U.S. patent application number 16/720176 was filed with the patent office on 2020-11-12 for nanoparticle immunoconjugates.
The applicant listed for this patent is Cornell University, The Curators of the University of Missouri, Memorial Sloan Kettering Cancer Center. Invention is credited to Michelle S. Bradbury, Feng Chen, Jason Lewis, Kai Ma, Thomas P. Quinn, Ulrich Wiesner, Barney Yoo.
Application Number | 20200353096 16/720176 |
Document ID | / |
Family ID | 1000004989253 |
Filed Date | 2020-11-12 |
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United States Patent
Application |
20200353096 |
Kind Code |
A1 |
Bradbury; Michelle S. ; et
al. |
November 12, 2020 |
NANOPARTICLE IMMUNOCONJUGATES
Abstract
Disclosed herein are nanoparticle immunoconjugates useful for
therapeutics and/or diagnostics. The immunoconjugates have diameter
(e.g., average diameter) no greater than 20 nanometers (e.g., as
measured by dynamic light scattering (DLS) in aqueous solution,
e.g., saline solution). In certain embodiments, the conjugates are
silica-based nanoparticles with single chain antibody fragments
attached thereto.
Inventors: |
Bradbury; Michelle S.; (New
York, NY) ; Quinn; Thomas P.; (Columbia, MO) ;
Chen; Feng; (New York, NY) ; Yoo; Barney; (New
York, NY) ; Lewis; Jason; (New York, NY) ;
Wiesner; Ulrich; (Ithaca, NY) ; Ma; Kai;
(Ithaca, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Memorial Sloan Kettering Cancer Center
Cornell University
The Curators of the University of Missouri |
New York
Ithaca
Columbia |
NY
NY
MO |
US
US
US |
|
|
Family ID: |
1000004989253 |
Appl. No.: |
16/720176 |
Filed: |
December 19, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15564315 |
Oct 4, 2017 |
10548989 |
|
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PCT/US16/26434 |
Apr 7, 2016 |
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16720176 |
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62151943 |
Apr 23, 2015 |
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62144278 |
Apr 7, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 51/1251 20130101;
A61K 49/0058 20130101; A61K 45/06 20130101; A61K 47/6923 20170801;
C07K 2317/569 20130101; C07K 2317/622 20130101; A61K 51/1093
20130101; C07K 2317/55 20130101; A61K 51/10 20130101; A61K 51/0478
20130101; A61K 49/1824 20130101; C07K 16/40 20130101 |
International
Class: |
A61K 47/69 20060101
A61K047/69; A61K 51/12 20060101 A61K051/12; A61K 51/04 20060101
A61K051/04; A61K 51/10 20060101 A61K051/10; A61K 45/06 20060101
A61K045/06; A61K 49/00 20060101 A61K049/00; A61K 49/18 20060101
A61K049/18; C07K 16/40 20060101 C07K016/40 |
Goverment Interests
GOVERNMENT FUNDING
[0002] This invention was made with government support under Grant
No. U54 CA199081-01 awarded by NIH. The government has certain
rights in this invention.
Claims
1-44. (canceled)
45. An immunoconjugate comprising: a nanoparticle coated with an
organic polymer; an antibody fragment conjugated to the organic
polymer-coated nanoparticle, a therapeutic agent conjugated to the
organic polymer-coated nanoparticle through a linker, wherein the
nanoparticle has a diameter no greater than 20 nanometers, wherein
the nanoparticle comprises a silica-based core and a silica shell
surrounding at least a portion of the core, wherein the antibody
fragment is a single chain variable fragment (scFv).
46. The immunoconjugate of claim 45, wherein the linker is a
cleavable linker.
47. The immunoconjugate of claim 46, wherein the cleavable linker
is selected from a group consisting of peptide, hydrazine and
disulfide linkers.
48. The immunoconjugate of claim 45, wherein the linker is a
peptide linker.
49. The immunoconjugate of claim 45, wherein the linker is a
peptide linker that is cleaved by lysosomal proteases.
50. The immunoconjugate of claim 49, wherein the lysosomal protease
is cathepsin-B.
51. The immunoconjugate of claim 45, wherein the linker is a
dipeptide linker.
52. The immunoconjugate of claim 51, wherein the dipeptide linker
is a valine-citrulline linker.
53. The immunoconjugate of claim 45, wherein the antibody fragment
is from about 25 kDa to about 30 kDa.
54. The immunoconjugate of claim 45, wherein the nanoparticle
comprises a fluorescent compound within the core.
55. The immunoconjugate of claim 45, wherein the nanoparticle has
from one to ten antibody fragments attached thereto.
56. The immunoconjugate of claim 45, wherein the nanoparticle has a
diameter no greater than 15 nanometers.
57. The immunoconjugate of claim 45, wherein the nanoparticle has a
diameter in a range from 1 nm to 20 nm.
58. The immunoconjugate of claim 45, wherein the antibody fragment
comprises anti-VEGF-A.
59. The immunoconjugate of claim 45, wherein the immunoconjugate
comprises one or more imaging agents.
60. The immunoconjugate of claim 59, wherein the one or more
imaging agents comprise a PET tracer.
61. The immunoconjugate of claim 59, wherein the one or more
imaging agents comprise a fluorophore.
62. The immunoconjugate of claim 4, wherein the therapeutic agent
comprises a chemotherapy drug.
63. The immunoconjugate of claim 45, wherein the therapeutic agent
comprises a radioisotope.
64. The immunoconjugate of claim 63, wherein the radioisotope is a
member selected from the group consisting of .sup.99mTc,
.sup.111In, .sup.64Cu, .sup.67Ga, .sup.186Re, .sup.188Re,
.sup.153Sm, .sup.177Lu, .sup.67Cu, .sup.123I, .sup.124I, .sup.125I,
.sup.11C, .sup.13N, .sup.15O, .sup.18F, .sup.186Re, .sup.188Re,
.sup.153Sm, .sup.166Ho, .sup.177Lu, .sup.149Pm, .sup.90Y,
.sup.213Bi, .sup.103Pd, .sup.109Pd, .sup.159Gd, .sup.140La,
.sup.198Au, .sup.199Au, .sup.169Yb, .sup.175Yb, .sup.165Dy,
.sup.166Dy, .sup.67Cu, .sup.105Rh, .sup.111Ag, .sup.89Zr,
.sup.225Ac, and .sup.192Ir.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Application Ser.
No. 62/144,278 filed on Apr. 7, 2015 and U.S. Application Ser. No.
62/151,943 filed on Apr. 23, 2015, the disclosures of which are
hereby incorporated by reference in their entireties.
FIELD OF THE INVENTION
[0003] This invention relates generally to nanoparticle
immunoconjugates (e.g., under 20 nanometers in diameter), useful,
for example, for the detection, prevention, and/or treatment of
cancer and other diseases.
BACKGROUND
[0004] Nano-therapeutic and/or -diagnostic delivery vehicles are
typically macro- or supra-molecular multicomponent systems, ranging
in size from 1-1,000 nm, that are either inherently therapeutic
(e.g., no active pharmaceutical ingredient) or function as
therapeutic or diagnostic delivery systems. To date, liposomal
nanoparticles and biologics comprise a large proportion of the
number of FDA-approved products or products in clinical trials used
to treat and/or detect a variety of cancer types, while a number of
polymer-based particle formulations are currently in early phase
trials.
[0005] Desirable candidates for nanotherapeutic delivery systems
share a common feature of incorporating and releasing a drug
compound in a controlled manner, which can favorably alter drug
bioavailability and pharmacokinetics, while minimizing off-target
toxicities. Ideally, an imaging label is incorporated therein to
assess their precise localization and retention at disease
sites.
[0006] However, these systems function using different mechanisms.
For example, antibody drug conjugates (ADCs) achieve lower drug
toxicity primarily through active targeting of tumor cells and
conditional release of drug molecules. Upon binding a cell surface
antigen, active drug release occurs after cellular internalization
and endosomal uptake. On the other hand, liposomes and
polymer-based drug delivery systems, which are typically much
larger assembled complexes (.about.20-150 nm diameters) passively
loaded with a greater payload (.about.10,000 drug molecules for
Doxil) or imaging agents, have generally lacked targeting
capabilities (BIND-014 is an exception). Therefore, these complexes
rely primarily on the well-known enhanced permeability and
retention (EPR) effect for the successful delivery of
nano-formulated drugs. While interstitial permeation of liposomes
may be poor due to their size, the free drug is released through
various mechanisms that are not entirely understood. For example,
Abraxane (.about.140 nm) relies on a different approach to enhance
the bioavailability of a hydrophobic compound. In this case, a
specific formulation of albumin and drug (paclitaxel) forms the
initial complex, which is in turn estimated to disperse into
smaller protein-drug aggregates upon injection.
[0007] Metastatic disease may effectively be treated with
immunotherapies; however, a significant subpopulation will not
respond due to lack of antigenic mutations or the immune-evasive
properties of cancer. In addition, although radiation therapy (RT)
is a standard treatment for cancer, local failures occur.
Preclinical data indicate that RT can potentiate the systemic
efficacy of immunotherapy, while activation of the innate and
adaptive immune system can enhance the local efficacy of RT.
[0008] There remains a need for a platform that can be used for the
detection, prevention, and/or treatment of cancer and other
diseases.
SUMMARY
[0009] Described herein are target-specific nanoparticle
immunoconjugates (e.g., single chain antibody fragments bound to
the particle surface) for targeted diagnostic and/or therapeutic
platforms. In certain embodiments, the nanoparticle
immunoconjugates are less than 20 nm (e.g., 6 to 10 nm) in
diameter. This small size is found to offer advantages in
therapeutic and/or imaging applications. For example, the disclosed
immunoconjugates may offer improved targeting of diseased tissue
and reduced non-specific uptake by organs (e.g., by the liver). The
smaller immunoconjugates may also demonstrate reduced immune
reactivity, thereby further improving efficacy.
[0010] Also described herein is a multi-therapeutic platform that
comprises an immunoconjugate and therapeutic radioisotopes. In
certain embodiments, immunoconjugates and therapeutic radioisotopes
are delivered in concert for synergistic effects of combined
radiation therapy and immunotherapy. In certain embodiments, an
antibody fragment and a therapeutic radioisotope are attached to
nanoparticles, thereby creating a target-specific nanoparticle
immunoconjugate. A given nanoparticle can have both radionuclides
(radioisotopes) and antibodies (and/or antibody fragments) attached
thereto (in which case, the immunoconjugate is a
radioimmunoconjugate). Also, in some embodiments, a portion of the
administered nanoparticles have radionuclides attached (covalently
or non-covalently bonded, or otherwise associated with the
nanoparticle) while other administered nanoparticles have antibody
fragments attached. Also included in various embodiments are
combination therapies in which either exiting (e.g., traditional)
radiotherapy is combined with administration of nanoparticle
immunoconjugates described herein, or existing (e.g., traditional)
immunotherapy is combined with administration of nanoparticle
radioconjugates (nanoparticles with bound radioisotopes),
[0011] The certain embodiments, the target-specific nanoparticle
immunoconjugates comprise a targeting peptide. In certain
embodiments, the therapeutic radioisotope is delivered separately
from the target-specific nanoparticle immunoconjugate (e.g., via
radiation therapy or via attached to a separate tareget-specific
nanoparticle). In certain embodiments, immunotherapy is delivered
separately from the target-specific immunoconjugate. In certain
embodiments, an antibody fragment is attached to one polyethylene
glycol (PEG) moiety (via a particular chelator) and a radioisotope
is attached to another PEG moiety (via another chelator). The PEG
moieties are then attached to nanoparticles.
