter, a retrovirus promoter sequence, or a minimal viral promoter sequence. One of ordinary skill in the art would be familiar with these and other constitutive promoter sequences.
[0204] In some embodiments, the constitutive promoter is a minimal viral promoter sequence. For example, the minimal viral promoter sequence may be a RNA virus promoter, DNA virus promoter, adenoviral promoter sequence, a baculoviral promoter sequence, a CMV promoter sequence, a parvovirus promoter sequence, a herpesvirus promoter sequence, a poxvirus promoter sequence, an adeno-associated virus promoter sequence, a semiliki forest virus promoter sequence, an SV40 promoter sequence, a vaccinia virus promoter sequence, a lentivirus promoter, or a retrovirus promoter sequence.
[0205] In further embodiments, the promoter sequence is a tissue selective promoter sequence. A “tissue selective promoter sequence” is defined herein to refer to a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled, wherein the sequence is active in cells of a particular lineage or tissue type. For example, the tissue-selective promoter sequence may be a promoter sequence that is active in normal and/or diseased heart, lung, esophagus, muscle, intestine, breast, prostate, stomach, bladder, liver, spleen, pancreas, kidney, neurons, myocytes, leukocytes, immortalized cells, neoplastic cells, tumor cells, cancer cells, duodenum, jejunum, ileum, cecum, colon, rectum, salivary glands, gall bladder, urinary bladder, trachea, larynx, pharynx, aorta, arteries, capillaries, veins, thymus, lymph nodes, bone marrow, pituitary gland, thyroid gland, parathyroid glands, adrenal glands, brain, cerebrum, cerebellum, medulla, pons, spinal cord, nerves, skeletal muscle, smooth muscle, bone, testes, epidiymides, prostate, seminal vesicles, penis, ovaries, uterus, mammary glands, vagina, skin, eyes, or optic nerve.
[0206] In some embodiments, the tissue-selective promoter sequence is an hTR promoter sequence, a hTERT promoter sequence, a CEA promoter sequence, a PSA promoter sequence promoter sequence, a probasin promoter sequence, a ARR2PB promoter sequence, an AFP promoter sequence, a MUC-I promoter sequence, a MUC-4 promoter sequence, a mucin-like glycoprotein promoter sequence, a C-erbB2/neu oncogene promoter sequence, a cyclo-oxygenase promoter sequence, a E2F transcription factor 1 promoter sequence, a tyrosinase related protein promoter sequence, a tyrosinase promoter sequence, a survivin promoter sequence, a Tcfl-alpha promoter sequence, a Ras promoter sequence, a Raf promoter sequence, a cyclin E promoter sequence, a Cdc25A promoter sequence, a HK II promoter sequence, a KRT 19 promoter sequence, a TFFl promoter sequence, a SELlL promoter sequence, or a CEL promoter sequence.
