Note: Descriptions are shown in the official language in which they were submitted.
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TUMOR-TARGETED NANODELI VERY SYSTEMS TO IMPROVE EARLY
MRI DETECTION OF CANCER
BACKGROUND OF THE INVENTION
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.
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 89:25-33 (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 (GUI) 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 (DTPA)
generates a well
tolerated, stable, strongly paramagnetic complex. This metal chelate is
metabolically
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inert. However, after i.v. injection of gadopentetate dimeglumine, the
meglumine ion
dissociates from the hydhophobic gadopentetate, which is distributed only in
the
extracellular water. It cannot cross an intact blood-brain barrier, and
therefore does not
accumulate in normal brain tissue, cysts, post-operative scars, etc, and is
rapidly excreted
in the urine. It has a mean half-life of about 1.6 hours. Approximately 80% of
the dose is
excreted in the urine within 6 hours.
[0004] 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.
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
preparing an antibody or antibody fragment, mixing the antibody or antibody
fragment
with a cationic liposome to form a cationic immunoliposome, wherein the
antibody or
antibody fragment is not chemically conjugated to the cationic liposome, and
mixing the
cationic immunoliposome with an imaging agent to form the antibody- or
antibody
fragment-targeted-cationic immunoliposome complex. Exemplary antibody
fragments
for use in the practice of the present invention include, single chain Fv
fragments, such as
an anti-transferrin receptor single chain Fv (TIRscFv) and anti-HER-2 antibody
or
antibody fragment. In additional embodiments, the methods further comprise
mixing the
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cationic immunoliposome with a peptide comprising the KM(H)KK.K15-K(H)KKC
(HoKC) (SEQ ID NO: 1) peptide.
[0006] Suitably, the antibody or antibody fragment is mixed with said
cationic liposome
at a ratio in the range of about 1:20 to about 1:40 (w:w). Suitably, the
cationic liposomes
comprise a mixture of dioleoyltrimethylammonium phosphate with
dioleoylphosphatidylethanolamine and/or cholesterol; or a mixture of
dimethyldioctadecylammonium bromide with dioleoylphosphatidylethanolamine
and/or
cholesterol.
[0007] In additional embodiments, the cationic immunoliposomes are mixed
with the
imaging agent at a ratio in the range of about 0-.1:10 to about 0.1:35 (mg
imaging agent:
liposome), suitably about 1:14 to about 1:28 (mg imaging agent:m liposome), or
about 1:21 (mg imaging agent:m liposome). Exemplary imaging agent for use in
the
practice of the present invention include, but are not limited to, magnetic
resonance
imaging (MRI) agents, such as gadolinium, gadopentetate dimeglumine, iopamidol
and
iron oxide. Also, barium, iodine and saline imaging agents for CT, 18F-2-deoxy-
2-fluoro-
D-glucose (FDG) and other imaging agents for PET can also be used.
[0008] The present invention also provides cationic immunoliposome
complexes
prepared by the methods of the present invention and antibody- or antibody
fragment-
targeted cationic immunoliposome complexes comprising a cationic liposome, an
antibody or antibody fragment, and an imaging agent, wherein the antibody or
antibody
fragment is not chemically conjugated to said cationic liposome.
[0009] In further embodiments, the present invention provides methods of
imaging an
organ or a tissue, and also for distinguishing between benign tissues/diseases
and
cancerous tissues/diseases in a patient comprising administering the cationic
immunoliposome complexes of the present invention to the patient prior to
performing
the imaging. Administration can occur via any route, for example, intravenous
administration, intramuscular administration, intradermal administration,
intraocular
administration, intraperitoneal administration, intratumoral administration,
intranasal
administration, intracereberal administration or subcutaneous administration.
Suitably,
the tissue that is imaged using the methods and complexes of the present
invention are
cancerous tissues, including cancerous metastasis.
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[0010] The present invention also provides methods of imaging and treating
a tumor
tissue in a patient suffering from cancer comprising administering the
cationic
immunoliposome complexes of the present invention to the patient to image the
tumor
tissue and administering an anti-cancer agent to the patient to treat the
tumor tissue.
Exemplary anti-cancer agents include nucleic acids, genes, proteins, peptides,
small
molecules, chemotherapeutic agents, such as docetaxel, mitoxantrone and
gemcitabine,
and antisense oligonucleotides or siRNA.
[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 1B show tumor-specific targeting of a CaPan-1
orthotopic
metastasis model by the TfRscFv-Liposome-DNA nanocomplex. The same tumor
nodule
in the liver indicated by an arrow in lA exhibits intense P-galactosidase
expression in 1B.
1A = gross necropsy; 1A = tissues after staining for [3-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. 1B = shows variation in relative intensity with the
amount of
Magnevist included in the complex (in RD. 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
=
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substrate and the mechanically compliant liposome are -3.5 , +8 , and +400.
The
interaction of the SPM tip and lipo some changes from attractive to repulsive
as the
setpoint is decreased.
[0016] Figure 5A-C show SPM and SEM images of liposome-encapsulated
Magnevist
(Lip+Mag). 5A is the Atomic Force Microscopy topographical image of the
Liposome
encapsulated Magnevist particle. The SPM phase image (setpoint = 1.6) (5B)
and 15
keV SEM (TB) [Transmission¨mode electron detector] image (5C) possess similar
contrast, although generated by entirely distinct complementary physical
mechanisms.
[0017] Figure 6A and 6B show SPM topographic and phase imaging of
TfRscFv+Lip+Mag nanocomplex. 6A is thel5 keV SEM (TB) [Transmission¨mode
electron detector] image of the full nanocomplex. 6B = A lower power image of
the field.
The boxed area is the image in 6A.
[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
topographic/magnetic phase image of the full TfRscFv-Lip-Mag nanocomplex. The
appearance of a double dipole-like signal in 7B consisting of attractive and
repulsive in-
plane magnetic interactions suggests that the cause of this interaction is the
nonuniform
toroidal distribution of Magnevist 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 Ligand-HIcLiposome-Mag nanocomplex. Human breast cancer MDA-MB-
435 (Figure 8E-811) and human prostate cancer cell line (DU145) (Figure 8A-
8D).
[0020] Figure 9A-C shows tumor-specific targeting of a CaPan-1
subcutaneous tumor
and orthotopic metastasis model by the TfRscFv-HK-Liposome-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
model, 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.
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[0024] Figure 13A-13D shows a greater enhancement in MR imaging of renal
cell
carcinoma lung metastases by Mag-comprising complexes of 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 the
present
invention.
[0026] Figure 15A-15B shows MR imaging of very small metastases by Mag-
comprising
complexes of the present invention, demonstrating the sensitivity of the
complexes of the
present invention.
[0027] Figure 16 shows sections of metastatic tissue confirming the
detection/imaging
seen by MRI using the Mag-comprising complexes of the present invention.
[0028] Figure 17 shows higher magnification of Figure 16.
[0029] Figure 18A-18F shows MR imaging of metastases in the subpleura of
the lung by
Mag-comprising complexes of the present invention.
[0030] Figure 19A-19B shows detection of BidFio 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 gadolinium, gadopentetate
dimeglumine (Magnevist ) and, iopamidol, 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 to targeted tissues, for example tumors. Scanning Electron Microscopy
(SEM) and
Scanning Probe Microscopy (SPM) (Wolfert, M.A., et al., Human Gene Therapy
7:2123-
2133 (1996); Dunlap, D.D., et aL, Nucleic Acids Research 25:3095-3101 (1997);
Kawaura, C., et aL, FEBS Letters 421:69-72 (1998); Choi, Y.H., et al., Human
Gene
Therapy /0:2657-2665 (1999); Diebel, C.E., et al., Nature 406:299-302 (2000);
Rasa, M.,
et al., J. Coll. Interface Sci 250:303-315 (2002)) have been used to examine
the physical
structure and size of these imaging agent-carrying nanocomplexes. In the case
of
gadolinium, a high-atomic number element which possess a large magnetic
moment,
these properties can be exploited in a variety of ways to enhance contrast in
both SEM
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and SPM. The findings presented herein demonstrate that the liposome
nanocomplexes of
the present invention do indeed encapsulate imaging agents, such as Magnevist
, and
that intravenous administration of these complexes result in enhanced tumor
imaging.
The present invention provides the unexpected and surprising results of
detection of very
small metastases, include pleural metastases in the lung, as well as the
ability to
differentiate between benign and cancerous tissues.
[00321 In one embodiment, the present invention provides tumor-targeting
delivery
systems comprising contrast agents, for example magnetic resonance imaging
(MR1)
contrast agents. U.S. Published Patent Application No. 2003/0044407
discloses these nano-sized,
cationic liposome encapsulating various agents. Decorating the surface of
these liposomes
are targeting molecules which can be a ligand, such as folate or trans ferrin,
or an antibody
or an antibody fragment directed against a cell surface receptor. The presence
of the
ligand/antibody on the liposomes facilitates the entry of the complexes into
the cells
through binding of the targeting molecule by its receptor followed by
internalization of
the bound complex via receptor mediated endocytosis, a highly efficient
internalization
pathway (Cristiano, R.I., and Curiel, D.T., Cancer Gene Therapy 3:49-57
(1996); Cheng,
P.W., Hunzan Gene Therapy 7:275-282 (1996)). This modification of the
liposomes
results in their being able not only to selectively deliver their payload to
tumor cells, but
also increases the transfection efficacy of the liposome. Transferrin receptor
(TfR) levels
are elevated in various types of cancer including oral, prostate, breast, and
pancreas
(Keer, FLN., et al., Journal of Urolo,D, /43:381-385 (1990); Rossi, M.C., and
Zetter,
B.R., Proc. Natl. Acad. Sci. (USA) 89:6197-6201 (1992); Elliott, R.L., et al.,
Ann. N.Y.
Acad. Sci. 698:159-166 (1993); Thorstensen, K., and Romslo, I., Scand. J.
