U.S. patent application number 17/069305 was filed with the patent office on 2021-08-26 for methods and compositions for localized agent delivery.
The applicant listed for this patent is MASSACHUSETTS INSTITUTE OF TECHNOLOGY. Invention is credited to Anna BERSHTEYN, Darrell J. IRVINE, Jaehyun MOON, Matthias STEPHAN.
Application Number | 20210259968 17/069305 |
Document ID | / |
Family ID | 1000005568460 |
Filed Date | 2021-08-26 |
United States Patent
Application |
20210259968 |
Kind Code |
A1 |
IRVINE; Darrell J. ; et
al. |
August 26, 2021 |
METHODS AND COMPOSITIONS FOR LOCALIZED AGENT DELIVERY
Abstract
The invention provides compositions and methods for delivering
agents to localized regions, tissues, or organs in vivo by
conjugating agent-loaded nanoparticles to cells having homing
capability. The agents may be therapeutic or diagnostic agents such
as cancer chemotherapeutic agents and imaging agents
respectively.
Inventors: |
IRVINE; Darrell J.;
(Arlington, MA) ; STEPHAN; Matthias; (Boston,
MA) ; MOON; Jaehyun; (Cambridge, MA) ;
BERSHTEYN; Anna; (Seattle, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MASSACHUSETTS INSTITUTE OF TECHNOLOGY |
Cambridge |
MA |
US |
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Family ID: |
1000005568460 |
Appl. No.: |
17/069305 |
Filed: |
October 13, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15625479 |
Jun 16, 2017 |
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17069305 |
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15015464 |
Feb 4, 2016 |
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15625479 |
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13130845 |
Aug 16, 2011 |
9283184 |
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PCT/US09/06290 |
Nov 24, 2009 |
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15015464 |
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61200160 |
Nov 24, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 5/0647 20130101;
C12N 15/87 20130101; A61K 9/127 20130101; A61K 9/19 20130101; A61K
2035/124 20130101; A61K 38/2086 20130101; A61K 31/711 20130101;
A61K 9/5153 20130101; C12N 5/0006 20130101; C12N 15/113 20130101;
A61K 47/6901 20170801; A61K 45/06 20130101; A61K 35/28 20130101;
B82Y 5/00 20130101; A61K 9/5146 20130101; A61K 38/19 20130101; A61K
9/146 20130101; A61K 39/395 20130101; A61K 47/6937 20170801; A61K
39/39 20130101; A61K 39/00 20130101; C07K 16/00 20130101; A61K
38/20 20130101; C12N 2310/14 20130101; A61K 47/6911 20170801; A61K
31/7105 20130101; A61K 47/46 20130101; C12N 5/0636 20130101 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 9/51 20060101 A61K009/51; A61K 31/7105 20060101
A61K031/7105; A61K 31/711 20060101 A61K031/711; A61K 38/20 20060101
A61K038/20; A61K 45/06 20060101 A61K045/06; B82Y 5/00 20060101
B82Y005/00; C12N 5/00 20060101 C12N005/00; C12N 5/0783 20060101
C12N005/0783; C12N 5/0789 20060101 C12N005/0789; C12N 15/87
20060101 C12N015/87; A61K 47/69 20060101 A61K047/69; A61K 39/00
20060101 A61K039/00; A61K 39/395 20060101 A61K039/395; A61K 47/46
20060101 A61K047/46; A61K 9/14 20060101 A61K009/14; A61K 9/19
20060101 A61K009/19; A61K 35/28 20060101 A61K035/28; A61K 38/19
20060101 A61K038/19; A61K 39/39 20060101 A61K039/39; C07K 16/00
20060101 C07K016/00; C12N 15/113 20060101 C12N015/113 |
Goverment Interests
FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with Government support under grant
number RGY0058/2006-C from the Human Frontier Science Program and
grant numbers CA140476 and EB007280-02 from the National Institutes
of Health. The Government has certain rights to this invention.
Claims
1. A biodegradable nanoparticle comprising maleimide groups on its
exterior surface.
2. The nanoparticle of claim 1, wherein the nanoparticle further
comprises a lipid bilayer surface.
3. The nanoparticle of claim 1, wherein the nanoparticle comprises
a poly(lactide-co-glycolide) (PLGA) core.
4. The nanoparticle of claim 1, wherein the nanoparticle further
comprises an agent.
5. The nanoparticle of claim 1, wherein the nanoparticle further
comprises a plurality of agents.
6. The nanoparticle of claim 4, wherein the agent is an
antibody.
7. The nanoparticle of claim 4, wherein the agent is an
antigen.
8. The nanoparticle of claim 4, wherein the agent is an
adjuvant.
9. The nanoparticle of claim 8, wherein the adjuvant is a TLR
ligand.
10. The nanoparticle of claim 4, wherein the agent is a nucleic
acid.
11. The nanoparticle of claim 10, wherein the nucleic acid is an
siRNA.
12. The nanoparticle of claim 4, wherein the agent is an
anti-cancer agent.
13. The nanoparticle of claim 4, wherein the agent is a
cytokine.
14. The nanoparticle of claim 4, wherein the agent is an
interleukin.
15. The nanoparticle of claim 5, wherein the plurality of agents
comprises an antigen and an adjuvant.
16. The nanoparticle of claim 5, wherein the plurality of agents
comprises an adjuvant and an anti-cancer agent.
17. The nanoparticle of claim 1, wherein the nanoparticle is 50-500
nanometers in diameter.
18. The nanoparticle of claim 1, wherein the nanoparticle is about
150 nanometers in diameter.
19. The nanoparticle of claim 1, in a lyophilized form.
20. A method for delivering an agent comprising administering to a
subject a nucleated cell bound to a nanoparticle that comprises an
agent, wherein the agent is released from the particle in vivo.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S patent application
Ser. No. 15/625,479, filed on Jun. 16, 2017, pending, which
application is a continuation of U.S. patent application Ser. No.
15/015,464, filed on Feb. 4, 2016, abandoned, which application is
a divisional of U.S. patent application Ser. No. 13/130,845, filed
on Aug. 16, 2011, now issued as U.S. Pat. No. 9,283,184, which
claims priority under 35 U.S.C. .sctn. 371 of International
Application No. PCT/US2009/006290, filed on Nov. 24, 2009, which
claims priority under 35 U.S.C. .sctn. 119 from U.S. provisional
application Ser. No. 61/200,160, filed Nov. 24, 2008, the entire
contents of which are incorporated by reference herein.
STATEMENT REGARDING SEQUENCE LISTING
[0003] The Sequence Listing associated with this application has
been submitted electronically in ASCII format, and is hereby
incorporated by reference into the specification in its entirety.
The name of the text file containing the sequence Listing is
MITN1-001USDVCN2_Sequence-Listing. The text file is 1464 bytes, was
created on October 13, 2020 and is being submitted electronically
via EFS-Web.
BACKGROUND OF THE INVENTION
1. Field of Invention
[0004] The invention relates to the delivery of agents to localized
regions, tissues, or cells in the body using nanoparticles and
cells.
2. Discussion of Related Art
[0005] Cell-based immunotherapies are in active development for
treatment of cancer, and adoptive cell therapy (ACT) of cancer with
ex vivo activated/expanded T cells is one of the more promising
treatments currently being tested in patients. (Rosenberg et al.,
Nat Rev Cancer 8(4): 299, 2008; Dudley et al., Science 298(5594):
850, 2002; June et al., J Clin Invest 117(5): 1204, 2007; Stephan
et al., Nat Med 13(12): 1440, 2007; Yee et al., Proc Natl Acad Sci
U S A 99(25): 16168, 2002.) These approaches involve the use of
autologous T cells taken from patients that are activated/expanded
ex vivo and then reinfused to combat tumors such as metastatic
tumors. Strategies that enhance the persistence, in vivo expansion,
and effector functions of ACT T cells should increase the frequency
of objective responses. (Rosenberg S A et al., Nat Rev Cancer 8(4):
299, 2008; June C H et al., J Clin Invest 117(5): 1204, 2007.) One
way to enhance the function of ACT T cells is via genetic
engineering of the cells themselves, introducing chimeric receptors
or costimulatory molecules. (Stephan et al., Nat Med 13(12): 1440,
2007; Morgan et al., Science 314(5796): 126, 2006; Gade et al.,
Cancer Res 65(19): 9080, 2005.)
[0006] Interleukin-family cytokines such as IL-2 and IL-15 have
been of particular interest for promoting the effector functions
and proliferation of anti-tumor T cells. IL-2 and IL-15 share some
of their properties in triggering T cell proliferation/effector
function, and systemic IL-2 has been used to support adoptively
transferred T cells in both mouse models and human clinical trials
of cancer treatment.
[0007] However, IL-2 expands regulatory T cells that can suppress
anti-tumor immune responses, is known to promote activation-induced
cell death (AICD) in T cells, and has substantial toxicity when
administered systemically. (Antony et al., J Immunol 176(9): 5255,
2006; Fontenot et al., Nat Immunol 6(11): 1142, 2005; Oh et al.,
Proc Natl Acad Sci U S A 100(6): 3392, 2003; Waldmann, Nat Rev
Immunol 6(8): 595, 2006; Waldmann et al., Immunity 14(2): 105,
2001.)
[0008] In contrast, IL-15 supports T cell proliferation and
effector functions without promoting AICD. (Oh et al., Proc Natl
Acad Sci U S A 100(6): 3392, 2003; Waldmann, Nat Rev Immunol 6(8):
595, 2006; Waldmann et al., Immunity 14(2): 105, 2001.) IL-15
signals through a heterotrimeric receptor composed of a dedicated
.alpha. chain, a shared IL-2/IL-15R.beta. chain, and the common
.gamma. chain used by several interleukins. In an unusual mode of
function, physiologic IL-15 signaling has been shown to be largely
mediated by presentation of the cytokine in trans: cells bearing
the IL-15R.alpha. chain bind the cytokine with high affinity and
present the cytokine to T cells bearing the .beta. and .gamma.
chains. As a result, IL-15R.alpha. chain expression by the
responding cells is unnecessary in this context. (Dubois et al.,
Immunity 17(5): 537, 2002; Stoklasek et al., J Immunol 177(9):
6072, 2006.)
[0009] Recently, strategies for re-activating or maintaining the
activity of anti-tumor T cells ex vivo have been described, based
on striking effects of IL-15 on anti-tumor CD8.sup.+ T cells. IL-15
has been used interchangeably with IL-2 as a systemic therapy in
preclinical models of ACT, promoting destruction of large melanoma
tumors when combined with booster vaccination to drive expansion of
adoptively transferred tumor-specific T cells. (Klebanoff et al.,
Proc Natl Acad Sci U S A 101(7): 1969, 2004.) Teague et al. showed
that culture of non-functional T cells recovered from tumors with
IL-15 overcomes the anergic state observed in these cells, allowing
them to proliferate and regain potent effector functions. (Teague
et al., Nat Med 12(3): 335, 2006.) However, systemically injected
IL-15 has been shown to have a short half life of only .about.1 hr,
and has limited potency in vivo, triggering limited proliferation
of T cells compared to responses observed during prolonged in vitro
culture. (Stoklasek et al., J Immunol 177(9): 6072, 2006.) This
result may reflect the protein's short half-life and/or limiting
availability of free IL-15R.alpha. chains for binding and
trans-presentation of the cytokine.
[0010] As a strategy to overcome this limitation, several
independent studies recently demonstrated that pre-complexation of
IL-15 with soluble recombinant IL-15R.alpha. enhances the systemic
potency of IL-15 by .about.50-fold, and also raises the half life
of the cytokine in serum following systemic injection to .about.20
hrs. (Stoklasek et al., J Immunol 177(9): 6072, 2006; Dubois et
al., J Immunol 180(4): 2099, 2008; Rubinstein et. al. Proc Natl
Acad Sci U S A 103(24): 9166, 2006.) Following on these findings,
long-term daily injections of IL-15/IL-15R.alpha. complexes have
been shown to prolong the survival of mice in a spontaneous mouse
model of pancreatic cancer, by reactivating the cytolytic activity
of tumor-resident T cells. (Epardaud et al., Cancer Res 68(8):
2972, 2008.) Notably, in these in vivo studies of
IL-15/IL-15R.alpha. superagonist (IL-15 SA) complex treatment, not
only memory CD8.sup.+ T cells but also naive CD8.sup.+ T cells were
shown to proliferate, upregulate activation markers, and gain
effector functions in response to IL-15/11,-15R.alpha. complex,
leading to gross splenomegaly in mice receiving prolonged IL-15 SA
treatment. (Stoklasek et al., J Immunol 177(9): 6072, 2006; Dubois
et al., J Immunol 180(4): 2099, 2008; Rubinstein et. al. Proc Natl
Acad Sci U S A 103(24): 9166, 2006.) This non-specific polyclonal T
cell activation elicited by systemic IL-15 SA may raise the risk of
autoimmunity if treatment is prolonged.
[0011] Cytokines such as IL-2 and IL-15 act primarily by acting on
T cells, NK cells, and NK T cells to promote immune responses.
Complementary to these signals, Toll-like receptor (TLR) ligands
have been used in cancer immunotherapy by driving activation of
dendritic cells (DCs) and other APCs both in tumor-draining lymph
nodes and directly in the tumor microenvironment. TLRs are pattern
recognition receptors that have evolved to detect a variety of
molecules associated with pathogens ranging from bacteria to fungi
to viruses. TLR ligands trigger DCs to upregulate costimulatory
receptors and secrete pro-immunity cytokines such as IL-12.
(Beutler, Nature 430(6996): 257, 2004; Iwasaki et al., Nat Immunol
5(10): 987, 2004; Pulendran, Immunol Rev 199: 227, 2004; Reis e
Sousa, Semin Immunol 16(1): 27, 2004.) Thus, these factors are
under study as potential adjuvants for vaccines. TLR signaling is
implicated in breaking regulatory T cell-mediated tolerance (Pasare
et al., Science 299(5609): 1033, 2003), and sustained delivery of
TLR ligands to lymph nodes has been shown to break tolerance of
tumor self-antigen specific T cells in an adoptive therapy model.
(Yang et al., Nat Immunol 5(5): 508, 2004.) Regression of large
established melanoma tumors achieved by adoptive therapy augmented
with a viral vector vaccination boost may function in part through
the sustained TLR engagement provided by viral vector immunization.
(Yang et al., Nat Immunol 5(5): 508, 2004; Overwijk et al., J Exp
Med 198(4): 569, 2003.) In other studies, repeated injections of
TLR ligands directly into tumors has been used to promote the
activation of tumor-resident APCs and drive effective local immune
responses. (Heckelsmiller et al., Eur J Immunol 32(11): 3235, 2002;
Furumoto et al., J Clin Invest 113(5): 774, 2004; Currie et al., J
Immunol 180(3): 1535, 2008.) TLR ligands in combination with IL-10
blockade have also been shown to convert dysfunctional DCs in the
tumor microenvironment into a pro-immunity functional state.
(Vicari et al., J Exp Med 196(4): 541, 2002.)
[0012] Drug-loaded synthetic biodegradable polymer nanoparticles
are becoming of more interest for treating a variety of diseases,
as they may offer a low-cost, readily manufacturable means to
achieve sustained drug delivery at selected target tissue sites and
concentrate drugs where they are needed in the body. (Davis et al.,
Nat Rev Drug Discov 7(9): 771, 2008.) In the delivery of protein
therapeutics, synthetic drug delivery particles (particles with
sizes in the 50-500 nm range, typically) may be able to achieve
results comparable to other means of delivery such as viral vectors
(Green et al., Advanced Materials 19(19): 2836, 2007) without the
associated side effects of such biological vectors, such as
anti-vector immune responses or dangers of viral integration.
(Donsante et al., Science 317(5837): 477, 2007; Kresge, IAVI Rep
9(4): 18, 2005; Mingozzi et al., Nat Med 13(4): 419, 2007; Watkins
et al., Nat Med 14(6): 617, 2008.) In cancer therapy, passive
accumulation of nanoparticles at tumor sites via the enhanced
permeation and retention effect (Maeda et al., J Control Release
65(1-2): 271, 2000; Matsumura et al., Cancer Res 46(12 Pt 1): 6387,
1986) (referring to the combined effects of leaky tumor vasculature
and poor lymphatic drainage often observed at solid tumor sites)
has been exploited for therapeutic and imaging agent delivery to
solid tumors. (Davis et al., Nat Rev Drug Discov 7(9): 771, 2008;
Shi et al., Advanced Materials 20(9): 1671, 2008; von Maltzahn et
al., Bioconjugate Chemistry 19(8): 1570, 2008; Drummond et al.,
Pharmacol Rev 51(4): 691, 1999; Kirpotin et al., Cancer Res 66(13):
6732, 2006; Park et al., Clin Cancer Res 8(4): 1172, 2002.)
[0013] However, treatment of metastatic disease via systemic
injection of nanoparticle drug carriers is limited by the rapid
clearance of typical nanoparticles. Thus, the half-life of
systemically injected nanoparticles or liposomes is typically a few
hours or less and accumulation of particles at tumor sites is often
only a very small fraction (.about.1%) of the total injected dose.
(Owens, Int J Pharm 307(1): 93, 2006; Vonarbourg et al.,
Biomaterials 27(24): 4356, 2006; Moghimi et al., Pharmacol Rev
53(2): 283, 2001.) Attachment of poly(ethylene glycol) (PEG) to the
surface of liposomes or nanoparticles to create so-called `stealth`
carriers can increase the circulation time of particles up to
.about.24-48 hrs (Owens, Int J Pharm 307(1): 93, 2006; Vonarbourg
et al., Biomaterials 27(24): 4356, 2006; Moghimi et al., Pharmacol
Rev 53(2): 283, 2001), but by far the greatest majority of injected
dose (often >80%) is still scavenged by the spleen and liver,
even when targeting antibodies are employed. (Kirpotin et al.,
Cancer Res 66(13): 6732, 2006.) Thus, a substantial quantity of
drug cargo is degraded without effect or worse, may elicit liver
toxicity.
SUMMARY OF THE INVENTION
[0014] The invention relates to the use of nanoparticles conjugated
to cell carriers to deliver agents in a controlled and localized
manner. The invention is based in part on the unexpected finding
that certain reactive groups exist at sufficient levels on the
surface of certain unmodified cell types that facilitate
conjugation to nanoparticles having complementary reactive groups.
The invention is further based in part on the unexpected finding
that nanoparticles can be maintained on the surface of certain
cells without internalization of the nanoparticles, which would
interfere with the controlled release of the agents comprised
within the nanoparticles. T cells are an example of cells that fail
to endocytose nanoparticles in the .about.150 nm size range
covalently conjugated to its surface even after many days or
through several rounds of cell division. The result is that T cells
could maintain nanoparticles and release agents in their local
environment for prolonged periods. Other cells which have been
found to be particularly suited to conjugation to nanoparticles via
their cell surface chemistry are B cells and hematopoietic
progenitor cells. The cell carriers may be eukaryotic (e.g.,
mammalian cells) or prokaryotic (e.g., bacterial cells), and they
may be naturally occurring or engineered (or modified). If the
carrier cells are bacterial or other prokaryotic cells, they may be
attenuated in order to reduce or eliminate the risk of infection to
the recipient.
[0015] Thus, in one aspect the invention provides a method for
delivering an agent comprising administering to a subject a
nucleated cell bound to a nanoparticle that comprises an agent,
wherein the cell does not internalize the nanoparticle, and wherein
the agent is released from the nanoparticle in vivo.
[0016] Various embodiments apply equally to the preceding aspect of
the invention as well as other aspects recited below, and for the
sake of brevity these will be recited only once. However it is to
be understood that combinations of these aspects and embodiments
are contemplated by the invention.
[0017] Thus, in some embodiments, the cell is a T cell. In some
embodiments, the cell is a B cell, an NK cell, or an NKT cell. In
other embodiments, the cell is a hematopoietic progenitor including
without limitation a pluripotent stem cell (i.e., a long-term
reconstituting cell), a multipotent progenitor cell (e.g., a CFU-S
or a CFC-GEMM), a unipotential progenitor cell (e.g., a BFU-E). An
example of a murine hematopoietic progenitor is a cell lacking
lineage marker cell surface expression, and having Sca-1 and/or
c-kit cell surface expression, as described herein.
[0018] In some embodiments, the subject has a tumor. In related
embodiments, the cell is a tumor-reactive T cell. In other related
embodiments, the cell homes to the tumor or to the tissue in which
the tumor exists (e.g., lymphoid tissue).
[0019] In some embodiments, the subject has an autoimmune disease.
In some embodiments, the subject has an infection.
[0020] In some embodiments, the subject is in need of hematopoietic
reconstitution as a result of, for example, myeloablative
chemotherapy and/or radiation.
[0021] In some embodiments, the cell is a gut-specific T cell. In
some embodiments, the cell is a skin-specific T cell.
[0022] In some embodiments, the cell is autologous to the subject.
In some embodiments, the cell is activated prior to administration
to the subject. In some embodiments, the cell is genetically
engineered. In other embodiments, the cell is naturally
occurring.
[0023] In some embodiments, the cell is a eukaryotic cell such as a
mammalian cell. In important embodiments, the mammalian cell is a
human cell. In other embodiments, the cell is a prokaryotic cell
such as a bacterial cell. The bacterial cell may be a Salmonella
bacterial cell. In related embodiments, the prokaryotic cell, such
as a bacterial cell, may be attenuated so as to prevent an
infection in the subject.
[0024] In some embodiments, the nanoparticle is 20-500 nm in
diameter, or 100-300 nm in diameter. In some embodiments, the
nanoparticle is about 150 nm in diameter, or about 200 nm in
diameter, or 250 nm in diameter.
[0025] In some embodiments, the nanoparticle comprises maleimide
reactive groups on its surface. In some embodiments, the
nanoparticle comprises a lipid coating.
[0026] In some embodiments, the nanoparticle is a DNA nanoparticle
(also referred to herein as a DNA-gel nanoparticle) comprising a
crosslinked DNA core and optionally a lipid coating.
