WO2010059253A2 - Procédés et compositions pour la délivrance localisée d'agents - Google Patents

Procédés et compositions pour la délivrance localisée d'agents Download PDF

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Publication number
WO2010059253A2
WO2010059253A2 PCT/US2009/006290 US2009006290W WO2010059253A2 WO 2010059253 A2 WO2010059253 A2 WO 2010059253A2 US 2009006290 W US2009006290 W US 2009006290W WO 2010059253 A2 WO2010059253 A2 WO 2010059253A2
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WO
WIPO (PCT)
Prior art keywords
cell
nanoparticle
agent
cells
nanoparticles
Prior art date
Application number
PCT/US2009/006290
Other languages
English (en)
Other versions
WO2010059253A9 (fr
WO2010059253A3 (fr
Inventor
Darrell J. Irvine
Matthias Stephan
Jaehyun Moon
Anna Bershteyn
Original Assignee
Massachusets Institute Of Technology
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Massachusets Institute Of Technology filed Critical Massachusets Institute Of Technology
Priority to EP21157030.4A priority Critical patent/EP3936122A1/fr
Priority to ES09827900T priority patent/ES2866623T3/es
Priority to US13/130,845 priority patent/US9283184B2/en
Priority to EP09827900.3A priority patent/EP2398466B1/fr
Publication of WO2010059253A2 publication Critical patent/WO2010059253A2/fr
Publication of WO2010059253A9 publication Critical patent/WO2010059253A9/fr
Publication of WO2010059253A3 publication Critical patent/WO2010059253A3/fr
Priority to US13/910,937 priority patent/US9393199B2/en
Priority to US15/015,464 priority patent/US20160256386A1/en
Priority to US15/625,479 priority patent/US20180110733A1/en
Priority to US17/069,305 priority patent/US20210259968A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.

Definitions

  • the invention relates to the delivery of agents to localized regions, tissues, or cells in the body using nanoparticles and cells.
  • 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.
  • 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.
  • AICD activation-induced cell death
  • IL-15 supports T cell proliferation and effector functions without promoting AICD.
  • IL-15 signals through a heterotrimeric receptor composed of a dedicated ⁇ chain, a shared IL- 2/IL-15R ⁇ chain, and the common ⁇ chain used by several interleukins.
  • 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.
  • 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 ⁇ 1 hr, and has limited potency in vivo, triggering limited proliferation of T cells compared to responses observed during prolonged in vitro culture.
  • 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.
  • ToIl- 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.
  • DCs dendritic cells
  • 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.
  • TLR signaling is - A - 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.
  • 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.
  • 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.
  • TLR ligands directly into tumors has been used to promote the activation of tumor-resident APCs and drive effective local immune responses.
  • 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.
  • 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.
  • synthetic drug delivery particles particles with sizes in the 50-500 nm range, typically
  • synthetic drug delivery particles 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.
  • 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 -150 nm size range covalently conjugated to its surface even after many days or through several rounds of cell division.
  • 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.
  • 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.
  • the cell is a T cell.
  • the cell is a B cell, an NK cell, or an NKT cell.
  • 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).
  • 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.
  • the subject has a tumor.
  • the cell is a tumor-reactive T cell.
  • the cell homes to the tumor or to the tissue in which the tumor exists (e.g., lymphoid tissue).
  • the subject has an autoimmune disease. In some embodiments, the subject has an infection.
  • the subject is in need of hematopoietic reconstitution as a result of, for example, myeloablative chemotherapy and/or radiation.
  • the cell is a gut-specific T cell. In some embodiments, the cell is a skin-specific T cell.
  • 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.
  • the cell is a eukaryotic cell such as a mammalian cell.
  • the mammalian cell is a human cell.
  • the cell is a prokaryotic cell such as a bacterial cell.
  • the bacterial cell may be a Salmonella bacterial cell.
  • the prokaryotic cell, such as a bacterial cell may be attenuated so as to prevent an infection in the subject.
  • 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.
  • the nanoparticle comprises maleimide reactive groups on its surface. In some embodiments, the nanoparticle comprises a lipid coating.
  • 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.
  • 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 ⁇ . 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.
  • 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.
  • the agents are anti-cancer agents including anticancer antibodies, cancer antigens, anti-cancer chemotherapeutic agents, and the like.
  • the agents may be used at doses that are below doses required to achieve the same effects in vivo following systemic administration.
  • 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.
  • the cell is covalently bound to a plurality of nanoparticles.
  • the plurality of nanoparticles comprise an identical agent.
  • the plurality of nanoparticles comprise different agents.
  • 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.
  • the method further comprises binding the nanoparticle to the cell. In some embodiments, the method further comprises providing the cell bound to the nanoparticle.
  • the cell is covalently bound to the nanoparticle.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • the agent is an anti-cancer agent.
  • the agent is an adjuvant.
  • the agent is an antigen.
  • the antigen is a tumor antigen.
  • 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.