Clin. Lab.
Investig. (Supp) 2/5:113-120 (1993); Miyamoto, T., et al., Ina. J. Oral
Maxillofacial
Surg. 23:430-433 (1994); Ponka, P. and Lok, C.N., J Biochem. Cell
Biol. 31:1111-
1137 (1999)). Moreover, the TfR recycles during internalization in rapidly
developing
cells such as cancer cells (Ponka, P. and Lok, C.N., Ina. J. Biochein. Cell
Biol. 31:1111-
1137 (1999)), thus contributing to the uptake of these transferrin targeted
nanocomplexes
even in cancer cells where TER levels are not elevated. In suitable
embodiments, the
nanocomplexes described herein employ an anti-transferrin receptor single
chain anti-
body fragment (TfRseFv) as the targeting moiety (Haynes, B.F., et al., J
Immunol.
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/27:347-351 (1981); Batra, J.K., et al., Molecular & Cellular Biology 11:2200-
2205
(1991)). TfRscFv contains the complete antibody binding site for the epitope
of the DR
recognized by the monoclonal antibody 5E9 (Batra, J.K., et al., Molecular &
Cellular
Biology 11:2200-2205 (1991)). TfRscFv has advantages over the Tf molecule
itself, or an
entire Mab, in targeting liposomes to cancer cells with elevated TfR levels:
1) the size of
the scFv (28 kDa) 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 tumors. 2) the smaller scFv has a
practical
advantage related to the scaled-up 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 Pc receptors is eliminated
(Jain, R.K.
and Baxter, L.T., Cancer Res. 48:7022-7032 (1988)). Such an anti-TfR single
chain
antibody molecule can target an intravenously administered cationic liposome-
DNA
nanocomplex preferentially to tumors (See U.S. Published Patent Application
No.
2003/0044407; Xu,L., et al., Molecular Medicine 7:723-734 (2001); Xu L, et
al.,
Molecular Cancer Therapeutics 1:337-346 (2002)). Encapsulating Magnevist8
(Mag)
within such a tumor-targeted nanocomplexes offers advantages for enhanced
sensitivity
and detection of tumor metastases and diagnosis of cancer. Gadolinium,
gadopentetate
dimeglumine (MagnevistO), iopamidol, 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 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 MIRE,
CT, PET, SPECT, etc.) can also be encapsulated within the imtnunoliposomes of
the
present invention.
[00331 Antibody- or antibody fragment- targeted cationic liposome
complexes in
accordance with this invention are made by a simple and efficient non-chemical
conjugation method in which the components of the desired complex are mixed
together
in a defined ratio and in a defined order (see, U.S. Published Patent
Application
No. 2003/0044407). The resultant complexes are as effective as, or more
effective than,
similar complexes in which the antibody or antibody fragment is chemically
conjugated
to the liposome or polymer. The terms "immunocomplex," "immunoliposome,"
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"complex," "nanocomplex," "immunonanocomplex," "liposome complex" are used
interchangeably throughout to refer to the cationic liposomes of the present
invention.
[00341 Either a whole antibody or an antibody fragment can be used to make
the
complexes of this invention. In suitable embodiments, an antibody fragment is
used.
Preferably, the antibody fragment is a single chain Fv fragment of an
antibody. One
preferred antibody is an anti-TM monoclonal antibody and a preferred antibody
fragment
is an scFv based on an anti-TfR. monoclonal antibody. A suitable anti-TfR.
monoclonal
antibody is 5E9 (see, e.g., Hayes, B.F., et al., "Characterization of a
Monoclonal
Antibody (5E9) that Defines a Human Cell Surface Antigen of Cell Activation,"
J.
ImmunoL /27:347-352 (1981); Batra, J.K., et aL, "Single-chain Immunotoxins
Directed at
the Human Transferring Receptor Containing Pseudomonas Exotoxin A or
Diphtheria
Toxin: Anti-TFR(Fv)-PE40 and DT388-Anti- 11-R(Fv)," Mot. Cell. Biol. 11:2200-
2205
(1991)). An scFv based on
5E9 antibody contains the complete antibody binding site for the epitope of
the TfR
recognized by this MAb as a single polypeptide chain of approximate molecular
weight
26,000. An scFv is formed by connecting the component VII and 'IL variable
domains
from the heavy and light chains, respectively, with an appropriately designed
linker
peptide, which bridges the C-terminus of the first variable region and N-
terminus of the
second, ordered as either VH-linker-VL or VL-linker-VH. Another 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.
[00351 In suitable embodiments, a cysteine moiety is added to the C-
terminus of the scFv.
Although not wishing to be bound by theory, it is believed that the cysteine,
which
provides a free sulfhydryl 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 E.coll inclusion bodies and then
refolded to
produce the antibody fragment in active form.
[00361 Unless it is desired to use a sterically stabilized immunoliposome
in the formation
of the complex, a first step in making the complex comprises mixing a cationic
liposome
or combination of liposomes or small polymer with the antibody or antibody
fragment of
choice (see Examples herein and in U.S. Published Patent Application
No. 2003/0044407). A wide variety of cationic liposomes are useful in the
preparation of
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the complexes of this invention. Published PCT application W099/25320
describes the
preparation of several cationic liposomes. Examples of desirable liposomes
include those
that comprise a mixture of dioleoyltrimethylammonium phosphate (DOTAP) and
dioleoylphosphatidylethanolamine (DOPE) and/or cholesterol (chol), 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
therapeutic molecule
for the specific target cell type. 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 1:(0.5-3), preferably 1:(1-2)
(molar ratio).
[0037] The present invention also provides for targeted-cationic polymers
for delivery of
imaging agents. Suitable polymers are DNA binding cationic polymers that are
capable
of mediating DNA compaction and can also mediate endosome release. A preferred
polymer is polyethyleneimine. Other useful polymers include polysine,
protamine and
polyamidoamine dendrimers.
[0038] The antibody or antibody fragment is one which will bind to the
surface of the
target cell, and preferably to a receptor that is differentially expressed on
the target cell.
The antibody or antibody fragment is mixed with the cationic liposome or
polymer at
room temperature and at a protein:lipid ratio in the range of about 1:20 to
about 1:40
(w:w) 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 liposome or polymer are
allowed to
incubate at room temperature for a short period of time, typically for about
10-15
minutes, 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
liposome include genes, high molecular weight DNA (genomic DNA), plasmid DNA,
antisense oligonucleotides, peptides, ribozymes, nucleic acids (including
siRNA and
antisense), viral particles, immunomodulating agents, proteins, small
molecules and
chemical agents. Preferred therapeutic molecules include genes encoding p53,
Rb94 or
Apoptin. RB94 is a variant of the retinoblastoma tumor suppressor gene.
Apoptin is a
gene that induces apoptosis in tumor cells only. In another preferred
embodiment, the
agent is an antisense oligonucleotide or an siRNA molecule, such as a HER-2
antisense
or siRNA 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,
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but are not limited to, Gadolinium, gadopentetate dimeglurnine (MagnevistO),
iopamidol,
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. If the agent is DNA, such as
the
coding region of p53, it can be positioned under the control of a strong
constitutive
promoter, such as an RSV or a CMV promoter.
100401 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
(fig of
agent:nmole of total lipid) or 1:10 to 1:40 (ug of agent:nmole of total
polymer) and
incubated at room temperature for a short period of time, typically about 10
to 15
minutes. The size of the liposome complex is typically within the range of
about 50-400
urn as measured by dynamic light scattering using a Malvern Zetasizer 3000.
[0041] In one embodiment of this invention, the liposome used to form the
complex is a
sterically stabilized liposome. Sterically stabilized liposomes are liposomes
into which a
hydrophilic polymer, such as PEG, poly(2-ethylacrylic acid), or poly(n-
isopropylacrylamide (PNIPAM) have been integrated. Such modified liposornes
can be
particularly useful when complexed with therapeutic or diagnostic agents, as
they
typically are not cleared from the blood stream by the reticuloendothelial
system as
quickly as are comparable liposomes that have not been so modified. To make a
sterically stabilized liposome complex of the 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 cationic liposome
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 (lag of agent:nrnole of lipid). To this lipoplex is added a
solution of a PEG
polymer in a physiologically acceptable buffer and the resultant solution is
incubated at
room temperature for a time sufficient to allow the polymer to integrate into
the liposome
complex. The antibody or antibody fragment then is mixed with the stabilized
liposome
complex at room temperature and at a protein:lipid ratio in the range of about
1:5 to about
1:30 (w:w).
[0042] The liposomal or polymer complexes prepared in accordance with the
present
invention can be formulated as a pharmacologically acceptable formulation for
in vivo
administration. The complexes can be combined with a pharmacologically
compatible
vehicle or carrier. The compositions can be formulated, for example, for
intravenous
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administration to a human patient to be benefited by administration of the
therapeutic or
diagnostic molecule of the complex. The complexes are sized appropriately so
that they
are distributed throughout the body following i.v. administration.
Alternatively, the
complexes can be delivered via other routes of administration, such as
intratumoral (IT),
intralesional (IL), aerosal, percutaneous, endoscopic, topical, intramuscular
(IN4),
intradermal (ID), intraocular (JO), intraperitoneal (IP), intranasal (IN),
intracereberal (IC)
or subcutaneous administration. Preparation of formulations for delivery via
such
methods, and delivery using such methods, are well known in the art.
[0043] In one embodiment, compositions comprising the antibody- or
antibody fragment-
targeted liposome (or polymer) and therapeutic agent complexes are
administered to
effect 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 systemic
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, both 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 fragment and liposome to the therapeutic or diagnostic agent, and the
choice of
antibody or antibody fragment and therapeutic or diagnostic agent.
[0045] In 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,
including glioblastoma, cervical, lung, liver, lipo sarcoma, rhabdomyosarcoma,
choriocarcinoma, melanoma, retinoblastoma, ovarian, urogenital, gastric and
colorectal
cancers are suitable targets.
