U.S. patent application number 16/046748 was filed with the patent office on 2018-11-29 for therapeutic and diagnostic probes.
This patent application is currently assigned to LI-COR, Inc.. The applicant listed for this patent is LI-COR, Inc.. Invention is credited to David L. Dilley, Joy Kovar.
Application Number | 20180339048 16/046748 |
Document ID | / |
Family ID | 54700548 |
Filed Date | 2018-11-29 |
United States Patent
Application |
20180339048 |
Kind Code |
A1 |
Dilley; David L. ; et
al. |
November 29, 2018 |
Therapeutic and Diagnostic Probes
Abstract
The present invention provides compositions and methods of use
of nanoparticle-based probes for in vivo imaging and therapy. The
probes can be used to track diseased target cells by non-invasive
imaging in the near-infrared range. Additionally, the probes can
induce cell death of the target cells via photodynamic
treatment.
Inventors: |
Dilley; David L.; (Lincoln,
NE) ; Kovar; Joy; (Lincoln, NE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LI-COR, Inc. |
Lincoln |
NE |
US |
|
|
Assignee: |
LI-COR, Inc.
Lincoln
NE
|
Family ID: |
54700548 |
Appl. No.: |
16/046748 |
Filed: |
July 26, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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14728247 |
Jun 2, 2015 |
10064943 |
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16046748 |
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62006790 |
Jun 2, 2014 |
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62017165 |
Jun 25, 2014 |
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62066807 |
Oct 21, 2014 |
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62082052 |
Nov 19, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 41/0071 20130101;
A61K 47/6415 20170801; A61P 17/06 20180101; A61M 1/3683 20140204;
A61K 51/0497 20130101; A61K 49/0036 20130101; A61K 47/6849
20170801; A61K 49/0032 20130101; A61K 51/082 20130101; A61P 9/10
20180101; A61P 17/10 20180101; A61P 31/04 20180101; A61K 47/64
20170801; A61P 35/00 20180101; A61P 9/00 20180101; A61P 27/02
20180101; A61M 1/3686 20140204; A61K 51/0446 20130101; A61K 49/005
20130101; A61P 17/00 20180101; A61P 43/00 20180101; A61K 41/10
20200101; A61K 51/083 20130101; A61K 47/642 20170801; A61K 51/088
20130101; A61N 2005/0658 20130101; A61N 5/062 20130101; A61M 1/3618
20140204 |
International
Class: |
A61K 41/00 20060101
A61K041/00; A61N 5/06 20060101 A61N005/06; A61M 1/36 20060101
A61M001/36; A61K 51/08 20060101 A61K051/08; A61K 51/04 20060101
A61K051/04; A61K 49/00 20060101 A61K049/00; A61K 47/68 20170101
A61K047/68; A61K 47/64 20170101 A61K047/64 |
Claims
1. A probe comprising a nanocarrier attached to both (1) an imaging
agent and (2) a therapeutic agent, which is IRDYE700DX.
2. The probe of claim 1, wherein said nanocarrier is selected from
a group consisting of a virus-like particle, a nanoparticle, a
liposome, a quantum dot, and a combination thereof.
3. The probe of claim 2, wherein said nanoparticle is selected from
the group consisting of a gold nanoparticle, a magnetic
nanoparticle, a polymeric nanoparticle, a carbon nanotube, an
inorganic nanoparticle, and a combination thereof.
4. The probe of claim 2, wherein said virus-like particle comprises
a L1 capsid protein, L2 capsid protein, or a combination
thereof.
5. The probe of claim 1, wherein said nanocarrier further comprises
a targeting agent.
6. The probe of claim 5, wherein said targeting agent is an
antibody or fragment thereof, a nanobody, an Affibody.RTM., a
diabody, a minibody, an antigen, a ligand, a protein, a eptide, a
nucleic acid or a small molecule.
7. The probe of claim 1, wherein said therapeutic agent is attached
to said nanocarrier via a first linker and wherein said imaging
agent is attached to said nanocarrier via a second linker.
8. The probe of claim 1, wherein said imaging agent is a cyanine
dye.
9. A method for detecting a target cell in an individual, said
method comprising: (a) contacting said target cell with a probe
comprising a nanocarrier attached to both (1) an imaging agent and
(2) a therapeutic agent, which is IRDYE700DX, wherein the probe
selectively associates with the said target cell; and (b) detecting
said imaging agent thereby providing an indication of the presence
and/or location of said target cell in the individual.
10. The method of claim 9, wherein said imaging agent is a cyanine
dye.
11. The method of claim 9, wherein said contacting comprises
systemic administration to a subject, local administration to a
tumor, or administration to a surgical site.
12. The method of claim 9, wherein said target cell is a cell of a
solid tumor.
13. The method claim 9, further comprises exposing said therapeutic
agent to a photoactivating light, thereby inducing apoptosis and/or
necrosis of said target cell in the individual.
14. A method for inducing cell death in a target cell in an
individual, said method comprising: (a) contacting said target cell
with a probe comprising a nanocarrier attached to both (1) an
imaging agent and (2) a therapeutic agent, which is IRDYE700DX,
wherein the probe selectively associates with said target cell; and
(b) exposing said therapeutic agent to a photoactivating light,
thereby inducing apoptosis and/or necrosis of said target cell in
the individual.
15. The method of claim 14, wherein said contacting comprises
systemic administration to a subject, local administration to a
tumor, or administration to a surgical site.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S. Ser. No.
14/728,247 filed Jun. 2, 2015 (Allowed); which claims priority to
U.S. Provisional Patent Application Nos. 62/006,790 filed Jun. 2,
2014; 62/017,165 filed Jun. 25, 2014; 62/066,807 filed Oct. 21,
2014; and 62/082,052 filed Nov. 19, 2014, the teachings all of
which are hereby incorporated by reference in their entireties for
all purposes.
BACKGROUND OF THE INVENTION
[0002] Photodynamic therapy (PDT) is a clinically approved and
rapidly evolving treatment regimen for disease including cancer,
cardiovascular disease, dermatological diseases and ophthalmic
disease. PDT traditionally involves administration of a
photosensitizer that preferentially accumulates within a target
tissue site. Following illumination of the tissue site with light
of an appropriate wavelength, in the presence of molecular oxygen,
reactive oxygen species, such as singlet oxygen, free radicals and
peroxides are produced which, in turn, damage cellular structures
containing the photo-sensitizer. The effects are localized to the
vicinity of the target tissue and within a few millimeters of the
light source, which minimizes systemic and normal tissue
toxicity.
[0003] Both disease treatment and detection depend on the selective
delivery of appropriate agents to the affected tissue site. Current
photodynamic therapeutic agents lack effective tissue localization.
There remains a need for compositions and methods to detect and
treat target cells and/or tissues in a non-invasive manner. The
present invention satisfies these and other needs.
BRIEF SUMMARY OF THE INVENTION
[0004] In one aspect, provided herein is a probe comprising a
nanocarrier attached to an imaging agent and a therapeutic agent.
In some embodiments, the nanocarrier is selected from a group of a
virus-like particle, a nanoparticle, a liposome, a quantum dot, or
a combination thereof. In some instances, the nanoparticle is
selected from the group consisting of a gold nanoparticle, a
magnetic nanoparticle, a polymeric nanoparticle, a carbon nanotube,
an inorganic nanoparticle, or a combination thereof.
[0005] In certain instances, the probe is a nanocarrier comprising
both an imaging agent and a therapeutic agent. In other instances,
the present invention provides a composition comprising two probes,
wherein the first probe is a nanocarrier with an imaging agent and
a second probe is a nanocarrier comprising a therapeutic agent.
[0006] In certain instances, the present invention provides methods
for treatment, wherein for example, a tumor is treated using the
therapeutic agent and thereafter, imaged to ascertain the extent of
treatment. The treatment can be repeated until the tumor is
destroyed or the site of treatment is satisfactorily complete. In
certain instances, the methods include, injecting the probe or
composition, treating the tumor using photodynamic therapy and
thereafter imaging to ascertain the extent of treatment.