[0207] Other examples of tissue-selective promoter sequences include an immunoglobulin heavy chain promoter sequence, an immunoglobulin light chain promoter sequence, a T-cell receptor promoter sequence, an HLA DQ a promoter sequence, an HLA DQ beta promoter sequence, a beta-interferon promoter sequence, an interleukin-2 promoter sequence, an interleukin-2 receptor promoter sequence, an MHC Class II 5 promoter sequence, an MHC Class II HLA-Dra promoter sequence, a muscle creatine kinase (MCK) promoter sequence, a prealbumin (transthyretin) promoter sequence, an albumin promoter sequence, an alpha-fetoprotein promoter sequence, a gamma-globin promoter sequence, a beta-globin promoter sequence, a, an insulin promoter sequence, a neural cell adhesion molecule (NCAM) promoter sequence, an alpha-1-antitrypsin promoter sequence, a growth hormone promoter sequence, a human serum amyoid A (SAA) promoter sequence, a troponin I (TN I) promoter sequence, a Duchenne Muscular Dystrophy promoter sequence, an SV40 promoter sequence, a Hepatitis B virus promoter sequence, a Gibbon Ape Leukemia Virus promoter sequence, a somatostatin receptor promoter sequence, a human CD4 promoter sequence, a human alpha-lactalbumin promoter sequence, a human Y promoter sequence, an alpha fetoprotein promoter sequence, a monocyte receptor for bacterial LPS promoter sequence, a leukocyte common antigen promoter sequence, a Desmin promoter sequence, a VEGF receptor promoter sequence, a glial fibrillary acidic protein promoter sequence, an interferon beta promoter sequence, a myoglobin promoter sequence, an osteocalcin 2 promoter sequence, a prostate specific antigen promoter sequence, a prostate specific membrane antigen promoter sequence, a surfactant protein B promoter sequence, a Synapsin promoter sequence, a tyrosinase related protein promoter sequence, a tyrosinase promoter sequence, a functional hybrid, functional portion, or a combination of any of the aforementioned promoter sequences. Some promoters may be both lineage specific and functional (e.g., albumin). Other examples of promoter sequences include a collagenase promoter sequence, an H2B (TH2B) histone promoter sequence, a type I collagen promoter sequence, a GRP94 promoter sequence, a GRP78 promoter sequence, a glucose-regulated protein promoter sequence, a Human Immunodeficiency Virus promoter sequence, a human LIMK2 gene promoter sequence, a murine epididymal retinoic acid-binding gene promoter sequence, a mouse alpha2 (XI) collagen promoter sequence, a D1A dopamine receptor promoter sequence, an insulin-like growth factor II promoter sequence, a human platelet endothelial cell adhesion molecule-1 promoter sequence, a 7SL promoter sequence, a human MRP-7-2 promoter sequence, a leukosialin promoter sequence, a Sialophorin promoter sequence, a Macrosialin or human analogue of macrosialin promoter sequence, and an Endoglin promoter sequence. To overcome weak expression, promoters and nucleic acids of the invention may be included in an amplified promoter system. An amplified promoter system typically includes a nucleic acid sequence encoding a transactivator under the control of a promoter such as a tissue specific promoter or a core promoter. The encoded transactivator is coupled or present in a cell with a second nucleic acid including a nucleic acid(s) of interest, such as a reporter or therapeutic, which is under the control of a promoter activated by the transactivator. In certain embodiments the transactivator is a GalVP 16 transactivator. The GalVP 16 may include a varying number of GAL and/or VP 16 within the construct. The GalVP 16 may include one or more genes linked to the GalVP 16.
[0208] The nucleic acids of the amplified promoter system may or may not be operatively coupled to a core promoter sequence. A core promoter sequence is defined herein to refer to a nucleotide sequence that maintains the ability to bind and locate a transactivator or a component of a transcription complex to a particular location in a nucleic acid. In some embodiments, tissue-selective promoter sequence is operatively coupled to a core promoter sequence. The core promoter sequence can be any core promoter sequence known to those of ordinary skill in the art, such as a minimal viral promoter sequence. A minimal viral promoter sequence can be any minimal viral promoter sequence known to those of ordinary skill in the art. For example, the minimal viral promoter sequence may be an RNA virus promoter, DNA virus promoter, adenoviral promoter sequence, a baculoviral promoter sequence, a CMV promoter sequence, a parvovirus promoter sequence, a herpesvirus promoter sequence, a poxvirus promoter sequence, an adeno-associated virus promoter sequence, a semiliki forest virus promoter sequence, an SV40 promoter sequence, a vaccinia virus promoter sequence, a lentivirus promoter, a reovirus promoter, or a retrovirus promoter sequence. In particular embodiments, the minimal viral promoter sequence is a mini-CMV promoter sequence.