[0012] In one aspect, the invention is directed to An
immunoconjugate comprising: a nanoparticle; and an antibody
fragment conjugated to the nanoparticle, wherein the nanoparticle
has a diameter (e.g., average diameter) no greater than 20
nanometers (e.g., as measured by dynamic light scattering (DLS) in
aqueous solution, e.g., saline solution) (e.g., wherein the average
nanoparticle diameter is from 1 to 20 nm, e.g., from 1 to 15 nm,
e.g., from 1 to 10 nm, e.g., from 1 to 8 nm, e.g., from 4 to 10 nm,
e.g., from 4 to 8 nm) (e.g., wherein the immunoconjugate has an
average diameter no greater than 50 nm, e.g., no greater than 40
nm, e.g., no greater than 30 nm, e.g., no greater than 20 nm, e.g.,
no greater than 15 nm, e.g., no greater than 10 nm).
[0013] In certain embodiments, the antibody fragment is covalently
or non-covalently bonded to the nanoparticle via a linker or
covalently or non-covalently bonded directly to the nanoparticle,
or associated with the nanoparticle or a composition surrounding
the nanoparticle, e.g., via van der Waals forces.
[0014] In certain embodiments, the nanoparticle is coated with an
organic polymer (e.g., polyethylene glycol (PEG)) (e.g., wherein
immunoconjugate comprises a chelator).
[0015] In certain embodiments, a targeting peptide (e.g., alphaMSH,
any peptide known to be immunomodulatory and anti-inflammatory in
nature).
[0016] In certain embodiments, the antibody fragment is in a range
from about 5 kDa to about 25 kDa (e.g., from about 10 kDa to about
20 kDa, e.g., about 15 kDa) (e.g., wherein the antibody fragment
comprises a functional single domain antibody fragment).
[0017] In certain embodiments, the antibody fragment is from about
20 kDa to about 45 kDa (e.g., from about 25 kDa to about 30 kDa)
(e.g., wherein the antibody fragment comprises a functional single
chain antibody fragment).
[0018] In certain embodiments, the antibody fragment is from about
40 kDa to about 80 kDa (e.g., from about 50 kDa to about 70 kDa,
e.g., about 60 kDa) (e.g., wherein the antibody fragment comprises
a functional fab fragment).
[0019] In certain embodiments, the nanoparticle comprises
silica.
[0020] In certain embodiments, the nanoparticle comprises a
silica-based core and a silica shell surrounding at least a portion
of the core.
[0021] In certain embodiments, the nanoparticle comprises a
fluorescent compound within the core.
[0022] In certain embodiments, the antibody fragment is a member
selected from the set consisting of a recombinant antibody fragment
(fAbs), a single chain variable fragment (scFv), and a single
domain antibody (sdAb) fragment.
[0023] In certain embodiments, the antibody fragment is a single
chain variable fragment (scFv).
[0024] In certain embodiments, the antibody fragment is a single
domain (sdAb) fragment.
[0025] In certain embodiments, the nanoparticle (a single
nanoparticle) has from one to ten antibody fragments (e.g., from 1
to 7, e.g., from 1 to 5, e.g., from 2 to 7, e.g., from 2 to 5,
e.g., from 1 to 4, e.g., from 2 to 4) attached thereto.
[0026] In certain embodiments, the antibody fragment is conjugated
to the nanoparticle via a PEG moiety and a chelator.
[0027] In certain embodiments, the nanoparticle has a diameter
(e.g., average diameter) no greater than 15 nanometers (e.g., no
greater than 13 nanometers, e.g., no greater than 10
nanometers).
[0028] In certain embodiments, the nanoparticle has a diameter
(e.g., average diameter) in a range from 1 nm to 20 nm (e.g., from
2 nm to 15 nm, e.g., from 5 nm to 15 nm, e.g., from 1 nm to 10 nm,
e.g., from 2 nm to 10 nm, e.g., from 5 nm to 10 nm).
[0029] In certain embodiments, the antibody fragment comprises a
member selected from the set consisting of anti-CEA scFv,
anti-GPIIb/IIIa, anti-VEGF-A, and anti-TNF-.alpha. (e.g.,
PEGylated).
[0030] In certain embodiments, the immunoconjugate comprises one or
more imaging agents (e.g., within the nanoparticle, attached to the
nanoparticle, and/or attached to the antibody fragment).
[0031] In certain embodiments, the one or more imaging agents
comprise a PET tracer (e.g., .sup.89Zr, .sup.64Cu, and/or
[.sup.18F] fluorodeoxyglucose).
[0032] In certain embodiments, the one or more imaging agents
comprise a fluorophore (e.g., a cyanine).
[0033] In certain embodiments, the immunoconjugate further
comprises a therapeutic agent (e.g., wherein the therapeutic agent
is attached to the nanoparticle, or to the antibody fragment, or to
both the nanoparticle and the antibody fragment, e.g., wherein the
attachment is covalent or non-covalent).
[0034] In certain embodiments, the therapeutic agent comprises a
chemotherapy drug (e.g., sorafenib, paclitaxel, docetaxel, MEK162,
etoposide, lapatinib, nilotinib, crizotinib, fulvestrant,
vemurafenib, bexorotene, and/or camptotecin).
[0035] In certain embodiments, the therapeutic agent comprises a
radioisotope (e.g., wherein the radioisotope is attached to the
nanoparticle via a second chelator) (e.g., wherein the radioisotope
is a therapeutic radioisotope).
[0036] In certain embodiments, the radioisotope is a member
selected from the group consisting of .sup.99mTc, .sup.111In,
.sup.64Cu, .sup.67Ga, .sup.186Re, .sup.188Re, .sup.153Sm,
.sup.177Lu, .sup.67Cu, .sup.123I, .sup.124I, .sup.125I, .sup.11C,
.sup.13N, .sup.15O, .sup.18F, .sup.186Re, .sup.188Re, .sup.153Sm,
.sup.166Ho, .sup.177Lu, .sup.149Pm, .sup.90Y, .sup.213Bi,
.sup.103Pd, .sup.109Pd, .sup.159Gd, .sup.140La, .sup.198Au,
.sup.199Au, .sup.169Yb, .sup.175Yb, .sup.165Dy, .sup.166Dy,
.sup.67Cu, .sup.105Rh, .sup.111Ag, .sup.89Zr, .sup.225Ac, and
.sup.192Ir.
[0037] In another aspect, the invention is directed to a method of
treating a disease or condition, the method comprising
administering to a subject a pharmaceutical composition comprising
the immunoconjugate (e.g., to target a particular type of tissue,
e.g., cancer).
[0038] In certain embodiments, the method comprises administering a
therapeutic radioisotope (e.g., wherein the therapeutic
radioisotope is attached to a second nanoparticle having a diameter
(e.g., average diameter) no greater than 20 nanometers (e.g., as
measured by dynamic light scattering (DLS) in aqueous solution,
e.g., saline solution) (e.g., wherein the radioisotope is attached
to the second nanoparticle via a second chelator)) (e.g., wherein
the second nanoparticle has a diameter from 1 to 20 nm, e.g., from
1 to 15 nm, e.g., from 1 to 10 nm, e.g., from 1 to 8 nm, e.g., from
4 to 10 nm, e.g., from 4 to 8 nm).
[0039] In another aspect, the invention is directed to a method of
treating a disease or condition, the method comprising
administering to a subject a pharmaceutical composition comprising
the immunoconjugate (e.g., to target a particular type of tissue,
e.g., cancer) (e.g., for combined radiation therapy and
immunotherapy).
[0040] In certain embodiments, the pharmaceutical composition
further comprises a carrier.
[0041] In another aspect, the invention is directed to a method of
in vivo imaging (e.g., intraoperative imaging), the method
comprising: administering to a subject a composition comprising the
immunoconjugate (e.g., such that the immunoconjugate preferentially
collects in a particular region, e.g., near or within a particular
tissue type, e.g., cancer), wherein the immunoconjugate comprises
an imaging agent; and detecting (e.g., via PET, X-ray, MRI, CT,
etc.) the imaging agent.
[0042] In another aspect, the invention is directed to a method of
making the immunoconjugate, the method comprising: contacting a
nanoparticle-PEG-thiol with a protein-maleimide, thereby producing
the immunoconjugate.
[0043] In certain embodiments, the method further comprises
reacting the nanoparticle with one or more compounds, the one or
more compounds comprising a thiol moiety and an amine moiety (e.g.,
cysteine methyl ester or cysteamine-HCl), thereby producing a
nanoparticle-PEG-amine; reacting the nanoparticle-PEG-amine with
SPDP, then removing a pyridine 2-thione from the product (e.g.,
using TCEP), thereby producing the nanoparticle-PEG-thiol.
[0044] In another aspect, the invention is directed to a method of
making the immunoconjugate, the method comprising: modifying the
antibody fragment (protein) with a first click reactive group
(e.g., methyltetrazine-PEG4-NHS ester; modifying a
nanoparticle-PEG-amine with a click partner of the first click
reactive group (e.g., TCO-PEG4-NHS ester); and reacting the
modified antibody fragment with the modified nanoparticle-PEG,
thereby producing the immunoconjugate.
[0045] In certain embodiments, the method further comprises
reacting the nanoparticle with one or more compounds, the one or
more compounds comprising a thiol moiety and an amine moiety (e.g.,
cysteine methyl ester or cysteamine-HCl), thereby producing the
nanoparticle-PEG-amine.
[0046] In another aspect, the invention is directed to a method of
treating a disease or condition, the method comprising
administering to a subject a composition (e.g., a pharmaceutical
composition) comprising: a nanoparticle; and a therapeutic
radioisotope conjugated to the nanoparticle (e.g., covalently or
non-covalently bonded to the nanoparticle via a linker or
covalently or non-covalently bonded directly to the nanoparticle,
or associated with the nanoparticle or a composition surrounding
the nanoparticle, e.g., via van der Waals forces), wherein the
nanoparticle has a diameter (e.g., average diameter) no greater
than 20 nanometers (e.g., as measured by dynamic light scattering
(DLS) in aqueous solution, e.g., saline solution) (e.g., wherein
the average nanoparticle diameter is from 1 to 20 nm, e.g., from 1
to 15 nm, e.g., from 1 to 10 nm, e.g., from 1 to 8 nm, e.g., from 4
to 10 nm, e.g., from 4 to 8 nm) (e.g., to target a particular type
of tissue, e.g., cancer).