[0046] The complexes made by the method of this invention also can be used
to target
non-tumor cells for delivery of a therapeutic molecule or any nucleic acid.
While any
normal cell can be a target, preferred cells are dendritic cells, endothelial
cells of the
blood vessels, lung cells, breast cells, bone marrow cells, thymus cells and
liver cells.
Undesirable, but benign, cells can be targeted, such as benign prostatic
hyperplasia cells,
over-active thyroid cells, lipoma cells, and cells relating to autoimmune
diseases, such as
CA 02638899 2013-04-09
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B cells that produce antibodies involved in arthritis, lupus, myasthenia
gravis, squarnous
metaplasia, macular degeneration, cardiovascular disease, neurologic disease
such as
Alzheimer's disease, dysplasia arid the like.
[00471 The complexes can be administered in combination with another
therapeutic
treatment, such as either a radiation treatment or chemotherapeutic agent. The
therapeutic
treatments, or a combination of therapeutic treatments, can be administered
before or
subsequent to the administration of the complex, for example within about 12
hours to
about 7 days. Chemotherapeutic agents include, for example, doxotubicin, 5-
fluorouracil
(5FU), cisplatin (CDDP), docetaxel (TAXOTERE ), gemcitabine (GEMZAR ),
pacletaxel, vinblastine, etoposide (VP-16), carnptothecia, actinomycin-D,
rnitoxantrone
and rnitomycin C. Radiation therapies/treatments include gamma radiation
(137Cs), X-
rays, UV irradiation, microwaves, electronic emissions and the like.
Additional
therapeutic agents include small molecules, peptides, proteins and the like.
[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 administration. Exemplary methods for
detecting,
visualizing, imaging or observing diagnostic and imaging agents are well }mown
in the art
and include, for example, optical imaging such as fluorescent imaging
(fluorimeters) or
bioluminescent imaging, positron emission tomography (PET) scanning, single
photon
emission computed tomography (SPECT) scanning, magnetic resonance imaging
(MRI),
x-ray, radionucleotide imaging (e.g., gamma camera, computed tomography (CT),
quantitative autoradiography, etc.) and the like. Exemplary diagnostic agents
include
electron dense materials, iron, magnetic resonance imaging agents and
radiopharmaceuticals. Radionuclides useful for imaging include radioisotopes
of copper,
gallium, indium, rhenium, and technetium, including isotopes Cu,64 Cu,67
95"TC,
67Ga or 68Ga. MRI agents such as a Gd-DTPA agent, gadolinium, or MagnevistO
(Gadopentetate Dimeglumine) (Mag) (Berlex Imaging, Montville, NJ). Imaging
agents
disclosed by Low et al. in U.S. Patent 5,688,488 are
useful in the present invention. Additional imaging agents include, but are
not limited to,
iopamidol (e.g., ISOVUE , Regional Health Limited, Aukland, AU), iron oxide;
barium,
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iodine and saline imaging agents for CT; and I8F-2-deoxy-2-fluoro-D-g1ucose
(FUG) and
other imaging agents for PEI.
10049) The complexes made in accordance with the method of this invention
can be
provided in the form of kits for use in the systemic delivery of a therapeutic
or diagnostic
molecule by the complex. Suitable kits can comprise, in separate, suitable
containers (or
in a single container), the liposome, the antibody or antibody fragment, and
the
therapeutic or diagnostic agent. The components can be mixed under sterile
conditions in
the appropriate order and administered to a patient within a reasonable period
of time,
generally from about 30 minutes to about 24 hours, after preparation. The kit
components
preferably are provided as solutions or as dried powders. Components provided
in
solution form preferably are formulated in sterile water-for-injection, along
with
appropriate buffers, osmolarity control agents, etc.
Encapsulation and Delivery of Imaging Agents
[00501 In 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,
complexed/associated with the inner and/or outer monolayer (e.g., via static
interaction or
chemical/covalent interaction), or a combination of any or all of these
possibilities.
Suitably, the imaging agents will be encapsulated within the interior of the
liposome
and/or associated with an inner and/or outer monolayer.
[0051] As used herein, the terms "diagnostic agents" and "imaging agents"
refer to agents
which can be detected, visualized, imaged or observed in vivo following
administration.
Exemplary imaging agents include electron dense materials, iron, magnetic
resonance
imaging agents and radiopharmaceuticals. Radionuclides useful for imaging
include
radioisotopes of copper, gallium, indium, rhenium, and technetium, including
isotopes
6701, I I IIII, 99mTc, 670a or aGa. MRI agents such as a gadolinium, Gd-DTPA
agent, or Magnevistit4 (Gadopentetate Dimeglumine) (Mag) (Berlex Imaging,
Montville,
NI). Imaging agents disclosed by Low et al. in T.J.S. Patent 5,688,488
are also useful in the present invention. Additional imaging agents
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include, but are not limited to, iopamidol (e.g., ISOVIJE , 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.
[0052] 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 0.1:10 to about 0.1:35 (mg imaging agent: pg liposome),
more
suitably about 1:14 to about 1:28 (mg imaging agentw liposome), or about 1:21
(mg
imaging agent:lag liposome).
[0053] 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
1:(0.5-3),
preferably about 1:(1-2) (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
Lip C DDAB/DOPE 1:2 molar ratio
LipD DOTAP/Chol 1:1 molar ratio
LipE DDAB/Chol 1:1 molar ratio
Lip G 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).
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[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 0.1:10 to about 0.1:35 (mg imaging agent: [t.g
liposome), more
suitably about 1:14 to about 1:28 (mg imaging agent:pg liposome), or about
1:21 (mg
imaging agentw 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 18F-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
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lipid. The HoKC liposomes are prepared as previously described (Yu, W. et al.
Enhanced
transfection efficiency of a systemically delivered tumor-targeting
immunolipoplex by
inclusion of a pH-sensitive histidylated oligolysine peptide, Nucleic Acids
Research 32,
e48 (2004)).
[0058] In a further embodiment, the present invention provides antibody-
or antibody
fragment-targeted cationic immunoliposome complexes comprising a cationic
liposome,
an antibody or antibody fragment, and one or more imaging agents, wherein the
antibody
or antibody fragment is not chemically conjugated to the cationic liposome.
The antibody
or antibody fragment is suitably associated with the liposome via an
interaction (e.g.,
electrostatic, van der Walls, or other non-chemically conjugated interaction)
between the
antibody or antibody fragment and the liposome, suitably between a cystein
residue on
the antibody or antibody fragment and the liposome surface. In general, a
linker or spacer
molecule (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 cationic
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 immunoliposomes of the present invention are unilamellar
liposomes (i.e. a
single bilayer), though multilamellar liposomes which comprise several
concentric
bilayers can also be used. Single bilayer cationic immunoliposomes of the
present
invention comprise an interior aqueous volume in which agents (e.g., imaging
agents) can
be encapsulated. They also comprise a single bilayer which has a hydrocarbon
chain
region (i.e., the lipid chain region of the lipids) in which agents (e.g.,
imaging agents) can
be contained. In addition, agents (e.g., imaging agents) can be complexed or
associated
with either, or both, the inner monolayer and/or the outer monolayer of the
liposome
membrane (i.e., the headgroup region of the lipids), e.g., via a charge-charge
interaction
between the negatively charged imaging agents and the positively charged
cationic
liposomes.
In further embodiments, agents (e.g., imaging agents) can be
encapsulated/associated/complexed in any or all of these regions of the
cationic
immunoliposome complexes of the present invention.
[0059] In a still further embodiment, the present invention provides
methods of imaging
an organ or a tissue in a patient comprising administering the imaging agent-
comprising
cationic immunoliposome complexes of the present invention to the patient
prior to
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performing the imaging. The immunoliposome complexes can be administered via
any
desired route, including, but not limited to, intravenous (IV), oral, topical,
via inhalation,
intramuscular (IM) injection, intratumoral (IT) injection, intradermal (ID)
injection,
intraperitoneal (M) injection, intranasal (IN) injection, intraocular (10)
injection,
intracranial (IC) injection, or other routes. As used herein, the term patient
includes both
animal patients (e.g., non-human mammals such as dogs, cats, pigs, sheep,
etc,) as well as
humans. Methods for imaging tissues of patients are well known in the art and
include,
but are not limited to, PET scanning, SPECT scanning, MRI imaging and the
like. Any
tissue or organ in a patient can be imaged using the methods and complexes of
the present
invention. Simply by modifying the targeting ligand on the liposomes, any over-
expressed protein or molecule can be targeted.
[00601 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 distinguish
cancerous
tissues from non-cancerous (benign) tissues.
[0061] In further embodiments, the present invention provides methods
of imaging and
treating a tumor tissue in a patient suffering from, or predisposed to, cancer
comprising
administering the imaging-agent comprising immunoliposome complexes of the
present
invention to image the tumor tissue, and administering an anti-cancer agent to
the patient
to treat the tumor tissue.
(00621 Examples of anti-cancer agents that can be administered include,
but are not
limited to small molecules, proteins, peptides, and chemotherapeutic agents
such as those
described herein, genes, antisense oligonuclotides and siRNA. Exemplary
chemotherapeutic agents include, but are not limited to, doxorubicin, 5-
fluorouracil
(5FU), cisplatin (CDDP), docetaxel (TAXOTE.Re), gemcitabine (GEMZAR ),
pacletaxel, vinblastine, etoposide (VP-16), oamptothecin, actinomycin-D,
mitoxantrone
and mitomycin C, and an antibody therapy, such as a monoclonal antibody, e.g.,
HERCEPTINI (Genentech, San Francisco CA). Examples of antisense
oligonucloetides
and sjRNA molecules for use in the practice of the present invention include,
but are not
limited to, those disclosed in U.S. Published Patent Application No.