  • 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.
  • the tissue homing cell is a T cell.
  • the T cell is a gut-homing T cell.
  • the T cell is a skin-homing T cell.
  • the biodegradable nanoparticle is covalently bound to the tissue homing cell.
  • 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.
  • the invention provides a biodegradable nanoparticle comprising maleimide groups on its exterior surface.
  • the nanoparticle further comprises a lipid bilayer surface.
  • the nanoparticle comprises a poly(lactide-co-glycolide) (PLGA) core.
  • the nanoparticle further comprises an agent.
  • the agent is an immunostimulatory agent.
  • the agent is an antigen.
  • the agent is an antibody.
  • the agent is an adjuvant.
  • the adjuvant is a TLR ligand.
  • the TLR ligand is an immunostimulatory agent in the presence or absence of antigen.
  • the agent is a nucleic acid.
  • the nucleic acid is an siRNA.
  • the agent is an anti-cancer agent.
  • the agent is a cytokine.
  • the agent is an interleukin.
  • the nanoparticle further comprises a plurality of agents.
  • the plurality of agents comprises an antigen and an adjuvant.
  • the plurality of agents comprises an adjuvant and an anti-cancer agent.
  • the plurality of agents comprises an immunostimulatory agent and an anti-cancer agent.
  • 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.
  • the invention provides a composition comprising an isolated T cell comprising a biodegradable nanoparticle at its cell surface, wherein the nanoparticle comprises an agent.
  • the biodegradable nanoparticle is covalently conjugated to the surface of the T cell.
  • 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.
  • 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.
  • FIG. IA Nanoparticle-functionalized T cells for ACT.
  • FIG. IB 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.
  • 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.
  • FIGs. 3A-B Maleimide-functionalized nanoparticles stably link to the surface of T cells without toxicity.
  • Pmel-1 CD8 + 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.
  • 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 + T cells (2500 nanoparticles/cell), then particle-conjugated T cells (bottom panel) or control 'bare' T cells (top panel) were stimulated with mature hgplOO peptide-pulsed bone marrow- derived dendritic cells at a 2:1 T cell:DC ratio; cultures were supplemented with IL-2 every 2 days.
  • FIGs. 5A-C Protein and TLR ligand incorporation in lipid-coated PLGA nanoparticles.
  • 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.
  • 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°C was assessed over 8 days, by measuring fluorescence of the released compounds.
  • FIGs. 8A-D Whole-animal bioluminescence/fluorescence imaging of nanoparticles, nanoparticle-conjugated T cells, and B 16 melanoma tumor models.
  • 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.
  • FIGs. 9A-D Melanoma-targeting Pmel-1 T lymphocytes vehicle surface- conjugated nanoparticles into the tumor microenvironment.
  • A-D 500,000 Bl 6F10 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 x 10 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).
  • T lymphocytes were incubated with lmg/ml Thiol-PEG for 30 min to avoid non-specific uptake of surface-bound nanoparticles by macrophages.
  • 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 (Thyl .1) and DiD nanoparticles (D).
  • FIGs. 10A-B Nanoparticle-decorated T cells function normally and efficiently carry surface-tethered nanoparticles into antigen-expressing tumors.
  • OT-I ova-specific CD8 + 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 Thyl.
  • I + effector OT-I T cells (with or without attached DiD-labeled nanoparticles), or an equivalent number of free nanoparticles.
  • Thy 1.I + OT-I 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.
  • FIGs. HA-C Tumor antigen-specific T cells transport surface-bound nanoparticles into established TRAMP prostate adenocarcinomas.
  • A Six month-old TRP-SIY mice (Bai 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 x 10 6 CBR-luciferase-expressing SIY-specific 2C transgenic CD8 + T cells. T cells were left unmodified or were surface-conjugated with DiD-tagged DNA- gel nanoparticles.
  • FIGs. 12A-F Na ⁇ ve 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 x 10 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 ⁇ -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.
  • FIGs. 13A-D Pmel-1 T cells conjugated with IL- 15Sa/IL-21 -releasing nanoparticles robustly proliferate in vivo and eradicate established B 16 melanomas.
  • A Dual in vivo bioluminescence imaging of Gaussia luciferase-expressing Bl 6F10 lung melanomas and CBR-luciferase-expressing Pmel-1 T cells in sublethally irradiated C57B1/6 mice. Lung tumors established by tail vein injection of B16F10 cells were treated after 6 days by i.v. infusion of 10 x 10 6 V ⁇ l3 + CD8 + Pmel-1 T cells.
  • mice received Pmel-1 T cells conjugated with 100 DNA-gel nanoparticles/cell carrying a total dose of 5 ⁇ g IL-15Sa/IL-21 (4.03 ⁇ g IL-15Sa + 0.93 ⁇ 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 ⁇ l3 + CD8 + 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.