[0047] In certain embodiments, the method comprises administering
immunotherapy (e.g., wherein the immunotherapy comprises
administering to a subject a pharmaceutical composition comprising
the immunoconjugate).
[0048] In another aspect, the invention is directed to an
immunoconjugate comprising: a nanoparticle; and an antibody
fragment conjugated to the nanoparticle (e.g., covalently or
non-covalently bonded to the nanoparticle via a linker or
covalently or non-covalently bonded directly to the nanoparticle,
or associated with the nanoparticle or a composition surrounding
the nanoparticle, e.g., via van der Waals forces), wherein the
nanoparticle has a diameter (e.g., average diameter) no greater
than 20 nanometers (e.g., as measured by dynamic light scattering
(DLS) in aqueous solution, e.g., saline solution) (e.g., wherein
the average nanoparticle diameter is from 1 to 20 nm, e.g., from 1
to 15 nm, e.g., from 1 to 10 nm, e.g., from 1 to 8 nm, e.g., from 4
to 10 nm, e.g., from 4 to 8 nm) (e.g., wherein the immunoconjugate
has an average diameter no greater than 50 nm, e.g., no greater
than 40 nm, e.g., no greater than 30 nm, e.g., no greater than 20
nm, e.g., no greater than 15 nm, e.g., no greater than 10 nm)
(e.g., wherein the nanoparticle is coated with an organic polymer
(e.g., polyethylene glycol (PEG)) (e.g., wherein immunoconjugate
comprises a chelator), for use in a method of treating a disease or
condition in a subject, wherein the treating comprises: delivering
the immunoconjugate to the subject; and delivering a therapeutic
radioisotope (e.g., wherein the therapeutic radioisotope is
attached to a second nanoparticle having a diameter (e.g., average
diameter) no greater than 20 nanometers (e.g., as measured by
dynamic light scattering (DLS) in aqueous solution, e.g., saline
solution) (e.g., wherein the radioisotope is attached to the second
nanoparticle via a second chelator)).
[0049] In another aspect, the invention is directed to an
immunoconjugate comprising: a nanoparticle; a therapeutic
radioisotope (e.g., wherein the radioisotope is attached to the
nanoparticle via a second chelator) (e.g., wherein the radioisotope
is a therapeutic radioisotope); and an antibody fragment conjugated
to the nanoparticle (e.g., covalently or non-covalently bonded to
the nanoparticle via a linker or covalently or non-covalently
bonded directly to the nanoparticle, or associated with the
nanoparticle or a composition surrounding the nanoparticle, e.g.,
via van der Waals forces), wherein the nanoparticle has a diameter
(e.g., average diameter) no greater than 20 nanometers (e.g., as
measured by dynamic light scattering (DLS) in aqueous solution,
e.g., saline solution) (e.g., wherein the average nanoparticle
diameter is from 1 to 20 nm, e.g., from 1 to 15 nm, e.g., from 1 to
10 nm, e.g., from 1 to 8 nm, e.g., from 4 to 10 nm, e.g., from 4 to
8 nm) (e.g., wherein the immunoconjugate has an average diameter no
greater than 50 nm, e.g., no greater than 40 nm, e.g., no greater
than 30 nm, e.g., no greater than 20 nm, e.g., no greater than 15
nm, e.g., no greater than 10 nm) (e.g., wherein the nanoparticle is
coated with an organic polymer (e.g., polyethylene glycol (PEG))
(e.g., wherein immunoconjugate comprises a chelator) for use in a
method of treating a disease or condition in a subject, wherein the
treating comprises: delivering the immunoconjugate to the
subject.
[0050] In another aspect, the invention is directed to an
immunoconjugate comprising a nanoparticle; and an antibody fragment
conjugated to the nanoparticle (e.g., covalently or non-covalently
bonded to the nanoparticle via a linker or covalently or
non-covalently bonded directly to the nanoparticle, or associated
with the nanoparticle or a composition surrounding the
nanoparticle, e.g., via van der Waals forces), wherein the
nanoparticle has a diameter (e.g., average diameter) no greater
than 20 nanometers (e.g., as measured by dynamic light scattering
(DLS) in aqueous solution, e.g., saline solution) (e.g., wherein
the average nanoparticle diameter is from 1 to 20 nm, e.g., from 1
to 15 nm, e.g., from 1 to 10 nm, e.g., from 1 to 8 nm, e.g., from 4
to 10 nm, e.g., from 4 to 8 nm) (e.g., wherein the immunoconjugate
has an average diameter no greater than 50 nm, e.g., no greater
than 40 nm, e.g., no greater than 30 nm, e.g., no greater than 20
nm, e.g., no greater than 15 nm, e.g., no greater than 10 nm)
(e.g., wherein the nanoparticle is coated with an organic polymer
(e.g., polyethylene glycol (PEG)) (e.g., wherein immunoconjugate
comprises a chelator), and wherein the immunoconjugate comprises an
imaging agent, for use in a method of in vivo diagnosis of a
disease or condition in a subject, wherein the in vivo diagnosis
comprises: delivering the immunoconjugate to the subject; and
detecting (e.g., via PET, X-ray, MRI, CT, etc.) the imaging
agent.
[0051] In another aspect, the invention is directed to an
immunoconjugate comprising: a nanoparticle; and an antibody
fragment conjugated to the nanoparticle (e.g., covalently or
non-covalently bonded to the nanoparticle via a linker or
covalently or non-covalently bonded directly to the nanoparticle,
or associated with the nanoparticle or a composition surrounding
the nanoparticle, e.g., via van der Waals forces), wherein the
nanoparticle has a diameter (e.g., average diameter) no greater
than 20 nanometers (e.g., as measured by dynamic light scattering
(DLS) in aqueous solution, e.g., saline solution) (e.g., wherein
the average nanoparticle diameter is from 1 to 20 nm, e.g., from 1
to 15 nm, e.g., from 1 to 10 nm, e.g., from 1 to 8 nm, e.g., from 4
to 10 nm, e.g., from 4 to 8 nm) (e.g., wherein the immunoconjugate
has an average diameter no greater than 50 nm, e.g., no greater
than 40 nm, e.g., no greater than 30 nm, e.g., no greater than 20
nm, e.g., no greater than 15 nm, e.g., no greater than 10 nm)
(e.g., wherein the nanoparticle is coated with an organic polymer
(e.g., polyethylene glycol (PEG)) (e.g., wherein immunoconjugate
comprises a chelator), and wherein the immunoconjugate comprises an
imaging agent, for use in (a) a method of treating a disease or
condition in a subject or (b) a method of in vivo diagnosis of a
disease or condition in a subject, wherein the method comprises:
administering to a subject a pharmaceutical composition comprising
the immunoconjugate (e.g., to target a particular type of tissue,
e.g., cancer); and optionally, detecting (e.g., via PET, X-ray,
MRI, CT, etc.) the imaging agent.
[0052] In another aspect, the invention is directed to an
immunoconjugate comprising a nanoparticle; and an antibody fragment
conjugated to the nanoparticle (e.g., covalently or non-covalently
bonded to the nanoparticle via a linker or covalently or
non-covalently bonded directly to the nanoparticle, or associated
with the nanoparticle or a composition surrounding the
nanoparticle, e.g., via van der Waals forces), wherein the
nanoparticle has a diameter (e.g., average diameter) no greater
than 20 nanometers (e.g., as measured by dynamic light scattering
(DLS) in aqueous solution, e.g., saline solution) (e.g., wherein
the average nanoparticle diameter is from 1 to 20 nm, e.g., from 1
to 15 nm, e.g., from 1 to 10 nm, e.g., from 1 to 8 nm, e.g., from 4
to 10 nm, e.g., from 4 to 8 nm) (e.g., wherein the immunoconjugate
has an average diameter no greater than 50 nm, e.g., no greater
than 40 nm, e.g., no greater than 30 nm, e.g., no greater than 20
nm, e.g., no greater than 15 nm, e.g., no greater than 10 nm)
(e.g., wherein the nanoparticle is coated with an organic polymer
(e.g., polyethylene glycol (PEG)) (e.g., wherein immunoconjugate
comprises a chelator) for use in therapy.
[0053] In another aspect, the invention is directed to an
immunoconjugate comprising: a nanoparticle; a therapeutic
radioisotope (e.g., wherein the radioisotope is attached to the
nanoparticle via a second chelator) (e.g., wherein the radioisotope
is a therapeutic radioisotope); and an antibody fragment conjugated
to the nanoparticle (e.g., covalently or non-covalently bonded to
the nanoparticle via a linker or covalently or non-covalently
bonded directly to the nanoparticle, or associated with the
nanoparticle or a composition surrounding the nanoparticle, e.g.,
via van der Waals forces), wherein the nanoparticle has a diameter
(e.g., average diameter) no greater than 20 nanometers (e.g., as
measured by dynamic light scattering (DLS) in aqueous solution,
e.g., saline solution) (e.g., wherein the average nanoparticle
diameter is from 1 to 20 nm, e.g., from 1 to 15 nm, e.g., from 1 to
10 nm, e.g., from 1 to 8 nm, e.g., from 4 to 10 nm, e.g., from 4 to
8 nm) (e.g., wherein the immunoconjugate has an average diameter no
greater than 50 nm, e.g., no greater than 40 nm, e.g., no greater
than 30 nm, e.g., no greater than 20 nm, e.g., no greater than 15
nm, e.g., no greater than 10 nm) (e.g., wherein the nanoparticle is
coated with an organic polymer (e.g., polyethylene glycol (PEG))
(e.g., wherein immunoconjugate comprises a chelator) for use in
therapy.
[0054] In another aspect, the invention is directed to an
immunoconjugate comprising: a nanoparticle; and an antibody
fragment conjugated to the nanoparticle (e.g., covalently or
non-covalently bonded to the nanoparticle via a linker or
covalently or non-covalently bonded directly to the nanoparticle,
or associated with the nanoparticle or a composition surrounding
the nanoparticle, e.g., via van der Waals forces), wherein the
nanoparticle has a diameter (e.g., average diameter) no greater
than 20 nanometers (e.g., as measured by dynamic light scattering
(DLS) in aqueous solution, e.g., saline solution) (e.g., wherein
the average nanoparticle diameter is from 1 to 20 nm, e.g., from 1
to 15 nm, e.g., from 1 to 10 nm, e.g., from 1 to 8 nm, e.g., from 4
to 10 nm, e.g., from 4 to 8 nm) (e.g., wherein the immunoconjugate
has an average diameter no greater than 50 nm, e.g., no greater
than 40 nm, e.g., no greater than 30 nm, e.g., no greater than 20
nm, e.g., no greater than 15 nm, e.g., no greater than 10 nm)
(e.g., wherein the nanoparticle is coated with an organic polymer
(e.g., polyethylene glycol (PEG)) (e.g., wherein immunoconjugate
comprises a chelator), and wherein the immunoconjugate comprises an
imaging agent, for use in in vivo diagnosis.