[0027] In some embodiments, the agent is an imaging agent. In some
embodiments, the agent is an immunostimulatory agent. In some
embodiments, the agent is a cytokine. In some embodiments, the
cytokine is IL-15/IL-15R.alpha.. In some embodiments, the agent is
an antigen. In some embodiments, the agent is an adjuvant. In some
embodiments, the adjuvant is a TLR ligand. The TLR ligand may
function to stimulate antigen-specific immune responses (typically
in the presence of exogenous or endogenous antigens) and/or
antigen-non-specific immune responses. Thus, the TLR ligand may be
used in the presence or absence of an antigen. In some embodiments,
the agent is an antibody or an antibody fragment. In some
embodiments, the agent is a drug. In some embodiments, the agent is
a chemical compound. In some embodiments, the agent is a nucleic
acid. In some embodiments, the nucleic acid is an siRNA.
[0028] In some important embodiments, the agents are anti-cancer
agents including anti-cancer antibodies, cancer antigens,
anti-cancer chemotherapeutic agents, and the like.
[0029] In various embodiments, the agents may be used at doses that
are below doses required to achieve the same effects in vivo
following systemic administration. In some instances, the doses are
at least 2 times less, at least 5 times less, at least 10 times
less, at least 20 times less, at least 50 times less, or at least
100 times less than the required systemic dose.
[0030] In some embodiments, the cell is covalently bound to a
plurality of nanoparticles. In some embodiments, the plurality of
nanoparticles comprise an identical agent. In some embodiments, the
plurality of nanoparticles comprise different agents. In some
embodiments, the plurality of nanoparticles is 50-10,000, or
100-10,000. In some embodiments, the plurality of nanoparticles is
about 50, or about 100, or about 150, or about 200, or about 250,
or about 500.
[0031] In some embodiments, the method further comprises binding
the nanoparticle to the cell. In some embodiments, the method
further comprises providing the cell bound to the nanoparticle.
[0032] In some embodiments, the cell is covalently bound to the
nanoparticle.
[0033] In some embodiments, the agent acts in an autocrine manner
(i.e., it acts upon the cell carrier itself). In some embodiments,
the agent acts in a paracrine manner (i.e., it acts upon cells
other than the cell carrier). In still other embodiments, the agent
acts in both an autocrine and a paracrine manner.
[0034] In another aspect, the invention provides a method for
delivering an agent comprising administering to a subject a
liposome covalently bound to a nanoparticle that comprises an
agent, wherein the agent is released from the nanoparticle in
vivo.
[0035] In another aspect, the invention provides a method for
delivering an agent to a tumor comprising administering to a
subject having a tumor a tumor-reactive T cell covalently bound to
a nanoparticle that comprises an agent, wherein the agent is
released from the nanoparticle in vivo. The tumor may be a
lymphoma, and the agent may be an anti-lymphoma agent (i.e., an
agent having therapeutic effect on lymphoma). An example of such an
agent is an antibody such as rituximab.
[0036] In another aspect, the invention provides a method for
delivering an agent to a tumor comprising administering to a
subject having a tumor a tumor-reactive T cell covalently bound to
a maleimide-coated nanoparticle that comprises an agent, wherein
the agent is released from the nanoparticle in vivo. In some
embodiments, the agent is an anti-cancer agent. In some
embodiments, the agent is an adjuvant. In some embodiments, the
agent is an antigen. In some embodiments, the antigen is a tumor
antigen.
[0037] In another aspect, the invention provides a method for
delivering an agent comprising administering to a subject a cell
covalently bound to a nanoparticle that comprises an agent, wherein
the cell does not internalize the nanoparticle, and wherein the
agent is released from the nanoparticle in vivo.
[0038] In another aspect, the invention provides a method for
locally delivering an agent within a subject comprising
administering to a subject having a tissue homing cell bound to a
biodegradable nanoparticle that comprises an agent, wherein the
agent is released from the biodegradable nanoparticle in vivo. In
some embodiments, the tissue homing cell is a T cell. In some
embodiments, the T cell is a gut-homing T cell. In some
embodiments, the T cell is a skin-homing T cell. In some
embodiments, the biodegradable nanoparticle is covalently bound to
the tissue homing cell.
[0039] In another aspect, the invention provides a method for
delivering an agent to a lymphoma within a subject comprising
administering to a subject having a lymphoma a B or T cell (e.g., a
central memory T cell) bound to a nanoparticle or a liposome that
comprises an agent, wherein the agent is released from the
nanoparticle in vivo and the nanoparticle is not internalized into
the cell. The agent may be an antibody, such as an anti-CD20
antibody, or it may be a chemotherapy, such as fludaribine. Other
agents having therapeutic effect on lymphoma may be used in place
of or in addition to anti-CD20 antibody or fludaribine.
[0040] In another aspect, the invention provides a biodegradable
nanoparticle comprising maleimide groups on its exterior
surface.
[0041] In some embodiments, the nanoparticle further comprises a
lipid bilayer surface. In some embodiments, the nanoparticle
comprises a poly(lactide-co-glycolide) (PLGA) core. In some
embodiments, the nanoparticle further comprises an agent. In some
embodiments, the agent is an immunostimulatory agent. In some
embodiments, the agent is an antigen. In some embodiments, the
agent is an antibody. In some embodiments, the agent is an
adjuvant. In some embodiments, the adjuvant is a TLR ligand. In
some embodiments, the TLR ligand is an immunostimulatory agent in
the presence or absence of antigen. In some embodiments, the agent
is a nucleic acid. In some embodiments, the nucleic acid is an
siRNA. In some embodiments, the agent is an anti-cancer agent. In
some embodiments, the agent is a cytokine. In some embodiments, the
agent is an interleukin.
[0042] In some embodiments, the nanoparticle further comprises a
plurality of agents. In some embodiments, the plurality of agents
comprises an antigen and an adjuvant. In some embodiments, the
plurality of agents comprises an adjuvant and an anti-cancer agent.
In still other embodiments, the plurality of agents comprises an
immunostimulatory agent and an anti-cancer agent.
[0043] In some embodiments, the nanoparticle is 50-500 nanometers
in diameter, or 100-300 nanometers in diameter. In some
embodiments, the nanoparticle is about 50 nanometers in diameter,
or about 100 nanometers in diameter, or about 150 nanometers in
diameter, or about 200 nanometers in diameter, or about 250
nanometers in diameter. In some embodiments, the nanoparticle is in
a lyophilized form.
[0044] In another aspect, the invention provides a composition
comprising an isolated T cell comprising a biodegradable
nanoparticle at its cell surface, wherein the nanoparticle
comprises an agent. In some embodiments, the biodegradable
nanoparticle is covalently conjugated to the surface of the T
cell.
[0045] In another aspect, the invention provides a composition
comprising an isolated hematopoietic progenitor cell comprising a
biodegradable nanoparticle at its cell surface, wherein the
nanoparticle comprises an agent. In some embodiments, the
biodegradable nanoparticle is covalently conjugated to the surface
of the hematopoietic progenitor cell. The agent may be an agent
that stimulates the proliferation of hematopoietic progenitor
cells, and optionally their self-renewal or their differentiation
towards one or more hematopoietic lineages. A non-limiting example
of such an agent is a GSK3beta inhibitor.
[0046] These and other aspects and embodiments will be described in
greater detail herein.
[0047] Each of the limitations of the invention can encompass
various embodiments of the invention. It is therefore anticipated
that each of the limitations of the invention involving any one
element or combinations of elements can be included in each aspect
of the invention. This invention is not limited in its application
to the details of construction and/or the arrangement of components
set forth in the following description or illustrated in the
drawings. The invention is capable of other embodiments and of
being practiced or of being carried out in various ways.
BRIEF DESCRIPTION OF THE DRAWINGS
[0048] The accompanying drawings are not intended to be drawn to
scale. In the drawings, each identical or nearly identical
component that is illustrated in various figures is represented by
a like numeral. For purposes of clarity, not every component may be
labeled in every drawing. In the drawings:
[0049] FIG. 1A. Nanoparticle-functionalized T cells for ACT.
[0050] FIG. 1B. Examples of modes of action for nanoparticles bound
to cells, including an autocrine mode of action in which the
nanoparticles (and their agent payload) act on the carrier cell and
a paracrine mode of action in which the nanoparticles (and their
agent payloads) act on cells in the environment.
[0051] FIGS. 2A-D. Liposomes and lipid-coated PLGA nanoparticles
for linkage to T cells. (A, B) Unstained cryo-electron microscopy
images of lipid-enveloped nanoparticles, illustrating surface
lipids. (B is magnified view of A inset.) Arrows highlight evidence
for bilayer formation at the surface of the enveloped
nanoparticles. (C) Size histograms of lipid-coated PLGA
nanoparticles and liposomes from cryoEM. (D) Schematic of
maleimide-based conjugation to T cell surface thiols.
[0052] FIGS. 3A-B. Maleimide-functionalized nanoparticles stably
link to the surface of T cells without toxicity. Pmel-1 CD8.sup.+ T
cells were incubated with 2500 fluorescent DiD-labeled
nanoparticles per cell for conjugation, washed, and cultured for 6
days in the presence of IL-2. (A) Confocal microscopy of live cells
on day 0 and day 6, showing nanoparticle fluorescence (purple). (B)
Viability of particle-conjugated or control T cells assessed by
annexin V and propidium iodide staining followed by flow cytometry
analysis.
[0053] FIGS. 4A-C. Nanoparticles conjugated via thiols to T cell
surfaces do not inhibit T cell proliferation, cytokine production,
or target cell killing. (A, B) DiD-labeled PLGA-core nanoparticles
were attached to CFSE-labeled pmel-1 CD8.sup.+ T cells (2500
nanoparticles/cell), then particle-conjugated T cells (bottom
panel) or control `bare` T cells (top panel) were stimulated with
mature hgp100 peptide-pulsed bone marrow-derived dendritic cells at
a 2:1 T cell:DC ratio; cultures were supplemented with IL-2 every 2
days. (A) The cells were analyzed by flow cytometry on day 6:
Scatter plots of DiD (nanoparticle label) vs. CFSE fluorescence
gated on live CD8.sup.+ cells are shown in the upper panel, and the
corresponding mean nanoparticle fluorescence as a function of the
number of cell divisions determined from CFSE are shown in the
right panel. (B) Cytokines secreted by nanoparticle-conjugated T
cells (.circle-solid.) or `bare` T cells (.smallcircle.) were
measured by ELISA on sups collected at 24 hrs (IL-2) or 48 hrs
(IFN-.gamma. and TNF-.alpha.). (C) Pmel-1 T cell blasts were
conjugated with 2500 nanoparticles/cell or left unmodified, and
co-cultured with hgp100 antigen-pulsed (Mingozzi F et al., Nat Med
13(4): 419, 2007) Cr-labeled EL4 target cells or unpulsed control
EL4 cells, and the % of specific target cell killing was quantified
by measuring (Mingozzi F et al., Nat Med 13(4): 419, 2007) Cr
release after 4 hrs for varying pmel-1 effector cell:target cell
ratios.
[0054] FIGS. 5A-C. Protein and TLR ligand incorporation in
lipid-coated PLGA nanoparticles. (A) Confocal image (left) and
cryoEM image (right) of lipid-coated nanoparticles loaded with
fluorescent ova in the particle cores. Note that the particles in
the confocal image are artificially aggregated here by drying on a
coverslip for imaging. (B) Kinetics of IL-15 release from
lipid-coated PLGA particles in vitro in complete medium at
37.degree. C. (C) Bone marrow-derived DCs were incubated with 3
mg/mL lipid-nanoparticles containing 1 mole % or 10 mole % MPLA in
the lipid coating, equivalent amounts of soluble MPLA (30 .mu.g/mL
or 3 .mu.g/mL), or soluble LPS (1 .mu.g/mL) as a positive control.
At 24 hrs, the maturation status of the cells was assessed by flow
cytometry analysis of cell surface MHC II, CD80, and CD40 (not
shown). Particle-MPLA was equivalent to or more potent than soluble
MPLA in triggering DC maturation.
[0055] FIG. 6. Bioactivity of cytokine IL-15 released from
cell-bound nanoparticles. T cells were conjugated with lipid-coated
PLGA nanoparticles loaded with IL-15 or IL-15 complexed with
soluble IL-15Ralpha-human Fc fusion protein (IL-15 superagonist).
The number of viable T cells after 6 days in culture was assessed
by cell counting after trypan blue staining. T cells carrying
IL-15-loaded nanoparticles exhibited enhanced survival and/or
proliferation.
[0056] FIG. 7. Lipid-coated PLGA nanoparticles can encapsulate and
then exhibit sustained release of TLR7/8 compounds. Toll-like
receptor ligands gardiquimod or resiquimod were encapsulated in
lipid-coated PLGA particles, and release into BSA-containing saline
at 37.degree. C. was assessed over 8 days, by measuring
fluorescence of the released compounds.
[0057] FIGS. 8A-D. Whole-animal bioluminescence/fluorescence
imaging of nanoparticles, nanoparticle-conjugated T cells, and B16
melanoma tumor models. (A) T cells polarize surface-bound
nanoparticles (red fluorescence) to the uropod during migration.
Primed T cells were conjugated with nanoparticles and then observed
migrating on glass coverslips by time-lapse fluorescence
videomicroscopy. Migrating cells clustered the nanoparticles to the
uropod (arrows in first frame denote direction of migrating cells).
Cells that halted and de-polarized even momentarily redistributed
the nanoparticles over the cell surface, indicating a lack of
aggregation among nanoparticles. (B) DiR-labeled nanoparticles (1
mg) were injected s.c. in the flank of an anesthetized mouse and
imaged by whole-animal fluorescence (shown in false color on the
right flank). (C) Bioluminescence imaging of
gaussia-luciferase-tagged nanoparticles attached to
4.times.10.sup.6 pmel-1 T cells, 4 hrs after tail vein injection of
particle-conjugated T cells. Red arrows denote T cells accumulated
in lungs, while white arrows highlight what may represent T cell
homing to axillary lymph nodes. (D) Bioluminescence imaging of
Gaussia-luciferase-expressing B16F10 melanoma cells, illustrating
metastasis 14 days following kidney capsule injection.
[0058] FIGS. 9A-D. Melanoma-targeting Pmel-1 T lymphocytes vehicle
surface-conjugated nanoparticles into the tumor microenvironment.
(A-D) 500,000 B 16F10 tumor cells, transduced with Gaussia
luciferase, were injected into the right femur of C57BL/6 mice.
After three weeks, tumor burden was visualized by IVIS imaging (A).
Animals were treated with 15.times.10.sup.6 effector Pmel-1 T
lymphocytes, transgenic for Firefly luciferase (A-D, left panel),
or effector Pmel-1 T cells conjugated with nanoparticles containing
the fluorescent tag DiD (A-D, right panel). Before adoptive
transfer, T lymphocytes were incubated with 1 mg/ml Thiol-PEG for
30 min to avoid non-specific uptake of surface-bound nanoparticles
by macrophages. Four days after T cell treatment, the
biodistribution of adoptively transferred Pmel-1 T cells was imaged
with bioluminescence (B), surface-bound nanoparticles were tracked
by fluorescent IVIS imaging for DiD (C). The right femurs were
flushed and analyzed by multicolor flow cytometry for T cell
infiltrates (Thy1.1) and DiD nanoparticles (D).
[0059] FIGS. 10A-B. Nanoparticle-decorated T cells function
normally and efficiently carry surface-tethered nanoparticles into
antigen-expressing tumors. OT-1 ova-specific CD8.sup.+ effector T
cells were conjugated with 100 DNA-gel nanoparticles per cell or
left unmanipulated as controls. (A and B) Comparative in vivo
bioluminescence (tumors, T cells) and fluorescence imaging
(nanoparticles) of mice bearing subcutaneous Gaussia luc-expressing
EG7-OVA and control EL4 tumors on opposite flanks, 2 days after
i.v. infusion of firefly luc-transgenic Thy1.1.sup.+ effector OT-1
T cells (with or without attached DiD-labeled nanoparticles), or an
equivalent number of free nanoparticles. Thy1.1.sup.+ OT-1 T cells
recovered from the EG7-OVA tumors were analyzed for surface-bound
DiD nanoparticles by flow cytometry (A), and the mean
bioluminescent T cell and fluorescent nanoparticle signals from
groups of 6 mice are graphed shown in (B). Shown is 1 of 2
independent experiments.
[0060] FIGS. 11A-C. Tumor antigen-specific T cells transport
surface-bound nanoparticles into established TRAMP prostate
adenocarcinomas. (A) Six month-old TRP-SIY mice (Bal et al., PNAS
USA, 105:13003, 2008), with established spontaneous prostate
adenocarinomas expressing a short linear peptide SIY (SIYRYYGL; SEQ
ID NO:1) were injected with 15.times.10.sup.6
CBR-luciferase-expressing SIY-specific 2C transgenic CD8.sup.+ T
cells. T cells were left unmodified or were surface-conjugated with
DiD-tagged DNA-gel nanoparticles. Alternatively, an equivalent
number of free DiD-labeled particles was injected intravenously.
Ventral whole body bioluminescent acquisitions are shown directly
after T cell injection (upper panel), and 3 days later (lower
panel). (B) On day 3, prostates were isolated from euthanized mice,
and DiD tissue fluorescence was quantified using the IVIS Spectrum
imaging system. (C) Flow cytometry analysis of a single cell
suspension prepared from recovered prostates shows a substantial
fraction of 2C transgenic T cells still physically attached to
DiD-labeled DNA-gel nanoparticles.
[0061] FIGS. 12A-F. Naive B lymphocytes transport
surface-conjugated nanoparticles into secondary lymphoid organs.
(A) Comparative in vivo bioluminescence (upper panel) and
fluorescence (lower panel) imaging of C57B1/6 mice 2 days after
infusion of 10.times.10.sup.6 Firefly-luciferase-transgenic naive B
lymphocytes, labeled with CellTracker Green and decorated with DiD
fluorescent DNA gel nanoparticles. Alternatively, mice were
systemically injected with an equivalent number of free DiD
nanoparticles only. One representative mouse out of 3 injected
mice/group is shown. (B) Strong DiD tissue fluorescence was
detected in isolated cervical lymph nodes by IVIS imaging. (C)
Histology of the removed cervical lymph node. High-magnification
confocal microscopy (left panel) shows lymph node homing B
lymphocytes with surface-attached DiD-fluorescent nanoparticles.
(D) Biodistribution analysis of B cells carrying Indium-loaded
liposomes and empty liposomes. (E) Histology section showing B
cells that have homed into lymph nodes still have nanoparticles
(blue) attached to their surfaces. (F) E.mu.-myc lymphoma cells
were injected into mice and allowed to establish tumors in systemic
lymphoid organs for 14 days prior to injection of particle-carrying
normal B cells. Flow cytometry shows the liposome-carrying B cells
have entered the lymph nodes.
[0062] FIGS. 13A-D. Pmel-1 T cells conjugated with
IL-15Sa/IL-21-releasing nanoparticles robustly proliferate in vivo
and eradicate established B16 melanomas. (A) Dual in vivo
bioluminescence imaging of Gaussia luciferase-expressing B16F10
lung melanomas and CBR-luciferase-expressing Pmel-1 T cells in
sublethally irradiated C57B1/6 mice. Lung tumors established by
tail vein injection of B 16F10 cells were treated after 6 days by
i.v. infusion of 10.times.10.sup.6 V.beta.13.sup.+ CD8.sup.+ Pmel-1
T cells. One group of mice received Pmel-1 T cells conjugated with
100 DNA-gel nanoparticles/cell carrying a total dose of 5 .mu.g
IL-15Sa/IL-21 (4.03 .mu.g IL-15Sa+0.93 .mu.g IL-21), control groups
received unmodified Pmel-1 cells and a single systemic injection of
the same doses of IL-15Sa/IL-21 or Pmel-1 cells alone. (B)
Frequencies of V.beta.13.sup.+ CD8.sup.+ Pmel-1 T cells recovered
from pooled lymph nodes of representative animals 16 days after T
cell transfer. (C) CBR-luc signal intensities from sequential
bioluminescence imaging every 2 days after T cell transfer. Every
line represents one animal with each dot showing the whole animal
photon count. (D) Survival of animals following T cell therapy
illustrated by Kaplan-Meier curves. Shown are 6 mice/treatment
group pooled from 3 independent experiments.
[0063] FIGS. 14A-D. Hematopoietic progenitor cells (referred to in
the Figure as HSCs) carrying GSK-3.beta. inhibitor-loaded
nanoparticles reconstitute recipient animals with rapid kinetics
following bone marrow transplants without affecting multilineage
differentiation potential. (A, B) Engraftment kinetics of
luciferase-transgenic HSC grafts in lethally-irradiated
nontransgenic syngeneic recipients. Mice were treated with a single
bolus injection of the GSK-3.beta. inhibitor TWS119 (1.6 ng) on the
day of transplantation, an equivalent TWS119 dose encapsulated in
HSC-attached DNA-gel nanoparticles, or no exogenous agent.
Transplanted mice were imaged for whole-body bioluminescence every
7 days for 3 weeks. Shown are representative IVIS images (A) and
whole animal photon counts (B) for 9 mice total/treatment
condition. (C) Percentage of donor-derived cells in recipient mice
2 weeks after transplantation of GFP.sup.+ HSCs with or without
TWS119. *P<0.001. (D) Average frequency of donor-derived
GFP.sup.+ B cells, T cells, and myeloid cells in recipient mice 3
months after transplantation. 5 mice/group were analyzed.
[0064] FIG. 15. Liposome conjugation to pmel-1 T cells. Confocal
image of liposomes (blue) conjugated to the surfaces of pmel-1 T
cells (CFSE-stained in green). Shown are 3D projections of optical
sections taken by confocal microscopy.
