Patent Application
20200384135
The instant application contains a Sequence Listing which has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 26, 2020, is named 110551-0146_SL.txt and is 5.479 bytes in size.
The invention generally relates to genetic constructs and methods for their use in cancer imaging, cancer treatment, and combined imaging and treatment protocols, e.g. for imaging and/or treating spontaneous metastasis. In particular, transcription of genes in the constructs is driven by cancer specific or cancer selective promoters.
Targeted imaging of cancer remains an important but elusive goal. Such imaging could provide early diagnosis, detection of metastasis, aid treatment planning and benefit therapeutic monitoring. By leveraging the expanding list of specific molecular characteristics of tumors and their microenvironment, molecular imaging also has the potential to generate tumor-specific reagents. But many efforts at tumor-specific imaging are fraught by nonspecific localization of the putstive targeted agents, eliciting unacceptably high background noise.
While investigators use many strategies to provide tumor-specific imaging agents—largely in the service of maintaining high target-to-background ratios—they fall into two general categories, namely direct and indirect methods1. Direct methods employ an agent that reports directly on a specific parameter, such as a receptor, transporter or enzyme concentration, usually by binding directly to the target protein. Indirect methods use a reporter transgene strategy, in analogy to the use of green fluorescent protein (GFP) in vitro, to provide a read-out on cellular processes occurring in vivo by use of an external imaging device, Molecular-genetic imaging employs an indirect technique that has enabled the visualization and quantification of the activity of a variety of gene promoters, transcription factors and key enzymes involved in disease processes and therapeutics in vivo including Gli2, E2Fl3, telomerase4,5, and several kinases, including one that has proved useful in human gene therapy trials6,7. Unfortunately, to date, none of these techniques has provided sufficient specific localization of imaging agents, and unacceptably high background noise is still prevalent.
Cancer therapies have also advanced considerably during the last few decades. However, they are also still hampered by nonspecific delivery of and-tumor agents to normal cells, resulting in horrendous side effects for patients. This lack of specificity also results in lower efficacy of treatments due to the want of a capability to deliver active agents in a focused manner where they are most needed, i.e., to cancer cells alone.
In addition, there is currently no efficacious way to image and/or treat cancerous cells and tissues which are caused by spontaneous metastasis. Spontaneous metastasis refers to cancer cells that escape from the primary tumor, enter the bloodstream or lymphatics, that settle in tissue remote from the primary tumor and grow and thrive in the new location(s). For example, prostate cancer often metastasizes to bone.
U.S. Pat. No. 6,737,523 (Fisher et al.), the complete contents of which is hereby incorporated by reference, describes a progression elevated gene-3 (PEG-3) promoter, which is specific for directing gene expression in cancer cells. The patent describes the use of the promoter to express genes of interest in cancer cells in a specific manner. However, imaging and combined imaging and treatment are not discussed.
United States patent application 2009/0311664 describes cancer cell detection and imaging using viral vectors that are conditionally competent for expression of a reporter gene only in cancer cells. However, the technique is not used in vivo, combined methods of imaging and treatment are not discussed, and only herpes and vaccinia viruses are discussed in detail.
There is an ongoing need to develop improved methods of cancer imaging and treatment that are highly specific for cancer cells, and it would be a boon for patients and physicians to have available methods which combine a means of cancer imaging and a means of therapeutically treating cancer in a single method.
The invention generally relates to genetic constructs and methods for their use in i) cancer imaging, and ii) cancer treatment; and iii) combined treatment and imaging. Combined treatment and imaging may be referred to herein as a “theranostic” approach to cancer. The gene constructs used in these methods comprise a promoter that is specifically or selectively active in cancer cells. These promoters may be referred to herein as “cancer promoters” or “cancer specific/selective promoters” or simply as “specific/selective promoters”. Due to the specificity afforded by these promoters, compositions, which include the constructs of the invention, can be advantageously administered systemically to a subject that is in need of cancer imaging or cancer treatment, or both. The promoters, e.g., AEG-Prom, are advantageously highly effective for imaging and treating spontaneous metastases, as demonstrated in Example 3 below.
The treatment aspect of the invention provides a high level of precise delivery of anti-tumor agents to cancer cells, even when delivery is made systemically, since the anti-tumor agents associated with the methods are only expressed within cancer cells. This advantageously results in few or no side effects for patients being treated by the method.
Similarly, the imaging aspect of the invention provides a high level of precise imaging of cancer cells and tumors with little or no background signal. Importantly, since there is little or no background “noise”, the imaging techniques of the invention enable the facile detection of metastatic cancer, even metastatic cancer that is not detectable with other methods due to e.g., the very small size of a newly developing tumor, or the diffuse pattern of cancer cells which do not actually form, a tumor. As is well known in the art, early detection of tumors can significantly improve the outcome of tumor treatment. Similarly, detection of cancerous tissues before formation of a tumor will provide significant benefits.
The combined imaging and treatment methods are advantageous over the prior art in many ways. A combined approach to imaging and therapy is more efficient and requires fewer procedures, and hence less effort, on the part of the patient and the cancer specialist. Since activity is confined to cancer cells, side effects are reduced. In addition, the combined imaging and treatment method provides the ability to accurately monitor the effects of prior treatment concomitantly with providing treatment and this provides a cancer treatment specialist with an invaluable and accurate window on the progress of therapy, permitting therapeutic parameters to be fine-tuned in close conjunction with treatment.
In addition, the invention provides transgenic animals that have been genetically engineered to contain nucleotide sequences encoding a reporter gene operably linked to a cancer specific or cancer selective promoter, and their use for clinical evaluation of therapies. In some embodiments, the transgenic animals have a propensity for developing cancer.
It is an object of this invention to provide a method of imaging tumors or cancerous cells or tissue in a subject. The method comprises the steps of 1) administering to said subject a nucleic acid construct comprising an imaging reporter gene operably linked to a cancer specific or cancer selective promoter; 2) administering to said subject an imaging agent that is complementary to said imaging reporter gene; and 3) imaging tumors or cancerous tissues or cells in said subject by detecting a detectable signal from said imaging agent. In some embodiments, the imaging reporter gene is selected from the groups consisting of luciferase and herpes simplex virus 1 thymidine kinase (HSV1-tk); the subject may be a cancer patient. The imaging agent may be a radiolabeled nucleoside analog such as 2′-fluoro-2′deoxy-β-D-5-[125I]iodouracil-arabinofuranoside. The step of imaging may be carried out via single photon emission computed tomography (SPECT) or by positron emission tomography (PET) The imaging reporter gene may be luciferase and said subject is a laboratory animal, in which case the imaging agent is a luciferase substrate. In some embodiments, the nucleic acid construct is present in a polyplex with a cationic polymer such as polyethylemeinine. One or both of the steps of administering may be carried out systemically. The step of administering a nucleic acid construct may be carried out by intravenous injection. In some embodiments, the tumors, cancerous tissues or cells include cancer cells of a type selected from groups consisting of breast cancer, melanoma, carcinoma of unknown primary (CUP), neuroblastoma, malignant glioma, cervical, colon, hepatocarcinoma, ovarian, lung, pancreatic, and prostate cancer. In some embodiments, the nucleic acid construct is present in a plasmid. In other embodiments, the nucleic acid construct is present in a viral vector such as a conditionally replication-competent adenovirus. In some embodiments, the cancer specific or cancer selective promoter is the progression elevated gene-3 (PEG-3) promoter.
The invention also provides a method of both imaging and treating tumors, or cancerous tissues or cells in a subject. The method includes the steps of 1) administering to said subject one or more nucleic acid constructs comprising an imaging reporter gene operably linked to a cancer specific or cancer selective promoter and a gene encoding an anti-tumor agent; 2) administering to said subject an imaging agent that is complementary to said imaging reporter gene; and 3) imaging tumors or cancerous tissues or cells in said subject by detecting a detectable signal from said imaging agent, wherein said gene encoding said anti-tumor agent is expressed by cells in said tumors or cancerous tissues or cells to act on said cells. In some embodiments, at least one, and possibly both, of the steps of administering may be carried out systemically. In some embodiments, the gene encoding an anti-tumor agent is operably linked to a tandem gene expression element, for example, an internal ribosomal entry site (IRES). In other embodiments, the gene encoding an anti-tumor agent is operably linked to a cancer specific or cancer selective promoter. The and-tumor agent may be inda-7/IL-24 or tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL).
The invention also provides a cancer specific or cancer selective gene expression imaging system, comprising a nucleic acid construct comprising an imaging reporter gene operably linked to a cancer specific or cancer selective promoter. In some embodiments, the cancer specific or cancer selective promoter is PEG-Prom. In some embodiments, the system is suitable for systemic administration.
The invention further provides a transgenic animal genetically engineered to contain and express a reporter gene linked to a cancer specific or cancer selective promoter. In some embodiments, the transgenic animal is also predisposed to develop cancer.
(16E) Comparison of Luc plasmid delivery to high tumor burden and low tumor burden areas in liver and lungs of the PCa group (n=3, PCa-1-3) and liver and lung sections of control (n=3, Ctrl-1-3). The absolute amount of pAEG-Luc in lung and liver tissues of each animal was quantified by quantitative real-time PCR. The differences in transfection efficiency between areas of high tumor burden vs. those of low tumor burden within liver in same animal (*P<0.0005), as well as between areas of high tumor burden within liver vs, normal liver (**P=0.0078), were significant. No significant difference was observed in transfection efficiency in the lungs and liver tissue between the control group and the PCa group. Error bars represent mean ± standard deviation (SD).
An embodiment of the invention provides nucleic acid constructs and methods for their use in cancer imaging, cancer treatment, and in methods which combine cancer imaging and treatment. Constructs designed for therapy generally comprise a cancer-specific or cancer selective promoter and a recombinant gene that encodes a therapeutic agent (e.g. a protein or polypeptide whose expression is detrimental to cancer cells, and/or or one which cart stimulate the immune system to attack the cancer) operably linked to the cancer-specific promoter. Thus, targeted killing of cancer cells and/or immunoregulation occurs even when the constructs are administered systemically. Constructs designed for imaging comprise a cancer-specific/selective promoter and a recombinant gene that encodes a reporter molecule (and optionally, a complement of the reporter molecule) operably linked to the cancer-specific promoter. The reporter molecule is either detectable in its own right, and hence when it is expressed in a cancer cell renders the cancer cell detectable; or the reporter is capable of associating or interacting with a “complement” (e.g. a substrate) that is detectable or becomes detectable due to the interaction. Constructs designed for both imaging and therapy contain both a recombinant gene that encodes a reporter molecule operably linked to a cancer-specific/selective promoter and a therapeutic agent operably linked to the same copy of a cancer-specific/selective promoter, or to a second copy of the same cancer-specific/selective promoter, or to a different type of cancer-specific/selective promoter. Because the reporter is expressed only in cancer cells, the constructs encoding a reporter and the complement of the reporter can be safely administered systemically even though both are distributed widely throughout the body of a subject, the complement encounters and interacts with the reporter only within cancer cells. In some applications, direct injection into a tumor could also be employed. In some embodiments, the reporter-complement association results in both imaging potential and lethality to the cancer cells. These constructs and methods, and various combinations and permutations thereof, are discussed in detail below.
The constructs described herein are highly effective for imaging and treating spontaneous metastasis.