[0007] In some embodiments, the virus-like particle comprises a L1
capsid protein, a L2 capsid protein, or a combination thereof. In
some instances, the ratio of L1:L2 capsid protein is about 15:1. In
other instances, the ratio of L1:L2 capsid protein is about 10:1.
In yet other instances, the ratio of L1:L2 capsid protein is about
5:1.
[0008] In some embodiments, the nanocarrier further comprises a
targeting agent. In some instances, the targeting agent is an
antibody or fragment thereof, a nanobody, an Affibody , diabody, a
minibody, an antigen, a ligand, a protein, a peptide, a nucleic
acid or a small molecule.
[0009] In some embodiments, the therapeutic agent is attached to
the nanocarrier via a first linker. In some embodiments, the
imaging agent is attached to the nanocarrier via a second
linker.
[0010] In some embodiments, the therapeutic agent is a
photosensitizer. In some instances, the photosensitizer is
IRDye.RTM. 700DX. In some embodiments, the imaging agent is a
cyanine dye. In some instances, the cyanine dye is IRDye.RTM.
800CW.
[0011] In another aspect, provided herein is a method for detecting
a target cell in an individual. The method comprises (a) contacting
the target cell with a probe comprising a nanocarrier attached to
an imaging agent and a therapeutic agent, wherein the probe
selectively associates with the target cell; and (b) detecting the
imaging agent thereby providing an indication of the presence
and/or location of the target cell in the individual. In some
instances, the step of contacting comprises systemic administration
to a subject, local administration to a target site, or
administration to a surgical site. In some embodiments, the method
further comprises exposing the therapeutic agent to a
photoactivating light, thereby inducing apoptosis and/or necrosis
of the target cell.
[0012] In some embodiments, the imaging agent is a cyanine dye. In
some instances, the cyanine dye is IRDye.RTM. 800CW.
[0013] In some embodiments, the target cell is a cell of a solid
tumor. In other embodiments, the target cell is a circulating tumor
cell.
[0014] In another aspect, provided herein is a method for inducing
cell death in a target cell in an individual. The method comprises
(a) contacting the cell with a probe comprising a nanocarrier
attached to an imaging agent and a therapeutic agent, wherein the
probe selectively associates with the target cell; and (b) exposing
said therapeutic agent to a photoactivating light, thereby inducing
apoptosis and/or necrosis of the target cell in the individual. In
some instances, the step of contacting comprises systemic
administration to a subject, local administration to a target site,
or administration to a surgical site. In some embodiments, the
method further comprises detecting the imaging agent thereby
providing an indication of the presence and/or location of the
target cell in the individual.
[0015] In some embodiments, the therapeutic agent is a
photosensitizer. In some instances, the photosensitizer is
IRDye.RTM. 700DX.
[0016] In some embodiments, the target cell is a cell of a solid
tumor. In other embodiments, the target cell is a circulating tumor
cell.
[0017] Other objects, features, and advantages of the present
invention will be apparent to one of skill in the art from the
following detailed description and figures.
BRIEF DESCRIPTION OF THE DRAWING
[0018] The figure provides an exemplary embodiment of the
therapeutic-diagnostic probe 100 described herein. A nanocarrier
110 is attached to a photosensitizing agent 120 through a first
linker 125. The nanocarriers is also attached to an imaging and/or
detecting agent 130 through a second linker 135.
DETAILED DESCRIPTION OF THE INVENTION
I. Introduction
[0019] The present invention provides a method of photodynamically
treating a target site of an individual, e.g., a human. The method
includes the step of administering a therapeutic-diagnostic probe
comprising a nanocarrier, a detecting agent and a photosensitizing
agent to the individual. Once the probe has been administered, the
tissue site is treated with a photoactivating light. Photons from
the light are absorbed by the photosensitizing agent which
activates oxygen in the site to produce reactive oxygen species
that results in death of the target cells within the tissue site.
The location of the probe can be determined by detecting the
imaging agent. Compositions of the probe are also provided
herein.
[0020] In certain instances, the therapeutic and diagnostic
portions can be separate components. In such cases, a diagnostic
probe delivery can be conducted either before or after the therapy
component.
II. Definitions
[0021] The terms "a," "an," or "the" as used herein not only
include aspects with one member, but also include aspects with more
than one member. For instance, the singular forms "a," "an," and
"the" include plural referents unless the context clearly dictates
otherwise. Thus, for example, reference to "a cell" includes a
plurality of such cells and reference to "the agent" includes
reference to one or more agents known to those skilled in the art,
and so forth.
[0022] The term "capsid" is meant to refer to the protein shell of
the virus. In particular embodiments, the capsid refers to the
protein shell of the papillomavirus or adenovirus. A viral capsid
may consist of multimers of oligomeric protein subunits. In certain
embodiments, the capsid comprises the papillomavirus L1 and L2
proteins.
[0023] The term "targeting agent" refers generally to a molecule or
compound that binds to a particular target molecule and forms a
bound complex. The binding can be highly specific binding. For
example, the targeting agent and its corresponding target molecule
can form a specific binding pair. Examples include, but are not
limited to small organic molecules, sugars, lectins, nucleic acids,
proteins, peptides, antibodies, nanobodies, Affibodies.RTM.,
diabodies, minibodies, antigens, ligands, cytokines, receptor
proteins, growth factors, nucleic acid binding proteins, small
molecules, and the like which specifically bind desired target
molecules, target collections of molecules, target receptors,
target cells, and the like.
[0024] The term "nanoparticle" refers to a particle having a
sub-micron (.mu.m) size. In various embodiments, microparticles
have a characteristic size (e.g., diameter) less than about 1
.mu.m, 800 nm, or 500 nm, preferably less than about 400 nm, 300
nm, or 200 nm, more preferably about 100 nm or less, about 50 nm or
less or about 30 or 20 nm or less.
[0025] The term "linker" refers to the atoms joining the agent
(e.g., imaging agent, therapeutic agent, and targeting agent) to
the nanocarrier or biomolecule.
III. Detailed Descriptions of Embodiments
[0026] In some embodiments, the present invention pertains to
nanocarrier-based probes that are useful as diagnostic agents
and/or therapeutics. In some instances, the probes can be utilized
to simultaneously image and treat diseased cells and tissues.
[0027] A. Nanocarriers
[0028] The therapeutic-diagnostic probes of the present invention
comprise a nanocarrier, such as a virus-like particle, a
nanoparticle, a liposome, a quantum dot, or a combination thereof,
that is attached to a detecting agent and a photosensitizing agent.
The nanocarrier can be directed to the target cell or tissue of
interest by passive targeting or directed targeting. With passive
targeting the probe is transported to the target by convection
(e.g., movement within fluids) or passive diffusion (e.g., movement
across the cell membrane according to, for example, a concentration
gradient or without the use of cellular energy) within the body.
For directed targeting, a targeting agent is attached to the
surface of the nanocarrier for binding to its corresponding binding
partner expressed at the target site.
[0029] 1. Virus-Like Particles
[0030] Provided herein are virus-like particles that can be used
for delivering imaging agents and therapeutics (e.g., photodynamic
therapy) to cells and tissues of the body. These particles can be
produced from recombinant proteins that mimic specific viruses. The
particles can be loaded with imaging agents and photosensitizing
agents and targeted to specific cells. For instance, the virus-like
particles can deliver the agents to tumor cells, such as tumor
cells from the lung, colon, ovary, kidney, skin, central nervous
system, blood, prostate, breast and the like.
[0031] Viron-derived nanocarriers can be produced from
papillomavirus capsids composed of L1 (major capsid protein) and L2
capsid (minor capsid protein) proteins. The viral-like particles of
the present invention can be formed from about multiple assembled
capsomers wherein each capsomer comprises L1 and L2 capsid
proteins. The particles can have a stochiometry of L1:L2 of about
15:1, about 10:1, or about 5:1. In other instances, the ratio is
15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1,
3:1, 2:1, or 1:1.