DETAILED DESCRIPTION OF THE INVENTION
[0065] The invention contemplates combined cell- and
nanoparticle-mediated delivery of agents including drugs. This
delivery strategy involves the conjugation of nanoparticles that
comprise one or more agents to a cell that can home to a region,
tissue or organ in vivo, thereby resulting in localized and
controlled delivery of agents in vivo. This approach offers
significant advantages over the prior art approaches of
administering agents alone or in non-cell bound delivery vehicles
such as nanoparticles. The former approaches suffer from systemic
toxicity problems. The latter approaches suffer from rapid
clearance of nanoparticles via the reticuloendothelial system
including macrophages and Kupffer cells of the spleen and liver (as
shown in the Examples), and limitations in biodistribution based on
size-mediated exclusion/inclusion from tissues. The clearance
mechanisms prevent prolonged release and thus sustained presence of
the agent of interest in vivo. Moreover clearance is potentially
associated with toxicity in the liver due to the accumulation of
nanoparticles at that site, also as shown in the Examples.
[0066] The invention therefore exploits the use of nanoparticles
and cells in the localized delivery of agents. The cells may
function simply as carriers that home to localized regions, tissues
or organs within the body and thereby deliver the agent more
specifically within the body (i.e., in a paracrine manner),
although in more preferred embodiments they also contribute
functionally at the ultimate target site and may be acted upon by
the agent they are carrying (i.e., in an autocrine manner). In
either event, the cells are referred to herein as carrier cells to
be distinguished from cells at target sites in vivo.
[0067] In one exemplary embodiment, the invention provides a
delivery method based on conjugation of nanoparticles to
tumor-reactive T cells, such as those used in adoptive cell therapy
(ACT). A functionalized biodegradable polymer nanoparticle,
liposome, or polymer vesicle may be loaded with one or more agents
which are released in vivo as the nanoparticle degrades in response
to its environment (typically an aqueous environment). This is
shown schematically in FIG. 1A. This approach offers several
potential advantages over systemic drug therapy including the
uniform exposure of ACT T cells to the released drugs, focused drug
action on the ACT T cells and other T cells at the target site,
reduced amounts of drugs administered to a subject as a result of a
biodistribution that follows the homing pattern of the T cells,
reduced exposure of non-target sites to the drug and thus reduced
probability of non-target toxicity, and extended and sustained
release of drug over the span of several days.
[0068] In a further exemplary embodiment, T cells are conjugated to
biodegradable nanoparticles that comprise immune stimulating agents
such as cytokines, antigens, antibodies, adjuvants or other
activation agents that function to stimulate or enhance immune
responses at the target site, maintain activation of the carrier
ACT T cells, and/or cause cell death directly or indirectly at the
target site. Exemplary agents are provided herein and include but
are not limited to IL-15/IL-15R.alpha. complexes (referred to
herein as an IL-15 superagonist, described by Rubenstein et al.,
PNAS 103(24):9166-9171, 2006, the teachings of which relating to
IL-15 SA are incorporated herein by reference) and TLR ligand
adjuvants such as MPLA and imiquimod. Such nanoparticles may
contain other agents such as anti-cancer agents, or they may be
used with other nanoparticles that contain such agents. TLR ligands
may act as immunostimulating agents independent of an antigen
effect, in some instances. In still other instances, the agents may
be immunomodulatory or even immunoinhibitory, if it is desired to
control or reduce an immune reaction in vivo, such as occurs in
autoimmune disorders as an example.
[0069] In some instances, the invention contemplates but is not
limited to enhancement (whether additive or super-additive) of the
therapeutic benefit that is provided by standard adoptive cell
therapy which involves transfer of cells that are not conjugated to
nanoparticles. This enhancement may be measured by reduction in
tumor load (or volume) in the case of a subject having a tumor, or
reduction in infectious agent load (for example in a bodily fluid)
or reduction in size, depth or volume of an infectious lesion in
the case of a subject having an infection.
[0070] It is to be understood that the agents carried by the
nanoparticles may function on cells or tissue at the target site
(i.e., a paracrine manner) and/or on the carrier cells themselves
(i.e., an autocrine manner), as depicted in FIG. 1B. Thus for
example where the cell is a T cell (or other cell with homing
capability), the agent may be one that stimulates the carrier cell
and optionally cells of the same type at the target site (e.g.,
other tumor-reactive T cells). In other embodiments, the agents
comprised within the nanoparticles are intended to act on cells
other than the carrier cell. Examples include anti-cancer agents
which act upon tumor cells and generally will have no effect on T
cells or other carrier cell types.
[0071] Targeting primarily the carrier cells, particularly carrier
T cells, serves to maintain their proliferation and effector
functions while limiting nonspecific activation of bystander T
cells. It has been reported that cytokines released in an autocrine
manner may be nearly quantitatively recaptured by the secreting
cell, due to the local high concentration of cytokine and its
corresponding upon release from the cell. (Monine et al., Biophys J
88(4): 2384, 2005; Lauffenburger et al., Proc Natl Acad Sci U S A
95(26): 15368, 1998; Joslin et al., J Cell Sci 120(Pt 20): 3688,
2007.) Cytokines such as IL-15 superagonist are therefore expected
to be more potent when released from nanoparticles conjugated to
carrier cells than when administered systemically in an
unconjugated form.
[0072] An example of a paracrine method involves the delivery of
adjuvant(s) to a target site alone or together with antigen(s).
Exemplary adjuvants are provided herein, and these include TLR
ligands such as MPLA and imiquimod. These agents can act on
dendritic cells and other antigen presenting cells present at a
target site (e.g., a tumor site or a site of infection or at a
secondary lymphoid organ or tissue including but not limited to
spleen and lymph nodes). The cell-mediated delivery methods of the
invention will both increase the local concentration of agents at
the relevant target sites and limit the overall systemic exposure
that occurs when the same agents are injected in an unconjugated
form.
[0073] Thus, as another example of paracrine-acting agent(s), in
instances where the target site is a tumor, the nanoparticles may
comprise an anti-cancer agent and/or an adjuvant. Once delivered to
the target tumor site, via tumor-reactive T cells, the anti-cancer
agent is gradually released resulting in the death of tumor cells
whether by necrosis or apoptosis. Such cell death is usually
accompanied by fragmentation and release of cellular components
including antigens specific to the tumor cells. The gradual release
of adjuvant from nanoparticles delivered to the target site will
enhance the body's antigen-specific immune response to the released
cancer antigens. The presence of activated tumor-reactive T cells
will serve to localize and enhance the immune response as well.
Tumor-reactive T cells have been described previously and include
without limitation melanoma reactive T cells (e.g., Melan A
specific T cells described by Li et al., J Immunother. 31(1):81-8,
2008, the teachings of which relating to melanoma-specific T cells
are incorporated herein by reference).
[0074] Other tumor targets include without limitation lymphomas. In
these instances, B cells and/or T cells such as central memory T
cells may be conjugated to nanoparticles comprising anti-lymphoma
agents. Such agents are known in the art and include without
limitation anti-CD20 antibodies, such as rituximab. As shown in the
Examples, B cells and central memory T cells are able to home
nanoparticles into lymphoid organs, in particular the spleen and
lymph nodes, and reduce the amount of nanoparticles that would
otherwise home and/or deposit in liver and bone. The use of
lymphocytes as carrier cells is advantageous because the cells are
easily obtained from peripheral blood of a subject and they can
naturally home (or be manipulated ex vivo to home) to certain
tissues (e.g., lymphoid tissues) or tumors. The invention
contemplates that other blood diseases including without limitation
leukemia may also be treated in a like manner.
[0075] The foregoing embodiments are intended for illustration and
are not to be construed to limit the invention simply to
tumor-specific ACT. Instead, the invention contemplates various
other applications where localized delivery of one or more agents
and optionally particular cells would be beneficial. As an example,
the invention contemplates delivery of imaging agents to various
distinct regions, tissues and/or organs. As another example, T
cells may be conjugated to nanoparticles carrying any variety
and/or combination of agents and can be targeted to any number of
sites in vivo. In this embodiment, T cells can be exploited for
their demonstrated tropism to different tissues. Examples include
naive T cells that can carry agent-loaded nanoparticles to lymphoid
organs and spleen (e.g., for vaccination), gut-homing T cells that
can carry agent-loaded nanoparticles to the gut (e.g., for
treatment of cancer or autoimmune disorders), skin-homing T cells
that can deliver agent-loaded nanoparticles to the skin layers
(e.g., for treatment of cutaneous lesions or autoimmune disease),
etc. These tissue sites can be targeted simply by isolating T cells
with the appropriate homing receptors from blood. It is to be
understood that the methods provided herein may be used to
stimulate (or enhance) immune responses (e.g., against tumors or
infections) or suppress immune responses (e.g., by promoting
tolerance to allergens or transplanted tissues).
[0076] As a further exemplary embodiment of the invention,
hematopoietic progenitor cells may be loaded with nanoparticles
that stimulate proliferation and, in some instances, self-renewal.
The Examples demonstrate the ability to conjugate nanoparticles
comprising the glycogen synthase kinase 3 beta (GSK3-beta)
inhibitor TWS119 to lineage-negative, Sca-1-positive,
c-kit-positive, and the delivery of such cells to a subject.
Biodistribution of the administered cells to the femur, humerus,
sternum and spleen of recipients was observed, as was a normal
differentiative potential of such cells several months
post-transplant.
[0077] The methods described herein may be combined with other
therapeutic or diagnostic strategies or methods including without
limitation surgery, radiation and/or chemotherapy including
immunotherapy.
[0078] It has been found, surprisingly, that the method is
straightforward to implement and thus could be easily incorporated
into any clinical process. The method requires simple mixing of the
functionalized and agent-loaded nanoparticles with the cells of
interest, as detailed in the Examples. Nanoparticles can be
prepared and stored in a convenient format prior to use (e.g.,
lyophilized powder). Nanoparticles are then reconstituted in a
suitable carrier and incubated with the cell population for a brief
period of time. Incubation times may range from 1-5 minutes, 1-10
minutes, 5-10 minutes, 5-15 minutes, 5-20 minutes, 5-30 minutes, or
5-60 minutes. The mixture is then washed, in some instances
incubated with a blocking agent to quench the reactive groups on
the nanoparticle and optionally on the cell, washed again, and then
formulated for administration. Administration typically will occur
via through parental routes, most preferably intravenous
injection.
Carrier Cells
[0079] The carrier cells are the cells to which the nanoparticles
are conjugated and which when administered in vivo preferably home
to target site(s). Suitable target cells are chosen based on their
homing potential, their cell surface phenotype (for conjugation to
the nanoparticles), and their ability to carry but not
significantly endocytose the nanoparticles. In some embodiments
described herein, T cells are suitable carrier cells. The T cells
may be CD4+ or CD8+ T cells. Other suitable cells include B cells,
NK cells, NK T cells, and hematopoietic progenitor cells including
without limitation murine lineage-negative, Sca-l-positive and
c-kit-positive cells and their human counterparts. B cells for
example can be used to carry antigen-loaded nanoparticles into
lymphoid organs to promote antibody responses or to regulate
allergic reactions. Macrophages and dendritic cells typically are
not suitable carriers for nanoparticles because of their
internalizing/phagocytosing capabilities. Substantial levels of
free thiol (--SH) groups exist on the surfaces of T cells, B cells
and hematopoietic progenitor cells (data not shown), thereby
facilitating conjugation of nanoparticles to such cells.
[0080] Carrier cells preferably also are able to extravasate from
the blood vessels (particularly when administered by intravenous
injection) and thereby enter target tissues or organs. Red blood
cells typically are not able to exit the blood stream. Accordingly,
one important class of carrier cells is nucleated cells. This class
by definition excludes red blood cells.
[0081] Some embodiments of the invention refer to isolated carrier
cells. Isolated carrier cells are cells that have been separated
from the environment in which they naturally occur (i.e., they are
not present in vivo). T cells in vitro are an example of an
isolated cell.
[0082] The carrier cells preferably are autologous to the subject
being treated, however some embodiments of the invention
contemplate non-autologous (yet preferably MHC matched cells).
[0083] The carrier cells preferably have half-lifes in vivo,
following administration (or re-infusion, in some instances) of at
least 48 hours, more preferably at least, at least 3 days, at least
4 days, at least 5 days, at least 6 days, at least 7 days, or
more.
[0084] The cells may be genetically engineered to express one or
more factors including without limitation costimulatory molecules
or receptors including chimeric receptors. In other embodiments,
the cells are not genetically engineered. In some such embodiments,
the carrier cells are isolated and naturally occurring (i.e., they
have not been genetically or otherwise engineered).
[0085] Depending on their nature and function, the cells may be
manipulated prior to conjugation with the nanoparticles. The cells
however need not be surface-modified in order to facilitate
conjugation of the nanoparticles. The invention in some of its
embodiments instead takes advantage of reactive groups that
normally exist on the cell surface without having to incorporate
reactive groups or other entities onto the cell surface. As a
result, such cells do not require the presence of exogenous
entities such as antibodies or antibody fragments, among others, on
their surface in order to conjugate to nanoparticles.
[0086] Such manipulation may also involve activation of the cells,
as is routinely performed for T cells. The cells may be expanded
and/or activated (or stimulated, as the terms are used
interchangeably herein) in vitro prior to mixing with the
nanoparticles (or liposomes). Expansion and activation protocols
will vary depending on the cell type but can include incubation
with one or more cytokines, incubation with one or more cell types,
incubation with one or more antigens, etc. If the carrier cell is a
T cell, then activation may be performed by incubating the cells
with IL-2, IL-15, IL-15 superagonist, costimulatory molecules such
as B7, B7.2, CD40, antibodies to various T cell surface molecules
including antibodies to cell surface receptors, anti-CD3
antibodies, anti-CD28 antibodies, anti-CTLA-4 antibodies,
anti-CD4OL antibodies, and the like. In some embodiments, the cells
and more particularly the T cells are not coated with exogenous
antibodies on their cell surface (i.e., the cells have not been
contacted with antibodies or antibody fragments in vitro prior to
administration).
[0087] Expansion may be measured by proliferation assays involving
incorporation of radiolabeled nucleotides such as tritiated
thymidine. Activation may be measured by production of cytokines
such as IL-2, gamma-IFN, IL-1, IL-4, IL-6, and TNF, among others.
Other ways of measuring expansion and activation are known in the
art.
[0088] Carrier cells may be selected prior to administration to a
subject in order to enrich and thus administer higher numbers of
such cells in smaller volumes and/or to remove other, potentially
unwanted, cells from the administered composition. Selection may
involve positive or negative selection, including for example
column or plate based enrichment protocols that are known in the
art.
[0089] T and B cells may be harvested from the peripheral blood of
a subject.
[0090] Hematopoietic progenitor cells may be obtained from a number
of sources including but not limited to cord blood, bone marrow,
mobilized peripheral blood, and in some instances differentiated
embryonic stem cells.
[0091] Hematopoietic progenitor cells have been characterized in
the art. Such cells in the human generally have minimally a CD34+
phenotype, although they may also be CD59.sup.+, Thy1/CD90.sup.+,
CD38.sup.lo/neg, CD33.sup.-, and/or c-kit/CD117.sup.+. They also
are characterized as not expressing lineage specific markers. They
can be harvested from bone marrow, cord blood or peripheral blood
using affinity columns, magnetic beads, fluorescence activated cell
sorting (FACS), some combination thereof, and the like. These cells
have the ability to repopulate one or more hematopoietic lineages
upon transplantation. Preferably, these cells repopulate more than
one lineage, and even more preferably, all lineages. Repopulation
or population of lineages as used herein refers to the
differentiation of the stem cell into one or more lineages such
that progeny of the stem cell contribute to the make up of that
lineage in the subject. It does not however require that the entire
lineage compartment derive from the transplanted cells, however in
some instances this may occur.
[0092] Isolated stem cells may be obtained by fractionating a
heterogenous cell population according to one or more markers,
including by not limited to cell surface markers.
[0093] The carrier cells may be eukaryotic cells, such as mammalian
cells (e.g., human cells). Alternatively, they may be non-mammalian
cells. In still other embodiments, the carrier cells may be
prokaryotic cells (e.g., bacterial cells). Several bacterial cell
types are of particular interest. For example, attenuated
salmonella typhimurium is under study as a candidate vector for
oral vaccine delivery (Xiang et al., Immunol Rev 222:117, 2008; and
Iweala et al., J Immunol 183(4):2252, 2009) and engineered E. coli
bacteria have been shown to be capable of specific homing to poorly
oxygenated tumors (Cheong et al., Science 314(5803):1308, 2006).
Bacteria offer new modes of administration and tissue site
targeting possibilities, such as oral administration and the
ability to target therapeutics to the gut and gut-associated
lymphoid tissues. Such microbial vectors may offer advantages
relative to autologous host cells in terms of creating
off-the-shelf ready-to-use cell-nanoparticles systems. Particles
conjugation to microbes can be achieved using the same suite of
chemical strategies described for mammalian cells. In some
instances, temporary removal of flagellar coats of microbes (e.g.,
via simple mechanical shearing as described by Rosu et al., J
Bacteriol 188(14):5196, 2006) can be used to achieve optimal
conjugation of particles to microbe cell bodies. The ability to
enhance the activity of these cells by conjugating drug-loaded
nanoparticles or microparticles to them for co-transport to their
target tissue sites can be used to alter their therapeutic efficacy
or alter the biodistribution of the synthetic particles as
described herein with other cell carriers. The ability of synthetic
drug particles to be loaded with small-molecule therapeutics makes
this approach complementary to genetic engineering of the
microbe.
Nanoparticles
[0094] As used herein, nanoparticles are solid colloidal particles
used to deliver agent. Nanoparticles are not liposomes, as used
herein. The nanoparticles are not viruses or particles thereof. The
nanoparticles are also to be distinguished from films or other
structurally layered polymers matrices, since the nanoparticles are
comprised of one or more solidified polymer(s) that is arranged in
a random manner. The nanoparticles are preferably biodegradable and
thus typically are not magnetic. Biodegradable nanoparticles may be
synthesized using methods known in the art including without
limitation solvent evaporation, hot melt microencapsulation,
solvent removal, and spray drying. Exemplary methods for
synthesizing nanoparticles are described herein in the Examples as
well as by Bershteyn et al., Soft Matter 4:1787-1787, 2008 and in
US 2008/0014144 A1, the specific teachings of which relating to
nanoparticle synthesis are incorporated herein by reference.
[0095] In some embodiments, the nanoparticles are comprised of a
nucleic acid internal core. Such "DNA nanoparticles" (or DNA-gel
nanoparticles) are described in greater detail in published U.S.
application no. US 20070148246. It is to be understood that the
nucleic acid core of such particles may act as a scaffold for the
agents being delivered in vivo and/or it may act as the agent
itself. An exemplary protocol for synthesizing DNA nanoparticles is
provided in the Examples.
[0096] The nanoparticles release their agent "payload" over a
number of days as a function of their degradation profile in vivo.
As discussed herein, the nanoparticles are biodegradable in nature
and thus they gradually degrade in an aqueous environment such as
occurs in vivo. If the agents are dispersed throughout the
nanoparticles then their release will occur as the outermost layers
of the nanoparticle degrade or as the pores within the nanoparticle
enlarge. Release kinetic studies have been performed and they
demonstrate that protein and small-molecule drugs can be released
from such nanoparticles over time-courses ranging from 1 day to at
least 2 weeks. The nanoparticles are preferably not engulfed by
either their carrier cells or other cells at the target site. They
function rather by gradually releasing their payload into the
environment of the target site(s).
[0097] The nanoparticles' diameter ranges from 1-1000 nanometers
(nm). In some embodiments, their diameter ranges in size from
20-750 nm, or from 20-500 nm, or from 20-250 nm. In some
embodiments, their diameter ranges in size from 50-750 nm, or from
50-500 nm, or from 50-250 nm, or from about 100-300 nm. In some
embodiments, their diameter is about 100, about 150, about 200 nm,
about 250 nm, or about 300 nm. As used in the context of
nanoparticle diameters, the term "about" means +/-5% of the
absolute value stated. Thus, it is to be understood that although
these particles are referred to herein as nanoparticles, the
invention intends to embrace microparticles as well.
[0098] As discussed herein, the nanoparticles may be synthesized to
comprise one or more reactive groups on their exterior surface for
reaction with reactive groups on cell carriers (e.g., leukocytes).
These nanoparticle reactive groups include without limitation
thiol-reactive maleimide head groups, haloacetyl (e.g., iodoacetyl)
groups, imidoester groups, N-hydroxysuccinimide esters, pyridyl
disulfide groups, and the like. These reactive groups react with
groups on the carrier cell surface and thus the nanoparticles are
bound to the cell surface. It will be understood that when surface
modified in this manner, the nanoparticles are intended for use
with specific carrier cells having "complementary" reactive groups
(i.e., reactive groups that react with those of the nanoparticles).
In some embodiments, the nanoparticles will not integrate into the
lipid bilayer that comprises the cell surface. Typically, the
nanoparticles will not be phagocytosed (or internalized) by the
carrier cells.
[0099] In some embodiments the nanoparticles do not comprise
antibodies or antibody fragments on their surface, while in other
embodiments they do. In some embodiments the nanoparticles do not
comprise antibodies or antibody fragments that are specific to T
cell surface moieties (or exogenous moieties coated onto a T cell
surface such other antibodies or antibody fragments), while in
other embodiments they do. Thus, in some embodiments the
nanoparticles themselves do not stimulate carrier cell activation
simply by binding to the carrier cell. In other embodiments however
the nanoparticles do stimulate carrier cell activation by binding
to the carrier cell (e.g., binding of the nanoparticle results in
crosslinking of cell surface moieties and this activates the
carrier cell).
[0100] The nanoparticles may be covalently conjugated (or attached
or bound, as the terms are used interchangeably herein), or they
may be non-covalently conjugated to the carrier cells. Covalent
conjugation typically provides a more stable (and thus longer)
association between the nanoparticles and the carrier cells.
Covalent conjugation in some embodiments also can provide stability
and thus more sustained localized delivery of agents in vivo.
Non-covalent conjugation includes without limitation absorption
onto the cell surface and/or lipid bilayer of the cell
membrane.