2003/0044407 and
U.S. Patent Application No. 11/520,796, filed September 14, 2006. Additional
anti-
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cancer 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). The anti-cancer agent (e.g., the chemotherapeutic agent, small
molecule, gene
or the antisense or siRNA, etc.) can be associated with the cationic
iramurtoliposome that
also comprises the imaging agent, or it can be delivered separately, either in
a different
immunoliposome 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] In suitable embodiments, the methods of the present invention
comprise
administering an immunoliposome complex comprising an imaging agent (e.g., KRI
imaging agent such as gadopentetate dirneglumine), and an anti-tumor agent at
different
times (i.e., 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 immunoliposome 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., administration of the cationic
immunoliposome
complex). In still further embodiments, the methods of imaging and treating a
tumor
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
timing for
administration in humans are easily determined by those of skill in the art,
based on
information contained herein and that is readily available in the art.
Furthermore, such
amounts can be estimated by extrapolating from experiments performed on
animals, e.g.,
mouse, rat, dog or other studies.
[0065] Exemplary benefits of utiliting the nanoimmunolipoosme complexes of
the
present invention (scL and scL-HoKC) 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
differentiation of
vascular flow and diffusion into interstitial space (as seen with the non-
complexed free
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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 permits delayed imaging using the complexes of the present
invention. The
complexes and methods can be used to image tissues of interest at various
depths.
[0066] Thus, 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 nodules. This helps to accelerate the decision on when
to begin
treatment. Currently, this is delayed to determine if the nodule increases
since it is not
certain if is malignant or not. However, since the complexes of this invention
preferentially and specifically transfect tumor cells, this would also serve
as a
confirmation of malignancy, for example if the small nodules seen on lung CT
are small
malignancies or not. These last two points are of particular significance in
lung and
pancreatic cancer.
[0067] Exemplary types of cancer imaging problems addressed by use of the
imaging
agent-comprising complexes of the present invention include, in pancreatic
cancer, early
detection and differentiation from chronic pancreatitis; early detection of
metastatic
disease to lungs; classification of solitary pulmonary nodules 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
that, using
this delivery system, therapeutic 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 that delivery of therapeutic genes or other agents associated
with the
complexes of the present invention will also enter these specific cancer
cells.
[0069] It will be readily apparent to one of ordinary skill in the
relevant arts that other
suitable modifications and adaptations to the methods and applications
described herein
may be made without departing from the scope of the invention or any
embodiment
thereof. Having now described the present invention in detail, the same will
be more
clearly understood by reference to the following examples, which are included
herewith
for purposes of illustration only and are not intended to be limiting of the
invention.
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Example 1
Immunoliposonte Complexes Comprising Magnevist
Materials and Methods
Cell lines
[00701 Human lymphoblastic leukemia cell line 1(562 was obtained from the
Lombardi
Comprehensive Cancer Center Tissue Culture core facility. These suspension
cells were
maintained in RPMI1640 supplemented with 10% Heat Inactivated FBS plus 2 m.M L-
Glutamine, and 50 pig/m1 each of penicillin, streptomycion 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 Iscov's
Modified
D ul becco's Medium containing 4m1v1 L-Glutiunine and Sodium Bicarbonate,
supplemental with 20% non-Heat Inactivated FBS, 2inM L-Glutamine and 50
ug/rni.
each of penicillin, streptomycin and neomycin. Human prostate cancer cell line
DU145
(ATCC, Manassas, VA) was originally derived from a lesion in the brain of a
patient with
widespread metastatic carcinoma of the prostate. It was maintained in Minimum
Essential
Medium with Earle's salts (EMEM) supplemented with 10% heat inactivated FBS
plus L-
glutamine and antibodies as above.
Nanocomp lex Formation
[00711 Cationic liposome (DOTAP:DOPE) was prepared by the ethanol injection
method
as previously described (see U.S. Published Patent Application No.
2003/0044407; Xu L,
et al., Molecular Cancer Therapeutics /:337-346 (2002)).
When delivering plasmid DNA, the full complex
was formed in a manner identical to that previously described (see U.S.
Published Patent
Application No. 2003/0014107). To encapsulate the imaging agent for in vitro
use, the
TIRscEv was mixed with the liposome at a specific ratio and incubated at room
temperature for 10 minutes. Magnevist was added to this solution, mixed and
again
incubated at room temperature for 10 minutes. When stored at 2-8 C the complex
is
stable for at least 8 days, as determined by size measurements using a Malvem
Zetasizer
3000H. The cumulants (Z average) average of measurements over this time frame
is
112.3 4.67 (S.E.) while the polydispersity (representing the reproducibility
of the values
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during repeat scans) is 0.445 0.03. A range of acceptable sizes for the
nanocomplexes is
from about 20 to 1000 nm, suitably about 50 to 700 rim and more suitably about
100 to
500 rim. For in vitro transfection, 2 ml of serum-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 0.33-1.17 pg TfRscFv to 10-35 pg Liposome (suitably 1 mg imaging
agent to 0.5
to 1.0 1,kg TfRScFv to 14-28 Kg Liposome, most suitably 1 mg imaging agent to
0.71 gg
TfRscFv to 21 [ig Liposome) using the above procedure. When prepared for in
vivo use,
dextrose was added to a final concentration of 5%.
In Vitro Transfection
[0072] To transfect suspension cells K562, 15x106 cells in a total volume
of 4.0 ml of
medium 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,
containing
varying amounts of Magnevist , was added to the cell suspension. The plate was
incubated at 37 C with gentle rocking for the length of time given in the
Results section
(up to 90 min), after which the cells were gently pelleted (600xg for 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 solution and placed on wet ice until imaged.
In Vivo Tumor Targeting
[0073] To assess the tumor selective targeting of the TfRscFv-Lip
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 athymic nude mice by injection of 1x107 CaPan-1 cells
suspended in
MatrigelTM 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 x107 cells, also suspended in MatrigelTM were injected into
the
surgically exposed pancreas of female athymic nude mice. Five weeks post-
surgery, the
complex carrying the LacZ gene was i.v. injected 3X over 24 hrs (at 40 jig
DNA/injection). 60 hrs later the animals were sacrificed and examined for the
presence of
metastases and organs stained for 13-galactosidase expression using a
previously described
procedure (Xu, L., et al., Human Gene Therapy /0:2941-2952 (1999)).
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MR1 Imaging
[0074] For in vitro MRI imaging, the cell pellets in microcentrifuge tubes
were
positioned at the center of the magnet. The MR imaging was performed at Howard
University using a 4.7T horizontal bore NMR machine (Varian Inc, Palo Alto,
CA). The
imaging protocols consist of a multi-slice Ti -weighted spin echo imaging
sequence and a
saturation-recovery sequence. For the Ti -weighted imaging technique, the
repetition time
(TR) was 1000 ms, and the echo time (TE) was 13 ms. The Ti-weighted spin-echo
imaging technique was applied to verify the positive image enhancement. The
saturation-
recovery MR sequence with variable echo times was used for the Ti measurement.
The
slice thickness of images was 0.5mm. The RF coil employed was a 30 mm single
loop
coil. The RF coil serves as an RF transmitter and receiver. The RF pulse was a
selective
ms sinc pulse. The number of phase encoding steps was 256. The field-of-view
was
mm x 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
microcentrifuge tube. The height of the cell 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
Image
Browser software. The signal is taken from a region-of-interest, which is big
enough to
cover two thirds of the image from each microcentrifuge tube. The relative
image
intensities of the pellets from these tubes were applied for contrast
enhancement
evaluation and the Ti measurements.
[0075] For the in vivo studies, mice bearing CaPan-1 orthotopic tumors or
DU145
subcutaneous xenograft tumors were employed. The CaPan-1 tumors were induced
as
described above. DU145 tumors were induced by the subcutaneous inoculation of
7x106
cells in Matrigel. These studies were performed at Georgetown University.
Animals to be
imaged were anesthetized and placed in a proprietary, in-house designed,
animal
management system. This system incorporates a warm water heating system that
maintains the temperature at 37 C, as well as a four channel thermal optical
monitoring
system used to monitor animal's skin temperature, ambient temperature and wall
temperature of the device. For imaging, anesthesia was induced using
isoflurane at 4%,
with the remaining gas comprised of a 66% oxygen and 30% nitrous oxide
mixture.
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Maintenance of anesthesia was achieved with 1.5% isofiurane under similar
gaseous
conditions of oxygen and nitrous oxide as noted. The anesthetized animal was
positioned
inside of a cylindrical variable radiofi-equency 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 Ti-weighted Turbo RARE (rapid acquisition with rapid
enhancement)
three-dimensional imaging sequences performed on a 7T Bruker BioSpin
(Germany/USA) imaging console. The imaging parameters used were: Ti-weighted
Turbo-RARE 3D (3-dimensional), TE 13.3 ms, TR 229.5, Flipback on, 4 echoes
with a
field of view of 8.0/3.5/3.5 cm and a 256 x 256 x 256 matrix. After a baseline
image was
acquired, the animal was kept immobilized in the animal holder and the
Magnevist only
(diluted to 400 ul with lx Phosphate Bufferred Saline pH-7.4) or the Magnevist
-
comprising immunoliposome complex (TfRscFv-Lip-Mag) (total volume 400 1) was
systemically administered using a 27G needle by intravenous injection into the
tail vein
of the animal and the 3-D imaging sequence was immediately initiated. The
imaging with
the two solutions were performed on sequential days.
