IL190773A - Antibody or antibody fragment-targeted cationic immunolysome complex and methods of preparing the same - Google Patents

Description

AN ANTIBODY OR ANTIBODY FRAGMENT-TARGETED CATIONIC IMMUNOLYSOME COMPLEX AND METHODS OF PREPARING THE SAME ix pa la Ivan »3i»op DITID^1 1D X v&xn insDn nitrwi pma νυ 190,773/2 1 Field of the Invention

[0001] 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.

It is to be noted that only the subject matter embraced in the scope of the claims appended hereto, whether in the manner defined in the claims or in a manner similar thereto and involving the main features as defined in the claims, is intended to be included in the scope of the present invention, while subject matter described and exemplified to provide background and better understanding of the invention, is not intended for inclusions as part of the present invention.

Background of the Invention

[0002] 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 59:25-33 (2003)).

[0003] One of the most common paramagnetic imaging agents employed in cancer diagnostics is Magnevist® (Gadopentetate Dimeglumine) (Mag) (Berlex Imaging, Montville, NJ). Gadolinum is a rare earth element. It shows paramagnetic properties since its ion (Gd++) has seven unpaired electrons. The contrast enhancement observed in MRI scans is due to the strong effect of Gd++ 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 (DTP A) generates a well tolerated, stable, strongly paramagnetic complex. This metal chelate is metabolically 190,773/2 2 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 US 2003/0044407 describes a cationic immunoliposome complex having a ratio lmg active agent: lOmg lipid to 1 mg active agent:20 nmol.

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. 190,773/3 3 BRIEF SUMMARY OF THE INVENTION

[0005] In one embodiment, the present invention provides methods of preparing an antibody- or antibody fragment-targeted cationic immunoliposome complex comprising: (a) preparing an antibody or antibody fragment; (b) mixing said antibody or antibody fragment with a cationic liposome to form a cationic immunoliposome, wherein said antibody or antibody fragment is not chemically conjugated to said cationic liposome; and (c) mixing said cationic immunoliposome with an imaging agent at a ratio in the range of about 1:10 to about 1:35 (mg imaging agent: μg liposome) to form said antibody- or antibody fragment-targeted-cationic immunoliposome complex.

This invention also provides an antibody- or antibody-fragment targeted cationic immunoliposomes complex obtained by a method as herein described. 190,773/2 4

[0011] Additional embodiments of the present invention will be familiar to one of ordinary skill in the art.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

[0012] Figure 1A and IB 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 β-galactosidase expression in IB. 1 A = gross necropsy; 1A = tissues after staining for β-galactosidase.

[0013] Figure 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. IB = shows variation in relative intensity with the amount of Magnevist® included in the complex (in μΐ). 1C = Comparison of relative intensity of the TfRscFv-Lip-Mag complex versus free Magnevist®. The small circles in all images are markers for sample orientation.

[0014] Figure 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.

[0015] Figure 4A-C show SPM phase images of liposomes without Magnevist®.

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 subsbate and the mfichanicalTy compliant liposome are -3.5°, +8°, and +40°. The mteraction of the SFM tip and liposome changes from attractive to repulsive as the setpoint is decreased. [0016| Figure 5A-C show SPM and SEM images of nrxiscene-encapsulated Magnevist® (Lip+Mag). SA is me Atomic Farce Microscopy topographical image of the liposome encapsulated Magnevist® particle. The SPM phase image (setpoint = 1.6) (SB) and IS keV SEM (ΓΕ) [Tiansnnssioi-HEaode election detector] image (SQ possess similar contrast, although generated by entirely distinct complementary physical mechanisms.

[0017] Figure 6A and 6B show SFM topographic and phase imaging of TSLscFv+Iip+Mag naiiccomplex. 6A is thel5 keV SEM (TE) [Transmission-mode electron detector] image of the fiifl nanDcoinplex.6B = A lower power image of the field The boxed area is the image in 6A.

[0018] Figure 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 topograpbicAmagnetic phase image of the mil TfRscFv-Iip-Mag nanocomplex. The appearance of a double dipole-Bke signal in 7B consisting of attractive and repulsive in- plane magnetic mteractians suggests mat the cause of this interaction is the nommirurm toroidal distribution of Magnevist within the NDS, consistent with SEM and nonmagnetic SPM phase images.

[0019] Figures 8A-8H show improved MR imaging in two different models of cancer using the Ugand-HK-Ijposome- ag nanocomplex. Human breast cancer MDA-MB- 435 (Figure 8E-8H) and human prostate cancer cell line (DU145) (Figure SA-8D).

[0020] Figure 9A-C shows tumor-specific targeting of a CaPan-1 subcutaneous tumor and orthotopic metastasis model by the TfRsiFv-HK-Iiposome-Mag nanocomplex.

[0021] Figure 10 shows dynamic MRI showing the increase in intensity using Mag- delivered by the complexes of the present invention in a pancreatic carcinoma dri, as compared to free Mag.

[0022] Figure 11A-11C shows MR imaging of pancreatic cancer metastases by Mag- comprising complexes of the present invention. [0023} Figure 12A-12E shows a greater enhancement in MR imaging of lung metastases by Mag-comprising complexes of the present invention- (00241 Figure 13A-13D shows a greater enhancement in MR imaging of renal cell ¾πτηηοτχ«ι Inpg rPF*¾fftaffgP by ^g-^wfppgjnE <*Mwple*es Qf the present invention. [0025} . Figure 14A-14D shows greater sensitivity of detection by MR imaging of small renal cell carcinoma lung metastases by Mag-comprising complexes of die present invention. 10026 Figure 15A-15B shows MR imaging of very small metastases by Mag-coiaprising complexes of the present invention, denK>nstrating the sensitivity of the complexes of the present invention. [00271 Figure 16 shows sections of metastatic tissue confirming the detectioii/iiiiagmg seen by MRI using die Mag-conipiising complexes of the present iitventiot-. [0028) Figure 17 shows higher magnification of Figure 16.

[0029] Figure 18A-1SF ε-hows MR imaguig of metastases m ite ag- xnnprising complexes of the present invention. 10030] Figure 19A-19B shows detection of Bie/Fjo melanoma lung metastases by Mag- comprising complexes of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0031] The present invention fulfills a critical need, that is, enhanced sensitivity and tumor-cell specificity for early detection and differential diagnosis of tumor versus benign tissue, by providing nanocomplexes for systemic delivery of imaging agents, such as magnetic resonance imaging (MRI) agents, such as gadotirmmi, gadopentetate dhnegtrn ine (Magnevist®) and, icpamidol, iron oxide; barium, iodine and saline imaging agents for CT; and l^-2-deoxy-2-fln rro-I ¾lucose (FDG) and other imaging agents for PET to targeted tissues, for example tumors. Scanning Electron Microscopy (SE ) and Scanning Probe Microscopy (SPM) (Wolfert, MA, et aL, Human Gene Therapy 72123- 2133 (1996); Dunlap, D >, et aL, Nucleic Acids Research 2*3095-3101 (1997) Kawaura, O, et aL, FEBS Letters 421:69-72 (1998); Choi, YJEL, et aL, Human Gene Therapy J0-.2657-2665 (1999); Diebel, C.E-, et aL, Nature 406299-302 (2000); Rasa, WL, et aL, J. CoJL Interface Sd 250:303-315 (2002)) have been used to etiarnine me physical structure- and size of these imaging age it-carryiBg nanocomplexes. In the case of gadohmitm, a high-atomic nuniber element which possess a large magnetic moment, these properties can be exploited in a variety of ways to enhance contrast in both SEM myf In one embodiment, the present invention provides tumor-targeting delivery systems comprising contrast agents, for example magnetic resonance imaging ( RT) contrast agents. US. Published Patent Application No. 2003 0044407 (the disclosure of which is incorporated herein by reference in its entirety) discloses these nano-sized, cationic liposome encapsulating various agents. Decorating the surface of these liposomes are targeting molecnles which can be a hganri, such as folate or transferrin, or an antibody or an antibody fragment directed against a cell surface receptor. The presence of the ligand/aritibody on the liposomes faraiitgtae the entry of the complexes into the cells through landing of the targeting mnl«?nlf- by its receptor followed by interaalization of the bound complex via receptor mediated endocytosis, a highly efficient internalization pathway (Qistiano, J-, and Curiel, D.T-, Cancer Gene Therapy 3:49-57 (1996); Cheng, P.W., Human Gene Therapy 7:275-282 (1996)). This modification of the liposomes results in their being able not only to selectively dekver their payioad to tumor cells, but also increases the transfection efficacy of the liposome. Transform receptor (Tf ) levels axe elevated in various types of cancer mchKHng oral, prostate, breast, and pancreas (Beer, ELN, et aL, Journal of Urology 143-3&1-3Z5 (1990); Rossi, M.C, and Zetter, BJL, Proa Nad. Acad. Set (USA) «9:6197-6201 (1992) EHiott, ILL, et aL, Ann. Ν.Ύ. Acad. ScL 698:159-166 (1993); Tbflrstensen, and Romslo, L, Scand. J. Can. Lab. Invesng- (&ΨΡ-) 2/5:113-120 (1993); ryamoto, T^ et aL, infl J. Oral Maxillofacial Surg. 25:430-433 (1994); Ponka, P. and Lok, N., Ml J. Biochem. Cett Biol 57:1111- 1137 (1999)). Moreover, the TfR recycles during internalization in rapidly devdciping cells such as cancer cells (Ponka, P. and Lok, C.N., Ml J. Biochem. Cett Biol 57:1111- 1137 (1999)), thus contributing to the uptake of these transferrin targeted nanocoinplexes even in cancer cells where TfR levels are not elevated, m suitable embodiments, die nanocomplexes described herein employ an arui-transierrin receptor single chain antibody fragment (TfRscFv) as the targeting moiety (Haynes, B-F, et aL, J. Immunol 727:347-351 (1981); Batra, JJ , et aL. Moleadar & Cellular Biology 11.2200-2205 (1991)). TfRscFv contains the complete antibody binding site for the epitope of the TfR recognized by the monoclonal antibody SE9 (Batra, JJL, et aL, Molecular & Cellular Biology 7/2200-2205 (1991)). TfRscFv has advantages over me Tf molecule itself, or an entire Mab, in targeting liposomes to cancer cells with elevated TfR levels: 1) foe size of the scFv (28 fcDa) is much smaller than the Tf molecule (80 kDa) or the parental Mab (155 kDa). The scFv-liposome-DNA complex may thus exhibit better penetration into small capillaries characteristic of solid trrmors. 2) me smaller scFv has a practical advantage related to the scaled-op production necessary for the clinical trials. 3) the scFv is a recombinant molecule and not a blood product like Tf and thus presents no danger of a potential contamination by blood borne pathogens.4) without the Fc region of the Mab, the issue of non-antigen-specific binding through Fc receptors is riiimnatrid (Tain, ILK. and Baxter, L.T., Cancer Res. 4&7022-7032 (1988)). Such an anri-TfR single chain antibody molecule can target an intravenously ad^ninistBred catianic hposcine-D A nanccomplex preferentially to tumors (See U-S. Published Patent Application No. 200370044407; uJ^ et aL, Moleadar Medicine 7:723-734 (2001); Xu L, et aL, Moleadar Cancer Therapeutics 1337-346 (2002)). Encapsulating Magnevist* (Mag) within such a tumor-targeted nanocomplexes offers advantages for enhance*! sensitivity and detection of tumor metastases and diagnosis of cancer. Gadoiin-um, gadopentetate dxtneglatnine (Magnevist®), iopamidol, iron oxide barium, iodine and saline imaging agents for CT; and 18F-2-deoxy-2-ilitoro-r ^ncose (FDG) and other imaging agents for PET, as well as any other current contrast agent known to one of ordinary skill in the art, as well as any future contrast agent or imaging agent yet to be developed (e.g^ for MR], CT, PET, SPECT, etc.) can also be encapsulated within the mwmimnposomes of the present invention.