The constructs of the invention include at least one transcribable element (e.g. a gene composed of sequences of nucleic acids) that is operably connected or linked to a promoter that specifically or selectivdy drives transcription within cancer cells. Expression of the transcribable element may be inducible or constitutive. Suitable cancer selective/specific promoters (and or promoter/enhancer sequences) that may be used include but are not limited to: PEG-PROM, astrocyte elevated gene 1 (AEG-1) promoter (AEG-Prom), survivin-Prom, human telomerase reverse transcriptase (hTERT)-Prom, hypoxia-inducible promoter (HIF-1-alpha), DNA damage inducible promoters (e.g. GADD promoters), metastasis-associated promoters (metalloproteinase, collagenase, etc.), ceruloplasmin promoter (Lee et al., Cancer Res Mar. 1, 2004 64; 1788), mucin-1 promoters such as DF3/MUC1 (see U.S. Pat. No. 7,247,297), HexII promoter as described in US patent application 2001/00111128; prostate-specific antigen enhancer/promoter (Rodriguez et al, Cancer Res., 57: 2559-2563, 1997); α-fetoprotein gene promoter (Hallenbeck et al. Hum. Gene Ther., 10: 1721-1733, 1999); the surfactant protein B gene promoter (Doronin et al. J. Virol., 75; 3314-3324, 2001); MUC1 promoter (Kurihara et al, J. Clin. Investig., 106: 763-771, 2000); H19 promoter as per U.S. Pat. No. 8,034,914; those described in issued U.S. Pat. Nos. 7,816,131, 6,897,024, 7,321,030, 7,364,727, and others; etc., as well as derivative forms thereof. Any promoter that is specific for driving gene expression only in cancer cells, or that is selective for driving gene expression in cancer cells, or at least in cells ola particular type of eancer (so as to treat and image e.g. prostate, colon, breast, etc. primary and metastatic cancer) may be used in the practice of the invention. By “specific for driving gene expression in cancer cells” we mean that the promoter, when operably linked to a gene, functions to promote transcription of the gene only when located within a cancerous, malignant cell, but not when located within normal, non-cancerous cells. By “selective for driving gene expression in cancer cells” we mean that the promoter, when operably linked to a gene, functions to promote transcription of the gene to a greater degree when located within a cancer cell, than when located within non-cancerous cells. For example, the promoter drives gene expression of the gene at least about 2-fold, or about 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold, or even about 20-, 30-, 40-, 50-, 60-,70-, 80-, 90- or 100 -fold or more (e.g. 500- or 1000-fold) when located within a cancerous cell than when located within a non-cancerous cell, when measured using standard gene expression measuring techniques that are known to those of skill in the art.
In one embodiment, the promoter is the PEG-PROM promoter (see
Vectors which comprise the constructs described herein are also encompassed by embodiments of the invention and include both viral and non-viral vectors. Exemplary non-viral vectors that may be employed include but are not limited to, for example: cosmids or plasmids; and, particularly for cloning large nucleic acid molecules, bacterial artificial chromosome vectors (BACs) and yeast artificial chromosome vectors (YACs); as well as liposomes (including targeted liposomes); cationic polymers; ligand-conjugated lipoplexes; polymer-DNA complexes; poly-L-lysine-molossin-DNA complexes; chitosan-DNA nanoparticles; polyethylenimine (PEI, e.g. branched PEI)-DNA complexes; various nanoparticles and/or nanoshells such as multifunctional nanoparticles, metallic nanoparticles or shells (e.g. positively, negatively or neutral charged gold particles, cadmium selenide, etc.); ultrasound-mediated microbubble delivery systems; various dendrimers (e.g. polyphenylene and poly(amidoamine)-based dendrimers; etc.
In addition, viral vectors may be employed. Exemplary viral vectors include but are not limited to: bacteriophages, various baculoviruses, retroviruses, and the like. Those of skill in the art are familiar with viral vectors that are used in “gene therapy” applications, which include but are not limited to: Herpes simplex virus vectors (Geller et al., Science, 241:1667-1669 (1988)); vaccinia virus vectors (Piccini et al., Meth. Enzymology, 153:545-563 (1987)); cytomegalovirus vectors (Mocarski et al., in Viral Vectors, Y. Gluzman and S. H. Hughes, Eds., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1988, pp. 78-84)); Moloney murine leukemia virus vectors (Danos et al., Proc. Natl. Acad. Sci, USA, 85:6460-6464 (1988); Blaese et al., Science, 270:475-479 (1995); Onodera et al., J. Virol., 72:1769-1774 (1998)); adenovirus vectors (Berkner, Biotechniques, 6:616-626 (1988); Cotten et al., Proc. Natl. Acad. Sci. USA, 89:6094-6098 (1992); Graham et al., Meth. Mol. Biol., 7:109-127 (1991); Li et al., Human Gene Therapy, 4:403-409 (1993); Zabner et al., Nature Genetics, 6:75-83 (1994)); adeno-associated virus vectors (Goldman et al., Human Gene Therapy, 10:2261-2268 (1997); Greelish et al., Nature Med., 5:439-443 (1999); Wang et al., Proc. Natl. Acad. Sci, USA, 96:3906-3910 (1999); Snyder et al., Nature Med., 5:64-70 (1999); Herzog et al., Nature Med., 5:56-63 (1999)); retrovirus vectors (Donahue et al., Nature Med., 4:181-186 (1998); Shackleford et al., Proc. Natl. Acad. Sci. USA, 85:9655-9659 (1988); U.S. Pat. Nos. 4,405,712, 4,650,764 and 5,252,479, and WIPO publications WO 92/07573, WO 90/06997, WO 89/05345, WO 92/05266 and WO 92/14829; and lentivirus vectors (Kafri et al., Nature Genetics, 17:314-317 (1997), as well as viruses that are replication-competent conditional to a cancer cell such as oncolytic herpes virus NV 1066 and vaccinia virus GLV-1h68, as described in United States patent application 2009/0311664. In particular, adenoviral vectors may be used, e.g. targeted viral vectors such as those described in published United States patent application 2008/0213220.
Those of skill in the art will recognize that the choice of a particular vector will depend on its precise usage. Typically, one would not use a vector that integrates into the host cell genome due to the risk of insertional mutagenesis, and one should design vectors so as to avoid or minimize the occurrence of recombination within a vector's nucleic acid sequence or between vectors.
Host cells which contain the constructs and vectors of the invention are also encompassed, e.g. in vitro cells such as cultured cells, or bacterial or insect cells which are used to store, generate or manipulate the vectors, and the like. The constructs and vectors may be produced using recombinant technology or by synthetic means.
Imaging Constructs and vectors
In some embodiments, the invention provides gene constructs for use in imaging of cancer cells and tumors. The constructs include at least one transcribable element that is either directly detectable using imaging technology, or which functions with one or more additional molecules in a manner that creates a signal that is detectable using imaging technology. The transcribable element is operably linked to a cancer selective/specific promoter as described above, and is generally referred to as a “reporter” molecule. Reporter molecules can cause production of a detectable signal in any of several ways: they may encode a protein or polypeptide that has the property of being detectable in its own right; they may encode a protein or polypeptide that interacts with a second substance and causes the second substance to be detectable; they may encode a protein or polypeptide that sequesters a detectable substance, thereby increasing its local concentration sufficiently to render the surrounding environment (e.g. a cancer cell) detectable. If the gene product of the reporter gene interacts with another substance to generate a detectable signal, the other substance is referred to herein as a “complement” of the reporter molecule.
Examples of reporter proteins or polypeptides that are detectable in their own right (directly detectable) include those which exhibit a detectable property when exposed to, for example, a particular wavelength or range of wavelengths of energy. Examples of this category of detectable proteins include but are not limited to: green fluorescent protein (GFP) and variants thereof, including mutants such as blue, cyan, and yellow fluorescent proteins; proteins which are engineered to emit in the near-infrared regions of the spectrum; proteins which are engineered to emit in the short-, mid-, long-, and far-infrared regions of the spectrum; etc. Those of skill in the art will recognize that such detectable proteins may or may not be suitable for use in humans, depending on the toxicity or immunogenicity of the reagents involved. However, this embodiment has applications in, for example, laboratory or research endeavors involving animals, cell culture, tissue culture, various ex vivo procedures, etc.
Another class of reporter proteins is those which function with a complement molecule. In this embodiment, a construct comprising a gene encoding a reporter molecule is administered systemically to a subject in need of imaging, and a molecule that is a complement of the reporter is also administered systemically to the subject, before, after or together with the construct. If administered prior to or after administration of the construct, administration of the two may be timed so that the diffusion of each entity into cells, including the targeted cancer cells, occurs in a manner that results in sufficient concentrations of each within cancer cells to produce a detectable signal, e.g. typically within about 1 hour or less. If the two are administered “together”, then separate compositions may be administered at the same or nearly the same time (e.g. within about 30, 20, 15, 10, or 5 minutes or less), or a single composition comprising both the construct and the complement may be administered. In any case, no interaction between the reporter and the complement can occur outside of cancer cells, because the reporter is not produced and hence does not exist in any other location, since its transcription is controlled by a cancer specific/selective promoter.
One example of this embodiment is the oxidative enzyme luciferase and various modified forms thereof, the complement of which is luciferin. Briefly, catalysis of the oxidation of its complement, luciferin, by luciferase produces readily detectable amounts of light. Those of skill in the art will recognize that this system is not generally used in humans due to the need to administer the complement, luciferin to the subject. However, this embodiment is appropriate for use in animals, and in research endeavors involving cell culture, tissue culture, and various ex vivo procedures.
Another exemplary protein of this type is thymidine kinase (TK), e.g. TK from herpes simplex virus 1 (HSV 1), or from other sources. TK is a phosphotransferase enzyme (a kinase) that catalyzes the addition of a phosphate group from ATP to thymidine, thereby activating the thymidine for incorporation into nucleic acids, e.g. DNA. Various analogs of thymidine are also accepted as substrates by TK, and radiolabeled forms of thymidine or thymidine analogs may be used as the complement molecule to reporter protein TK. Without being bound by theory, it is believed that once phosphorylated by TK, the radiolabeled nucleotides are retained intracellularly because of the negatively charged phosphate group; or, alternatively, they may be incorporated into e.g. DNA in the cancer cell, and thus accumulate within the cancer cell. Either way, they provide a signal that is readily detectable and distinguishable from background radioactivity. Also, the substrate that is bound to TK at the time of imaging provides additional signal in the cancer cell. In fact, mutant TKs with very low Kms for substrates may augment this effect by capturing the substrate. The radioactivity emitted by the nucleotides is detectable using a variety of techniques, as described herein. This aspect of the use of TK harnesses the labeling potential of this enzyme; the toxic capabilities of TK are described below.
Various TK enzymes or modified or mutant forms thereof may be used in the practice of the invention, including but not limited to: HSV1-TK, HSV1-sr39TK, mutants with increased or decreased affinities for various substrates, temperature sensitive TK mutants, codon-optimized TK, the mutants described in U.S. Pat. No. 6,451,571 and US patent application 2011/0136221, both of which are herein incorporated by reference; various suitable human TKs and mutant human TKs, etc.
Detectable TK substrates that may be used include but are not limited to: thymidine analogs such as: “fialuridine” i.e. [1-(2-deoxy-2-fluoro-1-D-arabinofuranosyl)-5-iodouracil], also known as “FIAU” and various forms thereof, e.g. 2′-fluoro-2′-deoxy-β-D-5-[125I] iodouracil-arabinofuranoside ([125I] FIAU), [124I]FIAU; thymidine analogs containing o-carboranylalkyl groups at the 3-position, as described by Al Mahoud et al., (Cancer Res Sep. 1, 2004 64; 6280), which may have a dual function in that they mediate cytotoxicity as well, as described below; hydroxymethyl]butyl)guanine (HBG) derivatives such as 9-(4-18F-fluoro-3-[hydroxymethyl] butyl)guanine (18F-FHBG); 2′-deoxy-2′-[18F]-fluoro-1-beta-D-arabinofuranosyl-5-iodouracil (18F-FEAU), 2′-deoxy-2′-[18F]-fluoro-5-methyl-1-β-L-arabinofuranosyluracil (18F-FMAU),1-(2′-deoxy-2′-fluoro-beta-D-arabinofuranosyl)-5-[18F] iodouracil (18F-FIAU), 2′-deoxy-2′-[18F]-fluro-1-beta-D-arabinoluranosyl-5-iodouracil (18F-FIAC, see, for example, Chan et al., Nuclear Medicine and Biology 38 (2011) 987-995; and Cai et al., Nuclear Medicine and Biology 38 (2011) 659-666); various alkylated pyrimidine derivatives such as a C-6 alkylated pyrimidine derivative described by Muller et al. (Nuclear Medicine and Biology, 2011, in press); and others.
