By a News Reporter-Staff News Editor at Life Science Weekly — From Washington, D.C., NewsRx journalists report that a patent application by the inventors CHANG, Esther H. (Potomac, MD); Pirollo, Kathleen F. (Rockville, MD), filed on December 6, 2013, was made available online on July 3, 2014 (see also Georgetown University).
The patent’s assignee is Georgetown University.
News editors obtained the following quote from the background information supplied by the inventors: “The present invention is in the fields of drug delivery, cancer treatment and diagnosis and pharmaceuticals. This invention provides a method of making antibody- or antibody fragment-targeted immunoliposomes for the systemic delivery of molecules to treat and image diseases, including cancerous tumors. The invention also provides immunoliposomes and compositions, as well as methods of imaging various tissues. The liposome complexes are useful for encapsulation of imaging agents, for example, for use in magnetic resonance imaging. The specificity of the delivery system is derived from the targeting antibodies or antibody fragments.
“The ability to detect cancer, both primary and metastatic disease, at an early stage would be a major step towards the goal of ending the pain and suffering from the disease. The development of tumor targeted delivery systems for gene therapy has opened the potential for delivery of imaging agents more effectively than is currently achievable. Magnetic resonance imaging (MRI) can acquire 3-Dimensional anatomical images of organs. Coupling these with paramagnetic images results in the accurate localization of tumors as well as longitudinal and quantitative monitoring of tumor growth and angiogenesis. (Gillies, R. J., et al., Neoplasia 2:139-451 (2000); Degani, H., et al., Thrombosis & Haemostasis 89:25-33 (2003)).
“One of the most common paramagnetic imaging agents employed in cancer diagnostics is MAGNEVIST.RTM.. (Gadopentetate Dimeglumine) (Mag) (Berlex Imaging, Montville, N.J.). Gadolinum is a rare earth element. It shows paramagnetic properties since its ion (Gd.sup.++) has seven unpaired electrons. The contrast enhancement observed in MRI scans is due to the strong effect of Gd.sup.++ primarily on the hydrogen-proton spin-lattice relaxation time (Ti). While free gadolinium is highly toxic, and thus unsuitable for clinical use, chelation with diethylenetriamine pentacetic acid (DTPA) generates a well tolerated, stable, strongly paramagnetic complex. This metal chelate is metabolically inert. However, after i.v. injection of gadopentetate dimeglumine, the meglumine ion dissociates from the hydhophobic gadopentetate, which is distributed only in the extracellular water. It cannot cross an intact blood-brain barrier, and therefore does not accumulate in normal brain tissue, cysts, post-operative scars, etc, and is rapidly excreted in the urine. It has a mean half-life of about 1.6 hours. Approximately 80\% of the dose is excreted in the urine within 6 hours.
“However, there are significant limitations with current contrast media, including that they are mainly based on perfusion and diffusion labels, and glucose uptake. With these free (non-complexed) agents, changes are seen in tumors, in inflammatory disease, and even with hormonal effects (in breast) (e.g. most gadolinium based and iodine based contrast agents document perfusion and diffusion into interstitial space, FDG-PET demonstrates glucose uptake). Thus, tumors are not specifically targeted by these contrast agents. In addition, active benign processes cannot always be separated from malignant, e.g. benign enhancing areas on breast MRI, chronic pancreatitis vs pancreatic carcinoma. There is also insufficient uptake by small tumors of these agents, and thus poor sensitivity and lack of early detection which is particularly critical in diseases like lung cancer. It may not be possible to detect solitary pulmonary nodules or pleural nodules. What is a needed, therefore, is a mechanism for delivering such agents to specific tissues within the body, for example, to tumor tissues and metastases.”
As a supplement to the background information on this patent application, NewsRx correspondents also obtained the inventors’ summary information for this patent application: “In one embodiment, the present invention provides methods of preparing an antibody- or antibody fragment-targeted cationic immunoliposome complex comprising preparing an antibody or antibody fragment, mixing the antibody or antibody fragment with a cationic liposome to form a cationic immunoliposome, wherein the antibody or antibody fragment is not chemically conjugated to the cationic liposome, and mixing the cationic immunoliposome with an imaging agent to form the antibody- or antibody fragment-targeted-cationic immunoliposome complex. Exemplary antibody fragments for use in the practice of the present invention include, single chain Fv fragments, such as an anti-transferrin receptor single chain Fv (TfRscFv) and anti-HER-2 antibody or antibody fragment. In additional embodiments, the methods further comprise mixing the cationic immunoliposome with a peptide comprising the K[K(H)KKK].sub.5-K(H)KKC(HOKC) (SEQ ID NO: 1) peptide.