[0101] In some instances, covalent attachment can be achieved in a
two-step process in which carrier cells are first incubated with
maleimide-bearing nanoparticles to allow conjugation to the cell
surface, followed by in situ PEGylation with thiol-terminated
poly(ethylene glycol) (PEG) to cap remaining maleimide groups of
the particles and avoid particle-mediated crosslinking of cells.
With this approach, substantial numbers of nanoparticles with
diameters in the 100-300 nm range have been conjugated to cell
types used commonly in cell therapy, including CD8.sup.+ T
lymphocytes and lineage.sup.-Sca-1.sup.+c-kit.sup.+ murine
progenitor cells (data not shown). This strategy allows particles
ranging from simple liposomes (e.g., with an aqueous drug-loaded
core) to more complex lipid-coated polymer or DNA-based
nanoparticles to be stably attached to live cells. Importantly, the
linkage chemistry is benign and non-toxic as evidenced in part by
the conjugation of up to 139 (.+-.29).about.200 nm-diameter
lipid-coated nanoparticles to the surface of cells without any
deleterious effect (data not shown).
[0102] Although liposomes and lipid-coated polymer particles are
able to spontaneously adsorb to cell surfaces, in some instances
covalent conjugation is preferred due to the increased stability it
achieves.
[0103] Nanoparticles bound to carrier cells, such as lymphocytes or
hematopoietic progenitor cells, remain localized at the cell
surface as revealed by optical sectioning with confocal microscopy,
scanning electron microscopy and by flow cytometry internalization
assays, even following extended in vitro stimulation (data not
shown). Phagocytic cells, such as immature dendritic cells, are
able to efficiently internalize maleimide-functionalized
nanoparticles after a short incubation (data not shown), and thus
they are not suitable as carrier cells.
[0104] Exemplary synthetic polymers which can be used to form the
biodegradable nanoparticles include without limitation aliphatic
polyesters, poly (lactic acid) (PLA), poly (glycolic acid) (PGA),
co-polymers of lactic acid and glycolic acid (PLGA),
polycarprolactone (PCL), polyanhydrides, poly(ortho)esters,
polyurethanes, poly(butyric acid), poly(valeric acid), and
poly(lactide-co-caprolactone), and natural polymers such as
alginate and other polysaccharides including dextran and cellulose,
collagen, chemical derivatives thereof, including substitutions,
additions of chemical groups such as for example alkyl, alkylene,
hydroxylations, oxidations, and other modifications routinely made
by those skilled in the art), albumin and other hydrophilic
proteins, zein and other prolamines and hydrophobic proteins,
copolymers and mixtures thereof. In general, these materials
degrade either by enzymatic hydrolysis or exposure to water in
vivo, by surface or bulk erosion.
[0105] The nanoparticles also preferably comprise a lipid bilayer
on their outermost surface. This bilayer may be comprised of one or
more lipids of the same or different type. Examples include without
limitation phospholipids such as phosphocholines and
phosphoinositols. Specific examples include without limitation
DMPC, DOPC, DSPC, and various other lipids such as those recited
below for liposomes.
Liposomes
[0106] The invention also contemplates the use of liposomes in
place of nanoparticles in the various embodiments described herein.
Liposomes are small closed vesicles comprising at least one lipid
bilayer and an internal aqueous compartment. As used herein,
liposomes are not nanoparticles. Liposomes may be anionic, neutral
or cationic. They may be unilamellar or multilamellar. Liposome may
comprise without limitation unilamellar vesicle lipids,
multilamellar vesicle lipids and extruded lipids including DOTMA,
DOTAP, DOTIM, DDAB, alone or together with cholesterol to yield
DOTMA and cholesterol, DOTAP and cholesterol, DOTIM and
cholesterol, and DDAB and cholesterol. Methods for preparation of
multilamellar vesicle lipids are known in the art (see for example
U.S. Pat. No. 6,693,086, the teachings of which relating to
multilamellar vesicle lipid preparation are incorporated herein by
reference). Extruded lipids are prepared in a similar manner but
are then extruded through filters of decreasing size, as described
in Templeton et al., Nature Biotech, 15:647-652, 1997, the
teachings of which relating to extruded lipid preparation are
incorporated herein by reference.
[0107] Liposomes may be surface modified during or after synthesis
to include reactive groups complementary to the reactive groups on
the carrier cells. Such reactive groups include without limitation
maleimide groups. As an example, liposomes may be synthesized to
include maleimide conjugated phospholipids such as without
limitation DSPE-MaL-PEG2000.
[0108] An exemplary synthesis protocol for liposomes is provided in
the Examples.
Agents
[0109] The invention contemplates the delivery of agents to
localized regions, tissues or cells in vivo. As used herein, an
agent is any atom or molecule or compound that can be used to
provide benefit to a subject (including without limitation
prophylactic or therapeutic benefit) or that can be used for
diagnosis and/or detection (for example, imaging) in vivo.
[0110] Any agent may be delivered using the methods of the
invention provided that it can be loaded into the nanoparticles
provided herein. For example, the agent must be able to withstand
the nanoparticle synthesis and optionally storage process. The
nanoparticles may be synthesized and stored in, for example, a
lyophilized form. The agents, if incorporated into the
nanoparticles during synthesis, should be stable during such
storage procedures and times.
[0111] The agent may be without limitation a protein, a
polypeptide, a peptide, a nucleic acid, a virus-like particle, a
steroid, a proteoglycan, a lipid, a carbohydrate, and analogs,
derivatives, mixtures, fusions, combinations or conjugates thereof.
The agent may be a prodrug that is metabolized and thus converted
in vivo to its active (and/or stable) form.
[0112] The agents may be naturally occurring or non-naturally
occurring. Naturally occurring agents include those capable of
being synthesized by the subjects to whom the nanoparticles are
administered. Non-naturally occurring are those that do not exist
in nature normally, whether produced by plant, animal, microbe or
other living organism.
[0113] One class of agents is peptide-based agents such as (single
or multi-chain) proteins and peptides. Examples include antibodies,
single chain antibodies, antibody fragments, enzymes, co-factors,
receptors, ligands, transcription factors and other regulatory
factors, some antigens (as discussed below), cytokines, chemokines,
and the like. These peptide-based agents may or may not be
naturally occurring but they are capable of being synthesized
within the subject, for example, through the use of genetically
engineered cells.
[0114] Another class of agents that can be delivered in a localized
manner using the nanoparticles of the invention includes those
agents that are not peptide-based and which could not be
synthesized by the transferred cells. Examples include chemical
compounds that are non-naturally occurring, or chemical compounds
that are not naturally synthesized by mammalian (and in particular
human) cells.
[0115] A variety of agents that are currently used for therapeutic
or diagnostic purposes can be delivered according to the invention
and these include without limitation imaging agents,
immunomodulatory agents such as immunostimulatory agents and
immunoinhibitory agents, antigens, adjuvants, cytokines,
chemokines, anti-cancer agents, anti-infective agents, nucleic
acids, antibodies or fragments thereof, fusion proteins such as
cytokine-antibody fusion proteins, Fc-fusion proteins, and the
like.
[0116] Imaging Agents. As used herein, an imaging agent is an agent
that emits signal directly or indirectly thereby allowing its
detection in vivo. Imaging agents such as contrast agents and
radioactive agents that can be detected using medical imaging
techniques such as nuclear medicine scans and magnetic resonance
imaging (MRI). Imaging agents for magnetic resonance imaging (MRI)
include Gd(DOTA), iron oxide or gold nanoparticles; imaging agents
for nuclear medicine include .sup.201Tl, gamma-emitting
radionuclide 99 mTc; imaging agents for positron-emission
tomography (PET) include positron-emitting isotopes,
(18)F-fluorodeoxyglucose ((18)FDG), (18)F-fluoride, copper-64,
gadoamide, and radioisotopes of Pb(II) such as 203 Pb, and 11In;
imaging agents for in vivo fluorescence imaging such as fluorescent
dyes or dye-conjugated nanoparticles. In other embodiments, the
agent to be delivered is conjugated, or fused to, or mixed or
combined with an imaging agent.
[0117] Immunostimulatory Agents. As used herein, an
immunostimulatory agent is an agent that stimulates an immune
response (including enhancing a pre-existing immune response) in a
subject to whom it is administered, whether alone or in combination
with another agent. Examples include antigens, adjuvants (e.g., TLR
ligands such as imiquimod, imidazoquinoline, nucleic acids
comprising an unmethylated CpG dinucleotide, monophosphoryl lipid A
or other lipopolysaccharide derivatives, single-stranded or
double-stranded RNA, flagellin, muramyl dipeptide), cytokines
including interleukins (e.g., IL-2, IL-7, IL-15 (or
superagonist/mutant forms of these cytokines), IL-12, IFN-gamma,
IFN-alpha, GM-CSF, FLT3-ligand, etc.), immunostimulatory antibodies
(e.g., anti-CTLA-4, anti-CD28, anti-CD3, or single chain/antibody
fragments of these molecules), and the like.
[0118] Antigens. The antigen may be without limitation a cancer
antigen, a self antigen, a microbial antigen, an allergen, or an
environmental antigen. The antigen may be peptide, lipid, or
carbohydrate in nature, but it is not so limited.
[0119] Cancer Antigens. A cancer antigen is an antigen that is
expressed preferentially by cancer cells (i.e., it is expressed at
higher levels in cancer cells than on non-cancer cells) and in some
instances it is expressed solely by cancer cells. The cancer
antigen may be expressed within a cancer cell or on the surface of
the cancer cell. The cancer antigen may be MART-1/Melan-A, gp100,
adenosine deaminase-binding protein (ADAbp), FAP, cyclophilin b,
colorectal associated antigen (CRC)--C017-1A/GA733,
carcinoembryonic antigen (CEA), CAP-1, CAP-2, etv6, AML1, prostate
specific antigen (PSA), PSA-1, PSA-2, PSA-3, prostate-specific
membrane antigen (PSMA), T cell receptor/CD3-zeta chain, and CD20.
The cancer antigen may be selected from the group consisting of
MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7,
MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-Al2, MAGE-Xp2 (MAGE-B2),
MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3,
MAGE-C4, MAGE-C5). The cancer antigen may be selected from the
group consisting of GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6,
GAGE-7, GAGE-8, GAGE-9. The cancer antigen may be selected from the
group consisting of BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4,
tyrosinase, p53, MUC family, HER2/neu, p21ras, RCAS1,
.alpha.-fetoprotein, E-cadherin, .alpha.-catenin, .beta.-catenin,
.gamma.-catenin, p120ctn, gp100.sup.Pmel117, PRAME, NY-ESO-1,
cdc27, adenomatous polyposis coli protein (APC), fodrin, Connexin
37, Ig-idiotype, p15, gp75, GM2 ganglioside, GD2 ganglioside, human
papilloma virus proteins, Smad family of tumor antigens, lmp-1,
P1A, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen
phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5,
SCP-1 and CT-7, CD20, and c-erbB-2.
[0120] Microbial Antigens. Microbial antigens are antigens derived
from microbial species such as without limitation bacterial, viral,
fungal, parasitic and mycobacterial species. As such, microbial
antigens include bacterial antigens, viral antigens, fungal
antigens, parasitic antigens, and mycobacterial antigens. Examples
of bacterial, viral, fungal, parasitic and mycobacterial species
are provided herein. The microbial antigen may be part of a
microbial species or it may be the entire microbe.
[0121] Allergens. An allergen is an agent that can induce an
allergic or asthmatic response in a subject. Allergens include
without limitation pollens, insect venoms, animal dander dust,
fungal spores and drugs (e.g. penicillin). Examples of natural,
animal and plant allergens include but are not limited to proteins
specific to the following genera: Canine (Canis familiaris);
Dermatophagoides (e.g. Dermatophagoides farinae); Felis (Felis
domesticus); Ambrosia (Ambrosia artemiisfolia; Lolium (e.g. Lolium
perenne or Lolium multiflorum); Cryptomeria (Cryptomeria japonica);
Alternaria (Alternaria alternata); Alder; Alnus (Alnus gultinoasa);
Betula (Betula verrucosa); Quercus (Quercus alba); Olea (Olea
europa); Artemisia (Artemisia vulgaris); Plantago (e.g. Plantago
lanceolata); Parietaria (e.g. Parietaria officinalis or Parietaria
judaica); Blattella (e.g. Blattella germanica); Apis (e.g. Apis
multiflorum); Cupressus (e.g. Cupressus sempervirens, Cupressus
arizonica and Cupressus macrocarpa); Juniperus (e.g. Juniperus
sabinoides, Juniperus virginiana, Juniperus communis and Juniperus
ashei); Thuya (e.g. Thuya orientalis); Chamaecyparis (e.g.
Chamaecyparis obtusa); Periplaneta (e.g. Periplaneta americana);
Agropyron (e.g. Agropyron repens); Secale (e.g. Secale cereale);
Triticum (e.g. Triticum aestivum); Dactylis (e.g. Dactylis
glomerata); Festuca (e.g. Festuca elatior); Poa (e.g. Poa pratensis
or Poa compressa); Avena (e.g. Avena sativa); Holcus (e.g. Holcus
lanatus); Anthoxanthum (e.g. Anthoxanthum odoratum); Arrhenatherum
(e.g. Arrhenatherum elatius); Agrostis (e.g. Agrostis alba); Phleum
(e.g. Phleum pratense); Phalaris (e.g. Phalaris arundinacea);
Paspalum (e.g. Paspalum notatum); Sorghum (e.g. Sorghum
halepensis); and Bromus (e.g. Bromus inermis).
[0122] Adjuvants. The adjuvant may be without limitation alum
(e.g., aluminum hydroxide, aluminum phosphate); saponins purified
from the bark of the Q. saponaria tree such as QS21 (a glycolipid
that elutes in the 21st peak with HPLC fractionation; Antigenics,
Inc., Worcester, Mass.); poly[di(carboxylatophenoxy)phosphazene
(PCPP polymer; Virus Research Institute, USA), Flt3 ligand,
Leishmania elongation factor (a purified Leishmania protein; Corixa
Corporation, Seattle, Wash.), ISCOMS (immunostimulating complexes
which contain mixed saponins, lipids and form virus-sized particles
with pores that can hold antigen; CSL, Melbourne, Australia),
Pam3Cys, SB-AS4 (SmithKline Beecham adjuvant system #4 which
contains alum and MPL; SBB, Belgium), non-ionic block copolymers
that form micelles such as CRL 1005 (these contain a linear chain
of hydrophobic polyoxypropylene flanked by chains of
polyoxyethylene, Vaxcel, Inc., Norcross, Ga.), and Montanide IMS
(e.g., IMS 1312, water-based nanoparticles combined with a soluble
immunostimulant, Seppic)
[0123] Adjuvants may be TLR ligands. Adjuvants that act through
TLR3 include without limitation double-stranded RNA. Adjuvants that
act through TLR4 include without limitation derivatives of
lipopolysaccharides such as monophosphoryl lipid A (MPLA; Ribi
ImmunoChem Research, Inc., Hamilton, Mont.) and muramyl dipeptide
(MDP; Ribi) andthreonyl-muramyl dipeptide (t-MDP; Ribi); OM-174 (a
glucosamine disaccharide related to lipid A; OM Pharma SA, Meyrin,
Switzerland). Adjuvants that act through TLR5 include without
limitation flagellin. Adjuvants that act through TLR7 and/or TLR8
include single-stranded RNA, oligoribonucleotides (ORN), synthetic
low molecular weight compounds such as imidazoquinolinamines (e.g.,
imiquimod, resiquimod). Adjuvants acting through TLR9 include DNA
of viral or bacterial origin, or synthetic oligodeoxynucleotides
(ODN), such as CpG ODN. Another adjuvant class is phosphorothioate
containing molecules such as phosphorothioate nucleotide analogs
and nucleic acids containing phosphorothioate backbone
linkages.
[0124] Immunoinhibitory Agents. As used herein, an immunoinhibitory
agent is an agent that inhibits an immune response in a subject to
whom it is administered, whether alone or in combination with
another agent. Examples include steroids, retinoic acid,
dexamethasone, cyclophosphamide, anti-CD3 antibody or antibody
fragment, and other immunosuppressants.
[0125] Anti-Cancer Agents. As used herein, an anti-cancer agent is
an agent that at least partially inhibits the development or
progression of a cancer, including inhibiting in whole or in part
symptoms associated with the cancer even if only for the short
term. Several anti-cancer agents can be categorized as DNA damaging
agents and these include topoisomerase inhibitors (e.g., etoposide,
ramptothecin, topotecan, teniposide, mitoxantrone), DNA alkylating
agents (e.g., cisplatin, mechlorethamine, cyclophosphamide,
ifosfamide, melphalan, chorambucil, busulfan, thiotepa, carmustine,
lomustine, carboplatin, dacarbazine, procarbazine), DNA strand
break inducing agents (e.g., bleomycin, doxorubicin, daunorubicin,
idarubicin, mitomycin C), anti-microtubule agents (e.g.,
vincristine, vinblastine), anti-metabolic agents (e.g., cytarabine,
methotrexate, hydroxyurea, 5-fluorouracil, floxuridine,
6-thioguanine, 6-mercaptopurine, fludarabine, pentostatin,
chlorodeoxyadenosine), anthracyclines, vinca alkaloids. or
epipodophyllotoxins.
[0126] Examples of anti-cancer agents include without limitation
Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine;
Adozelesin; Aldesleukin; Altretamine; Ambomycin; Ametantrone
Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin;
Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin;
Batimastat; Benzodepa; Bicalutamide; Bisantrene Hydrochloride;
Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate; Bortezomib
(VELCADE); Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin;
Calusterone; Caracemide; Carbetimer; Carboplatin (a
platinum-containing regimen); Carmustine; Carubicin Hydrochloride;
Carzelesin; Cedefingol; Chlorambucil; Cirolemycin; Cisplatin (a
platinum-containing regimen); Cladribine; Crisnatol Mesylate;
Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin;
Daunorubicin; Decitabine; Dexormaplatin; Dezaguanine; Diaziquone;
Docetaxel (TAXOTERE); Doxorubicin; Droloxifene; Dromostanolone;
Duazomycin; Edatrexate; Eflornithine; Elsamitrucin; Enloplatin;
Enpromate; Epipropidine; Epirubicin; Erbulozole; Erlotinib
(TARCEVA), Esorubicin; Estramustine; Etanidazole; Etoposide;
Etoprine; Fadrozole; Fazarabine; Fenretinide; Floxuridine;
Fludarabine; 5-Fluorouracil; Flurocitabine; Fosquidone; Fostriecin;
Gefitinib (IRESSA), Gemcitabine; Hydroxyurea; Idarubicin;
Ifosfamide; Ilmofosine; Imatinib mesylate (GLEEVAC); Interferon
alpha-2a; Interferon alpha-2b; Interferon alpha-n1; Interferon
alpha-n3; Interferon beta-I a; Interferon gamma-I b; Iproplatin;
Irinotecan; Lanreotide; Lenalidomide (REVLIMID, REVIMID);
Letrozole; Leuprolide; Liarozole; Lometrexol; Lomustine;
Losoxantrone; Masoprocol; Maytansine; Mechlorethamine; Megestrol;
Melengestrol; Melphalan; Menogaril; Mercaptopurine; Methotrexate;
Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin;
Mitogillin; Mitomalcin; Mitomycin; Mitosper; Mitotane;
Mitoxantrone; Mycophenolic Acid; Nocodazole; Nogalamycin;
Ormaplatin; Oxisuran; Paclitaxel; Pemetrexed (ALIMTA),
Pegaspargase; Peliomycin; Pentamustine; Pentomone; Peplomycin;
Perfosfamide; Pipobroman; Piposulfan; Piritrexim Isethionate;
Piroxantrone; Plicamycin; Plomestane; Porfimer; Porfiromycin;
Prednimustine; Procarbazine; Puromycin; Pyrazofurin; Riboprine;
Rogletimide; Safingol; Semustine; Simtrazene; Sitogluside;
Sparfosate; Sparsomycin; Spirogermanium; Spiromustine; Spiroplatin;
Streptonigrin; Streptozocin; Sulofenur; Talisomycin; Tamsulosin;
Taxol; Taxotere; Tecogalan; Tegafur; Teloxantrone; Temoporfin;
Temozolomide (TEMODAR); Teniposide; Teroxirone; Testolactone;
Thalidomide (THALOMID) and derivatives thereof; Thiamiprine;
Thioguanine; Thiotepa; Tiazofurin; Tirapazamine; Topotecan;
Toremifene; Trestolone; Triciribine; Trimetrexate; Triptorelin;
Tubulozole; Uracil Mustard; Uredepa; Vapreotide; Verteporfin;
Vinblastine; Vincristine; Vindesine; Vinepidine; Vinglycinate;
Vinleurosine; Vinorelbine; Vinrosidine; Vinzolidine; Vorozole;
Zeniplatin; Zinostatin; Zorubicin.
[0127] The anti-cancer agent may be an enzyme inhibitor including
without limitation tyrosine kinase inhibitor, a CDK inhibitor, a
MAP kinase inhibitor, or an EGFR inhibitor. The tyrosine kinase
inhibitor may be without limitation Genistein
(4',5,7-trihydroxyisoflavone), Tyrphostin 25
(3,4,5-trihydroxyphenyl), methylene]-propanedinitrile, Herbimycin
A, Daidzein (4',7-dihydroxyisoflavone), AG-126,
trans-1-(3'-carboxy-4'-hydroxyphenyl)-2-(2'',5''-dihydroxy-phenyl)ethane,
or HDB A (2-Hydroxy5-(2,5-Dihydroxybenzylamino)-2-hydroxybenzoic
acid. The CDK inhibitor may be without limitation p21, p27, p57,
p15, p16, p18, or p19. The MAP kinase inhibitor may be without
limitation KY12420 (C.sub.23H.sub.24O.sub.8), CNI-1493, PD98059, or
4-(4-Fluorophenyl)-2-(4-methylsulfinyl phenyl)-5-(4-pyridyl)
1H-imidazole. The EGFR inhibitor may be without limitation
erlotinib (TARCEVA), gefitinib (IRESSA), WHI-P97 (quinazoline
derivative), LFM-Al2 (leflunomide metabolite analog), ABX-EGF,
lapatinib, canertinib, ZD-6474 (ZACTIMA), AEE788, and AG1458.