Scanning Electron Microscopy (SEM)
[00761 Sample solutions of liposome-encapsulated Magnevist contrast
agent, and
complete nanocomplex consisting of a tumor-targeting single-chain transferrin
receptor
protein coating the liposome-encapsulated complex, TfRscFv-Lip-Mag, were
prepared at
GLTMC, delivered to NIST and were stored under dark and refrigeration. For
each
imaging session, a fresh dilution 1:3 by volume with deionized water was
prepared and a
I, droplet was micropipetted onto a standard 200-mesh TEM grid consisting of
30-
60 nm formvar and 15-20 mu carbon. The droplet was allowed to dry on the grid
in air for
5 minutes before loading into the vacuum chamber of the microscope. Imaging
was
performed using an Hitachi S-4800 field-emission microscope at NIST. Of
particular
interest to applications of SEM to NDA imaging is a comparison of upper and
lower
secondary electron detectors [SE(U) and SEM} -- using the SEM in its usual
mode -- to
the addition of a transmitted electron (TE) detector, transforming the
instrument into a
low voltage STEM.
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Scanning Probe Microscopy (SPM)
[0077] Samples solutions of liposome-encapsulated Magnevise contrast
agent, and
complete nanocomplex were prepared at GUMC, delivered to NEST and were stored
under dark and refrigeration. For each imaging session, a fresh dilution 1:3
by volume
with deionized water was prepared and a 5 ul droplet was micropipetted onto an
untrasonically cleaned silicon substrate used with native oxide or with a poly-
L lysine
coating. SPM imaging were obtained using a Veeco MultiMode microscope with a
Nanoscope W controller. Topography by tapping mode with Z control (Veeco RTESP
cantilevers for ¨320-360 kHz and k 20-60 Wm), phase imaging, and magnetic
force
microscopy using magnetic coated tips (Veeco MESP 68 kHz) were performed in
life
mode. Dynamic imaging of dewetting and surface energy "phase separation" as
the
solution evaporates to expose isolated nanop articles 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 Ligand-Liposome Nanocomplex Carrying a
Reporter Gene
[0078] To assess selective targeting of the TfRscFv-LipA nanocomplex to
primary tumor
and metastases an orthotopic metastasis model, a closer approximation of the
clinical
situation, using human PanCa cell line CaPan-1 was employed. Surgical
orthotopic
implantations of CaPan-1 xenograft tumor sections into nude mice have been
shown to
produce within 56 days, metastases in liver and spleen (Alisauskus,R., et al.,
Cancer
Research 55:5743s-5748s (1995)). Orthotopic tumors of CaPan-1 were induced in
female
athymic nude mice as described in Methods. Approximately 5 weeks later, the
animals
were euthanized and necropsied to look for tumor in the pancreas and other
organs. As
shown in Figure 1A, extensive tumor growth is evident throughout the pancreas.
The
same tumor nodule in the liver indicated by an arrow in lA exhibits intense f3-
galactosidase expression in 1B. lA = gross necropsy; 1A = tissues after
staining for p-
galactosidase. Metastases were present in various organs in four of five mice
including
the spleen, liver, lung, adrenal gland and even within the diaphragm. This
experiment was
repeated with similar results.
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[0079] To establish selective targeting tumor and metastasis, prior to
sacrificing the mice,
the TfRscFv-LipA complex carrying pSVb (LacZ) plasmid DNA for 13-galactosidase
expression was i.v. injected into the mice three times over a 24 hour period
(40 ,g of
plasmid DNA per injection). All five mice were sacrificed 60 hours post-
injection and
various organs including the liver, lung, spleen, pancreas and diaphragm were
harvested
and examined for the presence of metastasis and tumor specific staining. Fresh
samples,
sliced at 1 mm thickness, were stained with X-gal to produce a blue color
where the gene
is expressed. The tumor targeting ability and high transfection efficiency of
the complex
is demonstrated by the presence of the reporter gene in the various organs
from this
animal (Figure 1B). In the liver, lung, adrenal gland and diaphragm it is
clearly shown
that the reporter gene is highly expressed only in the metastases, while no
blue color is
evident in the adjacent normal tissue. The metastasis visible in the liver in
Figure lA
(arrow) is the same tumor nodule strongly expressing 13-galactosidase in
Figure 1B
(arrow) confirming the tumor specific nature of this nanocomplex. In some of
the mice,
growth of the tumor in pancreas also resulted in extrusion of tumor through
the original
incision site used for implantation. In Figure 1B 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 the rest of the mice, and in the
repeat
experiment. Thus, this systemically administrAed nanocomplex will target tumor
cells
both primary and metastatic, wherever they occur in the body, and efficiently
deliver
plasmid DNA. We wished to expand the 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-Lip Complex to Deliver Magnevise'
[0080] As Magnevist is one of the most frequently employed contrast agent
in the clinic,
it was chosen as for use in these studies. In these initial experiments, it
was examined
whether the complex could be prepared with Magnevise and if doing so would
enhance
the MRI signal. Since trypsinization could lead to membrane damage and leakage
of
contrast agent from the cells, adherent cells were not employed in these
studies. Instead, a
human lymphoblastic leukemia cell line, K562, which grows as a suspension
culture was
used. Moreover, gentle pelleting and washing of the cells would remove any
excess
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Magnevist or complex prior to imaging, allowing only cell associated signal
to be
detected.
1. Time Dependent Image Enhancement by the TfRscFv-Lip-Mag
Nanocomplex
[0081] The optimal time for transfection of the TfRscFv-Lip-Magnevist
nanocomplex
was examined. The suggested clinical dose of Magnevist is 0.1 mMole/kg. In
these
initial studies a dose of 0.3 mMole/kg was used (corrected for the smaller
weight and
blood volume of mouse versus man) in the complex per 250 IA of transfection
solution.
K562 cells were transfected for times ranging from 20 to 90 minutes. Twenty
minutes
showed very low transfection activity based upon the image intensity. However,
as shown
in Figure 2A, by sixty minutes 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 than that of an empty marker
tube (194
43) indicating that the cells themselves do not contribute to the signal
detected. More
importantly, the transfection efficiency plateaus at approximately 60 minutes
since the
relative intensity of the cells transfected for 60 and 90 minutes were
identical (317 46
and 317 47, respectively).
2. Magnevist Dose Dependent Image Enhancement
[0082] Using 60 minutes as the transfection time, the effect of increasing
amounts of
Magnevist on the TfRscFv-Lip-Mag complex image enhancement was then assessed.
The doses tested were 0.05, 0.3 and 0.9 mMole/kg. Corrected for size and blood
volume
of the mouse, the volumes of Magnevist used in the complex per 250u1 of
transfection
solution were 0.25 p1, 1.5 p.1 and 4.5 p.1. As shown in Figure 2B and Table 1,
the image
intensity increases and the Ti relaxation time shortens as a function of the
amount of
contrast agent included in the complex.
Table 1: Relative Intensity and Ti Relaxation Time as a Function of Magnevist
in the
Immunoliposome Complex
Dose of Contrast Agent Relative Intensity Ti (seconds)
(m114/kg)
0.05 (0.25 j,t1) 293 50 1.43 0.007
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0.3 (1.5 IA) 379 1 43 1.16 0.004
0.9 (4.5 1) 454 1 51 1.01 0.004
3. Image Enhancement by TfRscFv-Lip-Mag as Compared to Free
Magnevist
[0083] Based upon the above experiments, it was shown that the TfRscFv-
Liposome can
complex with Magnevist and deliver it to the cells for image enhancement. To
assess the
level of enhancement of the complexed contrast agent as compared to the agent
alone and
demonstrate that the signal obtained is not due to the presence of
unincorporated
Magnevist , K562 cells were treated with either free Magnevist or the TiRscFv-
Lip-
Mag nanocomplex. The identical amount of contrast agent (0.3 ttM/kg or 1.5
t1/250
transfection volume) and transfection time (60 minutes) was used for both
solutions.
While free Magnevist showed enhanced contrast relative to the untreated cells
as
expected, the cells treated with the TfRscv-Lip-Mag complex demonstrated a
much
greater increase in image intensity and shortened Ti relaxation time compared
to both
untreated and free Magnevist V 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 that the observed signal is likely not due to
uncomplexed
Magnevist .
Table 2: Comparison of the Relative Intensity and Ti Relaxation Time Between
Free and
Immunoliposome Complexed Magnevist
Treatment Relative Intensity Ti (seconds)
Untreated 455 1 47 1.80 0.009
Free Magnevist 538 50 1.51
01007
Immunliposome Complexed 662 52 1.40
0.004
Magnevist
In Vivo Image Enhancement With TiRscFv-Lip-Mag
[0084] The above studies established that the nanocomplex could more
efficiently image
tumor cells in vitro than Magnevist alone. However, to have potential for
clinical use,
the complex must exhibit a similar effect in vivo. The same human pancreatic
cancer
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orthotopic mouse model (CaPan-1) was used for these studies as was used above
to
demonstrate tumor specific targeting of the complex carrying a reporter gene.
In addition,
a second tumor model, a subcutaneous prostate xenograft mouse model (DU145)
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
TI-weighted Turbo RARE (rapid acquisition with rapid enhancement) three-
dimensional
imaging sequence. To facilitate image alignment, after baseline acquisition
the animal
was maintained in 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 included in the complex was 10 id. This amount is equivalent to 0.2
mM/Kg or
twice what is used in humans. This amount 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 administration 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
adjusted to correct for an automatic windowing feature of the scanner.
[0085] Images of the Magnevist and nanocomplex-Mag in three separate mice
are show
in Figure 3A-I. In 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 site of the incision. A small abdominal mass was
also detected
by palpation. Not only is the signal in the subcutaneous tumor more enhanced
after
administration 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 . Reconstruction and
quantitation was
performed on the images in Figure 3 B, 3E and 311 and 3 C, 3F and 31,
representing the
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two different tumor models, Pancreatic cancer (CaPan-1) and Prostate cancer
(DU145). In
both instances, there is an increased intensity (pixels) by the free Magnevist
over the
baseline, as expected. However, delivery of the imaging agent by the tumor
targeting
nanocomplex results in an almost three-fold further increase in signal
intensity in both of
these tumor models. These studies thus demonstrate that when Magneviste is
incorporated within the TiRscFv-Liposome complex there is an improved tumor
visualization in an in vivo situation, and they suggest the potential benefit
of further
developing this means of tumor detection for clinical use.