“Suitably, the antibody or antibody fragment is mixed with said cationic liposome at a ratio in the range of about 1:20 to about 1:40 (w:w). Suitably, the cationic liposomes comprise a mixture of dioleoyltrimethylammonium phosphate with dioleoylphosphatidylethanolamine and/or cholesterol; or a mixture of dimethyldioctadecylammonium bromide with dioleoylphosphatidylethanolamine and/or cholesterol.
“In additional embodiments, the cationic immunoliposomes are mixed with the imaging agent at a ratio in the range of about 1:10 to about 1:35 (mg imaging agent:.mu.g liposome), suitably about 1:14 to about 1:28 (mg imaging agent:.mu.g liposome), or about 1:21 (mg imaging agent:.mu.g liposome). Exemplary imaging agent for use in the practice of the present invention include, but are not limited to, magnetic resonance imaging (MRI) agents, such as gadolinium, gadopentetate dimeglumine, iopamidol and iron oxide. Also, barium, iodine and saline imaging agents for CT, .sup.18F-2-deoxy-2-fluoro-D-glucose (FDG) and other imaging agents for PET can also be used.
“The present invention also provides cationic immunoliposome complexes prepared by the methods of the present invention and antibody- or antibody fragment-targeted cationic immunoliposome complexes comprising a cationic liposome, an antibody or antibody fragment, and an imaging agent, wherein the antibody or antibody fragment is not chemically conjugated to said cationic liposome.
“In further embodiments, the present invention provides methods of imaging an organ or a tissue, and also for distinguishing between benign tissues/diseases and cancerous tissues/diseases in a patient comprising administering the cationic immunoliposome complexes of the present invention to the patient prior to performing the imaging. Administration can occur via any route, for example, intravenous administration, intramuscular administration, intradermal administration, intraocular administration, intraperitoneal administration, intratumoral administration, intranasal administration, intracerebral administration or subcutaneous administration. Suitably, the tissue that is imaged using the methods and complexes of the present invention are cancerous tissues, including cancerous metastasis.
“The present invention also provides methods of imaging and treating a tumor tissue in a patient suffering from cancer comprising administering the cationic immunoliposome complexes of the present invention to the patient to image the tumor tissue and administering an anti-cancer agent to the patient to treat the tumor tissue. Exemplary anti-cancer agents include nucleic acids, genes, proteins, peptides, small molecules, chemotherapeutic agents, such as docetaxel, mitoxantrone and gemcitabine, and antisense oligonucleotides or siRNA.
“Additional embodiments of the present invention will be familiar to one of ordinary skill in the art.
BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
“FIGS. 1A and 1B show tumor-specific targeting of a CaPan-1 orthotopic metastasis model by the TfRscFv-Liposome-DNA nanocomplex. The same tumor nodule in the liver indicated by an arrow in 1A exhibits intense .beta.-galactosidase expression in 1B. 1A=gross necropsy; 1A=tissues after staining for .beta.-galactosidase.
“FIG. 2A-2C show In Vitro MR Imaging of K564 cells after transfection with the TfRscFv-Lip-Mag nanocomplex. 1A=time dependent transfection. The values given are relative intensity. 1B=shows variation in relative intensity with the amount of MAGNEVIST.RTM. included in the complex (in .mu.l). 1C=Comparison of relative intensity of the TfRscFv-Lip-Mag complex versus free MAGNEVIST.RTM.. The small circles in all images are markers for sample orientation.
“FIG. 3A-I show improved MR imaging in two different models of cancer using the Ligand-Liposome-Mag nanocomplex. 3A, D, and G show the differences in MRI signal in a large pancreatic orthotopic tumor (arrow) (4 months after surgical implantation of the tumor) between the i.v. administered free contrast agent and the TfRscFv-Lip-Mag complex. 3B, E, and H show a similar effect in a second mouse with a subcutaneous pancreatic tumor and a much smaller abdominal pancreatic tumor (arrows). 3C, F and I are the images of a third animal with a subcutaneous prostate tumor (arrow) in which the same effect is evident.