  • FIGs. 14A-D Hematopoietic progenitor cells (referred to in the Figure as HSCs) carrying GSK-3 ⁇ inhibitor-loaded nanoparticles reconstitute recipient animals with rapid kinetics following bone marrow transplants without affecting multilineage differentiation potential.
  • HSCs Hematopoietic progenitor cells
  • 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 ⁇ inhibitor TWSl 19 (1.6 ng) on the day of transplantation, an equivalent TWSl 19 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.
  • 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.
  • 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.
  • clearance is potentially associated with toxicity in the liver due to the accumulation of nanoparticles at that site, also as shown in the Examples.
  • 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).
  • the cells are referred to herein as carrier cells to be distinguished from cells at target sites in vivo.
  • 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).
  • ACT adoptive cell therapy
  • 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. IA.
  • 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.
  • 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.
  • 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.
  • agents include but are not limited to IL-15/IL-15R ⁇ 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.
  • IL-15/IL-15R ⁇ 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
  • 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
  • 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.
  • 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. IB.
  • 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).
  • 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.
  • carrier cells particularly carrier T cells
  • 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.
  • 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.
  • An example of a paracrine method involves the delivery of adjuvant(s) to a target site alone or together with antigen(s).
  • adjuvants 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).
  • 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.
  • the nanoparticles may comprise an anti-cancer agent and/or an adjuvant.
  • tumor-reactive T cells Once delivered to the target tumor site, via tumor-reactive T cells, the anticancer 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(l):81-8, 2008, the teachings of which relating to melanoma-specific T cells are incorporated herein by reference).
  • B cells and/or T cells such as central memory T cells may be conjugated to nanoparticles comprising anti-lymphoma agents.
  • agents are known in the art and include without limitation anti-CD20 antibodies, such as rituximab.
  • 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.
  • 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.
  • tissue e.g., lymphoid tissues
  • the invention contemplates that other blood diseases including without limitation leukemia may also be treated in a like manner.
  • the invention contemplates various other applications where localized delivery of one or more agents and optionally particular cells would be beneficial.
  • the invention contemplates delivery of imaging agents to various distinct regions, tissues and/or organs.
  • 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).
  • 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 TWSl 19 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.
  • GSK3-beta glycogen synthase kinase 3 beta
  • 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.
  • 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.
  • 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, Sea- 1 -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 internal izing/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.
  • 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. 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. 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).
  • 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.
  • the cells may be genetically engineered to express one or more factors including without limitation costimulatory molecules or receptors including chimeric receptors.
  • the cells are not genetically engineered.
  • the carrier cells are isolated and naturally occurring (i.e., they have not been genetically or otherwise engineered).
  • the cells may be manipulated prior to conjugation with the nanoparticles.
  • the cells 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.
  • 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.
  • the carrier cell is a T cell
  • 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-CD40L antibodies, and the like.
  • 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).
  • 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-I, IL-4, IL-6, and TNF, among others.
  • cytokines such as IL-2, gamma- IFN, IL-I, IL-4, IL-6, and TNF, among others.
  • Other ways of measuring expansion and activation are known in the art.
  • 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.
  • T and B cells may be harvested from the peripheral blood of a subject.
  • 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.
  • 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 + , Thyl/CD90 + , CD38 lo/neg , CD33 , and/or c-kit/CDl 17 + . 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.
  • FACS fluorescence activated cell sorting
  • 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.
  • 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.
  • the carrier cells may be eukaryotic cells, such as mammalian cells (e.g., human cells). Alternatively, they may be non-mammalian cells.
  • the carrier cells may be prokaryotic cells (e.g., bacterial cells).
  • prokaryotic cells e.g., bacterial cells.
  • 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: 1 17, 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.
  • 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
  • Nanoparticles 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.
  • the nanoparticles are comprised of a nucleic acid internal core.
  • DNA nanoparticles or DNA-gel nanoparticles
  • 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.
  • the nanoparticles release their agent "payload” over a number of days as a function of their degradation profile in vivo.
  • the nanoparticles are biodegradable in nature and thus they gradually degrade in an aqueous environment such as occurs in vivo.
  • 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).
  • 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.
  • 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).
  • 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.
  • 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).
  • the nanoparticles will not integrate into the lipid bi layer that comprises the cell surface.
  • the nanoparticles will not be phagocytosed (or internalized) by the carrier cells.
  • 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).
  • 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.
  • 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.
  • PEG poly(ethylene glycol)
  • 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.
  • the linkage chemistry is benign and non-toxic as evidenced in part by the conjugation of up to 139 ( ⁇ 29) -200 nm-diameter lipid-coated nanoparticles to the surface of cells without any deleterious effect (data not shown).
  • 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.
  • Nanoparticles bound to carrier 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.
  • 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
  • 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
  • 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 US 6693086, 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.
  • Liposomes may be surface modified during or after synthesis to include reactive groups complementary to the reactive groups on the carrier cells.
  • reactive groups include without limitation maleimide groups.
  • liposomes may be synthesized to include maleimide conjugated phospholipids such as without limitation DSPE-MaL-PEG2000.