Antibody- or antibody fragment- targeted cationic liposome complexes in accordance with this invention are made by a simple and efficient lKm-chemical conjogation method in which the camipanents of the desired complex are mixed together in a defined ratio and in a defined order (see, U.S. Published Patent AppHeation No, 2003/0044407). The resultant complexes are. as effective as, or more effective man, similar complexes in which the aiuibbdy or antibody fragment is chemically conjugated to the liposome or polymer. The terms "immiinocemplex," "iiiiiiiuitonposrime," "complex," "nanocomplex," "immmioiiariocc^rmlex," "liposome complex1' ate used interchangeably throughout to refer to the cafionic liposomes of the present inventioii.

Either a whole antibody or an antibody fragment can be used to make me complexes of this invention. In suitable embod ments, an antibody fragment is used. Preferably, me antibody fragment is a single chain Fv fragment of an antibody. One preferred antibody is an anti-TfR monoclonal antibody and a uiefeued antibody fragment is an scFv based on an anti-TfR mmwv.innai antibody. A suitable anti-TfR monoclonal antibody is 5E9 (see, &g-, Hayes, B.F., et al^ "Cbaritclerizarion of a Monoclonal Antibody (5E9) mat Defines a Hitman Cell Surface Antigen of Cell Activation," J. Immunol 127M7-352 (1981); Batra, JJL, et aL, "Single-chain mitmmotDxins Directed at the Human Transferring Receptor Containing Pseudomonas Exotoxin A or Diphtheria Toxin: Anti-TFR{Fv>PE40 and DT388-Anti-TER(Pv)," Mol CeH Biol 77:2200-2205 (1991); the disclosures of which are incuipo aled herein by reference). An scFv based on 5E9 antibody contains the complete antibody binding she for the epitope of the TfR recognized by mis MAb as a single polypeptide chain of approximate molecular weight 26,000. An scFv is formed by connecting the compoTiBnt. VH and VL variable domains from the heavy and light t-ham^ respectively, with an appropriately dftgjgpwH hnker peptide, which bridges the C-tenninus of the first variable region and N-tenninus of me second, ordered as either VH-hnkar-VL or VL-lrnlcer-Vrl Ariother preferred antibody is an anti-HER-2 monoclonal antibody, and another preferred antibody fragment is an scFv based on an anti-HER-2 monoclonal antibody.

In suitable embodiments, a cysteine moiety is added to the C-Uoiuiuus of the scFv. Although not wishing to be bound by theory, it is believed mat the cysteine, which provides a free sulmydryl group, may enhance the formation of the complex between the antibody and the liposome, for example via a charge-charge interaction. With or without the cysteine, the protein can be expressed in Kcoli inclusion bodies and then refolded to produce the antibody fragment in active form.

Unless it is desired to use a stericauy srahinTed irormmoliposarne in me formation of the complex, -a first step in making the nTpp^prr fyimpr'g**<: mining « rsitirmii*. liposome or combination of hposomes or smalt polymer with the antibody or antibody nagment of choice (see Examples herein and in ILS. Published Patent Application No.2003/0044407). A wide, variety of catiomc liposomes are useful in the preparation of the complexes of tins invention. Published PCT applicaliaa WO99/25320 describes the preparation of several cationic liposomes. Examples of desirable liposomes include those mat comprise a mixture of dioleoyicri-nethylaiimioiiium phosphate (DOTAP) and dioleoylphosphat vleithaTrolamrne (DOPE) and/or cholesterol (choi), a mixture of dimethyldioctadec^rlainTnonium bromide (DDAB) and DOPE and or choL The ratio of me lipids can be varied to optimize the efficiency of uptake of the therapeutic molecule for the specific target cell type. The liposome cam comprise a mixture of one or more cationic lipids and one or more neutral or helper lipids. A desirable ratio of cationic lipid(s) to neutral or helper lipid(s) is about 1 :(0-5-3), preferably l:(l-2) (molar ratio).

[0037] The present rrrvention also provides tor targeted-catianic polymers tor delivery of imaging agents. Suitable polymers are DNA brndin& cationic polymers that are capable of mediating DNA coinpaction and can also mediate endosome release. A preferred polymer is polyemyleneirnine. Other useful t»lyrners include polysine, protarnine and polyarnidoamme dendrimers.

[0038] The antibody or antibody f agment is one which will bind to the surface of the target cell, and preferably to a receptor mat is differentially expressed on the target celL The antibody or antibody fragment is mixed with the ranardc hposome or polymer at room temperature and at a protemdipid ratio in the range of about 1:20 to about 1:40 (wrw) or a protein polymer ratio in the range of about 0.1:1 to 10:1 (molar ratio).

[0039] The antibody or antibody fragment and the hposome or polymer are allowed to incubate at room temperature for a short period of time, typically for about 10-15 itiimitRgj then the mixture is mixed with a therapeutic or diagnostic agent of choice. Examples of therapeutic molecules or agents which can be complexed to the antibody and hposome include genes, high molecular weight DNA (genomic DNA), plasnrid DNA, antisense ohgpnucleotides, peptides, ribozymes, rmcleic acids (rnchirnrig siRNA and antisense), viral particles, rnmimnmnriwiatrng agents, proteins, small molecules and chemical agents. Preferred therapeutic molecules include genes encoding p53, Kb94 or Apoptin. KB94 is a variant of the retinoblastoma, tumor suppressor gene. Apoptin is a gene/that Tiw¾ir » apoptosis in tumor cells only.■ m another piereiied embodiment, the agent is an antisense oHgonucleotide or an sIRNA molecule, such as a HE -2 antisense or -dE-NA molecule. A third type of preferred agent is a diagnostic imaging agent, such as an MRI imaging agent, such as a Gd-DTPA agent. Additional imaging agents include, but are not limited to, Gadolinium, gadopentetate dimftghimme (Magnevist®), iopamidol, iron oxide; barium, iodine and saline imaging agents for CT; and 18F-2-deoxy-2-fluoro- D-glncose (FDG) and other imaging agents for PET. If the agent is DNA, such as the coding region of p53, it can be positioned under the control of a strong constitutive promote, such as an RSV or a CMV promoter.

[0040] The antibody or antibody fragment and liposome combination is mixed with the therapeutic or diagnostic agent at a ratio in the range of about 1:10 to 1:20 Qig of agenimmole of total lipid) or 1:10 to 1:40 (ug of agentnmole of total polymer) and mcuhfftftd at room temperature for a short period of time, typically about 10 to IS nriimtrs, The size of the liposome complex is typically within me range of about 50-400 nm as measured by dynamic light scattering using a Malvern Zetasizer 3000.

[0041] In one embodiment of mis invention, the liposome used to form the complex is a .stoically stabilised liposome. Sterically stabilized liposomes are liposomes into which a hydrophilic polymer, soch as PEG, pory(2-etrrylacrvric acid), or poly(n- isopropviaciylamide (PNIPAM) have been, integrated. Such modified liposomes can be - particularly useful when complexed with therapeutic or diagnostic agents, as they typically are not cleared from the blood stream by the reticnloendothelial system as qmcJdy as are comparable liposomes mat have not been so modified. To make a sterically stabilized liposome complex of die present invention, the order of mixing the antibody or antibody fragment, the liposome and the therapeutic or diagnostic agent is reversed from the order set forth above. In a first -step, a canonic Hposome is first mixed with a therapeutic or diagnostic agent as described above at a ratio in the range of about 1:10 to 1.20 (jig of agentnmole of lipid). To Ibis lipoplex is added a solution of a PEG polymer in a physiologically acceptable buffer and the resonant solution is incubated at roam temperature tor a time sufficient to allow the polymer to integrate into the hposome mmfAtsx. The antibody or aiitibody fragment then is mixed with the stabilized liposome complex at room temperature and at a protemilrpid ratio in the range of about 1:5 to about 1:30 (war).

[0042] · The hpasomal or polymer complexes prepared in accoidance with tile present inventio caa be formulated as a pharmacologically acceptable fbrinnlation for in vivo athniuistration. The complexes can be combined with a phaanacologtcalry compatible, vehicle or carrier. The compositions can be tbrrrrnlated, for example. &r intravenous !wTiiNiiiuiiiiiififi o a l»iminn fftiffn† to be benefiied by adnmiis£ration of the therapeutic or diagnostic molecule of the complex. The complexes are sized appropriately so that they are distributed throughout the body following ίν. administration. Alternatively, the complexes can be delivered via other routes of admmistrarioii, such as intratumoral (IT), intralesional (IL), asrosal, percutaneous, endoscopic, topical, jntrnnnscnlar (UVf), intrademrial (ID), intraocular (IO), iiitraperitoneal (IP), intranasal (IN), intracereberal (IC) or subcutaneous admimstrarjan. Preparation of fonnulatious for delivery via such iprfh/yi^ mri delivery ngfng such methods, are well known in me art.

[0043] In one embodimcrit, compositions cornprising the antibody- or antibody fragment- targeted hposome (or polymer) and therapeutic agent complexes are administered to snect human gene therapy. The therapeutic agent component of the complex comprises a therapeutic gene under the control of an appropriate regulatory sequence. Gene therapy for various forms of human cancers can be accomplished by the systeonc delivery of antibody or antibody fragment-targeted liposome or polymer complexes which contain a nucleic acid encoding wt p53 or RB94. The complexes can specifically target and sensitize tumor cells, bom primary and metastatic tumors, to radiation and/or chemotherapy both in vitro and in vivo.

[0044] The complexes can be optimized for target cell type through the choice and ratio of lipids, the ratio of antibody or antibody fragment to liposome, the ratio of antibody or antibody f agment and hposome to the therapeutic or diagnostic agent, and the choice of antibody or antibody fragment and therapeutic or diagnostic agent

[0045] one embodiment, the target cells are cancer cells. Although any tissue having malignant cell growth can be a target, head and neck, breast, prostate, pancreatic, brain, jjrmhuting glioblastoma, cervical, hmg, liver, Iiposarcama, rhahri ryjosarcoma, choriocarcinoma, melanoma, retinoblastoma, ovarian, urogenital, gastric and colorectal

[0046] The complexes made by the method of mis invention also can be used to target non-tumor cells for deliver of a therapeutic molecule or any nucleic arid. While any normal cell can be a target, preferred cells are dendritic cells, endothelial cells of the blood vessels, hmg cells, breast cells, bone marrow cells, thymus cells and fiver cells. Undesirable, but benign, cells can be targeted, such as benign prostatic hyperplasia cells, over-active thyroid cells, lipoma ceDs, and cefls relating to autoimmune diseases, such as B cells mat produce antibodies involved in arthritis, bpus, myasthenia gravis, squamous metaplasia, macular degeneration, cardiovascular disease, neurologic disease such as AMieimer's disease, dysplasia and the Kke.

[0047] The complexes can be administered in combination with another merapentic tr«g*mfiitj fffrh"" * radiation fawrtmmt err hfmnmerajHailic agent. The therapeutic treatments, or a combination of therapeutic treatments, can be administered before or subsequent to the admmistratian of the complex, for example within about 12 hours to about 7 days. Q-xmc-therapeutic agents include, for example, doxorubicin, 5-Suoronracil (5FU), dsplatin (CDDP), docetaxel (TAXOTERE*), gemcitabine (QEMZAR*), pacletaxel, vinblastine, etoposide (VT-16), cam tothecia, aetinomycin-D, mitDxantrone and mitomycin C. Radiation therapies treatments include gamma radiation (^Cs), X- rays, UV irradiation, microwaves, electronic emissions and me like. Additional therapeutic agents inrfr" * small mol^ilfts, peptides, proteins and the hke.

[0048] Diagnostic or imaging agents also can be delivered to targeted cells via the liposome or polymer complexes. The terms "diagnostic agents" and "imaging agents" are used interchangeably throughout to refer to agents which can be detected, visualized, imaged or observed in vivo following admini-rtration. Exemplary methods for detecting, ranmn m^ imaging or observing diagnostic and imaging agents are well lninwn in the ait and include, for example, optical imaging such as fluorescent imaging (flnorimeters) or biobminescent i aging, positron emission tomography (PET) snaiming, single photon emission computed tomography (SPECT) grsmm , magnetic resonance a in (MRI), x-ray, radionncleotide hnagmg (e.g^ gamma camera, computed tomography (CT), quantitative autoradiography, etc.) and the tike. Exemplary diagnostic agents include electron dense materials, iron, magnetic resonance imaging agents and iatiopbarmaceuticals. Radionuclides useful for imaging include radioisotopes of copper, gaTlimnj in ium, ifrftninm, and technetium, including isotopes ^Cu, ^Cu, mm, "Tc, ^Ga or aGa. MRI agents such as a Gd-DTPA agent, gadoHnhim, or Magnevist® (Gadopentetate Dimeglninine) (Mag) (Beriex Imaging, Montvflle, NJ). Imaging agents disclosed by Low et aL m U& Pa^ useful in the present -invention. Additional imaging agents mnhide, but are not limited to, • iopamidol (e.g., ISOyUE*, Regional Health limited, Aukland, AU), iron oxide; barium, iodine and saline imaging agents for CT; and 18F-2-deoxy-2-flnoro-I>-glncose (FOG) and other imaging agents for PET.