B. Internal Ribosome Entry Sites (IRES)
[0209] In certain embodiments of the invention, internal ribosome entry site (IRES) elements are used to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5′ methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988). IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), as well an IRES from a mammalian message (Macejak and Sarnow, 1991) and further sequences as well as modified versions are envisioned in this application for invention. IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message (U.S. Pat. Nos. 5,925,565 and 5,935,819; and PCT application PCT/US99/05781) and are envisioned in this application for invention. The order (upstream or downstream of the IRES) of the reporter and gene(s) of interest is not important for the invention. More than one gene of interest may be linked.
C. Selectable Markers
[0210] In certain embodiments of the invention, a nucleic acid construct of the present invention may be isolated or selected for in vitro or in vivo by including a selectable marker in the expression vector. Such selectable markers would confer an identifiable characteristic to the cell permitting easy identification, isolation and/or selection of cells containing the expression vector. A positive selectable marker is one in which the presence of the marker allows for its selection, while a negative selectable marker is one in which its presence prevents its selection. An example of a positive selectable marker is a drug resistance marker. Examples of selectable and screenable markers are well known to one of skill in the art.
D. Other Elements of Expression Cassettes
[0211] Most transcribed eukaryotic RNA molecules will undergo RNA splicing to remove introns from the primary transcripts. Vectors containing genomic eukaryotic sequences may require donor and/or acceptor splicing sites to ensure proper processing of the transcript for protein expression (Chandler et al, 1997).
[0212] One may include a polyadenylation signal in the expression construct to effect proper polyadenylation of the transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and/or any such sequence may be employed. Specific embodiments include the SV40 polyadenylation signal and/or the bovine growth hormone polyadenylation signal, convenient and/or known to function well in various target cells. Also contemplated as an element of the expression cassette is a transcriptional termination site. These elements can serve to enhance message levels and/or to minimize read through from the cassette into other sequences. The vectors or constructs of the present invention may comprise at least one termination signal. A “termination signal” or “terminator” is comprised of the DNA sequences involved in specific termination of an RNA transcript by an RNA polymerase. Thus, in certain embodiments a termination signal that ends the production of an RNA transcript is contemplated. A terminator may be necessary in vivo to achieve desirable message levels.
[0213] In eukaryotic systems, the terminator region may also comprise specific DNA sequences that permit site-specific cleavage of the new transcript so as to expose a polyadenylation site. This signals a specialized endogenous polymerase to add a stretch of about 200 A residues (polyA) to the 3′ end of the transcript. RNA molecules modified with this polyA tail appear to more stable and are translated more efficiently. Thus, in other embodiments involving eukaryotes, the terminator may comprise a signal for the cleavage of the RNA, and it is more specific that the terminator signal promotes polyadenylation of the message. The terminator and/or polyadenylation site elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.
[0214] Terminators contemplated for use in the invention include any known terminator of transcription described herein or known to one of ordinary skill in the art, including but not limited to, for example, the termination sequences of genes, such as for example the bovine growth hormone terminator or viral termination sequences, such as for example the SV40 terminator. In certain embodiments, the termination signal may be a lack of transcribable or translatable sequence, such as due to a sequence truncation.
[0215] In order to propagate a vector in a host cell, it may contain one or more origins of replication sites (often termed “ori”), which is a specific nucleic acid sequence at which replication is initiated. Alternatively an autonomously replicating sequence (ARS) can be employed if the host cell is yeast.
IX. Gene Delivery
[0216] Aspects of the invention include transferring into a cell an expression construct comprising a nucleic acid sequence encoding a somatostatin receptor or its mutant. Techniques pertaining to the transfer of expression constructs into cells are well-known to those of ordinary skill in the art. Exemplary techniques are discussed below.
A. Viral Vectors
[0217] In certain embodiments of the present invention, transfer of an expression construct into a cell is accomplished using a viral vector. Techniques using “viral vectors” are well-known in the art. A viral vector is meant to include those constructs containing viral sequences sufficient to (a) support packaging of the expression cassette and (b) to ultimately express a recombinant gene construct that has been cloned therein.
[0218] In particular embodiments, the viral vector is a lentivirus vector. Lentivirus vectors have been successfully used in infecting stem cells and providing long term expression.