  • 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.
  • any agent may be delivered using the methods of the invention provided that it can be loaded into the nanoparticles provided herein.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • agents that are not peptide-based and which could not be synthesized by the transferred cells include chemical compounds that are non-naturally occurring, or chemical compounds that are not naturally synthesized by mammalian (and in particular human) cells.
  • 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.
  • 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 (NfRI).
  • Imaging agents for magnetic resonance imaging include Gd(DOTA), iron oxide or gold nanoparticles; imaging agents for nuclear medicine include 201 Tl, 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 1 Hn; imaging agents for in vivo fluorescence imaging such as fluorescent dyes or dye-conjugated nanoparticles.
  • the agent to be delivered is conjugated, or fused to, or mixed or combined with an imaging agent.
  • 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-CD
  • 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.
  • 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, gplOO, adenosine deaminase-binding protein
  • ADAbp colorectal associated antigen
  • CRC colorectal associated antigen
  • CEA carcinoembryonic antigen
  • CAP-I CAP-I
  • CAP-2 etv ⁇
  • AMLl prostate specific antigen
  • PSA prostate specific antigen
  • PSA-I PSA-2
  • PSA-3 prostate-specific membrane antigen
  • PSMA T cell receptor/CD3-zeta chain
  • CD20 CD20.
  • the cancer antigen may be selected from the group consisting of MAGE-Al, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-AlO, MAGE-Al 1, MAGE-A 12, MAGE- Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-Cl , MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5).
  • the cancer antigen may be selected from the group consisting of GAGE-I, 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-I, NAG, GnT-V, MUM-I, CDK4, tyrosinase, p53, MUC family, HER2/neu, p21ras, RCASl, ⁇ -fetoprotein, E-cadherin, ⁇ -catenin, ⁇ - catenin, ⁇ -catenin, pl20ctn, gpl00 Pmel1 17 , PRAME, NY-ESO-I, cdc27, adenomatous polyposis coli protein (APC), fodrin, Connexin 37, lg-idiotype, pi 5, gp75, GM2 ganglioside, GD2 ganglioside, human pap
  • 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.
  • 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 Lolium perenne or Lolium multiflorum); Cryptomeria (Cryptomeria japonica); Alternaria (Alternaria alternat ⁇ ); Alder; Alnus (Alnus gultinoasa); Betula (Betula verrucosa); Quercus ⁇ Quercus alba); Olea (Olea europ ⁇ ); Artemisia (Artemisia vulgaris); Plantago (e.g. Plantago lanceolata); Parietaria (e.g. Parietaria officinalis or Parietaria judaica); Blattella (e.g. Blattella germanic ⁇ ); Apis (e.g. Apis multiflorum); Cupressus (e.g.
  • Festuca e.g. Festuca elatior
  • Poa e.g. Poa pratensis or Poa compressa
  • 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
  • Adjuvants may be TLR ligands.
  • Adjuvants that act through TLR3 include without limitation double-stranded RNA.
  • Adjuvants that act through TLR4 include wihtout 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.
  • 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.
  • 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.
  • DNA damaging agents 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-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;
  • Eflornithine Elsamitrucin; Enloplatin; Enpromate; Epipropidine; Epirubicin; Erbulozole;
  • Erlotinib (TARCEVA), Esorubicin; Estramustine; Etanidazole; Etoposide; Etoprine;
  • Fadrozole Fazarabine; Fenretinide; Floxuridine; Fludarabine; 5-Fluorouracil;
  • Flurocitabine Flurocitabine; Fosquidone; Fostriecin; Gefitinib (IRESSA), Gemcitabine; Hydroxyurea; Idarubicin; Ifosfamide; Ilmofosine; Imatinib mesylate (GLEEVAC); Interferon alpha-2a;
  • Interferon alpha-2b Interferon alpha-nl; Interferon alpha-n3; Interferon beta-I a;
  • Interferon gamma-I b Interferon gamma-I b; Iproplatin; Irinotecan; Lanreotide; Lenalidomide (REVLLMlD,
  • Mitocarcin Mitocromin; Mitogillin; Mitomalcin; Mitomycin; Mitosper; Mitotane;
  • Pentomone Peplomycin; Perfosfamide; Pipobroman; Piposulfan; Piritrexim Isethionate; Piroxantrone; Plicamycin; Plomestane; Porfimer; Porfiromycin; Prednimustine;
  • Procarbazine Puromycin; Pyrazofurin; Riboprine; Rogletimide; Safingol; Semustine;
  • Taxotere Tecogalan; Tegafur; Teloxantrone; Temoporfin; Temozolomide (TEMODAR); Teniposide; Teroxirone; Testolactone; Thalidomide (THALOMID) and derivatives thereof; Thiamiprine; Thioguanine; Thiotepa; Tiazofurin; Tirapazamine; Topotecan;
  • Vinepidine Vinepidine; Vinglycinate; Vinleurosine; Vinorelbine; Vinrosidine; Vinzolidine; Vorozole; Zeniplatin; Zinostatin; Zorubicin.