[0032] In some embodiments, the papillomavirus is from a non-human
vertebrates such as, but not limited to, an ungulate, canine,
lapine, avian, rodent, simian, marsupial or marine mammal. In some
embodiments, the papillomavirus is from a human. In other
embodiments, the papillomavirus is selected from HPV-1, HPV-2,
HPV-5, HPV-6, HPV-11, HPV-18, HPV-31, HPV-45, HPV-52, and HPV-58,
bovine papillomavirus-1, bovine papillomavirus-2, bovine
papillomavirus-4, cottontail rabbit papillomavirus, and rhesus
macaque papillomavirus.
[0033] In some embodiments, the agent, such as an imaging agent or
a therapeutic agent, is encapsulated within the virus-like
particle.
[0034] The virus-like particle can be generated by isolating and
purifying capsid proteins produced in a host cell system, such as
bacterial cell, yeast cell, insect cell or mammalian cell system.
In some embodiments, the L1 and L2 proteins are intracellularly
assembled. Alternatively, the particle can be generated by
purifying the capsid proteins produced in an in vitro cell-free
protein synthesis system. The capsid proteins can readily
self-assemble into particles. For instance, L1 can spontaneously
self-assemble into a 60 nm, 72-pentamer icosahedral structure that
closely resembles a papillomavirus virion.
[0035] In some embodiments, viral capsid proteins L1 and/or L2 or
fragments thereof (e.g., L1 peptides and/or L2 peptides) are
coupled to a nanocarrier. In some instances, the peptide is coupled
to the external surface of the nanocarrier covalently or
non-covalently. In some instances, the coupling comprises a
covalent linker such as, but not limited to, an amide linker, a
disulfide linker, a thioether linker, a hydrazone linker, a
hydrazide linker, an imine or oxime linker, an urea or thiourea
linker, an amidine linker, an amine linker, or a sulfonamide
linker.
[0036] 2. Polymeric Nanoparticles
[0037] Biodegradable or non-biodegradable polymers may be used to
form nanoparticles of the present invention. In certain
embodiments, synthetic polymers are used, although natural polymers
may be used and may have equivalent or even better properties,
especially some of the natural biopolymers which degrade by
hydrolysis, such as some of the polyhydroxyalkanoates. Examples of
synthetic polymers include, but are not limited to, poly(hydroxy
acids) such as poly(lactic acid), poly(glycolic acid), and
poly(lactic acid-co-glycolic acid), poly(lactide), poly(glycolide),
poly(lactide-co-glycolide), polyanhydrides, polyorthoesters,
polyamides, polycarbonates, polyalkylenes such as polyethylene and
polypropylene, polyalkylene glycols such as poly(ethylene glycol),
polyalkylene oxides such as poly(ethylene oxide), polyalkylene
terepthalates such as poly(ethylene terephthalate), polyvinyl
alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides
such as poly(vinyl chloride), polyvinylpyrrolidone, poly siloxanes,
poly(vinyl alcohols), poly(vinyl acetate), polystyrene,
polyurethanes and co-polymers thereof, derivativized celluloses
such as alkyl cellulose, hydroxyalkyl celluloses, celluklose
ethers, cellulose esters, nitro celluloses, methyl cellulose, ethyl
cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl
cellulose, hydroxybutyl methyl cellulose, cellulose acetate,
cellulose propionate, cellulose acetate butyrate, cellulose acetate
phthalate, carboxylethyl cellulose, cellulose triacetate, and
cellulose sulfate sodium salt, polymers of acrylic acid,
methacrylic acid or copolymers or derivatives thereof including
esters, poly(methyl methacrylate), poly(ethyl methacrylate),
poly(butylmethacrylate), poly(isobutyl methacrylate),
poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl
methacrylate), poly(phenyl methacrylate), poly(methyl acrylate),
poly(isopropyl acrylate), poly(isobutyl acrylate), and
poly(octadecyl acrylate), poly(butyric acid), poly(valeric acid),
and poly(lactide-co-caprolactone), cyclodextrins, and copolymers
and blends thereof. As used herein, the term "derivatives" includes
polymers having substitutions, additions of chemical groups and
other modifications routinely made by those skilled in the art.
[0038] In particular embodiments, PLGA is used as the biodegradable
polymer. Examples of biodegradable polymers useful in the present
invention include polymers of hydroxy acids such as lactic acid and
glycolic acid, and copolymers with PEG, polyanhydrides,
poly(ortho)esters, polyurethanes, poly(butyric acid), poly(valeric
acid), poly(lactide-co-caprolactone), and blends and copolymers
thereof. Natural polymers include, but are not limited to, proteins
such as albumin, collagen, gelatin and prolamines, for example,
zein, and polysaccharides such as alginate, cellulose derivatives
and polyhydroxyalkanoates, for example, polyhydroxybutyrate. The in
vivo stability of the particles can be adjusted during the
production by using polymers such as poly(lactide-co-glycolide)
copolymerized with polyethylene glycol (PEG). Examples of
non-biodegradable polymers include, but are not limited to,
ethylene vinyl acetate, poly(meth)acrylic acid, polyamides, or
copolymers or mixtures thereof.
[0039] B. Liposomes
[0040] Liposomes are artificial vesicles composed of concentric
lipid bilayers separated by water-compartments and have been
extensively investigated as drug delivery vehicles. Due to their
structure, chemical composition and colloidal size, all of which
can be well controlled by preparation methods, liposomes exhibit
colloidal size, i.e., rather uniform particle size distributions in
the range from 10 nm to 10 and useful membrane and surface
characteristics. Liposomes can deliver therapeutics to diseased
tissues, for example, in circulation, and also rapidly enter the
liver, spleen, kidneys and reticuloendothelial systems.
[0041] In some embodiments, the liposome comprises synthetic
phospholipids such as, but not limited to, phosphatidyl cholines,
e.g., dipalmitoylphosphatidy choline (DPPC), dimyristoyl
phosphatidyl choline (DMPC), and distearoyl phosphatidyl choline
(DSPC), and phosphatidyl glycerols, e.g., as dipalmitoyl glycerol
(DPPG) or dimyristoyl phosphatidyl glycerol (DMPG). The liposome
can also include a monosaccharide such as glucose or fructose. In
some embodiments, the phospholipids are conjugated to a
polyethylene glycol (PEG) molecule.
[0042] C. Quantum Dots
[0043] Quantum dots are small molecular clusters having up to about
a few hundred atoms. Quantum dots can have a size range of about 1
nm to about 20 nm in diameter. They are typically only a few
nanometers in size. A quantum dot is typically composed of a
semiconductor material or materials, metal(s), or metal oxides
exhibiting a certain energy. A variety of materials may be utilized
for construction of nanoparticles, including, but not limited to,
TiO.sub.2, Al.sub.2O.sub.3, AgBr, CdSe, CdS, CdSlSel, CuCl,
CdTexS.sub.1-x, ZnTe, ZnSe, ZnS, GaN, InGaN, InP, CdS/HgS/CdS,
InAs/GaAs, Group II-VI, Groups III-V, and Groups I-VII
semiconductors as well as Group IV metals and alloys. A quantum dot
may also be surrounded by a material or materials having wider
bandgap energies (for example, ZnS-capped CdS), and especially may
be surrounded by those materials that improve biocompatibility of
the nanoparticles.
[0044] Quantum dots can photoluminesce when stimulated by light
having a wavelength in which the energy of a photon is at least
equal to the energy of the light-emitting material forming the
quantum dot. Consequently, quantum dots absorb light of a first
wavelength and emit light at a second wavelength that is shorter
than the first wavelength. The pump light supplied by, e.g., a
laser or light-emitting diode ("LED") array or other light source
in which photons have an energy at least equal to the band-gap
energy of the quantum dot is therefore absorbed by the quantum dot.
The quantum dot re-emits energy in the form of light at a different
wavelength and in a multidirectional fashion.