[0055] In another aspect, the invention is directed to a
composition (e.g., pharmaceutical composition) comprising: a
nanoparticle; and a therapeutic radioisotope conjugated to the
nanoparticle (e.g., covalently or non-covalently bonded to the
nanoparticle via a linker or covalently or non-covalently bonded
directly to the nanoparticle, or associated with the nanoparticle
or a composition surrounding the nanoparticle, e.g., via van der
Waals forces), wherein the nanoparticle has a diameter (e.g.,
average diameter) no greater than 20 nanometers (e.g., as measured
by dynamic light scattering (DLS) in aqueous solution, e.g., saline
solution) (e.g., wherein the average nanoparticle diameter is from
1 to 20 nm, e.g., from 1 to 15 nm, e.g., from 1 to 10 nm, e.g.,
from 1 to 8 nm, e.g., from 4 to 10 nm, e.g., from 4 to 8 nm) (e.g.,
wherein the nanoparticle is coated with an organic polymer (e.g.,
polyethylene glycol (PEG)) (e.g., wherein immunoconjugate comprises
a chelator)) for use in a method of treating a disease or condition
in a subject, wherein the treating comprises: delivering the
composition to the subject; and delivering immunotherapy (e.g.,
wherein the immunotherapy comprises administering to a subject a
pharmaceutical composition comprising the immunoconjugate).
[0056] Elements of embodiments involving one aspect of the
invention (e.g., methods) can be applied in embodiments involving
other aspects of the invention (e.g., systems), and vice versa.
Definitions
[0057] In order for the present disclosure to be more readily
understood, certain terms are first defined below. Additional
definitions for the following terms and other terms are set forth
throughout the specification.
[0058] In this application, the use of "or" means "and/or" unless
stated otherwise. As used in this application, the term "comprise"
and variations of the term, such as "comprising" and "comprises,"
are not intended to exclude other additives, components, integers
or steps. As used in this application, the terms "about" and
"approximately" are used as equivalents. Any numerals used in this
application with or without about/approximately are meant to cover
any normal fluctuations appreciated by one of ordinary skill in the
relevant art. In certain embodiments, the term "approximately" or
"about" refers to a range of values that fall within 25%, 20%, 19%,
18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%,
4%, 3%, 2%, 1%, or less in either direction (greater than or less
than) of the stated reference value unless otherwise stated or
otherwise evident from the context (except where such number would
exceed 100% of a possible value).
[0059] "Administration": The term "administration" refers to
introducing a substance into a subject. In general, any route of
administration may be utilized including, for example, parenteral
(e.g., intravenous), oral, topical, subcutaneous, peritoneal,
intraarterial, inhalation, vaginal, rectal, nasal, introduction
into the cerebrospinal fluid, or instillation into body
compartments. In certain embodiments, administration is oral.
Additionally or alternatively, in certain embodiments,
administration is parenteral. In certain embodiments,
administration is intravenous.
[0060] "Antibody": As used herein, the term "antibody" refers to a
polypeptide that includes canonical immunoglobulin sequence
elements sufficient to confer specific binding to a particular
target antigen. Intact antibodies as produced in nature are
approximately 150 kD tetrameric agents comprised of two identical
heavy chain polypeptides (about 50 kD each) and two identical light
chain polypeptides (about 25 kD each) that associate with each
other into what is commonly referred to as a "Y-shaped" structure.
Each heavy chain is comprised of at least four domains (each about
110 amino acids long)--an amino-terminal variable (VH) domain
(located at the tips of the Y structure), followed by three
constant domains: CH.sub.1, CH.sub.2, and the carboxy-terminal
CH.sub.3 (located at the base of the Y's stem). A short region,
known as the "switch", connects the heavy chain variable and
constant regions. The "hinge" connects CH.sub.2 and CH.sub.3
domains to the rest of the antibody. Two disulfide bonds in this
hinge region connect the two heavy chain polypeptides to one
another in an intact antibody. Each light chain is comprised of two
domains--an amino-terminal variable (VL) domain, followed by a
carboxy-terminal constant (CL) domain, separated from one another
by another "switch". Intact antibody tetramers are comprised of two
heavy chain-light chain dimers in which the heavy and light chains
are linked to one another by a single disulfide bond; two other
disulfide bonds connect the heavy chain hinge regions to one
another, so that the dimers are connected to one another and the
tetramer is formed. Naturally-produced antibodies are also
glycosylated, typically on the CH.sub.2 domain. Each domain in a
natural antibody has a structure characterized by an
"immunoglobulin fold" formed from two beta sheets (e.g., 3-, 4-, or
5-stranded sheets) packed against each other in a compressed
antiparallel beta barrel. Each variable domain contains three
hypervariable loops known as "complement determining regions"
(CDR1, CDR2, and CDR3) and four somewhat invariant "framework"
regions (FR1, FR2, FR3, and FR4). When natural antibodies fold, the
FR regions form the beta sheets that provide the structural
framework for the domains, and the CDR loop regions from both the
heavy and light chains are brought together in three-dimensional
space so that they create a single hypervariable antigen binding
site located at the tip of the Y structure. The Fc region of
naturally-occurring antibodies binds to elements of the complement
system, and also to receptors on effector cells, including for
example effector cells that mediate cytotoxicity. Affinity and/or
other binding attributes of Fc regions for Fc receptors can be
modulated through glycosylation or other modification. In certain
embodiments, antibodies produced and/or utilized in accordance with
the present invention include glycosylated Fc domains, including Fc
domains with modified or engineered such glycosylation. For
purposes of the present invention, in certain embodiments, any
polypeptide or complex of polypeptides that includes sufficient
immunoglobulin domain sequences as found in natural antibodies can
be referred to and/or used as an "antibody", whether such
polypeptide is naturally produced (e.g., generated by an organism
reacting to an antigen), or produced by recombinant engineering,
chemical synthesis, or other artificial system or methodology. In
certain embodiments, an antibody is polyclonal; in certain
embodiments, an antibody is monoclonal. In certain embodiments, an
antibody has constant region sequences that are characteristic of
mouse, rabbit, primate, or human antibodies. In certain
embodiments, antibody sequence elements are humanized, primatized,
chimeric, etc, as is known in the art. Moreover, the term
"antibody" as used herein, can refer in appropriate embodiments
(unless otherwise stated or clear from context) to any of the
art-known or developed constructs or formats for utilizing antibody
structural and functional features in alternative presentation. For
example, embodiments, an antibody utilized in accordance with the
present invention is in a format selected from, but not limited to,
intact IgG, IgE and IgM, bi- or multi-specific antibodies (e.g.,
Zybodies.RTM., etc), single chain Fvs, polypeptide-Fc fusions,
Fabs, cameloid antibodies, masked antibodies (e.g.,
Probodies.RTM.), Small Modular ImmunoPharmaceuticals ("SMIPs.TM."),
single chain or Tandem diabodies (TandAb.RTM.), VHHs,
Anticalins.RTM., Nanobodies.RTM., minibodies, BiTE.RTM.s, ankyrin
repeat proteins or DARPINs.RTM., Avimers.RTM., a DART, a TCR-like
antibody, Adnectins.RTM., Affilins.RTM., Trans-bodies.RTM.,
Affibodies.RTM., a TrimerX.RTM., MicroProteins, Fynomers.RTM.,
Centyrins.RTM., and a KALBITOR.RTM.. In certain embodiments, an
antibody may lack a covalent modification (e.g., attachment of a
glycan) that it would have if produced naturally. In certain
embodiments, an antibody may contain a covalent modification (e.g.,
attachment of a glycan, a payload [e.g., a detectable moiety, a
therapeutic moiety, a catalytic moiety, etc], or other pendant
group [e.g., poly-ethylene glycol, etc.]).
[0061] "Antibody fragment": As used herein, an "antibody fragment"
includes a portion of an intact antibody, such as, for example, the
antigen-binding or variable region of an antibody. Examples of
antibody fragments include Fab, Fab', F(ab')2, and Fv fragments;
triabodies; tetrabodies; linear antibodies; single-chain antibody
molecules; and multi specific antibodies formed from antibody
fragments. For example, antibody fragments include isolated
fragments, "Fv" fragments, consisting of the variable regions of
the heavy and light chains, recombinant single chain polypeptide
molecules in which light and heavy chain variable regions are
connected by a peptide linker ("ScFv proteins"), and minimal
recognition units consisting of the amino acid residues that mimic
the hypervariable region. In many embodiments, an antibody fragment
contains sufficient sequence of the parent antibody of which it is
a fragment that it binds to the same antigen as does the parent
antibody; in certain embodiments, a fragment binds to the antigen
with a comparable affinity to that of the parent antibody and/or
competes with the parent antibody for binding to the antigen.
Examples of antigen binding fragments of an antibody include, but
are not limited to, Fab fragment, Fab' fragment, F(ab')2 fragment,
scFv fragment, Fv fragment, dsFv diabody, dAb fragment, Fd'
fragment, Fd fragment, and an isolated complementarity determining
region (CDR) region. An antigen binding fragment of an antibody may
be produced by any means. For example, an antigen binding fragment
of an antibody may be enzymatically or chemically produced by
fragmentation of an intact antibody and/or it may be recombinantly
produced from a gene encoding the partial antibody sequence.
Alternatively or additionally, antigen binding fragment of an
antibody may be wholly or partially synthetically produced. An
antigen binding fragment of an antibody may optionally comprise a
single chain antibody fragment. Alternatively or additionally, an
antigen binding fragment of an antibody may comprise multiple
chains which are linked together, for example, by disulfide
linkages. An antigen binding fragment of an antibody may optionally
comprise a multimolecular complex. A functional single domain
antibody fragment is in a range from about 5 kDa to about 25 kDa,
e.g., from about 10 kDa to about 20 kDa, e.g., about 15 kDa; a
functional single-chain fragment is from about 10 kDa to about 50
kDa, e.g., from about 20 kDa to about 45 kDa, e.g., from about 25
kDa to about 30 kDa; and a functional fab fragment is from about 40
kDa to about 80 kDa, e.g., from about 50 kDa to about 70 kDa, e.g.,
about 60 kDa.
[0062] "Associated": As used herein, the term "associated"
typically refers to two or more entities in physical proximity with
one another, either directly or indirectly (e.g., via one or more
additional entities that serve as a linking agent), to form a
structure that is sufficiently stable so that the entities remain
in physical proximity under relevant conditions, e.g.,
physiological conditions. In certain embodiments, associated
moieties are covalently linked to one another. In certain
embodiments, associated entities are non-covalently linked. In
certain embodiments, associated entities are linked to one another
by specific non-covalent interactions (e.g., by interactions
between interacting ligands that discriminate between their
interaction partner and other entities present in the context of
use, such as, for example streptavidin/avidin interactions,
antibody/antigen interactions, etc.). Alternatively or
additionally, a sufficient number of weaker non-covalent
interactions can provide sufficient stability for moieties to
remain associated. Exemplary non-covalent interactions include, but
are not limited to, electrostatic interactions, hydrogen bonding,
affinity, metal coordination, physical adsorption, host-guest
interactions, hydrophobic interactions, pi stacking interactions,
van der Waals interactions, magnetic interactions, electrostatic
interactions, dipole-dipole interactions, etc.