  • 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 HDBA (2- Hydroxy5-(2,5-Dihydroxybenzylamino)-2-hydroxybenzoic acid.
  • the CDK inhibitor may be without limitation p21, p27, p57, pl5, pl6, pl8, or pl9.
  • the MAP kinase inhibitor may be without limitation KY12420 (C 23 H 24 O 8 ), CNI-1493, PD98059, or 4-(4- Fluorophenyl)-2-(4-methylsulfinyl phenyl)-5-(4-pyridyl) lH-imidazole.
  • the EGFR inhibitor may be without limitation erlotinib (TARCEVA), gefitinib (IRESSA), WHI- P97 (quinazoline derivative), LFM-A12 (leflunomide metabolite analog), ABX-EGF, lapatinib, canertinib, ZD-6474 (ZACTIMA), AEE788, and AG1458.
  • 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.
  • AVASTIN bevacizumab
  • ranibizumab LCENTIS
  • MACUGEN pegaptanib
  • sorafenib sunitinib
  • SUTENT sunitinib
  • ZACTIMA ZACTIMA
  • anecortave squalamine lactate
  • semaphorin semaphorin
  • 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),
  • 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, Wntl IA, and the like.
  • 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, Notch 1 activators, Lnk inhibitors, prostaglandin E2 (PGE2) and agents that stimulate the PGE2 pathway including PGE2, PGI2, Linoleic Acid, 13(s)-HODE, LYl 71883, Mead Acid, Eicosatrienoic Acid, Epoxyeicosatrienoic Acid, ONO-259, Cay 1039, 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-di
  • 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.
  • the agent may be an anti-infective agent including without limitation an anti-bacterial agent, an anti-viral agent, an anti-parasitic agent, an antifungal agent, and an anti-mycobacterial agent.
  • Anti-bacterial agents may be without limitation ⁇ -lactam antibiotics, penicillins
  • cephalosporins first generation, second generation, and third generation cephalosporins
  • other ⁇ -lactams such as imipenem, monobactams
  • ⁇ - lactamase inhibitors vancomycin, aminoglycosides and spectinomycin, tetracyclines, chloramphenicol, erythromycin, lincomycin, clindamycin, rifampin, metronidazole, polymyxins, sulfonamides and trimethoprim, or quinolines.
  • 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;
  • Cefepime Hydrochloride Cefetecol; Cefixime; Cefmenoxime Hydrochloride;
  • Cefmetazole Cefmetazole Sodium; Cefonicid Monosodium; Cefonicid Sodium;
  • Cefprozil Cefroxadine; Cefsulodin Sodium; Ceftazidime; Ceftibuten; Ceftizoxime
  • Cetocycline Hydrochloride Cetophenicol; Chloramphenicol; Chloramphenicol
  • Chlorhexidine Phosphanilate Chloroxylenol; Chlortetracycline Bisulfate;
  • Chlortetracycline Hydrochloride Cinoxacin; Ciprofloxacin; Ciprofloxacin Hydrochloride; Cirolemycin; Clarithromycin; Clinafloxacin Hydrochloride;
  • Dicloxacillin Sodium Dihydrostreptomycin Sulfate; Dipyrithione; Dirithromycin;
  • Doxycycline Doxycycline Calcium; Doxycycline Fosfatex; Doxycycline Hyclate;
  • Floxacillin Fludalanine; Flumequine; Fosfomycin; Fosfomycin Tromethamine; Fumoxicillin; Furazolium Chloride; Furazolium Tartrate; Fusidate Sodium; Fusidic
  • Meclocycline Meclocycline Subsalicylate; Megalomicin Potassium Phosphate;
  • Methenamine Hippurate Methenamine Mandelate; Methicillin Sodium; Metioprim;
  • Metronidazole Hydrochloride Metronidazole Hydrochloride; Metronidazole Phosphate; Mezlocillin; Mezlocillin Sodium; Minocycline; Minocycline Hydrochloride; Mirincamycin Hydrochloride;
  • Neomycin Natamycin; Nebramycin; Neomycin Palmitate; Neomycin Sulfate; Neomycin
  • Oxytetracycline Oxytetracycline Calcium; Oxytetracycline Hydrochloride; Paldimycin;
  • Penicillin G Benzathine; Penicillin G Potassium; Penicillin G Procaine; Penicillin G Sodium; Penicillin V; Penicillin V Benzathine; Penicillin V Hydrabamine; Penicillin V
  • 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 Rosithromycin
  • Sancycline Rosafetrinem Sodium
  • Sarmoxicillin Rosaramicin Propionate
  • Rosaramicin Sodium Phosphate Rosaramicin Stearate
  • Rosoxacin Rosoxacin
  • Roxarsone Rosithromycin
  • Sancycline Sanfetrinem Sodium
  • Sarmoxicillin Sarmoxicillin
  • Sulfamerazine Sulfameter; Sulfamethazine; Sulfamethizole; Sulfamethoxazole;
  • Sulfamonomethoxine Sulfamoxole; Sulfanilate Zinc; Sulfanitran; Sulfasalazine;
  • Tetracycline Tetracycline Hydrochloride; Tetracycline Phosphate Complex;
  • Tobramycin Tobramycin; Tobramycin Sulfate; Tosufloxacin; Trimethoprim; Trimethoprim Sulfate; Trisulfapyrimidines; Troleandomycin; Trospectomycin Sulfate; Tyrothricin;
  • Vancomycin Vancomycin Hydrochloride
  • Virginiamycin Vancomycin Hydrochloride
  • Zorbamycin Vancomycin Hydrochloride
  • Anti-mycobacterial agents may be without limitation Myambutol (Ethambutol)
  • 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),
  • Anti-viral agents may be without limitation amantidine and rimantadine, ribivarin, acyclovir, vidarabine, trifluorothymidine, ganciclovir, zidovudine, retinovir, and interferons. Anti-viral agents may be without limitation further include Acemannan;
  • Acyclovir Acyclovir Sodium; Adefovir; Alovudine; Alvircept Sudotox; Amantadine