[0045] There are a number of methods of making quantum dots. The
synthesis of small semiconductor clusters in trioctylphosphine
oxide (TOPO) at 300.degree. C. has been shown to yield highly
fluorescent (quantum yields >50%) small particles of a number of
semiconductor materials, such as CdSe, InP and InAs. Growth
conditions such as the length of time of crystallization,
concentration of monomer, and temperature establish the size of the
quantum dot and therefore the color of the light emitted from the
quantum dot. (See, Green and O'Brien, Chem. Commun., 1999, 2235-41;
and U.S. Pat. Nos. 5,909,670, 5,943,354, and 5,882,779). Quantum
dots are commercially available from manufacturers such as Life
Technologies, Nanoco Technologies, and Sigma-Aldrich.
[0046] In some embodiments, the nanoparticle comprises an inorganic
material and/or a quantum dot. In some instances, the nanoparticle
includes one or more materials selected from the group consisting
of cadmium, zinc, magnesium, mercury, aluminum, gallium, indium,
and thallium. Optionally, the nanoparticle can contain one or more
materials selected from the group of cadmium, zinc, magnesium,
mercury, aluminum, gallium, indium, or thallium.
[0047] In particular embodiments, the nanoparticle comprising an
inorganic material can comprises a core and a shell, where the
shell comprises a semiconductor overcoating the core. In certain
embodiments the shell comprises a group II, III, IV, V, or VI
semiconductor. In particular embodiments the shell comprises one or
more materials selected from the group consisting of ZnO, ZnS,
ZnSe, ZnTe, CdO, CdS, CdSe, CdTe, MgO, MgS, MgSe, MgTe, HgO, HgS,
HgSe, HgTe, AlN, AlP, AlAs, AlSb, GaN, GaP, GaAs, GaSb, InN, InP,
InAs, InSb, TlN, TlP, TlAs, and TlSb. In certain embodiments the
nanoparticle comprises a CdSe core and a ZnS shell and a SiO.sub.2
hydrophilic coating.
[0048] D. Imaging Agents
[0049] The compositions and methods described herein are useful for
non-invasive imaging in the near-infrared spectral range. In some
embodiments, the imaging agent can be a detecting agent. In some
embodiments, the imaging agent for the probe of the present
invention is a cyanine dye. In preferred embodiments, the cyanine
dye is IRDye.RTM. 800CW or an equivalent thereof. Detailed
description of other useful cyanine dyes are found in, for example,
U.S. Pat. Nos. 6,995,274; 7,597,878, 7,504, 089; and 8,303,939, the
disclosures are hereby incorporated by reference in their entirety
for all purposes.
[0050] The imaging and/or detecting agent can be directly attached
to the nanocarriers described herein. In some embodiments, the
agent is attached to a biomolecule, including, but not limited to,
a viral particle, a nanoparticle, a liposome, a quantum dot, a
protein, a peptide, a ligand, an enzyme substrate, a hormone, an
antibody, an antigen, a hapten, an avidin, a streptavidin, a
carbohydrate, an oligosaccharide, a polysaccharide, an
oligosaccharide, a nucleic acid, a deoxy nucleic acid, a fragment
of DNA, a fragment of RNA, nucleotide triphosphates, acyclo
terminator triphosphates, or PNA
[0051] In other embodiments, the biomolecule is a ligand that has
an affinity for a receptor expressed by a cell or tissue of
interest. In some instances, the receptor is selected from the
group consisting of EGFR, HER2, PDGFR, IGFR, c-Ryk, c-Kit, CD24,
integrins, FGFR, KFGR, VEGFR, TRAIL decoy receptors, retinoid
receptor, growth receptor, PPAR, vitamin receptor,
glucocorticosteroid receptor, retinoid-X receptor, RHAMM, high
affinity folate receptors, Met receptor, estrogen receptor and
Ki67.
[0052] In yet other embodiments, the biomolecule is selected from
the group of somatostatin, endostatin, a carbohydrate, a
monosaccaride, a disaccharide, a trisaccharide, an oligosaccharide,
aptamer, liposome and PEG. Alternatively, the biomolecule is
2-deoxy-D-glucose, 2-deoxy-D-glucosamine, a glucose derivative,
glyceraldehyde, erythrose, threose, ribose, arabinose, xylose,
lyxose, allose, altrose, glucose, mannose, gulose, idose,
galactose, talose, erythrulose, ribulose, xylulose, psicose,
fructose, sorbose, or tagatose.
[0053] In still other embodiments, the biomolecule is selected from
the group of angiopoietins, angiostatin, angiotensin II,
.alpha..sub.2-antiplasmin, annexin V, .beta.-cyclodextrin
tetradecasulfate, endoglin, endosialin, endostatin, epidermal
growth factor, fibrin, fibrinopeptide .beta., fibroblast growth
factor, FGF-3, basic fibronectin, fumagillin heparin, hepatocyte
growth factor, hyaluronan, insulin-like growth factor,
interferon-.alpha.,.beta. inhibitors, IL inhibitor, laminin,
leukemia inhibitory factor, linomide, matrix metalloproteinase-2,
metalloproteinases, metalloproteinase inhibitors, antibodies or
fragments, monoclonal antibodies or fragments, cyclic RGDD FV,
placental growth factor, placental proliferin-related protein,
plasminogen, plasminogen activator, plasminogen activator
inhibitor-1, platelet activating factor antagonists,
platelet-derived growth factor, platelet-derived growth factor
receptors, platelet-derived endothelial cell growth factor,
pleiotropin, proliferin, proliferin-related protein, selectins:
E-selectin, SPARC, snake venoms, substance P, suramin, tissue
inhibitor of metalloproteinases, thalidomide, thrombin,
thrombin-receptor-activating tetradecapeptide, transforming growth
factor-.alpha.,.beta., transforming growth factor receptor, tumor
growth factor-.alpha., tumor necrosis factor, vitronectin, avidin
or streptavidin.
[0054] In some embodiments, the probes of the present invention are
used to directly label a cell or tissue so that the cell or tissue
can be identified or quantitated. For instance, such probes can be
added as part of a detection assay for a target cell or tissue, as
a detectable tracer element in a biological or non-biological
fluid; or for such purposes as photodynamic therapy of diseased
tissues, in which a dyed cell is irradiated to selectively destroy
the diseased cells, usually through the photosensitized production
of singlet oxygen.
[0055] E. Photosensitizing Agents
[0056] In some embodiments, the photosensitizing agent of the probe
is a phthalocyanine dye. In some instances, the phthalocyanine dye
comprises a luminescent fluorophore moiety having at least one
silicon containing aqueous-solubilizing moiety, wherein the
phthalocyanine dye has a core atom selected from Si, Ge, Sn, and
Al. Preferably, the phthalocyanine dye exists as a single core
isomer, essentially free of other isomers and has a reactive or
activatible group. The core atom is preferably Si. In preferred
embodiments, the phthalocyanine dye is IRDye.RTM. 700DX or an
equivalent thereof. Detailed description of phthalocyanine dyes are
found in, for example, U.S. Pat. No. 7,005,518, the disclosure is
herein incorporated by reference in its entirety for all
purposes.
[0057] The term "IRDye.RTM. 700DX " or "IR700" refers to a dye
having the preferred NHS ester linkage to allow for conjugation.