Other exemplary reporter molecules may retain or cause retention of a delectably labeled complement by any of a variety of mechanisms. For example, the reporter molecule may bind to the complement very strongly (e.g. irreversibly) and thus increase the local concentration of the complement within cancer cells; or the reporter molecule may modify the complement in a manner that makes egress of the complement from the cell difficult, or at least slow enough to result in a net delectable accumulation of complement within the cell; or the reporter may render the complement suitable for participation in one or more reactions which “trap” or secure the complement, or a modified form thereof that still includes the detectable label, within the cell, as is the case with the TK example presented above.
One example of such a system would be an enzyme-substrate complex, in which the reporter is usually the enzyme and the complement is usually the substrate, although this need not always be the case: the reporter may encode a polypeptide or peptide that is a substrate for an enzyme that functions as the “complement”. In some embodiments, the substrate is labeled with a detectable label (e.g. a radio-, fluorescent-, phosphoresent-, colorimetric-, light emitting-, or other label) and accumulates within cancer cells due to, for example, an irreversible binding reaction with the enzyme (i.e. it is a suicide substrate), or because it is released from the enzyme at a rate that is slow enough to result in a detectable accumulation within cancer cells, or the reaction with the enzyme causes a change in the properties of the substrate so that it cannot readily leave the cell, or leaves the cell very slowly (e.g. due to an increase in size, or a change in charge, hydrophobicity or hydrophilicity, etc.); or because, as a result of interaction or association with the enzyme, the substrate is modified and, then engages in subsequent reactions which cause it (together with its detectable tag or label) to be retained in the cells, etc.
Other proteins that may function as reporter molecules in the practice of the invention are transporter molecules which are located on the cell surface or which are transmembrane proteins, e.g. ion pumps which transport various ions across cells membranes and into cells. An exemplary ion pup is the sodium-iodide symporter (NIS) also known as solute carrier family 5, member 5 (SLC5A5). In nature, this ion pump actively transports iodide (I−) across e.g. the basolateral membrane into thyroid epithelial cells, and the corresponding imaging agent [124I]NaI can be detected using positron emission tomography (PET) scanning, or the corresponding imaging agent [123I]NaI or [125]NaI can be detected using single photon emission computed tomography (SPECT). Recombinant forms of the transporter encoded by sequences of the constructs described herein may be selectively transcribed in cancer cells, and transport radiolabeled iodine into the cancer cells. Other examples of this family of transporters that may be used in the practice of the invention include but are not limited to norepinephrine transporter (NET); dopamine receptor; various estrogen receptor systems), ephrin proteins such as membrane-anchored ephrin-A (EFNA) and the transmembrane protein ephrin-B (EFNB); epidermal growth factor receptors (EGFRs); insulin-like growth factor receptors (e.g. IGF-1 IGE-2), etc.); transforming growth factor (TGF) receptors such as TGFα; etc. In these cases, the protein or a functional modified form thereof is expressed by the vector of the invention and the ligand molecule is administered to the patient. Usually, the ligand is labeled with a detectable label as described herein, or becomes detectable upon association or interaction with the transporter. In some embodiments, detection may require the association of a third entity with the ligand, e.g. a metal ion. The ligand may also be a protein, polypeptide or peptide.
In addition, antibodies may be utilized in the practice of the invention. For example, the vectors of the invention may be designed to express proteins, polypeptides, or peptides which are antigens or which comprise antigenic epitopes for which specific antibodies have been or can be produced. Exemplary antigens include but are not limited to tumor specific proteins that have an abnormal structure due to mutation (protooncogenes, tumor suppressors, the abnormal products of ras and p53 genes, etc.); various tumor-associated antigens such as proteins that are normally produced in very low quantities but whose production is dramatically increased in tumor cells (e.g. the enzyme tyrosinase, which is elevated in melanoma cells); various oncofetal antigens (e.g. alphafetoprotein (AEI') and carcinoembryonic antigen (CEA); abnormal proteins produced by cells infected with oncoviruses, e.g. EBV and HPV; various cell surface glycolipids and glycoproteins which have abnormal structures in tumor cells; etc. The antibodies, which may be monoclonal or polyclonal, are labeled with a detectable label and are administered to the patient after or together with the vector. The antibodies encounter and react with the expressed antigens or epitopes, which are produced only (or at least predominantly) in cancer cells, thereby labeling the cancer cells. Conversely, the antibody may be produced by the vector of the invention, and a labeled antigen may be administered to the patient. In this embodiment, an antibody or a fragment thereof, e.g. a Fab (fragment, antigen binding) segment, or others that are known to those of skill in the art, are employed. In this embodiment, the antigen or a substance containing antigens or epitopes for which the antibody is specific is labeled and administered to the subject being imaged.
Other examples of such systems include various ligand binding systems such as reporter proteins/polypeptides that bind ligands which can be imaged, examples of which include but are not limited to: proteins (e.g. metalloenzymes) that bind or chelate metals with a detectable signal; ferritin-based iron storage proteins such as that which is described by Iordanova et al, (Engineered Mitochondrial Ferritin as a Magnetic Resonance Imaging Reporter in Mouse Olfactory Epithelium. 2013, PLoS ONE 8(8): e72720. doi:10.1371/journal.pone.0072720), and others. Such systems of reporter and complement may be used in the practice of the invention, provided that the reporter or the complement can be transcribed under control of a cancer promoter, and that the other binding partner is detectable or can be detectably labeled, is administrable to a subject, and is capable of diffusion into cancer cells. Those of skill in the art will recognize that some such systems are suitable for use e.g. in human subjects, while others are not due to, for example, toxicity. However, systems in the latter category may be well-suited for use in laboratory settings.
In yet other aspects, the cancer-specific or cancer-selective promoters in the vectors of the invention drive expression of a secreted protein that is not normally found in the circulation. In this embodiment, the presence of the protein may be detected by standard (even commercially available) methods with high sensitivity in serum or urine. In other words, the cancer cells that are detected are detected in a body fluid.
In yet other embodiments, the cancer-specific or cancer-selective promoters in the vectors of the invention drive transcription of a protein or antigen to be expressed on the cell surface, which can then be tagged with a suitable detectable antibody or other affinity reagent. Candidate proteins for secretion and cell surface expression include but are not limited to: β-subunit of human chorionic gonadotropin (β hCG); human α-fetoprotein (AFP), and streptavidin (SA).
βhCG is expressed in pregnant women and promotes the maintenance of the corpus luteum during the beginning of pregnancy. The level of β hCG in non-pregnant normal women and men is 0-5 mIU/mL. hCG is secreted into the serum and urine and β hCG has been used for pregnancy test since the α-subunit of hCG is shared with other hormones. Urine β hCG can be easily detected by a chromatographic immunoassay (i.e. pregnancy test strip, detection threshold is 20-100 MlU/mL) at home- physician's office- and laboratory-based settings. The serum level can be measured by chemiluminescent or fluorescent immunoassays using 2-4 mL of venous blood for more quantitative detection. β hCG has been shown to secreted into the media when it was expressed in monkey cells. Human AFP is an oncofetal antigen that is expressed only during fetal development and in adults with certain types of cancers. AFP in adults can be found in hepatocellular carcinoma, testicular tumors and metastatic liver cancer. AFP can be detected in serum, plasma, or whole blood by chromatographic immunoassay and by enzyme immunoassay for the quantitative measurement.
Strepavadin (SA) can also be used as a cell surface target in the practice of the invention. The unusually high affinity of SA with biotin provides very efficient and powerful target for imaging and therapy. To bring SA to the plasma membrane of the cancer cells, SA can be fused to glycosylphosphatidylinositol (GPI)-anchored signal of human CD14. GPI-anchoring of SA will be suitable for therapeutic applications since GPI-anchor proteins can be endocytosed to the recycling endosomes. Once expressed on the cell surface, SA can then be bound by avidin conjugates that contain a toxic or radiotoxic warhead. Toxic proteins and venoms such as ricin, abrin, Pseudomonas exotoxin (PE, such as PE37, PE38, and PE40), diphtheria toxin (DT), saporin restrictocin, cholera toxin, gelonin, Shigella toxin, and pokeweed antiviral protein, Bordetella perutssis adenylate cyclase toxin, or modified toxins thereof, or other toxic agents that directly or indirectly inhibit cell growth or kill cells may be linked to avidin; as could toxic low molecular weight species, such as doxombicin or taxol or radionuclides such as 125I, 131I, 111In, 177Lu, 211At, 225Ac, 213Bi and 90Y; antiangiogenic agents such as thalidomide, angiostatin, antisense molecules, COX-2 inhibitors, integrin antagonists, endostatin, thrombospondin-1, and interferon alpha, vitaxin, celecoxib, rofecoxib; as well as chemotherapeutic agents such as: pyrimidine analogs (5-fluorouracl, floxuridine, capecitabine, gemcitabine and cytarabine) and purine analogs, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine (cladribine)); antiproliferative/antimitotic agents including natural products such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disruptors such as taxane (paclitaxel, docetaxel), vincristin, vinblastin, nocodazole, epothilones and navelbine, epidipodophyllotoxins (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamineox aliplatin, iph.osphamide, melphalan, merchlomhtamine, mitomycin, mitoxantrone, nitrosourea, plicarnycin, procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramide and etoposide (VP16)); antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin; enzymes (L-asparaginase which systemically metabolizes L-asparagine and deprives cells which do not have the capacity to synthesize their own asparagine); antiplatelet agents; antiproliferative/antimitotic alkylating agents such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), trazenes-dacarbazinine (DTIC); antiproliferative/antimitotic antimetabolites such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogen, tamoxifen, goserelin bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts and other inhibitors of thrombin); fibrinolytic agents (such as tissue plasminogen activator, streptokinase and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents; antisecretory agents (breveldin); immunosuppressives (cyclosporine tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); anti-angiogenic compounds (TNF'-470, genistein) and growth factor inhibitors (vascular endothelial growth factor (VEGF) inhibitors, fibroblast growth factor (FGF) inhibitors); angiotensin receptor blocker; nitric oxide donors; anti-sense oligonucleotides; antibodies (trastuzumab, rituximab); cell cycle inhibitors and differentiation inducers (tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, irinotecan (CPT-11) and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylpednisolone, prednisone, and prenisolone); growth factor signal transduction kinase inhibitors; mitochondrial dysfunction inducers; caspase activators; and chromatin disruptors, especially those which can be conjugated to nanoparticles
The detectable components of the system (usually a complement or substrate) used in the imaging embodiment of the invention may be labeled with any of a variety of detectable labels, examples of which are described above. In addition, especially useful detectable labels are those which are highly sensitive and can be detected non-invasively, such as the isotopes 125I, 124I, 123I, 99mTc, 18F, 11C, 125I, 64Cu, 67Ga, 68Ga, 201TI, 76Br, 75Br, 111In, 82Rb, 13N, and others.
Those of skill in the art will recognize that many different detection techniques exist which may be employed in the practice of the present invention, and that the selection of one particular technique over another generally depends on the type of signal that is produced and also the medium in which the signal is being detected, e.g. in the human body, in a laboratory animal, in cell or tissue culture, ex vivo, etc. For example, bioluminescence imaging (BLI); fluorescence imaging; magnetic resonance imaging [MRI, e.g. using lysine rich protein (LRp) as described by Gilad et al., Nature Biotechnology, 25,2 (2007); or creatine kinase, tyrosinase, β-galactosidase, iron-based reporter genes such as transferrin, ferritin, and MagA; low-density lipoprotein receptor-related protein (LRP; polypeptides such as poly-L-lysine, poly-L-arginine and poly-L-threonine; and others as described, e.g. by Gilad et al., J. Nucl. Med. 2008; 49(12):1905-1908); computed tomography (CT); positron emission tomography (PET); single-photon emission computed tomography (SPECT); boron neutron capture; for metals: synchrotron X-ray fluorescence (SXRF) microscopy, secondary ion mass spectrometry (SIMS), and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) for imaging metals; photothermal imaging (using for example, magneto-plasmonic nanoparticles, etc.