  • Cipamfylline Cytarabine Hydrochloride
  • Delavirdine Mesylate Desciclovir
  • Ganciclovir Ganciclovir Sodium; Idoxuridine; Kethoxal; Lamivudine; Lobucavir;
  • 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.
  • 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
  • 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; Fluda
  • Nucleic Acid Agents 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 US 6194388 (the teachings of which relating to immunostimulatory CpG nucleic acids are inco ⁇ orated 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.
  • DNA including cDNA, genomic DNA, nuclear DNA, mitochondrial DNA
  • RNA including mRNA, rRNA
  • 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; antianxiety; anti-arthritic; anti-asthmatic including ⁇ -adrenergic agonists, methylxanthines, mast cell stabilizing agents, anticholinergics, adrenocortical steroids such as glucocorticoids; anti-atherosclerotic; anticholelithic
  • Subjects 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • cancer causing agents i.e., carcinogens
  • 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.
  • 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.
  • 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.
  • the nanoparticles may comprise an antimicrobial 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.
  • 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.
  • 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.
  • the agents may be self- renewal agents (i.e., agents that drive the progenitor cells to self-renew).
  • 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.
  • 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).
  • 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.
  • NSCLC non-small cell lung cancer
  • Sarcomas are rare mesenchymal neoplasms that arise in bone (osteosarcomas) and soft tissues (fibrosarcomas).
  • Sarcomas include without limitation liposarcomas (including myxoid liposarcomas and pl omorphic 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,
  • Melanomas are tumors arising from the melanocyte 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.
  • the cancer may be a solid tumor lymphoma. Examples include Hodgkin's lymphoma, Non-Hodgkin's lymphoma, and B cell lymphoma.
  • 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.
  • 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.
  • an infection such as a bacterial infection, a viral infection, a fungal infection, a parasitic infection or a mycobacterial infection.
  • the bacterial infection may be without limitation an E.
  • the mycobacterial infection may be without limitation tuberculosis or leprosy respectively caused by the M. tuberculosis and M. leprae species.
  • 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, HlNl (swine flu) infection, respiratory syncytial virus infection, varicella-zoster virus infections, small pox infection, monkey pox infection, SARS infection or avian flu infection.
  • a Herpes simplex virus 1 infection a Herpes simplex virus 2 infection
  • cytomegalovirus infection hepatitis A virus infection
  • hepatitis B virus infection hepatitis B virus infection
  • hepatitis C virus infection human papilloma virus infection
  • Epstein Barr virus infection Epstein Barr virus infection
  • rotavirus infection adenovirus infection
  • the fungal infection may be without limitation candidiasis, ringworm, histoplasmosis, blastomycosis, paracoccidioidomycosis, crytococcosis, aspergillosis, chromomycosis, mycetoma infections, pseudallescheriasis, or tinea versicolor infection.
  • 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.
  • 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 ThI cytokines, such as IL-12 and IFN-gamma, according to the invention can be used to treat allergy or asthma.
  • Autoimmune Disease 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.
  • an immune response is mounted against a subject's own antigens, referred to as self antigens.
  • Autoimmune diseases are generally considered to be ThI biased.
  • Th2 immune response or Th2 like cytokines can be beneficial.
  • Such cytokines include IL-4, IL-5 and IL-IO.
  • 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.
  • SLE systemic
  • Transplant Therapy 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.
  • the invention contemplates administration of the nanoparticle-cell conjugates to subjects that are going to undergo, are undergoing, or have undergone a transplant.
  • 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.
  • 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.
  • 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.
  • compositions are sterile compositions that comprise cells, nanoparticles and/or agent(s), preferably in a pharmaceutically-acceptable carrier.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ 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 ⁇ 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).