Physical Characterization Studies
[0086] While the in vitro studies offered evidence that complexed
Magneviste is
encapsulated within the liposome, sophisticated microscopy techniques (SEM and
SPM)
have confirmed this and further characterize (e.g. complex size) the TiRscFv-
Lip-Mag
complex.
1.= e
Imaging of liposomes without Magnevist
[0087] High-resolution imaging implies narrow depth of focus and so
requires relatively
thin 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.
(Foo, J.J., et al., Annals of Biomedical Engineering 31:1279-1286 (2003)). So
an
understanding of reliable methods for preparing and characterizing the
dimensional and
mechanical 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 that the contrast agent is indeed
contained within
the nanoparticle and not simply associated externally with the liposomes.
[0088] 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 maintains the oscillation of the cantilever at a constant amplitude.
This constant
amplitude is given a by a set point which is somewhat smaller than that of the
freely
oscillating cantilever. Since the SPM tip interacts with the surface 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 inhomogeneous surface, the tip-surface
interactions will vary
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according to surface charge, steep topographical changes, and mechanical
stiffness
variations, for example. By changing the set point and observing how certain
features
respond to softer or harder tapping, we can correlate this with the response
expected for a
specific structure such as a liposome. (The free oscillation amplitude signal
is
approximately 1.78 V.) A sequence of SPM 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
indicates that the tip-sample interaction is attractive for the liposome,
given by a phase
value of -3.5 degrees. In the 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 the effect of reducing the set point to
1.45 V: The
liposome now appears bright since the tip-sample interaction becomes
repulsive, and in
this case the phase difference between the liposome and substrate is +8
degrees. Finally,
Figure 4C shows that the phase difference recorded at a set point of 1.35 V
increases
further, becoming +35 degrees.
2. Imaging of liposome-encapsulated Magnevist
[00891 Figure 5A-C presents SPM and SEM images of isolated liposome-
encapsulated
Magnevist (Lip+Mag) nanoparticles. The size distribution of single Lip+Mag
particles is
in the range of 100-200 nm diameter and scales according to optical
measurements that
indicate that payload-encapsulating liposomes are approximately 50% larger
than
liposomes alone in their spherical state.
[0090] The SPM topograph appearing in Figure 5A indicates that liposomes
containing
Magnevist have a bimodal surface shape after drying that is more complex than
that of
the simple elliptical surface of a liposome containing no payload (not shown).
The SPM
phase behavior differs markedly from that 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 both attractive and repulsive tip-sample interaction
appear at
moderate set point values. A correlation between the SPM phase image obtained
at a set
point of 1.6 and the SEM image in TE mode is evident in Figures 5B and 5C.
Liposomes
appear uniformly bright across the entire particle in SEM images (not shown),
similar to
the uniform 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
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instabilities due to foreign material on the tip. Thus, they are changed
frequently. Since
each cantilever is somewhat different with respect to its resonance
properties, the set
points used in Figures 4 and 5 are different.
3. Imaging of TfRscFv-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 6B indicate
that the
solvent film 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 scanned several times before
beam damage
becomes significant. The appearance of the full complex is different from that
of the
(Lip+Mag) only. The shape is less regular, and considerable texturing of the
liposome
surface following drying is consistent with protein denaturation. Also, SEM TE
images
indicate that the well-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 film 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 gadolinium-containing Magneviste is quite large, it should
be
possible using a magnetized SPM tip to interact with the oriented Magneviste
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
capabilities of the SPM it is established that the produced image is truly
magnetic in
nature. In this mode, a topographic image under normal tapping mode conditions
is
obtained. The reference surface information is then 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 the influence of topography on the signal. MFM images obtained in lift-
mode at
a height of 15 nm or 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 7A is an SPM topographic/magnetic phase image of the full
TfRscFv-
Lip-Mag nanocomplex. The appearance of a double dipole-like signal in Figure
7B
consisting of attractive and repulsive in-plane magnetic interactions suggests
that the
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cause of this interaction is the nonuniform toroidal distribution of Magnevist
within the
NDS, 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 both in vitro and in
an
orthotopic animal model and in doing so produce a more defined and intense
image than
seen with uncomplexed Magnevist.
[0094] As shown in Figure 1, the nanocomplexes of the present invention
can target
metastatic disease, thereby enhancing detection sensitivity for metastases.
Using SEM
and SPM it has been demonstrated that the TfRscFv-liposome complex maintains
its
nanometer size when Magnevist is encapsulated (particles of approximately 100-
200 nm
are shown in Figure 6 and 7). It has also been demonstrated that the
structural and
mechanical properties of liposomes containing a payload are sufficiently
different from
those without one, thereby confirming that Magnevist is indeed encapsulated
with the
liposome. This was further confirmed by MFM imaging of the 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 liposome-confined phase separation, i.e., formation of a dense
Magnevist
lipidtoroidal distribution around the periphery of the particle with an
preferential aqueous
phase at the particle's center. This response is probably attributable to
several important
factors: First, the properties of Magnevist solution are pH 6.5-8, an
osmolality of
1,960 and viscosity of 4.9 at 20 C according to the manufacturer. A plausible
chemical
basis for this separation of the solution noted in the Magnevist data sheet:
The
meglumine salts dissociate completely from the complex, so changes in the
local osmotic
conditions. Coupled with the charge interaction of the gadolinium complex and
cationic
lipid, these interactions may provide a strong driving force for a hypertonic
phase
separation within the liposome. The charge distribution between the cationic
lipid and
Magnevist solution is effective at stabilizing the liposome and providing
structural
support in solution, and apparently in the bloodstream. This enhanced
structural support is
an important benefit for these studies since it allows most particles to
remain intact during
the film drying process, in contrast to the extensive decomposition observed
with the
liposome only solutions.
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[00961 The foregoing Examples demonstrate the successful encapsulation of
an MR
contrast agent in the immunoliposome complexes of the present invention. The
image
enhancement demonstrated by the complexes over conventionally delivered
Magneviste
indicates the ability of this system to improve early detection of cancer via
MR1.
Example 2
Comparison of Imaging in Different Cell Lines
[00971 Figures 8A-8H show improved MR imaging in two different models of
cancer
using the Ligand-HK-Liposome-Mag nanocomplex. Nanocomplexes for use in this
Example were prepared using the same ratios and procedures as set forth in
Example 1.
Human breast cancer MDA-MB-435 (Figure 8E-8H) or human prostate cancer cell
line
(DU145) (Figure 8A-8D) cells were subcutaneously injected on the lower back,
of female
athymic nude mice. Free Magnevist -, or the TfRscFv-liposome nanocomplex
(scLip-
Mag), or the TIRscFv-HK-liposome nanocomplex (scLip-HK-Mag) comprising the
HoKc
peptide, containing the same dose of Magnevist were i.v. 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 that would be administered to a human patient.
The total
volume of solution administered in all cases was 400 I. A baseline scan was
performed
just prior to administration of both nanocomplexes to confirm that all of the
Magneviste
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
mouse with a subcutaneous tumor in which the increased definition and contrast
are
evident in both 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 scLip-HK-Mag.
[00981 Figure 9A-9C shows tumor-specific targeting of a CaPan-1
subcutaneous tumor
and orthotopic metastasis model by the TfRscFv-HK-Liposome-Mag nanocomplex.
Subcutaneous CaPan-1 xenograft tumors were induced in female athyrnic nude
mice as
described in Methods in Example 1. The tumors were harvested and a single cell
suspension in MATRIGELe was injected into the surgically exposed pancreas.
Eight
weeks post injection the TfRscFv-Liposome complex with or without HoKC (HK)
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peptide carrying Magnevise was injected into the mouse on two consecutive
days. The
total volume of solution administered in all cases was 400 iii. A baseline
scan was
performed just prior to administration of the nanocomplex to confirm that all
of the
Magnevise 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
imaging 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 DU14S
% Increase Over Baseline
Complexed Maguevist 99 215
Free Magnemt 34.5 70
Example 3
Comparison of Dynamic MM Scans of Subcutaneous PANC-I Tumors after Systemic
Injection of Free (Unconzplexed) or TfRScFv-Lip-Magnevist
[00991 The following experiments were performed to compare the 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
athymic nude mice by injection of 1 to 2x107 PANC-1 cells suspended in
Matrigel Tt
collagen basement membrane matrix (BD Biosciences). Approximately 2.5-3 weeks
later, the animals were used for imaging. Cationic liposome (DOTAP:DOPE) was
prepared by the ethanol injection method as previously described (see U.S.
Published
Patent Application No. 2003/0044407; Xu L, et al, Molecular Cancer
Therapeutics
/:337-346 (2002)).
The targeting moiety used in these studies is the anti-transferrin receptor
single chain
antibody fragment (TfRscFv).
[0100] To encapsulate the imaging agent, the TIRscFv was mixed with the
liposorue at a
specific ratio and incubated at room temperature for 1- 30 minutes, suitably 5-
20 minutes,
most suitably 10-12 minutes. Magrievist was added to this solution, mixed and
again
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incubated at room temperature for 1- 30 minutes, suitably 5-20 minutes, most
suitably 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 minutes, suitably 5-25
minutes,
most suitably 15-20 minutes. The complex is formed at a ratio of lmg imaging
agent to
0.33-1.17 ug TfRscFv to 10-35 ug Liposome (suitably lmg imaging agent to 0.5
to 1.0ug
TfRScFv to 14-28 ug Liposome, most suitably lmg imaging agent to 0.71 ug
TfRscFv to
2lug Liposome) using the above procedure. A range of acceptable sizes of the
complex
is from about 20 to 1000 nm, suitably about 50 to 700 urn and most suitably
about 100 to
500 urn. Here the complex was formed using 4.7 mg Magnevist, 99 ug Liposome
and 3.3
ug TfRscFv with dextrose to a final concentration of 5%.