The complexes made in accordance with the method of fins invention can be provided in die form of kits for use in the systemic delivery of a therapeutic or diagnostic molecule by the complex. Suitable kits can comprise, in separate, statable containers (or in a single container), the liposome, the antibody or antibody fragment, and the therapeutic or diagnostic agent The cornponents can be mixed nnder sterile conditions in the appropriate order and adnmnstered to a patient within a reasonable period of time, generally from about 30 τητρπ ρ* to about 24 hours, after preparation. The lat cornponents preferably are provided as solutions or as dried powders. Components provided in solution form preferably are ibmnilated in sterile aier-ibr-injection, along with appropriate buffers, osmolality control agents, etc.

Encapsulation and Delivery of Imaging Agenis certain embodiments, the present invention provides cationic liposomal complexes wherein one or more imaging agents are encapsulated within the interior of the liposome, contained within, the hydrocarbon chain region of the bilayer, complexedVassociated with the inner and/or outer monolayer (e.g_, via static interaction or cherrrical/covalent interaction), or a ranffiiw-i^iMtii of any or all of these possibilities. Suitably, the imaging agents will be encapsulated within the interior of the liposome and/or assoc a ed with an inner and or outer monolayer.

As used herein, the terms "diagnostic agents" and "imaging agents" refer to agents which can be detected, visualized, imaged or observed in vivo following ».'l......lTj .i Exemplary imaging agents inclnde electron dense materials, iron, magnetic resonance imaging agents and raninpharmacemicals. adionucndes useful for imaging include radioisotopes of copper, gallium, indium, rhenium, and technetmm, mcmdrng isotopes 6 Cn, CTCu, mIn, ""Tc, CTGa or eGa. RI agents such as a gnn^lrrmnn, Gd-DTPA agent, or Magnevist® CGadopentetate Dimeglumine) (Mag) (Beriex Tmagmg. bntviQe, NT). Imaging agents disclosed by low et aL in U.S. Patent 5,688,488, incorporated herein by reference, are also useful in the present invention. Additional imaging agents 190,773/2 include, but are not limited to, iopamidol (e.g., ISOVUE , Regional Health Limited, Aukland, AU), iron oxide; barium, iodine and saline imaging agents for CT; and 18F-2- deoxy-2-fluoro-D-glucose (FDG) and other imaging agents for PET.

As described herein, imaging agents are suitably encapsulated, contained or complexed/associated with the liposome complexes of the present invention by simply mixing the one or more imaging agents with the liposomes during processing. Suitable ratios of imaging agents:liposome complexes are readily determined by the ordinarily skilled artisan. For example, the ratio of imaging agents to liposome complex is suitably in the range of about 1:10 to about 1 :35 (mg imaging agent: μg liposome), more suitably about 1 :14 to about 1 :28 (mg imaging agent^g liposome), or about 1 :21 (mg imaging agent^g liposome).

As described throughout, examples of desirable cationic liposomes for delivery/encapsulation of imaging agents include those that comprise a mixture of dioleoyltrimethylammonium phosphate (DOTAP) and dioleoylphosphatidylethanolamine (DOPE) and/or cholesterol (chol); and a mixture of dimethyldioctadecylammonium bromide (DDAB) and DOPE and/or chol. The ratio of the lipids can be varied to optimize the efficiency of uptake of the imaging agents. The liposome can comprise a mixture of ' one or more cationic lipids and one or more neutral or helper lipids. A desirable ratio of cationic lipid(s) to neutral or helper lipid(s) is about l :(0.5-3), preferably about l:(l-2) (molar ratio). Examples of ratios of various lipids useful in the practice of the present invention include, but are not limited to: LipA DOTAP/DOPE 1 :1 molar ratio LipB DDAB/DOPE 1 :1 molar ratio LipC DDAB/DOPE 1 :2 molar ratio LipD DOTAP/Chol 1 :1 molar ratio LipE DDAB/Chol 1 :1 molar ratio LipG DOTAP DOPE/Chol 2:1 :1 molar ratio LipH DDAB/DOPE/Chol 2:1 :1 molar ratio (DOTAP = dioleoyltrimethylaminnonium phosphate, DDAB = dimethyldioctadecylammonium bromide; DOPE = dioleoylphosphatidylethanolamine; chol = cholesterol). , 16

[0054] In one embodiment, the present invention provides methods of preparing imaging agent-comprising antibody- or antibody fragment-targeted cationic immunoliposome complexes 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 one or more imaging agents to form the antibody- or antibody fragment-targeted-cationic immunoliposome complex.

[0055] In suitable embodiments, the antibody fragment is a single chain Fv fragment, for example, an anti-transferrin receptor single chain Fv (TfRscFv) or an anti-HER-2 antibody or antibody fragment. Examples of suitable lipids for use in preparing the imaging agent-comprising cationic immunoliposomes are described herein, and include, mixtures of dioleoyltrimethylammonium phosphate with dioleoylphosphatidylethanolamine and/or cholesterol; and mixtures of dimethyldioctadecylammonium bromide with dioleoylphosphatidylethanolamine and/or cholesterol. Suitably the antibody or antibody fragment is mixed with the cationic liposome at a ratio in the range of about 1 :20 to about 1:40 (w:w) to form a cationic immunoliposome. Suitably, the cationic immunoliposome is mixed with the imaging agent in the range of about 1 :10 to about 1 :35 (mg imaging agent: μg liposome), more suitably about 1 :14 to about 1 :28 (mg imaging agent: μ g liposome), or about 1 :21 (mg imaging agent: μg liposome).

[0056] Exemplary imaging agents include those described herein and known in the art.

Suitably, the imaging agent is an MRI imaging agent, such as gadolinium, gadopentetate dimeglumine, iopamidol (e.g., ISOVUE®, Regional Health Limited, Aukland, AU), or iron oxide; barium, iodine and saline imaging agents for CT; and l8F-2-deoxy-2-fluoro- D-glucose (FDG) and other imaging agents for PET.

[0057] In additional embodiments, the methods and immunoliposome complexes of the present invention further comprise mixing the cationic immunoliposome with a peptide comprising the K[K(H)KKK]5-K(H)KKC (HoKC or HK) (SEQ ID NO: 1) peptide. The HoKC peptide carries a terminal cysteine to permit conjugation to a maleimide group. Thus, when the HoKC peptide is used, the liposome formulations also suitable include N- maleimide-phenylbutyrate-DOPE (MPB-DOPE) at 0.1 to 50 molar percent of total lipid, more preferably 1-10 molar percent of total lipid, most preferably 5 molar percent of total Iipid. TheHoKC liposomes are prepared as previoiisly described (Yu, W. etaL Enhanced transfection efficiency of a systemically delivered tmnor argetmg inmmnolipoplex by inclusion of a pB-sensttive histkryiated oligolysme peptide, Nucleic Acids Research 32, e48 (2004)).

In a further embodiment, the present invention provides, antibody-' or antibody fragment-targeted catiomc immnriolrposome complexes comprising a catiomc liposome, an antibody or antibody f™g «"t, and one or more imaging agents, wherein the antibody or antibody fragment is not dienncally conjugated to the cationic liposome. The antibody or antibody fragment is suitably associated with the liposome via an interaction (e-g^ electrostatic, van der WaDs, or other iira-chemically conjugated interaction) between the antibody or antibody fragment and the liposome, suitably between a cystein residue on the aiitibody or antibody fragment and the liposome surface, in general, a linTcra- or spacer tflnlfff-nto (e.g., a polymer or other molecule) is not used to attach the antibodies and the liposome. The imaging agent(s) can be encapsulated within the nationic liposome, contained with a hydrocarbon chain region of the cationic liposome, associated with an inner or outer monolayer of the cationic liposome, or any combination thereof Suitably, the cationic immunohposomes of the present invention are unilamellar liposomes (Le. a single bilayjer), though multilamellar liposomes which comprise several concentric bilayers can also be used. Single bflayer cationic OTmanoliposomes of the present invention comprise an interior aqneons volume in which agents (e.g^ imaging agents) can be -yrvpariatii They also comprise a single bflayer which has a hydrocarbon chain region (Le-, the hpid chain region of the lipids) in which agents (e.g., imaging agents) can be contained. In addition, agents (cg^ imaging agents) can be complexed or associat id with either, or both, me inner monolayer and/or the outer monolayer of the liposome membrane (Le-, the headgroup region of the lipids), eg-, via a charge-charge interaction between the negatively charged imaging agents and the positively charged catiomc liposomes. In further embodiments, agents (e.g-, imaging agents) can be CT^g mli>tp>t/ap^g<^ rfi^1eareil jn any or all of these regions of the cationic immonoliposome complexes of the present invention.

I In a still farther erabc

[0060] Suitably, the methods of the present invention are used to image a cancerous tissue in a patient suffering from, or predisposed to, cancer. Cancerous tissues that can be imaged using the methods of the present invention include solid tumors, as well as metastasic lesions. The methods of the present invention can also riistingnish cancerous ticOTipg from non-cancerous (benign) tissues.

[0061] In fhrther embodiments, the present invention provides methods of imaging and treating a tumor tissue in a patient suffering from, or predisposed to, cancer comprising mluiiiiiini Frin the ima i -a Hnt comprising fmimirwliposome complexes of the present wivHfififin to nriflg ft ft tiirnor tissue, an ^Mliinnig^'ing an anti-cancer fl wit to ma patient to treat the honor tissue.

[0062] Examples of ann-cancer agents that can be administered include, but are not hrnited to small molecules, proteins, peptides, and chemotherapeutic agents such as those described herein, genes, antisense oUgonucbtides and siRNA. Exemplary chemotherapeutic agents momde, but are not limited to, doxorubicin, 5-flnorouracil (SFU), casplatin (CDDPX docetazel (TAXOTERE*), gemcitabine (GEMZAR*), pacletaxel, vinblastine, etoposide (VP-16), camptothecin, actinomycin-D, initoxaiitrone and mrtcmycin C, and an antibody therapy, such as a monoclonal antibody, cg^ HERUfcfil * (Genentech, San Francisco CA). Examples of antisense ohgoniicloetides and siRNA molecules for use in the practice of the present invention include, bu are not limited to, those disclosed in U.S. Published Patent Application No.20030044407 and U_S. Patent Application No. 117520,796, filed September 14, 2006, the disclosures of each of which are incorporated heron by reference in their entireties. Additional anticancer agents include peptides, proteins and small molecules (see, e.g, U.S. Provisional Patent Application Nos. 60 800,163, filed May 15, 2006 and 60/844,352, filed September 14, 2006, me disclosures of each of which are incorporated herein by reference in their entireties). The anti-cancer agent (e.g-, the chemothtsapeutic agent, small molecule, gene or the antisense or siRNA, etc.) can be associated with the cationic immunohposome that also comprises the imagine agent, or it can be delivered separately, either in a different immrmoli tosorne in accordance with the present invention, or via another carrier or delivery system (for example, IV injection of a chemotherapeutic per normal clinical standards).

[0063] m suitable einbodiments, the methods of the present invention comprise art ministering an inrrnimoliposome complex comprising an imaging agent (e.g, MRI ima ing agent such as gadopentetate drmeghirn ne), and an anti-tumor agent at different times (La, the complex and the agent can be given at the same time or at different times). Suitably, the anti-cancer agent is administered either before or after the imaging agent- comprising immonoliposome complex, (e.g., at least 1 hour before or after, at least 6 hours before or after, at least 12 hours before or after, at least 24 hours before or after, at least 48 hours before or after, etc, adiiiinistialion of the canonic immunohposome complex), in still former embodiments, the methods of imaging and treating a tumo tissue in a patient suffering from cancer can further comprise administering radiation treatment to the patient.