  • PLGA poly(lactide-co-glycolide)
  • 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°C (short term storage) or lyophilized in the presence of trehalose and stored at 4°C until used.
  • Simple variations in the processing conditions e.g., use of homogenization instead of sonication
  • DLS dynamic light scattering
  • X-DNA monomer was then admixed to 6.7 ⁇ l T4 DNA ligase (3 Weiss units/ ⁇ l, Promega), 20 ⁇ l T4 ligase buffer (Promega) and nuclease-free water (IDT) to a total volume of 200 ⁇ 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.
  • T4 DNA ligase 3 Weiss units/ ⁇ l, Promega
  • 20 ⁇ l T4 ligase buffer Promega
  • IDT nuclease-free water
  • the resulting DNA gel-lipid mixture was sonicated on ice (5 min total, alternating power cycles of 1 W and 5 Watts every 30s 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°C and overnight incubation at 4°C to allow ligase-mediated X-DNA crosslinking, 4 ⁇ l Exonuclease III (New England Biolabs), 20 ⁇ l Buffer 1 (New England Biolabs) and nuclease-free water to a total volume of 200 ⁇ l was were added and incubated at 37°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 10 DNA gel nanoparticles in the 200-250 nm diameter range was measured using a 90Plus Particles Size Analyzer (Brookhaven Instruments).
  • IL-15Sa/IL-21 encapsulation in DNA-gel nanoparticles
  • 30 ⁇ g recombinant mouse IL-15R ⁇ /Fc chimera was precomplexed with 10 ⁇ 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 ⁇ g mouse IL-21 (Peprotech) and blended with the X-DNA/T4 ligase mixture for DNA-gel particle synthesis, following the procedure described above.
  • TWSl 19 (Cayman Chemical) 1 mg TWS was resuspended in 250 ⁇ l DMSO, before adding it to the X- DNA/T4 ligase mixture for DNA-gel particle synthesis.
  • 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).
  • lipid-like dyes such as carbocyanine dyes (DiD, Invitrogen)
  • 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 + T cells express a T cell receptor which recognizes a peptide from murine gplOO, a melanoma self-antigen expressed by B16 melanoma tumor cells that is also used as a T cell target in human melanoma vaccines.
  • 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 gplOO.
  • 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.
  • 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.
  • T cells, B cells and hematopoietic progenitor cells were found to have high levels of free thiols at the cell surface, though red blood cells did not (not shown). Based on these results, we developed the strategy outlined in FIG. 2D. Nanoparticle carriers were prepared which included lipids with maleimide-terminated headgroups.
  • CD8 + 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 x 10 6 cells/mL) with maleimide-functionalized- nanoparticles (at varying concentrations) at 37°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 x 10 6 /mL) with 1 mg/mL thiol-terminated 2 KDa poly(ethylene glycol) (PEG, Laysan Bio) at 37°C for 30 min in complete RPMI medium, followed by two washes to remove unbound PEG.
  • PEG poly(ethylene glycol)
  • FIG. 3A nanoparticles were readily attached to cells using this thiol-reaction strategy. 2500 nanoparticles per cell during conjugation as shown in FIG.
  • 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.
  • a key issue for these studies was the localization of the particles.
  • 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.
  • 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 ⁇ M CpG oligonucleotide (a ligand for TLR 9) and pulsed with 1 ⁇ M hgpl00 25O3 peptide (a peptide recognized by pmel-1 T cells in the context of H-2D 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.
  • Cytokine Secretion or CTL Activity Activated CD8 + T cells secrete cytokines such as IFN- ⁇ and TNF- ⁇ and directly kill antigen-bearing target cells as part of their anti-tumor activity.
  • cytokines such as IFN- ⁇ and TNF- ⁇
  • 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.
  • pmel-1 T cells decorated with nanoparticles produced equivalent amounts of IL-2, IFN- ⁇ and TNF- ⁇ in response to antigen stimulation as unmodified 'bare' T cells.
  • substantial quantities of nanoparticles can be bound to cells without blocking effector cytokine secretion.
  • 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 51 Cr-labeled EL4 target cells pulsed with hgpl002 5 - 33 peptide at varying effecto ⁇ target ratios for 4 hrs at 37°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).
  • TCR-Transgenic OT-I CD8+ TCeIl Analysis Similar results were found with other T cells.
  • TCR-transgenic OT-I CD8 + 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).
  • 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.
  • TLR Toll-like receptor
  • MPLA vaccine antigens
  • 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.
  • 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 HCl, and measuring released ova fluorescence calibrated against ova solution standards exposed to the same base treatment conditions. By these measurements, we found that ⁇ 1 ⁇ g of ova per mg nanoparticles was encapsulated (-25% encapsulation efficiency).
  • 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.
  • IL-15 interleukin-15
  • the kinetics of IL-15 release from the particles was determined by incubating the particles in complete RPMI medium containing 10% FCS at 37°C with gentle agitation and taking aliquots of the supernatant at staggered timepoints for ELISA analysis of cytokine content. As shown in FIG. 5B, -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.