Typically, the nanocarrier, agent or biomolecule has a primary
amine (e.g., an amino group) wherein the NHS ester and the amino
group react to form an amide bond, linking the targeting moiety
such as an antibody to 700DX. The NHS ester IRDye.RTM. 700DX has
the following formula:
##STR00001##
[0058] The dye is commercially available from LI-COR (Lincoln,
Nebr.). Amino-reactive IRDye.RTM. 700DX is a relatively hydrophilic
dye and can be covalently conjugated with an antibody using the NHS
ester of IRDye.RTM. 700DX. Other variations of IRDye.RTM. 700Dx are
disclosed in U.S. Pat. Nos. 7,005,518 (incorporated herein by
reference), and those too are useful in the present invention. The
carboxylate derivative has the following name and structure,
silicate(5-),
bis[N-[3-[(hydroxy-.kappa.O)dimethylsilyl]propyl]-3-sulfo-N,N-bis(3-sulfo-
propyl)-1-propanaminiumato(4-)][6-[[[3-[(29H,31H-
phthalocyanin-yl-.kappa.N29, .kappa.N30, .kappa.N31,
.kappa.N32)oxy]propoxy]carbonyl]amino]hexanoato(3-)]-, sodium
(1:5); CAS Registry Number: [1623074-46-3]:
##STR00002##
[0059] The photosensitizing agent can be directly attached to the
nanocarriers described herein. In some embodiments, the agent is
attached to a biomolecule, including, but not limited to, a viral
particle, a nanoparticle, a liposome, a quantum dot, a small
molecule, a cell, a drug (e.g., small molecule), a liposome, a
protein, a peptide, an enzyme substrate, a hormone, an antibody, an
antigen, a hapten, an avidin, a streptavidin, biotin, a
carbohydrate, an oligosaccharide, a polysaccharide, a nucleic acid,
a deoxy nucleic acid, a fragment of DNA, a fragment of RNA,
nucleotide triphosphates, acyclo terminator triphosphates, peptide
nucleic acid (PNA) biomolecules, or a combination thereof. The
photosensitizing agent can be linked to a material, such as a
biomolecule, for example, by using phosphoramidite chemistry,
ultimately forming a phosphate linkage between the dye and the
biomolecule. For examples of such labeling methods, see U.S. Pat.
No. 6,027,709, which discloses many preferred linking groups,
linking methods, and biomolecules that can be readily labeled. Many
methods of linking dyes to various types of biomolecules are well
known in the art. For a thorough review of oligonucleotide labeling
procedures, see, e.g., R. Haugland in Excited States of
Biopolymers, Steiner ed., Plenum Press (1983), Fluorogenic Probe
Design and Synthesis: A Technical Guide, PE Applied Biosystems
(1996), and G. T. Herman, Bioconjugate Techniques, Academic Press
(1996).
[0060] In some embodiments, the photosensitizing agent is reacted
with a biomolecule to form a covalent bond between the dye and the
biomolecule. The bond is for example, an amide, a secondary or
tertiary amine, a carbamate, an ester, an ether, an oxime, a
phosphate ester, a sulfonamide, a thioether, a thiourea, or a urea.
Preferably, the bond is covalent, such as an amide or carbamate
bond. Non-limiting examples of reactive functionalities useful for
attaching the dye to the biomolecule are described in, for example,
U.S. Pat. No. 7,005,518.
[0061] F. Linkers
[0062] The detecting agent and/or photosensitizing agent can be
attached to the nanocarrier or a biomolecule of the nanocarriers
via a linker. In some embodiments, the linker is a direct link or a
covalent linkage, wherein the covalent linkage is linear or
branched, cyclic or heterocyclic, saturated or unsaturated, having
1-60 atoms selected from the group consisting of C, N, P, O, and S,
wherein the linker can have additional hydrogen atoms to fill
valences, and wherein the linker contains any combination of ether,
thioether, amine, ester, carbamate, urea, thiourea, oxy or amide
bonds; or single, double, triple or aromatic carbon-carbon bonds;
or phosphorus-oxygen, phosphorus-sulfur, nitrogen-nitrogen,
nitrogen-oxygen, or nitrogen-platinum bonds; or aromatic or
heteroaromatic bonds. The linker can include phosphoramidite
groups, NHS ester, activated carboxylic acid, thiocyanate,
isothiocyanate, maleimide and iodoacetamide. The linker may
comprise a terminal amino, carboxylic acid, or sulfhydryl group
covalently attached to the ring. In certain instances, the terminal
amino, carboxylic acid, or sulfhydryl group is shown and is
represented as --L--NH.sub.2, or --L--C(O)OH or --L--SH.
[0063] In certain embodiments, the linker arm is
--(CH.sub.2).sub.n--, wherein r is an integer from 1 to 10,
preferably n is an integer from 1 to 5, such as 1 to 4, or 1, 2, 3,
4, or 5.
[0064] The detecting agent and/or photosensitizing agent can be
reacted with a biomolecule or a carrier molecule using conjugation
chemistry well known in the art. For example, an activated ester
(an NHS ester) can react with a primary amine to make a stable
amide bond. A maleimide and a thiol can react together and make a
thioether. Alkyl halides react with amines and thiols to make
alkylamines and thioethers, respectively. Any derivative providing
a reactive moiety that can be conjugated to a protein can be
utilized herein. As is known in the art, moieties comprising a free
amino group, a free carboxylic acid group, or a free sulfhydryl
group provide useful reactive groups for protein conjugation. For
example, a free amino group can be conjugated to proteins via
glutaraldehyde cross-linking, or via carbodiimide cross-linking to
available carboxy moieties on the protein. Also, a linker with a
free sulfhydryl group can be conjugated to proteins via maleimide
activation of the protein, e.g., using
sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate
(Sulfo-SMCC), then linkage to the sulfhydryl group.
[0065] When linking a biomolecule having a carboxylic acid group
for attachment to an amine containing molecule, the carboxylic acid
can first be converted to a more reactive form using an activating
reagent, to form for example, a N-hydroxy succinimide (NHS) ester
or a mixed anhydride. The amine-containing metabolite is treated
with the resulting activated acid to form an amide linkage. One of
skill in the art will recognize that alternatively, the NHS ester
can be on the metabolite and the amine can be on the carrier
protein.
[0066] In other embodiments, the linker is a member selected from
the group of a PEG, a block copolymer of PEG-polyurethane and a
PEG-polypropylene. In yet other embodiments, the linker is a member
selected from the group of a polysaccharide, a polypeptide, an
oligosaccharide, a polymer, a co-polymer and an
oligonucleotide.
[0067] The linker can have the formula:
--X.sup.1--Y.sup.1--X.sup.2--
wherein: X is a member selected from the group of a bivalent
radical, a direct link, oxygen, an optionally substituted nitrogen
and sulfur; Y.sup.1 is a member selected from the group of a direct
link and C.sub.1-C.sub.10 alkylene optionally interrupted by a
heteroatom; and X.sup.2 is a member selected from the group of a
bivalent radical, a direct link, oxygen, an optionally substituted
nitrogen and sulfur.
[0068] Preferably, the bivalent radical of X.sup.1 and X.sup.2 are
each independently selected from the group of a direct link,
optionally substituted alkylene, optionally substituted
alkyleneoxycarbonyl, optionally substituted alkylenecarbamoyl,
optionally substituted alkylenesulfonyl, optionally substituted
alkylenesulfonylcarbamoyl, optionally substituted arylene,
optionally substituted arylenesulfonyl, optionally substituted
aryleneoxycarbonyl, optionally substituted arylenecarbamoyl,
optionally substituted arylenesulfonylcarbamoyl, optionally
substituted carboxyalkyl, optionally substituted carbamoyl,
optionally substituted carbonyl, optionally substituted
heteroarylene, optionally substituted heteroaryleneoxycarbonyl,
optionally substituted heteroarylenecarbamoyl, optionally
substituted heteroarylenesulfonylcarbamoyl, optionally substituted
sulfonylcarbamoyl, optionally substituted thiocarbonyl, a
optionally substituted sulfonyl, and optionally substituted
sulfinyl.
[0069] Alternatively, the linker is --(CH.sub.2).sub.r--, wherein r
is an integer from 1 to 50.
[0070] Selected example of reactive functionalities useful for the
attaching of the agent to the nanocarriers or biomolecule are shown
in Table 1, wherein the bond results from the reaction of an agent
(e.g., detecting agent or photosensitizing agent) with the
nanocarriers or biomolecule. Those of skill in the art will know of
other bonds suitable for use in the present invention.