[0128] The anti-cancer agent may be a VEGF inhibitor including
without limitation bevacizumab (AVASTIN), ranibizumab (LUCENTIS),
pegaptanib (MACUGEN), sorafenib, sunitinib (SUTENT), vatalanib,
ZD-6474 (ZACTIMA), anecortave (RETAANE), squalamine lactate, and
semaphorin.
[0129] The anti-cancer agent may be an antibody or an antibody
fragment including without limitation an antibody or an antibody
fragment including but not limited to bevacizumab (AVASTIN),
trastuzumab (HERCEPTIN), alemtuzumab (CAMPATH, indicated for B cell
chronic lymphocytic leukemia,), gemtuzumab (MYLOTARG, hP67.6,
anti-CD33, indicated for leukemia such as acute myeloid leukemia),
rituximab (RITUXAN), tositumomab (BEXXAR, anti-CD20, indicated for
B cell malignancy), MDX-210 (bispecific antibody that binds
simultaneously to HER-2/neu oncogene protein product and type I Fc
receptors for immunoglobulin G (IgG) (Fc gamma RI)), oregovomab
(OVAREX, indicated for ovarian cancer), edrecolomab (PANOREX),
daclizumab (ZENAPAX), palivizumab (SYNAGIS, indicated for
respiratory conditions such as RSV infection), ibritumomab tiuxetan
(ZEVALIN, indicated for Non-Hodgkin's lymphoma), cetuximab
(ERBITUX), MDX-447, MDX-22, MDX-220 (anti-TAG-72), IOR-C5, IOR-T6
(anti-CD1), IOR EGF/R3, celogovab (ONCOSCINT OV103), epratuzumab
(LYMPHOCIDE), pemtumomab (THERAGYN), and Gliomab-H (indicated for
brain cancer, melanoma).
[0130] Hematopoietic Differentiating Agents. The agent may be one
that stimulates the differentiation of hematopoietic progenitor
cells towards one or more lineages. Examples include without
limitation IL-3, G-CSF, GM-CSF, M-CSF, thrombopoeitin,
erythropoietin, Wnt5A, Wnt11A, and the like.
[0131] Hematopoietic Self-Renewing Agents. The agent may be one
that stimulates the self-renewal of hematopoietic progenitor cells.
Examples include without limitation kit ligand, GSK3-beta
inhibitors, Wnt5A together with SLF, Notch1 activators, Lnk
inhibitors, prostaglandin E2 (PGE2) and agents that stimulate the
PGE2 pathway including PGE2, PGI2, Linoleic Acid, 13(s)-HODE,
LY171883, Mead Acid, Eicosatrienoic Acid, Epoxyeicosatrienoic Acid,
ONO-259, Cay1039, a PGE2 receptor agonist, of 16,16-dimethyl PGE2,
19(R)-hydroxy PGE2, 16,16-dimethyl PGE2 p-(p-acetamidobenzamido)
phenyl ester, 11-deoxy-16,16-dimethyl
PGE2,9-deoxy-9-methylene-16,16-dimethyl PGE2, 9-deoxy-9-methylene
PGE2, Butaprost, Sulprostone, PGE2 serinol amide, PGE2 methyl
ester, 16-phenyl tetranor PGE2,15(S)-15-methyl PGE2,15(R)-15-methyl
PGE2, BIO, 8-bromo-cAMP, Forskolin, Bapta-AM, Fendiline,
Nicardipine, Nifedipine, Pimozide, Strophanthidin, Lanatoside,
L-Arg, Sodium Nitroprusside, Sodium Vanadate, Bradykinin,
Mebeverine, Flurandrenolide, Atenolol, Pindolol, Gaboxadol,
Kynurenic Acid, Hydralazine, Thiabendazole, Bicuclline, Vesamicol,
Peruvoside, Imipramine, Chlorpropamide,
1,5-Pentamethylenetetrazole, 4-Aminopyridine, Diazoxide,
Benfotiamine, 12-Methoxydodecenoic acid, N-Formyl-Met-Leu-Phe,
Gallamine, IAA 94, Chlorotrianisene, and derivatives thereof, and
the like.
[0132] Anti-Infective Agents. The agent may be an anti-infective
agent including without limitation an anti-bacterial agent, an
anti-viral agent, an anti-parasitic agent, an anti-fungal agent,
and an anti-mycobacterial agent.
[0133] Anti-bacterial agents may be without limitation
.beta.-lactam antibiotics, penicillins (such as natural
penicillins, aminopenicillins, penicillinase-resistant penicillins,
carboxy penicillins, ureido penicillins), cephalosporins (first
generation, second generation, and third generation
cephalosporins), other .beta.-lactams (such as imipenem,
monobactams), .beta.-lactamase inhibitors, vancomycin,
aminoglycosides and spectinomycin, tetracyclines, chloramphenicol,
erythromycin, lincomycin, clindamycin, rifampin, metronidazole,
polymyxins, sulfonamides and trimethoprim, or quinolines.
[0134] Other anti-bacterials may be without limitation Acedapsone;
Acetosulfone Sodium; Alamecin; Alexidine; Amdinocillin;
Amdinocillin Pivoxil; Amicycline; Amifloxacin; Amifloxacin
Mesylate; Amikacin; Amikacin Sulfate; Aminosalicylic acid;
Aminosalicylate sodium; Amoxicillin; Amphomycin; Ampicillin;
Ampicillin Sodium; Apalcillin Sodium; Apramycin; Aspartocin;
Astromicin Sulfate; Avilamycin; Avoparcin; Azithromycin;
Azlocillin; Azlocillin Sodium; Bacampicillin Hydrochloride;
Bacitracin; Bacitracin Methylene Disalicylate; Bacitracin Zinc;
Bambermycins; Benzoylpas Calcium; Berythromycin; Betamicin Sulfate;
Biapenem; Biniramycin; Biphenamine Hydrochloride; Bispyrithione
Magsulfex; Butikacin; Butirosin Sulfate; Capreomycin Sulfate;
Carbadox; Carbenicillin Disodium; Carbenicillin Indanyl Sodium;
Carbenicillin Phenyl Sodium; Carbenicillin Potassium; Carumonam
Sodium; Cefaclor; Cefadroxil; Cefamandole; Cefamandole Nafate;
Cefamandole Sodium; Cefaparole; Cefatrizine; Cefazaflur Sodium;
Cefazolin; Cefazolin Sodium; Cefbuperazone; Cefdinir; Cefepime;
Cefepime Hydrochloride; Cefetecol; Cefixime; Cefmenoxime
Hydrochloride; Cefmetazole; Cefmetazole Sodium; Cefonicid
Monosodium; Cefonicid Sodium; Cefoperazone Sodium; Ceforanide;
Cefotaxime Sodium; Cefotetan; Cefotetan Disodium; Cefotiam
Hydrochloride; Cefoxitin; Cefoxitin Sodium; Cefpimizole;
Cefpimizole Sodium; Cefpiramide; Cefpiramide Sodium; Cefpirome
Sulfate; Cefpodoxime Proxetil; Cefprozil; Cefroxadine; Cefsulodin
Sodium; Ceftazidime; Ceftibuten; Ceftizoxime Sodium; Ceftriaxone
Sodium; Cefuroxime; Cefuroxime Axetil; Cefuroxime Pivoxetil;
Cefuroxime Sodium; Cephacetrile Sodium; Cephalexin; Cephalexin
Hydrochloride; Cephaloglycin; Cephaloridine; Cephalothin Sodium;
Cephapirin Sodium; Cephradine; Cetocycline Hydrochloride;
Cetophenicol; Chloramphenicol; Chloramphenicol Palmitate;
Chloramphenicol Pantothenate Complex; Chloramphenicol Sodium
Succinate; Chlorhexidine Phosphanilate; Chloroxylenol;
Chlortetracycline Bisulfate; Chlortetracycline Hydrochloride;
Cinoxacin; Ciprofloxacin; Ciprofloxacin Hydrochloride; Cirolemycin;
Clarithromycin; Clinafloxacin Hydrochloride; Clindamycin;
Clindamycin Hydrochloride; Clindamycin Palmitate Hydrochloride;
Clindamycin Phosphate; Clofazimine; Cloxacillin Benzathine;
Cloxacillin Sodium; Cloxyquin; Colistimethate Sodium; Colistin
Sulfate; Coumermycin; Coumermycin Sodium; Cyclacillin; Cycloserine;
Dalfopristin; Dapsone; Daptomycin; Demeclocycline; Demeclocycline
Hydrochloride; Demecycline; Denofungin; Diaveridine; Dicloxacillin;
Dicloxacillin Sodium; Dihydrostreptomycin Sulfate; Dipyrithione;
Dirithromycin; Doxycycline; Doxycycline Calcium; Doxycycline
Fosfatex; Doxycycline Hyclate; Droxacin Sodium; Enoxacin;
Epicillin; Epitetracycline Hydrochloride; Erythromycin;
Erythromycin Acistrate; Erythromycin Estolate; Erythromycin
Ethylsuccinate; Erythromycin Gluceptate; Erythromycin Lactobionate;
Erythromycin Propionate; Erythromycin Stearate; Ethambutol
Hydrochloride; Ethionamide; Fleroxacin; Floxacillin; Fludalanine;
Flumequine; Fosfomycin; Fosfomycin Tromethamine; Fumoxicillin;
Furazolium Chloride; Furazolium Tartrate; Fusidate Sodium; Fusidic
Acid; Gentamicin Sulfate; Gloximonam; Gramicidin; Haloprogin;
Hetacillin; Hetacillin Potassium; Hexedine; Ibafloxacin; Imipenem;
Isoconazole; Isepamicin; Isoniazid; Josamycin; Kanamycin Sulfate;
Kitasamycin; Levofuraltadone; Levopropylcillin Potassium;
Lexithromycin; Lincomycin; Lincomycin Hydrochloride; Lomefloxacin;
Lomefloxacin Hydrochloride; Lomefloxacin Mesylate; Loracarbef;
Mafenide; Meclocycline; Meclocycline Sulfosalicylate; Megalomicin
Potassium Phosphate; Mequidox; Meropenem; Methacycline;
Methacycline Hydrochloride; Methenamine; Methenamine Hippurate;
Methenamine Mandelate; Methicillin Sodium; Metioprim; Metronidazole
Hydrochloride; Metronidazole Phosphate; Mezlocillin; Mezlocillin
Sodium; Minocycline; Minocycline Hydrochloride; Mirincamycin
Hydrochloride; Monensin; Monensin Sodium; Nafcillin Sodium;
Nalidixate Sodium; Nalidixic Acid; Natamycin; Nebramycin; Neomycin
Palmitate; Neomycin Sulfate; Neomycin Undecylenate; Netilmicin
Sulfate; Neutramycin; Nifuradene; Nifuraldezone; Nifuratel;
Nifuratrone; Nifurdazil; Nifurimide; Nifurpirinol; Nifurquinazol;
Nifurthiazole; Nitrocycline; Nitrofurantoin; Nitromide;
Norfloxacin; Novobiocin Sodium; Ofloxacin; Ormetoprim; Oxacillin
Sodium; Oximonam; Oximonam Sodium; Oxolinic Acid; Oxytetracycline;
Oxytetracycline Calcium; Oxytetracycline Hydrochloride; Paldimycin;
Parachlorophenol; Paulomycin; Pefloxacin; Pefloxacin Mesylate;
Penamecillin; Penicillin G Benzathine; Penicillin G Potassium;
Penicillin G Procaine; Penicillin G Sodium; Penicillin V;
Penicillin V Benzathine; Penicillin V Hydrabamine; Penicillin V
Potassium; Pentizidone Sodium; Phenyl Aminosalicylate; Piperacillin
Sodium; Pirbenicillin Sodium; Piridicillin Sodium; Pirlimycin
Hydrochloride; Pivampicillin Hydrochloride; Pivampicillin Pamoate;
Pivampicillin Probenate; Polymyxin B Sulfate; Porfiromycin;
Propikacin; Pyrazinamide; Pyrithione Zinc; Quindecamine Acetate;
Quinupristin; Racephenicol; Ramoplanin; Ranimycin; Relomycin;
Repromicin; Rifabutin; Rifametane; Rifamexil; Rifamide; Rifampin;
Rifapentine; Rifaximin; Rolitetracycline; Rolitetracycline Nitrate;
Rosaramicin; Rosaramicin Butyrate; Rosaramicin Propionate;
Rosaramicin Sodium Phosphate; Rosaramicin Stearate; Rosoxacin;
Roxarsone; Roxithromycin; Sancycline; Sanfetrinem Sodium;
Sarmoxicillin; Sarpicillin; Scopafungin; Sisomicin; Sisomicin
Sulfate; Sparfloxacin; Spectinomycin Hydrochloride; Spiramycin;
Stallimycin Hydrochloride; Steffimycin; Streptomycin Sulfate;
Streptonicozid; Sulfabenz; Sulfabenzamide; Sulfacetamide;
Sulfacetamide Sodium; Sulfacytine; Sulfadiazine; Sulfadiazine
Sodium; Sulfadoxine; Sulfalene; Sulfamerazine; Sulfameter;
Sulfamethazine; Sulfamethizole; Sulfamethoxazole;
Sulfamonomethoxine; Sulfamoxole; Sulfanilate Zinc; Sulfanitran;
Sulfasalazine; Sulfasomizole; Sulfathiazole; Sulfazamet;
Sulfisoxazole; Sulfisoxazole Acetyl; Sulfisoxazole Diolamine;
Sulfomyxin; Sulopenem; Sultamicillin; Suncillin Sodium;
Talampicillin Hydrochloride; Teicoplanin; Temafloxacin
Hydrochloride; Temocillin; Tetracycline; Tetracycline
Hydrochloride; Tetracycline Phosphate Complex; Tetroxoprim;
Thiamphenicol; Thiphencillin Potassium; Ticarcillin Cresyl Sodium;
Ticarcillin Disodium; Ticarcillin Monosodium; Ticlatone; Tiodonium
Chloride; Tobramycin; Tobramycin Sulfate; Tosufloxacin;
Trimethoprim; Trimethoprim Sulfate; Trisulfapyrimidines;
Troleandomycin; Trospectomycin Sulfate; Tyrothricin; Vancomycin;
Vancomycin Hydrochloride; Virginiamycin; or Zorbamycin.
[0135] Anti-mycobacterial agents may be without limitation
Myambutol (Ethambutol Hydrochloride), Dapsone
(4,4'-diaminodiphenylsulfone), Paser Granules (aminosalicylic acid
granules), Priftin (rifapentine), Pyrazinamide, Isoniazid, Rifadin
(Rifampin), Rifadin IV, Rifamate (Rifampin and Isoniazid), Rifater
(Rifampin, Isoniazid, and Pyrazinamide), Streptomycin Sulfate or
Trecator-SC (Ethionamide).
[0136] Anti-viral agents may be without limitation amantidine and
rimantadine, ribivarin, acyclovir, vidarabine, trifluorothymidine,
ganciclovir, zidovudine, retinovir, and interferons.
[0137] Anti-viral agents may be without limitation further include
Acemannan; Acyclovir; Acyclovir Sodium; Adefovir; Alovudine;
Alvircept Sudotox; Amantadine Hydrochloride; Aranotin; Arildone;
Atevirdine Mesylate; Avridine; Cidofovir; Cipamfylline; Cytarabine
Hydrochloride; Delavirdine Mesylate; Desciclovir; Didanosine;
Disoxaril; Edoxudine; Enviradene; Enviroxime; Famciclovir; Famotine
Hydrochloride; Fiacitabine; Fialuridine; Fosarilate; Foscarnet
Sodium; Fosfonet Sodium; Ganciclovir; Ganciclovir Sodium;
Idoxuridine; Kethoxal; Lamivudine; Lobucavir; Memotine
Hydrochloride; Methisazone; Nevirapine; Penciclovir; Pirodavir;
Ribavirin; Rimantadine Hydrochloride; Saquinavir Mesylate;
Somantadine Hydrochloride; Sorivudine; Statolon; Stavudine;
Tilorone Hydrochloride; Trifluridine; Valacyclovir Hydrochloride;
Vidarabine; Vidarabine Phosphate; Vidarabine Sodium Phosphate;
Viroxime; Zalcitabine; Zidovudine; Zinviroxime or integrase
inhibitors.
[0138] Anti-fungal agents may be without limitation imidazoles and
triazoles, polyene macrolide antibiotics, griseofulvin,
amphotericin B, and flucytosine. Antiparasites include heavy
metals, antimalarial quinolines, folate antagonists,
nitroimidazoles, benzimidazoles, avermectins, praxiquantel,
ornithine decarboxylase inhbitors, phenols (e.g., bithionol,
niclosamide); synthetic alkaloid (e.g., dehydroemetine);
piperazines (e.g., diethylcarbamazine); acetanilide (e.g.,
diloxanide furonate); halogenated quinolines (e.g., iodoquinol
(diiodohydroxyquin)); nitrofurans (e.g., nifurtimox); diamidines
(e.g., pentamidine); tetrahydropyrimidine (e.g., pyrantel pamoate);
or sulfated naphthylamine (e.g., suramin).
[0139] Other anti-infective agents may be without limitation
Difloxacin Hydrochloride; Lauryl Isoquinolinium Bromide; Moxalactam
Disodium; Ornidazole; Pentisomicin; Sarafloxacin Hydrochloride;
Protease inhibitors of HIV and other retroviruses; Integrase
Inhibitors of HIV and other retroviruses; Cefaclor (Ceclor);
Acyclovir (Zovirax); Norfloxacin (Noroxin); Cefoxitin (Mefoxin);
Cefuroxime axetil (Ceftin); Ciprofloxacin (Cipro); Aminacrine
Hydrochloride; Benzethonium Chloride: Bithionolate Sodium;
Bromchlorenone; Carbamide Peroxide; Cetalkonium Chloride;
Cetylpyridinium Chloride: Chlorhexidine Hydrochloride; Clioquinol;
Domiphen Bromide; Fenticlor; Fludazonium Chloride; Fuchsin, Basic;
Furazolidone; Gentian Violet; Halquinols; Hexachlorophene: Hydrogen
Peroxide; Ichthammol; Imidecyl Iodine; Iodine; Isopropyl Alcohol;
Mafenide Acetate; Meralein Sodium; Mercufenol Chloride; Mercury,
Ammoniated; Methylbenzethonium Chloride; Nitrofurazone;
Nitromersol; Octenidine Hydrochloride; Oxychlorosene; Oxychlorosene
Sodium; Parachlorophenol, Camphorated; Potassium Permanganate;
Povidone-Iodine; Sepazonium Chloride; Silver Nitrate; Sulfadiazine,
Silver; Symclosene; Thimerfonate Sodium; Thimerosal; or Troclosene
Potassium.
[0140] Nucleic Acid Agents. Nucleic acids that can be delivered to
a subject according to the invention include naturally or
non-naturally occurring DNA (including cDNA, genomic DNA, nuclear
DNA, mitochondrial DNA), RNA (including mRNA, rRNA, tRNA),
oligonucleotides, a triple-helix forming molecule,
immunostimulatory nucleic acids such as those described in U.S.
Pat. No. 6,194,388 (the teachings of which relating to
immunostimulatory CpG nucleic acids are incorporated herein by
reference), small interfering RNA (siRNA) used to modulate gene
expression, antisense oligonucleotides used to modulate gene
expression, aptamers, ribozymes, a gene or gene fragment, a
regulatory sequence, including analogs, derivatives, and
combinations thereof. These nucleic acids may be administered neat
or complexed to another entity, for example in order to facilitate
their binding to and/or uptake by target tissues and/or cells.
[0141] Other Agents. The agent may be without limitation adrenergic
agent; adrenocortical steroid; adrenocortical suppressant; alcohol
deterrent; aldosterone antagonist; ammonia detoxicant; amino acid;
amylotropic lateral sclerosis agent; anabolic; analeptic;
analgesic; androgen; anesthetic; anorectic; anorexic; anterior
pituitary activator; anterior pituitary suppressant; anthelmintic;
anti-acne agent; anti-adrenergic; anti-allergic; anti-amebic;
anti-androgen; anti-anemic; anti-anginal; anti-anxiety;
anti-arthritic; anti-asthmatic including .beta.-adrenergic
agonists, methylxanthines, mast cell stabilizing agents,
anticholinergics, adrenocortical steroids such as glucocorticoids;
anti-atherosclerotic; anticholelithic; anticholelithogenic;
anticholinergic; anticoagulant; anticoccidal; anticonvulsant;
antidepressant; antidiabetic; antidiarrheal; antidiuretic;
antidote; antidyskinetic; anti-emetic; anti-epileptic;
anti-estrogen; antifibrinolytic; antiglaucoma; antihemorrhagic;
antihemorrheologic; antihistamine; antihyperlipidemic;
antihyperlipoproteinemic; antihypertensive; antihypotensive;
anti-infective; anti-inflammatory; antikeratinizing agent;
antimigraine; antimitotic; antimycotic; antinauseant;
antineutropenic; antiobsessional agent; antioxidant;
antiparkinsonian; antiperistaltic; antipneumocystic; antiprostatic
hypertrophy agent; antiprotozoal; antipruritic; antipsoriatic;
antipsychotic; antirheumatic; antischistosomal; antiseborrheic;
antisecretory; antispasmodic; antithrombotic; antitussive;
anti-ulcerative; anti-urolithic; appetite suppressant; blood
glucose regulator; bone resorption inhibitor; bronchodilator;
carbonic anhydrase inhibitor; cardiac depressant; cardioprotectant;
cardiotonic; cardiovascular agent; cerebral ischemia agent;
choleretic; cholinergic; cholinergic agonist; cholinesterase
deactivator; coccidiostat; cognition adjuvant; cognition enhancer;
conjunctivitis agent; contrast agent; depressant; diagnostic aid;
diuretic; dopaminergic agent; ectoparasiticide; emetic; enzyme
inhibitor; estrogen; estrogen receptor agonist; fibrinolytic;
fluorescent agent; free oxygen radical scavenger; gastric acid
suppressant; gastrointestinal motility effector; geriatric agent;
glucocorticoid; gonad-stimulating principle; hair growth stimulant;
hemostatic; herbal active agent; histamine H2 receptor antagonists;
hormone; hypocholesterolemic; hypoglycemic; hypolipidemic;
hypotensive; HMGCoA reductase inhibitor; impotence therapy adjunct;
inflammatory bowel disease agent; keratolytic; LHRH agonist; liver
disorder agent; luteolysin; memory adjuvant; mental performance
enhancer; mineral; mood regulator; mucolytic; mucosal protective
agent; multiple sclerosis agent; mydriatic; nasal decongestant;
neuroleptic; neuromuscular blocking agent; neuroprotective; NMDA
antagonist; non-hormonal sterol derivative; nutrient; oxytocic;
Paget's disease agent; plasminogen activator; platelet activating
factor antagonist; platelet aggregation inhibitor; post-stroke and
post-head trauma agents; progestin; prostaglandin; prostate growth
inhibitor; prothyrotropin; psychotropic; radioactive agent;
relaxant; rhinitis agent; scabicide; sclerosing agent; sedative;
sedative-hypnotic; selective adenosine Al antagonist; sequestering
agents; serotonin antagonist; serotonin inhibitor; serotonin
receptor antagonist; steroid; stimulant; suppressant; thyroid
hormone; thyroid inhibitor; thyromimetic; tranquilizer; unstable
angina agent; uricosuric; vasoconstrictor; vasodilator; vulnerary;
wound healing agent; or xanthine oxidase inhibitor.