[0064] Appropriate dosages of the anti-cancer agents (e.g., chemotherapy, genes, small •molecules, proteins, peptides, antisense oligonucleotides or siRNA, etc.) and tinting for adininistration in humane are easOy determined by those of skill in the art, based on information r*yfltameA herein and that is readily available in the art. Furthermore, such gmoimtg can be estimated by extrapolating from experiments performed on nimalw^ e-g^ moose, rat, dog or other studies. • [0065] Exemplary benefits of utilising the nanoirnmimolipoosme complexes of the present invention (scL and scL-HoKQ to encapsulate and delivery imaging agents include higher concentration in cancer tissues due to the tumor targeting nature' of the complexes. As the complex accumulates in cancer cells, there is diflerenuatioa of vascular flow and diffusion into interstitial space (as seen with the non-complexed free imaging agents as currently in use in the clinic) from cancer specific imaging There is also differential enhancement of cancer vs benign processes. Longer vascular and tissue half life pennits delayed hnagmg using the complexes of the present invention. The complexes and methods can be used to image tissues of interest at various depths. [0066) T ms, the methods and complexes of the present invention result not only in enhanced signal in the tumor, but also greater definition of the internal structure of the tumors. More significantly, smaller tumors can be detected leading to earlier detection and thus improved response/survival. These complexes can also be used to distinguish benign from malignant nodnles. This helps to accelerate me decision on 'when to begin treatment Currently, mis is delayed to determine if the nodule increases since it is not certain if is malignant or not However, since the complexes of mis invention preferentially and specifically transfect tumor cells, mis would also serve as a confirmation of malignancy, for example if the small nodnles seen on mg CT are small malignancies or not These last two points are of particular significance in hmg and pancreatic cancer.

[0067] Exemplary types of cancer imaging problems addressed by use of the imaging agent-comprising complexes of me present invention inclndfi, in pancreatic cancer, early detection and differentiation from chronic pancreatitis; early detection of metastatic disease to lungs classification of solitary pulmonary nodnles as benign or malignant; classification of small focal areas of increased MR enhancement in breast as benign or malignant

[0068] The complexes of the present invention can also be used to confirm mat, using this delivery system, merapentic genes will likely enter patient specific cancer cells. That is, the fact that the imaging agent-comprising complexes are able to enter cells provides an indication mat delivery of therapeutic genes or other agents associated with the complexes of the present invention wQl also enter these specific cancer cells.

[0069] It will be readily apparent to one of ordinary skill in the rele ant arts mat other suitable modifications and adaptations to the methods and applications described herein ma be made without departing from the scope of the mvention or any embodiment thereof Having now described ate present invention in detail, the same will be more clearly understood by reference to the following examples, which are included herewith for purposes of ilhistration only and are not intended to"be mmtingof me invention. · ' Example 1 Immuiwiiposome Complexes Comprising MagnevisP Materials and Methods Cell lines

[0070] Human lymphoblastic leukemia cell line K562 was obtained from the Lombardi Comprehensive Cancer Center Tissue Culture core facility- These suspension cells were maintained m RPMT1640 supplemented with 10% Heat hiactivated FBS plus 2mM L- Glntarnme, and SO pg/rnl each of penicillin, streptomyckm and neomycin. Human pancreatic cancer cell line CaPan-1 (obtained from ATCC Manassas, VA) was derived from a metastatic adenocarcinoma of the pancreas. It was maintained in IscoVs Modified Dulbecco's Medium containing 4mM L^hitam e and Sodium Bicarbonate, supplemental with 20% non-Heat Inactivated FBS, 2mM L-Orhxtarnme and 50 ug/mL each of penicillin, streptomycin and neomycin. Hnman prostate cancer cell line DU145 (ATCC, Manassas, VA) was originally derived from a lesion in me brain of a patient with widespread metastatic carcinoma of the prostate. It was maintained in Minimum Essential Medium with Earie's salts (EMEM) supplemented with 10% heat inactivated FBS plus L- glutarmne and antibodies as above.

Naiiocomplex Formation

[0071] Canonic liposome DOTAP.DOPE) was prepared by the ethanol ir'ection method as previously described (see US. Published Patent Application No.2003/0044407; Xu L, et aL, Molecular Cancer Therapeutics 7:337-346 (2002) me disclosures of each of which are incorporated herein by reference). When delivering plasmid DNA, the mil camplex was fbnned in a manner identical to mat previously described (see ILS. Published Patent Application No. 2003/0044407). To rmrapsnlatn the imaging agent for m vitro use, the TfRscFv was mixed with the liposome at a specific ratio and incubated at room temperantre for 10 minutes. Magnevist® was added to mis solution, mized and again incubated at room temperature for 10 nrnnrtes. When stored at 2-§°C the complex is stable fqr at least 8 days, as AMnrfph^ by size measurements using a Malvern Zetasizer 3000H. The cumulants (Z average) average of measurements over this time frame is 1123 ± 4.67 (SJJ.) while me por tnspersiry (representing die reproducibifity of the values during repeat scans) is 0.445 ± 0.03. A range of acceptable sizes for the nanoconmlexes is from about 20 to 1000 nm, suitably about SO to 700 nm and mare suitably about 100 to 500 am. For in vitro transfection, 2 ml of scrum-free media was added to the complex prior to transfection. For in vivo use the complex is formed at a ratio of 1 mg imaging agent to 033-1.17 ug TfRscFv to 10-35 g Liposome (suitably 1 mg imaging agent to 05 to 1.0 ug TfRScFv to 14-28 ug Liposome, most suitably I mg imaging agent to 0.71 ug TfRscFv to 21 ug Liposome) using the above procedure. When prepared for in vivo use, dextrose was added to a final coiicentratian of 5%.

In Vitro Transfection [00721 To transfect suspension cells 562, 15x10s cells in a total volume of 4.0 ml of ρηρΛϊιττη with all supplements except serum (serum free medium) were placed into a 100 mm2 tissue culture dish. Two ml of the transfection solution from above, coruaining varying amounts of Magnevist®. was added to the cell ^its^nginn. The plate was ipraifiatwrf at 37°C with gentle rocking for the length of time given in the Results section (up to 90 wan), after which the cells were gendy pelleted (600xg tor 7 minutes) at 4°C in 0.5 ml microcentrifuge tubes and washed three times with 10 ml of serum free medium to remove any excess transfection sohraon and placed on wet ice until imaged In FhwTnmor Targeting

[0073] To assess me tumor selective targeting of the TfRscFv-Iip nanocomplex to primary and metastatic tumors, an orthotopic metastases model using human pancreatic cancer cell line CaPan-1 was used. Subcutaneous xenograft tumors of CaPan-1 were induced in female amymic node mice by injection of lxlO7 CaPan-1 ceBs suspended in Matrigel™ collagen basement membrane matrix (BD Biosciences). Approximately eight weeks later the tumors were harvested and a single cell suspension of the tumor was prepared. 1-2-1-5 xlO7 cells, also suspended in Matdgel™ were injected into the surgically exposed pancreas of female athymic nude mice. Five weeks post-surgery, the complex carrying the LacZ gene was Lv. injected 3X over 241ns (at 40 ug - DNA injection).60 hrs later the animals were sacrificed and examined for the presence of metastases and organs stained for β-galactosidase expression using a previously described procedure (Xu, L, t aL. Human Gene Τηβηφγ 10^9413962 (I999) MRI Imaging For in vitro MRI imaging, the cell pellets in nriraocenlrifuge tubes were positioned at the center of the magnet The MR imaging was performed at Howard University using a 4.7T horizontal bore MMR m rine (Varian mc, Palo Alto, CA). The imaging protocols consist of a mu -sEce Tl-weighted spin echo ima in sequence and a saturation-recovery sequence. For the Tl -weighted imaging technique, the repetition time (TR) was 1000ms, and the echo time (TE) was 13 ms. The Tl-weighted spin-echo imaging technique was applied to verify the positive image enhancement. The saluralion-lecovery MR sequence with variable echo times was used for the Tl measurement The slice fra rness of images was 03mm. The RF coil employed was a 30 mm single loop cofl. The RF coil serves as an RF transmitter and receiver. The RF pulse was a selective 5 ms sine pulse. The number of phase encoding steps was 256. The field-of-view was lS mmz 15 mm. The image area chosen in the study was located at the center of the RF coil for RF homogeneity. The MR images were taken in the cross-section direction of the miaocemrifuge tube. The height of the cdD pellet was 12 mm. The range of the multi-slice images covers the whole pellet The center slice images, which were not influenced by the image distortion due to the susceptibility effect from the air-pellet boundary, were utilized for the studies. The image intensity was measured using the Varian linage Browser software. The signal is taken from a region-of-mterest, winch is big enough to cover two thirds of the image from each nucrocentrifuge tube. The relative image intensities of the pellets from these tubes were applied for contrast enhancement evaluation and the Tl measurements.

For the in vivo studies, mice bearing CaPan-1 orthotopic tumors or DU14S subcutaneous xenograft tumors were employed. The CaPan-1 tnmors were Tndncnd as described above. DU14S tnmors were induced by the subcutaneous inoculation of 7x1ο5 cells in MatrigeL These studies were performed at Georgetown IMversity. Animals to be imaged were anesthetized and placed in a proprietary, in-house designed, animal management system. This system mcorporates a. warm water heating system mat maintains the temperature at 37°C, as well as a four channel thermal- optical momtoring system used to monitor animal's skin temperature, ambient temperature and wall temperatuie of the device. For imaging, anesthesia' was induced using isoilurane at 4%, with the remaining gas comprised of a 66% oxygen and 30% nitrons oxide mixture.

Maintenance of «ne«tfr«m» was achieved wife 1.5% isoflorane under similar gaseous conditions of oxygen a nirro us oxide as noted. The anesthetized animal was positioned inside of a cyHndrical variable ladiofieqaency resonant antenna (bird cage resonator volume coil) and toned to a center frequency of apporoximatery 300 MHz (the resonant frequency of water molecules when sobject to a field strength of 7 Tesla). The imaging protocol used was T weighted Turbo RAKE (rapid acqmsmon with rapid enhancement) mree-dinifinsional imaging sapiences performed on a 71 Broker BioSpin (Germany/USA) imaging console. The imaging paianirters used were: Tl -weighted Turbo-RARE 3D (^-dimensional), TE 133: ms, TR 7295, Fhpback on, 4 echoes with a field of view of 8.03.5/3.5. cm and a256 x 256 x 256 matri-L Afier base£ne nage was acquired, the anjma] was kept ,""ΐ«τΜ*'ΐίΪ7"^ in the holder and fee Magnevisl® only (dinned to 400 ul with lx Phosphate Bo Eerred Saline pH=7.4) or the Magpevist®-cojnpristng immmioliposome complex (TfRscFv-Iip-Mag) (total volume 400 μΐ) was svstemically administered using a 27G needle by intravenous injection into fee tail vein of fee animal and fee 3-D imaging sequence was Tmmfiriiatery undated. The imaging wife fee two solutions were performed on sequential days.

Sample solutions of liposoxne-encapsolated Magnevist® contrast agent, and complete nanocomplez consisting of a tomor-targeting single-cham transferrin receptor protein coating fee hposome-encapsulated complex, TfRscFv-Iip-Mag, were prepared at GUMC, delivered to NET and were stored under dark and refrigeration. For each imaging session, a fresh dilution 13 by volume wife f rinm7eA water was prepared and a 5 uL droplet was irnaopipetted onto a standard 200-mesh TEM grid consisting of 30-60 nm formvar and 15-20 nm carbon. The droplet was allowed to dry on fee grid in air for 5 mmntes before loading into fee vacuum chamber of fee microscope, imagi g was performed using an Hitachi S-4800 fie -emission microscope at NET. Of particular interest to apphcatians of SEM to NDA imaging is a comparison of upper and lower' secondary electron detectors [SE(U) and SE(L)} - using fee SEM in its usual mode - to fee addition of a transmitted electron. (TE) detector, transforming fee instrumerit into a low voltage STEM. ' Scanning Probe Microscopy (SPM) Samples solutions of trposome-encapsnlated Magnevist* conttast agent, and complete nanocomplez were piepaied at GUMC, delivered to NET and were stored under dark and ieMgeiation. For each imaging session, a fresh dOntion 1:3 by volume with deionized water was prepared and a 5 ul droplet was rmcropipetted onto an imtrasonically cleaned silicon substrate used with native oxide or with a poty-L lysine coating. SPM imaging were obtained using a Veeco MnttiMode microscope with a Nanoscope IV controller. Topography by tapping mode with Z control (Veeco RTESP cantilevers for—320-360 kHz and k ~ 20-60 m), phase "n»fi "gt arM* magnetic force microscopy using magnetic coated tips (Veeco MESP 68 kHz) were performed in life mode. Dyrtamic imaging of dewetting and surface energy "phase separation" as the solution evaporates to expose isolated nanoparticles and aggregates were used to understand the consequences of solvent drying on the stability of the particles and its effect on the various SPM contrast mechanisms available with the SPM system.