[0063] "Biocompatible": The term "biocompatible", as used herein is
intended to describe materials that do not elicit a substantial
detrimental response in vivo. In certain embodiments, the materials
are "biocompatible" if they are not toxic to cells. In certain
embodiments, materials are "biocompatible" if their addition to
cells in vitro results in less than or equal to 20% cell death,
and/or their administration in vivo does not induce inflammation or
other such adverse effects. In certain embodiments, materials are
biodegradable.
[0064] "Biodegradable": As used herein, "biodegradable" materials
are those that, when introduced into cells, are broken down by
cellular machinery (e.g., enzymatic degradation) or by hydrolysis
into components that cells can either reuse or dispose of without
significant toxic effects on the cells. In certain embodiments,
components generated by breakdown of a biodegradable material do
not induce inflammation and/or other adverse effects in vivo. In
certain embodiments, biodegradable materials are enzymatically
broken down. Alternatively or additionally, in certain embodiments,
biodegradable materials are broken down by hydrolysis. In certain
embodiments, biodegradable polymeric materials break down into
their component polymers. In certain embodiments, breakdown of
biodegradable materials (including, for example, biodegradable
polymeric materials) includes hydrolysis of ester bonds. In certain
embodiments, breakdown of materials (including, for example,
biodegradable polymeric materials) includes cleavage of urethane
linkages.
[0065] "Carrier": As used herein, "carrier" refers to a diluent,
adjuvant, excipient, or vehicle with which the compound is
administered. Such pharmaceutical carriers can be sterile liquids,
such as water and oils, including those of petroleum, animal,
vegetable or synthetic origin, such as peanut oil, soybean oil,
mineral oil, sesame oil and the like. Water or aqueous solution
saline solutions and aqueous dextrose and glycerol solutions are
preferably employed as carriers, particularly for injectable
solutions. Suitable pharmaceutical carriers are described in
"Remington's Pharmaceutical Sciences" by E. W. Martin.
[0066] "Imaging agent": As used herein, "imaging agent" refers to
any element, molecule, functional group, compound, fragments
thereof or moiety that facilitates detection of an agent (e.g., a
polysaccharide nanoparticle) to which it is joined. Examples of
imaging agents include, but are not limited to: various ligands,
radionuclides (e.g., .sup.3H, .sup.14C, .sup.18F, .sup.19F,
.sup.32P, .sup.35S, .sup.135I, .sup.125I, .sup.123I, .sup.131I,
.sup.64Cu, .sup.68Ga, .sup.187Re, .sup.111In, .sup.90Y, .sup.99mTc,
.sup.177Lu, .sup.89Zr etc.), fluorescent dyes (for specific
exemplary fluorescent dyes, see below), chemiluminescent agents
(such as, for example, acridinum esters, stabilized dioxetanes, and
the like), bioluminescent agents, spectrally resolvable inorganic
fluorescent semiconductors nanocrystals (i.e., quantum dots), metal
nanoparticles (e.g., gold, silver, copper, platinum, etc.)
nanoclusters, paramagnetic metal ions, enzymes (for specific
examples of enzymes, see below), colorimetric labels (such as, for
example, dyes, colloidal gold, and the like), biotin, dioxigenin,
haptens, and proteins for which antisera or monoclonal antibodies
are available. The radionuclides may be attached via click
chemistry, for example.
[0067] "Protein": As used herein, the term "protein" refers to a
polypeptide (i.e., a string of at least 3-5 amino acids linked to
one another by peptide bonds). Proteins may include moieties other
than amino acids (e.g., may be glycoproteins, proteoglycans, etc.)
and/or may be otherwise processed or modified. In certain
embodiments "protein" can be a complete polypeptide as produced by
and/or active in a cell (with or without a signal sequence); in
certain embodiments, a "protein" is or comprises a characteristic
portion such as a polypeptide as produced by and/or active in a
cell. In certain embodiments, a protein includes more than one
polypeptide chain. For example, polypeptide chains may be linked by
one or more disulfide bonds or associated by other means. In
certain embodiments, proteins or polypeptides as described herein
may contain L-amino acids, D-amino acids, or both, and/or may
contain any of a variety of amino acid modifications or analogs
known in the art. Useful modifications include, e.g., terminal
acetylation, amidation, methylation, etc. In certain embodiments,
proteins or polypeptides may comprise natural amino acids,
non-natural amino acids, synthetic amino acids, and/or combinations
thereof. In certain embodiments, proteins are or comprise
antibodies, antibody polypeptides, antibody fragments, biologically
active portions thereof, and/or characteristic portions
thereof.
[0068] "Pharmaceutical composition": As used herein, the term
"pharmaceutical composition" refers to an active agent, formulated
together with one or more pharmaceutically acceptable carriers. In
certain embodiments, active agent is present in unit dose amount
appropriate for administration in a therapeutic regimen that shows
a statistically significant probability of achieving a
predetermined therapeutic effect when administered to a relevant
population. In certain embodiments, pharmaceutical compositions may
be specially formulated for administration in solid or liquid form,
including those adapted for the following: oral administration, for
example, drenches (aqueous or non-aqueous solutions or
suspensions), tablets, e.g., those targeted for buccal, sublingual,
and systemic absorption, boluses, powders, granules, pastes for
application to the tongue; parenteral administration, for example,
by subcutaneous, intramuscular, intravenous or epidural injection
as, for example, a sterile solution or suspension, or
sustained-release formulation; topical application, for example, as
a cream, ointment, or a controlled-release patch or spray applied
to the skin, lungs, or oral cavity; intravaginally or
intrarectally, for example, as a pessary, cream, or foam;
sublingually; ocularly; transdermally; or nasally, pulmonary, and
to other mucosal surfaces.
[0069] "Substantially": As used herein, the term "substantially",
and grammatic equivalents, refer to the qualitative condition of
exhibiting total or near-total extent or degree of a characteristic
or property of interest. One of ordinary skill in the art will
understand that biological and chemical phenomena rarely, if ever,
go to completion and/or proceed to completeness or achieve or avoid
an absolute result.
[0070] "Subject": As used herein, the term "subject" includes
humans and mammals (e.g., mice, rats, pigs, cats, dogs, and
horses). In many embodiments, subjects are be mammals, particularly
primates, especially humans. In certain embodiments, subjects are
livestock such as cattle, sheep, goats, cows, swine, and the like;
poultry such as chickens, ducks, geese, turkeys, and the like; and
domesticated animals particularly pets such as dogs and cats. In
certain embodiments (e.g., particularly in research contexts)
subject mammals will be, for example, rodents (e.g., mice, rats,
hamsters), rabbits, primates, or swine such as inbred pigs and the
like.
[0071] "Therapeutic agent": As used herein, the phrase "therapeutic
agent" refers to any agent that has a therapeutic effect and/or
elicits a desired biological and/or pharmacological effect, when
administered to a subject.
[0072] "Therapeutically effective amount": as used herein, is meant
an amount that produces the desired effect for which it is
administered. In certain embodiments, the term refers to an amount
that is sufficient, when administered to a population suffering
from or susceptible to a disease, disorder, and/or condition in
accordance with a therapeutic dosing regimen, to treat the disease,
disorder, and/or condition. In certain embodiments, a
therapeutically effective amount is one that reduces the incidence
and/or severity of, and/or delays onset of, one or more symptoms of
the disease, disorder, and/or condition. Those of ordinary skill in
the art will appreciate that the term "therapeutically effective
amount" does not in fact require successful treatment be achieved
in a particular individual. Rather, a therapeutically effective
amount may be that amount that provides a particular desired
pharmacological response in a significant number of subjects when
administered to patients in need of such treatment. In certain
embodiments, reference to a therapeutically effective amount may be
a reference to an amount as measured in one or more specific
tissues (e.g., a tissue affected by the disease, disorder or
condition) or fluids (e.g., blood, saliva, serum, sweat, tears,
urine, etc.). Those of ordinary skill in the art will appreciate
that, in certain embodiments, a therapeutically effective amount of
a particular agent or therapy may be formulated and/or administered
in a single dose. In certain embodiments, a therapeutically
effective agent may be formulated and/or administered in a
plurality of doses, for example, as part of a dosing regimen.
[0073] "Treatment": As used herein, the term "treatment" (also
"treat" or "treating") refers to any administration of a substance
that partially or completely alleviates, ameliorates, relives,
inhibits, delays onset of, reduces severity of, and/or reduces
incidence of one or more symptoms, features, and/or causes of a
particular disease, disorder, and/or condition. Such treatment may
be of a subject who does not exhibit signs of the relevant disease,
disorder and/or condition and/or of a subject who exhibits only
early signs of the disease, disorder, and/or condition.
Alternatively or additionally, such treatment may be of a subject
who exhibits one or more established signs of the relevant disease,
disorder and/or condition. In certain embodiments, treatment may be
of a subject who has been diagnosed as suffering from the relevant
disease, disorder, and/or condition. In certain embodiments,
treatment may be of a subject known to have one or more
susceptibility factors that are statistically correlated with
increased risk of development of the relevant disease, disorder,
and/or condition.
[0074] Drawings are presented herein for illustration purposes, not
for limitation.
BRIEF DESCRIPTION OF DRAWINGS
[0075] The foregoing and other objects, aspects, features, and
advantages of the present disclosure will become more apparent and
better understood by referring to the following description taken
in conduction with the accompanying drawings, in which:
[0076] FIG. 1 shows a schematic illustration showing the synthesis
of .sup.89Zr-labeled C'dot radioimmunoconjugate using a
chelator-based radiolabeling technique. PEGylated and
maleimide-functionalized C' dot (C' dot-PEG-Mal, 1) was first
reacted with reduced glutathione (GSH) to introduce the --NH.sub.2
groups for the following-up bioconjugates, forming C' dot-PEG-GSH
(2). Then the nanoparticle was conjugated with DBCO-PEG4-NHS ester
and DFO-NCS, forming C' dot-PEG-DBCO (3) and DFO-C' dot-PEG-DBCO
(4), respectively. Azide-functionalized small targeting ligands,
such as single-chain variable fragment (scFv-azide) (or
single-domain antibody, sdAb-azide), was conjugated to the
nanoparticle based on strain-promoted azide-alkyne cycloaddition,
forming DFO-C' dot-PEG-scFv (5). The final C'dot
radioimmunoconjugate (.sup.89Zr-DFO-C' dot-PEG-scFv, 6) was by
labeling it with .sup.89Zr-oxalate. The embodiments illustrated in
FIG. 1 are not limited to scFv and can include various types of
antibody fragments, e.g., sdAbs.