TABLE-US-00001 TABLE 1 A B Reactive functionality Complementary
group (either on the (either on the C agent or the
nanocarrier/biomolecule The resulting nanocarrier/biomolecule) or
the agent) bond activated esters* amines/anilines carboxamides
acrylamides thiols thioethers acyl azides** amines/anilines
carboxamides acyl halides amines/anilines carboxamides acyl halides
alcohols/phenols esters acyl nitriles alcohols/phenols esters acyl
nitriles amines/anilines carboxamides aldehydes amines/anilines
imines aldehydes or ketones hydrazines hydrazones aldehydes or
ketones hydroxylamines oximes alkyl halides amines/anilines alkyl
amines alkyl halides carboxylic acids esters alkyl halides thiols
thioethers alkyl halides alcohols/phenols ethers anhydrides
alcohols/phenols esters anhydrides amines/anilines carboxamides/
imides aryl halides Thiols thiophenols aryl halides amines aryl
amines aziridines thiols thioethers boronates glycols boronate
esters activated carboxylic acids amines/anilines carboxamides
activated carboxylic acids alcohols esters activated carboxylic
acids hydrazines hydrazides carbodiimides carboxylic acids
N-acylureas or anhydrides diazoalkanes carboxylic acids esters
epoxides thiols (amines) thioethers (alkyl amines) epoxides
carboxylic acids esters haloacetamides Thiols thioethers
haloplatinate amino platinum complex haloplatinate heterocycle
platinum complex halotriazines amines/anilines aminotriazines
halotriazines alcohols/phenols triazinyl ethers imido esters
amines/anilines amidines isocyanates amines/anilines ureas
isocyanates alcohols/phenols urethanes isothiocyanates
amines/anilines thioureas maleimides thiols thioethers
phosphoramidites alcohols phosphite esters silyl halides alcohols
silyl ethers sulfonate esters amines/anilines alkyl amines sulfonyl
halides amines/anilines sulfonamides *Activated esters, as
understood in the art, generally have the formula --COM, where M is
a good leaving group (e.g. succinimidyloxy
(--OC.sub.4H.sub.4O.sub.2) sulfosuccinimidyloxy
(--OC.sub.4H.sub.3O.sub.2SO.sub.3H), -1-oxybenzotriazolyl
(--OC.sub.6H.sub.4N.sub.3); 4-sulfo-2,3,5,6-tetrafluorophenyl; or
an aryloxy group or aryloxy substituted one or more times by
electron withdrawing substituents such as nitro, fluoro, chloro,
cyano, or trifluoromethyl, or combinations thereof, used to form
activated aryl esters; or a carboxylic acid activated by a
carbodiimide to form an anhydride or mixed anhydride --OCOR.sup.a
or OCNR.sup.aNHR.sup.b, where R.sup.a and R.sup.b, which may be the
same or different, are C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6
perfluoroalkyl, or C.sub.1-C.sub.6 alkoxy; or cyclohexyl,
3-dimethylaminopropyl, or N-morpholinoethyl). **Acyl azides can
also rearrange to isocyanates.
[0071] In some embodiments, the covalent linkage between the linker
and biomolecule is selected from the group consisting of a direct
bond, an amide bond, an ester bond, an ether bond, an oxime bond, a
phosphate ester bond, a sulfonamide bond, a thioether bond, a
thiourea bond, and an urea bond.
[0072] G. Targeting Molecules
[0073] In certain embodiments, the external surface of the
nanocarrier is attached to a 1) targeting molecule, as well as 2) a
therapeutic agent and 3) a detecting agent. The targeting molecule
can be entrapped within the particle, associated with the surface
of the nanocarrier (e.g., adsorbed or conjugated (directly or
indirectly) to the nanocarrier surface), and/or otherwise
associated with the nanocarrier to varying degrees (e.g., admixed
with nanocarrier in a liquid suspension, admixed with the
nanocarrier in a solid composition, for instance, co-lyophilized
with the nanocarrier, etc.), among other possibilities. In some
embodiments, at least two different targeting molecules are
attached to the nanocarrier.
[0074] In some embodiments, the targeting molecule specifically
binds to an antigen such as a tumor antigen, bacterial antigen
viral antigen, and fungal antigen. The targeting molecule can
recognize tumor antigens such as, but not limited to: (a)
cancer-testis antigens such as NY-ESO-1, SSX2. SCP1 as well as
RAGE, BAGE, GAGE and MAGE family polypeptides, for example, GAGE-1,
GAGE-2, MAGE-1. MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, and MAGE-12
(which can be used, for example, to address melanoma, lung, head
and neck, NSCLC, breast, gastrointestinal, and bladder tumors), (b)
mutated antigens, for example, p53 (associated with various solid
tumors, e.g., colorectal, lung, head and neck cancer), p21/Ras
(associated with, e.g. melanoma, pancreatic cancer and colorectal
cancer), CDK4 (associated with, e.g. melanoma), MUM1 (associated
with, e.g., melanoma), caspase-8 (associated with, e.g., head and
neck cancer), CIA 0205 (associated with, e.g., bladder cancer),
HLA-A2-R 1701, beta catenin (associated with, e.g., melanoma), TCR
(associated with, e.g., T-cell non-Hodgkins lymphoma), BCR-abl
(associated with, e.g., chronic myelogenous leukemia),
triosephosphate isomerase, KIA 0205, CDC-27, and LDLR-FUT. (c)
over-expressed antigens, for example, Galectin 4 (associated with,
e.g., colorectal cancer), galectin 9 (associated with, e.g.,
Hodgkin's disease), proteinase 3 (associated with, e.g., chronic
myelogenous leukemia), WT 1 (associated with, e.g., various
leukemias), carbonic anhydrase (associated with, e.g. renal
cancer), aldolase A (associated with, e.g., lung cancer), PRAIVIE
(associated with, e.g. melanoma). HER-2/neu (associated with, e.g.,
breast, colon, lung and ovarian cancer), alpha-fetoprotein
(associated with, e.g., hepatoma), KSA (associated with, e.g.,
colorectal cancer), gastrin (associated with, e.g., pancreatic and
gastric cancer), telomerase catalytic protein, MUC-1 (associated
with, e.g., breast and ovarian cancer), G-250 (associated with,
e.g., renal cell carcinoma), and carcinoembryonic antigen
(associated with, e.g., breast cancer, lung cancer, and cancers of
the gastrointestinal tract such as colorectal cancer), (d) shared
antigens, for example, melanoma-melanocyte differentiation antigens
such as MART-1/Melan A, gp100, MC1R, melanocyte-stimulating hormone
receptor, tyrosinase, tyrosinase related protein-1/TRP1 and
tyrosinase related protein-2/TRP2 (associated with, e.g.,
melanoma), (e) prostate associated antigens such as PAP, PSA, PSMA,
PSH-P1, PSM-P1, PSM-P2, associated with e.g. prostate cancer, (f)
immunoglobulin idiotypes (associated with myeloma and B cell
lymphomas, for example), and (g) other tumor antigens, such as
polypeptide- and saccharide-containing antigens including (i)
glycoproteins such as sialyl Tn and sialyl Lex (associated with,
e.g., breast and colorectal cancer) as well as various mucins;
glycoproteins may be coupled to a carrier protein (e.g., MUC-1 may
be coupled to KLH); (ii) lipopolypeptides (e.g., MUC-1 linked to a
lipid moiety); (iii) polysaccharides (e.g., Globo H synthetic
hexasaccharide), which may be coupled to a carrier proteins (e.g.,
to KLH), (iv) gangliosides such as GM2, GM12, GD2, GD3 (associated
with, e.g., brain, lung cancer, melanoma), which also may be
coupled to carrier proteins (e.g., KLH).
[0075] Other tumor antigens include p15, Hom/Mel-40, H-Ras,
E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens,
EBNA, human papillomavirus (HPV) antigens, including E6 and E7,
hepatitis B and C virus antigens, human T-cell lymphotropic virus
antigens, TSP-180, p185erbB2, p180erbB-3, c-met, mn-23H1, TAG-72-4,
CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, p16, TAGE, PSCA, (CT7,
43-9F, 5T4, 791 Tgp72, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 (CA
27.29\BCAA). CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5,
Ga733 (EpCAM), 1HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K,
NY-CO-1. RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein\cyclophilin
C-associated protein), TAAL6, TAG72, TLP, TPS, and the like.