Targeted cancer therapy is carried out by administering the constructs, vectors, etc. as described herein to a patient in need thereof. In this embodiment, a gene encoding a therapeutic molecule, e.g. a protein or polypeptide, which is deleterious to cancer cells is operably linked to a cancer-specific promoter as described herein in a “therapeutic construct” or “therapeutic vector”. The therapeutic protein may kill cancer cells (e.g. by initiating or causing apoptosis), or may slow their rate of growth (e.g. may slow their rate of proliferation), or may arrest their growth and development or otherwise damage the cancer cells in some manner, or may even render the cancer cells more sensitive to other anti-cancer agents, to immune recognition, etc.
Genes encoding therapeutic molecules that may be employed in the present invention include but are not limited to suicide genes, including genes encoding various enzymes; oncogenes; tumor suppressor genes; toxins; cytokines; oncostatins; TRAIL, etc. Exemplary enzymes include, for example, thymidine kinase (TK) and various derivatives thereof; TNF-related apoptosis-inducing ligand (TRAIL), xanthine-guanine phosphoribosyltransferase (GPT); cytosine deaminase (CD); hypoxanthine phosphoribosyl transferase (HPRT); etc. Exemplary tumor suppressor genes include neu, EGF, ras (including H, K, and N ras), p53, Retinoblastoma tumor suppressor gene (Rb), Wilm's Tumor Gene Product, Phosphotyrosine Phosphatase (PTPase), AdE1A and nm23. Suitable toxins include Pseudomonas exotoxin A and S; diphtheria toxin (DT); E. coli LT toxins, Shiga toxin, Shiga-like toxins (SLT-1-2), ricin, abrin, supporin, gelonin, etc. Suitable cytokines include interferons and interleukins such as interleukin 1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-18, β-interferon, α-interferon, γ-interferon, angiostatin, thrombospondin, endostatin, GM-CSF, G-CSF, M-CSF, METH 1, METH 2, tumor necrosis factor, TGFβ, LT and combinations thereof. Other anti-tumor agents include: GM-CSF interleukins, tumor necrosis factor (TNF); interferon-beta and virus-induced human Mx proteins; TNFα and TNFβ; human melanoma differentiation-associated gene-7 (mda-7), also known as interleukin-24 (IL-24), various truncated versions of mda-7/IL-24 such as M4; siRNAs and shRNAs targeting important growth regulating or oncogenes which are required by or overexpressed in cancer cells; antibodies such as antibodies that are specific or selective for attacking cancer cells; etc.
When the therapeutic agent is TK (e.g. viral TK), a TK substrate such as acyclovir; ganciclovir; various thymidine analogs (e.g. those containing o-carboranylalkyl groups at the 3-position [Cancer Res Sep. 1, 2004 64; 6280]) is administered to the subject. These drugs act as prodrugs, which in themselves are not toxic, but are converted to toxic drugs by phosphorylation by viral TK. Both the TK gene and substrate must be used concurrently to be toxic to the host cancer cell.
In some embodiments, the invention provides cancer treatment protocols in which imaging of cancer cells and tumors is combined with treating the disease, i.e.. with killing, destroying, slowing the growth of, attenuating the ability to divide (reproduce), enhancing immune recognition or otherwise damaging the cancer cells. These protocols may be referred to herein as “theranostics” or “combined therapies” or “combination protocols”, or by similar terms and phrases,
In some embodiments, the combined therapy involves administering to a cancer patient a gene construct (e.g. a plasmid) that comprises, in a single construct, both a reporter gene (for imaging) and at least one therapeutic gene of interest (for treating the disease). In this embodiment, expression of either the reporter gene or the therapeutic gene, or preferably both is mediated by a cancer cell specific or selective promoter as described herein. Preferably, two different promoters are used in this embodiment in order to prevent or lessen the chance of crossover and recombination within the construct. Alternatively, tandem translation mechanisms may be employed, for example, the insertion of one or more internal ribosomal entry site (IRES) into the construct, which permits translation of multiple mRNA transcripts from a single mRNA. In this manner, both a reporter proteinlpolypeptide and a protein/polypeptide that is lethal or toxic to cancer cells are selectively or specifically produced within the targeted cancer cells.
Alternatively, the polypeptides encoded by the constructs of the invention (e.g. plasmids) may be genetically engineered to contain a contiguous sequence comprising two or more polypeptides of interest (e.g. a reporter and a toxic agent) with an intervening sequence that is cleavable within the cancer cell, e.g. a sequence that is enzymatically cleaved by intracellular proteases, or even that is susceptible to non-enzymatic hydrolytic cleavage mechanisms. In this case, cleavage of the intervening sequence results in production of functional polypeptides, i.e. polypeptides which are able to carry out their intended function, e.g. they are at least 50, 60, 70, 80, 90, or 100% (or possible more) as active as the protein sequences on which they are modeled or from which they are derived (e.g. a sequence that occurs in nature), when measured using standard techniques that are known to those of skill in the art.
In other embodiments of combined imaging and therapy, two different vectors may be administered, one of which is an “imaging vector or construct” as described herein, and the other of which is a “therapeutic vector or construct” as described herein.
In other embodiments of combined imaging and therapy, the genes of interest are encoded in the genome of a viral vector that is capable of transcription and/or translation of multiple mRNAs and/or the polypeptides or proteins they encode, by virtue of the properties inherent in the virus. In this embodiment, such viral vectors are genetically engineered to contain and express genes of interest (e.g. both a reporter gene and a therapeutic gene) under the principle control of one or more cancer specific promoters.
The present invention provides compositions, which comprise one or more vectors or constructs as described herein and a pharmacologically suitable carrier. The compositions are usually for systemic administration. The preparation of such compositions is known to those of skill in the art. Typically, they are prepared either as liquid solutions or suspensions, or as solid forms suitable for solution in, or suspension in, liquids prior to administration. The preparation may also be emulsified. The active ingredients may be mixed with excipients, which are pharmaceutically acceptable and compatible with the active ingredients. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol and the like, or combinations thereof. In addition, the composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and the like. If it is desired to administer an oral form of the composition, various thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders and the like may be added. The composition of the present invention may contain any of one or more ingredients known in the art to provide the composition in a form suitable for administration. The final amount of vector in the formulations may vary. However, in general, the amount in the formulations will be from about 1-99%.
The vector compositions (preparations) of the present invention are typically administered systemically, although this need not always be the case, as localized administration (e.g. intratumoral, or into an exteri al orifice such as the vagina, the nasopharygeal region, the mouth; or into an internal cavity such as the thoracic cavity, the cranial cavity, the abdominal cavity, the spinal cavity, etc.) is not excluded. For systemic distribution of the vector, the preferred routes of administration include but are not limited to: intravenous, by injection, transdermal, via inhalation or intranasally, or via injection or intravenous administration of a cationic polymer-based vehicle (e.g. vivo-jet.PEI™). Liposomal delivery, which when combined with targeting moieties will permit enhanced delivery. The ultrasound-targeted microbubble-destruction technique (UTMD) may also be used to deliver imaging and theranostic agents (Dash et al. Proc Natl Acad Sci USA. 2011 May 24;108(21):8785-90, Epub 2011 May 9]; hydroxyapatite-chitosan nanocomposites (Venkatesan et al. Biomaterials. 2011 May; 32(15):3794-806); and others (Dash et al. Discov Med. 2011 Jan;11(56);46-56. Review); etc. Any method that is known to those of skill in the art, and which is commensurate with the type of construct that is employed, may be utilized. In addition, the compositions may be administered in conjunction with other treatment modalities known in the art, such as various chemotherapeutic agents such a Pt drugs, substances that boost the immune system, antibiotic agents, and the like; or with other detections and imaging methods (e.g. to confirm or provide improved or more detailed imaging, e.g. in conjunction with mammograms, X-rays, Pap smears, prostate specific antigen (PSA) tests, etc.
Those of skill in the art will recognize that the amount of a construct or vector that is administered will vary from patient to patient, and possibly from administration to administration for the same patient, depending on a variety of factors, including but not limited to: weight, age, gender, overall state of health, the particular disease being treated, and other factors, and the amount and frequency of administration is best established by a health care professional such as a physician. Typically, optimal or effective tumor-inhibiting or tumor-killing amounts are established e.g. during animal trials and during standard clinical trials. Those of skill in the art are familiar with conversion of doses e.g. from a mouse to a human, which is generally done through body surface area, as described by Freireich et al— (Cancer Chemother Rep 1966; 50(4):219-244); and see Tables 1 and 2 below, which are taken from the website located at dtp,nci.nih.gov.
For example, given a dose of 50 mg/kg in the mouse, and appropriate does in a monkey would be 50 tog/kg X 1/4=13 mg/kg/; or a dose of about 1.2 mg/kg is about 0.1 mg/kg fora human.
To express the dose as the equivalent mg/sq.m, dose, multiply the dose by the appropriate factor. In adult humans, 100 mg/kg is equivalent to 100 mg/kg×37 kg/sq.m.=3700 mg/sq.m.
In general, for treatment methods, the amount of a vector such as a plasmid will be in the range of from about 0.01 to about 5 mg/kg or from about 0.05 to about 1 mg/kg (e.g. about 0.1 mg/kg), and from about 105 to about 1020 infectious units (IUs), or from about 108 to about 1013 viral-based vector. In general, for imaging methods, the amount of a vector will be in the range of from about 0.01 to about 5 mg/kg or from about 0.05 to about 1 mg/kg (e.g. about 0.1 mg/kg) of e.g. a plasmid and from about 105 to about 1020 infectious units (IUs), or from about 108 to about 1013 IUs for a viral-based vector. For combined imaging and therapy, the amounts of a vector will be in the ranges described above. Those of skill in the art are familiar with calculating or determining the level of an imaging signal that is required for adequate detection. For example, for radiopharmaceuticals such as [124]FIAU, an injection on the order or from about 1 mCi to about 10 mCi, and usually about 5 mCi, (i.e.. about 1 mg of material) is generally sufficient.
Further, one type of vector or more than one type of vector may be administered in a single administration, e.g. a therapy vector plus an imaging vector, or two (or more) different therapy vectors (e.g. each of which have differing modes of action so as to optimize or improve treatment outcomes), or two or more different imaging vectors, etc.
Typically cancer treatment requires repeated administrations of the compositions. For example, administration may be daily or every few days, (e.g, every 2, 3, 4, 5, or 6 days), or weekly, bi-weekly, or every 3-4 weeks, or monthly, or any combination of these, or alternating patterns of these. For example, a “round” of treatment (e.g. administration one a week for a month) may be followed by a period of no administration for a month, and then followed by a second round of weekly administration for a month, and so on, for any suitable time periods, as required to optimally treat the patient.
Imaging methods also may be carried out on a regular basis, especially when a subject is known or suspected to be at risk for developing cancer, due to e.g., the presence of a particular genetic mutation, family history, exposure to carcinogens, previous history of cancer, advanced age, etc. For example, annual, semi-annual, or bi-annual, or other periodic monitoring may be considered prudent for such individuals. Alternatively individuals with no risk factors may simply wish to be monitored as part of routine health care, in order to rule out the disease.
For embodiments of the invention, which encompass both treatment and imaging, the administration protocols may be any which serve the best interest of the patient. For example, initially, an imaging vector alone may be administered in order to determine whether or not the subject does indeed have cancer, or to identify the locations of cancer cells in a patient that has already been diagnosed with cancer. Of note, the present method is very specific so that even very small masses of cancer cells can be visualized using the methods. If cancer is indeed indicated, then compositions with therapeutic vectors are then administered are needed to treat the disease. Usually a plurality of administrations is required as discussed above, and at least one, usually more, and sometimes all of these include at least one imaging vector together with a least one therapeutic vector; or optionally, a single vector with both capabilities. The ability to alternate between therapy and imaging, or to concomitantly carry out both, is a distinct boon for the field of cancer treatment. This methodology allows a medical professional to monitor the progress of treatment in a tightly controlled manner, and to adjust and/or modify the therapy as necessary for the benefit of the patient. For example, administration of a therapeutic and an imaging vector may be alternated; or, during early stages of treatment, initially an imaging vector may be administered, followed by therapy and imaging vectors together until the tumors are no longer visible, followed by imaging vector alone for a period of time deemed necessary to rule out or detect recurrence or latent disease.