Subjects
[0142] The invention can be practiced in virtually any subject type
that is likely to benefit from localized delivery of agents as
contemplated herein. Human subjects are preferred subjects in some
embodiments of the invention. Subjects also include animals such as
household pets (e.g., dogs, cats, rabbits, ferrets, etc.),
livestock or farm animals (e.g., cows, pigs, sheep, chickens and
other poultry), horses such as thoroughbred horses, laboratory
animals (e.g., mice, rats, rabbits, etc.), and the like. Subjects
also include fish and other aquatic species.
[0143] The subjects to whom the agents are delivered may be normal
subjects. Alternatively they may have or may be at risk of
developing a condition that can be diagnosed or that can benefit
from localized delivery of one or more particular agents.
[0144] Such conditions include cancer (e.g., solid tumor cancers),
infections (particularly infections localized to particular regions
or tissues in the body), autoimmune disorders, allergies or
allergic conditions, asthma, transplant rejection, and the
like.
[0145] Tests for diagnosing various of the conditions embraced by
the invention are known in the art and will be familiar to the
ordinary medical practitioner. These laboratory tests include
without limitation microscopic analyses, cultivation dependent
tests (such as cultures), and nucleic acid detection tests. These
include wet mounts, stain-enhanced microscopy, immune microscopy
(e.g., FISH), hybridization microscopy, particle agglutination,
enzyme-linked immunosorbent assays, urine screening tests, DNA
probe hybridization, serologic tests, etc. The medical practitioner
will generally also take a full history and conduct a complete
physical examination in addition to running the laboratory tests
listed above.
[0146] A subject having a cancer is a subject that has detectable
cancer cells. A subject at risk of developing a cancer is a subject
that has a higher than normal probability of developing cancer.
These subjects include, for instance, subjects having a genetic
abnormality that has been demonstrated to be associated with a
higher likelihood of developing a cancer, subjects having a
familial disposition to cancer, subjects exposed to cancer causing
agents (i.e., carcinogens) such as tobacco, asbestos, or other
chemical toxins, and subjects previously treated for cancer and in
apparent remission.
[0147] Subjects having an infection are those that exhibit symptoms
thereof including without limitation fever, chills, myalgia,
photophobia, pharyngitis, acute lymphadenopathy, splenomegaly,
gastrointestinal upset, leukocytosis or leukopenia, and/or those in
whom infectious pathogens or byproducts thereof can be
detected.
[0148] A subject at risk of developing an infection is one that is
at risk of exposure to an infectious pathogen. Such subjects
include those that live in an area where such pathogens are known
to exist and where such infections are common. These subjects also
include those that engage in high risk activities such as sharing
of needles, engaging in unprotected sexual activity, routine
contact with infected samples of subjects (e.g., medical
practitioners), people who have undergone surgery, including but
not limited to abdominal surgery, etc.
[0149] The subject may have or may be at risk of developing an
infection such as a bacterial infection, a viral infection, a
fungal infection, a parasitic infection or a mycobacterial
infection. In these embodiments, the nanoparticles may comprise an
anti-microbial agent such as an anti-bacterial agent, an anti-viral
agent, an anti-fungal agent, an anti-parasitic agent, or an
anti-mycobacterial agent and the cell carriers (e.g., the T cells)
may be genetically engineered to produce another agent useful in
stimulating an immune response against the infection, or
potentially treating the infection.
[0150] In some instances, the subjects to whom the carrier
cell-nanoparticle conjugates are administered are in need of
hematopoietic reconstitution. Such subjects may have been exposed
to a deliberate or accidental myeloablative event, including
without limitation myeloablative chemotherapy and/or whole body
radiation, as may be given as part of a therapeutic regimen for
non-solid cancers or metastatic cancers. The invention contemplates
administering to such subjects hematopoietic progenitor cells
conjugated to nanoparticles that comprise agents capable of
stimulating the proliferation of the progenitor cells. In some
instances, the agents may also be differentiating agents (i.e.,
agents that drive the progenitor cells and their progeny to
differentiate, optionally towards all lineages or a subset of
lineages. In other instances, the agents may be self-renewal agents
(i.e., agents that drive the progenitor cells to self-renew). In
yet other instances, the carrier cells may be conjugated to
nanoparticles that comprise both types of agents, whether such
agents be in the same nanoparticle or in different nanoparticles.
Moreover, the invention further contemplates that exposure of the
subject to these different agents may be staggered (e.g., exposure
to the self-renewing agents may occur before exposure to the
differentiating agents).
Cancer
[0151] The invention contemplates administration of the
nanoparticle-cell conjugates to subjects having or at risk of
developing a cancer including for example a solid tumor cancer. The
cancer may be carcinoma, sarcoma or melanoma. Carcinomas include
without limitation to basal cell carcinoma, biliary tract cancer,
bladder cancer, breast cancer, cervical cancer, choriocarcinoma,
CNS cancer, colon and rectum cancer, kidney or renal cell cancer,
larynx cancer, liver cancer, small cell lung cancer, non-small cell
lung cancer (NSCLC, including adenocarcinoma, giant (or oat) cell
carcinoma, and squamous cell carcinoma), oral cavity cancer,
ovarian cancer, pancreatic cancer, prostate cancer, skin cancer
(including basal cell cancer and squamous cell cancer), stomach
cancer, testicular cancer, thyroid cancer, uterine cancer, rectal
cancer, cancer of the respiratory system, and cancer of the urinary
system.
[0152] Sarcomas are rare mesenchymal neoplasms that arise in bone
(osteosarcomas) and soft tissues (fibrosarcomas). Sarcomas include
without limitation liposarcomas (including myxoid liposarcomas and
pleiomorphic liposarcomas), leiomyosarcomas, rhabdomyosarcomas,
malignant peripheral nerve sheath tumors (also called malignant
schwannomas, neurofibrosarcomas, or neurogenic sarcomas), Ewing's
tumors (including Ewing's sarcoma of bone, extraskeletal (i.e., not
bone) Ewing's sarcoma, and primitive neuroectodermal tumor),
synovial sarcoma, angiosarcomas, hemangiosarcomas,
lymphangiosarcomas, Kaposi's sarcoma, hemangioendothelioma, desmoid
tumor (also called aggressive fibromatosis), dermatofibrosarcoma
protuberans (DFSP), malignant fibrous histiocytoma (MFH),
hemangiopericytoma, malignant mesenchymoma, alveolar soft-part
sarcoma, epithelioid sarcoma, clear cell sarcoma, desmoplastic
small cell tumor, gastrointestinal stromal tumor (GIST) (also known
as GI stromal sarcoma), and chondrosarcoma.
[0153] Melanomas are tumors arising from the melanocytic system of
the skin and other organs. Examples of melanoma include without
limitation lentigo maligna melanoma, superficial spreading
melanoma, nodular melanoma, and acral lentiginous melanoma.
[0154] The cancer may be a solid tumor lymphoma. Examples include
Hodgkin's lymphoma, Non-Hodgkin's lymphoma, and B cell
lymphoma.
[0155] The cancer may be without limitation bone cancer, brain
cancer, breast cancer, colorectal cancer, connective tissue cancer,
cancer of the digestive system, endometrial cancer, esophageal
cancer, eye cancer, cancer of the head and neck, gastric cancer,
intra-epithelial neoplasm, melanoma neuroblastoma, Non-Hodgkin's
lymphoma, non-small cell lung cancer, prostate cancer,
retinoblastoma, or rhabdomyosarcoma.
Infection
[0156] The invention contemplates administration of the
nanoparticle-cell conjugates to subjects having or at risk of
developing an infection such as a bacterial infection, a viral
infection, a fungal infection, a parasitic infection or a
mycobacterial infection.
[0157] The bacterial infection may be without limitation an E. coli
infection, a Staphylococcal infection, a Streptococcal infection, a
Pseudomonas infection, Clostridium difficile infection, Legionella
infection, Pneumococcus infection, Haemophilus infection,
Klebsiella infection, Enterobacter infection, Citrobacter
infection, Neisseria infection, Shigella infection, Salmonella
infection, Listeria infection, Pasteurella infection,
Streptobacillus infection, Spirillum infection, Treponema
infection, Actinomyces infection, Borrelia infection,
Corynebacterium infection, Nocardia infection, Gardnerella
infection, Campylobacter infection, Spirochaeta infection, Proteus
infection, Bacteriodes infection, H. pylori infection, or anthrax
infection.
[0158] The mycobacterial infection may be without limitation
tuberculosis or leprosy respectively caused by the M. tuberculosis
and M. leprae species.
[0159] The viral infection may be without limitation a Herpes
simplex virus 1 infection, a Herpes simplex virus 2 infection,
cytomegalovirus infection, hepatitis A virus infection, hepatitis B
virus infection, hepatitis C virus infection, human papilloma virus
infection, Epstein Barr virus infection, rotavirus infection,
adenovirus infection, influenza A virus infection, H1N1 (swine flu)
infection, respiratory syncytial virus infection, varicella-zoster
virus infections, small pox infection, monkey pox infection, SARS
infection or avian flu infection.
[0160] The fungal infection may be without limitation candidiasis,
ringworm, histoplasmosis, blastomycosis, paracoccidioidomycosis,
crytococcosis, aspergillosis, chromomycosis, mycetoma infections,
pseudallescheriasis, or tinea versicolor infection.
[0161] The parasite infection may be without limitation amebiasis,
Trypanosoma cruzi infection, Fascioliasis, Leishmaniasis,
Plasmodium infections, Onchocerciasis, Paragonimiasis, Trypanosoma
brucei infection, Pneumocystis infection, Trichomonas vaginalis
infection, Taenia infection, Hymenolepsis infection, Echinococcus
infections, Schistosomiasis, neurocysticercosis, Necator americanus
infection, or Trichuris trichuria infection.
Allergy and Asthma
[0162] The invention contemplates administration of the
nanoparticle-cell conjugates to subjects having or at risk of
developing an allergy or asthma. An allergy is an acquired
hypersensitivity to an allergen. Allergic conditions include but
are not limited to eczema, allergic rhinitis or coryza, hay fever,
bronchial asthma, urticaria (hives) and food allergies, and other
atopic conditions. Allergies are generally caused by IgE antibody
generation against harmless allergens. Asthma is a disorder of the
respiratory system characterized by inflammation, narrowing of the
airways and increased reactivity of the airways to inhaled agents.
Asthma is frequently, although not exclusively, associated with
atopic or allergic symptoms. Administration of Thl cytokines, such
as IL-12 and IFN-gamma, according to the invention can be used to
treat allergy or asthma.
Autoimmune Disease
[0163] The invention contemplates administration of the
nanoparticle-cell conjugates to subjects having or at risk of
developing an autoimmune disease. Autoimmune disease is a class of
diseases in which a subject's own antibodies react with host tissue
or in which immune effector T cells are autoreactive to endogenous
self peptides and cause destruction of tissue. Thus an immune
response is mounted against a subject's own antigens, referred to
as self antigens. Autoimmune diseases are generally considered to
be Th1 biased. As a result, induction of a Th2 immune response or
Th2 like cytokines can be beneficial. Such cytokines include IL-4,
IL-5 and IL-10.
[0164] Autoimmune diseases include but are not limited to
rheumatoid arthritis, Crohn's disease, multiple sclerosis, systemic
lupus erythematosus (SLE), autoimmune encephalomyelitis, myasthenia
gravis (MG), Hashimoto's thyroiditis, Goodpasture's syndrome,
pemphigus (e.g., pemphigus vulgaris), Grave's disease, autoimmune
hemolytic anemia, autoimmune thrombocytopenic purpura, scleroderma
with anti-collagen antibodies, mixed connective tissue disease,
polymyositis, pernicious anemia, idiopathic Addison's disease,
autoimmune-associated infertility, glomerulonephritis (e.g.,
crescentic glomerulonephritis, proliferative glomerulonephritis),
bullous pemphigoid, Sjogren's syndrome, insulin resistance, and
autoimmune diabetes mellitus.
Transplant Therapy
[0165] The methods provided herein may also be used to modulate
immune responses following transplant therapy. Transplant success
is often limited by rejection of the transplanted tissue by the
body's immune system. As a result, transplant recipients are
usually immunosuppressed for extended periods of time in order to
allow the transplanted tissue to survive. The invention
contemplates localized delivery of immunomodulators, and
particularly immunoinhibitory agents, to transplant sites in order
to minimize transplant rejection. Thus, the invention contemplates
administration of the nanoparticle-cell conjugates to subjects that
are going to undergo, are undergoing, or have undergone a
transplant.
[0166] The foregoing lists are not intended to be exhaustive but
rather exemplary. Those of ordinary skill in the art will identify
other examples of each condition type that are amenable to
prevention and treatment using the methods of the invention.
Effective Amounts, Regimens, Formulations
[0167] The agents are administered in effective amounts. An
effective amount is a dosage of the agent sufficient to provide a
medically desirable result. The effective amount will vary with the
particular condition being treated, the age and physical condition
of the subject being treated, the severity of the condition, the
duration of the treatment, the nature of the concurrent or
combination therapy (if any), the specific route of administration
and like factors within the knowledge and expertise of the health
practitioner. It is preferred generally that a maximum dose be
used, that is, the highest safe dose according to sound medical
judgment.
[0168] For example, if the subject has a tumor, an effective amount
may be that amount that reduces the tumor volume or load (as for
example determined by imaging the tumor). Effective amounts may
also be assessed by the presence and/or frequency of cancer cells
in the blood or other body fluid or tissue (e.g., a biopsy). If the
tumor is impacting the normal functioning of a tissue or organ,
then the effective amount may be assessed by measuring the normal
functioning of the tissue or organ.
[0169] The invention provides pharmaceutical compositions.
Pharmaceutical compositions are sterile compositions that comprise
cells, nanoparticles and/or agent(s), preferably in a
pharmaceutically-acceptable carrier. The term
"pharmaceutically-acceptable carrier" means one or more compatible
solid or liquid filler, diluents or encapsulating substances which
are suitable for administration to a human or other subject
contemplated by the invention. The term "carrier" denotes an
organic or inorganic ingredient, natural or synthetic, with which
the cells, nanoparticles and agent(s) are combined to facilitate
administration. The components of the pharmaceutical compositions
are commingled in a manner that precludes interaction that would
substantially impair their desired pharmaceutical efficiency.
[0170] The nanoparticle-cell conjugates, when it is desirable to
deliver them systemically, may be formulated for parenteral
administration by injection, e.g., by bolus injection or continuous
infusion. Formulations for injection may be presented in unit
dosage form, e.g., in ampoules or in multi-dose containers.
Pharmaceutical parenteral formulations include aqueous solutions of
the ingredients. Aqueous injection suspensions may contain
substances which increase the viscosity of the suspension, such as
sodium carboxymethyl cellulose, sorbitol, or dextran.
Alternatively, suspensions of ingredients may be prepared as
oil-based suspensions. Suitable lipophilic solvents or vehicles
include fatty oils such as sesame oil, or synthetic fatty acid
esters, such as ethyl oleate or triglycerides, or liposomes.
EXAMPLES
Example 1. Nanoparticle Synthesis, Characterization and Conjugation
to T Cells
[0171] 1.1. Nanoparticle Synthesis. We recently developed a
strategy to prepare `lipid-enveloped` biodegradable polymer
nanoparticles. (Bershteyn A et al., Soft Matter 4: 1787, 2008.)
These particles have a biodegradable poly(lactide-co-glycolide)
core and a surface coating of a phospholipid bilayer (FIGS. 2A and
B, arrows). These nanoparticles can encapsulate drug molecules in
their core and/or incorporate drugs in the surface lipid bilayer,
enabling sustained release of proteins, peptides, or small-molecule
compounds. Nanoparticles were synthesized by a double
emulsion/solvent evaporation process: 200 .mu.L water was
emulsified in 1 mL chloroform containing 2 mg of a lipid mixture
(4:1 mole:mole DOPC:DOPG with varying quantities of dioleoyl
maleimidophenyl phosphoethanolamine (MPB PE), with or without 25
.mu.g 1,1'-dioctacdecyl-3,3,3',3'-tetramethylindodicarbocyanine
(DiD) or DiR lipid-like fluorescent dye (Invitrogen)) and 30 mg
poly(lactide-co-glycolide) (PLGA, 50:50 wt:wt lactide:glycolide, 13
KDa, Lakeshore biopolymers). Inclusion of the maleimide-headgroup
MPB PE lipid in the lipid fraction enables cell conjugation, as
described below. The resulting water-in-oil emulsion was sonicated
on ice (1 min, 7 Watts with a Misonix Microson XL probe tip
sonicator) then added to 6 mL deionized water on ice with
sonication (5 min, 12 Watts), forming a water-in oil-in water
double emulsion. Chloroform was evaporated from the double emulsion
by stirring at 20.degree. C. under atmospheric pressure for 6 hrs
to form solid nanoparticles. During solvent evaporation, the lipids
in the organic phase self-assemble at the oil-water interface and
form a bilayer coating around the nascent PLGA-core particles
(FIGS. 2A and B); excess lipid is also present in the particle
bulk. The particles were purified from free lipid by centrifugation
through a 60 wt % sucrose cushion, dialyzed to remove sucrose, and
stored at 4.degree. C. (short term storage) or lyophilized in the
presence of trehalose and stored at 4.degree. C. until used. Simple
variations in the processing conditions (e.g., use of
homogenization instead of sonication) allowed particles of
different size to be prepared, as determined by dynamic light
scattering (DLS, data not shown).
[0172] To synthesize DNA-gel nanoparticles, we first generated
four-armed DNA junctions, X-DNA monomers, by annealing the
following oligonucleotides (Integrated DNA Technology, IDT):
TABLE-US-00001 (SEQ ID NO: 2) 1)
5'-p-ACGTCGACCGATGAATAGCGGTCAGATCCGTACCTACTCG-3' (SEQ ID NO: 3) 2)
5'-p-ACGTCGAGTAGGTACGGATCTGCGTATTGCGAACGACTCG-3' (SEQ ID NO: 4) 3)
5'-p-ACGTCGAGTCGTTCGCAATACGGCTGTACGTATGGTCTCG-3' (SEQ ID NO: 5) 4)
5'-p-ACGTCGAGACCATACGTACAGCACCGCTATTCATCGGTCG-3'
[0173] These oligos self-assemble into three-dimensional "X"
nanostructures with complementary overhangs at the end or each arm.
As recently described (Um et al., Nat Mater, 5:797, 2006), addition
of ligase to a solution of these DNA macromers leads to covalent
crosslinking and the formation of DNA-base hydrogels. To form
nanoparticles, 1.667 mg X-DNA monomer was then admixed to 6.7 .mu.l
T4 DNA ligase (3 Weiss units/.mu.l, Promega), 20 .mu.l T4 ligase
buffer (Promega) and nuclease-free water (IDT) to a total volume of
200 .mu.l, which was subsequently vortexed with a dry lipid film
containing 0.396 mg DOPC, 0.101 mg DOPG, 0.63 mg MPB and 0.04 mg
DiD. The resulting DNA gel-lipid mixture was sonicated on ice (5
min total, alternating power cycles of 1 W and 5 Watts every 30 s
with a Misonix Microson XL probe tip sonicator), and extruded 21
times through a polycarbonate filter (200 nm pore size, Whatman).
Following a 3 hour incubation at 25.degree. C. and overnight
incubation at 4.degree. C. to allow ligase-mediated X-DNA
crosslinking, 4 .mu.l Exonuclease III (New England Biolabs), 20
.mu.l Buffer 1 (New England Biolabs) and nuclease-free water to a
total volume of 200 .mu.l was were added and incubated at
37.degree. C. for 90 minutes. DNA-gel nanoparticles were purified
from free lipids and DNA by centrifugation through a 10 wt %
sucrose cushion, and washed three times with nuclease-free water. A
typical yield of 10.sup.10 DNA gel nanoparticles in the 200-250 nm
diameter range was measured using a 90Plus Particles Size Analyzer
(Brookhaven Instruments).
[0174] For IL-15Sa/IL-21 encapsulation in DNA-gel nanoparticles, 30
.mu.g recombinant mouse IL-15R.alpha./Fc chimera (R&D systems)
was precomplexed with 10 .mu.g mouse IL-15 (Peprotech) in
nuclease-free water for 1 hour at room temperature to generate
superagonist IL-15 (IL-15Sa), combined with 10 .mu.g mouse IL-21
(Peprotech) and blended with the X-DNA/T4 ligase mixture for
DNA-gel particle synthesis, following the procedure described
above.