[0076] In some embodiments, the targeting molecule is an antibody
that binds an antigen selected from the group consisting of, a
gastrointestinal cancer cell surface antigen, a lung cancer cell
surface antigen, a brain tumor cell surface antigen, a glioma cell
surface antigen, a breast cancer cell surface antigen, an
esophageal cancer cell surface antigen, a common epithelial cancer
cell surface antigen, a common sarcoma cell surface antigen, an
osteosarcoma cell surface antigen, a fibrosarcoma cell surface
antigen, a melanoma cell surface antigen, a gastric cancer cell
surface antigen, a pancreatic cancer cell surface antigen, a
colorectal cancer cell surface antigen, a urinary bladder cancer
cell surface antigen, a prostatic cancer cell surface antigen, a
renal cancer cell surface antigen, an ovarian cancer cell surface
antigen, a testicular cancer cell surface antigen, an endometrial
cancer cell surface antigen, a cervical cancer cell surface
antigen, a Hodgkin's disease cell surface antigen, a lymphoma cell
surface antigen, a leukemic cell surface antigen and a
trophoblastic tumor cell surface antigen.
[0077] In some embodiments, targeting moiety is an antibody that
binds an antigen selected from the group consisting of 5 alpha
reductase, a-fetoprotein, AM-1, APC, APRIL, BAGE, .beta.-catenin,
Bc12, bcr-abl (b3a2), CA-125, CASP-8/FLICE, Cathepsins, CD19, CD20,
CD21, CD23, CD22, CD38, CD33, CD35, CD44, CD45, CD46, CDS, CD52,
CD55, CD59 (791Tgp72), CDC27, CDK4, CEA, c-myc, Cox-2, DCC, DcR3,
E6/E7, EGFR, EMBP, Ena78, FGF8b and FGF8a, FLK-1/KDR, folic acid
receptor, G250, GAGE-Family, gastrin 17, GD2/GD3/GM2, GnRH, GnTV,
gp100/Pme117, gp-100-in4, gp15, gp75/TRP-1, hCG, Heparanase,
Her2/neu, HER3, Her4, HMTV, HLA-DR10, Hsp70, hTERT , IGFR1, IL-13R,
iNOS, Ki 67, KIAA0205, K-ras, H-ras, N-ras, KSA, (C017-1A),
LDLR-FUT, MAGE Family (MAGE1, MAGE3, etc.), mammaglobin, MAP17,
Melan-A/, MART-1, mesothelin, MIC A/B, MT-MMP's, such as MMP2,
MMP3, MMPI, MMP9, Mox1, MUC-1, MUC-2, MUC-3, and MUC-4, MUM-1,
NY-ESO-1, Osteonectin, p15, P170/MDR1, p53, p97/melanotransferrin,
PAI-1, PDGF, plasminogen (uPA), PRAIVIE, probasin, progenipoietin,
PSA, PSM, RAGE-1, Rb, RCAS1, SART-1, SSX gene, family, STAT3, STn,
TAG-72, TGF-.alpha., TGF-.beta., and thymosin (3, 15, nucleolin,
Ca15-3, astro Intestinal Tumor Antigen (Ca19-9), ovarian tumor
antigen (Ca125), tag72-4 antigen (CA72-4) and carcinoembryonic
antigen (CEA).
[0078] In some embodiments, the targeting molecule is a
carbohydrate. Carbohydrates may be natural or synthetic. A
carbohydrate may be a derivatized natural carbohydrate. In some
embodiments, the carbohydrate comprises monosaccharide or
disaccharide, including but not limited to, glucose, fructose,
galactose, ribose, lactose, sucrose, maltose, trehalose, cellbiose,
mannose, xylose, arabinose, glucoronic acid, galactoronic acid,
mannuronic acid, glucosamine, galatosamine, or neuramic acid. In
some embodiments, the carbohydrate is a polysaccharide, such as,
but not limited to, pullulan, cellulose, microcrystalline
cellulose, hydroxypropyl methylcellulose (HPMC), hydroxycellulose
(HC), methylcellulose (MC), dextran, cyclodextran, glycogen,
starch, hydroxyethylstarch, carageenan, glycon, amylose, chitosan,
N,O-carboxylmethylchitosan, algin and alginic acid, starch, chitin,
heparin, konjac, glucommannan, pustulan, heparin, hyaluronic acid,
curdlan, and xanthan. In some embodiments, the carbohydrate is a
sugar alcohol, such as, but not limited to mannitol, sorbitol,
xylitol, erythritol, maltitol, or lactitol.
[0079] H. Diagnostic Uses
[0080] The probe can be combined with the sample in any way that
facilitates contact between the probe and the sample of interest.
The probe typically forms a covalent or non-covalent association or
complex with an element of the sample, or is simply present within
the bounds of the sample or portion of the sample. The sample is
then illuminated at a wavelength selected to elicit the optical
response. Equipment that is useful for illuminating the dye
compounds of the invention includes, but is not limited to,
hand-held ultraviolet lamps, mercury arc lamps, xenon lamps, lasers
and laser diodes. These illumination sources are optionally
integrated into laser scanners, fluorescence microplate readers,
standard or minifluorometers, or chromatographic detectors.
Preferred embodiments of the invention are dyes that are be
excitable at or near the wavelengths 633-636 nm, 647 nm, 660 nm,
680 nm and beyond 700 nm, such as 780 nm, 810 nm and 850 nm as
these regions closely match the output of relatively inexpensive
excitation sources.
[0081] The optical response is optionally detected by visual
inspection, or by use of any of the following devices: CCD cameras,
video cameras, photographic film, laser-scanning devices,
fluorometers, photodiodes, quantum counters, epifluorescence
microscopes, scanning microscopes, flow cytometers, fluorescence
microplate readers, or by means for amplifying the signal such as
photomultiplier tubes. In certain other aspects, the probes are
used as in vivo optical imaging agents of tissues and organs in
various biomedical applications including, but not limited to,
tomographic imaging of organs, monitoring of organ functions,
coronary angiography, fluorescence endoscopy, imaging of tumors,
laser guided surgery, photoacoustic and sonofluorescence methods,
and the like. The probes of the present invention are particularly
useful for imaging tumors, tissues, and organs in a subject. In one
embodiment, the probes are useful for the detection of the presence
of tumors and other abnormalities by monitoring the blood clearance
profile of the dyes. In another embodiment, the probes are useful
for laser assisted guided surgery for the detection of
micro-metastases of tumors upon laparoscopy.
[0082] The probes may be administered either systemically or
locally to the organ or tissue to be imaged, prior to the imaging
procedure. In one embodiment, the probes are administered
intravenously. In another embodiment, the probes are administered
parenterally. In yet another embodiment, the probes are
administered enterally. The compositions used for administration of
the probe typically contain an effective amount of the detecting
agent along with conventional pharmaceutical carriers and
excipients appropriate for the type of administration contemplated.
For example, parenteral formulations advantageously contain a
sterile aqueous solution or suspension of the dye compound or
conjugate according to the invention. Compositions for enteral
administration typically contain an effective amount of the dye in
aqueous solution or suspension that may optionally include buffers,
surfactants, thixotropic agents, flavoring agents, and the
like.
[0083] The compositions can be administered in doses effective to
achieve the desired optical image of a tumor, tissue, or organ.
Such doses may vary widely, depending upon the particular dye
compound or conjugate employed, the tumor, tissue, or organ
subjected to the imaging procedure, the imaging equipment being
used, and the like.