[0077] FIGS. 2A and 2B show in vivo (FIG. 2A) coronal and (FIG. 2B)
sagittal PET images of .sup.89Zr-DFO-C' dot-PEG at different
post-injection time points (10 min, 1 h, Day 1, Day 3 and Day 6) in
a healthy nude mouse. The reaction ratio between C' dot-PEG-Mal and
GSH was kept at 1:20. The PET images were acquired by using a Focus
120 MicroPET scanner.
[0078] FIG. 3 shows biodistribution data of .sup.89Zr-DFO-C'
dot-PEG in a healthy nude mouse on Day 6. Less than 2% ID/g of bone
(and joint) uptake was observed.
[0079] FIGS. 4A and 4B show a chelator-free .sup.89Zr radiolabeling
experimental example.
[0080] FIG. 4A shows .sup.89Zr labeling yields of C' dot-PEG-Mal
under varied pH conditions at 75.degree. C.
[0081] FIG. 4B shows .sup.89Zr labeling yields of C' dot-PEG-Mal
using varied combinations of C' dot to .sup.89Zr-oxalate ratio.
[0082] FIGS. 5A and 5B show in vivo coronal PET images of [89Zr]C'
dot-PEG at different post-injection time points (10 min, Day 1, Day
3 and Day 6) in a healthy nude mouse. [.sup.89Zr]C' dot-PEG was
synthesized by using a chelator-free radiolabeling technique. The
PET images were acquired by using a Focus 120 MicroPET scanner.
[0083] FIG. 5A shows PET images acquired without EDTA
(ethylenediaminetetraacetic acid).
[0084] FIG. 5B shows PET images acquired with EDTA
[0085] FIG. 6 shows biodistribution data of [.sup.89Zr]C' dot-PEG
in healthy nude mice (n=3) on Day 7. Over 10% ID/g of bone (and
joint) uptake was observed in this case, indicating a less stable
radiolabeling using a chelator-free method (when compared with that
of chelator-based method).
[0086] FIG. 7 shows biodistribution data of .sup.89Zr-DFO-C' dot,
.sup.89Zr-DFO-C' dot-DBCO and .sup.89Zr-DFO-C' dot-PEG-sdAb in
healthy nude mice at 48 h post-injection. An improved
pharmacokinetic profile (with prolonged blood circulation half-life
and lower liver uptake) can be achieved by optimizing the number of
DFO, DBCO and sdAb from each C' dot.
[0087] FIG. 8 shows an exemplary schematic of thiol-maleimide
chemistry.
[0088] FIG. 9 shows an exemplary schematic of alkene-tetrazine
chemistry.
DETAILED DESCRIPTION
[0089] Throughout the description, where compositions are described
as having, including, or comprising specific components, or where
methods are described as having, including, or comprising specific
steps, it is contemplated that, additionally, there are
compositions of the present invention that consist essentially of,
or consist of, the recited components, and that there are methods
according to the present invention that consist essentially of, or
consist of, the recited processing steps.
[0090] It should be understood that the order of steps or order for
performing certain action is immaterial so long as the invention
remains operable. Moreover, two or more steps or actions may be
conducted simultaneously.
[0091] The mention herein of any publication, for example, in the
Background section, is not an admission that the publication serves
as prior art with respect to any of the claims presented herein.
The Background section is presented for purposes of clarity and is
not meant as a description of prior art with respect to any
claim.
[0092] Molecular therapeutics (e.g., antibodies) can modulate the
immune system toward antitumor activity by manipulating immune
checkpoints (e.g., the monoclonal antibody ipilimumab inhibits
CTLA4, a negative regulatory molecule that inhibits function of the
immune system). The rationale is to trigger preexisting, but
dormant, antitumor immune responses. Other molecules and pathways
have acted as immune switches. PD-1, another negative regulatory
receptor expressed on T cells, has also been targeted. Switching a
single immune checkpoint may not be sufficient to induce an
antitumor response, explaining some of the failures of targeting
single immune regulatory checkpoints like PD-1 or CTLA4. However,
without wishing to be bound to any theory, treatment can be
bolstered by the addition of RT, which is thought, in some cases,
to have immunomodulatory properties. In these cases, tumors outside
of RT treatment fields have been found to shrink as a result of a
putative systemic inflammatory or immune response provoked by RT,
highlighting the potential for radiation to spark a systemic
antitumor immune response. Augmenting immune activity may also
potentiate the local effects of RT.
[0093] By raising the concentration alone of these
immunoconjugates, disease can be treated. A therapeutic radiolabel
can also be added to further treat disease. In certain embodiments,
the immunoconjugate act as a therapeutic at high concentrations,
and without a therapeutic radiolabel. In certain embodiments, the
radiolabel is attached to the same nanoparticle in an all-in-one
multi-therapeutic platform. Alternatively, therapeutic
radioisotopes can be administered independently.
[0094] Described herein are target-specific nanoparticle
immunoconjugates (e.g., single chain antibody fragments bound to
the particle surface) for targeted diagnostic and/or therapeutic
platforms. In certain embodiments, the nanoparticle
immunoconjugates are less than 20 nm (e.g., 6 to 10 nm) in
diameter. This small size is found to offer advantages in
therapeutic and/or imaging applications. For example, the disclosed
immunoconjugates may offer improved targeting of diseased tissue
and reduced non-specific uptake by organs (e.g., by the liver). The
smaller immunoconjugates may also demonstrate reduced immune
reactivity, thereby further improving efficacy.
[0095] In certain embodiments, the nanoparticle comprises silica,
polymer (e.g., poly(lactic-co-glycolic acid) (PLGA)), and/or metal
(e.g., gold, iron).
[0096] In certain embodiments, the silica-based nanoparticle
platform comprises ultrasmall nanoparticles or "C dots," which are
fluorescent, organo-silica core shell particles that have diameters
controllable down to the sub-10 nm range with a range of modular
functionalities. C dots are described by U.S. Pat. No. 8,298,677 B2
"Fluorescent silica-based nanoparticles", U.S. Publication No.
2013/0039848 A1 "Fluorescent silica-based nanoparticles", and U.S.
Publication No. US 2014/0248210 A1 "Multimodal silica-based
nanoparticles", the contents of which are incorporated herein by
reference in their entireties. Incorporated into the silica matrix
of the core are near-infrared dye molecules, such as Cy5.5, which
provides its distinct optical properties. Surrounding the core is a
layer or shell of silica. The silica surface is covalently modified
with silyl-polyethylene glycol (PEG) groups to enhance stability in
aqueous and biologically relevant conditions. These particles have
been evaluated in vivo and exhibit excellent clearance properties
owing largely to their size and inert surface. Among the additional
functionalities incorporated into C dots are chemical sensing,
non-optical (PET) image contrast and in vitro/in vivo targeting
capabilities, which enable their use in visualizing lymph nodes for
surgical applications, and melanoma detection in cancer.
[0097] C dots are synthesized via an alcohol-based modified Stober
process. C'dots are synthesized in water.
[0098] C dots or C'dots provide a unique platform for drug delivery
due to their physical properties as well as demonstrated human in
vivo characteristics. These particles are ultrasmall and benefit
from EPR effects in tumor microenvironments, while retaining
desired clearance and pharmacokinetic properties. To this end,
described herein is a nanoparticle drug delivery system in which,
in certain embodiments, drug constructs are covalently attached to
C dots or C'dots (or other nanoparticles).
[0099] C dots or C'dots can serve as highly specific and potent
multi-therapeutic targeted particle probes to combine antibody
fragments with therapeutic radiolabels (e.g., .sup.177Lu,
.sup.225Ac, .sup.90Y, .sup.89Zr) on a single platform.
Alternatively, C dot or C'dot coupling of targeting peptides, such
as alphaMSH, known to be immunomodulatory and anti-inflammatory in
nature, can also be combined with C dot or C'dot radiotherapeutic
(and/or other particle-based) platforms to achieve enhanced
efficacy. In certain embodiments, the concentration of the
radioisotope and/or antibody fragment is higher in therapeutic
applications compared to diagnostic applications.
[0100] In contrast to other multimodal platforms, immunoconjugates
can comprise different moieties that are attached to the
nanoparticle itself. For example, in certain embodiments, a
radioisotope is attached to the nanoparticle and an antibody
fragment is attached to the nanoparticle--that is, in these
embodiments, the radiolabel is not attached to the antibody
fragment itself. As another example, immunoconjugates can comprise
a targeting ligand attached to the nanoparticle, a radioisotope
attached to the nanoparticle, and an antibody fragment attached to
the nanoparticle. The stoichiometric ratios of different moieties
attached to the C dot will affect the biodistribution of the
nanoparticle immunoconjugate.
[0101] The immunoconjugates, e.g., C dot-antibody (mAb) and
-antibody-fragment (vFab) conjugates, can be prepared using either
of two approaches. Scheme 1 comprises thiol-maleimide chemistry, as
shown in FIG. 8. Scheme 1 is designed around proteins modified to
contain maleimide groups. Scheme 2 comprises alkene-tetrazine
chemistry as shown in FIG. 9.
[0102] In Scheme 1 as shown in FIG. 8, C dots containing Cy5 dye,
surface functionalized with PEG and maleimide groups (C
dots-(Cy5)-PEG-mal) were prepared as previously described in
Bradbury et al., 2014. Silanes modified with the Cy5 fluorophore
were prepared and titrated with tetramethylorthosilane (TMOS) into
a dilute solution of NH.sub.4OH (molar ratio TMOS:Cy5:NH3:H20 is
1:0.001:0.44:1215) and allowed to mix for 24 hours (Urata C, Aoyama
Y, Tonegawa A, Yamauchi Y, Kuroda K. Dialysis process for the
removal of surfactants to form colloidal mesoporous silica
nanoparticles. Chem Commun (Camb). 2009; (34):5094-6) (Yamada H,
Urata C, Aoyama Y, Osada S, Yamauchi Y, Kuroda K. Preparation of
Colloidal Mesoporous Silica Nanoparticles with Different Diameters
and Their Unique Degradation Behavior in Static Aqueous Systems,
Chem. Mater. 2012; 24(8):1462-71.) (Wang J, Sugawara-Narutaki A,
Fukao M, Yokoi T, Shimojima A, Okubo T. Two-phase synthesis of
monodisperse silica nanospheres with amines or ammonia catalyst and
their controlled self-assembly. ACS Appl Mater Interfaces. 2011;
3(5):1538-44.) This resulted in a Cy5 encapsulated silica particle,
the surface of which was further PEGylated and functionalized with
maleimide groups by treatment with PEG-silane (500 g/mole) (Suzuki
K, Ikari K, Imai H. Synthesis of silica nanoparticles having a
well-ordered mesostructured using a double surfactant system. J Am
Chem Soc. 2004; 126(2):462-3) and maleimide-PEG-silane (molar ratio
PEG-silane:TMOS:mal-PEG-silane of 1:2.3:0.006). The maleimide
groups can then be effectively transformed into amine groups by
reacting the particles with compounds that contain a thiol and
amine (e.g., cysteine methyl ester or cysteamine-HCl). The
resulting C dot-(Cy5)-PEG-amine can then be subsequently modified
with a succinimidyl 3-(2-pyridyldithio)propionate (SPDP). The
pyridyldithiol serves at least two purposes: one, it can be used to
quantitate conjugation efficiencies; two, it may serves as a
`protecting group` to minimize oxidation of thiol groups; etc. TCEP
can then be used to remove the group releasing a pyridine 2-thione,
which can be measured by HPLC or UV-absorption for quantitation.