[0219] Another method for delivery of a nucleic acid involves the use of an adenovirus vector. Adenovirus vectors are known to have a low capacity for integration into genomic DNA. Adenovirus vectors result in highly efficient gene transfer.
[0220] Adenoviruses are currently the most commonly used vector for gene transfer in clinical settings. Among the advantages of these viruses is that they are efficient at gene delivery to both nondividing and dividing cells and can be produced in large quantities. The vector comprises a genetically engineered form of adenovirus (Grunhaus et al, 1992). In contrast to retrovirus, the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity. Also, adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification.
[0221] Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized genome, ease of manipulation, high titer, wide target-cell range and high infectivity. A person of ordinary skill in the art would be familiar with experimental methods using adenoviral vectors.
[0222] The adenovirus vector may be replication defective, or at least conditionally defective, and the nature of the adenovirus vector is not believed to be crucial to the successful practice of the invention. The adenovirus may be of any of the 42 different known serotypes or subgroups A-F and other serotypes or subgroups are envisioned. Adenovirus type 5 of subgroup C is the starting material in order to obtain the conditional replication-defective adenovirus vector for use in the present invention. This is because Adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector. Adenovirus growth and manipulation is known to those of skill in the art, and exhibits broad host range in vitro and in vivo. Modified viruses, such as adenoviruses with alteration of the CAR domain, may also be used. Methods for enhancing delivery or evading an immune response, such as liposome encapsulation of the virus, are also envisioned. The retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse-transcription (Coffin, 1990). The resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins. The integration results in the retention of the viral gene sequences in the recipient cell and its descendants. The retroviral genome contains two long terminal repeat (LTR) sequences present at the 5′ and 3′ ends of the viral genome. These contain strong promoter and enhancer sequences and are also required for integration in the host cell genome (Coffin, 1990).
[0223] In order to construct a retroviral vector, a nucleic acid encoding a nucleic acid or gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. A person of ordinary skill in the art would be familiar with well-known techniques that are available to construct a retroviral vector. Adeno-associated virus (AAV) is an attractive vector system for use in the present invention as it has a high frequency of integration and it can infect nondividing cells, thus making it useful for delivery of genes into mammalian cells in tissue culture (Muzyczka, 1992). AAV has a broad host range for infectivity (Tratschin et al., 1984; Laughlin et al, 1986; Lebkowski et al, 1988; McLaughlin et al, 1988), which means it is applicable for use with the present invention. Details concerning the generation and use of rAAV vectors are described in U.S. Pat. Nos. 5,139,941 and 4,797,368, each incorporated herein by reference.
[0224] Typically, recombinant AAV (rAAV) virus is made by cotransfecting a plasmid containing the gene of interest flanked by the two AAV terminal repeats (McLaughlin et al, 1988; Samulski et al, 1989; each incorporated herein by reference) and an expression plasmid containing the wild-type AAV coding sequences without the terminal repeats, for example pIM45 (McCarty et al., 1991; incorporated herein by reference). A person of ordinary skill in the art would be familiar with techniques available to generate vectors using AAV virus.
[0225] Herpes simplex virus (HSV) has generated considerable interest in treating nervous system disorders due to its tropism for neuronal cells, but this vector also can be exploited for other tissues given its wide host range. Another factor that makes HSV an attractive vector is the size and organization of the genome. Because HSV is large, incorporation of multiple genes or expression cassettes is less problematic than in other smaller viral systems. In addition, the availability of different viral control sequences with varying performance (temporal, strength, etc) makes it possible to control expression to a greater extent than in other systems. It also is an advantage that the virus has relatively few spliced messages, further easing genetic manipulations.
[0226] HSV also is relatively easy to manipulate and can be grown to high titers. Thus, delivery is less of a problem, both in terms of volumes needed to attain sufficient MOI and in a lessened need for repeat dosings. For a review of HSV as a gene therapy vector, see Glorioso et al. (1995). A person of ordinary skill in the art would be familiar with well-known techniques for use of HSV as vectors.