  • the lipid-coated particles can be loaded with protein and release encapsulated material over a -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.
  • 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 ⁇ .
  • Particle- conjugated or control T cells were then co-cultured with EL4 target cells pulsed with hgplOO 2 5-33 peptide at effecto ⁇ target ratios of 20:1 at 37°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 ⁇ 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 IL 15 and IL-15/IL-15R ⁇ controls.
  • IL- 15 or its superagonist complexed with IL-15R ⁇ continuously released from nanoparticles maintain its bioactivity and is able to support T cell survival and/or proliferation in vitro.
  • 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.
  • 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.
  • 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.)
  • 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 ⁇ 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.
  • 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-I, 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.
  • 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(l):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.
  • gardiquimod for encapsulation of gardiquimod in nanoparticles, 200 ⁇ L water in the synthesis protocol described in section 3.1 was replaced with 1.8 mg of gardiquimod dissolved in 200 ⁇ 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.
  • T lymphocytes 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" mo ⁇ hology, 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.
  • 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).
  • 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.
  • FIG. 8C 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.
  • 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.
  • nanoparticles can be tracked using near-IR dyes and fluorescence.
  • Gaussia-luciferase-expressing Bl 6F10 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.
  • T cell receptor-transgenic for the melanoma antigen gp-100 into hosts with established Bl 6F10 tumors in their right femur (FIG. 9A).
  • Animals were treated with 15 x 10 6 Pmel-l 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).
  • 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).
  • 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).
  • FIG. 9D multicolor flow cytometry of tumor single cell suspensions
  • 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-I 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.
  • F-luc Firefly luciferase
  • FIG. 10A Particle-carrying OT-I T cells specifically trafficked to pre-established EL4-OVA tumors.
  • FIG. 10B 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-I T cells compared to systemically infused free nanoparticles, which were rapidly scavenged by the liver and the spleen.
  • 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).
  • 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. 11 A-C).
  • 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.
  • DNA-gel particles were linked to resting CCR7 + CD62L + B cells (FIGs. 12A-C) or central memory CD8 + 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. 12A-C resting CCR7 + CD62L + B cells
  • central memory CD8 + T cells data not shown
  • 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.
  • 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.
  • DNA-gel particles -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 + Pmel-1 effector T cells which recognize a peptide from the melanocyte differentiation antigen gplOO.
  • CBR Click bettle red
  • Particle-conjugated or control T cells were infused into lympho-depleted mice bearing established Gaussia luciferase- expressing Bl 6F 10 melanoma lung tumors (FIG. 13A).
  • 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 + CD62L + central memory T cells to lymph nodes and spleen (FIGs. 13A and B, and 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).
  • 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.
  • 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-Sea- 1 microbeads resulted in an average 92% purity of lin " Sca " l + c-kit + HSCs.
  • Cells were kept in serum-free StemSpan (Stem Cell Technologies) for 3 hours before further modification.
  • 1 x 10 4 unmodified or nanoparticle-decorated HSCs were transplanted by retroorbital injection into lethally irradiated (1300 cGy of total body irradiation from a 137 Cs source as a split dose with 3-hr interval between) nontransgenic recipients.
  • HSCs retrovirally transduced with NUP98- HOXA lOhd, were cultured in DMEM supplemented with 15% FBS and cytokines (6 ng/mL of IL-3, 10 ng/mL of IL-6, lOOng/mL of SCF, all Preprotech).
  • glycogen synthase kinase-3 ⁇ (GSK-3 ⁇ ) inhibitor TWSl 19 (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).
  • a DOPC/DOPG/MPB PE/DiD lipid film (lipid ratios as in polymer nanoparticles) was hydrated with 185 ⁇ l PBS for a one-hour period with vigorous vortexing every 10 minutes. After six cycles of freezing (liquid N2) 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).
  • 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.
  • inventive 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.
  • 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.
  • 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.
  • “at least one of A and 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.
  • 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.

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Abstract

La présente invention concerne des compositions et des procédés visant à délivrer des agents dans des régions localisées, des tissus ou des organes in vivo en conjuguant des nanoparticules chargées en agent à des cellules possédant une capacité d’écotropisme. Il peut s’agir d’agents thérapeutiques ou diagnostiques, par exemple des agents chimiothérapeutiques ou des agents d'imagerie, respectivement.
PCT/US2009/006290 2008-11-24 2009-11-24 Procédés et compositions pour la délivrance localisée d'agents WO2010059253A2 (fr)

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US20180110733A1 (en) 2018-04-26
US9393199B2 (en) 2016-07-19
US20110293705A1 (en) 2011-12-01
WO2010059253A9 (fr) 2010-10-14
US20210259968A1 (en) 2021-08-26
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EP2398466B1 (fr) 2021-02-17
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US20160256386A1 (en) 2016-09-08

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