The subjects or patients to whom the compositions of the invention are administered are typically mammals, frequently humans, but this need not always be the case. Veterinary applications are also contemplated.
Exemplary Types of Cancer that can be Treated
The constructs and methods of the invention are not specific for any one type of cancer. By “cancer” we mean malignant neoplasms in which cells divide and grow uncontrollably, forming malignant tumors, and invade nearby parts of the body. Cancer may also spread or metastasize to more distant parts of the body through the lymphatic system or bloodstream. The constructs and methods of the invention may be employed to image, diagnose, treat, monitor, etc. any type of cancer, tumor, neoplastic or tumor cells including but not limited to: osteosarcoma, ovarian carcinoma, breast carcinoma, melanoma, hepatocarcinoma, lung cancer, brain cancer, colorectal cancer, hematopoietic cell, prostate cancer, cervical carcinoma, retinoblastoma, esophageal carcinoma, bladder cancer, neuroblastoma, renal cancer, gastric cancer, pancreatic cancer, and others.
In addition, the invention may also be applied to imaging and therapy of benign tumors, which are generally recognized as not invading nearby tissue or metastasizing, for example, moles, uterine fibroids, etc.
The invention also encompasses transgenic non-human animals that have been genetically engineered to contain nucleotide sequences encoding a reporter gene operably linked to a cancer-specific or cancer-selective promoter, and their use for clinical evaluation of therapies. In the transgenic animals, the nucleotide sequences are stably integrated into the genome of the animal. In healthy animals, the promoter is not active and the reporter gene is not expressed. However, if such an animal develops cancer, then the promoter is induced or activated, and the reporter gene is expressed. Upon administration of the reporter complement to the animal, the development, location and fate of cancer cells can be monitored in detail. Such animals may be used for laboratory purposes, e.g. for testing carcinogenicity of substances, evaluating chemoprevention strategies and monitoring therapy. The animals can be exposed to potential carcinogens, administered complement, and then monitored to observe the effects of the potential carcinogen. Likewise, the effects of candidate anti-cancer agents can be tested or screened in the animals by administering the candidate either before attempting, to induce cancer, or after cancer is established, and the effectiveness of the agent can be tracked and measured. Those of skill in the art are familiar with methods of evaluating the efficacy of drug candidates, including, for example, monitoring tumor location, stage, size, volume, appearance, frequency, duration, etc.
In other aspects, the PEG-PROM (or another cancer-specific or cancer-selective promoter) animals of the invention are further genetically altered to have a predisposition to the development of cancer. This may be done, for example, by cross breeding the animals with animals who already have the predisposition for cancer development (for example, any one of the number of mice that have been selected or genetically engineered to serve as model systems for various cancers). Alternatively, this may be accomplished by inducing desired genetic mutations in the cancer-specific or cancer-selective promoter, e.g. mutations which are associated with cancer development, or by further genetically engineering the animals to have a tendency to develop cancer.
Exemplary types of cancer-prone animals include any of those which are susceptible (or certain to develop) a cancer such as: breast cancer (e.g. mice such as mouse mammary tumor virus (MMTV)-neu transgenic mice; mouse MMTV-PyMT transgenic mice); prostate cancer mice such as Hi-Myc, TRAMP, etc.); C3(1)/SV40 T antigen transgenic mouse model of prostate and mammary cancer; as well as animals which are models for melanoma, brain cancer, colorectal and intestinal cancer, etc. Such mice are available for example, from Jackson Labs in Bar Harbor, Me.
The animals that are genetically modified in this manner include but are not limited to: mice, rats, guinea pigs, rabbits, dogs, pigs, chickens, goats, primates such as marmosets, etc. Those of skill in the art are well acquainted with methods of genetically engineering and/or cross breeding and selecting animals for use in research.
Molecular-genetic imaging is advancing from a valuable preclinical tool to guiding patient management. The strategy involves pairing an imaging reporter gene with a complementary imaging agent in a system that can be used to measure gene expression, protein interaction or track gene-tagged cells in vivo. Tissue-specific promoters can be used to delineate gene expression in certain tissues, particularly when coupled with an appropriate amplification mechanism. Here we show that the progression elevated gene-3 promoter (PEG-Prom), derived from a rodent gene mediating the malignant phenotype, can be used to drive imaging reporters selectively to enable detection of micrometastatic disease in marine models of human melanoma and breast cancer using bioluminescence and radionuclide-based molecular imaging techniques. Because of its strong promoter, tumor specificity and capacity for clinical translation, PEG-Prom-driven gene expression may represent a practical, new system by which to facilitate cancer imaging and imaging in combination with therapy.
A minimal promoter region of progression elevated gene-3 (PEG-3), a rodent gene, was previously identified for its association with malignant transformation and tumor progression using subtraction hybridization. PEG-Prom drives downstream gene expression in a tumor-specific manner and has been tested in cancer cell lines of various tissues such as brain, prostate, breast and pancreas9-11, as well as in metastatic melanoma12. Transcription factors AP-1 and E1AF/PEA3 (ETS-1) are known to mediate the cancer-specific activity of PEG-Prom8,9,13. Previous studies have demonstrated the utility of PEG-Prom for cancer gene therapy through intratumoral delivery9-12, 14. Here we describe a novel method for imaging a variety of metastatic cancers through systemic delivery of PEG-Prom. Based on these experiments it can be seen that the systemic delivery of PEG-Prom-driven imaging constructs will enable tumor-specific expression of reporter genes, not only within primary tumor, but also in associated metastases in a manner broadly applicable to tumors of different tissue origin or subtype.
Additional detail regarding experimental procedures and results can be found above under “Brief Description of the Drawings”.
Plasmids. pPEG-Luc was constructed as described previously9. The Luc-encoding gene in pPEG-Luc was replaced by the HSV1-tk-encoding sequence from pORF-HSV1tk plasmid (InvivoGen) to generate pPEG-HSV1tk. pDNA were prepared with the EndoFree Plasmid Kit (Qiagen) and DNA pellets were dissolved in endotoxin-free water (Lonza). Endotoxin level was ensured as <2.5 endotoxin unit (EU)/mg pDNA with the ToxinSensor Gel Clot Endotoxin Assay Kit (GenSeript).
Systemic DNA delivery. Low molecular weight 1-PEI-based cationic polymer, in vivo-jetPEI™, (Polyplus-transfection) provided the gene delivery vehicle. DNA-polyplex was formed according to the Manufacturer's Instructions, 30 μg of pDNA and 3.6 μl of 150 mM in vivo-jetPEI™ were diluted in endotoxin-free 5% glucose separately and then mixed together to give an N:P ratio of 6:1 in a total volume of 400 μl. The DNA-polymer mixture was incubated at room temperature for 15 min. 400 μl were injected into the lateral tail vein of an animal as two 200 μl-injections, within a 5 minute-interval.
Generation of experimental metastasis models. Animal studies were undertaken in accordance with the rules and regulations of the Johns Hopkins Animal Care and Use Committee. BLI studies employed experimental metastasis models of human melanoma (Mel) and breast cancer (BCa). 5-6 week-old female NCR nu/nu mice (NCI-Frederick) received whole body irradiation (5 Gy) to ensure suppression of the residual immune system in nude mice. Within 24 h after irradiation, animals were randomly divided into three groups. One group was injected with 5×106 cells of the human malignant melanoma cell line McWo (ATCC) intravenously (IV) to generate Mel. Another group of mice received IV injection of 2×106 cells of the human breast cancer cell line MDA-MB-231 for BCa. Another group was maintained as a control. In both models metastatic nodule formation in the lung was confirmed by CT at 4-7 weeks after cell injection. For the SPECT-CT studies the Mel model was generated as described above except that whole body irradiation was omitted. As a control group, we used female NCR nu/nu mice of the same age. MeWo and MDA-MB-231 cell lines were maintained in MEM and RPMI-1640 media, respectively, supplemented with 10% FBS and 1% penicillin/streptomycin.
In vivo bioluminescence imaging. At 24 and 48 h after gene delivery, animals were imaged with the IVIS Spectrum (Xenogen/Caliper). For each imaging session mice were injected intraperitoneally with D-luciferin (150 mg/kg) under anesthesia using 1.5-2.5% isollurane/oxygen mixture. Images were acquired serially from 5-35 minutes after injection of D-luciferin. In order to compensate the limitation of 2D images, most animals were imaged in four different positions: ventral, left- and right-sided, dorsal. ROIs of the same size and shape, covering the entire thoracic cavity, were applied to the images to account for intra-group variations in metastatic site localization. Total Flux (p/s) in the ROIs was measured. One NCR nu/nu female mouse that did not receive any reagent was imaged with the same settings including binning and exposure time. The identical ROIs were applied to the images and the quantified total flux was used as background signal, which was subtracted from the measured counts from experimental animals. Image acquisition and BLI data analysis were done using Living Image softwares (Caliper Life Sciences).
SPECT-CT imaging and data analysis. At 46 h after injection of pPEG-HSV1tk/PEI polyplex animals were injected intravenously with 51.8 mBq (1.4 mCi) of [125I]FIAU. 24 and 48 h after radiotracer injection image data were acquired with the X-SPECT small-animal SPECT-CT system (Gamma Medica-Ideas, Inc.) using the low-energy single pinhole collimator (1.0 mm aperture). Focused lung imaging was acquired with a radius of rotation (ROR) of 3.35 cm and the whole body imaging with ROR of 6.75 cm. At 24 h after injection, animals were imaged in 64 projections with 5.625 degree increments and 30 sec of acquisition per projection, and at 48 h after injection with 60 sec per projection. SPECT images were co-registered with the 512-slice CT images. Tomographic image datasets were reconstructed with the 2D ordered subsets-expectation maximum (OS-EM) algorithm with two iterations and four subsets, and AMIDE38 and Amira (Visage imaging) software was used for analysis.
PET-CT imaging and data analysis. At 1 h after 9.25 mBq (0.25 mCi) of IV administration of FDG, whole body images were acquired with the eXplore Vista small animal PET scanner (GE Healthcare) using the 250-700 keV energy window. Animals were fasted for 6-12 h prior to receiving FDG and were kept warm on the heating pad in order to minimize radiotracer accumulation in non-tumor tissues. PET images were co-registered with the 512-slice CT images. Tomographic image datasets were reconstructed with the 3D ordered subsets expectation maximization (OS-EM) algorithm with three iterations and twelve subsets and analyzed with AMIDE38 software.
Immunohistochemistry. After the BLI data acquisition at 48 h after the pPEG-Luc/PEI polyplex delivery, each organ demonstrating expression of Luc was harvested and fixed in 10% neutral buffered formalin. Paraffin-embedded 5 μm-thick slices and 25 μm-thick lung cryosections were stained with rabbit anti-luciferase polyclonal antibody (1:25 dilution of 50 μg/ml stock, Fitzgerald Industries international, Inc.) at room temperature for 1 h. Horseradish peroxidase (HRP)-conjugated polyclonal goat anti-rabbit antibody was used as a secondary antibody. HRP activity was detected with 3,3′-diaminobenzidine substrate-chromogen (EnVision™+Kit, Dako).
Statistical analysis. Error bars in graphical data represent means ±s.e.m. The two-tailed Student's t test was performed, with P<0.05 considered statistically significant.