[0175] For DNA-gel nanoparticle loading with the GSK3-.beta.
inhibitor TWS119 (Cayman Chemical), 1 mg TWS was resuspended in 250
.mu.l DMSO, before adding it to the X-DNA/T4 ligase mixture for
DNA-gel particle synthesis.
[0176] 1.2. Nanoparticle Characterization. Characterization of the
nanoparticles by DLS and cryoelectron microscopy showed that the
mean particle diameter obtained from this process is 161.+-.74 nm
(FIG. 2C). Labeling of PLGA-core or liposome nanoparticles with
lipid-like dyes such as carbocyanine dyes (DiD, Invitrogen) allowed
the particles to be easily detected in confocal microscopy or flow
cytometry analysis of particle-decorated cells (illustrated in the
data discussed below).
[0177] 1.3. TCR-Transgenic System for Modeling Adoptive Cell
Therapy in Murine Melanoma. To develop and test the concepts
proposed here, we used the pmel-1 TCR-transgenic mouse/B16F10
murine melanoma system developed at the NCI as a model of adoptive
cell therapy for melanoma. Pmel-1 CD8.sup.+ T cells express a T
cell receptor which recognizes a peptide from murine gp100, a
melanoma self-antigen expressed by B16 melanoma tumor cells that is
also used as a T cell target in human melanoma vaccines. (Overwijk
et al., J Exp Med 198(4): 569, 2003; Klebanoff et al., Proc Natl
Acad Sci USA 102(27): 9571, 2005; Overwijk et al., J Exp Med
188(2): 277, 1998.) Pmel-1 mice develop T cells tolerized to this
antigen, mimicking what is thought to be a common situation in the
immune response to human cancers, although these cells can be
activated and expanded by priming them with an altered peptide
ligand, a peptide from human gp100. (Overwijk et al., J Exp Med
198(4): 569, 2003.) This model serves as a mimic of human ACT where
tolerance must be broken to fully prime the immune response
following adoptive transfer of expanded T cells into recipient
tumor-bearing mice.
[0178] 1.4. Coupling of Nanoparticles to Live T cells Through Free
Surface Thiols.
[0179] Having developed a strategy for preparation of lipid-coated
particles, we next performed a number of studies using `blank`
nanoparticles (no encapsulated cytokine/TLR ligand compounds) to
evaluate the prospects of this approach. We first tested whether
nanoparticles could be simply adsorbed to T cell surfaces stably,
by incubating cells with nanoparticles at varying particle:cell
ratios for different durations at 4.degree. C. or 37.degree. C.
Though PLGA-core nanoparticles could be adsorbed to cells (in
varying quantities, depending on the surface charge of the
nanoparticles used), we found that in some instances physical
adsorption did not provide very stable binding to the cells, and an
increasing fraction of nanoparticles was removed from the cells
during repeated washing as assessed by flow cytometry analysis of
tagged cells (not shown). It is to be understood however that in
some embodiments linkage of nanoparticles to carrier cells through
non-covalent absorption may be sufficient for the particular
application. This may be useful for example in the delivery of
antigen-loaded nanoparticles that may be transferred to antigen
presenting cells in lymphoid organs after administration and
appropriate homing.
[0180] To obtain more stable binding of particles to T cells, we
developed a non-toxic strategy to covalently link the lipid-coated
nanoparticles to T cells. We exploited the substantial amounts of
free thiols available on cell-surface proteins of leukocytes.
(Sahaf et al., Proc Natl Acad Sci USA 100(7): 4001, 2003.) We
conjugated maleimide-functionalized dyes (which react with thiols
to form stable thioether linkages) to freshly-isolated T cells and
analyzed the cells by flow cytometry. T cells, B cells and
hematopoietic progenitor cells (e.g., murine Lin.sup.-,
Sca-1.sup.+, c-kit.sup.+ cells) were found to have high levels of
free thiols at the cell surface, though red blood cells did not
(not shown).
[0181] Based on these results, we developed the strategy outlined
in FIG. 2D. Nanoparticle carriers were prepared which included
lipids with maleimide-terminated headgroups. CD8.sup.+ T cells were
isolated from spleens of pmel-1 TCR-transgenic using magnetic bead
negative selection (Miltenyi Biotec) and expanded for 4 days in
vitro using anti-CD3/anti-CD28-coated beads in the presence of 200
IU/mL human IL-2, mimicking the preparation of tumor-specific T
cells for adoptive cell therapy. T cells were washed and incubated
(60.times.10.sup.6 cells/mL) with
maleimide-functionalized-nanoparticles (at varying concentrations)
at 37.degree. C. for 45 min at varying particle:cell ratios. Cells
were then separated from unbound particles by gentle
centrifugation. Residual maleimide groups present on particles
bound to the T cells were quenched by incubation of the cells
(3.times.10.sup.6/mL) with 1 mg/mL thiol-terminated 2 KDa
poly(ethylene glycol) (PEG, Laysan Bio) at 37.degree. C. for 30 min
in complete RPMI medium, followed by two washes to remove unbound
PEG. By varying the amount of maleimide-lipid incorporated, we
found that 50 mole % maleimide in the lipid fraction provided
optimal binding to T cells and retention of particles through
multiple washes (not shown).
[0182] As shown in FIG. 3A, nanoparticles were readily attached to
cells using this thiol-reaction strategy. 2500 nanoparticles per
cell during conjugation as shown in FIG. 3 gave .about.500
nanoparticles bound per cell as determined from particle counting
at high magnification in confocal microscopy; this corresponds to a
theoretical occlusion of .about.3.2% of the average T cell surface
area by 160 nm diameter particles. T cells cultured in IL-2 showed
a dilution of the density of nanoparticles bound to the cells over
the course of a week, due to proliferation of the cells (FIG. 3A,
day 6). Conjugation of nanoparticles to cells at this density led
to no loss of T cell viability over a week in culture (FIG. 3B),
and also did not trigger spontaneous activation of these cells.
[0183] A key issue for these studies was the localization of the
particles. If the cells internalize these particles then
encapsulated drug cargos may not be released into the local
microenvironment and/or drugs released from the nanoparticles may
be unable to access their target receptors on the T cell itself.
Importantly, we found that T cells do not internalize lipid-coated
PLGA nanoparticles (illustrated by FIG. 3A), even during extended
culture or following proliferation (discussed further below). This
is in stark contrast to what we observed with dendritic cells,
which phagocytosed the attached nanoparticles within minutes.
Example 2. Assessment of Nanoparticle Binding on T Cell
Functions
[0184] 2.1. Nanoparticles Bound to Cells Do Not Block Antigen
Recognition or T Cell Proliferation. Having found that thiol
coupling allowed stable non-toxic linkage of nanoparticles to
cells, we next sought to determine whether the coupling reaction
interfered with T cell behavior, and to find what dose of
nanoparticles could be attached to T cells without blocking key T
cell functions. We first tested whether T cell proliferation was
impacted by nanoparticle coupling. Pmel-1 T cells were
primed/expanded in vitro with anti-CD3/anti-CD28 beads and IL-2 as
described above. The expanded cells were labeled with
carboxyfluorescein succinimidyl ester (CFSE), and incubated with
2500 DiD-labeled lipid-coated PLGA nanoparticles per cell for
conjugation. In parallel, day 6 bone marrow-derived dendritic cells
from C57B1/6 mice prepared as described (Stachowiak et al., J
Immunol 177(4): 2340, 2006) were activated by incubation with 1
.mu.M CpG oligonucleotide (a ligand for TLR 9) and pulsed with 1
.mu.M hgp100.sub.25-33 peptide (a peptide recognized by pmel-1 T
cells in the context of H-2D.sup.b MHC I molecules) overnight.
Nanoparticle-conjugated or control `bare` T cells were co-cultured
with activated antigen-loaded DCs at a 2:1 T:DC ratio for 6 days,
and then analyzed by flow cytometry. As shown in FIG. 4A, the
degree of proliferation of control and nanoparticle-conjugated T
cells as determined by CFSE dilution in the dividing cells was
indistinguishable. In addition, analysis of the mean fluorescence
from nanoparticles bound to cells showed a steady decline in
nanoparticle fluorescence as the number of cell divisions
increased, reflecting segregation of particles to separate daughter
cells during division. Notably, when sorted CFSE-low divided cells
were examined in confocal microscopy, nanoparticles were found to
still be surface localized even on cells that had undergone 5 cell
divisions.
[0185] 2.2. High Densities of Nanoparticles Can Be Bound to T Cells
Without Inhibiting Cytokine Secretion or CTL Activity. Activated
CD8.sup.+ T cells secrete cytokines such as IFN-.gamma. and
TNF-.alpha. and directly kill antigen-bearing target cells as part
of their anti-tumor activity. To determine whether conjugation of
lipid-coated PLGA nanoparticles to T cells interferes with cytokine
secretion, particle-conjugated or control T cells were co-cultured
with antigen-pulsed DCs as described above, and the production of
several key cytokines by the T cells was assessed by ELISA. As
shown in FIG. 4B, pmel-1 T cells decorated with nanoparticles
produced equivalent amounts of IL-2, IFN-.gamma. and TNF-.alpha. in
response to antigen stimulation as unmodified `bare` T cells. Thus,
substantial quantities of nanoparticles can be bound to cells
without blocking effector cytokine secretion.
[0186] We next carried out a dose response analysis to determine
the maximal dose of nanoparticles could be attached to T cells
without inhibiting cytolytic activity of the lymphocytes. Pmel-1 T
cells were expanded in vitro as before, and then incubated with
varying doses of lipid-coated PLGA nanoparticles per cell ranging
from 100 nanoparticles/cell up to 10,000 nanoparticles per cell for
particle conjugation. Particle-tagged or control T cells were then
co-cultured with .sup.51Cr-labeled EL4 target cells pulsed with
hgp100.sub.25-33 peptide at varying effector:target ratios for 4
hrs at 37.degree. C. in complete medium. Specific target cell lysis
was determined by measurement of radioactive chromium released into
the culture supernatant. As shown in FIG. 4C, target cell killing
by nanoparticle-conjugated T cells was indistinguishable from
control T cells except at the two highest coupling doses tested
(10,000 or 5000 nanoparticles/cell).
[0187] 2.3. TCR-Transgenic OT-1 CD8.sup.+ T Cell Analysis. Similar
results were found with other T cells. TCR-transgenic OT-1
CD8.sup.+ T cells, which are specific for a peptide derived from
ovalbumin, and which were conjugated with up to 100 (.+-.21)
nanoparticles per cell, fully retained their physiological
proliferative response after co-culture with ovalbumin-pulsed
target dendritic cells. In some instances, higher surface densities
of the same nanoparticles began to inhibit T cell proliferation
(data not shown). During cell division, surface-attached
nanoparticles segregated equally to daughter cells, which was
reflected by a stepwise decrease in the mean fluorescent signal
from cell-conjugated nanoparticles with increasing number of cell
divisions (data not shown). Attachment of up to .about.100
particles/cell also did not impact T cell recognition/killing of
ovalbumin peptide-pulsed target cells or cytokine release profiles
(data not shown).
[0188] In summary, for conditions of up to 2500 nanoparticles/cell
during conjugation (nanoparticles with diameters .about.160 nm), no
inhibition of T cell antigen recognition, proliferation, cytokine
secretion, or target cell killing is observed. These results
together suggest that substantial quantities of submicron-sized
nanoparticles can be attached to T cells without blocking key cell
functions.
Example 3. Cytokine/Drug Loading in Lipid-Coated PLGA
Nanoparticles
[0189] Nanoparticles are conjugated to ACT T cells in two different
ways: (i) nanoparticles are loaded with cytokines designed to act
on the carrier T cells themselves to support their proliferation,
survival and effector function (e.g., IL-15 superagonist) or (ii)
nanoparticles will be used to deliver compounds designed to act on
other cells in the microenvironment, including Toll-like receptor
(TLR) ligands and vaccine antigens (e.g., imiquimod or MPLA). In
the previous studies, `empty` nanoparticles were used to assess the
impact of particle conjugation on T cell functions. Here we tested
the encapsulation/incorporation of proteins (e.g., IL-15
superagonist) and TLR ligands into the nanoparticles, in order to
deliver therapeutically relevant cargos.
[0190] PLGA nanoparticles have been explored in numerous prior
studies as vehicles for encapsulation and delivery of proteins,
peptides, and small molecule drug compounds, and notably vaccine
antigens/adjuvants. (Davis M E et al., Nat Rev Drug Discov 7(9):
771, 2008; Chacon Metal., International Journal of Pharmaceutics
141(1-2): 81, 1996; Diwan M et al., Curr Drug Deliv 1(4): 405,
2004; Elamanchili P et al., Vaccine 22(19): 2406, 2004; Li Y et
al., J Control Release 71(2): 203, 2001; Zhang Z P et al.,
Biomaterials 28(10): 1889, 2007; Heit A et al., Eur J Immunol
37(8): 2063, 2007.) We first tested whether protein encapsulation
was facile in our lipid-coated nanoparticles by adding protein to
the inner aqueous phase of the double emulsion synthesis: 200 .mu.L
water in the synthesis protocol described in section 3.1 was
replaced with 200 .mu.L of a solution of the model protein
Alexa488-labeled ovalbumin (100 .mu.g in PBS), and particles were
prepared and purified as before. As shown in FIG. 5A, ova
fluorescence was clearly detected in nanoparticles by confocal
microscopy, and cryoEM imaging of the nanoparticles showed that the
particle morphology was not disrupted by protein encapsulation and
the surface lipid layer was retained for protein-loaded particles
(FIG. 5B). Measurement of the amount of protein encapsulated was
performed by lysing the nanoparticles for 4 hrs in 0.02 M NaOH/2%
SDS, neutralizing the solution with 0.2 M HC1, and measuring
released ova fluorescence calibrated against ova solution standards
exposed to the same base treatment conditions. By these
measurements, we found that .about.1 .mu.g of ova per mg
nanoparticles was encapsulated (.about.25% encapsulation
efficiency).
[0191] Ova however is a model globular protein and as such it was
chosen to illustrate the behavior of other proteins such as
interleukin-15 (IL-15) superagonist molecules which can be used to
support ACT T cells. We encapsulated IL-15 (cytokine alone) in
lipid-coated PLGA to test the feasibility of cytokine loading in
these particles. IL-15 (5 .mu.g) in PBS was used in the inner
aqueous phase of the particle synthesis, and the resulting
cytokine-loaded particles were purified as described in section
3.1. The kinetics of IL-15 release from the particles was
determined by incubating the particles in complete RPMI medium
containing 10% FCS at 37.degree. C. with gentle agitation and
taking aliquots of the supernatant at staggered timepoints for
ELISA analysis of cytokine content. As shown in FIG. 5B, .about.80%
of the encapsulated cytokine was released by the end of this
incubation period. Other experiments with ova-loaded nanoparticles
showed continuous release of protein over a similar 7-10-day
period. Thus, the lipid-coated particles can be loaded with protein
and release encapsulated material over a .about.1 week period. The
release kinetics can be modulated to faster or slower rates by
altering the MW of the PLGA used in the particles.
[0192] Having found that cytokines can be successfully encapsulated
and released from nanoparticles, we tested whether survival of T
cells in vitro could be enhanced by cytokines released from
nanoparticles. Pmel-1 T cells were primed/expanded in vitro with
anti-CD3/anti-CD28 beads and IL-2 as described above. The expanded
cells were incubated with 2500 lipid-coated PLGA nanoparticles per
cells for conjugation. The nanoparticles were formulated with 10 mg
of IL-15 or IL-15 and IL-15R.alpha.. Particle-conjugated or control
T cells were then co-cultured with EL4 target cells pulsed with
hgp100.sub.25-33 peptide at effector:target ratios of 20:1 at
37.degree. C. in complete medium without exogenous IL-2 supplement.
After 6 days of culture, the number of live T cells was counted
after trypan blue staining to assess proliferation and survival of
T cells. As shown in FIG. 6, nanoparticles encapsulating IL-15 and
IL-15/IL-15R.alpha. significantly enhanced survival and/or
proliferation T cells compared to no treatment or empty
nanoparticle groups. Proliferation observed in T cells tagged with
cytokine-encapsulated nanoparticles was comparable to soluble IL15
and IL-15/IL-15R.alpha. controls. Thus, IL-15 or its superagonist
complexed with IL-15R.alpha. continuously released from
nanoparticles maintain its bioactivity and is able to support T
cell survival and/or proliferation in vitro.
[0193] Nanoparticles were also loaded with the TLR4 ligand MPLA
and/or the TLR7 ligand, imiquimod, as potent clinically-relevant
ligands for driving DC activation during T cell adoptive therapy.
MPLA is a synthetic lipopolysaccharide mimic that has shown promise
as a nontoxic analog of the potent immunostimulant
lipopolysaccharide (LPS). MPLA provides adjuvant activity in
vaccines comparable to LPS but has orders of magnitude reduced
systemic toxicity due to its selective engagement of downstream
signals in the TLR4 signaling pathway. (Mata-Haro et al., Science
316(5831): 1628, 2007.) Notably, LPS and its derivatives have shown
promise in breaking tolerance to tumors, and beneficial effects of
whole-body irradiation observed during adoptive T cell therapy
studies have been in part ascribed to LPS and other TLR signaling
occurring when the integrity of the gut epithelium is compromised.
(Yang et al., Nat Immunol 5(5): 508, 2004; Paulos et al., Clin
Cancer Res 13(18 Pt 1): 5280, 2007; Paulos et al., J Clin Invest
117(8): 2197, 2007.)
[0194] Imiquimod, a small-molecule imidazoquinoline ligand for
TLR7/8, is a promising pro-immunity factor for cancer therapy
approved for clinical use as a topical cream in the treatment of
certain skin cancers. In addition to its pro-immunity activation of
macrophages and dendritic cells (Hemmi et al., Nat Immunol 3(2):
196, 2002), imiquimod has recently been reported to activate
tumor-local dendritic cells to a direct tumor-killing phenotype in
humans. (Stary et al., J Exp Med 204(6): 1441, 2007.)
[0195] Imiquimod and MPLA however share challenges in their
application for cancer therapy. Systemic imiquimod delivered orally
has shown dose-limiting toxicity in humans (Goldstein et al., J
Infect Dis 178(3): 858, 1998) and has a short half-life following
injection of only .about.2 hrs (Soria et al., Int J Clin Pharmacol
Ther 38(10): 476, 2000). Topical administration of imiquimod
however has not been shown to be effective in systemic metastases
or non-cutaneous cancers. Both TLR4 and TLR7 have broad expression
patterns (expressed at low levels in endothelial cells and by
epithelial cells (Fan et al., J Clin Invest 112(8): 1234, 2003;
Gunzer et al., Blood 106(7): 2424, 2005)), raising concerns of
systemic toxicity in prolonged treatment. TLR4 and TLR7 ligands
however have been shown to induce expression of ICAM-1, ICAM-2, and
selectins on endothelial cells (Gunzer et al., Blood 106(7): 2424,
2005), and such effects if locally stimulated at tumor sites could
be used to enhance T cell trafficking into tumors. Thus, selective
delivery of these ligands to tumor sites and secondary lymphoid
organs might be used to enhance their anti-tumor activity while
limiting systemic side effects.
[0196] In parallel with protein encapsulation experiments, we thus
also tested incorporation of the Toll-like receptor ligand
monophosphoryl lipid A (MPLA) in lipid-coated PLGA. Due to its
lipid-like structure, MPLA is quantitatively incorporated into the
particles by simply co-dissolving this ligand with the other
phospholipids in the chloroform phase of the particle synthesis. To
test the ability of MPLA incorporated in lipid-coated PLGA
nanoparticles to activate dendritic cells (which express the LPS
receptor, TLR4), nanoparticles containing 1 mole % or 10 mole %
MPLA as part of the lipid fraction of the synthesis were added to
bone marrow-derived DCs for 24 hrs, and then the surface expression
of class II MHC molecules and costimulatory receptors was analyzed
by flow cytometry. DCs stained with antibodies against MHCII, CD80,
and CD40 showed upregulation of these markers when treated with
MPLA-containing nanoparticles comparable to DCs treated with
1.mu.g/mL LPS as a positive control; `blank` nanoparticles however
triggered no DC maturation (FIG. 5C). Thus, TLR ligands
incorporated in the lipid-coated nanoparticles are capable of
activating DCs.
[0197] Gardiquimod and resiquimod are imidazoquinoline derivatives
that, similar to imiquimod, are selective ligands for TLR7/8.
Gardiquimod and resiquimod have been suggested to have more potent
effect than imiquimod, based on findings that they induce stronger
cytokine production, macrophage activation, and enhanced cellular
immunity (Wager et al., Cell Immunol 191(1):10, 1999; Burns et al.,
Clin Immunol 94(1):13, 2004; Schon et al., Oncogene 27(2): 109,
2008.) Encapsulation of gardiquimod and resiquimod and detection of
their release from PLGA nanoparticles were carried out with minor
modifications. For encapsulation of gardiquimod in nanoparticles,
200 .mu.L water in the synthesis protocol described in section 3.1
was replaced with 1.8 mg of gardiquimod dissolved in 200 .mu.L of
water, and for encapsulation of resiquimod, 0.83 mg of resiquimod
was dissolved along with 30 mg of PLGA in organic solvent; the rest
of nanoparticle synthesis protocol outlined in section 3.1 was
followed thereafter. The kinetics of drug release from the
particles was determined by incubating the particles in water with
gentle agitation at room temperature and taking aliquots of the
supernatant at staggered timepoints for fluorescent detection of
drug release at excitation/emission of 260/340 nm. As shown in FIG.
7, continuous release of gardiquimod and resiquimod from
nanoparticles was observed over 8 days of incubation.