Results Tumor Specific Targeting by the Iigand-Iiposorne Nar-ocomplex Carrying a Reporter Gene To assess selective targeting of the TfRscFv-IjpA nanocornplex to primary tumor and metastases an orthotopic metastasis model, a closer approximation of the. Jimrsil situation, using human PanCa cell line CaPan-1 was employed. Surgical orthotopic implantations of CaPan-1 xenograft tnmor sections into nude mice have been shown to produce within 56 days, meas ases in liver and spleen (AlisanaknsJL, et aL, Cancer Research 5J:5743s-5748s (1995)). Orthotopic tumors of CaPan-1 were tn^n !ftd in female athymic nude mice as described in Methods. Approximately 5 weeks later, the animals were enrnanized and necropsied to took for tumor in the pancreas and other organs. As shown in Figure 1A, extensive tnmor growth is evident mroughout the pancreas. The same tumor nodule in the liver indicated by an arrow in 1A exhibits intense β-galfictosidase expression in IB: 1A = gross necropsy 1A - tissues after staining for β-galactosidase. Metastases were present in various organs in four of five mice mclndihg the spleen, fiver, rang, adrenal gland and even within the o¾aphragrrL This Kxpmimflnt was repeated with sirnilar results.

[0079] To establish selective targeting tumor and metastasis, prior to sacrificing the mice, the TfRscFv-IipA complex carrying pSVb (LacZ) plasmid DNA for β-galactosidase expression was Lv. injected into me mice three times over a 24 boor period (40ug of plasmid DNA per irrjecrion). All five mice were sacrificed 60 hoars post-injection and various organs rnchiding the liver, hmg, spleen, pancreas and diaphragm were harvested and examined tor the presence of metastasis and tumor specific staining. Fresh samples, sliced at 1 mm trrLckness, were stained with X-gal to produce a bhie color where the gene is expressed. The tumor targeting ability and high transaction efficiency of the complex is demonstrated by fhe presence of the reporter gene in die various organs from this animal (Figure IB). In the liver, lung, adrenal gland and diaphragm it is clearly shown mat fhe reporter gene is highly expressed only in the metastases, while no bhie color is evident in fhe adjacent ncxmal tissue. The metastasis visible in fhe over in Figure 1A (arrow) is fbe same tumor nodule strongly expressing f^galactosidase in Figure IB (arrow) cccfinnmg fhe tumor specific nature of this nanocomplex. m some of fhe mice, growth of fhe tumor in pancreas also resulted in extrusion of tumor through fhe original incision site used for implantation, m Figure IB this strongly blue stained subcutaneous tumor, surrounded by normal non-stained skin is also shown, again showing tumor cell specificity. Similar results were observed in fhe rest of the mice, and in fhe repeat experiment Thus, this systemically administrated nanc«omplex will target tumor cells both primary and metastatic, wherever they occur in the body, and efficiently deliver plasmid DNA. We wished to expand fhe potential of this delivery system to include contrast agents. The ability to do so could result in improved imaging and cancer detection.

In vitro Studies Using TfRscFv-Iip Complex to Deliver Magnevist*

[0080] As Magnevist* is one of the most frequently employed contrast agent in the chnic, it was chosen as fin: use in these studies, m these initial experiments, it was examined whether fhe complex could be prepared with Magnevist? and if doing so would enhance fhe MRI signaL Since trypsirrization could lead to membrane damage and leakage of contrast agent from fhe cells, adherent cells were not employed in these studies. Instead, a human lymphoblastic Ierakeniia cell line, 562, which grows as a suspension xnilture was used. Moreover, gentle pelleting and washing of fhe cells would remove any excess Magnevist® or complex prior to imaging, allowing only ceS associated signal to be detected. 1. Time Dependent Image RnhnnrrRmprnt by the TfRscFv-Lip-Mag Nanocomplex

[0081] The optimal time for transfection of the TfRscFv-IJ^Magnevist® nanocomplex was examined. The suggested clinical dose of Magnevist* is 0.1 mMole/kg. In these initial studies a dose of 03 mMole kg was used (corrected for the smaller weight and blood volume of moose versos man) in the complex per 250 ul of txansfection solution. 562 cells were traxisfected for times ranging from 20 to 90™½η*« Twenty minutes showed very low transfection activity based upon the image intensity. However, as shown in Figure 2A, by sixty mimitwa the cells transfected with the complex showed a large increase in intensity as compared to the untreated cells. The intensity of the untreated cells (202 ± 48) was not significantly different man mat of an empty marker tube (194 ± 43) w ¾r«*mg that the cells themselves do not contribute to the signal detected. More iuipuitautly. the trawefcrtrinn efficiency plateaus at approximately 60 minutes since the relative intensity of the cells transfected for 60 and 90 rrorrntes were identical (317 ± 46 and 317 ± 47, respectively). 2. Magnevist* Dose Dependent Image Enhancement

[0082] Using 60 mmnteg as the transfection time, the effect of increasing mwimh! of Magnevist* on the TfRscFv-Lip-Mag complex image enhancement was then assessed.- The doses tested were 0.05, 03 and 0.9 mMole kg. Corrected for size and blood volume of the mouse, the volumes of Magnevist® used in the complex per 250ul of transfection solution were 0-25 ul, 1.5 ul and 4.5 uL As shown in Figure 2B and Table 1, the image intensity increases and the Tl relaxation time shortens as a function of the amount of contrast agent inr-ln ^d in the complex.

Table 1: Relative mtartsfy TiiniiiiimTipnBfWTMt f!rnnplflar Dose of Contrast Agent Relative Intensity - ΤΊ {seconds) (mM/kg) 0.05 (0.25 ul) 293 ±50 1.43 ±0.007 03 (1-5 3) 379 ±43 1.16 ± 0.004 0.9 (4.5 ul) 454 ± 51 1.01 ±0.004 3. Image Enhancement by Tf scFv-Iip-Mag as Compared to Free Magnevist*

[0083] Based upon tfae above exp^rhnents, it was shown mat the TfRscFv-Liposome can complex with Magnevist* and deliver it to tfae cefls for image enhancement To assess tfae level of enl-ancement of me complezed contrast agent as compared to me agent alone and demonstrate mat the signal obtained is not due to the presence of unincorporated Magnevist* K562 cells were treated with either tree Magnevist* or die TfRscFv-Lip- Mag nanocomplex. The identical amount of contrast agent (0-3 μΜkg or 1.5 ul/250 μΐ transfection vohnne) and transfection time (60 minutes) was used for bom solutions. While free Magnevist* showed contrast relative to tfae untreated cells as expected, tfae cells treated with tfae TfRscv-Up-Mag complex demonstrated a much greater increase in image intensity and shortened Ti relaxation time compared to both ttnt »fr¾i and free Magnevist® treated cells (Figure 2C, Table 2). These results not only demonstrate the increased efficiency of contrast agent uptake by means of the targeted nanocomplex, but also indicate mat tfae observed signal is Kkery not due to uncomplexed Magnevist*.

Table 2: Comparison of me Relative Intensity and TI Relaxation Time Between Free and Tmrrmnnl Treatment Relative Intensity TI (seconds) Untreated 455 ±47 1.80 ±0.009 Free Magnevist* 538 ± 50 131 ±01007 nmmmhposome Complexed 662 ± 52 1.40 ±0.004 Magnevist* In Vivo Image Enhancement With TfRscFv-Iip-Mag

[0084] The above studies established mat tfae nanocomplex could more efficiently image tumor cells in vitro man Magnevist* alone. However, to have potential for clinical use, the complex must exhibit a similar effect in vivo. The same human pancreatic cancer oit otopic mouse model (CaPan-1) was used for these studies as was used above to demonstrate tamor specific targeting of the complex carrying a reporter gene. In addition, a second tumor model, a subcutaneous prostate xenograft mouse model (Dm 45) was also used. Mice bearing CaPan-1 or DU145 tumors were imaged on a 7T Bruker NMR as described in Methods. Once positioned in the coil, a baseline image was obtained using a Ή-wdghted Turbo RAKE (rapid acquisition with rapid enhancement) three-dimensional imaging sequence. To facilitate image alignment, after baseline acquisition the animal was TTurmtahv^ m the animal holder while the imaging solution was administered via intravenous injection. Signal acquisition was begun within three minutes of the injection. The amount of Magnevist* administered to the mouse, either free (as is performed in the clinic) or iucluded in the complex was 10 uL This amount is equivalent to 0.2 mM Kg or twice what is used in humans. This amoant was selected since the standard human dose of 0.1 mM Kg Magnevist® alone gave a very poor signal in the mice. The imaging with free Magnevist* and the TfRscFv-Lip-Mag complex were performed on two consecutive days. A baseline scan was also performed prior to admmistration of nanocomplex to confirm that all of the Magnevist® from the previous day had been washed out MR technique and windows were consistent between the two sets of images with the windows urijinrtwH in mmri for an mitmrmrir. winAiwmg fenhlTB nf nSe arsnrner Images of the Magnevist® and naiiocomplex-Mag in three separate mice are show in Figure 3A-L La Figure 3 A, 3D and 3G, four months after surgical implantation of the CaPan-1 tumor cells, the animal is carrying a large orthotopic tumor. The increased resolution and signal intensity, as compared to the contrast agent alone is quite evident. Similar results are observed in the second mouse with a CaPan-1 tumor shown in Figure 3 B, 3E and 3H. This animal, only two months post-surgery, has a visible subcutaneous tumor growing through the s e of the incision. A small abdominal mass was also detected by palpation. Not only is the signal in lbs snbcataneous tmnar more mhimrrA after' admimstration of the complexed Magnevist*, but what appears to be the small orthotopic tumor (arrow) is evident in this scan and not in the one in which the animal received the free Magnevist*. Similarly, increased definition and contrast -are evident in the subcutaneous DU145 tumor (Figure 3 C, 3F and 31) after injection with the TfRscFv-Lip-Mag complex as compared to the free Magnevist®. Reconstniction and quantitation was performed on the images in Figure 3 B, 3E and 3H and 3 C, 3F and 31, representing the two different tumor models, Pancreatic cancer (CaPan-1) and Prostate cancer (DU145). in bom instances, mere is an increased intensity (pixels) by me free Magnevist® over the baseline, as expected. However, delivery of the imaging agent by the tnmor targeting uanocomplez results in an almost three-fold former increase in signal intensity in both of these tumor models. These studies thus demonstrate mat when Magnevist* is incorporated within the TfRscFv-I .iposome complex mere is an improved tumor visualization in an in vivo situation, and they suggest the potential benefit of farther developing mis means of tumor detection for dinical use.

Physical Characterization Studies While the in vitro studies offered evidence that complexed Magnevist* is encapsulated within the liposome, sophisticated microscopy *ρ*·¾ <ρι»» (SEM and SPM) have confirmed this and former characterize (e.g. complex size) the TfRscFv-Lip-Mag complez. 1. Tmngjng of liposomes without Magnevist® H-gh-resolntion imaging implies narrow depth of focus and so requires relatively mm and flat samples. How thin varies with technique, but surface and substrate effects— surface energy and symmetry lowering— often dominate the structural forces typical of biomaterials. This is particularly true in the case of liposomes given their tenuous nature. (Poo, JJ-, et al, Amah of Biomedical Engineering 5i:1279-1286 (2003)). So an ^irM¾^rgfam/fmg of reliable methods for prcpanng and characterizing e dnnensional smA Tnnr-hnniggl stability of isolated liposomes is an essential step. The goal of this characterization is to perform direct sensing of the mechanical stiffness and magnetic properties of nanoparticles to establish mat the contrast agent is indeed contained within the nanoparticle and not simply associated externally with the liposomes.