To test the specificity of PEG-Prom for tumor imaging in vivo, we used two different reporters, firefly luciferase (Luc) and the herpes simplex virus 1 thymidine kinase (HSV1-tk). Luc is often used with bioluminescence imaging (BLI) to establish proof-of-principle for imaging specific gene expression or gene-tagged cells in preclinical models, while HSV1-tk, also often used preclinically, has been translated to clinical studies. Accordingly, we generated two plasmid constructs, pPEG-Luc and pPEG-HSV1tk (
After confirmation of the presence of metastatic nodules in the lung by computed tomography (CT) at 4-6 weeks after IV administration of the human malignant melanoma cell line MeWo, or the human metastatic breast cancer cell line MDA-MB-231, animals received an IV dose of pPEG-Luc/PEI polyplex (
On average an approximately three-fold higher level of Luc expression was observed from the Mel group compared to the BCa group at 48 h. CT scans and gross anatomical views revealed very different patterns of metastatic nodule formation in the lung of those two models. While MeWo cells formed small nodules uniformly scattered throughout the lungs (
In order to exclude the possibility that tumor-specific expression of Luc by BLI might have resulted from the difference in transfection efficiency between normal and malignant mouse lung tissues, we quantified the amount of pDNA delivered to the lung of each animal. We performed quantitative real time PCR (qRT-PCR) using a primer set designed to amplify a region of the Luc-encoding gene in the pPEG-Luc plasmid. Total DNA extracted from the lung tissues was used as a template. The difference in transfection efficiency between the control group and the Mel group was not significant (
BLI with systemically administered pPEG-Luc also enabled imaging of small metastatic deposits, i.e., micrometastases, outside of the lung parenchyma in both the Mel and BCa models. That was confirmed through harvesting regions producing BLI signal above background and performing correlative histological analysis. Specifically, histological analysis on the tissue sections from a representative Mel model, Mel-2, confirmed that Luc expression was associated with the metastatic sites formed in the lung, adrenal glands, the chest cavity adjacent to the sternum and abdominal inguinal adipose tissues adjoining the bladder. Similarly, correlation between metastatic sites and PEG-Prom activity was observed in a representative BCa model, BCa-3 inside the lung, the peripancreatic area, the thoracic wall adjacent to the sternum, a lymph node located in the adipose connective tissues surrounding the bladder and the rib cage in the form of thin rows of micrometastatic deposits.
Although both malignant lung lesions and extrathoracic micrometastases could be detected with BLI, this technique is limited to preclinical studies. That is due to several factors, including the need to administer lucirase substrate, insufficient depth of penetration of BLI light output and difficulty in generating quantitative, tomographic BLI-based images. Accordingly, we generated a more clinically relevant PEG-Prom-driven gene expression imaging system, pPEG-HSV1tk (
Our goal was to develop a systemically deliverable construct that would enable molecular-genetic imaging of cancer. Necessary elements to provide such a construct include a sufficiently strong promoter with cancer specificity, potential for clinical translation and capacity to be linked to gene therapy. Promoters derived from human telomerase reverse transcriptase (hTERT)4, survivin19 and carcinoembryonic antigen (CEA)20 promoters and enhancer elements have been used in molecular-genetic imaging to provide tumor-specific reporter expression. However, because those studies employed adenoviral vectors, delivery was limited to local administration, systemic administration resulted in expression only within the liver. By contrast here we could delineate metastases with PEG-Prom after systemic delivery using a nonviral vector. Often promoter activity must be amplified to drive the downstream gene for purposes of imaging or therapy. One such strategy for doing so involves the two-step transcriptional amplification (TSTA) system12,22 using GAL4-VP16 fusion protein and the GAL4 response elements19,20,23-25. However, PEG-Prom did not require amplification to achieve high-sensitivity imaging. SPECT-CT imaging demonstrated a metastatic to normal lung signal ratio of 31 out to four days after administration of pPEG-HSV1tk (
Here we show how PEG-Prom can be used as an imaging agent for melanoma and breast cancer metastases in vivo and propose this promoter as potentially universal for this purpose. Such an agent could be used to detect tumors before their tissue of origin or subtype is identified, without concern for nonspecific expression in normal tissues. As with other imaging agents, PEG-Prom can be used not just for tumor detection, but also for preoperative planning, intraoperative management and therapeutic monitoring. The PEG-Prom imaging system can also be fashioned into a theranostic agent, through use of an internal ribosome entry site or other strategy enabling tandem gene expression. Promoters such as PSA (prostate-specific antigen) promoter2,243 for prostate cancer, mucin-1 promoter25,35 for breast cancer, and mesothelin promoter36 for ovarian cancer have been used to delineate primary tumors and lymph node metastasis through molecular-genetic imaging. Similarly, although hTERT, survivin and CEA promoters were reported to be of a less tissue- and more cancer-specific nature, their activity relies on the transcription level of the marker genes. Rather, PEG-Prom is responsive directly to transcription factors unique to tumor cells. The PEG-3 gene is a truncated mutant form of the rat growth arrest- and DNA damage-inducible gene, GADD34, which occurs uniquely during murine tumorigenesis and may function as a dominant-negative of GADD34 promoting the malignant phenotype37. No homolog to PEG-Prom is found in the human genome including the promoter/enhancer region of the human GADD homolog, which makes the use of PEG-Prom in human subjects likely to produce only minimal background signa19,37.
These studies demonstrate that PEG-Prom may possess all of the necessary elements to provide a practical strategy for imaging and potentially image-guided therapy of a variety of cancers.
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Targeted imaging of cancer remains a significant but elusive goal. Such imaging could provide early diagnosis, aid in treatment planning and profoundly benefit therapeutic monitoring. We identified the minimal promoter region of progression elevated gene-3 (PEG-Prom)1,2 derived from a rodent PEG-3 gene through subtraction hybridization', whose expression directly correlates with malignant transformation and tumor progression in rodent tumors3,4, as well as in human tumors, including cancer cell lines derived from tumors in the brain, prostate, breast, melanoma, and pancreas5-9. Based on these findings, we hypothesized and subsequently confirmed that systemic delivery of the PEG-Prom linked to and regulating an imaging construct would enable tumor-specific expression of reporter genes, not only within a primary tumor, but also in associated metastases in a manner broadly applicable to tumors of different tissue origin or subtype10. PEG-Prom is responsive directly to elevated transcription factors unique to tumor cells6-9, AP-1 and PEA-3, and no homolog has been found in the human genome, which makes the use of PEG-Prom in human subjects likely to produce only minimal background signal1,5. Thus, the PEG-Prom can be used not just for tumor detection, but also for preoperative planning, intra-operative management and therapeutic monitoring.
Construction of a PEG-3-Luc mouse: Based on the transformation-specificity of the PEG-Prom, we developed a PEG-Luc transgenic mouse. To generate the PEG-3/luc2 transgene construct, a 446-bp fragment of the rat PEG-3 promoter (from −252 to +194) was inserted upstream of the rabbit β-globin region of pBS/pKCR3. The vector pBS/pKCR3 contains β-globin intron 2 and its flanking exons for efficient transgene expression11. A PEG-3/β-globin composite fragment from the first construct was then inserted upstream of a synthetic firefly luciferase gene (luc2) in the pGL4.10[luc2] vector (Promega). To generate PEG-3/luc2 transgenic mice, a 3.4-kb SpeI/BamHI fragment was excised from the PEG-3/luc2 construct and evaluated for transgene expression. A PEG-3/β-globin composite fragment from the first construct was then inserted upstream of a synthetic firefly luciferase gene (luc2) in the pGL4.10[luc2] vector (Promega). To generate PEG-3/lue2 transgenic mice, a 3,4-kb SpellBarnifl fragment was excised from the PEG-3/luc2 construct and microinjected into the male pronucleus of fertilized single-cell mouse embryos obtained from mating CB6F1 (C57BL/6×Balb/C) males and females. The injected embryos were then reimplanted into the oviducts of pseudopregnant CD-1 female mice. Offspring were screened for the presence of the PEG-3/luc2 transgene by PCR analysis of genomic tail DNA using a rabbit β-globin intron 2 sense primer (5′-CCCTCTGCTAACCATGTTCATGC-3′, SEQ ID NO: 3) and a luc2 antisense primer (5′-TCTTGCTCACGAATACGACGGTG-3′, SEQ ID NO: 4). Four potential founders carrying the PEG-3/luc2 transgene have been established and colonies of PEG-Luc mice have been developed.
Mouse mammary tumor virus (MMTV)-neu transgenic mice: Mouse mammary tumor virus (MMTV)-neu transgenic mice overexpresses NEU protein, the moose homolog of the human her2gene12. This model carries an unactivated neu gene under the transcriptional control of the MMTV promoter/enhancer. Thus, the model simulates human her2-driven breast cancer by overexpression rather than point mutation of neu; resulting in focal mammary tumors and allowing for a realistic therapeutic study platform, MTV-neu transgenic mouse develop focal mammary tumors during lactation and have a latency period of 7-8 months.
Development of double transgenic mice (MMTV-neitIFEG-From-Luc; MnPp-Luc) for in vivo imaging: Based on the cancer specific expression of the PEG-prom in human breast cancer cell lines, we hypothesized that the activity of the PEG-Prom will increase as mammary cells become transformed into tumors and metastases. To establish the proof-of-principal, we have generated MMTV-neu/PEG-From-Luc (MnPp-Luc) mice through mating between the MMTV-neu females with PEG-luc transgenic males from multiple PEG-luc lines to develop complex (MMTV-neu/FEG-Prom-Luc; MnPp-Luc) transgenic mice. As anticipated, the mammary tumor bearing mice (
Of significance, this studies highlights the relevance of the Peg-Prom-Luc animal model in producing complex transgenic tumor animal models that can employ BLI for monitoring tumor development, progression to metastasis, and monitoring and evaluating various modes of therapeutic intervention (including treatment with cytotoxic, apoptosis-inducing, toxic autophagy-inducing and necrosis-inducting agents; viral therapeutic approaches; immune therapies, etc.). In addition, the PEG-Prom-Luc animals (or animals with other cancer-specific or cancer-selective promoters integrated into the germ line) could be used as single transgenic animals to look at processes such as skin carcinogenesis, organ carcinogenesis as a result of exposure to specific toxic agents and the role of ehemoprevention in preventing or limiting the severity of cancer induction and progression.
In conclusion, these studies are paradigm shifting, providing proof-of-pdriciple for developing cancer diagnostic mice (OncoView Mice). They further provide evidence for the utility of the PEG-Prom-Luc/double transgenic mouse approach for producing OncoView Mice in which cancer development and progression can be imaged using BLI. Moreover, this approach is not restricted to only breast cancer, since it can, in principle, be applied to any cancerous transgenic animal model including but not limited to pancreas, prostate, lung, colorectum, brain, ovary, esophagus, stomach, skin (melanoma) and others.
1. Su Z Z, Sarkar D, .Emdad L, Duigou G J, Young C S H, Ware J, Randolph A, Valerie K, and Fisher P B. Targeting gene expression selectively in cancer cells by using the progression-elevated gene-3 promoter. Proc Nati Acad Sci USA 2005;102(4):1059-1064.
2. Su Z, Shi Y, Fisher P B. Cooperation between API and PEA3 sites within the progression elevated gene-3 (PEG-3) promoter regulate basal and differential expression of PEG-3 during progression of the oncogenic phenotype in transformed rat embryo cells. Oncogene 2000;19(30):3411-21.
3. Su Z Z, Shi Y, Fisher P B. Subtraction hybridization identifies a transformation progression-associated gene PEG-3 with sequence homology to a growth arrest and DNA damage-inducible gene. Proc Nati Acad Sci USA 1997;94(17):9125-30.
4. Su Z Z. Goldstein N I, hang H, Wang M N, Diligent G J, Young C S, Fisher P B. PEG-3, a nontrans forming cancer progression gene, is a positive regulator of cancer aggressiveness and angiogenesis. Proc Nati Acad Sci USA 1999;96(26):15115-20.
5. Su Z Z., Emdad L, Sarkar D, Randolph A, Valerie K, Yacoub A, Dent P, Fisher P B. Potential molecular mechanism for rodent tumorigenesis: mutational generation of Progression Elevated Gene-3 (PEG-3). Oncogene 2005;24(13):2247-55.
6. Sarkar D, Su Z Z, Vozhilla N. Park E S, Gupta P, Fisher P B. Dual cancer-specific targeting strategy cures primary and distant breast carcinomas in nude mice. Proc Natl Acad Sci USA 2005;102(39);14034-9.
7. Sarkar D, Su Z Z, Vozhilla N, Park E S, Randolph A, Valerie K, Fisher, P B. Targeted virus replication plus immunotherapy eradicates primary and distant pancreatic tumors in nude mice. Cancer Res 2005;65(19):9056-63.