The resulting C dot-(Cy5)-PEG-thiol can then be reacted with
protein-maleimide leading to the desired C dot-(Cy5)-PEG-mAb or C
dot-(Cy5)-PEG-vFab.
[0103] In Scheme 2 as shown in FIG. 9, alkene-tetrazine chemistry
is utilized for protein attachment. Here, the mAb or vFab is
modified with a click reactive groups, such as
methyltetrazine-PEG.sub.4-NHS ester. The C dot-(Cy5)-PEG-amine, as
described in FIG. 8 (Scheme 1), is then modified with the
appropriate click partner, (e.g., TCO-PEG4-NHS ester). In the final
step, the methyltetrazine-mAb or -vFab can then be reacted with the
C dot-(Cy5)-PEG-TCO leading to the C dot-(Cy5)-PEG-mAb or C
dot-(Cy5)-PEG-vFab product.
[0104] Antibody fragments (fAbs) provide advantages (e.g., size, no
Fc region for reduced immunogenicity, scalability, and
adaptability) compared to standard monoclonal antibodies (mAbs).
fAbs are the stripped-down binding region of an antibody which is
usually expressed as a single continuous sequence in an expression
host (e.g., E. Coli). In certain embodiments, a fAb or mAb can be
as small as 15 kDa (+/-5 kDa) (e.g., about 3 nm). In other
embodiments, a fAb or mAb can be up to 150 kDa (e.g., up to 20 nm).
In one embodiment, a fAb is approximately 60 kDa (e.g., +/-15 kDa).
A fAb comprises an immunoglobin heavy-chain variable and constant
domain linked to the corresponding domains of an immunoglobin light
chain. In another embodiment, the antibody format can be a single
chain variable fragment (scFv) fragment that is approximately 30
kDa (e.g., +/-10 kDa). A scFv fragment comprises a heavy-chain
variable domain linked to a light-chain variable domain. In other
embodiments, the antibody format can be a single domain antibody
(sdAb) fragment that is approximately 15 kDa (e.g., +/-5 kDa). A
sdAb fragment comprises a single heavy-chain variable domain. In
certain embodiments, the antibody fragment is an anti-CEA scFv for
targeting different tumors.
[0105] In certain embodiments, various linkers are used. In certain
embodiments, a cleavable linker (e.g., peptide, hydrazine, or
disulfide) is used. In certain embodiments, a noncleavable linker
(e.g., thioether) is used. In certain embodiments, a peptide linker
is selectively cleaved by lysosomal proteases (e.g., cathepsin-B).
In certain embodiments, a valine-citrulline dipeptide linker is
used.
[0106] In certain embodiments, different linkers as described in
U.S. Pat. Nos. 4,680,338, 5,122,368, 5,141,648, 5,208,020,
5,416,064, 5,475,092, 5,543,390, 5,563,250 5,585,499, 5,880,270,
6,214,345, 6,436,931, 6,372,738, 6,340,701, 6,989,452, 7,129,261,
7,375,078, 7,498,302, 7,507,420, 7,691,962, 7,910,594, 7,968,586,
7,989,434, 7,994,135, 7,999,083, 8,153,768, 8,236,319, Zhao, R.; et
al, (2011) J. Med. Chem. 36, 5404; Doronina, S.; et al, (2006)
Bioconjug Chem, 17, 114; Hamann, P.; et al. (2005) Bioconjug Chem.
16, 346, the contents of which are hereby incorporated by reference
herein, are used.
[0107] In certain embodiments, the mAbs and/or fAbs are U.S.
approved for certain uses. Non-limiting examples of mAbs and fAbs
include anti-GPIIb/IIIa, anti-VEGF-A, and anti-TNF-.alpha.. ReoPro
(abciximab) is an anti-GPIIb/IIIa, chimeric fAb, IgG1-.kappa.
developed by Centocor/Eli Lilly as described by Nelson and
Reichert, "Development trends for therapeutic antibody fragments,"
Nature Biotechnology, 27(4), 2009. Lucentis (ranibizumab) is an
anti-VEGF-A, humanized Fab IgG1-.kappa. developed by Genentech
(Nelson and Reichert, 2009) that is used to prevent wet age-related
macular degeneration. Cimzia (certolizumab pegol), is an
Anti-TNF-.alpha., PEGylated humanized fAb developed by UCB (Nelson
and Reichert, 2009) that is used to prevent moderate to severe
Crohn's disease.
[0108] In certain embodiments, PET (Positron Emission Tomography)
tracers are used as imaging agents. In certain embodiments, PET
tracers comprise .sup.89Zr, .sup.64Cu, [.sup.18F]
fluorodeoxyglucose.
[0109] In certain embodiments, fluorophores comprise fluorochromes,
fluorochrome quencher molecules, any organic or inorganic dyes,
metal chelates, or any fluorescent enzyme substrates, including
protease activatable enzyme substrates. In certain embodiments,
fluorophores comprise long chain carbophilic cyanines. In other
embodiments, fluorophores comprise DiI, DiR, DiD, and the like.
Fluorochromes comprise far red, and near infrared fluorochromes
(NIRF). Fluorochromes include but are not limited to a carbocyanine
and indocyanine fluorochromes. In certain embodiments, imaging
agents comprise commercially available fluorochromes including, but
not limited to Cy5.5, Cy5 and Cy7 (GE Healthcare); AlexaFlour660,
AlexaFlour680, AlexaFluor750, and AlexaFluor790 (Invitrogen);
VivoTag680, VivoTag-S680, and VivoTag-5750 (VisEn Medical); Dy677,
Dy682, Dy752 and Dy780 (Dyomics); DyLight547, DyLight647 (Pierce);
HiLyte Fluor 647, HiLyte Fluor 680, and HiLyte Fluor 750 (AnaSpec);
IRDye 800CW, IRDye 800RS, and IRDye 700DX (Li-Cor); and ADS780WS,
ADS830WS, and ADS832WS (American Dye Source) and Kodak X-SIGHT 650,
Kodak X-SIGHT 691, Kodak X-SIGHT 751 (Carestream Health).
[0110] In certain embodiments, click reactive groups are used (for
`click chemistry`). Examples of click reactive groups include the
following: alkyne, azide, thiol (sulfydryl), alkene, acrylate,
oxime, maliemide, NHS (N-hydroxysuccinimide), amine (primary amine,
secondary amine, tertiary amine, and/or quarternary ammonium),
phenyl, benzyl, hydroxyl, carbonyl, aldehyde, carbonate,
carboxylate, carboxyl, ester, methoxy, hydroperoxy, peroxy, ether,
hemiacetal, hemiketal, acetal, ketal, orthoester, orthocarbonate
ester, amide, carboxyamide, imine (primary ketimine, secondary
ketamine, primary aldimine, secondary aldimine), imide, azo
(diimide), cyanate (cyanate or isocyanate), nitrate, nitrile,
isonitrile, nitrite (nitrosooxy group), nitro, nitroso, pyridyl,
sulfide, disulfide, sulfinyl, sulfonyl, sulfino, sulfo,
thiocyanate, isothiocyanate, caronothioyl, thione, thial,
phosphine, phosphono, phosphate, phosphodiester, borono, boronate,
bomino, borinate, halo, fluoro, chloro, bromo, and/or iodo
moieties.
[0111] Cancers that may be treated include, for example, prostate
cancer, breast cancer, testicular cancer, cervical cancer, lung
cancer, colon cancer, bone cancer, glioma, glioblastoma, multiple
myeloma, sarcoma, small cell carcinoma, melanoma, renal cancer,
liver cancer, head and neck cancer, esophageal cancer, thyroid
cancer, lymphoma, and/or leukemia.
[0112] In certain embodiments, targeting peptide ligands, such as
alpha-MSH, attached to C dots, can serve as immunomodulators
alongside other therapies to enhance treatment response.
[0113] In certain embodiments, in addition to administration of an
immunoconjugate described herein, a method of treatment may include
administration of antibodies, small molecule drugs, radiation,
pharmacotherapy, chemotherapy, cryotherapy, thermotherapy,
electrotherapy, phototherapy, ultrasonic therapy and/or
surgery.
[0114] In certain embodiments, the immunoconjugate comprises a
therapeutic agent, e.g., a drug (e.g., a chemotherapy drug) and/or
a therapeutic radioisotope. As used herein, "therapeutic agent"
refers to any agent that has a therapeutic effect and/or elicits a
desired biological and/or pharmacological effect, when administered
to a subject.
[0115] In certain embodiments, the radioisotope is a radiolabel
that can be monitored/imaged (e.g., via PET or single-photon
emission computed tomography (SPECT)). Example radioisotopes that
can be used include beta emitters (e,g. .sup.177Luteium) and alpha
emitters (e.g., .sup.225Ac). In certain embodiments, one or more of
the following radioisotopes are used: .sup.99mTc, .sup.111In,
.sup.64Cu, .sup.67Ga, .sup.186Re, .sup.188Re, .sup.153Sm,
.sup.177Lu, .sup.67Cu, .sup.123I, .sup.124I, .sup.125I, .sup.11C,
.sup.13N, .sup.15O, .sup.18F, .sup.186Re, .sup.188Re, .sup.153Sm,
.sup.166Ho, .sup.177Lu, .sup.149Pm, .sup.90Y, .sup.213Bi,
.sup.103Pd, .sup.109Pd, .sup.159Gd, .sup.140La, .sup.198Au,
.sup.199Au, .sup.169Yb, .sup.175Yb, .sup.165Dy, .sup.166Dy,
.sup.67Cu, .sup.105Rh, .sup.111Ag, .sup.89Zr, .sup.225Ac, and
.sup.192Ir.
[0116] In certain embodiments, the immunoconjugate comprises one or
more drugs, e.g., one or more chemotherapy drugs, such as
sorafenib, paclitaxel, docetaxel, MEK162, etoposide, lapatinib,
nilotinib, crizotinib, fulvestrant, vemurafenib, bexorotene, and/or
camptotecin.