The SPM images surface topography in tapping mode by oscillating the tip and cantilever to which it is attached close to the cantilever resonance frequency. A feedback circuit TnarntarnB the oscillation of the cantilever at a constant amplitude. This constant amplitude is given a by a set point which is somewhat smaller than mat of the freely oscillating cantilever. Since the SPM up interacts with the suriace through various small forces, there is a phase shift between the cantilever excitation and its response at a given point on the surface. For an inhomogeneons surface, the tip-surface interactions will vary ac ottting to surface charge, steep topographical changes, and inechanical stiffness variations, for example. By changing the set point and observing how certain features respond to sorter or ardtJ tapping, we can correlate Ms with the response expected for a specific structure such as a liposome. (The free oscillation amplitade signal is approximately 1.78 V.) A sequence of SP phase images of a pair of isolated liposomes without payload is shown in Figure 4A-C Figure 4A was imaged at a set point of 1.68 V and the corresponding negative phase difference between the substrate and liposome mttirarfwi that the tip-sample interaction is attractive for the liposome, given by a phase value of -3.5 degrees. In me case of an attractive interaction and negative phase, the phase image of the liposome appears dark, except for a topographically keyed ring at the liposome edge. Figure 4B demonstrates me effect of reducing the set point to 1.45 V: The liposome now appears bright since the tip-sample interaction becomes repulsive, and in mis case me phase difference between the liposome and substrate is 4-8 degrees. Finally, Figure 4C shows that the phase difference recorded at a set point of 135 V increases former, becoming +35 degrees. 2. Imaging of Hposome-eaicapsiilated Magnevist®

[0089] Figure 5A-C presents SPM and SEM images of isolated liposome-encapsulated Magnevist (Iip+Mag) nanoparticles. The size distribution of single Iip+Mag particles is in the range of 100-200 nm diaroffter and scales according to optical measurements that indicate mat paykiad-encapsulatmg liposomes are approximately 50% larger man liposomes alone in their spherical state. [00901 The SPM topograph appearing in Figure 5A indicates that liposomes containing Magnevist® have a bimodal surface shape after drying that is more complex man that of the simple elliptical surface of a liposome curtaining no payload (not shown). The SPM phase behavior differs markedly from mat of payloadless liposomes, the outer ring is repulsive relative to the center, and a corresponding 'SPM phase image is shown in Figure 5B. Regions of bom attractive and repulsive tip-sample interaction appear at moderate set point values. A correlation between die SPM phase image obtained at a set point of 1.6 and the SEM image in ΊΈ mode is evident in Figures 5B and 5C. liposomes appear mufonnly bright across fi½ entire particle in SEM images (not shown), similar to me wnifonp phase images we obtain by SPM. Tips and cantilevers change with time and usage. Moreover, it is important to verify that the images produced are not affected by tip instabih'ties doe to foreign matedal on the tip. Thus, they ate changed fieqaently. Since each cantilever is somewhat different with respect to its resonance properties, the set points used in Figures 4 and S are different 3. Imaging of TfR-^v-Lip-Mag Nanocomplex

[0091] The complete TfRscFv-Lip-Mag nanocomplex was prepared and imaged by SEM and SPM as described in Methods. Results, shown in Figure 6A and 6 indicate that the solvent firm undergoes phase separation; however, examples of isolated NDS can be readily observed on the dried film. Note that the SEM beam clearly causes some damage to the film, but the particles can be repeatedly erameA several times before beam damage becomes significant The appearance of the full complex is different from mat of the (Lrp+Mag) only. The shape is less regular, and considerable texturing of the liposome surface following drying is consistent with protein denatnration. Also, SEM TE images ηγΚ αίι», that the weQ-defined boundary between the outer ring and center of the liposome seen with the (Lip+Mag) particles is less apparent and the shape much more variable. This is consistent with the view that the presence of protein within the liposome has altered the osmotic outflow across the liposome during fim drying.

[0092] It is possible to obtain additional information about these NDS particles by using the magnetic force microscopy imaging capabilities of the SPM (MFM). Since the magnetic moment of gadolimirm-∞ntairiing Magnevist* is quite large, it should be possible using a magnetfyaert SPM tip to interact with the oriented. Magnevist* concentrated within the liposomes. This is shown in Figure 7A and 7B for MFM of several approximately 100-200 nm diameter nanocomplexes. By using the lift-mode opabilities of the SPM it is established mat the produced image is truly magnetic in nature, m this mode, a topographic image under normal tapping mode conditions is obtained. The reference surface mfbnnarion is men used to offset the tip by a specified height away from the surface and the surface is then scanned at this increased height This removes Que influence of topography on the signal. MEM images obtained in fift-mode at a height of 15 nm CT more from the surface are given by the magnetic phase image. The appearance of a signal confirms the presence of gadolinium encapsulated within the ■ complex. Figure 7 A is an SPM topograrrino'magnetic phase image of the fall TfRscFv- Lip-Mag nanocomplex. The appearance of a double dipole-rike signal in Figure 7B . consisting of attractive and repulsive in-plane magnetic interactions suggests mat the cause of this iateraction is the nommi&nii toroidal distribution of Magnevist within me DS, consistent with SEM and nonmagnetic SPM phase images.

Discussion

[0093] The results described herein demonstrate that we can encapsulate and deliver the commonly used MR imaging agent Magnevist*, to tumor cells bom in vitro and in an orthotopic animal model and in doing so produce a more defined and intense image man seen with uncomplexed Magnevist*.

[0094] As shown in Figure 1, the nanocomplexes of the present invention can target metastatic Ajyasn, thereby enhancing detection sensitivity for metastases. Using SEM and SPM it has been demonstrated that the TfRscFv-liposome complex numrtamg its . nanometer size when Magnevist* is encapsulated (particles of approximately 100-200 nm are shown in Figure 6 and 7). fi has also been demonstrated that the structural and mechanical properties of liposomes containing a payioad are sufficiently different f om those without one, thereby rem firming that Magnevist* is indeed encapsulated with the liposome. This was further cxmfemed by MFM imaging of me complex.

[0095] While not wishing to be bound by the following theory, a tentative explanation for the internal structure of (Lip+Mag) is that the slight bulge in the SPM topographic image, represents a Uposome-contlned phase separation, Le^ formation of a dense Magnevist*- lipid toroidal distribution around the periphery of the particle with an preferential aqueous phase at the particle's center. This response is probably attributable to several impottant factors: First, the properties of Magnevist* solution are pH ~ 6.5-8, an osmolality of 1,960 and viscosity of 43 at 20°C according to the mami&cfnrcr. A plausible ciiemical basis for mis separation of the solution noted in the Magnevist* data sheet The THf¾frm»nft salts dissociate completely from the complex, so changes in the local osmotic conditions. Coupled with the charge' interaction of the gadonrrhrrn complex and cationic lipid, these interactions may provide a strong driving force for a hypertonic phase separation within the hposome. The charge distribution. between the cationic hpid and Magnevist* solution is effective at stabilizing the hposome and providing structural support in solution, and apparently in me bloodstream. This enhanced ttuiicuirul support Is an important benefit for these studies since it allows most particles to remain intact dnrfng the film drying process, in contrast to the extensive decomposition observed with the liposome only solutions. 01096] The foregoing Examples demonstrate the successful encapsulation of an MR contrast agent in the inmiuno liposome complexes of the present invention. The image enhancement demonstrated by the complexes over conventionally delivered Magnevist* indicates the ability of this system to improve earry detection of cancer via MRL Example 2 Comparison of imaging in Different Cell Lines

[0097] Figures 8A-8H show improved MR imaging in two different models of cancer usmg the ligand-HK-Iiposome-Mag nanocomplex. Nanocomplexes for use in this Example 'were prepared using the same ratios and procedures as set form in Example 1. Human breast cancer MDA-MB-435 (Figure 8E-8H) or hrmnan prostate cancer cell line (DU145) (Figure 8A-8D) cells were subcutaneously injected on the lower back, of female afhymic nude mice. Free Magnevist® or the TiRscFv-hposome nanocomplex (sclip- Mag), or the TfRscFv-HK-liposome nanocomplex (scIip-HK-Mag) cornprisrng the HoKc peptide, containing the same dose of Magnevist® were Lv. injected (via the tail vein) into each of the three mice on three consecutive days. This amount of Magnevist* is equivalent to twice the dose at would be administered to a human patient The total volume of solution administered in all cases was 400 jiL A baseline scan was performed just prior to adnnmstration of bom nanocomplexes to> confirm mat all of the Magnevist* from the previous day had been washed out MR technique and windows were constant between the four sets of images with the windows adjusted to correct for an automatic windowing feature of the scanner. The panel shows the difference in MRI signal in a πιρτφβ with a subcutaneous tumor in which the increased defimuon. and contrast are evident in bom the prostate tumor (DU145) (Figure 8A-8D) and the breast tumor (435) (Figure 8E-8H) after injection with the sclip-Mag and even more so after injection with the scIip-HK-Mag. {0098] Figure 9A-9C shows tumor-specific targeting of a CaPan-1 subcutaneous tumor . and orthotopic metastasis model, by die TiSscFv-HK-Iip some-Mag nanocomplex. Subcutaneous CaPan-1 xenograft tumors were induced in female auiymic nude mice as described in Methods in Example 1. The tumors were' harvested and a single cell suspension in MATRIGEL® was injected into the surgically exposed pancreas. Eight weeks post injection the TfRscFv-Iiposame complex with or without HoKC (BE) peptide carrying Magnevist* was injected into the moose an two consecutive days. The total volume of solution admimstered in all cases was 400 uL A baseline scan was performed just prior to administration of the nanocomplex to confirm at all of the Magnevist* from the previous day had been washed out MR technique and windows were constant between the three sets of images with the windows adjusted to correct for an automatic windowing feature of the scanner. Similar to Figure 8A-8H, improved "tinging resolution of subcutaneous tumor (white arrow) and the metastatic lesions is observed, as shown in Table 3.

Table 3: Intensity Increase over Baseline by Free and Complexed Magnevist* Sample CaPan-1 DU1 5 % Increase Over Baseline Complexed Magnevist"' 99 215 Free Magnevist9 34_5 70 Example 3 Comparison of Dynamic MRI Scans of Subcutaneous PANC-1 Tumon after Systemic Injection of Free (Uncomplexed) or TfKScFv-I p-Magnevist

[0099] The following experiments were performed to compare me rate and level of uptake and washout between free (uncomplexed) and TfRscFv-Lip-Mag in tumors after systemic delivery. Subcutaneous xenograft tumors of PANC-1 were induced in female amynric nude mice by injection of 1 to 2xl07 PANC-1 cells suspended in Matrigel™ collagen basement membrane matrix (BD Biosciences). Approximately 15-3 weeks later, the animals were used for imaging. Canonic liposome (TDOTAPiDOPE) was prepared by the efhanol injection method as previously described (see US. Published Patent Application No. 2003 0044407; Xu L, et aL, Molecular Cancer Therapeutics 1 -337-346 (2002) the disclosures of each of which are incorporated herein by reference). The targeting moiety used in these studies is the anti-tamsfenm receptor single chain . antibody nrigment (TfSscFv).

[0100] To encapsulate the imaging agent, the TfRscFv was mixed with the nposome at a specific ratio and incubated at room teuipeiature for 1- 30 minntes, suitably 5-20 minutes, most suitably 10-12 imr tim. Magnevist* was added to mis solution, mixed and again iacubated at room tenmer-tture for 1- 30™'™*«, suitably 5-20 τπτηιτ*ρ¾ most suitably 10-12 TTimntpg When prepared for in vivo use, sucrose or dextrose was added to a final concentration of 03-50%, suitably 1-20%, most suitably 10% for sucrose and 5% for dextrose, and incubated at room ternperature for 1- 30 TT,÷T"lfp The imaging parametexs used were: Tl-weighted 2D (2-dimensioiiaI), TE 10.21 ms, TR 400 ms, Flipback off, 8 averages with a field of view of 3.84 z 3.84 cm and a 256 z 256 matrix. Afier a baseline image was acquired, me animal was kept immobilized in me animal holder and either the free (imcomplexed) Magnevist*(gad-d) or the TfRscFv-Iip- ag complex∞ntaimng the identical amount of Mag (total volume 50-1000 uL moie suitably 100-500uL most suitably 200-400ul) was systenricaDy administered using a 27G needle by intravenous injection into me tail vein of me animal and the imaging sequence was immediately undated. The pixel intensity of the images was measured and plotted.