Example 4. Whole Animal Imaging Reagents for Independently Tracking
Tumor Cells, Nanoparticles, and T Cells In Vivo
[0198] The data described in the previous two sections demonstrate
the protocol we have developed to attach nanoparticles to T cells
in a nontoxic manner that does not interfere with key T cell
functions. The particles can be loaded with protein or TLR ligands
as therapeutic cargos that will be explored in the proposed
research. A final key function that nanoparticle conjugation must
not interrupt is migration/tissue homing of T cells. We first
tested in vitro migration of particle-conjugated T cell blasts
plated on glass coverslips. As shown in FIG. 8A, migrating T cells
observed in time-lapse videomicroscopy polarized cell-surface-bound
nanoparticles to the uropod during migration, but when cells halted
migration, the particles re-dispersed over the cell surface
(`arrested cell`). Thus, particle-conjugated cells are able to
migrate and rearward polarization of the nanoparticles during
motility may help reduce the likelihood that the particles will
interfere with the patrolling function of these cells in vivo.
[0199] We next assessed the impact of cell surface-tethered
nanoparticles on the ability of their cellular carrier to
transmigrate across endothelial barriers, as a measure of the
ability of the cell carrier to infiltrate its target tissue. We
utilized an in vitro transwell co-culture system in which
unmanipulated or nanoparticle-conjugated effector T lymphocytes
migrate from the upper chamber across a membrane-supported
confluent TNF-a activated endothelial monolayer towards in response
to a T cell chemoattractant placed in the lower chamber. Unaltered
T cells carrying 100 nanoparticles/cell exhibited unaltered
transmigration efficiencies compared to unmodified cells (data not
shown). After crossing the endothelial barrier, T lymphocytes still
had retained 83% (.+-.3%) of the original nanoparticle cargo
physically attached. Confocal imaging revealed that T cells
migrating on the endothelial layer polarized to a characteristic
"hand-mirror" morphology, and localized their nanoparticle pool to
the uropod (data not shown), likely reflecting the uropodal
localization of many cell surface proteins on migrating T
cells.
[0200] Further experiments were conducted to show that
particle-conjugated T cells can home to their expected tissue
sites, and for therapy that such cells can enter solid tumors as
effectively as unmodified T cells. To aid in these studies, we use
multicolor bioluminescence/fluorescence whole-animal imaging to
simultaneously track the location of nanoparticles,
adoptively-transferred T cells, and tumor cells. These experiments
are performed using a Xenogen IVIS Spectrum
bioluminescence/fluorescence imaging instrument located in the Koch
Cancer Institute core facilities at MIT.
[0201] As shown in FIG. 8B, lipid-coated PLGA nanoparticles labeled
with DiR dye are readily detected in whole-animal fluorescence
following subcutaneous injection, due to the low absorption of
near-IR excitation light used for this dye (exc 750 nm/em 790 nm).
In an in vivo homing experiment, nanoparticles with
surface-conjugated recombinant Gaussia luciferase were prepared.
Pmel-1 T cells were then coupled with these luciferase-decorated
nanoparticles and injected i.v. (via tail vein) into a recipient
C57B1/6 mouse. Whole-animal bioluminescence imaging following the
injection of the Gaussia luciferase substrate coelentarizine 4 hrs
after T cell transfer via tail vein injection is shown in FIG. 8C.
At this timepoint, a majority of T cells are still localized in the
lungs as previously reported for effector T cells (Hamann A et al.,
Eur J Immunol 30(11): 3207, 2000) but nanoparticle/T cell
signatures were also detected at flank sites that may reflect
homing to inguinal lymph nodes and small intense spots of
bioluminescence were detected next to the lungs (white arrows) that
may reflect initial homing to axillary/brachial lymph nodes.
[0202] Because the nanoparticles can be tracked using near-IR dyes
and fluorescence, we crossed pmel-1 TCR-transgenic mice with
luciferase-transgenic mice, to obtain pmel-1-luc mice where the
pmel-1 CD8.sup.+ T cells express firefly luciferase (data not
shown). In parallel, Gaussia-luciferase-expressing B 16F10 melanoma
cells were prepared by retroviral transfection of B16 cells with a
luciferase construct. As illustrated in FIG. 8D, the B16-gaussia
luc cells were readily detected via bioluminescence imaging.
[0203] To assess the potential of tumor-reactive T lymphocytes to
vehicle surface-conjugated nanoparticles into the microenvironment
of established tumors, we adoptively transferred CD8 Pmel-1
effector T cells, T cell receptor-transgenic for the melanoma
antigen gp-100, into hosts with established B 16F10 tumors in their
right femur (FIG. 9A). Animals were treated with
15.times.10.sup.6Pmel-1 T lymphocytes, transgenic for Firefly
luciferase for in vivo bioluminescent T cell tracking. T cells were
either conjugated to nanoparticles tagged with the fluorescent dye
DiD (right panels) or left unmodified (left panels). In both
treatment groups, we incubated infused T lymphocytes with Thiol-PEG
to avoid nonspecific phagocytosis of nanoparticles by macrophages
and dendritic cells. Infused T cells of both groups displayed rapid
and effective homing to the tumor site, as monitored by
bioluminescent T cells imaging on day 4 after T cell transfer (FIG.
9B). Notably, the ex vivo surface conjugation of nanoparticles to T
cells, did neither alter their in vivo migration nor did it
constrain their potential to recognize tumor antigen. Tumor-homing
T cells, furthermore, efficiently aggregated surface-conjugated
nanoparticles at the tumor site, as shown by the largely amplified
fluorescent DiD signal of the isolated right tumor-infiltrated
femur, compared to the left tumor-free femur (FIG. 9C, right
panel). Importantly, nanoparticles at the tumor site were still
physically linked to tumor-infiltrating T cells, as measured by
multicolor flow cytometry of tumor single cell suspensions (FIG.
9D). In essence, we demonstrate that tumor-targeted T lymphocytes
effectively shuttle therapeutic nanoparticles to the tumor site.
The ex-vivo surface conjugation does not impair T cell viability,
migration or tumor recognition and, therefore, offers novel
prospect for the targeted biodistribution of nanoparticles and the
functional enhancement of tumor-reactive T lymphocytes.
[0204] We next evaluated the migratory and tumor-homing properties
of nanoparticle-conjugated lymphocytes in an another murine model
system. C57B1/6 mice were injected with EL4 tumor cells expressing
membrane-bound Gaussia luciferase (extG-luc) and ovalbumin
(EG7-OVA) s.c. on the right flank and control tumors EL4 cells
expressing extG-luc alone on the left flank. Tumors were allowed to
establish and then mice then received adoptive transfers of Firefly
luciferase (F-luc)-transgenic OT-1 T cells with or without
surface-conjugated red-fluorescent DNA-gel nanoparticles, or an
i.v. injection of an equivalent dose of fluorescent particles
alone. Particle-carrying OT-1 T cells specifically trafficked to
pre-established EL4-OVA tumors (FIG. 10A). No difference in the
tumor homing potential of particle-conjugated compared to plain
unmodified OT-1 T cells was observed (FIG. 10B, left panel).
Quantitative fluorescent particle imaging of EG7-OVA tumors
demonstrated that nanoparticles accumulated a mean 176-fold more
efficiently at the tumor site when surface-attached to OT-1 T cells
compared to systemically infused free nanoparticles, which were
rapidly scavenged by the liver and the spleen (FIG. 10B). Flow
cytometry analysis verified that T cell infiltration of EG7-OVA
tumors was quantitatively identical for particle-decorated and
control OT-1 cells, and that the majority of particle-conjugated
cells recovered from tumors still retained their nanoparticle cargo
(FIG. 10A).
[0205] The benefit of tumor-antigen-specific T lymphocytes as
cellular vectors for active nanoparticle delivery was also
evidenced in a spontaneous prostate cancer model (i.e., the TRAMP
prostate adenocarcinoma model). In this model system, prostate
tumor-specific T cells loaded with DNA-gel nanoparticles
efficiently homed to antigen-expressing hyperplastic TRAMP
prostates and aggregated surface-linked fluorescent particles at
the tumor site, whereas no fluorescent nanoparticle signal above
background was detected in the prostate following systemic
injection of an equivalent particle dose (FIG. 11A-C).
[0206] The ability of lymphocytes to efficiently transfer
surface-tethered nanoparticles across endothelial barriers in vivo
was not restricted to the abnormal, leaky and discontinuous
endothelial lining found in tumor vasculature. When DNA-gel
particles were linked to resting CCR7.sup.+CD62L.sup.+ B cells
(FIGS. 12A-C) or central memory CD8.sup.+ T cells (data not shown),
particles were transported across the intercellular boundaries of
high endothelial venules into lymph nodes, a poorly accessible
compartment for systemically infused free nanoparticles. FIGS.
12D-F show the biodistribution profile of nanoparticles conjugated
to B cells versus free nanoparticles, the presence of nanoparticles
on B cells harvested from subjects, and the localization of
administered B cells and conjugated nanoparticles to lymph
nodes.
[0207] In the studies above, nanoparticles without therapeutic
cargo were appended to cells possessing a defined tissue tropism to
demonstrate the utility of therapeutic cells as highly efficient
vectors for nanoparticle delivery to otherwise difficult-to-access
anatomical compartments.
[0208] We next tested whether cell-bound drug-loaded nanoparticles
could directly impart amplified therapeutic functions to their
cellular carriers, using a murine model of adoptive T cell therapy
for melanoma (Overwijk et al., J Exp Med, 188:277, 1998). We
encapsulated a mixture of IL-15, (converted to a superagonist
(IL-15Sa) by pre-complexing with soluble IL-15R.alpha. (Rubinstein
et al., PNAS USA, 103:9166, 2006)), in combination with IL-21 into
lipid-coated DNA-gel particles. IL-15 and IL-21 are known to
cooperatively promote in vivo T cell expansion and effector
function when administered daily at high doses. DNA-gel particles
.about.200 nm in diameter efficiently entrapped the IL-15Sa/IL-21
cytokine mixture and displayed slow release kinetics over a 7-day
period (data not shown). These cytokine-loaded particles were
conjugated to Click bettle red (CBR)-luciferase expressing
CD8.sup.+ Pmel-1 effector T cells which recognize a peptide from
the melanocyte differentiation antigen gp100. Particle-conjugated
or control T cells were infused into lympho-depleted mice bearing
established Gaussia luciferase-expressing B 16F10 melanoma lung
tumors (FIG. 13A). Serial imaging of non-conjugated Pmel-1 T cells
showed a gradual CBR-luc signal decline following T cell injection,
consistent with poor in vivo T cell expansion and persistence
(FIGS. 13B and C). Whereas a single systemic infusion of 5 .mu.g
free IL-15Sa/IL-21 (4.03 .mu.g IL-15Sa+0.93 .mu.g IL-21) given on
the day of adoptive transfer did not significantly boost Pmel-1
proliferation (1.4-fold-higher CBR-luc signal on day 6, P=0.32),
the same cytokine dose loaded in surface-attached nanoparticles
conferred markedly amplified proliferative capabilities on Pmel-1 T
cells (81-fold higher peak photon count relative to unmodified
Pmel-1 T cells on day 6, P<0.0001, FIGS. 13A and C). Subsequent
to a contraction period, IL-15Sa/IL-21 nanoparticle-carrying T
cells displayed enhanced long-term persistence (14.8-fold and
4.7-fold higher photon count than Pmel-1 T cells alone at 16 and 30
days after T cell infusion, respectively, P<0.0001) and homed as
CD44.sup.+CD62L.sup.+ central memory T cells to lymph nodes and
spleen (FIGS. 13A and B, and data not shown). There was no evidence
of progressive T cell clonality or leukemia formation in any
treated animal imaged at late time points (data not shown). Pmel-1
T cells conjugated with "empty" nanoparticles exhibited the same
expansion/decline in vivo as unmodified Pmel-1 cells (data not
shown). All mice receiving IL-15Sa/IL-21 nanoparticle-decorated
Pmel-1 T cells achieved complete tumor clearance (FIGS. 13A and D),
whereas treatment with Pmel-1 T cells with or without systemic
IL-15Sa/IL-21 infusion at the same doses yielded only modest
survival advantages (FIG. 13D).
Example 5. Conjugation of Nanoparticles to Hematopoietic Progenitor
Cells
[0209] We further examined the utility of this delivery approach in
the context of hematopoietic stem cell transplantations. We treated
C57B1/6 F-luc-transgenic mice or C57B1/6 GFP-transgenic mice with
5-fluorouracil (5-FU, Sigma Aldrich) (150 mg/kg, i.p) and
euthanized them 3 days later. Bone marrow cells were removed
aseptically from femurs and tibias. Bone marrow was pre-enriched
for progenitor cells using a lineage depletion kit (Miltenyi). A
subsequent positive selection with anti-Sca-1 microbeads (Miltenyi)
resulted in an average 92% purity of
lin.sup.-Sca.sup.-1.sup.+c-kit.sup.+ HSCs. Cells were kept in
serum-free StemSpan (Stem Cell Technologies) for 3 hours before
further modification.
[0210] 1.times.10.sup.4 unmodified or nanoparticle-decorated HSCs
were transplanted by retroorbital injection into lethally
irradiated (1300 cGy of total body irradiation from a .sup.137Cs
source as a split dose with 3-hr interval between) nontransgenic
recipients.
[0211] For in vitro HSC expansion, HSCs, retrovirally transduced
with NUP98-HOXA10hd, were cultured in DMEM supplemented with 15%
FBS and cytokines (6 ng/mL of IL-3, 10 ng/mL of IL-6, 100ng/mL of
SCF, all Preprotech).
[0212] We chose the glycogen synthase kinase-3.beta. (GSK-3.beta.)
inhibitor TWS119 (Gattinoni et al., Nat Med 15:808, 2009) as
therapeutic cargo, based on reports that repeated high-dose bolus
therapy of transplant recipients with glycogen synthase kinase-3
(GSK-3) inhibitors enhances the repopulation kinetics of donor HSCs
(Trowbridge et al., Nat Med 12:89, 2006). DNA-gel nanoparticles
efficiently encapsulated this small-molecule drug, and slowly
released it over a 7-day time window (data not shown). We evaluated
the in vivo repopulation capabilities of hematopoietic grafts
supported by cell-bound TWS119-loaded nanoparticles based on the
whole body photon emission from Firefly luciferase-transgenic donor
progenitor cells, and in separate experiments, by tracing the
frequencies of GFP.sup.+ donor progenitor cells by flow cytometry.
Following transplantation of murine
lineage.sup.-Sca-1.sup.+c-kit.sup.+ progenitor cells from
luciferase-transgenic donors into syngeneic recipients, a steady
increase in whole body bioluminescent emission was observed
originating from discrete foci over anatomical sites corresponding
to the femurs, humeri, sternum and the spleen (FIG. 14A). While a
systemic TWS119 bolus injection (1.6 ng) at the time of
transplantation did not significantly alter measured engraftment
kinetics (FIGS. 14A and B), the same TWS119 dose encapsulated in
nanoparticles surface-tethered to donor progenitor cells markedly
enhanced the proliferative reconstitution of progenitor cell grafts
(median 5.7-fold higher bioluminescence than systemic TWS119 after
1 week, P<0.0001, FIGS. 14A-C). Notably, animals in all
treatment groups initially engrafted progenitor cells in both
femurs and the sternum, indicating that nanoparticle conjugation
did not compromise the intrinsic homing properties of donor
progenitor cells. While increasing the rate of initial
reconstitution, conjugating TWS119 nanoparticles onto progenitor
cells did not affect their multilineage differentiation potential,
reflected by a similar frequency of donor-derived GFP.sup.+
reconstituted cell types compared to control progenitor cell grafts
three months after transplantation (FIG. 14D). Thus, this simple
approach for donor cell modification just prior to cell transfer
can also augment hematopoietic progenitor cell, including
hematopoietic stem cell, transplants, a procedure in routine
clinical practice.
Example 6. Conjugation of Liposomes to Cells
[0213] One exemplary protocol for synthesizing unilamellar
liposomes is as follows: A DOPC/DOPG/MPB PE/DiD lipid film (lipid
ratios as in polymer nanoparticles) was hydrated with 185 .mu.l PBS
for a one-hour period with vigorous vortexing every 10 minutes.
After six cycles of freezing (liquid N.sub.2) and thawing, the
liposomes were extruded 21 times through a polycarbonate filter
(200 nm pore size, Whatman) and purified using a Zeba Spin
Desalting Column (Thermo Scientific).
[0214] FIG. 15 shows liposome conjugation to pmel-1 T cells. The
confocal image shows liposomes (blue) conjugated to the surfaces of
pmel-1 T cells (CFSE-stained in green). Shown are 3D projections of
optical sections taken by confocal microscopy.
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EQUIVALENTS
[0294] While several inventive embodiments have been described and
illustrated herein, those of ordinary skill in the art will readily
envision a variety of other means and/or structures for performing
the function and/or obtaining the results and/or one or more of the
advantages described herein, and each of such variations and/or
modifications is deemed to be within the scope of the inventive
embodiments described herein. More generally, those skilled in the
art will readily appreciate that all parameters, dimensions,
materials, and configurations described herein are meant to be
exemplary and that the actual parameters, dimensions, materials,
and/or configurations will depend upon the specific application or
applications for which the inventive teachings is/are used. Those
skilled in the art will recognize, or be able to ascertain using no
more than routine experimentation, many equivalents to the specific
inventive embodiments described herein. It is, therefore, to be
understood that the foregoing embodiments are presented by way of
example only and that, within the scope of the appended claims and
equivalents thereto, inventive embodiments may be practiced
otherwise than as specifically described and claimed. Inventive
embodiments of the present disclosure are directed to each
individual feature, system, article, material, kit, and/or method
described herein. In addition, any combination of two or more such
features, systems, articles, materials, kits, and/or methods, if
such features, systems, articles, materials, kits, and/or methods
are not mutually inconsistent, is included within the inventive
scope of the present disclosure.
[0295] All definitions, as defined and used herein, should be
understood to control over dictionary definitions, definitions in
documents incorporated by reference, and/or ordinary meanings of
the defined terms.
[0296] The indefinite articles "a" and "an," as used herein in the
specification and in the claims, unless clearly indicated to the
contrary, should be understood to mean "at least one."
[0297] The phrase "and/or," as used herein in the specification and
in the claims, should be understood to mean "either or both" of the
elements so conjoined, i.e., elements that are conjunctively
present in some cases and disjunctively present in other cases.
[0298] Multiple elements listed with "and/or" should be construed
in the same fashion, i.e., "one or more" of the elements so
conjoined. Other elements may optionally be present other than the
elements specifically identified by the "and/or" clause, whether
related or unrelated to those elements specifically identified.
Thus, as a non-limiting example, a reference to "A and/or B", when
used in conjunction with open-ended language such as "comprising"
can refer, in one embodiment, to A only (optionally including
elements other than B); in another embodiment, to B only
(optionally including elements other than A); in yet another
embodiment, to both A and B (optionally including other elements);
etc.
[0299] As used herein in the specification and in the claims, "or"
should be understood to have the same meaning as "and/or" as
defined above. For example, when separating items in a list, "or"
or "and/or" shall be interpreted as being inclusive, i.e., the
inclusion of at least one, but also including more than one, of a
number or list of elements, and, optionally, additional unlisted
items. Only terms clearly indicated to the contrary, such as "only
one of" or "exactly one of," or, when used in the claims,
"consisting of," will refer to the inclusion of exactly one element
of a number or list of elements. In general, the term "or" as used
herein shall only be interpreted as indicating exclusive
alternatives (i.e. "one or the other but not both") when preceded
by terms of exclusivity, such as "either," "one of," "only one of,"
or "exactly one of." "Consisting essentially of," when used in the
claims, shall have its ordinary meaning as used in the field of
patent law.
[0300] As used herein in the specification and in the claims, the
phrase "at least one," in reference to a list of one or more
elements, should be understood to mean at least one element
selected from any one or more of the elements in the list of
elements, but not necessarily including at least one of each and
every element specifically listed within the list of elements and
not excluding any combinations of elements in the list of elements.
This definition also allows that elements may optionally be present
other than the elements specifically identified within the list of
elements to which the phrase "at least one" refers, whether related
or unrelated to those elements specifically identified. Thus, as a
non-limiting example, "at least one of A and B" (or, equivalently,
"at least one of A or B," or, equivalently "at least one of A
and/or B") can refer, in one embodiment, to at least one,
optionally including more than one, A, with no B present (and
optionally including elements other than B); in another embodiment,
to at least one, optionally including more than one, B, with no A
present (and optionally including elements other than A); in yet
another embodiment, to at least one, optionally including more than
one, A, and at least one, optionally including more than one, B
(and optionally including other elements); etc.
[0301] It should also be understood that, unless clearly indicated
to the contrary, in any methods claimed herein that include more
than one step or act, the order of the steps or acts of the method
is not necessarily limited to the order in which the steps or acts
of the method are recited.
[0302] In the claims, as well as in the specification above, all
transitional phrases such as "comprising," "including," "carrying,"
"having," "containing," "involving," "holding," "composed of," and
the like are to be understood to be open-ended, i.e., to mean
including but not limited to. Only the transitional phrases
"consisting of" and "consisting essentially of" shall be closed or
semi-closed transitional phrases, respectively, as set forth in the
United States Patent Office Manual of Patent Examining Procedures,
Section 2111.03.
Sequence CWU 1
1
518PRTartificial sequencesynthetic peptide 1Ser Ile Tyr Arg Tyr Tyr
Gly Leu1 5240DNAartificial sequencesynthetic nucleic acid
2acgtcgaccg atgaatagcg gtcagatccg tacctactcg 40340DNAartificial
sequencesynthetic nucleic acid 3acgtcgagta ggtacggatc tgcgtattgc
gaacgactcg 40440DNAartificial sequencesynthetic nucleic acid
4acgtcgagtc gttcgcaata cggctgtacg tatggtctcg 40540DNAartificial
sequencesynthetic nucleic acid 5acgtcgagac catacgtaca gcaccgctat
tcatcggtcg 40
* * * * *