“FIG. 4A-C show SPM phase images of liposomes without MAGNEVIST.RTM.. The images appearing in 4A, 4B and 4C were obtained at setpoints of 1.68 V, 1.45 V, and 1.35 V, respectively. The corresponding phase differences between the noncompliant substrate and the mechanically compliant liposome are -3.5.degree., +8.degree., and +40.degree.. The interaction of the SPM tip and liposome changes from attractive to repulsive as the setpoint is decreased.
“FIG. 5A-C show SPM and SEM images of liposome-encapsulated MAGNEVIST.RTM. (Lip+Mag). 5A is the Atomic Force Microscopy topographical image of the Liposome encapsulated MAGNEVIST.RTM. particle. The SPM phase image (setpoint=1.6) (5B) and 15 keV SEM (TE) [Transmission-mode electron detector] image (5C) possess similar contrast, although generated by entirely distinct complementary physical mechanisms.
“FIGS. 6A and 6B show SPM topographic and phase imaging of TfRscFv+Lip+Mag nanocomplex. 6A is the 15 keV SEM (TE) [Transmission-mode electron detector] image of the full nanocomplex. 6B=A lower power image of the field. The boxed area is the image in 6A.
“FIGS. 7A and 7B show cross-sectional comparison of SPM topographic and magnetic phase image in lift mode using 25-nm height displacement. 7A is an SPM topographic/magnetic phase image of the full TfRscFv-Lip-Mag nanocomplex. The appearance of a double dipole-like signal in 7B consisting of attractive and repulsive in-plane magnetic interactions suggests that the cause of this interaction is the nonuniform toroidal distribution of MAGNEVIST.RTM. within the NDS, consistent with SEM and nonmagnetic SPM phase images.
“FIGS. 8A-8H show improved MR imaging in two different models of cancer using the Ligand-HK-Liposome-Mag nanocomplex. Human breast cancer MDA-MB-435 (FIG. 8E-8H) and human prostate cancer cell line (DU145) (FIG. 8A-8D).
“FIG. 9A-C shows tumor-specific targeting of a CaPan-1 subcutaneous tumor and orthotopic metastasis model by the TfRscFv-HK-Liposome-Mag nanocomplex.
“FIG. 10 shows dynamic MRI showing the increase in intensity using Mag-delivered by the complexes of the present invention in a pancreatic carcinoma model, as compared to free Mag.
“FIG. 11A-11C shows MR imaging of pancreatic cancer metastases by Mag-comprising complexes of the present invention.
“FIG. 12A-12E shows a greater enhancement in MR imaging of lung metastases by Mag-comprising complexes of the present invention.
“FIG. 13A-13D shows a greater enhancement in MR imaging of renal cell carcinoma lung metastases by Mag-comprising complexes of the present invention.
“FIG. 14A-14D shows greater sensitivity of detection by MR imaging of small renal cell carcinoma lung metastases by Mag-comprising complexes of the present invention.
“FIG. 15A-15B shows MR imaging of very small metastases by Mag-comprising complexes of the present invention, demonstrating the sensitivity of the complexes of the present invention.
“FIG. 16 shows sections of metastatic tissue confirming the detection/imaging seen by MRI using the Mag-comprising complexes of the present invention.
“FIG. 17 shows higher magnification of FIG. 16.
“FIG. 18A-18F shows MR imaging of metastases in the subpleura of the lung by Mag-comprising complexes of the present invention.
“FIG. 19A-19B shows detection of B.sub.16/F.sub.10 melanoma lung metastases by Mag-comprising complexes of the present invention.”
For additional information on this patent application, see: CHANG, Esther H.; Pirollo, Kathleen F. Tumor-Targeted Nanodelivery Systems to Improve Early Mri Detection of Cancer. Filed December 6, 2013 and posted July 3, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=\%2Fnetahtml\%2FPTO\%2Fsearch-adv.html&r=3742&p=75&f=G&l=50&d=PG01&S1=20140626.PD.&OS=PD/20140626&RS=PD/20140626
Keywords for this news article include: Antibodies, Immunology, Blood Proteins, Immunoglobulins, Georgetown University.
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