[0101] A mouse bearing PANC-1 subcutaneous tumors induced as 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 mixture. Maintenance of anesthesia was achieved with 1.0 to 2.0%
isoflurane
(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 Tesla). The imaging protocol used was Ti-weighted two
dimensional
Turbo RARE (rapid acquisition with rapid enhancement) imaging sequences
performed
on a 7T Braker BioSpin (Germany/USA) imaging console. The imaging parameters
used
were: Ti-weighted 2D (2-dimensional), TE 10.21 ins, TR 420.3, Flipback off, 8
echoes
with a field of view of 5.12/5.12 cm. After a baseline image was acquired, the
animal
was kept immobilized in the animal holder and either the free (uncomplexed)
Magnevise
(Mag, or gad-d herein) or the TfRscFv-Lip-Mag complex containing the identical
amount
of Mag (total volume 50-1000 ul, suitably 100-50 Oul, most suitably 200-400
ul) was
systemically administered using a 27G needle by intravenous injection into the
tail vein
of the animal and the imaging sequence was immediately initiated. The scan (2
averages,
1.3min) was repeated periodically over two hours and the pixel intensity
measured and
plotted. The same mouse was used for imaging with both the free and the
complex. The
imaging was performed on sequential days, with the Free Mag first.
CA 02638899 2013-04-09
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[0102] As Shown in Figure 10, there is a significantly higher level signal
in the tumor
after intravenous injection of the complex as compared to the free imaging
agent. More
significantly, this higher level is maintained over the time course of the
experiment.
Example 4
Detection of CaPan-1 Liver Metastasis by TffiscFv-Lip-HoKC-Magnevist
[01031 The following experiments were performed to assess the ability of
the TIRseFv-
Lip-HoKC-Mag complex of this invention to detect and enhance imaging of
metastatic
tumors. As an example, metastases from a pancreatic cancer was examined,
however,
imaging of metastases from any type of cancer can be achieved using the
complexes and
methods of the present invention (e.g. prostate, melanoma, renal, breast,
gastric, liver,
ovarian, bladder, head and neck, brain, bone and any other type of solid
tumor).
Subcutaneous xenograft tumors of CaPan-1 were induced in female athymic nude
mice
by injection of 0.5 to 1x107 CaPan-1 cells suspended in Matrigelm 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
x107 cells, also
suspended in MatrigelTm, were injected into the surgically exposed pancreas of
female
athyrnic nude mice as previously described (Alisauslcus, R., Wong, G.Y., and
Gold, D.V.,
Initial studies of monoclonal antibody PAM4 targeting to xenografted
orthotopic
pancreatic cancer, Cancer Research 55, 5743s-5748s (1995)).
[01041 Cationic liposome (DOTAP:DOPE) was prepared by the ethanol injection
method
as previously described (see U.S. Published Patent Application No.
2003/0044407; Xu L,
et at., Molecular Cancer Therapeutics /:337-346 (2002)).
The HoKC peptide {K[K(H)ICKK15-K(H)KKC)
(SEQ JD NO:1) carries a terminal cysteine to permit conjugation to a maleimide
group.
Thus, when the HoKC peptide was used, the liposome formulation also included 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
lipid. The HoKC Liposomes were prepared as previously described (Yu, W. et al.
Enhanced transfection efficiency of a systemically delivered tumor-targeting
immunolipoplex by inclusion of a pH-sensitive histidylated oligolysine
peptide, Nucleic
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Acids Research 32, e48 (2004)). The targeting moiety used in these studies is
the anti-
transferrin receptor single chain antibody fragment (TfRscFv).
[0105] To encapsulate the imaging agent, the TfRscFv was mixed with the
liposome at a
specific ratio and incubated at room temperature for 1- 30 minutes, suitably 5-
20 minutes,
most suitably 10-12 minutes. Magnevist was added to this solution, mixed and
again
incubated at room temperature for 1- 30 minutes, suitably 5-20 minutes, most
suitably 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 minutes, suitably 5-25
minutes,
most suitably 15-20 minutes. The complex is formed at a ratio of lmg imaging
agent to
0.33-1.17 ug TfRscFv to 10-35 ug Liposome-HoKC (suitably lmg imaging agent to
0.5
to 1.0ug TfRScFv to 14-28 ug Liposome-HoKC, most suitably lmg imaging agent to
0.71
ug TfRscFv to 2lug Liposome-HoKC) using the above procedure. A range of
acceptable
sizes of the complex is from about 20 to 1000 urn, suitably about 50 to 700nm
and most
preferable 100 to 500 urn. Here the complex was formed using 4.7 mg Magnevist
, 99 ug
Liposome-HoKC and 3.3ug TfRscFv with dextrose to a final concentration of 5%.
[0106] Mice bearing CaPan-1 orthotopic tumors induced above (approximately
12 weeks
post-surgical implantation of the tumor cells) were 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 mixture. Maintenance of
anesthesia
was achieved with 1.0 to 2.0% isoflurane (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 Tesla). The
imaging
protocol used was Ti-weighted Turbo RARE (rapid acquisition with rapid
enhancement)
three-dimensional imaging sequences performed on a 7T Bruker BioSpin
(Germany/USA) imaging console. The imaging parameters used were: Ti-weighted
Turbo-RARE 3D (3-dimensional), TB 13.3 ms, TR 229.5 ms, Flipback on, 4 echoes
with
a field of view of 8.0/3.5/3.5 cm and a 256 x 256 x 256 matrix. After a
baseline image
was acquired, the animal was kept immobilized in the animal holder and the
TfRscFv-
Lip-HoKC-Mag complex (total volume 50-1000 ul, more preferably 100-500u1, most
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preferably 200-400u1) was systemically administered using a 27G needle by
intravenous
injection into the tail vein of the animal and the 3D imaging sequence was
immediately
initiated.
[0107] After imaging, the animal was euthanized and visually examined for
the presence
of metastases. The liver was removed, fixed in Formalin, paraffin embedded,
sectioned
and stained using H&E using standard procedures well know to one of ordinary
skill in
the art. The sections were examined by microscope and the observed metastasis
photographed.
[0108] Figure 11A-11A: Figure 11A: pre-contrast. Figure 1118: TfRcFv-Lip-
HoKC-Mag
injection, Figure 11C: histology. The orthotopic pancreatic cancer shows
enhancement
with TfRav-Lip-HoKC-Mag (short white arrows). The two areas identified with
the
short white arrows are connected on more posterior slices and represent the
primary
orthotopic placed tumor. A small metastasis (thick white arrows) enhances in
the same
pattern seen with the primary tumor. The thin extension of liver (long thin
arrow) lies
adjacent to the metastasis. Necropsy (not shown) and histology (right image)
confirm
presence of metastasis (black arrows) directly adjacent to long thin extension
of liver.
Note the similarity of shape of one of the pieces of metastatic tumor to the
appearance on
the MRI.
Example 5
Enhanced Detection of Lung Metastasis by TfRscFv-Lip-Magnevist
[0109] The following experiments were performed to demonstrate that when
administered intravenously (or via any other appropriate route, e.g., but not
limited to IT,
IM, IP) the complexes of the present invention carrying an imaging agent can
enhance
detection of metastases as compared to when the imaging agent is administered
without
the use of the complex. Lung tumors were induced in female Balb/C mice by the
intravenous injection of 1 to 10x104 RenCa cells. This method results in
metastases that
reside almost exclusively in the lungs of the animals and thus serves as a
model system
CA 02638899 2013-04-09
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for any other type of cancer that results in lung tumors either as primary
disease or as
metastases. Approximately 2-4 weeks later the animals were used for imaging.
[0110] Cationic liposome (DOTAP:DOPE) was prepared by the ethanol injection
method as previously described (see U.S. Published Patent Application No.
2003/0044407; Xu L, et at, Molecular Cancer Therapeutics /:337-346 (2002)).
The targeting moiety
used in these studies is the anti-trausferrin receptor single chain antibody
fragment
(TfRscFv).
[01111 To encapsulate the imaging agent, the TiltscRv was mixed with the
liposome at a
specific ratio and incubated at room temperature for 1- 30 minutes, suitably 5-
20 minutes,
most suitably 10-12 minutes. Magnevist* was added to this solution, mixed and
again
incubated at room temperature for 1- 30 minutes, suitably 5-20 minutes, most
suitably
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 minutes, more suitably 5-
25
minutes, most suitably 15-20 minutes. The complex is formed at a ratio of Img
imaging
agent to 0.33-1.17 ug TfRscFv to 10-35 ug Liposome (suitably lmg imaging agent
to 0.5.
to 1.Oug TfR.Sav to 14-28 ug Liposome, most suitably Img imaging agent to 0.71
ug
TfRscFv to 2Ing Liposome) using the above procedure. A range of acceptable
sizes of
the complex is from about 20 to 1000 nm, suitably about 50 to 700nrn and most
suitably
about 100 to 500 rim. Here the complex was formed using 4.7 mg Magnevist, 99ug
Liposome and 3.3ug TfRscFv with dextrose to a final concentration of 5%.
[01121 A mouse bearing lung 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 mixture.
Maintenance of
anesthesia was achieved with 1.0 to 2.0% isoflurane (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
Tesla). The
imaging protocol used was TI-weighted two dimensional Turbo Multislice-
Multiecho
imaging sequence performed on a 7T Bruker BioSpin (Germany/USA) imaging
console.
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The imaging parameters used were: Ti-weighted 2D (2-dimensional), TB 10.21 ms,
TR.
400 ms, Flipback off, 8 averages with a field of view of 3.84 x 3.84 cm and a
256 x 256
matrix. After a baseline image was acquired, the animal was kept immobilized
in the
animal holder and either the free (uncomplexed) Magnevise (gad-d) or the
TfRscFv-Lip-
Mag complex containing the identical amount of Mag (total volume 50-1000 ul,
more
suitably 100-500u1, most suitably 200-400u1) was systemically administered
using a 270
needle by intravenous injection into the tail vein of the animal and the
imaging sequence
was immediately initiated. The pixel intensity of the images was measured and
plotted.