[0227] Vaccinia virus vectors have been used extensively because of the ease of their construction, relatively high levels of expression obtained, wide host range and large capacity for carrying DNA. Vaccinia contains a linear, double-stranded DNA genome of about 186 kb that exhibits a marked “A-T” preference. Inverted terminal repeats of about 10.5 kb flank the genome.
[0228] Other viral vectors may be employed as constructs in the present invention. For example, vectors derived from viruses such as poxvirus may be employed. A molecularly cloned strain of Venezuelan equine encephalitis (VEE) virus has been genetically refined as a replication competent vaccine vector for the expression of heterologous viral proteins (Davis et al., 1996). Studies have demonstrated that VEE infection stimulates potent CTL responses and it has been suggested that VEE may be an extremely useful vector for immunizations (Caley et al., 1997). It is contemplated in the present invention, that VEE virus may be useful in targeting dendritic cells.
[0229] A polynucleotide may be housed within a viral vector that has been engineered to express a specific binding ligand. The virus particle will thus bind specifically to the cognate receptors of the target cell and deliver the contents to the cell. A novel approach designed to allow specific targeting of retrovirus vectors was developed based on the chemical modification of a retrovirus by the chemical addition of lactose residues to the viral envelope. This modification can permit the specific infection of hepatocytes via sialoglycoprotein receptors.
[0230] Another approach to targeting of recombinant retroviruses was designed in which biotinylated antibodies against a retroviral envelope protein and against a specific cell receptor were used. The antibodies were coupled via the biotin components by using streptavidin (Roux et al., 1989). Using antibodies against major histocompatibility complex class I and class II antigens, they demonstrated the infection of a variety of human cells that bore those surface antigens with an ecotropic virus in vitro (Roux et al., 1989).
B. Nonviral Gene Transfer
[0231] Several non-viral methods for the transfer of nucleic acids into cells also are contemplated by certain aspects of the present invention. These include calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al, 1990) DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et al, 1986; Potter et al, 1984), nucleofection (Trompeter et al, 2003), direct microinjection (Harland and Weintraub, 1985), DNA-loaded liposomes (Nicolau and Sene, 1982; Fraley et al, 1979) and lipofectamine-DNA complexes, polyamino acids, cell sonication (Fechheimer et al, 1987), gene bombardment using high velocity microprojectiles (Yang et al, 1990), polycations (Boussif et al, 1995) and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988). Some of these techniques may be successfully adapted for in vivo or ex vivo use. A person of ordinary skill in the art would be familiar with the techniques pertaining to use of nonviral vectors, and would understand that other types of nonviral vectors than those disclosed herein are contemplated by the present invention. In a further embodiment of the invention, the expression cassette may be entrapped in a liposome or lipid formulation. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. Also contemplated is a gene construct complexed with Lipofectamine (Gibco BRL). One of ordinary skill in the art would be familiar with techniques utilizing liposomes and lipid formulations.
X. Kits
[0232] The present invention provides kits, such as diagnostic and therapeutic kits based on cellular therapy and/or gene therapy. For example, a kit may comprise one or more pharmaceutical compositions as described herein and optionally instructions for their use. Kits may also comprise one or more devices for accomplishing administration of such compositions. For example, a subject kit may comprise a pharmaceutical composition and catheter for accomplishing direct intraarterial injection of the composition into a cancerous tumor. In other embodiments, a subject kit may comprise pre-filled ampoules of a protein isoform specific antibody construct, optionally formulated as a pharmaceutical, or lyophilized, for use with a delivery device.