Th* ffami'* I*""" imaging with hrrth fhr. ftae ami ftie ratmplmr The imaging was performed on sequential days.

[0113] As Shown in Figure 12A-12E, there is a significantly higher level signal in the tumor afier intravenous injection of the complex as compared to the free imaging agent. Thus, the complex of lids invention also enhances detection of relatively large metastases in the lung as compared to the currently used method of administenng free ima ing agent.

Example 6 Enhanced Detection of Small Lung Metastasis by TRscFv-U -Magnevist

[0114] The following experiments were performed to demonstrate mat when administered intravenously (or via any other appropriate route, e-g^ but not limited to IT, ID, ΊΜ, IP) the complexes of the present invention carrying an imaging agent can detect very small metastases that can not be detected when the imaging agent is administered without the use of the complex. Lung tumors were induced in female Balh C mice by the intravenous injection of 1 to 10x10* RenCa cells. This method results in metastases mat reside almost exclusively in the hmgs of the ammals and thus serves as a model system for any type of cancer that results in hmg tumors either as primary disease or as metastases. Appiuxiuiatery 7-9 days later the ammals were used far imaging. [0115} Catiorric liposome (DOTAPiDOPE) was prepared by the ethanol injection method as previously described (see U.S. Published Patent Application No.2003/0044407; Xu L, et dL, Molecular Cancer Therapeutics 337-346 (2002) the disclosures of each of which aie incorporated herein by reference). The targeting moiety used in these studies is the arm^ransfesrin receptor single cham antibody fragment (TfRscFv). [01161 To encapsnlate the imaging agent the TfRscFv was mixed with the liposome at a specific ratio and incubated at room Leuiptaaiure for 1- 30 nrimites, suitably 5-20 Ti i"*'*6) most suitabty 10-12 mrmites Magnevist9 was added to (his solution, mixed and again incubated at room temperature far 1- 30 mnrntes, suitably 5-20 mwmteg, most snitably 10- 12 minutes. When prepared for in vivo use, sucrose or dextrose was added to a final concentration of 0.5-50%, suitably 1-20%, most suitably 10% for sucrose and 5% for dextrose, and incubated at room temperature for 1- 30 mtmrtws, suitably 5-25 mimitee most suitably 15-20 The complex is formed at a ratio of lmg imaging agent to 033-1.17 ug TfRscFv to 10-35 ug Liposome (suitably lmg imaging agent to 0.5 to l.Oug TfRScFv to 14-28 ug Liposome, most suitably lmg imaging agent to 0.71 ug TfRscFv to 21ug liposome) using the above procedure. A range of acceptable sizes of the complex is from about 20 to 1000 mn, suitably about 50 to 700nm and most suitably about 100 to 500 ran. Here the complex was formed using 4.7 mg Magnevist, 99ug Liposome and 3-3ug TfRscFv with dextrose to a final concentration of 5%.

[8117] A mouse bearing rang tumors induced above was anesthetized and placed in an animal holder system. Anesthesia was induced using isoflurane at 4%, with the remaining gas comprising a 66% oxygen and 30% nitrous oxide rmxtare. Maintenance of anesthesia was achieved with 1.0 to 2.0% isofturane (preferably 1.5%) under similar gaseous conditions of oxygen and nitrous oxide as noted. The anesthetized animal was positioned inside of a cylindrical' variable rahofreqoency resonant antenna (bird cage resonator volume coil) and tuned to a center frequency of approximately 300 MHz (the resonant frequency of water molecules when subject to a field strength of 7 Tesla). The imaging protocol used was Tl-weighted two dimensional Turbo Mtiuiidice-MuMecho imaging sequence performed on a 7T Broker BioSpin (Germany/XJSA) imaging console. The imaging parameters used were: Tl-weighted 2D (2-dniiensionaI) imaging sequence, TE 1021 ms, TR 572.99ms, FBpback off, 8 averages with a field of view of 156 x 2÷56 cm and a 256 x 256 matrix. After a baseline image was acquired, the animal was kept . T xjjpn nM-TpA ]Q animal holder and either the free (uncornplexed) Magnevist* (gad-d) or the TfRscFv-Lip-Mag cornplex contamng the identical amount of Mag (total volume 50-1000 uL suitably 100-500uL .most snitably 200-400uI) was svstemically administered using a Z7G needle by intravenous injection into me tail vein of the animal and the imaging sequence was immediately initiated The pixel intensity of the images was measured. The same moose was used for imaging with bom the free and the complex. The imaging was performed on sequential days. At this field of view 5 pixels is equivalent to approximately a 3 inm human tumor detected by CT. [01181 As Shown in. Figure 13A-13D, a metastasis of 4 pixels (lower arrow) (which corresponds to a™*»<*"<"« of approximately 0.4 mm in diameter) was detectable afte injection with the complex bat not after me free gad-d. Moreover the signal was significantly enhanced in a second slightly larger metastasis (upper arrow) as compared to the free gad-d. Thus, the complex of mis invention also enhances detection of small metastases in the lung as compared to the currently used method of admmistering free imaging agent. An even smaller metastasis of approximately 3 pixels (equivalent to a tumor of approximately 03 mm in diameter) was also detected using the complex of mis invention, but was not detectable by the free gad-d (Figure 14A-14D). (0119] Employing the identical tumor model system as above, tumors of even smaller size can be detected after intravenous injection of the complex of the invention- Here the imaging parameters used were also Tl -weighted 2D (2-dirjM_asior-ai) Mutitisrice- Multiecho imaging sequence, TE 10-21 ms, with T = 630.8ms, Flipback oft, 8 averages with a field of view of 156 x 2-56 cm and a 256 x 256 matrix. As shown in Figure 15A- 15B, nodules of 1-2 pixels were detectable by me complex. Nodules Nl and N2 were visualized on the MRI scan. As they are so small (1-2 pixels) to determine if they were actually giving signal above background, intensity was measured using Image J software and the minimum, man ΐιιιιιιτι, mean values and standard deviation (SD) was-determined the two nodules. Statistically, if the max of the nodule was greater than the max of the base +2SD of the base, mere is a 95% confidence mat the nodule is not noise but is real Nodule 2 is clearly within mis 95% confidence and Nodule 1 is just at the limit, thus it too is most hkery a real tumor mass enhanced by the complex. After imaging the lungs from mis animal were removed, fixed in Formalin, paraffin embedded, sectioned and -4amed using H&E using standard procedures well know to one of cmiinary skill in the art. The sections were examined by microscope and the observed metastasis photographed. As shown if Figure 16 (low power, 2X) and Figure 17 (high power, 10X), two metastases with a size of approximately 0.1mm .were found in the same lobe and approxiiuate locatioii as expected based upon the MRL The distance between the two nodules was measured on the MRI image and was found to be eqmvalent (~6O0nm) to mat based upon the histology. Thus, these extremely small histological determined tumor mases do in fact represent the nodules detected on MRI using the complex of this invention. The level of sensitivity of detection found here for hmg metastases is greater man that c mi entry seen with. CT, the commonly used method of detection of primary tumors of the hmg and lung metastases derived from other cancer types. Clearly this represents and unexpected and snrprisnig result.

Example 7 Detection of Sub-Pl ral Lung Metastases by TfRscFv-Lip-Magnevist

[0120] Employing the identical tumor model system and imaging parameters as described above in Example 6 above for Figures 13 and 14, it is also possible to detect metastases in the sub pleura of the hmg as shown in Figure 18A-18F. This is unexpected and surprising since anient MRI imaging with non-complexed agents which do not actually enter the cell are not able to detect metastases in mis location. This provides a significant advantage in early detection and treatment of lung and other types of cancer.

Example 8 Enhanced Detection of Melanoma Lang Metastasis by TfRscFv-Lip-Magnevist

[0121] With respect to detec-tion treatment of pleural metastases, clinical control is very difficult to achieve and measurement of benefit is also difficnlt The results presented in the Examples herein indicate that the complexes of this invention can reach and transfect pleural metastases and therefore can also be used to treat them. Moreover, the complexes of mis invention could be the imaging tool employed to measure e Eectiveness of ™. or any other therapy.

[0122] The following experiments were performed to demonstrate mat when . administered intravenously (or via, any other appropriate route, e-g^ but not tj rtwd to IT, ID, IM, IP) the complexes of the present invention carrying an imaging agent can detect metastases mat are not limited to those from renal cell carcinomas. Lung tumors were induced in female C57/B1 6 mice by the intravenous injection of 0.1 to 5x10s B167F10 i I moose melannma cells. This method results in metastases that reside almost exclusively in me lungs of me animals and thus serves as a model system for any type of cancer that results in long tumors either as primary disease or as metastases. Appiuxiiiiatery 2-4 weeks later, the animals were used for imaging.

[0123] Canonic liposome (DOTAPiDOPE) was prepared by the ethanol injection method as as previously described {see U.S. Published Patent Application No. 2003 0044407; Xn I et oL, Molecular Cancer Therapeutics i 337-346 (2002) the disclosures of each of which are ir-corporated herein by reference). The targeting moiety used in these studies is the aru-trajisierrin receptor single chain antibody fragment (TfRscFv).

[0124] To encapsulate the imaging agent, the TfRscFv was mixed with the liposome at a specific ratio and incubated at room temperature for 1- 30 mmntp-g suitably 5-20· minutes, most suitably 10-12 minutes. Magnevist® was added to mis solution, mixed and again inrmhateri at ττνττη tampai-ahim ftrr 1 - ¾fl Tmmitp niitaMy S-7n mrnnte; mnet gntnhly 1 Π- 12 minutes. When prepared for in vivo use, sucrose or dextrose was added to a final cciacentration of 0.5-50%, suitably 1-20%, most suitably 10% for sucrose and 5% for dextrose, and incubated at room temperature for 1- 30 mrnn pg^ suitably 5-25 rn ""* t most suitably 15-20 minutes. The complex is formed at a ratio of lmg imaging agent to 033-1.17 ug TfRscFv to 10-35 ug Liposome (suitably lmg imaging agent to 0.5 to l.Oug TfRScFv to 14-28 ug Liposome, most suitably lmg imaging agent to 0.71 ug TfRscFv to 21ug Liposome) using the above procedure. A range of acceptable sizes of the complex is from about 20 to 1000 ran, suitably 50 to 70 run and most suitably 100 to 500 run. Here the complex was formed using 4.7 mg Magnevist, 9ug Liposome and 33ug TfRscFv with dextrose to a final concentration of 5%.

[0125] A mouse bearing hmg tumors induced above was anesthetized and placed in an animal holder system. Anesthesia was induced using isoflurane at 4%, with the nmaitimg gag iwmprigmg a 66% mr gm and n tmm mririe Tmxtim- Mantenance of anesthesia was achieved with 1.0 to 2.0% isoflnrane (preferably 1.5%) under similar gaseous conditions of oxygen and nitrous oxide as noted. The anesthetized animal was positioned inside of a cylindrical variable radiofrequency resonant antenna (bird cage, resonator volume coil) and tuned to a center frequency of approximately 300 MHz (the resonant frequency of water molecules when subject to a field strength of 7 Teste). The ½agmg protocol used was Tl-wedghted two dimensional Turbo Mulrishce-Multiecho I imaging sequence performed on a 7T Bniker BioSpin (Gennany/USA) imaging console. The imaging parameters used were: Tl-wedgfated 2D (2-dimensionaI) imagmg sequence, TE 10.21 ms, TR 1418.13ms, Flipback o¾ 8 averages with a field of view of 3.84 x 3.84 cm and a 256 x 256 matrix- Afier a baseline image was acquired, the animal was kept immobilized in the animal holder and either the free (uncomplexed) Magnevist* (gad-d) or the TfRsc v-Lip-Mag complex containing me identical amount of Mag (total volume 50-1000 ul, suitably 100-SOOul, most suitably 200400ul) was sy^teniicafly administered ng ng a 27G needle by intravenous injection witn the tail vein of the armtiaT and the "n« i" sequence was immediately initiated. The pixel intensity of the images was measured. The same mouse was used for imaging with both the free and the complex. The imagmg was performed on sequential days. {0126] As Shown in Figure 19A-19B, two small metastases (arrows) were detected in the . lungs afier injection with the complexed Magnevist* (Mag). The images represent two different slices through the lungs.