[0117] In certain embodiments, the immunoconjugate comprises a
chelator, for example,
1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diyl)diacetic acid
(CB-TE2A); desferoxamine (DFO); diethylenetriaminepentaacetic acid
(DTPA); 1,4,7, 10-tetraazacyclotetradecane-1,4,7, 10-tetraacetic
acid (DOTA); thylenediaminetetraacetic acid (EDTA); ethylene
glycolbis(2-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA);
1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA);
ethylenebis-(2-4 hydroxy-phenylglycine) (EHPG); 5-Cl-EHPG;
5Br-EHPG; 5-Me-EHPG; 5t-Bu-EHPG; 5-sec-Bu-EHPG;
benzodiethylenetriamine pentaacetic acid (benzo-DTPA);
dibenzo-DTPA; phenyl-DTPA, diphenyl-DTPA; benzyl-DTPA; dibenzyl
DTPA; bis-2 (hydroxybenzyl)-ethylene-diaminediacetic acid (HBED)
and derivatives thereof; Ac-DOTA; benzo-DOTA; dibenzo-DOTA;
1,4,7-triazacyclononane N,N',N''-triacetic acid (NOTA); benzo-NOTA;
benzo-TETA, benzo-DOTMA, where DOTMA is
1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetra(methyl tetraacetic
acid), benzo-TETMA, where TETMA is
1,4,8,11-tetraazacyclotetradecane-1,4,8,11-(methyl tetraacetic
acid); derivatives of 1,3-propylenediaminetetraacetic acid (PDTA);
triethylenetetraaminehexaacetic acid (TTHA); derivatives of
1,5,10-N,N',N''-tris(2,3-dihydroxybenzoyl)-tricatecholate (LICAM);
and 1,3,5-N,N',N''-tris(2,3-dihydroxybenzoyl)aminomethylbenzene
(MECAM), or other metal chelators.
[0118] In certain embodiments, the immunoconjugate comprises more
than one chelator.
[0119] In certain embodiments the radioisotope-chelator pair is
.sup.89Zr-DFO. In certain embodiments the radioisotope-chelator
pair .sup.177Lu-DOTA. In certain embodiments, the is
radioisotope-chelator pair is .sup.225Ac-DOTA.
[0120] In certain embodiments, the therapeutic agent (e.g., drug
and/or radioisotope) is attached to the nanoparticle or the
antibody fragment (protein), or both, using a bioorthogonal
conjugation approach (e.g., amine/NHS-ester, thiol/maleimide,
azide/alkyne click, or tetrazine/TCO click). For radiolabeling
using radiometals, the radiometal chelator can be first attached to
either particle or protein or both, followed by the radiometal.
Alternatively, the radiometal/chelator complex can be performed,
followed by attachment onto the particle or protein or both.
Radioiodination can also be achieved using standard approaches
where a tyrosine or phenolic group on the particle or protein or
both is modified by electrophilic addition chemistry.
[0121] In certain embodiments, the immunoconjugate is administered
to a subject suffering from a particular disease or condition
(e.g., cancer) for treatment of the disease or condition.
EXPERIMENTAL EXAMPLES
Preparation of the C Dot-(Cy5)-PEG-Maleimide
[0122] A maleimide and NHS ester functionalized polyethylene glycol
(mal-dPEG.sub.12-NHS) was conjugated with aminosilane (APTES) in
DMSO (molar ratio mal-PEG-NHS:APTES:DMSO 1:0.9:60). The reaction
mixture was left under nitrogen at room temperature for 48 hours to
generate silane functionalized mal-dPEG (mal-dPEG-APTES). A
maleimide functionalized Cy5 (mal-Cy5) was reacted with a
thiol-silane (MPTMS) in DMSO (molar ratio Cy5:MPTMS:DMOS
1:25:1150). The reaction was left under nitrogen at room
temperature for 24 hours to generate a silane functionalized Cy5
(Cy5-MPTMS). TMOS and Cy5-MPTMS were then titrated into an ammonia
hydroxide solution (.about.pH 8) (molar ratio TMOS:Cy5:NH3:H2O
1:0.001:0.44:1215). The solution was stirred at 600 rpm at room
temperature for 24 hours to form homogeneous Cy5 encapsulated
silica nanoparticles. The mal-dPEG-APTES and silane functionalized
polyethylene glycol (PEG-silane, MW around 500, Gelest) were then
added into the synthesis solution to PEGylate and
surface-functionalize the particles (PEG-silane:TMOS:mal-PEG-APTES
1:2.3:0.006). The solution was stirred at 600 rpm at room
temperature for 24 hours followed by incubation at 80.degree. C.
for another 24 hours without stirring. The solution was dialyzed in
2000 mL with deionized water for two days (10 k MWCO), filtered
with 200 nm syringe filters, and finally chromatographically
purified (Superdex 200) resulting in the desired mal-C dots.
Preparation of C Dot Immunoconjugates
[0123] Studies were performed to conjugate single chain antibody
fragments (scFv)s to the C dot core silica nanoparticles. An scFv
that bound matrix metalloproteinase 12 (MMP-12) was expressed in E.
coli. The construct contained C-terminal His and FLAG tags for
nickel affinity chromatography and immune-detection. A mutant scFv
was constructed in which the last amino acid of the polypeptide
chain was converted to a cysteine (Cys). The change was confirmed
by sequencing the mutant gene. Expression and nickel affinity
purification of the wild type scFv and the C-terminal Cys
containing mutant was confirmed by sodium dodecyl sulfate (SDS)
polyacrylamide gel electrophoresis (PAGE), visualized with
Coomassie blue stain at a molecular weight consistent with the
scFv. Western blot analysis of the scFv SDS PAGE gel was performed
with an anti-FLAG tag HRP conjugate. The Western blot analysis
confirmed that the identity of the gel band was the scFv.
[0124] The scFv were clones modified with azide containing
bifunctional linkers. The wild type scFv was modified with
N-hydroxy-succinimide (NHS) ester--polyethylene glycol
(PEG).sub.4-azide. Without wishing to be bound to any theory,
modification of wild type scFv with NHS ester-PEG.sub.4-azide
results in the random incorporation of PEG.sub.4-azide on to free
amines on surface lysine residues. The C-terminal scFv Cys
construct was conjugated with maleimide-PEG.sub.3-azide for site
specific PEG.sub.3-azide introduction on to the Cys sulfhydryl. The
scFv constructs were analyzed for azide incorporation by reaction
with a Dibenzocyclooctyne (DBCO)-PEG-Cy5 fluorescent probe. Azides
react with DBCOs via a metal free click chemistry reaction to form
a covalent linkage. Unreacted DBCO-Cy5 dye was removed from the
reaction mixtures by 40 kDa cutoff size exclusion spin columns. The
successful introduction of an azide group on the surface of the
scFvs was confirmed by visualizing the wild type and C-terminal Cys
scFv-PEG-Cy5 fluorescent dye constructs using a BioRad Versa-Doc
imager.
[0125] The azide conjugated scFv were then reacted with C dots
containing 1-3 DBCOs on their surfaces. The reaction was allowed to
continue for 12 h at room temperature. Unconjugated scFv was
purified from conjugated scFv-C dots using multiple techniques
including phosphate buffered saline washes in 50,000 molecular
weight cut off spin columns, G-200 size exclusion column
chromatography or size exclusion spin columns and velocity
sedimentation thought a sucrose cushion. Velocity sedimentation and
size exclusion chromatography appear to be the most scalable
methods of purification. The purified scFv C-dot conjugates were
analyzed by dot blot scFv immune-detection/particle fluorescence
assays, gel electrophoresis and fluorescent ELISAs with immobilized
MMP-12.
[0126] These methods can be applied to other types of antibody
fragments, e.g., sdAbs.
[0127] FIG. 1 shows a schematic illustration showing the synthesis
of .sup.89Zr-labeled C'dot radioimmunoconjugate using a
chelator-based radiolabeling technique. PEGylated and
maleimide-functionalized C' dot (C' dot-PEG-Mal, 1) was first
reacted with reduced glutathione (GSH) to introduce the --NH.sub.2
groups for the following-up bioconjugates, forming C' dot-PEG-GSH
(2). Then the nanoparticle was conjugated with DBCO-PEG4-NHS ester
and DFO-NCS, forming C' dot-PEG-DBCO (3) and DFO-C' dot-PEG-DBCO
(4), respectively. Azide-functionalized small targeting ligands,
such as single-chain variable fragment (scFv-azide) (or
single-domain antibody, sdAb-azide), was conjugated to the
nanoparticle based on strain-promoted azide-alkyne cycloaddition,
forming DFO-C' dot-PEG-scFv (5). The final C'dot
radioimmunoconjugate (.sup.89Zr-DFO-C' dot-PEG-scFv, 6) was by
labeling it with .sup.89Zr-oxalate. The schematic illustrated in
FIG. 1 is not limited to scFv and can include various types of
antibody fragments, e.g., sdAbs.
[0128] FIGS. 2A and 2B show in vivo (FIG. 2A) coronal and (FIG. 2B)
sagittal PET images of .sup.89Zr-DFO-C' dot-PEG at different
post-injection time points (10 min, 1 h, Day 1, Day 3 and Day 6) in
a healthy nude mouse. The reaction ratio between C' dot-PEG-Mal and
GSH was kept at 1:20. The PET images were acquired by using a Focus
120 MicroPET scanner.
[0129] FIG. 3 shows biodistribution data of .sup.89Zr-DFO-C'
dot-PEG in a healthy nude mouse on Day 6. Less than 2% ID/g of bone
(and joint) uptake was observed.
[0130] FIGS. 4A and 4B show a chelator-free .sup.89Zr radiolabeling
experimental example.
[0131] FIG. 4A shows .sup.89Zr labeling yields of C' dot-PEG-Mal
under varied pH conditions at 75.degree. C.
[0132] FIG. 4B shows .sup.89Zr labeling yields of C' dot-PEG-Mal
using varied combinations of C' dot to .sup.89Zr-oxalate ratio.
[0133] FIGS. 5A and 5B show in vivo coronal PET images of [89Zr]C'
dot-PEG at different post-injection time points (10 min, Day 1, Day
3 and Day 6) in a healthy nude mouse. [.sup.89Zr]C' dot-PEG was
synthesized by using a chelator-free radiolabeling technique. The
PET images were acquired by using a Focus 120 MicroPET scanner.
[0134] FIG. 5A shows PET images acquired without EDTA
(ethylenediaminetetraacetic acid).
[0135] FIG. 5B shows PET images acquired with EDTA
[0136] FIG. 6 shows biodistribution data of [.sup.89Zr]C' dot-PEG
in healthy nude mice (n=3) on Day 7. Over 10% ID/g of bone (and
joint) uptake (highlighted with a red box) was observed in this
case, indicating a less stable radiolabeling using a chelator-free
method (when compared with that of chelator-based method).
[0137] FIG. 7 shows biodistribution data of .sup.89Zr-DFO-C' dot,
.sup.89Zr-DFO-C' dot-DBCO and .sup.89Zr-DFO-C' dot-PEG-sdAb in
healthy nude mice at 48 h post-injection. An improved
pharmacokinetic profile (with prolonged blood circulation half-life
and lower liver uptake) can be achieved by optimizing the number of
DFO, DBCO and sdAb from each C' dot.
* * * * *