[0233] Kits may comprise a container with a label. Suitable containers include, for example, bottles, vials, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The container may hold a composition which includes an antibody that is effective for therapeutic or non-therapeutic applications, such as described above. The label on the container may indicate that the composition is used for a specific therapy or non-therapeutic application, and may also indicate directions for either in vivo or in vitro use, such as those described above. The kit of the invention will typically comprise the container described above and one or more other containers comprising materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
XI. Examples
[0234] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Example 1
[0235] In the following experiments, HA-tagged receptors were used. In vitro experiments demonstrate that the delta340 mutant inhibits cell growth without ligand in both transiently transfected cells and stably transfected cells, whereas cells expressing wild-type SSTR2 require ligand stimulation to inhibit growth and vector control cells do not inhibit growth regardless of ligand presence. Human HA-SSTR2D314 does not inhibit cellular growth upon ligand stimulation, whereas HA-SSTR2D340 inhibits growth even without ligand stimulation, similar to inhibition by wild-type receptor upon activation with ligand (FIG. 1 ).
[0236] Human HA-SSTR2D340 was confirmed to inhibit growth even without ligand stimulation (FIG. 2 ). Two days after transient transfection with HA-SSTR2D351 or HA-SSTR2D340, 3000 HT1080 cells were grown in the presence of 5% FBS with or without 100 nM of the somatostatin analogue sandostatin. Three days later, MTT assay was used to determine cell number. Stable transfectants, wild-type receptor (SSTR2FL) was used as a positive control for ligand responsive growth inhibition and HA-SSTR2D314 does not inhibit growth upon ligand stimulation. HA-SSTR2D351 or HA-SSTR2D340 demonstrated equivalent amounts of expression (data not shown). As shown in FIG. 12 , constitutively active SSTR2delta340 inhibits the growth of tumor cells in vivo.
[0237] In vitro experiments demonstrate that in a dose dependent manner, 90-Y-octreotate induces cell kill in cells expressing wild-type and signaling deficient mutant delta314 more than vector-transfected cells (FIGS. 3A-B ). Stably transfected HT1080 cells were exposed to different amounts of 90Y-DOTATATE for 1 hour and 72 hours. Dose dependent and time dependent killing was observed in HT1080 cells expressing wild-type or signaling deficient HA-SSTR2 receptors, but not vector transfected cells. Cell death was assessed by trypan blue uptake. *p<0.05 vs Vector.
[0238] Mixing mesenchymal stem cells expressing SSTR2 or the signaling deficient mutant delta 314 with untransfected tumor cells results in kill of rosettes of clusters of cells that are not seen if mesenchymal stem cells transfected with vector are used (FIG. 4 ), consistent with bystander killing. Human mesenchymal stem cells (HS5) stably expressing wild-type or signaling deficient HA-SSTR2 receptors or transfected with vector were admixed with untransfected HT1080 cells and then exposed to 90Y-DOTATATE for 24 hours. Rosettes (clusters) of dead cells were seen in wells containing mesenchymal stem cells stably expressing wild-type or signaling deficient HA-SSTR2 receptors but not in wells containing mesenchymal stem cells transfected with vector.
[0239] In vivo, addition of unlabeled somatostatin analogue octreotide inhibited growth of tumors expressing wild-type receptor, but not the signaling deficient receptor (FIG. 5 ). Stably transfected HT1080 cells were implanted subcutaneously in nude mice (three tumors, HA-wt, HA-D314, vector, per animal). When tumors were palpable, daily injections of the somatostatin analogue sandostatin were initiated and tumors were measured by calipers every two days. Receptor activation by octreotide inhibited growth of tumors expressing wild-type receptor, but no decrease in tumor size was seen with the signaling deficient receptor. Thus, a signaling active receptor can inhibit tumor growth.
[0240] The same finding was noted with 111-In octreotide implying that this imaging agent affects tumor growth via the biologic effect of octreotide and not the radioactivity (FIG. 6A ). The imaging agent 111-In-octreotide inhibited growth of tumors expressing the wild-type receptor, but not the signaling deficient receptor or tumors derived from vector transfected cells. Stably transfected HT1080 cells were implanted subcutaneously in nude mice (three tumors, HA-wt, HA-D314, vector, per animal). When tumors were palpable, 0.9 mCi of 111-In octreotide was given IV on day 4 and day 6. Tumors were measured by calipers every two days. *p<0.05 SSTR2FL vs Vector. This suggests that tumor growth may be inhibited via activation of SSTR signaling in cells with functional SSTR signaling.