[0127] Employing the identical tumor model system (B16 F10 melanoma) and imaging parameters as above, pixel intensity of a metastasis in another animal was measured using dynamic profiling in linage J software after baseline, afier Free Magnevist* and afier TfRscFv-Lip-Mag and the values compared. As shown in Table 4 below, the complex showed the greatest enhancement over the haserme value. The Standard Deviation shows that the difference between complex and baseline values is significant while that between Free Magnevist* and baseline is not Table 4: Comparison of Signal intensities in a B16/F10 Lung Metastasis Maxinnun Pixel Average Pixel Value Standard Deviation Value Baseline* 12888 7765.1 17572 Free Magnevist" 17959 129793 2976.8 Complexed Magnevist" 22351 143413 2384.6 REFERENCES Gillies, RJ., et oL, Neoplasia (New York) 2:139-451 (2000) Degani, IL,etaL, Thrombosis & Haemostasis 59.25-33 (2003) n, L, et aL. Human Gene Therapy 70.2941-2952 (1999) XOy L^ etaL, Tumor Targeting 4:92-104 (1999) XnJL, et aL. Molecular Medicine 7:723-734 (2001) XnL, ef of., Molecular Cancer Therapeutics 7:337-346 (2002) Rait, A^ et aL, Molecular Medicine 5:476-487 (2002) Rah, , etaL. Ann. N.T. Acad ScL 7002:1-12 (2003) Cristiano, X, and Cariel, D.T., Cancer Gene TSerqpy 5:49-57 (1996) Cheng, P.W., iftonan Gene Therapy 7:275-282 (1996) Keer, BLN-, ei al, ounuz/ of Oology 745:381-385 (1990) Rosa, M.C., and Zetter, BJL, P!roe. iVml Acad ScL (USA) 59^197-6201 (1992) EHiott, RX-, et aL. nn. KY. Acad ScL 698:159-166 (1993) ΊΤ-orstensen, K-, and Romslo, L, Sbmi CZm. Za6. bivestig. (Supp.) 275:113-120 (1993) Miyamoto, T-, et al. Ml J. Oral Maxillofacial Surg. 25:430-433 (1994) Pooka, P. andI^k, C.N.,/iirt £l^^ Haynes, BJ., et al. J. Immunol 727347-351 (1981) Batra, JJL, er o£, Molecular & Cellular Biology 112200-2205 (1991) Jariii, RJK. and Baxter, L.T, Cancer Res. 48:7022-7032 (1988) Wolfert, MA, ef ai, 7 tumoi Gene Therapy 7:2123-2133 (1996) Danlap, D-D, et al, Nucleic Acids Research 253095-3101 (1997) Kmum^ ., etaL, FEBS Letters 421*9-72 (1998) Choi, YJL, ei al, ΛΉτηαη Gene J¾er<9ry 7( 2657-2665 (1999) I&db^ C^ etal. Nature 406299-302 (2000) Rasa, M^ et aL, J. CoJL Interface Set 250-303-315 (2002) Yu, W, et aL Nucleic Acids Research, 32(5):e48(2Q04) AlisaiiRkns.R-, et aL, Cancer Research 55:5743s-5748s (1995) Foo, JJ, et aL, Annals of Biomedical Engineering 57:1279-1286 (2003) Xn, L, et aL, Human Gene Therapy 75:469-481 (2002) Freedman, M., et aL, SPIE Medical Imaging: Physiology and Function from Multidimensional bnagei 14327:163-167 (2001) .

Wisner, EJL, et aL, Investigative Radiology 57:232-239 (2002) Winter, P-*£, etal. Circulation 70&227O-2274 (2003) -48- 190,773/2 Morawski, A.M., et at., Magnetic Resonance in Medicine 51 :480-486 (2004) SEQUENCE LISTING <110> Intrexon Corporat Reed, Thomas D. <120> METHODS OF MAKING MODULAR FUSION PROTEIN EXPRESSION PRODUCTS <130> 2584.024PC02 <160> 2 <170> Patentln version 3.3 <210> 1 <211> 45 <212> DNA <213> artificial <220> <223> chimeric <400> 1 gccggcaaga agaaaaagaa gaagcccggg ggcggaggca tcgat <210> 2 <211> 15 <212> PRT <213> artificial <220> <223> chimeric <400> 2 Ala Gly Lys Lys Lys Lys Lys Lys Pro Gly Gly Gly Gly lie Asp

Claims (7)

190,773/3 49 What is Claimed is:

1. A method of preparing an antibody- or antibody fragment-targeted cationic immunoliposome complex comprising: (a) preparing an antibody or antibody fragment; (b) mixing said antibody or antibody fragment with a cationic liposome to form a cationic immunoliposome, wherein said antibody or antibody fragment is not chemically conjugated to said cationic liposome; and (c) mixing said cationic immunoliposome with an imaging agent at a ratio in the range of about 1 :10 to about 1 :35 (mg imaging agent: μg liposome) to form said antibody- or antibody fragment-targeted-cationic immunoliposome complex.

2. The method of claim 1 , wherein an antibody is mixed with said cationic liposome.

3. The method of claim 1, wherein an antibody fragment is mixed with said cationic liposome.

4. The method of claim 3, wherein said antibody fragment is a single chain Fv fragment.

5. The method of claim 4, wherein said antibody fragment is an anti-transferrin receptor single chain Fv (TfRscFv).

6. The method of claim 1 , wherein said antibody or antibody fragment is an anti-HER-2 antibody or antibody fragment.

7. The method of claim 1, wherein said antibody fragment comprises a cysteine moiety at a carboxy terminus prior to being mixed with said cationic liposome. 190,773/3 50 The method of claim 1 , further comprising mixing said cationic immunoliposome with a peptide comprising the K[K(H)KK ]5-K(H)KKC (HoKC) (SEQ ID NO: 1) peptide. The method of claim 1, wherein said cationic liposome comprises a mixture of one or more cationic lipids and one or more neutral or helper lipids. The method of claim 1, wherein said 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). The method of claim 1, wherein said cationic liposome comprises a mixture of dioleoyltrimethylammonium phosphate with dioleoylphosphatidylethanolamine and/or cholesterol; or a mixture of dimethyldioctadecylammonium bromide with dioleoylphosphatidylethanolamine and/or cholesterol. The method of claim 1, wherein said cationic immunoliposome is mixed with said imaging agent at a ratio of about 1 :14 to about 1 :28 (mg imaging agent^g liposome). The method of claim 1, wherein said cationic immunoliposome is mixed with said imaging agent at a molar ratio of about 1 :21 (mg imaging agent^g liposome). The method of claim 1, wherein said imaging agent is a magnetic resonance imaging (MRI) agent, a computed tomography (CT) imaging agent, or a positron emission tomography (PET) imaging agent. The method of claim 15, wherein said MRI agent is gadopentetate dimeglumine, iron oxide, or iopamidol, said CT imaging agent is barium, iodine or saline, or said PET imaging agent is 1KF-2-deoxy-2-fluoro-D-glucose (FDG). An antibody- or antibody fragment-targeted cationic immunoliposome complex prepared by the method of claim 1, comprising a cationic liposome, an antibody or antibody fragment, and an imaging agent, wherein said antibody or antibody fragment is not chemically conjugated to said cationic liposome and wherein said imaging agent and said 190,773/3 51 cationic immunoliposome are present at a ratio in the range of about 1 :10 to about 1 :35 (mg imaging agent: μg liposome). The cationic immunoliposome complex of claim 16, wherein said imaging agent is encapsulated within said cationic liposome. The cationic immunoliposome complex of claim 16, wherein said imaging agent is associated with an inner or outer monolayer of said cationic liposome. The cationic immunoliposome complex of claim 16, wherein said antibody fragment is a single chain Fv fragment. The cationic immunoliposome complex of claim 16, wherein said antibody fragment is an anti-transferrin receptor single chain Fv (TfRscFv). The cationic immunoliposome complex of claim 16, wherein said antibody or antibody fragment is an anti-HER-2 antibody or antibody fragment. The cationic immunoliposome complex of claim 16, wherein said cationic liposome comprises a mixture of one or more cationic lipids and one or more neutral or helper lipids. The cationic immunoliposome complex of claim 16, wherein said antibody or antibody fragment and said cationic liposome are present at a ratio in the range of about 1 :20 to about 1 :40 (w:w). The cationic immunoliposome complex of claim 16, wherein said cationic liposome comprises a mixture of dioleoyltrimethylammonium phosphate with dioleoylphosphatidylethanolamine and/or cholesterol; or a mixture of dimethyldioctadecylammonium bromide with dioleoylphosphatidylethanolamine and/or cholesterol. 190,773/3 52 The cationic immunoliposome complex of claim 16, wherein said imaging agent and said cationic immunoliposome are present at a molar ratio of about 1 :14 to about 1 :28 (mg imaging agent^g liposome). The cationic immunoliposome complex of claim 16, wherein said imaging agent and said cationic immunoliposome are present at a molar ratio of about 1 :21 (mg imaging agent^g liposome). The cationic immunoliposome complex of claim 16, wherein said imaging agent is an magnetic resonance imaging (MRI) agent, a computed tomography (CT) imaging agent, or a positron emission tomography (PET) imaging agent. The cationic immunoliposome complex of claim 27, wherein said MRI agent is gadopentetate dimeglumine, iron oxide, or iopamidol, said CT imaging agent is barium, iodine or saline, or said PET imaging agent is 18F-2-deoxy-2-fluoro-D-glucose (FDG). The cationic immunoliposome complex of claim 16, further comprising a K[K(H)K K|5-K(H)KKC (HoKC) (SEQ ID NO: 1) peptide associated with said complex. A method of imaging an organ or a tissue in a patient comprising administering the cationic immunoliposome complex of claim 16 to the patient prior to performing said imaging. The method of claim 30, wherein said administration comprises intravenous administration, intramuscular administration, intradermal administration, intraocular administration, intraperitoneal administration, intratumoral administration, intranasal administration, intracereberal administration or subcutaneous administration. A method of imaging a cancerous tissue in a patient comprising administering the cationic immunoliposome complex of claim 16 to the patient prior to performing said imaging. 190,773/3 53 A method of imaging a cancerous metastasis in a patient comprising administering the cationic immunoliposome complex of claim 16 to the patient prior to performing said imaging. The method of claim 32, wherein said administration comprises intravenous administration, intramuscular administration, intradermal administration, intraocular administration, intraperitoneal administration, intratumoral administration, intranasal administration, intracereberal administration or subcutaneous administration. The method of claim 33, wherein said administration comprises intravenous administration, intramuscular administration, intradermal administration, intraocular administration, intraperitoneal administration, intratumoral administration, intranasal administration, intracereberal administration or subcutaneous administration. Use of the cationic immunoliposome complex of claim 16 in the manufacture of a medicament for imaging a tumor tissue in a patient suffering from cancer comprising administering the cationic immunoliposome complex to the patient to image the tumor tissue. The use of claim 36, wherein said medicament further comprises an anti-cancer agent. The use of claim 36, wherein said administration comprises intravenous administration, intramuscular administration, intradermal administration, intraocular administration, intraperitoneal administration, intratumoral administration, intranasal administration, intracereberal administration or subcutaneous administration. The use of claim 37, wherein said anti-cancer agent is a chemotherapeutic agent, gene or small molecule. 190,773/3 54 The use of claim 39, wherein said chemotherapeutic agent is selected from the group consisting of docetaxel, mitoxantrone and gemcitabine. The use of claim 39, wherein said anti-cancer agent is associated with said cationic immunoliposome. The use of claim 37, wherein said anti-cancer agent is an antisense oligonucleotide or an siRNA. The use of claim 42, wherein said antisense oligonucleotide or said siRNA is associa with said cationic immunoliposome. For the Applicant WOLFF, B EGMAN AND GOLLER