8. Sarkar D, Lebedeva I V, Su Z Z, Park E S, Chatman L, Vozhilla N, Dent P, Curiel, D T, Fisher P B. Eradication of therapy-resistant human prostate tumors using a cancer terminator virus. Cancer Res 2007;67(11.):5434-5442.
9. Sarkar D, Su Z Z, Park E S, Vozhilla N, Dent P, Curiel D T, Fisher P B. A cancer terminator virus eradicates both primary and distant human melanomas. Cancer Gene Therapy 2008;15(5):293-302.
10. Bhang H E C, Gabrielson K L, Laterra J, Fisher P B, Pulver M G. Tumor-specific imaging through progression elevated gene-3 promoter-driven gene expression. Nature Medicine 2011;17(1):1.23-9.
11. Howes K A, Ransom N, Papermaster D S, Lasudry J G, Albert D M, Windle J J, Apoptosis or retinoblastoma: alternative fates of photoreceptors expressing the HPV-16 E7 gene in the presence or absence of p53. Genes Dev 1994;8(11):1300-10.
12. Jolicoeur P, Bouchard L, Guimond. A, Ste-Marie M, Hanna Z, Dievart A. Use of mouse mammary tumour virus (MMTV)/neu transgenic mice to identify genes collaborating with the c-erbB-2 oncogene in mammary tumour development. Biochem Soc Symp. 1998;63:159-65.
A transcription-based imaging, therapeutic or theranostic system can be considered for clinical translation if it meets certain criteria such as high tumor specificity, broad application and minimal toxicity (1). The first two criteria can be met through the choice of a strong and tumor-specific promoter. For example, cancer-specific gene therapy with the osteocalcin promoter, delivered through intra-lesional administration of an adenoviral vector, caused apoptosis in a subset of patients with prostate cancer (PCa) (2). We have previously shown that cancer-specific imaging could be accomplished in vitro and in vivo in experimental models by placing imaging reporters under the control of the progression elevated gene-3 promoter (PEG-Prom) (1, 3). Here we show that by employing the astrocyte elevated gene-1 promoter (AEG-Prom) (4) for cancer-specific imaging, focusing on metastatic models of PCa, for which there is no reliable clinical imaging agent.
AEG-1 was first identified using subtraction hybridization as an up-regulated gene in primary human fetal astrocytes infected with H1V-1 (5, 6). Subsequent studies identified AEG-1 as a metastasis-associated gene in the mouse, called metadherin (MTDH) (7), and as a lysine-rich CEACAM1 co-isolated gene in the rat, called LYRIC (8). Recent studies in multiple cancer indications confirm a significant role for AEG-1. as an oncogene (9) implicated in cancer development and progression in many organ sites (10) Based on the potentially diverse roles of AEG-1 in tumor progression, including transformation, growth regulation, cell survival, prevention of apoptosis, cell migration and invasion, metastasis, angiogenesis, and resistance to chemotherapy (11), this gene provides a viable target for therapies for diverse cancers. Expression of AEG-1 involves transcriptional regulation through defined sites in its promoter (4). A minimal promoter region of AEG-1 was identified by virtue of its association with oncogenic Ha-ras-induced transformation (4). AEG-1 is a downstream target of the firs-ras and c-myc oncogenes, accounting in part for its tumor-specific expression. We have previously shown that AEG-Prom is activated by the binding of the transcription factors c-Myc and its partner Max to the two E-box elements of the promoter in. Ha-ras-transformed rodent and immortalized transformed astrocyte cell lines (4). AEG-1 interacts with PLZF, the transcriptional repressor that regulates the expression of the genes involved in cell growth and apoptosis (12). AEG-Prom acts as a broadly applicable cancer sensor and has been tested in a spectrum of malignancies, including those involving brain, prostate, breast and pancreas, among others (P.B. Fisher, unpublished data).
Although molecular-genetic imaging with AEG-Prom should be generally applicable to a variety of malignancies, our initial study performed here was in part to demonstrate the utility of this system in a relevant and challenging application, namely, for molecular imaging of PCa. We also focus on PCa because positron emission tomography (PET) with [18F]fluorodeoxyglucosc (FDG), which is the current clinical standard for a wide variety of malignancies, does not work particularly well for this disease (13). Although a variety of new molecular imaging agents for PET with computed tomography (PET/CT) of PCa are emerging, such as [18F]NaF (NaF) (14, 15), [11C]- —and [18F]choline (16-18), [18F ]FDHT (19), anti- [18F]FACBC (20) and [18F]DCFBC (21), some are limited to detecting bone lesions (NaF), have significant overlap with normal prostate tissue (the cholines), or have not yet been extensively tested in the clinic. To maintain relevance to clinical translation, we used a linear polyethyleneimine (1-PEI) nanopartiele to deliver the construct systemically. Nanoparticles comprised of 1-PEI are being used in a variety of ongoing clinical trials (22-24). We describe AEG-Prom-mediated imaging in tumors derived from PC3-ML cells, a human androgen-independent invasive and metastatic model of PCa (25-27). We show that imaging with AEG-Prom delineates lesions from PCa as well as or with higher sensitivity than FDG- or NaF-PET/CT in this model system.
Cloning of plasmid constructs. pPEG-Luc and pAEG-Luc were generated as described previously (3, 28). The firefly luciferase-encoding gene in pAEG-Luc was replaced by the HSV1-tk-encoding sequence from pORF-HSVtk plasmid (InvivoGen, San Diego, Calif.) to generate pAEG-HSV1tk . Details of cloning by restriction enzyme digestion and other conditions are available upon request. The plasmid. DNA was purified with the EndoFree Plasmid Kit (Qiagen, Valencia, Calif.). Endotoxin level was ensured as <2.5 endotoxin units per mg of plasmid DNA.
Cell lines. PC3-ML-Luc (stable transfectants) and PC3-ML were kindly provided by Dr. Mauricio Reginato (Drexel University, Philadelphia, Pa.). These were routinely cultured in Dulbeeco's modified Eagle's medium (DME M) (Cellgro, Manassas, Va.) supplemented with 10% (vol/vol) FBS and 1% (vol/vol) antimycotic solution (Sigma-Aldrich, St. Louis, Mo.) and incubated at 37° C., 5% CO2. PrEC (normal prostate epithelium) cells were kindly provided by Dr. John T. Isaacs (Johns Hopkins School of Medicine, Baltimore, Md). Those were grown in keratinocyte serum-free medium (total [Ca2+] is 75±2 μmol/L) supplemented with bovine pituitary extract and recombinant epidermal growth factor (Invitrogen Life Technologies, Grand Island, N.Y.).
Transient transfection and luciferase assay. The following PCa cell lines: PC3-ML, LNCaP, DU145, PrEC (primary cells) were plated in 6-well plates (BD Biosciences, Bedford, Mass., USA) at 180×103−200×103 cells. Cells were transfected using in-vitro jetPRIME® (Polyplus-transfection, Illkrich, France) according to the manufacturer's instructions. The indicated cells were transfected with Luc under the experimental promoters AEG-Prom, PEG-Prom, and a promoter-less empty vector (control) as a pDNA-PEI polyplex. Luminescence was normalized for transfection efficiency by co-transfection with a vector expressing renilla luciferase, pGL4.74[hRluc/TK] (Promega, Madison, Wis.). After 48 h of transfection, the expression level of the Luc reporter was measured by the Dual Luciferase Reporter Assay kit (Promega). Luminescence was normalized for cell number (by μg total protein) using the BCA Protein Assay Kit (Pierce Biotechnology, Rockford, Ill.).
Construction of mutant AEG-Prom. The mEbox1 and mEbox2 sites were mutated in the wild-type pAEG-Luc plasmid to generate the pAEG-mEbox1&2-Luc plasmid. The consensus E-box sequence, CACGTG, was mutated into AGAGTG using the QuikChann Lightening Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, Calif.) and appropriate primers. The sequences of the forward (F) and reverse (R) primers for mutagenesis were F: 5′ CTGGTCTACAGTAACGGGTCC (SEQ ID NO: 5) and R: 5′ ATTCAGCCCATATCGTFTC (SEQ ID NO: 6). The mutated constructs were confirmed by sequencing (Integrated DNA Technologies, Coralville, Iowa). PC3-ML cells were transiently transfected with the wild-type and mutated plasmid for the subsequent luciferase assay, as described above.
Generation of an hi vivo experimental model of metastatic PCa. All protocols involving the use of animals were approved by the Johns Hopkins Animal Care and Use Committee. Four-to-six-week old male NOG (NOD/Shi-scid/IL-2Rγnull) mice were purchased from the Sidney Kimmel Comprehensive Cancer Center's Animal Resources Core (Johns Hopkins School of Medicine). PC3-ML and PC3-ML-Luc cells were expanded over three to five passages. The cells were harvested and diluted in sterile Dulbeeco's PBS lacking Ca2+ and Mg2+ (Invitrogen Life Technologies). For intravenous injection, mice were administered 1×106 PC3-ML cells in 100 μL of sterile Dulbecco's PBS via the tail vein. To ensure hematogenous dissemination, includine, to the bone, the cells were injected into the left ventricle of the heart (26, 27). For this intra-cardiac model mice were anesthetized with ketamine (100 mg/kg) and xylazine (20 mg/kg) and inoculated into the left ventricle with 5×104 PC3-ML-Luc enriched or PC3-ML cells in a total volume of 100 μL of sterile Dulbecco's PBS using a 263/4-gauge needle. To image the PC3-ML-Luc cells with BLI, mice were injected intraperitoneally (IP) with 100 μL of 25 mg/mL of D-luciferin solution (Caliper LifeScienees Hopkinton, Mass.), and BLI was performed 20 min after the intra-cardiac injection to detect the distribution of cells. Mice were imaged weekly. Images were acquired on an NIS Spectrum small animal imaging system (Caliper Life Sciences, Alameda, Calif.) and results were analyzed using Living Image software (Caliper Life Sciences). A group of age-matched healthy NOG mice served as a negative control for the PCa metastasis model.
Enrichment of PC3-ML-Luc cells. The PC3-ML-Luc cells were further selected for bone-homing tendency. Mice bearing PC3-ML-Luc tumors developed through the intra-cardiac injection method were monitored for tumor formation by BLI. After five weeks, following euthanasia the femur and tibia of the regions demonstrating clear signal were aseptically dissected. The tumor cells were established in culture by mincing the epiphysis and flushing the bone marrow with 1× PBS (Invitrogen Life Technologies) as described previously (25). The subpopulations of cells selected using a Transwell migration chamber with an 8 μm pore size (BD Falcon, San Jose, Calif.) were tested and confirmed for Luc expression as described previously (29), but using 1 mM of D-luciferin, potassium salt (Gold Biotechnology, St. Louis, Mo.). The radionuclide imaging experiments were performed with the enriched PC3-ML-Luc cell lines.
Systemic delivery of plasmid constructs. Low molecular weight 1-PEI-based cationic polymer, in vivo-jetPEI®(Polyplus Transfection), was used for gene delivery. The DNA-PEI polyplex was formed according to the manufacturer's instructions. For systemic delivery 40 μg or DNA and 4.2 μL of 150 mM in vivo-jetPEI® was diluted in endotoxin-free 5% (wt/vol) glucose separately. The glucose solutions of DNA and 1-PEI polymer were then mixed together to give an N:P ratio (the number of nitrogen residues of vivo-jetPEI® per number of phosphate groups of DNA) of 6:1 in a total volume of 400 μL. The DNA-PEI polyplex was injected IV as two 200 μL injections with a 5 min interval.
Bioluminescence imaging. In vivo BLI was conducted and 48 h after the systemic delivery of reporter genes. Mice were imaged with the IVIS Spectrum. For each imaging session mice were injected IP with 150 mg/kg of D-luciferin, potassium salt under anesthesia using a 2.0% isollurane/oxygen mixture. Ex vivo BLI was conducted within 10 min of necropsy. Living Image 2.5 and Living Image 3.1 software were used for image acquisition and analysis.