[0241] Expression of SSTR2 can be imaged in tumors expressing wild-type or signaling deficient HA-SSTR2 (FIG. 6B ). Gamma-camera imaging of mice at 24 hours and 48 hours after the first and 24 hours after the second 111-In octreotide injection. Uptake (degree of receptor expression) is similar in tumors expressing wild-type or signaling deficient HA-SSTR2 at 24 hours and wanes in both at 48 hours. Increased uptake is again seen 24 hours after the second 111-In octreotide injection. Minimal uptake is seen in tumors derived from cells transfected with vector. Thus, both wild-type and signaling deficient SSTR2 uptake 111-In octreotide.
[0242] In comparison, 90Y-octreotate inhibited growth of tumors expressing wild-type or signaling deficient receptors (FIGS. 7A-7B ). The latter was noted to be dose dependent. Stably transfected HT1080 cells were implanted subcutaneously in nude mice (three tumors, HA-wt, HA-D314, vector, per animal). When tumors were palpable, 90Y-octreotate was given once at a dose of 1 mCi (FIG. 7A ) or twice at doses of 2 mCi and 1 mCi (FIG. 7B ). Tumors were measured by calipers every two days. *p<0.05 HA-SSTR2FL or HA-D314 vs Vector. Tumors expressing the signaling deficient receptor decreased in size in a dose dependent manner due to the effect of radioactivity on the tumor. Reduction in size of tumors expressing wild-type HA-SSTR2 was due to a combination of effects of receptor activation by the somatoatatin analogue octreotate and effects of radioactivity. This suggests that tumor growth may be inhibited by other mechanisms such as radiation-induced damage in cells without functional SSTR signaling but expressing SSTR's.
[0243] Stem cells expressing the signaling deficient mutant SSTR2D314 incorporated into tumor and can be targeted with a therapeutic for inhibiting tumor growth (FIG. 8 ). 90Y-octreotate (primarily beta emitter) inhibited growth of human tumors incorporating HS5 human mesenchymal stem cells expressing SSTR2D314, but not control HS5 cells. Human ovarian cancer (Hey A8) cells were co-injected subcutaneously in nude mice with HS5 cells expressing or not expressing HA-SSTR2D314. (Previous experiments demonstrated that HS5 cells alone do not form tumors). Once tumors were established, 90Y-octreotate was delivered intravenously. Tumors were measured by calipers every two days. Tumors incorporating HS5 cells expressing the HA-SSTR2D314 mutant were growth inhibited compared to control (*p<0.05, HeyA8+HS5-SSTR2D314 vs HeyA8+HS5vector). The incorporated stem cells, but not the tumor cells, serve as a target for the therapeutic. Data suggest that human stem cells expressing a signaling deficient SSTR2 mutant that were incorporated into human tumor and can serve as a therapeutic target for inhibiting established tumor growth.
[0244] Stem cells expressing the signaling deficient mutant SSTR2D314 home to and incorporate into tumor and can be targeted with a therapeutic for inhibiting tumor growth (FIG. 9 ). 90Y-octreotate (primarily beta emitter) inhibited growth of human tumors to which human mesenchymal stem cells expressing HA-SSTR2D314 homed and incorporated, but not control HS5 cells. Human ovarian cancer (Hey A8) cells on one side and vehicle on the other side were injected subcutaneously in nude. Two days later, human mesenchymal stem cells (HS5 cells) expressing or not expressing HA-SSTR2D314 were injected systemically (intracardiac). No tumors were seen in the side injected with vehicle. Tumors were seen on the side injected with HeyA8 cells. Once tumors were established, 90Y-octreotate was delivered intravenously. Tumors were measured by calipers every two days. Tumors to which HS5 cells expressing the HA-SSTR2D314 mutant homed and incorporated were growth inhibited compared to control (*p<0.05, HeyA8+HS5-SSTR2D314 vs HeyA8+HS5vector). The incorporated stem cells not the tumor cells serve as a target for the therapeutic. Data suggest that systemically delivered human stem cells expressing a signaling deficient SSTR2 mutant home to and incorporate into human tumors and can serve as a therapeutic target for inhibiting established tumor growth. This suggests that established tumors and likely (micro)metastases can be targeted by this method.