The same mouse was used for imaging with both the free and the complex. The
imaging
was performed on sequential days.
[0113] As Shown in Figure 12A-12E, there is a significantly higher level
signal in the
tumor after intravenous injection of the complex as compared to the free
imaging agent.
Thus, the complex of this invention also enhances detection of relatively
large metastases
in the lung as compared to the currently used method of administering free
imaging agent.
Example 6
Enhanced Detection of Small Lung Metastasis by TfltscFv-Lip-Magnevist
[ana] The following experiments were performed to demonstrate that when
administered intravenously (or via any other appropriate route, e.g., but not
limited to IT,
ID, 1M, 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 Balb/C mice
by the
intravenous injection of 1 to 10x104 RenCa cells. This method results in
metastases that
reside almost exclusively in the lungs of the animals and thus serves as a
model system
for any type of cancer that results in lung tumors either as primary disease
or as
metastases. Approximately 7-9 days later the animals were used for imaging.
[0115] Cationic liposome (DOTAP:DOPE) was prepared by the ethanol injection
method
as previously described (see U.S. Published Patent Application No.
2003/0044407; Xu L,
et aL, Molecular Cancer Therapeutics 1:337-346 (2002)).
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The targeting moiety used in these studies is the
anti-transferrin receptor single chain antibody fragment (TiRscFv).
[01161 To encapsulate the imaging agent, the TfRscEv was mixed with the
liposome at a
specific ratio and incubated at room temperature for 1- 30 minutes, suitably 5-
20 minutes,
most suitably 10-12 minutes. Magnevise was added to this solution, mixed and
again
incubated at room temperature for 1- 30 minutes, suitably 5-20 minutes, most
suitably 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 I- 30 minutes, suitably 5-25
minutes,
most suitably 15-20 minutes. The complex is formed at a ratio of lmg imaging
agent to
0.33-1.17 ug TfascEv to 10-35 ug Liposome (suitably lmg imaging agent to 0.5
to 1.0ug
TIR.ScFv to 14-28 ug Liposome, most suitably lmg imaging agent to 0.71 ug
TfRscFv to
2lug Liposome) using the above procedure. A range of acceptable sizes of the
complex
is from about 20 to 1000 rim, suitably about 50 to 700nm and most suitably
about 100 to
500 em. Here the complex was formed using 4.7 mg Magnevist, 99ug Liposome and
3.3ug TfRscEv with dextrose to a final concentration of 5%.
[01171 A mouse bearing lung 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 mixture.
Maintenance of
anesthesia was achieved with 1.0 to 2.0% isoflurane (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
Tesla). The
imaging protocol used was TI-weighted two dimensional Turbo Multislice-
Multiecho
imaging sequence performed on a 7T Bruker BioSpin (Germany/USA) imaging
console.
The imaging parameters used were: TI -weighted 2D (2-dimensional) imaging
sequence,
TB 10.21 ms, TR 572.99ms, Flipback off, 8 averages with a field of view of
2.56 x 2.56
cm and a 256 x 256 matrix. After a baseline image was acquired, the animal was
kept
immobilized in the animal holder and either the free (uncomplexed) Magnevist
(gad-d)
or the TfR.scFv-Lip-Mag complex containing the identical amount of Mag (total
volume
50-1000 ul, suitably 100-500u1, most suitably 200-400u1) was systemically
administered
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using a 27G needle by intravenous injection into the tail vein of the animal
and the
imaging 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 imaging was performed on sequential days. At this field of view 5 pixels
is
equivalent to approximately a 3 mm human tumor detected by CT.
[0118] As Shown in Figure 13A-13D, a metastasis of 4 pixels (lower arrow)
(which
corresponds to a metastasis of approximately 0.4 mm in diameter) was
detectable after
injection with the complex but not after the 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 this invention also enhances detection of
small
metastases in the lung as compared to the currently used method of
administering free
imaging agent. An even smaller metastasis of approximately 3 pixels
(equivalent to a
tumor of approximately 0.3 mm in diameter) was also detected using the complex
of this
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 T1 -weighted 2D (2-dimensional) Mutltislice-
Multiecho imaging sequence, TE 10.21 ms, with TR = 630.8ms, Flipback off, 8
averages
with a field of view of 2.56 x 2.56 cm and a 256 x 256 matrix. As shown in
Figure 15A-
15B, nodules of 1-2 pixels were detectable by the complex. Nodules Ni 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, maximum, mean values and standard deviation (SD) was
determined
for the two nodules. Statistically, if the max of the nodule was greater than
the max of the
base +2SD of the base, there is a 95% confidence that the nodule is not noise
but is real.
Nodule 2 is clearly within this 95% confidence and Nodule 1 is just at the
limit, thus it
too is most likely a real tumor mass enhanced by the complex. After imaging
the lungs
from this animal were removed, fixed in Formalin, paraffin embedded, sectioned
and
stained using H&E using standard procedures well know to one of ordinary 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 0.1min were found in the same lobe
and
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approximate location as expected based upon the MRI. The distance between the
two
nodules was measured on the MRI image and was found to be equivalent (-600nm)
to
that 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 lung
metastases is greater
than that currently seen with CT, the commonly used method of detection of
primary
tumors of the lung and lung metastases derived from other cancer types.
Clearly this
represents and unexpected and surprising result.
Example 7
Detection of Sub-Pleural 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 lung as shown in Figure 18A-18F. This is unexpected and
surprising
since current MRI imaging with non-complexed agents which do not actually
enter the
cell are not able to detect metastases in this location. This provides a
significant
advantage in early detection and treatment of lung and other types of cancer.
Example 8
Enhanced Detection of Melanoma Lung Metastasis by TfRscFv-Lip-Magnevist
[0121] With respect to detection/treatment of pleural metastases, clinical
control is very
difficult to achieve and measurement of benefit is also difficult. 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 this invention could be the imaging tool employed to measure effectiveness
of this, or
any other therapy.
[0122] The following experiments were performed to demonstrate that when
administered intravenously (or via any other appropriate route, e.g., but not
limited to IT,
ID, IM, IP) the complexes of the present invention carrying an imaging agent
can detect
metastases that are not limited to those from renal cell carcinomas. Lung
tumors were
induced in female C57/131 6 mice by the intravenous injection of 0.1 to 5x105
B16/F10
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mouse melanoma cells. This method results in metastases that reside almost
exclusively
in the lungs of the animals and thus serves as a model system for any type of
cancer that
results in lung tumors either as primary disease or as metastases.
Approximately 2-4
weeks later, the animals were used for imaging.
[01231 Cationic liposome (DOTAP:DOPE) was prepared by the ethanol injection
method
as as previously described (see U.S. Published Patent Application No.
2003/0044407; Xu
L, et aL, Molecular Cancer Therapeutics 1:337-346 (2002)).
The targeting moiety used in these studies is
the anti-transferrin receptor single chain antibody fragment (TfRscFv).
[01241 To encapsulate the imaging agent, the TfRscFv was mixed with the
liposome at a
specific ratio and incubated at room temperature for 1- 30 minutes, suitably 5-
20 minutes,
most suitably 10-12 minutes. Magnevist was added to this solution, mixed and
again
incubated at room temperature for 1- 30 minutes, suitably 5-20 minutes, most
suitably 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 I- 30 minutes, suitably 5-25
minutes,
most suitably 15-20 minutes. The complex is formed at a ratio of lmg imaging
agent to
0.33-1.17 ug TfRscFv to 10-35 ug Liposome (suitably Img imaging agent to 0.5
to I.Oug
TfRScFv to 14-28 ug Liposome, most suitably lmg imaging agent to 0.71 ug
TfRscFv to
2 lug Liposome) using the above procedure. A range of acceptable sizes of the
complex
is from about 20 to 1000 um, suitably 50 to 70- nm and most suitably 100 to
500 nm.
Here the complex was formed using 4.7 mg Magnevist, 99ug Liposome and 3.3ug
TfRscFv with dextrose to a final concentration of 5%.
[01251 A mouse bearing lung 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 mixture.
Maintenance of
anesthesia was achieved with 1.0 to 2.0% isoflurane (preferably 1.5%) under
similar
gaseous conditions of oxygen and nitrous oxide as noted. The anesthetized
animal was
positioned inside of a cylindrical variable radio frequency 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
TesIa). The
imaging protocol used was Ti-weighted two dimensional Turbo Multislice-
Multiecho
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imaging sequence performed on a 7T Bruker BioSpin (Germany/USA) imaging
console.
The imaging parameters used were: Ti-weighted 2D (2-dimensional) imaging
sequence,
TE 10.21 ms, TR 1418.13ms, Flipback off, 8 averages with a field of view of
3.84 x 3.84
cm and a 256 x 256 matrix. After a baseline image was acquired, the animal was
kept
immobilized in the animal holder and either the free (uncomplexed) Magnevist
(gad-d)
or the TfRscFv-Lip-Mag complex containing the identical amount of Mag (total
volume
50-1000 ul, suitably 100-500u1, most suitably 200-400u1) was systemically
administered
using a 27G needle by intravenous injection into the tail vein of the animal
and the
imaging 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 imaging was performed on sequential days.
[0126] As Shown in Figure 19A-19B, two small metastases (arrows) were
detected in the
lungs after 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 Image J software after baseline, after Free Magnevist
and after
TfRscFv-Lip-Mag and the values compared. As shown in Table 4 below, the
complex
showed the greatest enhancement over the baseline 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
Treatment Maximum Pixel Average Pixel Value Standard Deviation
Value
Baseline 12888 7765.1 1757.2
Free Magnevist 17959 12979.3 2976.8
Complexed Magnevist 22351 14341.3 2384.6
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