SPECT-CT imaging and data analysis. At 48 h after injection of pAEG-HSV1tk/PEI polyplex, animals were injected IV with 37.0 .MBq (1.0 mCi) of [125I]FIAU. At 18-20 h after radiotracer injection, imaging data were acquired with the X-SPECT SPECT-CT system (Gamma Medica Ideas, Northridge, Calif.) using the low-energy single pinhole collimator (1.0 mm aperture). Focused lung and liver imaging were acquired with a radius of rotation of 3.35 cm and whole-body imaging was undertaken with a radius of rotation of 7.00 cm. Mice were imaged in 64 projections at 45 sec of acquisition per projection. SPECT images were co-registered with the corresponding 512-slice CT images. Tomographic image datasets were reconstructed with the 2D ordered subsets-expectation maximum (OS-EM) algorithm. AMIDE (30) and PMOD (v3.3, PMOD Technologies Ltd, Zurich, Switzerland) software were used for image quantification and analysis.
FDG- and NaF-PET/CT imaging and analysis. 9.25 MBq (0.25 mCi) of each imaging agent was injected via the tail vein. Animals were placed on a heating pad and were allowed mobility during the 1 h radiotracer uptake period. The animals were then subjected to isoflurane anesthesia. Whole-body images were acquired with the eXplore Vista small animal PET scanner (GE Healthcare, Milwaukee, Wisc.) using the 250-700 keV energy window. Acquisition time was 30 min (two bed positions, 15 min per bed position). Mice were fasted for 6-12 h before receiving FDG to minimize radiotracer accumulation in non-tumor tissues. FDG and NaF imaging was done between four and five weeks after injection of PC3-ML-L.uc cells. PET images were co-registered with the corresponding 512-slice CT images. Tomographic image datasets were reconstructed with the 3D OS-EM algorithm with three iterations and 12 subsets and were analyzed with AMIDE software (30).
Histological analysis. After BLI data acquisition at 48 h after pAEG-Luc-PEI delivery, each organ demonstrating expression of Luc was collected and fixed in 10% neutral buffered formalin. Tissues were embedded in paraffin blocks. Serial paraffin longitudinal sections were stained with goat anti-luciferase polyclonal antibody (Promega) or rabbit anti-Myc polyclonal antibody (Epitomics, Burlingame, Calif.). Horseradish peroxidase (HRP)-conjugated polyclonal rabbit anti-goat antibody was used as a secondary antibody. HRP activity was detected with 3, 3′-diaminobenzidine (DAB) substrate chromogen (EnVision™+Kit, Dako, Carpinteria, Calif.). Consecutive sections of each tissue sample were stained with hematoxylin and eosin (H & E) and were photographed with a Zeiss photomicroscope III.
Quantitative real-time PCR. After imaging experiments, animals were euthanized and their lung and liver tissue were harvested and snap frozen. Total DNA was extracted by using DNeasy Blood & Tissue Kit (Qiagen) following the manufacturer's instructions. 100 ng of purified total DNA form each animal was used as a template. Quantitative real-time PCR was performed in triplicate per template using the inventoried Taqman® Gene Expression Assays (Cat. #41331182, Life Technologies, Grand. Island, N.Y.) with the FAM dye labeled primer set for Luc. Reaction conditions were set as 50° C. for 2 min, 95° C. for 10 min and 50 cycles of 95° C. for 15 see, 60° C. for 1 min followed by the disassociation step of 95° C. for 15 sec, 60° C. for 15 sec, 95° C. for 15 sec in a Bio-Rad iQ™5 Multicolor Real-Time PCR Detection system (Bic-Rad Laboratories, Hercules, Calif.). Data were analyzed by the absolute quantification method using a standard curve by iQ5 v2.0 software (Bio-Rad). Quantified data was normalized relative to the amplification of mouse GAPDH (glyceraldehyde-3-phosphate dehydrogenase) DNA.
Radiographic and gross visualization of bone lesions. A Faxitron MX20 Specimen X-ray system (Faxitron Corp., Tuscan, Ariz.) with digital exposures of 25 kV, 17 sec was used. Films were obtained on Kodak Portal Pack Oncology X-ray film (25.4×30.5 cm) for 22 kV, 15 sec. For gross pathology, bone tissues were fixed in 10% neutral buffered formalin and were decalcified for 2 h in Decal® (Decal Chemical. Corp., Suffern, N.Y.) and cut in thin slices.
Statistical Analysis. For BLI error bars in graphical data represent mean ±standard deviation (SD). P-values<0.05 were considered to be statistically significant.
Comparison of cancer specificity of AEG-Prom and PEG-Prom by bioluminescence imaging (BLI) in PCa. To examine the cancer-specific activity of AEG-Prom we constructed two plasmids, pAEG-Luc, expressing firefly luciferase, and pAEG-HSV1tk, expressing the herpes simplex virus type I thymidine kinase (not shown). AEG-Prom drives the expression of the imaging reporter genes firefly luciferase (Luc) and HSV1-tk which enable BLI and radionuclide based-techniques, respectively. Given the high sensitivity and ease of BLI, our initial studies used this modality for proof of concept. The HSV1-tk reporter gene was used, as before (1), to provide a method that has a clear path to clinical translation. The PEG-Prom construct, namely, pPEG-Luc, was generated previously (1), and was used for the current studies.
Using BLI we tested the cancer specificity of AEG-Prom and PEG-Prom in different PCa cell lines, including PC3-ML, LNCaP, DU-145, and in the non-malignant counterpart cells of prostate epithelium. Robust expression from AEG-Prom and PEG-Prom was observed only in the malignant cell lines, whereas promoter activity was negligible in the normal prostate epithelial cells (PrEC) (
We then tested and compared the specificity of AEG-Prom and PEG-Prom in vivo in a relevant experimental model of PCa. To develop this model we used two human PCa sub-lines selected from initial metastases of the parental human PC3 cells that targeted the murine lumbar vertebrae, hence ML (metastasis lumbar). We used PC3-ML cells and the luciferase-tagged version of the PC3-ML cells, namely, PC3-ML-Luc (25-27), which were injected either intravenously (IV) or directly into the left ventricle of the heart to ensure widespread dissemination including to bone. BLI confirmed the presence of widespread metastases after IV injection of PC3-ML-Luc cells (not shown). We assumed a similar time course for the development of metastases from the PC3-ML cells that did not express Luc so that we could use them in conjunction with the AEG-Prom-driven system to identit metastatic lesions by BLI. Mice received an IV dose of pAEG-Luc-PEI and pPEG-Luc-PEI polyplexes (
Histological analysis of the photon-emitting regions within lung for the animals treated with pAEG-Luc or pPEG-Luc showed the presence of tumor and the correlative Luc expression in the cancer models, but not in the controls (
BLI signal intensity was significantly higher in the PCa group compared to controls within lung at both the 24 and 48 h time points (after administration of pAEG-Luc and pPEG-Luc) (P <0.0001;
To enable reliable formation of metastasis to bone, a tissue prominently involved in human PCa, we injected PC3-ML-Luc and PC3-ML cells through an intra-cardiac route (not shown). Once timing for the development of metastases was determined for the luciferase-expressing cells, we then studied metastases due to PC3-ML cells using the pAEG-Luc-PEI polyplex. At 48 h after plasmid delivery we observed AEG-Prom-mediated expression of Luc from the PC3-ML models, as shown for PCa-4 and PCa-2 and not from controls (
Ex vivo BLI of PCa-2, when imaged five weeks after cell injection, showed the presence of tumor in the lungs, liver, adrenals and kidneys, as also confirmed by gross pathology, histological analysis and Luc IHC (
Radionuclide imaging of cancer via AEG-Prom. BLI is limited to pre-clinical studies due to the dependence of signal on tissue depth, the need for administration of exogenous D-luciferin substrate at relatively high concentration for light emission, rapid consumption of D-luciferin leading to unstable signal, and low anatomic resolution (1). Accordingly we cloned pAEG-HSV1tk (not shown), which can be detected by the radionuclide-based techniques of PET or single photon emission computed tomography (SPECT), upon administration of a suitably radiolabeled nucleoside analog (32). We examined the SPECT-CT imaging capabilities of pAEG-HSV1tk for detection of bone and soft tissue metastases in the PC3-ML model. Approximately five weeks after receiving an intra-cardiac administration of PC3-ML-Luc cells, the PCa group and the corresponding controls received pAEG-HSV1tk-PEI polyplex. Forty-eight hours after plasmid delivery, mice received the known HSV1-TK substrate, 2′-fluoro-2′-deoxy-β-d-5-[125I] iodouracil-arabinofuranoside ([125I]FIAU) (29), and were imaged at 18-20 h after injection of radiotracer.
BLI performed ex vivo and gross pathology of lesions within the right shoulder, dorsal thoracic wall, ribs, sternum and the heart confirmed that tumor was the source of signal seen on the living images (
Our goal was to develop a systemically deliverable construct for molecular-genetic imaging of metastatic lesions within both soft tissue and bone in a relevant model of PCa. Ultimately the intent of this technology will be to utilize a radionuclide-based imaging system for clinical translation. As stated at the outset, current clinical methods to image PCa are sub-optimal, and a genetic method, which can be paired with concurrent therapy (theranostic), would be a useful addition to detecting (and treating) the disease.
The minor-specific promoters of PEG-3 and AEG-1 have certain features that might render them more specific and selective while at the same time instill them with greater utility than other promoters, namely to use them in a variety of cancers beyond PCa. PEG-Prom and AEG-Prom: [1] maintain universal cancer specificity regardless of the tissue of origin; [2] do not require amplification to achieve high sensitivity; and, [3] are systemically delivered using a non-viral delivery vehicle. To recapitulate the clinical characteristics of PCa metastasis, we implemented a bone metastatic model of PCa in which we tested AEG-Prom activity. In animals showing tibial lesions using BLI of PC3-ML-Luc cells, subsequent SPECT/CT was able to detect these lesions in all animals tested (
By using a biodegradable polymer, viva-jetPEI®, we addressed certain problems that may arise when employing viral vectors, such as immune-mediated toxicity, inflammation and liver tropism. We checked the ability of the non-viral delivery vehicle to provide widespread, systemic dissemination of plasmid by conducting quantitative PCR on sections of lung and liver and compared the transfection efficiency between controls and animals affected with PCa (
AEG-Prom enables a sensitive method for molecular-genetic imaging of PCa in vivo. From mutational analysis of AEG-Prom we have shown that its activation relies significantly on c-Myc binding to the two E-box elements discussed above, As Ras-mediated c-Myc signal transduction is a pathway present in nearly all malignancies yet is absent in normal tissue (36). AEG-Prom will enable imaging of a wide variety of cancers directly and specifically.
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All terms and phrases (e.g. nucleic acid, protein, polypeptide, etc.) used herein have the meaning as commonly understood in the art, unless otherwise indicated.
As used in this specification and the appended claims, the singular forms “a,” “an” and “the” include plural references unless the content clearly dictates otherwise.
All patents, patent applications and publications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.
While the invention has been described in terms of its preferred embodiments, those skilled in the art will recognize that the invention can be practiced with modification within the spirit and scope of the appended claims. Accordingly, the present invention should not be limited to the embodiments as described above, but should further include all modifications and equivalents thereof within the spirit and scope of the description provided herein.
The present application is a continuation of U.S. patent application Ser. No. 14/182,690, filed Feb. 18, 2014, which is a continuation-in-part of U.S. patent application Ser. No. 13/881,777, filed Jun. 13, 2013, which claims the benefit of and priority to U.S. Provisional Patent Application No. 61/766,473, filed Feb. 19, 2013, which are herein incorporated by reference in their entireties for all purposes. U.S. patent application Ser. No. 13/881,777 is a U.S. National Stage Application under 35 U.S.C. § 371 of International Application No. PCT/US2011/058249, filed Oct. 28, 2011, which claims priority to and the benefit of U.S. Provisional Patent Application No. 61/407,714, filed Oct. 28, 2010, which are herein incorporated by reference in their entireties for all purposes.
| Number | Date | Country | |
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| 61766473 | Feb 2013 | US | |
| 61407714 | Oct 2010 | US |
| Number | Date | Country | |
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| Parent | 14182690 | Feb 2014 | US |
| Child | 16730705 | US |
| Number | Date | Country | |
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| Parent | 13881777 | Jun 2013 | US |
| Child | 14182690 | US |