[0245] After intracardiac injection, human MSC's traffic to HeyA8 tumors; and expression of the signaling deficient SSTR2 mutant in such MSC's can be distinguished (FIG. 10 ). As can be seen in FIG. 10 , left panel, the 111-In-octreotate signal could be visualized in the “HS5-HA-hSSTRs-SD” treated panel, above left, whereas no such signal could be seen in the control vector panel, below left. In particular, HS5 cells expressing signaling deficient human SSTR2 (HS5-HA-hSSTR2-SD) injected intracardiac localized to Hey A8 tumors in mice and could be visualized one day after 111-In-octreotide injection (arrow, middle panel, top) whereas background signal was seen with control HS5 cells transfected with vector (arrow, middle panel, bottom). After imaging, tumors were removed and evaluated, confirming the imaging findings. Right panel: Biodistribution demonstrated increased uptake of 111-In-octreotide in tumors from animals injected intracardiac with HS5 cells expressing signaling deficient human SSTR2 (HA-hSSTR2-SD) compared to injected with HS5 cells transfected with vector. Inset: Western blot using an antibody to the HA domain of signaling deficient HA-SSTR2 demonstrated expression of the reporter by HS5 cells in tumors from animals injected intracardiac with HS5 cells expressing signaling deficient human SSTR2 (top arrow, left, Anti-HA-11) but not with HS5 cells transfected with vector (top arrow, right, Anti-HA-11). Beta-actin, a control house-keeping protein expected to be found in all tumors, was seen in both tumor lysates (bottom arrow, Anti-beta-actin) as expected.
[0246] After tumors were present in three locations per mouse in all 8 mice, the animals were injected i.v. with 90-Y labeled octreotate two days and 5 days later as on the chart. Tumor size was measured by calipers. Unlike many stem cells, embryonic stem cells (ES) more commonly form teratomas. Inhibition of growth of established teratomas was assessed. Growth of teratomas expressing HA-SSTR2 (ES-FL) or signaling deficient HA-SSTR2-delta 314 (ES+delta314) was inhibited, compared to wild type teratomas (FIG. 11 ). This adds a safety factor and suggests that if delivered cells become neoplastic, their growth may be inhibited by targeting SSTR or their mutants or chimeras.
[0247] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
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Claims
The invention claimed is:
1. A constitutively active somatostatin receptor type 2 (SSTR2) mutant, wherein the mutant is a C-terminal deletion mutant of a human somatostatin receptor, wherein mutant is SSTR2 delta 340 mutant with the sequence of amino acids 1-340 of SEQ ID NO:6.
2. A therapeutic composition comprising the mutant of claim 1 and a pharmaceutically acceptable carrier.
3. A nucleic acid molecule comprising a constitutively active somatostatin receptor (SSTR2) mutant of a human somatostatin receptor, having a nucleotide sequence that encodes the amino acids 1-340 of SEQ ID NO: 6.
4. A therapeutic composition comprising the nucleic acid of claim 3, and a pharmaceutically acceptable carrier.
5. An expression construct comprising the nucleic acid molecule of claim 3.
6. A therapeutic composition comprising the expression construct of claim 5, and a pharmaceutically acceptable carrier.
7. The expression construct of claim 5, wherein the nucleic acid further comprises a second coding sequence.
8. The expression construct of claim 7, wherein the second coding sequence is a protein tag gene, a reporter gene, a therapeutic gene, a signaling sequence, or a trafficking sequence.
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