[0084] I. Therapeutic Uses
[0085] In yet other aspects, the present invention provides methods
and compounds for photodynamic therapy (PDT) of target tumors,
tissues, and organs in a subject. PDT is a two-step treatment
process that may be used in a wide variety of cancers and diseased
tissue and organs. The first step in this therapy is carried out by
administering a photosensitizing agent systemically by ingestion or
injection, or topically applying the compound to a specific
treatment site on a subject, followed by illumination of the
treatment site with light having a wavelength or waveband
corresponding to a characteristic absorption waveband of the
photosensitizing agent. The light activates the photosensitizing
agent, causing singlet oxygen radicals and other reactive species
to be generated, leading to a number of biological effects that
destroy the abnormal or diseased tissue, which has absorbed the
photosensitizing agent. The depth and volume of the cytotoxic
effect on the abnormal tissue, such as a cancerous tumor, depend in
part on the depth of the light penetration into the tissue, the
photosensitizing agent concentration and its cellular distribution,
and the availability of molecular oxygen, which will depend upon
the vasculature system supplying the tumor, tissue, or organ.
[0086] In certain instances, the present invention provides methods
for treatment, wherein for example, a tumor is treated using the
therapeutic agent and thereafter, imaged to ascertain the extent of
treatment. The treatment can be repeated until the tumor is
destroyed or the site of treatment is satisfactorily complete. In
certain instances, the methods include, injecting the probe or
composition, treating the tumor using photodynamic therapy and
thereafter imaging to ascertain the extent of treatment.
[0087] The method of the present invention provides for
administering to the subject a therapeutically effective amount of
a targeted photosensitizing agent, such as a therapeutic-diagnostic
probe. The probe can be administered systemically by ingestion or
injection, or locally administered to a target tissue site or to a
surgical site. The photosensitizing agent of the probe can bind to
one or more types of target cells or tissues, such as circulating
tumor cells or cells of a solid tumor. When exposed to
photoactivating light of an appropriate waveband, the agent absorbs
the light, causing substances to be produced that impair or destroy
the target cells or tissues. Preferably, the compound is nontoxic
to the subject to which it is administered or is capable of being
formulated in a nontoxic composition that can be administered to
the subject. In addition, following exposure to light, the compound
in any resulting photodegraded form is also preferably
nontoxic.
[0088] The probes and activating light can be administered by any
means known in the art for PDT, including, but not limited to,
ingestion, injection, transcutaneous administration, transdermal
administration, and transillumination. Preferably, the light is
administered transcutaneously to a subject. For example,
"transcutaneous" as used herein refers to the passage of light
through unbroken tissue. Where the tissue layer is skin or dermis,
transcutaneous includes "transdermal" and it will be understood
that the light source is external to the outer skin layer. However,
the term "transillumination" as used herein refers to the passage
of light through a tissue layer, such as the outer surface layer of
an organ, e.g., the liver, and it will be apparent that the light
source is external to the organ, but internal or implanted within
the subject or patient.
[0089] In some embodiments, the forms of energy used for
administering PDT include, but are not limited to, light (i.e.,
radiation), thermal, sonic, ultrasonic, chemical, light, microwave,
ionizing (such as x-ray and gamma ray), mechanical, and electrical.
The term "radiation" as used herein includes all wavelengths and
wavebands. Preferably, the radiation wavelength or waveband is
selected to correspond with or at least overlap the wavelengths or
wavebands that excite the photosensitizing agent. Photosensitive
agents typically have one or more absorption wavebands that excite
them to produce the substances which damage or destroy target
cells, tissues, organs, or tumors. Preferably, the radiation
wavelength or waveband matches the excitation wavelength or
waveband of the photosensitizing agent and has low absorption by
the non-target cells and the rest of the subject, including blood
proteins.
[0090] In further embodiments, the target tumor, tissue, or organ
for treatment with PDT is selected from the group consisting of
vascular endothelial tissue, an abnormal vascular wall of a tumor,
a solid tumor, a tumor of the head, a tumor of the neck, a tumor of
a the gastrointestinal tract, a tumor of the liver, a tumor of the
breast, a tumor of the prostate, a tumor of the ovary, a tumor of
the uterus, a tumor of the testicle, a tumor of the lung, a
nonsolid tumor, malignant cells of one of a hematopoietic tissue
and a lymphoid tissue, lesions in the vascular system, a diseased
bone marrow, and diseased cells in which the disease is one of an
autoimmune and an inflammatory disease. In yet a further
embodiment, the target tissue is a lesion in the vascular system of
a type selected from the group consisting of atherosclerotic
lesions, arteriovenous malformations, aneurysms, and venous
lesions.
IV. Examples
[0091] The following examples are offered to illustrate, but not to
limit, the claimed invention.
Example 1
Therapeutic and Diagnostic Probes Containing a Viral Particle
[0092] This example provides an exemplary embodiment of a
therapeutic and diagnostic probe as described herein.
Producing a Viral Particle Based Probe
[0093] A recombinant DNA molecule containing a sequence encoding a
papillomavirus L1 protein, a L2 protein or a combination of L1 and
L2 proteins is constructed using standard molecular biology
methods. See, for example, Sambrook et al., Molecular cloning: a
laboratory manual, 3.sup.rd. ed., Cold Spring Harbor, N.Y., Cold
Spring Harbor Laboratory. A host cell such as a bacterial cell is
transfected with the recombinant DNA molecule. Thereafter, the L1
and L2 capsid proteins are purified from the transfected cells such
that they self-assemble into papillomavirus-like particles. See,
for example, U.S. Application Publication No. 2011/0091496. IRDye
800CW-n-hydroxysuccinimide (NHS) ester (LI-COR, Lincoln, Nebr.) and
IRDye.RTM. 700DX-NHS ester are conjugated to the viral particle
according to the manufacturer's instructions.
Using the Viral Particle Based Probe to Induce Cell Death of a
Target Cell in a Subject
[0094] The probe is injected intravenously into a subject, e.g., a
human subject. NIR infrared fluorescence is visualized at different
time points with an optical imager at excitation of 774 nm and
emission at 789 nm. The non-invasive deep-tissue imaging allows the
clinician to identify target cells, e.g., diseased cells, in
various tissues of the body.
[0095] After the location of the probe is determined, an activating
light is directed at the specific target cells bound to the probe.
The photosensitizer agent of probe is exposed to the light, thereby
generating a cytotoxic singlet oxygen. This, in turn, results in
cell death of the target cell via apoptosis and/or necrosis.
Example 2
Therapeutic and Diagnostic Probes Containing a Viral Particle and a
Targeting Moiety
[0096] This example provides an exemplary embodiment of the present
invention, in particular, a therapeutic and diagnostic probe based
on a viral particle that selectively binds to EGFR expressing
cells, such as cancer cells.
[0097] The virus-like particle based probe is generated according
to the method described in Example 1 with modifications to express
a targeting moiety, such as EGF on the surface of the probe. The
EGF protein can be labeled on free amine groups using an NHS ester
derivative of IRDye.RTM. 700DX or IRDye.RTM. 800CW (LI-COR,
Lincoln, Nebr.). The viral-like particle can also be linked to the
dye(s) through a linker as described above.
[0098] The probe is administered to the subject, e.g., the human
subject, by intravenous injection or by direct injection into a
solid tumor containing EGFR-overexpressing cells. The EGF probe
then binds to EGFR on the surface of cancer cells. Near-infrared
fluorescence imaging is performed at various time points to
determine the location of the IRDye.RTM.800CW dye of probe. An
activating light is directed to the IRDye.RTM.700DX of the probe
which in turn produces a cytotoxic singlet oxygen that induces cell
death in the EGFR-overexpressing cancer cells.
[0099] In summary, this example illustrates the use of a
therapeutic-diagnostic probe that contains an EGF targeting moiety
that selectively binds to EGFR expressing cells. Described herein
is a method for inducing cell death of EGFR expressing cancer cells
by administering the probe.
[0100] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, one of skill in the art will appreciate that
certain changes and modifications may be practiced within the scope
of the appended claims. In addition, each reference provided herein
is incorporated by reference in its entirety to the same extent as
if each reference was individually incorporated by reference.
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